The Pigmentary System,

The Pigmentary System: Physiology and Pathophysiology

The Pigmentary System: Physiology and Pathophysiology Edited by

James J. Nordlund Dermatologist and Professor Emeritus, Group Health Associates, Cincinnati, OH, USA

Raymond E. Boissy Professor of Dermatology and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA

Vincent J. Hearing Chief, Pigment Cell Biology Section, Laboratory of Cell Biology, National Institutes of Health, Bethesda, MD, USA

Richard A. King Director, Genetics Division, Department of Medicine, Institute of Human Genetics, University of Minnesota, Minneapolis, MN, USA

William S. Oetting Associate Professor, Genetics Division, Department of Medicine Institute of Human Genetics, University of Minnesota, Minneapolis, MN, USA

Jean-Paul Ortonne Professor of Dermatology and Chairman, Department of Dermatology, University of Nice-Sophia Antipolis, Nice, France

SECOND EDITION

© 2006 Blackwell Publishing Ltd Blackwell Publishing, Inc., 350 Main Street, Malden, Massachusetts 02148-5020, USA Blackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK Blackwell Publishing Asia Pty Ltd, 550 Swanston Street, Carlton, Victoria 3053, Australia The right of the Author to be identified as the Author of this Work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. First published 1998 by Oxford University Press Second edition 2006 1 2006 Library of Congress Cataloging-in-Publication Data The pigmentary system : physiology and pathophysiology / edited by James J. Nordlund . . . [et al.]. — 2nd ed. p. ; cm. Includes bibliographical references and index. 1. Pigmentation disorders. 2. Melanocytes. I. Nordlund, James J. [DNLM: 1. Pigmentation Disorders–physiopathology. 2. Melanocytes. 3. Pigmentation–physiology. WR 265 P6309 2006] RL790.P53 2006 616.5¢5–dc22 2005030369 A catalogue record for this title is available from the British Library Set in 9/12 pt Sabon by SNP Best-set Typesetter Ltd., Hong Kong Printed and bound in India by Replika Press PVT Ltd ISBN-13: 978-1-4051-2034-0 ISBN-10: 1-4051-2034-7 Commissioning Editor: Stuart Taylor Editorial Assistant: Saskia Van der Linden Development Editor: Rob Blundell Production Controller: Kate Charman For further information on Blackwell Publishing, visit our website: http://www.blackwellpublishing.com The publisher’s policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp processed using acid-free and elementary chlorine-free practices. Furthermore, the publisher ensures that the text paper and cover board used have met acceptable environmental accreditation standards. Blackwell Publishing makes no representation, express or implied, that the drug dosages in this book are correct. Readers must therefore always check that any product mentioned in this publication is used in accordance with the prescribing information prepared by the manufacturers. The author and the publishers do not accept responsibility or legal liability for any errors in the text or for the misuse or misapplication of material in this book.

Contents

Contributors, x Foreword, xv Preface, xvii Acknowledgements, xviii Frontispiece can be found between pages ii and iii Part I: The Physiology of the Pigmentary System Section 1: Historical and Comparative Perspectives of the Pigmentary System, 3 1 A History of the Science of Pigmentation, 5 Sidney N. Klaus 2 Comparative Anatomy and Physiology of Pigment Cells in Nonmammalian Tissues, 11 Joseph T. Bagnara & Jiro Matsumoto Section 2: The Science of Pigmentation, 61 3 General Biology of Mammalian Pigmentation, 63 Walter C. Quevedo Jr. & Thomas J. Holstein 4 Extracutaneous Melanocytes, 91 Raymond E. Boissy & Thomas J. Hornyak 5 Regulation of Melanoblast Migration and Differentiation, 108 David M. Parichy, Mark V. Reedy, & Carol A. Erickson 6 Melanoblast Development and Associated Disorders, 140 Richard A. Spritz 7 Biogenesis of Melanosomes, 155 Raymond E. Boissy, Marjan Huizing, & William A. Gahl 8 Melanosome Trafficking and Transfer, 171 Glynis A. Scott 9 Melanosome Processing in Keratinocytes, 181 H. Randolph Byers 10 The Regulation of Melanin Formation, 191 Vincent J. Hearing 11 The Tyrosinase Gene Family, 213 William S. Oetting & Vijayasaradhi Setaluri 12 Molecular Regulation of Melanin Formation: Melanosome Transporter Proteins, 230 Murray H. Brilliant 13 Transcriptional Regulation of Melanocyte Function, 242 Kazuhisa Takeda & Shigeki Shibahara 14 Enzymology of Melanin Formation, 261 Francisco Solano & José C. García-Borrón

15 Chemistry of Melanins, 282 Shosuke Ito & Kazumasa Wakamatsu 16 The Physical Properties of Melanins, 311 Tadeusz Sarna & Harold A. Swartz 17 Photobiology of Melanins, 342 Antony R. Young 18 Toxicological Aspects of Melanin and Melanogenesis, 354 Edward J. Land, Christopher A. Ramsden, & Patrick A. Riley 19 Regulation of Pigment Type Switching by Agouti, Melanocortin Signaling, Attractin, and Mahoganoid, 395 Gregory S. Barsh 20 Human Pigmentation: Its Regulation by Ultraviolet Light and by Endocrine, Paracrine, and Autocrine Factors, 410 Zalfa Abdel-Malek & Ana Luisa Kadekaro 21 Paracrine Interactions of Melanocytes in Pigmentary Disorders, 421 Genji Imokawa 22 Growth Factor Receptors and Signal Transduction Regulating the Proliferation and Differentiation of Melanocytes, 445 Ruth Halaban & Gisela Moellmann 23 Aging and Senescence of Melanocytes, 464 Debdutta Bandyopadhyay & Estela E. Medrano 24 The Genetics of Melanoma, 472 Vanessa C. Gray-Schopfer & Dorothy C. Bennett 25 The Transformed Phenotype of Melanocytes, 489 Dong Fang & Meenhard Herlyn Part II: The Pathophysiology of Pigmentary Disorders Section 3: An Overview of Human Skin Color and its Disorders, 497 26 A More Precise Lexicon for Pigmentation, Pigmentary Disorders, and “Chromatic” Abnormalities, 499 James J. Nordlund, Tania Cestari, Pearl Grimes, Henry Chan, & Jean-Paul Ortonne 27 The Normal Color of Human Skin, 504 James J. Nordlund & Jean-Paul Ortonne 28 Mechanisms that Cause Abnormal Skin Color, 521 Jean-Paul Ortonne & James J. Nordlund v

CONTENTS

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Section 4: Disorders of Hypopigmentation, Depigmentation and Hypochromia, 539 Genetic Hypomelanoses: Disorders Characterized by Congenital White Spotting — Piebaldism, Waardenburg Syndrome, and Related Genetic Disorders of Melanocyte Development — Clinical Aspects, 541 Richard A. Spritz Genetic Hypomelanoses: Acquired Depigmentation, 551 Rozycki Syndrome, 551 Jean L. Bolognia Vitiligo Vulgaris, 551 James J. Nordlund, Jean-Paul Ortonne, & I. Caroline Le Poole Genetic Hypomelanoses: Generalized Hypopigmentation, 599 Oculocutaneous Albinism, 599 Richard A. King & William S. Oetting Albinoid Disorders, 613 Philippe Bahadoran & Jean-Paul Ortonne Ataxia Telangiectasia, 621 Anne-Sophie Gadenne Hallerman–Streiff Syndrome, 623 James J. Nordlund Histidinemia, 623 Marnie D. Titsch Homocystinuria, 625 Allan D. Mineroff Oculocerebral Syndrome with Hypopigmentation, 626 Jean L. Bolognia Tietz Syndrome, 630 Jean-Paul Ortonne Kappa-Chain Deficiency, 631 Jean-Paul Ortonne Menkes’ Kinky Hair Syndrome, 631 Tanusin Ploysangam Phenylketonuria, 634 Allan D. Mineroff Genetic Hypomelanoses: Localized Hypopigmentation, 636 “Hypomelanosis of Ito” and Mosaicism, 636 Wolfgang Küster & Rudolf Happle Focal Dermal Hypoplasia, 645 James J. Nordlund Hypomelanosis with Punctate Keratosis of the Palms and Soles, 646 Jean L. Bolognia Darier–White Disease (Keratosis Follicularis; 124200), 647 Jean L. Bolognia Nevus Depigmentosus, 649 Stella D. Calobrisi Tuberous Sclerosis Complex, 652 Pranav B. Sheth Genetic Hypomelanoses: Disorders Characterized by Hypopigmentation of Hair, 657

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Bird-Headed Dwarfism (Seckel Syndrome), 657 Stan P. Hill Down Syndrome, 658 Rosemary Geary Fisch Syndrome, 659 Stan P. Hill Premature Canities, 659 James J. Nordlund Mandibulofacial Dysostosis (Treacher Collins Syndrome), 660 Rosemary Geary Myotonic Dystrophy, 660 Peggy Tong PHC Syndrome (Böök Syndrome), 661 Stan P. Hill Pierre Robin Syndrome, 661 James J. Nordlund Prolidase Deficiency, 662 Pranav B. Sheth Metabolic, Nutritional, and Endocrine Disorders, 664 Metabolic and Nutritional Hypomelanoses, 664 Peter S. Friedmann Hypomelanosis Associated with Endocrine Disorders, 667 Peter S. Friedmann Chemical, Pharmacologic, and Physical Agents Causing Hypomelanoses, 669 Chemical and Pharmacologic Agents Causing Hypomelanoses, 669 Stefania Briganti, Monica Ottaviani, & Mauro Picardo Physical Agents Causing Hypomelanoses, 683 Jean-Philippe Lacour Infectious Hypomelanoses, 686 Jean-Philippe Lacour Inflammatory Hypomelanoses, 699 Jean-Philippe Lacour Hypomelanoses Associated with Melanocytic Neoplasia, 705 Lieve Brochez, Barbara Boone, & Jean-Marie Naeyaert Miscellaneous Hypomelanoses: Depigmentation, 725 Alezzandrini Syndrome, 725 Wiete Westerhof, David Njoo, & Henk E. Menke Idiopathic Guttate Hypomelanosis, 726 Wiete Westerhof, David Njoo, & Henk E. Menke Leukoderma Punctata, 729 Wiete Westerhof, David Njoo, & Henk E. Menke Lichen Sclerosus et Atrophicus, 731 Philippe Bahadoran Vagabond Leukomelanoderma, 732 Wiete Westerhof, David Njoo, & Henk E. Menke Vogt–Koyanagi–Harada Syndrome, 734 Wiete Westerhof, David Njoo, & Henk E. Menke Westerhof Syndrome, 741 Wiete Westerhof, David Njoo, & Henk E. Menke

CONTENTS

40 Miscellaneous Hypomelanoses: Hypopigmentation, 745 Disseminated Hypopigmented Keratoses, 745 Wiete Westerhof, David Njoo, & Henk E. Menke Hypermelanocytic Punctata et Guttata Hypomelanosis, 746 Wiete Westerhof, David Njoo, & Henk E. Menke Progressive Macular Hypomelanosis, 748 Henk E. Menke, Germaine Relyveld, David Njoo, & Wiete Westerhof Sarcoidosis, 751 Wiete Westerhof, David Njoo, & Henk E. Menke 41 Miscellaneous Hypomelanoses: Extracutaneous Loss of Pigmentation, 754 Alopecia Areata, 754 Wiete Westerhof Heterochromia Irides, 756 Wiete Westerhof, David Njoo, & Henk E. Menke Senile Canities, 760 Wiete Westerhof, David Njoo, & Henk E. Menke Sudden Whitening of Hair, 764 Wiete Westerhof, David Njoo, & Henk E. Menke 42 Hypochromia without Hypomelanosis, 767 Jean-Philippe Lacour Section 5: Disorders of Hyperpigmentation and Hyperchromia, 769 43 Genetic Epidermal Syndromes: Disorders Characterized by Generalized Hyperpigmentation, 771 Adrenoleukodystrophy, 771 Sheila S. Galbraith & Nancy Burton Esterly Familial Progressive Hyperpigmentation, 774 Nancy Burton Esterly, Eulalia Baselga, Beth A. Drolet, Susan Bayliss Mallory, & Sharon A. Foley Fanconi Anemia, 776 Sheila S. Galbraith & Nancy Burton Esterly Gaucher Disease, 778 Sheila S. Galbraith & Nancy Burton Esterly 44 Genetic Epidermal Syndromes: Disorders Characterized by Reticulated Hyperpigmentation, 780 Berlin Syndrome, 780 Eulalia Baselga & Nancy Burton Esterly Cantu Syndrome, 781 Eulalia Baselga & Nancy Burton Esterly Kindler Syndrome, 781 Eulalia Baselga & Nancy Burton Esterly Dermatopathia Pigmentosa Reticularis, 784 Eulalia Baselga & Nancy Burton Esterly Dyschromatosis Universalis Hereditaria, 786 Eulalia Baselga & Nancy Burton Esterly Epidermolysis Bullosa with Mottled Pigmentation, 788 Eulalia Baselga & Nancy Burton Esterly Familial Mandibuloacral Dysplasia, 790 Eulalia Baselga & Nancy Burton Esterly Hereditary Acrokeratotic Poikiloderma, 792 Eulalia Baselga & Nancy Burton Esterly

Hereditary Sclerosing Poikiloderma, 795 Eulalia Baselga & Nancy Burton Esterly Mendes Da Costa Disease, 796 Eulalia Baselga & Nancy Burton Esterly Naegeli–Franceschetti–Jadassohn Syndrome, 798 Eulalia Baselga & Nancy Burton Esterly Reticulated Acropigmentation of Dohi (Dyschromatosis Symmetrica Hereditaria), 799 Eulalia Baselga & Nancy Burton Esterly Reticulate Acropigmentation of Kitamura, 802 Eulalia Baselga & Nancy Burton Esterly Rothmund–Thomson Syndrome, 804 Eulalia Baselga & Nancy Burton Esterly 45 Genetic Epidermal Syndromes with Café-au-lait Macules, 809 Familial Multiple Café-au-lait Spots, 809 Nancy Burton Esterly Neurofibromatosis, 809 Nancy Burton Esterly, Eulalia Baselga, & Sheila S. Galbraith Neurofibromatosis 1 with Noonan Syndrome, 816 Nancy Burton Esterly McCune–Albright Syndrome, 817 Sheila S. Galbraith & Nancy Burton Esterly Segmental Neurofibromatosis, 819 Nancy Burton Esterly & Eulalia Baselga Silver–Russell Syndrome, 820 Nancy Burton Esterly, Eulalia Baselga, & Sheila S. Galbraith Watson Syndrome, 823 Nancy Burton Esterly 46 Genetic Epidermal Pigmentation with Lentigines, 824 Lentigo Simplex, 824 Mary K. Cullen Lentigo Senilis et Actinicus, 829 Mary K. Cullen Centrofacial Lentiginosis, 837 Mary K. Cullen LEOPARD Syndrome, 842 Mary K. Cullen Carney Complex, 851 Mary K. Cullen Other Lentiginoses, 863 Mary K. Cullen 47 Genetic Epidermal Syndromes: Localized Hyperpigmentation, 873 Anonychia with Flexural Pigmentation, 873 Nancy Burton Esterly, Eulalia Baselga, & Beth A. Drolet Incontinentia Pigmenti, 873 Sheila S. Galbraith & Nancy Burton Esterly Periorbital Hyperpigmentation, 879 Nancy Burton Esterly, Eulalia Baselga, & Beth A. Drolet vii

CONTENTS

Pigmentary Demarcation Lines, 880 Sheila S. Galbraith & Nancy Burton Esterly Dowling–Degos Disease, 882 Sheila S. Galbraith & Nancy Burton Esterly 48 Genetic Epidermal Syndromes: Disorders of Aging, 884 Acrogeria, 884 Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, & Beth A. Drolet Metageria, 886 Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, & Beth A. Drolet Progeria, 886 Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, & Beth A. Drolet Xeroderma Pigmentosum, 889 Anita P. Sheth, Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, & Beth A. Drolet Werner Syndrome, 894 Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, Beth A. Drolet, & Cindy L. Lamerson 49 Congenital Epidermal Hypermelanoses, 898 Dyskeratosis Congenita, 898 Susan Bayliss Mallory, Peggy L. Chern, & Sharon A. Foley Ectodermal Dysplasias, 901 Susan Bayliss Mallory, Peggy L. Chern, & Sharon A. Foley Transient Neonatal Pustular Melanosis, 905 Susan Bayliss Mallory, Peggy L. Chern, & Sharon A. Foley Universal Acquired Melanosis, 906 Susan Bayliss Mallory, Peggy L. Chern, & Sharon A. Foley 50 Acquired Epidermal Hypermelanoses, 907 Acanthosis Nigricans, 907 Norman Levine & Cynthia Burk Acromelanosis Progressiva, 914 Norman Levine & Cynthia Burk Becker Nevus, 915 Norman Levine & Cynthia Burk Café-au-lait Spots, 917 Norman Levine & Cynthia Burk Carcinoid Syndrome, 919 Norman Levine & Cynthia Burk Confluent and Reticulated Papillomatosis, 922 Norman Levine & Cynthia Burk Cutaneous Amyloidosis, 924 Norman Levine & Cynthia Burk Dermatosis Papulosa Nigra, 928 Norman Levine & Cynthia Burk Ephelides (Freckles), 929 Norman Levine & Cynthia Burk Erythema ab Igne, 931 Norman Levine & Cynthia Burk viii

Erythema Dyschromicum Perstans, 933 Norman Levine & Cynthia Burk Erythromelanosis Follicularis Faciei et Colli, 935 Norman Levine & Cynthia Burk Erythrose Péribuccale Pigmentaire of Brocq, 937 Norman Levine & Cynthia Burk Extracutaneous Neuroendocrine Melanoderma, 938 Norman Levine & Cynthia Burk Felty Syndrome and Rheumatoid Arthritis, 939 Norman Levine & Cynthia Burk Hyperpigmentation Associated with Human Immunodeficiency Virus (HIV) Infection, 941 Philippe Bahadoran Melanoacanthoma, 946 Norman Levine & Cynthia Burk Phytophotodermatitis, 948 Norman Levine & Cynthia Burk Polyneuropathy, Organomegaly, Endocrinopathy, M Protein, and Skin Changes: POEMS Syndrome, 951 James J. Nordlund Urticaria Pigmentosum and Mastocytosis, 954 James J. Nordlund Poikiloderma of Civatte, 959 Vlada Groysman & Norman Levine Riehl’s Melanosis, 961 Scott Bangert & Norman Levine Atrophoderma of Pasini et Pierini, 963 James J. Nordlund, Norman Levine, Charles S. Fulk, & Randi Rubenzik Hyperpigmentation Associated with Scleromyxedema and Gammopathy, 965 Kazunori Urabe, Juichiro Nakayama, & Yoshiaki Hori Ichthyosis Nigricans, Keratoses, and Epidermal Hyperplasia, 965 James J. Nordlund Morphea and Scleroderma, 967 James J. Nordlund Pigmentary Changes Associated with Addison Disease, 969 Cindy L. Lamerson & James J. Nordlund Pigmentary Changes Associated with Cutaneous Lymphomas, 972 Debra L. Breneman 51 Hypermelanosis Associated with Gastrointestinal Disorders, 979 Porphyria Cutanea Tarda, 979 Eun Ji Kwon & Victoria P. Werth Cronkhite–Canada Syndrome, 983 Eun Ji Kwon, James J. Nordlund, & Victoria P. Werth Hemochromatosis and Hemosiderosis, 986 Joerg Albrecht & Victoria P. Werth Primary Biliary Cirrhosis, 992 Joerg Albrecht & Victoria P. Werth Inflammatory Bowel Disease and Pigmentation, 995 Joerg Albrecht & Victoria P. Werth

CONTENTS

Pellagra, 995 Eun Ji Kwon & Victoria P. Werth Peutz–Jeghers Syndrome, 999 Nancy Burton Esterly, Eulalia Baselga, & Beth A. Drolet 52 Acquired and Congenital Dermal Hypermelanosis, 1003 Sacral Spot of Infancy, 1003 Sang Ju Lee, Seung Kyung-Hann, & Sungbin Im Nevus of Ota, 1006 Sang Ju Lee, Seung Kyung-Hann, & Sungbin Im Nevus of Ito, 1012 Sang Ju Lee, Seung Kyung-Hann, & Sungbin Im Phakomatosis Pigmentovascularis, 1013 Sang Ju Lee, Seung Kyung-Hann, & Sungbin Im Other Congenital Dermal Melanocytosis, 1015 Sang Ju Lee, Seung Kyung-Hann, & Sungbin Im Acquired Dermal Melanocytosis, 1016 Sang Ju Lee, Seung Kyung-Hann, & Sungbin Im Carleton–Biggs Syndrome, 1017 Sang Ju Lee, Seung Kyung-Hann, & Sungbin Im Acquired Bilateral Nevus of Ota-like Macules (ABNOM), 1017 Sang Ju Lee, Seung Kyung-Hann, & Sungbin Im Blue Macules Associated with Progressive Systemic Sclerosis, 1018 Sang Ju Lee, Seung Kyung-Hann, & Sungbin Im 53 Mixed Epidermal and Dermal Hypermelanoses and Hyperchromias, 1020 Melasma, 1020 Sang Ju Lee, Seung Kyung-Hann, & Sungbin Im Melanosis from Melanoma, 1023 Sang Ju Lee, Seung Kyung-Hann, & Sungbin Im 54 Drug-induced or -related Pigmentation, 1026 Peter A. Lio & Arthur J. Sober Section 6: Disorders of Pigmentation of the Nails and Mucous Membranes, 1055 55 The Melanocyte System of the Nail and its Disorders, 1057 Robert Baran, Christophe Perrin, Luc Thomas, & Ralph Braun

56 Pigmentary Abnormalities and Discolorations of the Mucous Membranes, 1069 John C. Maize, Jr. & John C. Maize, Sr. Section 7: Benign Neoplasms of Melanocytes, 1091 57 Common Benign Neoplasms of Melanocytes, 1093 Pigmented Spindle Cell Nevi, 1093 Julie V. Schaffer & Jean L. Bolognia Speckled Lentiginous Nevus (Nevus Spilus), 1098 Julie V. Schaffer & Jean L. Bolognia Melanocytic (Nevocellular) Nevi and Their Biology, 1112 Julie V. Schaffer & Jean L. Bolognia 58 Rare Benign Neoplasms of Melanocytes, 1148 Nevus Aversion Phenomenon, 1148 James J. Nordlund Melanotic Neuroectodermal Tumor of Infancy, 1148 Julie V. Schaffer & Jean L. Bolognia Pilar Neurocristic Hamartoma, 1157 Julie V. Schaffer & Jean L. Bolognia Section 8: Treatment of Pigmentary Disorders, 1163 59 Topical Treatment of Pigmentary Disorders, 1165 Rebat M. Halder & James J. Nordlund 60 Chemophototherapy of Pigmentary Disorders, 1175 Rebat M. Halder & James J. Nordlund 61 UVB Therapy for Pigmentary Disorders, 1183 Thierry Passeron & Jean-Paul Ortonne 62 Sunscreens and Cosmetics, 1188 James J. Nordlund & Rebat M. Halder 63 Surgical Treatments of Pigmentary Disorders, 1191 Rebat M. Halder & James J. Nordlund 64 Laser Treatment of Pigmentary Disorders, 1198 Rebat M. Halder, Lori M. Hobbs, & James J. Nordlund Index, 1205 Color Plates, 1230

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Contributors

Zalfa Abdel-Malek

PhD Research Professor of Dermatology, Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, OH, USA

Joerg Albrecht MD Department of Dermatology, University of Pennsylvania, Philadelphia, PA, USA Mayra Alvarez-Franco

(deceased) Formerly of the Departments of Dermatology and Pathology, Yale University School of Medicine, New Haven, CT, USA

Joseph T. Bagnara PhD Professor Emeritus, Department Cell Biology and Anatomy, University of Arizona College of Medicine, Tucson, AZ, USA Philippe Bahadoran

MD PhD Assistant Professor of Medicine, Service de Dermatologie, Hôpital l’Archet, Nice, France

Debdutta Bandyopadhyay

PhD Instructor of Dermatology, Huffington Center on Aging, Baylor College of Medicine, Houston, TX, USA

Raymond E. Boissy PhD Professor of Dermatology and Cell Biology, Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Jean L. Bolognia MD Professor of Dermatology, Department of Dermatology, Yale University School of Medicine, New Haven, CT, USA Barbara Boone MD Resident in Dermatology, Universitair Ziekenhuis Ghent, Ghent, Belgium Ralph P. Braun

MD Assistant Professor, Pigmented Skin Lesion Unit, Department of Dermatology, University Hospital, Geneva, Switzerland

Debra L. Breneman

MD Associate Professor of Dermatology, University of Cincinnati, Cincinnati, OH, USA

Stefania Briganti

PhD San Gallicano Dermatological Institute, Rome, Italy

Scott D. Bangert

MD Banner Good Samaritan Hospital, Department of Medical Education, Phoenix, AZ, USA

Murray H. Brilliant PhD Lindholm Professor of Genetics, Department of Pediatrics, University of Arizona College of Medicine, Tucson, AZ, USA

Robert Baran MD Head of Nail Disease Center, Cannes, France

Lieve Brochez

Gregory S. Barsh MD PhD Professor of Genetics and Pediatrics, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, CA, USA

Cynthia J. Burk

Eulalia Baselga

Stella D. Calobrisi

MD Consultant in Pediatric Dermatology, Hospital de la Santa Creu I Sant Pau, Barcelona, Spain

Dorothy C. Bennett

MA PhD Professor of Cell Biology, St George’s Hospital, University of London, London, UK

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MD PhD Professor of Dermatology, Department of Dermatology, University Hospital of Ghent, Ghent, Belgium

MD Section of Dermatology, University of Arizona Health Sciences Center, Tucson, AZ, USA MD Medical Director, The Dermatology Clinic, Boca Raton, FL, USA

Tania Cestari

MD PhD Associate Professor of Dermatology, Department of Dermatology, University of Rio Grande do Sul, Hospital de Clínicas de Porto Alegre, Brazil

CONTRIBUTORS

Henry H. L. Chan MBBS MSc (Clin Derm) MD FRCP FRCP(Ed) FRCP(Glasg) FHKCP FHKAM (Med) Specialist in Dermatology and Honorary Clinical Associate Professor, University of Hong Kong and Chinese University of Hong Kong, Hong Kong Peter Man Hon Chan MD Clinical Research Fellow, University of Minnesota, Eagan, MN, USA Peggy L. Chern MD Department of Dermatology, University of Iowa Hospitals and Clinics, Iowa City, IA, USA Mary K. Cullen

MD Department of Cell Biology and Physiology, Clinical Instructor, Washington University School of Medicine, St Louis, MO, USA

José C. García-Borrón PhD Professor of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Murcia, Spain Rosemary J. Geary MD President, East Valley Dermatology Center, Chandler, AZ, USA Vanessa C. Gray-Schopfer PhD Signal Transduction Team, Cellular and Molecular Biology Section, Institute of Cancer Research, London, UK Pearl E. Grimes MD Vitiligo and Pigmentation Institute of Southern California, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA Vlada Groysman

Beth A. Drolet

MD Section of Dermatology, University of Arizona Health Sciences Center, Tucson, AZ, USA

Carol A. Erickson

Ruth Halaban PhD Senior Research Scientist, Department of Dermatology, Yale University School of Medicine, New Haven, CT, USA

MD Department of Dermatology, The Medical College of Wisconsin, Milwaukee, WI, USA PhD Professor of Molecular Cellular Biology, University of California at Davis, Davis, CA, USA

Nancy B. Esterly MD Professor of Dermatology and Pediatrics, Department of Dermatology, The Medical College of Wisconsin, Milwaukee, WI, USA Dong Fang MD PhD Staff Scientist, The Wistar Institute, Philadelphia, PA, USA Sharon A. Foley MD Department of Dermatology, Washington University School of Medicine, St Louis, MO, USA Peter S. Friedmann MD FRCP FMedSci Professor of Dermatology and Head of Division, Dermatopharmacology Unit, Southampton General Hospital, Southampton, UK Charles S. Fulk

MD FACP Department of Pathology, Vanderbilt University, Nashville, TN, USA

Rebat M. Halder

MD Professor and Chairman, Department of Dermatology, Howard University, Washington, DC, USA

Seung-Kyung Hann MD PhD Director, Korea Institute of Vitiligo Research, Yongsan-Gu, Seoul, Korea Rudolf Happle MD Professor of Dermatology, Department of Dermatology, Philipp’s University of Marburg, Marburg, Germany Vincent J. Hearing

PhD Chief, Pigment Cell Biology Section, Laboratory of Cell Biology, National Institutes of Health, Bethesda, MD, USA

Meenhard Herlyn DVM, DSc Professor and Chair of Program of Molecular and Cellular Oncogenesis, The Wistar Institute, Philadelphia, PA, USA Stan P. Hill

MD Denver, CO, USA

Anne-Sophie J. Gadenne MD Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, OH, USA

Lori M. Hobbs MD

William A. Gahl MD PhD Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA

Thomas J. Holstein

Sheila S. Galbraith MD Assistant Professor of Dermatology, Dermatology Department, Medical College of Wisconsin, Milwaukee, WI, USA

Assistant Professor, Department of Dermatology, Howard University, Washington, DC, USA PhD Professor Emeritus, Roger Williams University, Bristol, RI, USA

Yoshiaki Hori

MD PhD (deceased) Formerly Honorary Professor, Department of Dermatology, Kyushu University, Fukuoka, Japan

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CONTRIBUTORS

Thomas J. Hornyak MD PhD Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

I. Caroline Le Poole

Marjan Huizing

Norman Levine MD Professor of Medicine, Section of Dermatology, University of Arizona College of Medicine, Tucson, AZ, USA

PhD Head, Cell Biology of Metabolic Disorders Unit, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA

PhD Loyola University Medical Center Department of Pathology, Cancer Center, Maywood, IL, USA

Peter A. Lio Sungbin Im MD PhD Director, Kangnam Wootaeha Skin and Esthetic Clinic, Yeoksam-Dong, Kangnam-Gu, Seoul, Korea Genji Imokawa

PhD Director, Skin-Bio Assess Institute, Utsunomiya, Tochigi, Japan

Shosuke Ito

PhD Professor of Chemistry, Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan

Ana Luisa Kadekaro

PhD Research Instructor of Dermatology, University of Cincinnati College of Medicine, Cincinnati, OH, USA

Richard A. King MD PhD Director, Genetics Division, Department of Medicine and Associate Director, Institute of Human Genetics, University of Minnesota, Minneapolis, MN, USA Sidney N. Klaus

MD Professor of Dermatology, Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA

Wolfgang Küster MD Professor and Chief of Dermatology, TOMESA Clinic, Department of Dermatology, Bad Salzschlirf, Germany Eun Ji Kwon

BSc Department of Dermatology, University of Pennsylvania, Philadelphia, PA, USA

Jean-Philippe Lacour

MD Service de Dermatologie, Hôpital Archet 2, Nice, France

Cindy L. Lamerson MD Clinical Assistant Professor, University of Nevada, Reno, NV, USA

MD Instructor in Dermatology, Beth Israel Deaconess Medical Center, Boston, MA, USA

John C. Maize Sr MD Medical Director, DermPath Diagnostics, Maize Center for Dermatopathology, Mt Pleasant, SC, USA John C. Maize Jr

MD DermPath Diagnostics, Maize Center for Dermatopathology, Mt Pleasant, Clinical Assistant Professor of Dermatology, Medical University of South Carolina, SC, USA

Susan Bayliss Mallory MD FAAD FAAP Professor of Internal Medicine (Dermatology) and Pediatrics, Division of Dermatology, Washington University School of Medicine, St Louis, MO, USA Jiro Matsumoto

PhD Emeritus Professor, Department of Biology, Keio University, Kohoku-ku, Yokohama, Japan

Estela E. Medrano PhD Professor of Molecular and Cellular Biology and Dermatology, Huffington Center on Aging, Baylor College of Medicine, Houston, TX, USA Henk E. Menke MD PhD Dermatologist, Department of Dermatology, Sint Franciscus Gasthuis, Rotterdam, The Netherlands Allan D. Mineroff MD Lansdale, PA, USA

Gisela E. Moellmann

PhD Professor Emeritus, Yale University School of Medicine, New Haven, CT, USA

Jean-Marie Naeyaert

MD PhD Professor of Dermatology and Head, Department of Dermatology, University Hospital of Ghent, Ghent, Belgium

Edward J. Land

BSc PhD Honorary Professor, Lennard-Jones Laboratories, School of Geographical and Physical Sciences, Keele University, Keele, UK

Juichiro Nakayama

Sang Ju Lee MD PhD Director, Yonsei Star Skin and Laser Clinic, Chancheon-Dong, Seodaemun-Gu, Seoul, Korea

Marcelius Davy Njoo

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MD PhD Department of Dermatology, School of Medicine, Fukuoka University, Fukuoka, Japan

MD PhD Streekzierenhuis Middentwente, Hengelo, The Netherlands

CONTRIBUTORS

James J. Nordlund MD Dermatologist and Professor Emeritus, Group Health Associates, Cincinnati, OH, USA

Patrick A. Riley

William S. Oetting PhD Associate Professor, Genetics Division, Department of Medicine, Institute of Human Genetics, University of Minnesota, Minneapolis, MN, USA

Randi E. Rubenzik

Jean-Paul Ortonne

MD Professor of Dermatology and Chairman, Department of Dermatology, University of Nice-Sophia Antipolis, Nice, France

Monica Ottaviani PhD San Gallicano Dermatological Institute, Rome, Italy

MBBS PhD DSc FRCPath CBiol FIBiol FLS Professor Emeritus of Cell Biology and Director, Totteridge Institute for Advanced Studies, London, UK MD FAAD

Sun City West, AZ, USA

Tadeusz Sarna

PhD DSc Professor of Biophysics, Department of Biophysics, Faculty of Biotechnology, Jagiellonian University, Krakow, Poland

Julie V. Schaffer MD Assistant Professor of Dermatology and Pediatrics, New York University School of Medicine, New York, NY Glynis A. Scott

David M. Parichy

MD Associate Professor of Dermatology and Pathology, Department of Dermatology, University of Rochester School of Medicine, Rochester, NY, USA

Thierry Passeron

MD Clinical Assistant in Medicine, Nice, France

Vijayasaradhi Setaluri PhD Associate Professor of Dermatology, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA

Christophe Perrin

MD Laboratoire d’Anatomie Pathologique, Hôpital L. Pasteur, Nice, France

Anita P. Sheth

Mauro Picardo

Pranav B. Sheth MD Assistant Professor, Department of Dermatology, University of Cincinnati, Cincinnati, OH, USA

PhD Assistant Professor, Department of Biology, University of Washington, Seattle, WA, USA

MD San Gallicano Dermatological Institute, Rome, Italy

Tanusin Ploysangam

MD PhD Director, Institute of Beauty and Health Science, and Director, Belage Skin Care and Cosmetic Center, Nunthawan Prachacheun, Bangtalad, Nonthaburi, Thailand

Walter C. Quevedo Jr PhD Emeritus Professor of Biology, Department of Molecular and Cell Biology and Biochemistry, Brown University, Providence, RI, USA Christopher A. Ramsden BSc PhD DSc Cchem FRSC Professor of Organic Chemistry, Lennard-Jones Laboratories, School of Geographical and Physical Sciences, Keele University, Keele, UK

H. Randolph Byers MD PhD Professor of Dermatology, Boston University School of Medicine, Boston, MA, USA Mark V. Reedy

PhD Assistant Professor of Biology, Creighton University, Omaha, NE, USA

Germaine N. Relyveld

MD Department of Dermatology, Academic Medical Center, Amsterdam, The Netherlands

MD Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, OH, USA

Shigeki Shibahara MD PhD Professor, Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi, Japan Arthur J. Sober MD Professor of Dermatology, Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA Francisco Solano PhD Professor of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Spain Richard A. Spritz, MD Professor and Director, Human Medical Genetics Program, University of Colorado Health Sciences Center at Fitzsimons, Aurora, CO, USA Harold M. Swartz MD PhD Professor of Radiology, Dartmouth Medical School, Hanover, NH, USA Kazuhisa Takeda

MD PhD Assistant Professor of Molecular Biology, Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi, Japan

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CONTRIBUTORS

Luc Thomas MD PhD Professor and Chairman, Department of Dermatology, Lyon 1 University, Hotel Dieu, Lyon, France

Victoria P. Werth MD Professor of Dermatology and Chief, VA Dermatology, Department of Dermatology, University of Pennsylvania, Philadelphia, PA, USA

Marnie D. Titsch MD Dermatology Specialist, Oyster Point Dermatology Inc., Newport News, VA, USA

Wiete Westerhof

Peggy Tong MD Dermatology Associates, Milwaukee, WI, USA Kazunori Urabe

MD PhD Associate Professor, Department of Dermatology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan

Kazumasa Wakamatsu

PhD Professor, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan

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MD PhD Assistant Professor of Dermatology, Netherlands Institute for Pigment Disorders and Department of Dermatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Antony R. Young PhD Professor of Experimental Photobiology, King’s College London and St John’s Institute of Dermatology, St Thomas’ Hospital, London, UK

Foreword

In this second edition of The Pigmentary System: Physiology and Pathophysiology, the editors have expanded upon the dynamic field of pigment cell biology to reflect not only the rapidly moving pace of developments in this broad field but also the explosive growth of research and development across the basic life sciences. We have all benefited enormously from the recent growth in the research budgets of agencies such as the National Institutes of Health (NIH), and these benefits transcend beyond grant recipients to citizens who benefit from better treatments or management protocols for diseases that in the past were nearly always seriously debilitating. Never before have advances come so rapidly or with so much promise for improving the human condition. These improvements come as a result of better understanding of all aspects of the composition and function of living cells. From the molecules that direct activity to those that confer shape and phenotype, we know more today about the basic building blocks of living matter than at any time previously in our history. Perhaps even more astounding is the pace at which discoveries in the life sciences continue to be made. In recent years we have experienced near-exponential growth in the quantity and quality of information available across all sectors of the life sciences. There is little doubt that as we move toward the end of the first decade of the twenty-first century we will have answers to many questions, some of which we never imagined being able to ask. The pigmentary system, as a model system for studying basic questions about differentiating stem cells, has provided ample opportunities for study across a broad range of inter-

ests and expertise. Animal pigment has long been known to function protectively in some animals as well as decoratively in others. Pigment can provide camouflage for some and flamboyant advertisement for others. The embryonic origin of skin pigment cells, the neural crest, suggests that pigment cells share common traits with a wide variety of nerve cells. And yet, like all stem cells, neural crest cells can differentiate along a multitude of pathways, perhaps to become a sensory neuron or to become a melanocyte. These highly motile embryonic cells, even after settling down and differentiating, still retain a remarkable capacity for further motion and differentiation or reversion. Witness the havoc that can ensue if melanocytes become cancerous and migrate to all parts of the body to form tumors. The first edition of The Pigmentary System was unquestionably the definitive text of its time. The editors, who are themselves pre-eminent in this field, have worked with the authors to provide significant revisions to nearly every chapter, and a new chapter on melanocytes as malignant cells has been added. Virtually any topic related to pigmentary systems can be found in this single source, from animal chromatophores to human pigmentary disorders, from basic cell biology to clinical studies and results. Congratulations to the editors and to those researchers who are actively engaged in helping us better understand the complexities of this fascinating system! Sally Frost Mason PhD Purdue University West Lafayette, IN, USA

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Preface

This second edition of The Pigmentary System: Physiology and Pathophysiology presents the latest information about the biology of melanocytes and their disorders. The first section is dedicated to the science of pigmentation and has been completely rewritten, not just updated. The chapters have been reorganized to provide the most current and comprehensive review of the science of pigmentation. New chapters have been added and some old chapters in the first edition have been deleted. All of these were chapters on methodology that were rapidly outdated after publication. Some information and data remain constant and are unchanged in this edition. Other topics are entirely new or significantly enlarged since publication of the previous edition. Part II on the pathophysiology of pigmentary disorders is mostly updated. New chapters have been included. A lexicon on proper terminology for description of skin color has been included as the first chapter in this section. Unless the scientific and clinical communities use the same terminology to describe and identify scientific or clinical topics, there will be confusion about the nature and treatment of pigmentary or dyschromic disorders. We hope that this lexicon becomes standard in the dermatological and scientific communities. The comments that we made in the preface to the first edition are still very relevant. A few are worth repeating. This book is intended to be a reference book for both the scientific and the clinical communities. The fact that there is so much

ongoing scientific work related to pigmentation is an indicator of the importance of melanocytes. It also is a clue that melanocytes, melanotropins and related cells and factors are not just a mechanism for sun protection. It is obvious and certain that melanocytes and melanin are protective against sunlight. But the role of melanin in the eyes and ears is more than just sun protection. Melanocytes are involved in many processes within the epidermis. Melanotropins have effects on virtually every organ system in the body and control inflammation, appetite, some functions within the nervous system, and other critical functions. The pigmentary system, including melanin, melanocytes and melanotropins, is best described as a modulator that helps the body adapt to the environmental changes related to seasonal fluctuations in sunlight, temperature, and humidity. We hope that scientists, clinicians, students, and residents find this book useful in their studies on skin color, melanocytes, and melanin, and that it expands their perspective and view about the pigmentary system as a modulator, not just a method of sun protection. James J. Nordlund Raymond E. Boissy Vincent J. Hearing Richard A. King William S. Oetting Jean-Paul Ortonne

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Acknowledgments

The editors wish to acknowledge the very gracious and generous support of the following companies. Without their support, this project would not have been possible. Galderma International, Paris, France Proctor and Gamble, Cincinnati, OH, USA Combe Inc., White Plains, NY, USA

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

I

The Physiology of the Pigmentary System

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

1

Historical and Comparative Perspectives of the Pigmentary System

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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A History of the Science of Pigmentation Sidney N. Klaus

Introduction In Europe, prior to the seventeenth century, notions about the origin of human skin color were based largely on myths and fanciful stories passed down from the ancient world. The stories focused on providing explanations for the blackness of Africans. Europeans with an ethnocentric perspective thought that it was necessary to justify the darkness of outsiders rather than to explain their own paleness. In Europe, two theories about the origin of Africans’ color predominated. The first proposed that the black hue of African skin was a consequence of the intense sunlight and heat to which Africans were exposed. The second idea proposed that African color was a result of divine intervention, a concept that stemmed from the well-known passage in Genesis describing Noah’s curse on Ham and his progeny. Such traditional views of skin color were questioned during the Age of Discovery. Explorers were returning home from remote regions of the globe and bringing with them descriptions of black-, yellow-, or red-hued peoples. The array of colors fascinated scientists and excited them to investigate how these skin colors originated. During the sixteenth century, the scientific community prepared for this task. Scientists learned new techniques of human dissection and new methods of chemical analysis. More importantly, they adopted a new philosophical approach for the study of natural phenomena. Rather than analysis of phenomenon based on a priori assumptions, they based their analyses on firsthand observations and experimentation. By the seventeenth century, anatomists and physicians had begun to collect objective data about skin color. For the next two centuries, so many important observations had been collected about the seat of skin color and the causes of its diversity that, as one researcher boasted, “a good sized volume was scarcely large enough to contain them.” The furious pace of forward progress was slowed somewhat in the early part of the nineteenth century when skin color scientists in both Europe and America were drawn into acrimonious debates over social issues, especially slavery and the place of “peoples of color” in the family of man. By the end of the century, the slavery issue had been settled, and the discovery of the cell and improved methods for the microscopic examination of the skin had stimulated the discipline to set off in new directions.

In retrospect, it is clear that the science of skin color had completed its early phase of development by the 1840s. With the discovery of the cellular nature of the epidermis, the “modern” phase of skin color science had begun (Meirowsky, 1940). This chapter briefly summarizes the first 250 years of the earlier phases of pigment research, from its birth in the 1600s to its “coming of age” in the 1840s. A history of the modern era, from the 1840s to the present, may be found in reviews by Becker (1959) and Nordlund et al. (1989).

Early Anatomic Discoveries To the ancient Greeks and Romans who were acquainted with the peoples of Ethiopia, the black color of the Africans was regarded as their most characteristic and most curious feature. Greek and Roman scientists were not equipped to investigate the causes of dark complexions in a meaningful way. They understood little about dissection of individual tissues and were unable to sort out the anatomic details related to the skin. They regarded the skin as an amorphous membrane made from the congealing of “moist exhalations” driven to the surface of the body by internal heat. They believed that the color of skin was imposed either from outside by the sun or by some inner humor. These notions about skin color persisted through the Middle Ages and into the Renaissance. In 1543, Vesalius succeeded in splitting the skin into two layers by applying a burning candle to the abdomen of a living subject. He did not examine the skin layers for their content of color. It was not until 75 years later that the first “scientific” study of skin color was performed. In 1618, Jean Riolan the younger, a Parisian anatomist, was the first person to make a detailed analysis of skin color. He used a technique similar to that of Vesalius to separate the skin of a black subject into two layers. But he used a vesicant rather than a flame to produce a blister and carefully examined the two layers for their color and texture. He found that, while the top of the blister (the cuticle) was pigmented, the base (the cutis) remained white. “Color . . .” he announced “. . . lay in the outer layer, and did not go so deep as the true skin” (Riolan, 1618). To Riolan, this finding reinforced the idea that heat and sunlight were the ultimate causes of blackness. Riolan reasoned that, if the sun caused skin darkening, it was expected that the outer cuticle would be darker than the inner cutis. 5

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A few years later, Alexander Read, an anatomist working in London, repeated Riolan’s experiments and found the same results. However, Read offered a different interpretation. The skin pigment, he concluded, was not caused by singeing the body’s surface. He concluded rather that skin color was derived from the body’s inner humors. As they escaped through the skin, some of the elements became attached to the surface and dried, thus producing the pigment. Negroes, he argued, had darker skin because they had larger pores than whites, a morphologic variation allowing greater volumes of their humors to escape leaving behind larger amounts of black “remnants” of the humors (Read, 1642). Perhaps the most detailed analysis of skin color in the seventeenth century was carried out by the distinguished writer and physician, Sir Thomas Browne. Browne had listened carefully to the accounts of explorers who had found variously colored races, and he concluded from these accounts that color did not arise from the influence of climate. “If the fervor of the Sun were the cause of the Negroes’ complexion, . . .” he wrote, “. . . it is reasonable to assume that inhabitants of the same latitude, subjected unto the same vicinity of the Sun, partake of the same hue and complexion, which they do not.” In addition, he noted that Africans who had been transplanted into “. . . cold and phlegmatic habitations . . .” had not turned white, but “. . . continued their hue, both in themselves and their generations.” He rejected the idea that skin blackness was acquired from a “. . . power of imagination . . .” or from “. . . anointing with bacon and fat substances.” Instead, he thought that the tincture of the skin was based on an inborn trait, a trait that was passed from father to son by the sperm. Browne was not an armchair scientist. His diary records the outline of numerous experiments he carried out to determine for himself the seat of color. For example, he set out to make a “. . . blistering plaster in a negroes skinne and trie if the next skin will bee white . . .” and to see whether a “. . . vesication will do anything upon a dead cold body.” He even urged his son Edward, a medical student, to carry out additional studies. He wrote to him asking that he “. . . separate the skin of a black person with boiling water and take notice of the cuticula and cutis and observe how the scarres become whiter or less blacke than any other parts.” He also urged him to “. . . touche the skin with aqua fortis and see how it will alter the colour.” Unfortunately, Browne did not record the results of these experiments (Keynes, 1964). Another seventeenth century polymath, Robert Boyle, devoted a chapter of his book Experiments and Considerations Touching Colours to what he called “the Cause of the Blackness of those many nations which by one common Name we are wont to call Negroes.” He examined the various causes proposed at that time and denied that either Noah’s curse on Ham or “heat alone” could produce a true blackness. Instead, he focused on principles of inheritance, concluding that the “Principal Cause of the Blackness of Negroes is some Peculiar and Seminal Impression.” As evidence, he reported that “the off-spring of Negroes, trans6

planted out of Africa, retain still the complexion of their Progenitors” (Boyle, 1664).

Malpighi’s Innovation: the Rete Mucosum Riolan’s contention that the cuticle was the seat of color had been in place for less than a century when it was superseded by an entirely different notion proposed by the Italian anatomist, Marcello Malpighi. In 1667, Malpighi stated that the seat of color was not the cuticle. Rather, he proclaimed that color was located in a separate layer of the skin sandwiched between the cuticle and the cutis. He named this layer the rete mucosum. The discovery of rete came about in the following way. In 1665, Malpighi had been searching for the structures that mediated the sensation of taste, and he succeeded in finding large papillae just below the surface of the tongue. Turning to the skin to see if he could find similar structures that mediated touch, he found that he was unable to separate the skin’s layers properly by boiling, the method that he had used previously. He was forced to use putrefaction, an old dissection technique in which the separation of parts is enhanced by allowing a small piece of tissue to decompose partially. Using this method, he prepared skin from cadavers and found that he could easily lift the cuticle from the cutis. By this method, he was able to identify the dermal papillae. However, he also found a mucoid material draped over the papillae, which he regarded as a protective covering. The material had a netlike appearance when seen from above, so he called it the rete mucosum. Malpighi’s experiments were not directed at discovering the proximate cause of skin color. However, in one experiment in which he used skin from an Ethiopian cadaver, he noted that the mucoid material was tinged black. On the basis of this finding, he later wrote “It is certain that the cutis (of the Ethiopian) is white, as is the cuticula too; hence all their blackness arises from the underlying mucous and netlike body” (Malpighi, 1665). Malpighi did not follow up on his discovery of the “seat of color” but the putrefaction technique he used allowed the layers of the skin to be separated gently. The method produced reliable results, and other investigators soon expanded on Malpighi’s finding. In 1677, three years after Malpighi published his findings, Johann Pechlin, a Dutch anatomist, used Malpighi’s method of dissection to make a thorough study of Ethiopian skin. In one experiment, after he lifted off the cuticle, he scraped away the black mucous and reapplied the scarf to the cutis. He reported that “. . . a whiteness came forth immediately of the sort seen often enough in Europe. In truth the surface of the skin was scarcely unattractive.” Pechlin was concerned that his results obtained using cadaver skin might not be the same had he studied living skin. To his dismay, he was unable to convince living subjects to cooperate in his experiments “. . . because of I don’t know what sort of evil on the part of the Negroes” (Pechlin, 1677).

A HISTORY OF THE SCIENCE OF PIGMENTATION

A few years later, the Dutch microscopist Antoni van Leeuwenhoek, in a letter to the Royal Society of London, reported on his observations made using his microscope of the skin of a black Moorish girl. “I took from several parts of the arms the outer skin with a fine little instrument and found that it consisted of little scales. Putting these scales before my microscope I found them to be not as transparent as those of my skin.” Van Leeuwenhoek concluded that the black color of the skin was the result of the black scales, and that “the little vessels which form the scales of the Moors” may possibly develop a slightly darker color (Collected Letters of Antoni van Leeuwenhoek, 1683–1684; see Schierbeek, 1952). If one follows Malpighi’s directions for uncovering the rete, as the cuticle is lifted off one observes tiny mucoid threads bridging the angle between the two layers. William Hunter described them as “. . . an infinite number of filaments, as fine as the most delicate threads of a spider’s web, that pass between the cutis and the more external integuments” (Hunter, 1764). According to some anatomists, the threads were tiny vessels. This idea led some of the more inventive investigators to propose that the coloring matter in Africans’ skin was not the mucous material itself but was a black fluid contained within the web of small vessels. One contemporary observer wrote “. . . between the outward and inner skin of the corpse can be found a kind of vascular plexus, spread over the whole body like a web or net, which was fill’d with a Juice as black as Ink” (Marana, 1801). It became something of a challenge to prove unequivocally that these filaments were in fact “vessels.” In anatomic laboratories, the identification of small vessels in anatomic preparations relied on their visualization after they had been injected with colored glue or isinglass. Only a few investigators were skilled enough to inject these “. . . most delicate threads.” One anatomist who was given credit for the first demonstration of this vascular network was William Baynham, an American who had moved to London in 1769. Although Baynham never published his findings, he placed his specimens on exhibition in John Hunter’s anatomic museum. Not all Baynham’s colleagues were convinced by his discovery. William Cruickshank made a careful study of Baynham’s preparations and later wrote that he was “. . . not perfectly satisfied” with them. He concluded that Baynham had not injected the vessels of the rete, but rather a series of vessels that lay between the rete and the cutis (Cruickshank, 1795). In the eighteenth century, the attention of researchers began to move from the anatomy of the rete to the “nature” of the coloring matter itself. Early attempts to collect and analyze material from the rete were not productive. Alexis Littre, a French surgeon, soaked pieces of skin from a Negro cadaver for a week either in warm water or in spirits of wine but was unable to extract any of the coloring matter (Littre, 1720). A few years later, another French scientist, Pierre Barrere, in an essay submitted to the Academy of Bordeaux in 1741, denied Malpighi’s claim that the pigment of Africans’ skin arose from the “corps reticulaire.” It is evident, he wrote, the coloring matter in the skin of Africans was bile. He reported

that he had examined cadavers of Africans and observed that “the bile is black, and the blackness of the skin is in proportion to the blackness of the bile.” Later in the century, another investigator attempted to prove that the pigment of Africans was derived from bile. Samuel Stanhope Smith, a professor of moral theology at the College of New Jersey, had learned from an American colleague that bile exposed to the sun and air changed its color to black. Smith hypothesized that, in southern climates, the secretion of bile was augmented and, when the bile reached the skin, it became “. . . more languid and almost fixed.” In the skin, the aqueous parts of the bile easily escape through the pores of the skin by perspiration, while the more dense portion remained in a glutinous state within the rete. There, it received the repeated radiation from the sun and atmosphere and turned black. Smith believed that a cold environment would reverse the process of darkening and render the complexion “. . . clear and florid.” According to Smith, if Africans moved to temperate climates from Africa “. . . they would soon lose their imposed color and become the color of Europeans” (Smith, 1788). Johann Friedrich Blumenbach, the German ethnologist, also proposed that bile was the coloring agent of the skin. He had learned that bile was made of a mixture of carbon and hydrogen. He suggested that, as the bile reached the skin, the hydrogen it contained became more volatile than the carbon and combined with atmospheric oxygen. This reaction left behind a black carbon residue that became embedded in the Malpighian mucous and caused the skin to darken (Blumenbach, 1865). Another “chemical” theory was advanced by the young Humphrey Davy in 1799. Davy thought that light had a great affinity for oxygen and could extract it from other compounds. Davy had evidence that the mucous material of the rete was made of a colorless mixture of carbon, nitrogen, and oxygen. He proposed that sunlight extracted the oxygen from this mixture and left behind a black material made of carbon and nitrogen. To Davy, this explained differences in skin hues found throughout the world. “In northern latitudes, where the inhabitants are less exposed to light, the rete continues to contain its full proportion of oxygen, and the inhabitants are lighter in color. In the torrid zone, on the other hand, where the sun was especially intense, larger quantities of oxygen would be removed, and the blackness peculiar to the negroes will be found.” Davy had no direct proof that light could subtract oxygen from the skin, but he did cite other chemical experiments to support his claim. He reported that, when a compound that binds oxygen, such as sulfur of potash, is applied to the skin of a white person, the skin blackens. When a compound that gives off oxygen, such as muriatic acid, is applied to the skin of a Negro, it lightens (Davy, 1799). An even more imaginative notion about the nature of skin color was suggested by Immanuel Kant. Kant based his theory on the well-known capacity of phlogiston to turn blood a black color. Phlogiston was an imaginary element considered to be the essential principle of combustion. It was said to be 7

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released from the body through the lungs as part of the metabolic process. Kant believed that, in regions of the globe where the atmosphere was heavily phlogisticated, such as the coast of Africa, it would be impossible to eliminate all the body’s phlogiston by breathing. Kant proposed that the excess phlogiston would travel through the circulation and, on reaching the skin, it would precipitate in the ends of the small cutaneous vessels and turn the skin black (Lovejoy, 1959). Claude Le Cat, a French physician, claimed that the source of Africans’ blackness was neither bile nor blood. He said that the black mucous, which he called ethiops, was found only in Africans and that it was secreted directly into the skin through the tips of the cutaneous nerves (Le Cat, 1765). Although most early scientists believed that skin pigment was derived from some “internal” source such as bile or blood, a few investigators decided that the coloring matter was produced by glands within the skin itself. The earliest version of this hypothesis was offered by an English anatomist, Edward Tyson. He proposed that the black colour in Negroes’ skins came from glands that were full of a “black liquor.” He suggested that the climate might alter the glands so that “. . . they might separate from the mass of blood a differing humor from White, and by this means give a different hue to the inhabitants” (Montagu, 1943). Two French researchers, G. H. Breschet and Roussel de Vauzeme, reported that they had found a “. . . chromatogenous apparatus . . .” in the cutis which secreted the black mucous. They said that the mucous was deposited on the surface of the dermal papillae through short, excretory ducts emanating from glandular parenchyma located in the cutis (Plumbe, 1837). Another French investigator, M. Gaultier, also argued that the coloring matter was secreted within the skin but not from glands. The pigment of the skin, he said, was produced instead from hair bulbs. Gaultier’s claim was based on the results of an experiment he carried out on a living subject. He burned the skin of a Negro and then closely observed the pattern of repigmentation during the healing process. According to Gaultier, the pigment appeared first around the “pores” through which the hairs exited and only later did it radiate out to cover the entire area of the burn (Prichard, 1813).

Differences Between Blacks and Whites As a more complete picture of skin color began to emerge by the end of the eighteenth century, political and social forces required the disciplines of anatomy and biology to take on a social dimension. Advocates of black slavery argued that Africans were not the “brothers” of Europeans but rather were the product of a separate creation. Skin color, the most obvious marker of racial identity, was forced to the center of the debate over the unity of mankind. The polygenists began looking for “significant” anatomic differences between blacks and whites to prove they were separate species. Monogenists began looking for evidence of similarities. 8

Bolstering the polygenist cause was the English anatomist, John Gordon. Gordon claimed that whites, unlike blacks, had no rete mucosum. “I have satisfied myself by many dissection that in the Negro there is a Black membrane interposed between the epidermis and true skin upon which their dark color entirely depends . . . But after the strictest examination I have not been able to find any light colored rete mucosum in the inhabitants of Great Britain, nor in those of other nations resembling them in color” (Gordon, 1815). Several anatomists disagreed. Richard Harlan, a young lecturer in anatomy from Philadelphia, wrote that “. . . the existence of the rete mucosum in the white race, so frequently denied, has been demonstrated occasionally in the European by skilful anatomists and if not deceived I myself have discovered it several times in a living European subject, by raising the epidermis with a blister, especially upon the back of the hands and the neck” (Harlan, 1835). Charles Caldwell, who had moved from Philadelphia to become a professor in a medical college in Lexington, Kentucky, reported that he too found the rete in both races, although the structures were not identical in appearance. “The rete mucosum in blacks is comparatively thick, while in the Caucasian, the rete is present but it is much thinner.” In spite of this similarity, however, Caldwell concluded that Negroes and Caucasians were sufficiently distinct to be called separate biological species (Caldwell, 1830).

Experiments of Nature In addition to the work carried out by anatomists and chemists in dissecting rooms and laboratories, many physicians made important contributions to the fund of information about skin color through reporting and analyzing patients with clinical problems. John Josselyn, a physician from England who was visiting the Massachusetts Bay Colony in 1675, was one of the first physicians to advance the science of skin color by using observations based on a clinical case. While in Boston, Josselyn had been called upon to lance a “corruption” in the palm of a “Moor.” Later, he described his findings. “After I lanced it, I perceived that the Moor had one skin more than Englishmen, deeper in colour than our European veins, and upon it rests the epidermis” (Josselyn, 1675). Samuel Marcy, a physician from rural New Jersey, reported the case of an albino girl. After providing a full account of her family history, including the fact that she had two albino sisters, he went on to consider the cause of the disorder. “The mother accounts for the appearance of the child by attributing it to a severe fright she receive by the falling down of an old white mare she was driving,” wrote Marcy. “Although I was unwilling to admit at first that the Great Creator ever left his work in so loose a manner, that the imagination of the mother should alter or determine form or color of her children, the birth of two other albino children go further to strengthen the doctrine that the mind of the mother may affect the fetus in utero” (Marcy, 1839).

A HISTORY OF THE SCIENCE OF PIGMENTATION

John Morgan, from Philadelphia, reported the case of Adelaide, a pied Negro girl from the West Indies. Like Marcy, Morgan considered the possibility that maternal impression might cause the disorder. Morgan related that, while pregnant, the mother of the girl “. . . delighted in laying out all night in the open air, and contemplating the stars and planets. Whether the strong impression made upon the mother of Adelaide by the nightly view of the stars and planetary system may be the cause of the very extraordinary appearances in this girl, everyone will have to determine for themselves” (Morgan, 1784). During the seventeenth and eighteenth centuries, the pigment disorder that attracted most attention among the medical community, as well as the general public, was vitiligo vulgaris. In 1697, a remarkable case was presented to the Royal Society by William Byrd, a Virginian who had recently returned to London after a visit home. Under the title “An Account of a Negro-Boy that is dappeld in several Places of his Body with White Spots,” Byrd reported that the boy was born in Virginia of black parents, who was “well till 3 years old, and now was speckled of his breast and back and that no fancy had taken his Mother.” The spots he wrote are “wonderfully White, at least equal to the skin of the fairest Lady. His Spots grow continually larger and larger, and ’tis probable, if he lives, he may in time become all over white.” Thomas Jefferson described an African slave whom he encountered on his own plantation. He described the man as “A Negro man within my knowledge, born black and of black parents, developed when a boy, a white spot on his chin. This continued till he became a man, by which time it had extended over his chin, lips, and the neck on that side. He is robust and healthy and the change of color is not accompanied with any sensible disease, either general or topical” (Jefferson, 1904). Charles Peale, the painter and natural historian, also described a case under the title “An Account of a person born a Negro who afterwards became white” (Peale, 1791). The case of vitiligo that attracted most attention during this period from both lay and medical communities was Mr Henry Moss. At the age of 38 years, Moss first noted a change in the color of his skin. The change began on his fingers and hands and extended over his arms, legs, and face. Six years later, in the summer of 1796, Moss traveled from his home in Maryland to exhibit himself for money at the Black Horse Tavern in Philadelphia. He became a popular attraction and, according to one account, was so well known to the local citizens “. . . that his name was almost as familiar as John Adams, Thomas Jefferson, or James Madison” (Caldwell, 1855). Several prominent physicians from Philadelphia visited Mr Moss. Benjamin Barton, a professor at the medical school, was impressed with the distribution of the white spots. Noting that Moss’ color had disappeared completely from his armpits, Barton suggested initially that it was likely that the pigment had been washed away by perspiration. Charles Caldwell also examined Moss and was skeptical of Barton’s interpretation.

He decided to study Moss in detail. “Anxious to know as much of his case as possible, I took him in some measure under my care, . . . and . . . for a slight reward made on him such experiments as suited my purpose. While thousands visited and gazed at Moss as an object of curiosity and wonder, I alone endeavored to make him a source of scientific information.” Caldwell asked Moss “. . . to excite by exercise a copious perspiration to ascertain whether the fluid perspired by the colored portions of the skin was itself colored. And I found that it was not.” Caldwell examined Moss’ skin and found that the rete mucosum had disappeared from the depigmented areas. He concluded that the rete had been removed “. . . by means of absorption.” Moss’ sensitivity to low temperatures he thought supported his theory that Moss was losing his rete. He concluded that Moss’ true skin was now “protected” from a cold atmosphere by nothing but the cuticle (Caldwell, 1855). Samuel Stanhope Smith, who had earlier pioneered the idea that African blackness was caused by environmental factors, was another visitor. He was accompanied by two other gentlemen “. . . of whom none are more capable of observing a fact of this nature with a sound and accurate judgment.” Smith paid great attention to the pattern of Moss’ pigment loss and concluded that this provided elegant proof of his hypothesis. “Although there was evidently a strong and general tendency in the constitution of this negro to a change of color, yet this tendency was much longer resisted in those parts of the body which were most exposed to the immediate action of the sun’s rays. As he was a laboring man, wherever there were rents in the thin clothes which covered him there were generally seen the largest spots of black.” From this pattern, Smith inferred that “. . . where any dark color has been contracted by the human skin, the solar influence alone, and the free contact of the external air, will be sufficient to continue it a long time even in those climates which are most favorable to the fair complexion” (Smith, 1810). Benjamin Rush suggested perhaps the most unusual hypothesis to explain Moss’ pigment loss. In a paper published in 1799, Rush argued that it was the blackness of Moss, and not his white spots, that represented his disease. I shall prove, he wrote, that “. . . the color and figure of our fellow creatures who are known by the epithet of negroes are derived from a modification of that disease which is known by the name of Leprosy.” Rush went on to explain that the black color of the negroes, their thick lips, and their insensitivity to pain were all common signs of leprosy. He suggested that it was likely that leprosy was an infectious disorder “. . . since a white woman in North Carolina living with a black husband has not only acquired a dark color but also several features of a negro.” Rush proposed that Moss was not suffering from a disease but was actually undergoing a spontaneous cure of one. “If the color of negroes is a disease . . .” he added, “. . . let science and humanity combine their efforts and endeavor to discover a remedy for it.” From his experience with other ailments, Rush suggested that bleeding or purging be tried to lessen the black color (Rush, 1799). 9

CHAPTER 1

The Beginnings of Modern Pigment Research The “early” phase of pigment research ended in the 1840s when the cell theory ended the rete’s reign as a separate amorphous layer of the skin and assigned the seat of skin color to the lower portion of a cell-rich epidermis. Microscopic anatomists had also discovered that the pigment was not in the form of a liquid or mucoid material but was rather composed of tiny intracellular granules. With the discovery that a population of dendritic cells derived from the neural crest served as the source of the coloring matter for the skin and hair, the “modern” phase of pigment research moved into high gear. For the past 150 years, researchers have relied on a melanocyte-centered paradigm to lead them along new paths of discovery. To see how far their investigations have proceeded and how wide-ranging their interests and disciplines, one needs only to look through the remaining chapters in this volume.

References Becker, S. W. Historical background of research on pigmentary diseases of the skin. J. Invest. Dermatol. 32: 185–196, 1959. Blumenbach, J. F. The Anthropological Treatises of Johann Friedrich Blumenbach. London: Longman Green, 1865. Boyle, R. Experiments and Considerations Touching Colours. London: Henry Herringman, 1664, pp. 151–167. Caldwell, C. Thoughts on the Original Unity of the Human Race. New York: E. Bliss, 1830. Caldwell, C. Autobiography of Charles Caldwell, MD. Philadelphia: Lippincott, Grambo and Co., 1855. Cruickshank, W. Experiments on the Insensible Perspiration of the Human Body. London: George Nicol, 1795. Davy, H. Essays on heat and light. In: Contributions to Physical and Medical Knowledge Principally From the West of England, T. Beddoes (ed.). Bristol: Biggs and Cottle, 1799, pp. 193–198. Gordon, J. A System of Human Anatomy. Edinburgh: William Blackwood, 1815. Harlan, R. Medical and Physical Researches. Philadelphia: Lydia Bailey, 1835. Hunter, W. Remarks on the cellular membrane and some of its diseases. Med. Observ. Inquiries 2: 26–55, 1764. Jefferson, T. Notes on Virginia, in the Writings of Thomas Jefferson. Washington, DC: Thomas Jefferson Memorial Association, 1904.

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Josselyn, J. An Account of Two Voyages to New-England. London: G. Widdowes, 1675. Keynes, G. The Works of Sir Thomas Browne. Chicago: University of Chicago Press, 1964. Le Cat, C. N. Traite de la Couleur de la Peau Humaine en General, de celle des Negres en Particulier, et de la Metamorphose d’une de ces Coleurs en l’autre, soit de Naissance, soit Accidentellement. Amsterdam, 1765. Littre, A. La Peau in Histoire de l’Academie Royale des Sciences. Paris: Hochereau, 1720, pp. 30–32. Lovejoy, A. O. Kant and Evolution in Forerunners of Darwin. Baltimore: Johns Hopkins, 1959, pp. 173–206. Malpighi, M. De Externo Tactus Organo Anatomica Observatio. Naples: Apud Agidium Longu, 1665. Marana, G. P. Letters Written by a Turkish Spy, vol. 8. London: Vernor and Hood, 1801. Marcy, S. S. On the albino. Am. J. Med. Sci. 24: 517–518, 1839. Meirowsky, E. A critical review of pigment research in the last hundred years. Br. J. Dermatol. 52: 205–217, 1940. Montagu, M. F. A. Edward Tyson, MD, FRS, and the Rise of Human and Comparative Anatomy in England. Philadelphia: The American Philosophical Society, 1943, pp. 212–213. Morgan, J. Some account of a motley coloured, or pye, negro girl and mulatto boy. Trans. Am. Phil. Soc. 2: 392–395, 1784. Nordlund, J. J., Z. A. Abdel-Malek, R. E. Boissy, and L. A. Rheins. Pigment cell biology: An historical review. J. Invest. Dermatol. 92(Suppl.): 53s–60s, 1989. Peale, C. W. Account of a person born a negro, or a very dark mullato, who afterwards became white. Univ. Asylum Columbia Mag. 2: 409–410, 1791. Pechlin, J. N. Du Habitu & Colore Aethiopum Qui Vulgo Nigritae. Koln: Joach Reumani, 1677. Plumbe, S. A Practical Treatise on the Diseases of the Skin. London: Sherwood, Gilbert & Piper, 1837, pp. 9–16. Prichard, J. C. Researches into the Physical History of Man. London: John and Arthur Arch, 1813, pp. 163–164. Read, A. The Manual of the Anatomy or Dissection of the Body of Man. London: Francis Constable, 1642. Riolan, J. Anthropographia. Paris: Hadrianum Perier, 1618. Rush, B. Observations intended to favour a supposition that the black color (as it is called) of the negroes is derived from leprosy. Am. Philos. Soc. Trans. 4: 289–297, 1799. Schierbeek, A. (ed.). Collected Letters of Antoni van Leeuwenhoek, Vol. IV, 1683–1684, Amsterdam: Swets & Zeitlinger, 1952, pp. 245–251. Smith, S. S. An Essay on the Causes of the Variety of Complexion and Figure in the Human Species. Edinburgh: C. Elliot, 1788. Smith, S. S. An Essay on the Causes of the Variety of Complexion and Figure in the Human Species. New Brunswick: J. Simpson and Co., 1810.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

2

Comparative Anatomy and Physiology of Pigment Cells in Nonmammalian Tissues Joseph T. Bagnara and Jiro Matsumoto

Summary 1 The terms “chromatophore” and “pigment cell” are equivalent, although the former is usually applied to lower vertebrates and the latter to homeotherms (birds and mammals). 2 Epidermal melanophores (-cytes) are ubiquitous pigment cells present in all vertebrate classes. They are primarily elements of morphologic color change (the increase or decrease in skin pigments) and are thus responsible for the amounts of melanins found in the epidermis, feathers, and hair. 3 Dermal melanophores predominate in the dermis of lower vertebrates and are elements of both morphologic color change and physiologic color change (the rapid changes from pallor to darkening and vice versa). Other dermal chromatophores include iridophores (and leukophores) and xanthophores and erythrophores, which are also elements involved in both morphologic and physiologic color changes. The pigment composition of the three basic dermal chromatophore types is different from one another, and each contains unrelated pigments. Similarly, their cytoplasmic pigmentary organelles differ from one another in appearance, although they all seem to have a common origin from the endoplasmic reticulum. Dermal chromatophores function in background adaptation (cryptic coloration), thermal regulation, warning coloration, reproduction, and possibly in protection against solar radiation. 4 Morphologic and physiologic color changes are primarily mediated by hormones, especially melanocyte-stimulating hormone (MSH). Melatonin, melanophore-contracting hormone (MCH), catecholamines, and sex hormones play more particular roles. MSH release from the pituitary is controlled by the hypothalamus in response to background color and intensity. Melatonin is produced and released by the pineal gland (epiphysis cerebri) under conditions of darkness. While MSH causes dispersion of dermal and epidermal melanophores and aggregation of iridophores, melatonin causes aggregation of only dermal melanophores whereas epidermal melanophores and iridophores are unaffected by melatonin. Some chromatophores are directly affected by light and disperse or aggregate in response to the presence or absence of specific wavelengths of light. 5 All pigment cell types originate from the neural crest and are determined (in the embryonic sense) either in situ or by

extrinsic factors encountered during migration or after their final destination is reached. These factors may include elements of the extracellular matrix and local factors present in the integument, including a melanization-inhibiting factor (MIF) and a melanization-stimulating factor (MSF). The last two factors may be major elements in controlling the expression of pigmentation patterns. In the zebrafish model, a hostile relationship of xanthophores toward adult-type melanophores plays a key role in color pattern independent of dorsal/ventral regions. The organellogenesis of all pigmentary types seems to involve a primordial vesicle derived from the endoplasmic reticulum. 6 Such hormones as MSH, thyroid hormones, and sex hormones influence pigment cell development, but do not seem to determine pigmentation patterns. 7 The pigment cells of all vertebrate classes are homologous in that they have in common embryonic origin, pigment composition, morphology, and hormonal control. Chromatophores, usually thought to be restricted to lower vertebrates, are also present in homeotherms. Proteins or peptides that regulate pattern expression, such as MIF from amphibians and agouti protein from mammals, may be chemically and physiologically related. 8 Numerous anomalies and pathologies of lower vertebrate pigment cells exist including neoplasms such as erythrophoroma. These are not unlike malignant melanomas of higher forms. 9 As the burgeoning knowledge and technology derived from work on mammalian systems is applied to those of lower vertebrates, much can be learned about the fundamental cell biology and physiology of lower vertebrate pigmentation. In turn, because of the relative accessibility of lower vertebrates for experimentation, new knowledge about the normal and abnormal biology of mammalian pigmentation, including that of humans, will be accrued from continuing studies on these lower forms.

Historical Background For many years, knowledge, understanding, and experimentation on lower vertebrate pigmentation went on separately from that of higher vertebrates, including humans. Not until the middle of the twentieth century was it realized that the 11

CHAPTER 2

bright and often rapidly changing pigmentation of fishes, frogs, and lizards (poikilotherms) is fundamentally related to the more static and more uniform pigmentation of birds and mammals (homeotherms). This divergence in understanding was significantly registered in pigment cell terminology and nomenclature. For a detailed discussion, see Bagnara and Hadley (1973). No real confusion existed with respect to birds and mammals, in which melanin-bearing pigment cells seemed to be the only ones present and were called melanocytes without question. Among lower animals, the first term to be applied was chromoforo (Sangiovanni, 1819; according to Parker, 1948) and, thus, with translation from the Italian, the term chromatophore has precedence. This was a term of prevalence for both lower vertebrates and invertebrates, notwithstanding the use of the suffix -zyte as a substitute for -phore in much of the German literature. Thus, with respect to melanin-bearing pigment cells, the reference was to melanophores (or melanozyten). It was early realized that melanophores were present primarily in the dermis, where a variety of other brightly colored nonmelanin-bearing pigment cells were also often found, and their nomenclature provided a new source of confusion because of the discovery of specific nonmelanin pigments in or in association with these dermal pigment cells (Fox, 1953; Fox and Vevers, 1960). Thus, guanine-bearing chromatophores were often designated as guanophores based upon the pigment they contained; however, these same pigment cell types were designated by others as xantholeukophores, based upon what colors they manifested. By the end of the 1970s, much new information about structure, function, and composition of all pigment cell types had accumulated, and it was thus possible to establish a reasonable and unifying pigment cell terminology for both lower and higher vertebrates (Bagnara, 1966a). The unifying element in pigment cell terminology considered that pigment cells should be designated primarily by their appearance rather than their composition, and details of its application were presented earlier (Bagnara and Hadley, 1973). It was concluded that, among poikilotherms, there exist epidermal and dermal melanophores. The latter, together with iridophores (silvery to white) and xanthophores (yellow) and/or erythrophores (red), are also dermal. The pigmentary organelles of melanophores were referred to as melanosomes, those of iridophores as reflecting platelets, and those of erythrophores/xanthophores as pterinosomes and carotenoid vesicles. The embryonic origin of the various chromatophore types was addressed in early experiments performed mostly on amphibians, and on salamanders in particular. All poikilothermic chromatophores are derived from the neural crest, as was shown by the early elegant extirpation and transplantation experiments of DuShane (1935). From this site on the neural tube, chromatoblasts migrate to all regions of the integument and to other areas of the body to give rise to the particular and striking patterns of lower vertebrates. Among the early questions considered by investigators were: when were the specific chromatophores determined (in the embryonic sense); and how were the various circumscribed patterns 12

accomplished? These questions are discussed at length in an extraordinary book, Of Scientists and Salamanders, by Professor Victor T. Twitty (1966). Two of Professor Twitty’s students investigated these problems in detail. M. C. Niu (Twitty and Niu, 1954) considered the mutual inhibitory effects of individual chromatoblasts as they encountered one another during migration, while H. E. Lehman [see Lehman and Youngs (1959)] concluded that both extrinsic and intrinsic factors influenced chromatoblast expression and thus were responsible for pigmentation patterns. Later, Volpe (1964) concluded that pigmentation patterns of frogs were an autonomous function of neural crest cells, but this was disproved by Bagnara (1982) who had earlier proposed that the diverse pigment cell types of vertebrates had a common origin from a neural crest stem cell and were specified later (Bagnara et al., 1979). This concept has stood the test of time. The capacity for rapid color change (physiologic color change) among lower vertebrates has long attracted attention, and an enormous literature accumulated by the start of World War II [see Parker (1948)]. With the understanding that individual chromatophores brought about color change due to their capacity to “expand or contract,” the search was on for factors controlling these cells that allowed them to respond in an integrated fashion. Among the first candidates for chromatophore control was the nervous system and direct innervation of chromatophores. As early as 1876, Pouchet provided information that, in teleost fishes, the sectioning of peripheral nerves or the electrical stimulation of spinal nerves led, respectively, to darkening or pallor in appropriate areas. Even earlier, Brücke (1852) presented data to support the idea that color change in chameleons is under nervous control. A history of work demonstrating the control of fish chromatophores by direct innervation is well provided in Parker (1948) and Fujii (1993a). Chromatophore control by direct innervation is not a general phenomenon in lizards (Bagnara and Hadley, 1973), but such is surely the case for African chameleons (Whimster, 1971). While chromatophore control through direct innervation was shown not to be a general phenomenon among vertebrates, indirect control of chromatophores by neurotransmitter agents and hormones was shown to be significant in most lower vertebrates (see Bagnara and Hadley, 1973; Fujii, 1993a; Parker, 1948). Of particular importance are the catecholamines, epinephrine and norepinephrine, which affect melanophores directly by causing melanophore contraction, thus leading to the phenomenon of excitement pallor seen among many fishes, amphibians, and reptiles. Because these catecholamines are released by the adrenal medulla, excitement pallor can be viewed as a color change controlled by hormones. While this is the case, the role of catecholamines is minor compared with that of the hypophysial hormone, MSH. That the pituitary contains an important factor that influences pigment cells was first realized from the simultaneous early work of P. E. Smith (1916) and B. M. Allen (1916). Early on, this factor was designated “intermedin” (Zondek and Krohn, 1932) and later it received its current designation of

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

MSH (Shizume et al., 1954). The older literature on this subject has been extensively summarized by Waring (1963) and by Bagnara and Hadley (1973). More recently, the role of MSH on poikilotherm chromatophores has been reviewed by Fujii (1993a) for fishes and by Fernandez and Bagnara (1991, 1993) for amphibians. Almost 30 years ago, it became clear that MSH is the principal agent bringing about melanosome dispersion in lower vertebrates and that it does so through cyclic adenosine monophosphate (AMP) as a second messenger (Bagnara and Hadley, 1969; Goldman and Hadley, 1969). This hormone was also shown to bring about iridophore aggregation and xanthophore or erythrophore dispersion in amphibians (Bagnara, 1958, 1969). The same is now known to be true for fishes (Fujii, 1993a). Before the characterization of MSH, the pituitary was sometimes viewed as possessing two opposing chromatophorotropic hormones that controlled color changes of lower vertebrates. This “bihumoral” theory of hormonal control was originally championed by Sir Lancelot Hogben in the 1940s and was refined by his scholarly descendants, notably H. Waring in his monograph Color Change Mechanisms of Cold-Blooded Vertebrates (Waring, 1963). It considers that the pituitary stimulates melanophore dispersion through the elaboration of a “melanophore-dispersing hormone” (essentially MSH according to Bradshaw and Waring, 1969) and melanophore aggregation through the action of a melanophore-aggregating hormone, which had not yet been identified at that time. While the bihumoral theory failed to reach acceptance, it lurked in the background largely because of the discovery by Enami (1955) of a MCH in the pituitary of a catfish. Because of his untimely death, this putative hormone received little attention until its cause was revived by Professor Bridget Baker in the 1980s. Its nature and biological significance were reviewed by her (Baker, 1993). Among other hormones found to be of importance in the regulation of chromatophores is melatonin, a hormone produced and released by the pineal gland during darkness to bring about aggregation of dermal melanophores and thus lead to the body blanching reaction of amphibian embryos and larvae (Bagnara, 1960, 1963, 1966b). It was known from the early study of McCord and Allen (1917) that the mammalian pineal contained an agent that aggregated dermal melanophores, but it was not until the work of Lerner et al. (1958) that the agent was purified, characterized as an indole, and designated melatonin. Shortly thereafter, Bagnara (1960) announced that melatonin was elaborated from the pineal during darkness and was actually a hormone released to bring about a body blanching reaction that occurs in amphibian larvae at night in a circadian rhythm (Bagnara, 1960, 1963). Subsequently, this hormonal role for melatonin was extended to many other vertebrates including fishes (see Fujii, 1993a). It is significant that melatonin’s direct action in bringing about melanophore aggregation is restricted to dermal melanophores; thus, epidermal melanophores and dermal iridophores do not respond directly to the hormone (Bagnara, 1964a).

It has long been known that the integument of animals is directly sensitive to light in a little-understood phenomenon known as the “dermal light sense.” An excellent review of this subject has been presented by Steven (1963), who included numerous references to the possibility that pigment cells themselves are directly responsive to changes in illumination. This topic is addressed further in the later section on adaptation to darkness. Among the important concepts of pigment cell biology that have long commanded interest is that of the intracellular movements of pigment granules and, in particular, the melanosomes. Even to this day, this area of research continues to be most active and even controversial. One of the earliest theories of melanosomal movements was that of Marsland (1944), who considered that melanosomal movements were related to the solation and gelation of the chromatophore cytoplasm. Later, because it was known that colchicine causes melanosome dispersion (Wright, 1955) and reflecting platelet aggregation (Bagnara, 1969) in amphibians, Malawista’s (1965) conclusion that this result suggested solation of chromatophore cytoplasm seemed reasonable. Attention then shifted to piscine melanophores when Bikle et al. (1966) suggested that microtubules were important for the dispersion and aggregation of melanophores. Later, Malawista (1971) suggested a role for microfilaments in these processes, and thus the involvement of the cytoskeleton in melanosomal movement was established. From this period to today, most work on the translocation of pigmentary organelles has focused on fish chromatophores and especially on melanophores and erythrophores/xanthophores, and the issue has become exceedingly complicated by the many variables involved. There seem to be species differences, and there is a profound variation in the velocity of organellar movements, some occurring in seconds and others over minutes. Organellar movements involve different organellar types within the same cell, such as pterinosomes and carotenoid vesicles. In some cases, individual organellar types migrate differentially within the cell and thus preclude the possibility of passive movements during aggregation and dispersion. The actin–myosin system has been invoked in explanations of many types of organellar motility, but not all. Tubulin–dynein interactions have been considered to provide the forces that drive pigmentary organelles along the length of microtubules (Oshima and Fujii, 1987), but organellar motility can occur even in the absence of microtubules. Other force-generating proteins such as kinesin and dynamin have gained attention as possible movers of pigmentary organelles as they slide along microtubules in both aggregation and dispersion. To add to the complexity of the situation, the endoplasmic reticulum has now been invoked as a major player in organellar motility. The continuing history of this phenomenon is complicated, but discussions are available in numerous reviews that have emerged (Kimler and Taylor, 1995; Obika, 1986; Schliwa, 1984; Taylor, 1992) and in the subsequent section on molecular mechanisms for intracellular transport of pigment granules. 13

CHAPTER 2 Table 2.1. Chromatophore terminology and characterization. Chromatophore

Organelle

Pigment

Color

Source

Epidermal melanophore (-cyte) Melanophore

Melanosome Melanosome Pterino-melanosome

Eumelanin Eumelanin Pterorhodin–eumelanin

Black, brown Black, brown Black, red

Iridophore

Reflecting platelet (iridosome)

Structural colors and iridescence

Leukophore

Leukosome (refractosome) Pterinosome (xanthosome) Carotenoid vesicles Pterinosome (erythrosome) Carotenoid vesicles Cyanosome

Purines, especially guanine; some pteridine crystals Uric acid (?), other purines (?) Pteridines

Fishes, amphibians, reptiles Fishes, amphibians, reptiles Phyllomedusine frogs, rhacophorid frogs (?) Fishes, amphibians, reptiles

Structural white

Fishes

Yellow to orange

Fishes, amphibians, reptiles

Carotenoids Pteridines

Orange to red

Fishes, amphibians, reptiles

Carotenoids ?

Blue

Mandarin fish

Xanthophore

Erythrophore

Cyanophore

Epidermal melanin unit: an association of an epidermal melanophore (-cyte) with adjacent keratinocytes that serve to receive cytocrine melanin. Dermal chromatophore unit: an association of the dermal chromatophores (xanthophores or erythrophores, iridophores, and melanophores) to bring about integrated color changes. These are especially important in the expression of green colors in amphibians and reptiles.

Current Concepts It is impossible in one chapter alone to consider all the salient aspects of lower vertebrate pigment cells that should be of interest and importance to scholars of pigmentation. Thus, in this section, an attempt is made to focus on those topics that may be most valuable to current workers in the field. These topics are considered to be perhaps most relevant to the ongoing research of today. At the same time, it should be understood that much of the knowledge presented in the previous section on history remains current. Much of it will not be discussed further because, unfortunately, current relevance dictates the direction of current research support, even at the expense of allowing important concepts to lie fallow. In recent years, most pigmentary research on poikilotherms has centered on fishes and amphibians (Bagnara, 1976). The cytophysiology of fish chromatophores has been the subject of detailed review (Fujii, 1993a, b). Accordingly, amphibian examples will be stressed here.

Chromatophore Characterization Because of new knowledge gained during the past few years, it is time to re-examine chromatophore terminology and perhaps establish a nomenclature that can be used universally. Table 2.1 presents a suggested terminology to be used for the near future. It does not differ markedly from that which we presented earlier (Bagnara, 1966a; Bagnara and Hadley, 1973); however, other terms that have emerged more recently are included as are some lesser known synonyms that are indicated in parentheses. Figures 2.1–2.3 demonstrate some of the brightly colored pigment cells. 14

Fig. 2.1. Living red-backed salamander, Plethodon cinereus, showing the distribution of erythrophores and melanophores over capillaries between the skin glands of the dorsal surface. A few silvery iridophores are also visible (see also Plate 2.1, pp. 494–495). [From Bagnara, J. T. Color change. In: Physiology of the Amphibia, vol. 3, B. Lofts (ed.). New York: Academic Press, 1976, pp. 1–52 with permission.]

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

Fig. 2.2. A living scale from the red-colored variant of swordtail fish, Xiphophorus helleri. Note the presence of numerous erythro(xantho)phores with red pterinosomes in the periphery and yellow carotenoids in the center under a dispersed state (see also Plate 2.2, pp. 494–495). [From Bagnara, J. T., and M. E. Hadley. Chromatophores and Color Change: The Comparative Physiology of Animal Pigmentation. Englewood Cliffs, NJ: Prentice-Hall, 1973, with permission.]

Melanophores and Melanocytes Whether these cells should be designated -phores or -cytes is a question that has often been addressed, and there seems to be no answer other than to let use by the scientific community be the ultimate judge. In an earlier attempt to reconcile the problem, Fitzpatrick et al. (1966) suggested that “melanophore” be the term of preference for lower vertebrates because this pigment cell was important in rapid color change phenomena (physiologic color change) and involved the rather rapid and profound intracellular movements of melanosomes, the melanin-containing organelles. It was suggested that the term “melanocyte” be used for higher vertebrates primarily because their epidermal pigment cells were of a form different from that of dermal melanophores and presumably were not involved in physiologic color change. Actually, some of the rationale for their premise no longer holds, but their conclusion that melanocyte be used for birds and mammals seems justified because this term has come to be used almost exclusively for melanin-bearing pigment cells by current workers. Accordingly, although this chapter is devoted to the pigment cells of lower vertebrates, the mutuality of nomenclature for melanin-bearing pigment cells will be acknowledged by liberal use of the suffix -cyte, as in melanophore (-cyte). Epidermal Melanophores (-cytes) While the epidermal melanocytes of birds and mammals had long been recognized and reasonably characterized, it was not until the work of Hadley and Quevedo (1967) that it was generally realized that this very same pigment cell type exists in the epidermis of lower vertebrates where it functions as it does in homeotherms. Indeed, it has been observed to be a prominent pigment cell of most lower vertebrates, from primitive fishes to reptiles, where it exhibits its typical spindle shape (Fig. 2.4) and

Fig. 2.3. Melanophore and xanthophores from the teleost, Fundulus heteroclitus. Note that the xanthophores appear as aggregated oil droplets (see also Plate 2.3, pp. 494–495). [From Bagnara, J. T., and M. E. Hadley. Chromatophores and Color Change: The Comparative Physiology of Animal Pigmentation. Englewood Cliffs, NJ: Prentice-Hall, 1973, with permission.]

Fig. 2.4. Epidermal melanophores of adult Rana pipiens, dispersed at left and aggregated at right. Note cytocrine melanin in adjacent epidermal cells. Each epidermal melanocyte with its surrounding epidermal cells comprises an epidermal melanin unit (courtesy of Professor M. E. Hadley).

15

CHAPTER 2

V pre

mel

V mel pre

Fig. 2.6. Dispersed (left) and aggregated (right) dermal melanophores in the tail fin of Xenopus larvae.

Fig. 2.5. Dermal melanophores from larvae of Pachymedusa dacnicolor. Left, melanosomes (mel) and fibrillar premelanosomes (pre) are typical of early stages. Right, at metamorphic climax, melanosomes begin to develop into the adult form and, at this stage, are characterized by elevation of the limiting membrane and the rough appearance of the eumelanin mass; prominent Golgi (G) and numerous vesicles (V) are found within the cytoplasm together with premelanosomes (pre) (from Bagnara et al., 1978b).

serves to deposit considerable quantities of cytocrine melanin into adjacent epidermal cells (Bagnara and Hadley, 1973). Each epidermal melanophore (-cyte), together with the epidermal cells that it serves, comprise a true “epidermal melanin unit” as first described by Fitzpatrick and Breathnach (1963). Much of what is known about organellogenesis of lower vertebrate epidermal melanophores is extrapolated from studies on mammals (see Chapter 7); however, there are probably few differences among all vertebrate groups with respect to melanosome formation. The melanin-containing organelles of poikilotherms are similar in shape and size to those of mammals and include fibrillar premelanosome stages (Fig. 2.5). The similarity between epidermal melanophores among the various taxonomic groups is also registered in their responses to hormones. Epidermal melanophores of lower vertebrates respond to MSH by dispersing their melanosomes just as do mammalian epidermal melanocytes in the presence of the hormone (Snell, 1967). This response to MSH is rather interesting in view of the fact that their thin spindle shape precludes much of a role in physiologic color change. In contrast, epidermal melanophores of lower vertebrates play an important role in morphologic color change, as is manifested by the significant amounts of melanin that they deposit in the epidermis (see Figs 2.4 and 2.8). Obviously, there is an increase in melanogenesis in these cells in response to MSH stimulation, but it is not known how this occurs. Presumably, MSH affects melanogenesis as it does in mammals (see Chapter 19). In contrast to dermal melanophores, epidermal melanophores are not sensitive to melatonin. They do not aggregate in response to melatonin in any vertebrate studied so far (Bagnara and Hadley, 1970a). 16

Fig. 2.7. Dispersed dermal melanophores from the dorsal surface of a Xenopus larva demonstrating the broad flattened form of this dermal pigment cell.

Dermal Melanophores These prominent black or brown chromatophores of lower vertebrates are profoundly important not only because they provide most of the dark pigmentation of fishes, amphibians, and reptiles, but because they are exceedingly active in physiologic color change in these animal groups (Fig. 2.6). They are large cells with broad dendrites; they usually reside in the upper portion of the dermis, just below the basal lamina. Often, they are flat and thus occupy a broad area of the dermal surface (Fig. 2.7). This is especially true of organisms in larval or embryonic stages. Flat melanophores seem more prevalent when the dermis is relatively thin. In some adults, especially those of amphibians and reptiles, which possess a thicker dermis, the form of dermal melanophores is more threedimensional. Such forms often possess “dermal chromatophore units,” to be discussed subsequently, composed of an upper layer of xanthophores (yellow pigment cells) lying

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

EM X I

M

Fig. 2.8. Transverse section of the dorsal skin from an MSH-treated adult R. pipiens showing basket-shaped dermal melanophore (M). Iridophores are in a punctate state (I), and the clear xanthophore layer (X) is also visible. An epidermal melanin unit is seen in the epidermis with an epidermal melanophore (EM) and surrounding epidermal cells containing cytocrine melanin (from Bagnara and Hadley, 1969).

just below the basal lamina and above a layer of iridophores, and a basal layer of melanophores. These dermal melanophores extend processes upward to terminate between the xanthophores and iridophores (reflecting cells) in amphibians (Bagnara et al., 1968) or above the xanthophores in lizards (Taylor and Hadley, 1970). In both cases, the form of the melanophore becomes basket-like (Fig. 2.8). Melanophores of the dermal type are not restricted to the dermis, but are often present on and in internal organs such as the liver, kidney, heart, thymus, and gonads, on blood vessels, in the peritoneum, and on the meninges (see Figs 2.25, 2.26, and 2.30). That these are of the dermal type is inferred from their sensitivity to hormones such as MSH or melatonin. For example, in hypophysectomized larvae of Xenopus, these deep melanophores are aggregated (see Fig. 2.26), in contrast to those of intact larvae, which are dispersed. Similarly, such deep melanophores aggregate in the presence of melatonin (see Fig. 2.34). Melanosomes of dermal melanophores are not unlike those of epidermal melanophores except for the fact that they are often larger than those of the latter. Generally, they are more spherical in form and about 0.5 mm in diameter. Most poikilotherm dermal melanosomes, especially those of amphibians, possess typical fibrillar premelanosomes (Fig. 2.5) and form in the usual way from the Golgi–endoplasmic reticulum (GERL). In contrast, piscine melanosomes, such as those of the goldfish, form in a different manner (Turner et al., 1975). Here, a multivesicular body of endoplasmic reticular origin forms the premelanosome and no fibrillar substructure is observed. Whereas melanosomes of all vertebrates seem consistently similar, there are remarkably unusual types found in some taxonomic groups. The most notable of these are the melanosomes of the phyllomedusine frogs (leaf frogs) of the New World (Bagnara et al., 1973; Bagnara, 2003). Dermal

Fig. 2.9. Wholemount of portion of brown dorsal skin of P. dacnicolor that had been masked and photographed with epiillumination. The sharp transition between the masked (above) and unmasked (below) skin is evident. The preparation involved passage through solvents, thus the iridophore surfaces are exposed because of the loss of yellow pigments and blue coloration is expressed. Note the red color of the melanophore pigment (see also Plate 2.4, pp. 494–495).

melanophores of adults of these species are a red color (Fig. 2.9) and, as shown in Figure 2.10, they may contain large fibrillar melanosomes (Taylor and Bagnara, 1969). They may or may not contain eumelanin, but their predominant pigment is pterorhodin, a pteridine dimer (Misuraca et al., 1977). They are unquestionably melanophores on the basis of their form, position, and function (Figs 2.9 and 2.11); moreover, in at least the Mexican leaf frog, Pachymedusa dacnicolor, adult melanosomes are derived from typical melanin-containing melanosomes (Bagnara et al., 1978a). Although these unusual organelles have never been named formally, we have designated them as pterino-melanosomes in Table 2.1. At metamorphic climax, the typical larval melanosomes undergo a “redifferentiation” such that pterorhodin fibers begin to deposit upon the eumelanin core of the melanosome to form a compound organelle containing a central kernel of eumelanin surrounded by a concentric mass of pterorhodin (Bagnara and Ferris, 1974). It is likely that such unusual organelles are not restricted to the phyllomedusine frogs (Bagnara and Ferris, 1975), but may be present in taxonomically unrelated frogs such as the rhacophorids (J. T. Bagnara, personal observations; P. Schwalm, personal communication). Iridophores and Leukophores Pigment cells that function primarily through the reflection of light from the surface of orderly distributed organelles, or reflecting platelets, are called iridophores (Fig. 2.12) (Bagnara, 1966a; Taylor, 1969). Although not true pigments, the purines guanine, uric acid, hypoxanthine, and adenine have long been considered to be deposited in crystalline form in the reflecting platelets, which are usually arranged in oriented stacks. More recently, studies of pure iridophores from frog larvae grown 17

CHAPTER 2

A

0.1 µ

B

0.1 µ

Fig. 2.10. Unusual large melanosomes in dermal melanophores of two phyllomedusine species, P. dacnicolor (A) and Agalychnis callidryas (B). The melanosomes of the two species are each larger than the typical vertebrate melanosome and contain the pigment pterorhodin. Each melanosome is composed of a fibrous mass enclosed by a limiting membrane, but that of A. callidryas lacks the electron-dense eumelanin kernel typical of the P. dacnicolor melanosome (from Bagnara et al., 1973).

Fig. 2.11. An adult Pachymedusa dacnicolor in a “semi-natural” outdoor cage in full illumination on a light background. The black plastic letter masks, UA (University of Arizona), to the left and right of this brown-adapted frog were removed after having been in place on the frog’s back for 1 hour. Note the green color that developed under the mask (see also Plate 2.5, pp. 494–495). [From Iga, T., and J. T. Bagnara. An analysis of color change phenomena in the leaf frog, Agalychnis dacnicolor. J. Exp. Zool. 192:331–342, 1975.]

in cell culture have revealed that guanine was the sole purine present; moreover, it was discovered that some pteridine pigments were also present in these cells (Bagnara et al., 1988). It seems that reflecting platelets may contain both crystalline purines and pteridines. The orientation of these stacks of platelets determines the nature of the pigmentary function of the iridophore. Normally, when viewed with reflected light, iridophores appear to contribute a metallic gold or silver luster (see Figs 2.1 and 2.47–2.49). When viewed with transmitted light, iridophores exhibit blues, greens, and reds. These are structural colors probably arising from thin-layer interference and the scatter or the diffraction of light from the stacks of reflecting platelets. Some iridophores examined with reflected light appear to be blue, while others appear to be brown or tan in color. These colors probably relate to the size, shape, orientation, and conformation of reflecting platelets. A pene18

trating analysis of the optics of iridophores in fishes has been presented by Fujii (1993a), and a more specific study of the structural colors of spiny lizards has been made by Morrison (1995). The latter study emphasizes the role of thin-layer interference in establishing the various iridescent colors in these lizards. A complete analysis and understanding of the structural colors of any individual iridophore or group of iridophores is very difficult for numerous reasons, not least of which is the size and shape of reflecting platelets. Among the more common types found in amphibians and fishes are those represented by long, thin profiles (Fig. 2.12). Among the leaf frogs, small bead-like or cubic reflecting platelets prevail (Fig. 2.13) as they often do in some lizards (Fig. 2.14). Distances between reflecting platelets are very important, and only slight changes in spacing can markedly alter the

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

ians and reptiles (see Bagnara and Hadley, 1973; Bagnara et al., 1978b). An elementary scheme of how these colors are brought about is shown in Figure 2.17.

Fig. 2.12. An iridophore from the dorsum of an adult R. pipiens showing stacks of reflecting platelets (actually spaces previously occupied by purine crystals that shattered and dissolved during sectioning and staining). An individual reflecting platelet is trapped between two lobes of the nucleus. Scale = 1 mm (from Bagnara, 1976).

apparent color, as was shown for the tree frog Hyla arborea by Nielsen (1978). Another chromatophore active in structural coloration has been recognized in fishes (see Fujii, 1993a). This chromatophore, the leukophore, seems to be somewhat different from the iridophore; however, it functions much as iridophores and probably utilizes purines as pigments in its pigmentary organelles, as do the reflecting platelets of iridophores. The introduction of another chromatophore designation may add to nomenclatural confusion; however, out of deference to recent investigators of piscine chromatophores who have described these cells, the leukophore is acknowledged as a distinctive chromatophore (Menter et al., 1979). Very likely, it is a particular type of iridophore found primarily in fishes or in anomalous situations in other vertebrates, for example, as in the periodic albino mutant of Xenopus (Fukuzawa, 2004). With the acceptance of the existence of both iridophores and leukophores, new names for their inclusions (Table 2.1) must be recognized; these include “leukosome” or “refractosome.” Despite the existing differences between iridophores and leukophores, their similarities are such that it warrants their being grouped together as a basic pigment cell type. Iridophores play an important role in imparting blue coloration as a structural color (Figs 2.15 and 2.16). Blue coloration is relatively uncommon among most poikilotherms and blue pigments are rare; however, blue as a structural color is important because it plays a major role in the elaboration of green coloration, which is very common among amphib-

Xanthophores and Erythrophores Dermal chromatophores that range in color from pale yellow to bright red are designated xanthophores (yellow) or erythrophores (red) depending upon how yellow or red they appear (Figs 2.1–2.3). Despite their differences in color, xanthophores and erythrophores should be considered as the same basic pigment cell type (Table 2.1). At the beginning of the twentieth century, they were called lipophores because many of these cells contain fat-soluble yellow, orange, or red carotenoid pigments that are localized in fatty vesicles (Bagnara, 1966a; Bagnara and Hadley, 1973). It was not until much later (Bagnara, 1961; Bagnara and Obika, 1965; Obika, 1963; Obika and Bagnara, 1964) that another group of pigments, pteridine in nature, was found to be even more important pigments of xanthophores and erythrophores. Soon afterwards, it was discovered by Matsumoto (1965a) that pteridine pigments are localized in organelles called pterinosomes and that both carotenoid vesicles and pterinosomes are often found within the same cell, be it a xanthophore or an erythrophore (Figs 2.2, 2.13, and 2.18). The specific color manifested by the cell often depends upon the pattern of carotenoids and pteridines it contains. In addition, it is possible that there exist other pigments so far undiscovered. All these facts were important considerations in the decision to designate chromatophores in accordance with their color rather than their chemical composition (Bagnara, 1966a). Although the association of carotenoid pigments with xanthophores or erythrophores has a long history, there have been relatively few definitive studies on their specific carotenoid content. Moreover, it has been only recently that attempts have been made to correlate carotenoid composition with xanthophore or erythrophore ultrastructure. Matsui et al. (2002) have observed that the bright red ventral integument of the Japanese newt, Cynops pyrrogaster, contains beta-carotene and at least five other carotenoids including canthaxanthin and lutein. They suggest that these pigments are contained in either thin-walled vesicles or thicker-walled “ring-type” vesicles. The precise localization of carotenoids in these vesicles remains unclear. In a general sense, perhaps the most important of the xanthophore and erythrophore pigments is the pteridine group. Pteridines are a group of yellow, orange, or red compounds having the structure of pyrimido[4,5-b]pyradine and are known to be synthesized from guanosine triphosphate (GTP) by GTP-cyclohydrolase I (GTP-CHI) that has a variety of isoforms (Thony et al., 2000). The vast majority of naturally occurring pteridines have the structure of 2-amino-4oxopteridine and are called unconjugated pteridines collectively. This terminology is convenient to discriminate them from folic acid or its related compounds, which have a long side-chain at position 6 of the pteridine ring. Thus far in medical research, much attention has been focused on 19

PT

CV

RP

MS

5 Fig. 2.13. Transverse section through the dorsal dermis of P. dacnicolor. Xanthophores are filled with pterinosomes (PT) and carotenoid vesicles (CV). Iridophores contain reflecting platelets with round or oval profiles, which seem to be strung together in a bead-like fashion. Melanosomes (MS) are typical for this species. The organelle indicated by an arrow may be a pterinosome. Magnification ¥ 6000 (from Taylor and Bagnara, 1972).

Fig. 2.14. Iridophores of the lizard, Urosaurus ornatus, at low magnification at left (¥ 2000) and high magnification at right (¥ 75 000). Note the rectangular profiles of reflecting platelets and their orderly arrangement. The image on the left includes views of melanophores (bottom) and xanthophores (top) (courtesy of Dr Randall Morrison).

20

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

Fig. 2.15. Melanophores (aggregated) and iridophores (dispersed) in a wholemount dorsal skin preparation of Rana pipiens photographed with transmitted light. Note the varied structural colors evident in the iridophores (see also Plate 2.6, pp. 494–495). [From Bagnara, J. T., and M. E. Hadley. Chromatophores and Color Change: The Comparative Physiology of Animal Pigmentation. Englewood Cliffs, NJ: Prentice-Hall, 1973, with permission.]

biopterin, particularly its tetrahydro form, because of its role as a cofactor for hydroxylation of phenylalanine and tryptophan (Nicol et al., 1985). In lower vertebrates, large amounts of unconjugated pteridines accumulate specifically in xanthophores and erythrophores. Some, such as sepiapterin, serve as yellow pigments by themselves, whereas others, such as biopterin, 2-amino-4-oxopterin, 7-oxobiopterin (ichthyopterin), and isoxanthopterin, are colorless in nature. The roles of these colorless pteridines are unknown, but it is possible that they function in the absorption of ultraviolet light as their spectrophotometric characteristics include strong absorption in the 340–360 nm region (Mori et al., 1960; Tschesche and Glaser, 1958). It is also known that some unconjugated colorless pteridines, such as drosopterins, form dimers that act as chromophores (Pfleiderer, 2001). Biosynthetic pathways of pteridines common to lower vertebrates are well established, as summarized in Figure 2.19

Fig. 2.16. Wholemount of portion of brown dorsal skin of P. dacnicolor that had been masked and photographed with epiillumination. The sharp transition between the masked (above) and unmasked (below) skin is evident. The preparation involved passage through solvents, thus the iridophore surfaces are exposed because of the loss of yellow pigments and blue coloration is expressed. Note the red color of the melanophore pigment (see also Plate 2.7, pp. 494–495).

(Masada et al., 1990; Ziegler et al., 2000). As seen in this scheme, it is apparent that pteridine metabolism is regulated by three key enzymes including GTP-CHI, 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SPR) (Masuda et al., 1990; Ziegler et al., 2000). Therefore, mutations in genes associated with these enzymes, particularly GTP-CHI, would inevitably affect development and differentiation of xanthophores and thus yield pigmentary mutants. This seems to be the case in zebrafish where there exists a variety of mutants, three of which are associated with xanthophore pigmentation and pteridine composition, including one that displays an alteration in pteridine metabolism similar to a mutation in Drosophila (Odenthal et al., 1996). The autonomy of xanthophores and erythrophores for pteridine biosynthesis was shown by elegant techniques of classic embryology. Obika (1963) demonstrated by in vitro 21

CHAPTER 2

Fig. 2.17. Diagrammatic interpretation for the basis of green coloration in amphibians and other vertebrates. As light strikes the surface of an animal such as a frog, short wavelengths of light (blue–violet) are largely absorbed by the filtering xanthophore or yellow pigment layer; the rest are scattered by the iridophore or scattering layer. Long wavelengths (red–orange) largely pass through the filtering and scattering layers of the skin and are absorbed by the melanophore or melanin layer. Intermediate wavelengths (yellow–green) pass through the filtering layer and are scattered from the surface of the iridophore layer (Tyndall scattering) and pass back through the filtering layer. Thus, the light reflected from the surface contains a high proportion of yellow–green wavelengths and the animal appears green (from Bagnara and Hadley, 1973).

Fig. 2.18. Schematic interpretation of the dermal chromatophore unit based upon observations from several anurans. Adaptation to a dark background is represented. Processes from basally located melanophores, between overlying xanthophores and iridophores, become filled with melanosomes and thus obscure light reflection from the iridophore surface (from Bagnara et al., 1968).

hanging drop culture of urodele (Triturus and Hynobius) neural crest that yellow pigmentation of larval xanthophores occurs simultaneously with formation of pteridines in simple physiological salt (Holtfreter) solution. Richards and Bagnara (1967) further demonstrated autonomous formation of species-specific pteridines, such as 6-hydroxymethylpteridine, by larval urodele xanthophores through the use of xenoplastic transplantation of neural crest between Axolotl and Pleurodeles. These experiments also revealed the autonomous expression of “Pleurodeles blue,” a pteridine relative and a presumed yellow pigment in larval xanthophores. This novel 22

pyridone N-glycoside (Yoshida et al., 1988) seems to be newt specific (J. T. Bagnara, unpublished). Combined cytochemistry and chemical analytical studies on skin preparations provided proof that pteridines function as pigments in these brightly colored pigment cells. Their intracellular localization in particular cytoorganelles, as in the aforementioned pterinosomes, was ascertained by ultracentrifugal fractionation of subcellular components of xanthophores and erythrophores in sucrose density gradients (Matsumoto, 1965a; Matsumoto and Obika, 1968; Obika and Matsumoto, 1968). Skin samples laden solely with these pigment cells were collected from fishes or frogs by careful dissection of homogeneously colored spots or stripes. Combined pteridine assay and electron microscopy on separated fractions disclosed that the vast majority of pteridines were detected in a fraction composed of large cytoplasmic granules, the pterinosomes, although certain amounts were present in fractions composed of membranous elements, mostly those of smooth endoplasmic reticula or Golgi complexes. Carotenoids, when present, were detectable in fractions composed of small vesicles or membrane fragments, indicating an apparent disparate distribution of pteridines and carotenoids within the cells. The morphology of pterinosomes is characterized by a spherical shape, slightly larger than melanosomes, and a distinct boundary of unit membrane enclosing an internal structure consisting of thin concentric lamellae or deposits of thin fibrils (Matsumoto, 1965a; Matsumoto and Obika, 1968; Obika and Matsumoto, 1968). Little is known about the relationship between the internal structure of pterinosomes and the composition of pteridines deposited. In developing xanthophores or erythrophores, multivesicular bodies are present

GTP GTP-CHI O

OH OH N

HN H2N

N

PHO

N H

H

O

C

C CH2O

P O

H

H

OH

3

H

Dihydroneopterin-3-phosphate PTPS

O HN H2N

N

O

OH OH N

C

C CH2OH

H

H

HN H2N

N

N

H N

HO O C C CH3 H N H H

H2N

N

H HH O N C C CH3 OH H N H H

HN H 2N

6-Lactoyltetrahydropterin

N

N H

H

Dihydropterin

SPR O

H HO H N C C CH3 H OH N H H

H H

H2N

XOR

AR O

N HN

6-Pyruvoyltetrahydropterin

Neopterin

HN

O

N

6-(1’-Hydroxy, 2’-oxopropyl) tetrahydropterin

O

O

H N

HN

O

H

N

HN

H H2N

N H

N

H

H2N

Dihydroxanthopterin

N

N

Pterin

SPR

XOR SPR

N

HN H2N

N

O

O H

O

N H

C C CH3 OH H H

H2N

N

O

O

O HN

H2N

N

N

H2N

Xanthopterin

N

N

H

N H

O

Isoxanthopterin

H H H C C CH3 OH OH H N H H N

HN HN

O

H N

HN

Tetrahydrobiopterin

Sepiapterin

SPR

H H H H N C C CH3 OHOH N HH H

HN

N

Quinoiddihydrobiopterin

O N

HN H2N

N

N H

H H C C CH3 OH OH H H

Dihydrobiopterin

O N

HN

COOH

O N

HN

H H C C CH3

O N

HN

OHOH H2N

N

N

Pterin-6-carboxylic acid

H 2N

N

N

Biopterin

H 2N

N

N H

H H C C CH3 OHOH O

Ichthyopterin

Fig. 2.19. Biosynthetic pathways of unconjugated pteridines common to xanthophores and their related pigment cells of lower vertebrates. Abbreviations of enzymes: AR, aldose reductase; GTP-CHI, GTP-cyclohydrolase I; PHO, phosphatase; PTPS, 6-pyruvoyl-tetrahydropterin synthase; SPR, sepiapterin reductase; XOR, xanthine oxidoreductase (adapted from Katoh and Akino, 1986; Masada et al., 1990; Takigawa and Nakagoshi, 1994; Ziegler et al., 2000).

23

CHAPTER 2

in the vicinity of Golgi complexes or GERL (J. Matsumoto et al., unpublished data). The similarity of these multivesicular bodies to Stage I melanosomes suggests that pterinosome genesis shares an origin common to that of melanosomes. These observations are consistent with the concept of the common origin of pigment cells (Bagnara et al., 1979). Cyanophores A most remarkable discovery was made a decade ago by Goda and Fujii (1995) who found blue dermal chromatophores that they designated cyanophores. These chromatophores contain a true blue pigment of unknown chemical nature. The blue pigment is found in fibrous organelles that they termed cyanosomes. Whereas cyanophores have been found, so far, in only two species of callionymid fish, it may be premature to afford them legitimate status. However, they are real and unique and must be recognized. Accordingly, they are included in Table 2.1.

Fig. 2.20. Green eggs (yolk plug stage) of P. dacnicolor. Eggs normally vary in color from blue-green to yellow-green (see also Plate 2.8, pp. 494–495).

Other Unknown Chromatophores Among some of the less studied groups of amphibians, there exist unusual pigmentary organelles in chromatophores that may be xanthophores or erythrophores. Schwalm and McNulty (1980) described some unusual chromatophores that seem to contain large vesicles with pigments that have not been elucidated. The dendrobatid (poison arrow) frogs also contain brightly colored chromatophores with vesicular organelles, the compositions of which are also unresolved (J. T. Bagnara, personal observations).

The Amphibian Egg as a Pigment Cell One would hardly think that there could be any relationship between the pigmentation of the integument and that of the eggs. On the contrary, the pigmentation of the eggs of frogs and skin have much in common both by homology and by analogy (Bagnara, 1985). The frog integumental pigmentary pattern of a dark dorsum and light ventrum is paralleled in the animal and vegetal poles, respectively, of frog eggs. Moreover, melanin is the dark pigment in both cases, and the pigment in each is contained in melanosomes. It appears that this pattern of pigmentation serves protective functions in both cases. In addition to dark pigmentation, the bright colors of the integument have much in common with the comparable colors that are found in the eggs of some species. In particular, the eggs of leaf frogs, the Pyllomedusinae, which are usually laid on green vegetation above the surface of pools of water, are often bright green (Fig. 2.20). A fundamental difference in egg pigmentation is that the green color is arrived at in a manner different from that of the skin. In both cases, the green color derives from a combination of blue and yellow; however, the blue element of the skin is a structural color whereas that of eggs is provided by the blue pigment, biliverdin IXa. This can be seen in Fig. 2.21, in which a blue area from a portion of P. dacnicolor skin is manifested when the overlying yellow pigment is leached from normally green skin. In contrast, as shown in Fig. 2.21, a blue pigment 24

Fig. 2.21. Pigments extracted from P. dacnicolor eggs. Left, biliverdin IXa; right, lutein (see also Plate 2.9, pp. 494–495).

(biliverdin) and a yellow pigment (lutein) provide the green color of P. dacnicolor eggs (Marinetti and Bagnara, 1982, 1983). In the absence of yellow pigments from xanthophores in the integument, frogs become blue. In Fig. 2.22, a blue individual of P. dacnicolor is shown. This frog was apparently deficient in xanthine dehydrogenase, a key enzyme in pteridine biosynthesis, and thus failed to produce pteridine pigments (Frost, 1978; Frost and Bagnara, 1978). In cases of carotenoid deficiency, the skin of this species also becomes bluish. This is a consequence of the absence of yellow carotenoid pigments from the xanthophores. In Fig. 2.23, sibling juveniles of P. dacnicolor are shown, one green and the other blue. The latter was raised on carotenoid-deficient crickets (potato-fed) and the former on carrot-fed crickets (J. T. Bagnara and G. V. Marinetti, unpublished). Analysis of the crickets revealed a preponderance of carotenes in the carrot-fed individuals and very low levels of any carotenoid in crickets fed potatoes. Of special interest here is the fact that the integumental

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

Fig. 2.22. “Blue Boy,” a young male P. dacnicolor, which lacked xanthophore pigments from his metamorphosis until sexual maturity when he gradually began to turn green. Presumably, he was xanthine dehydrogenase-deficient, but eventually produced enough pteridine pigments to restore normal green coloration (see also Plate 2.10, pp. 494–495).

Fig. 2.23. Sibling froglets of P. dacnicolor. The green frog was raised on crickets fed carrots exclusively, while the blue frog was fed potato-raised crickets that were carotenoid-deficient (see also Plate 2.11, pp. 494–495).

carotenoids of P. dacnicolor appear to be exclusively acidic xanthophylls. Thus, it appears that the frog liver must undertake major metabolic steps to convert carotenes to xanthophylls. How this occurs in frogs is unknown. Moreover, the transport of the xanthophylls from the liver to the skin is a mystery. Probably, this transport to the integument is similar to that of lutein, an acidic xanthophyll, to the egg wherein vitellogenin seems to serve as a transport protein for both biliverdin and lutein (Bagnara, 1985). Such transport in insects, that of biliverdin and lutein, involves specific transport proteins (see Kanost et al., 1990; Kawooya et al., 1985) and thus, by analogy, it seems reasonable to consider that the same may hold for amphibians.

Chromatophore Function A major function of integumental chromatophores is in background adaptation (cryptic coloration). Most lower vertebrates exhibit rapid body color changes for use in camouflage or background matching, as is well exemplified by the tropical flounder (Ramachandran et al., 1996). The simplest type, adaptation to black or white backgrounds, mainly involves the participation of both melanophores and iridophores. Thus, on black surfaces, melanosomes within melanophores become widely dispersed within the dendritic processes of the cell. At the same time, iridophores are minimally visible as their reflecting platelets aggregate to the center of the cell. In other words, black coloration is maximized and white is minimized. 25

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Fig. 2.24. In vitro response of R. pipiens skin photographed with reflected light. Left, in Ringer solution, iridophores, which appear silvery, are expanded; dermal melanophores are punctate. Right, in the presence of caffeine, intracellular levels of cyclic AMP become elevated; thus, iridophores are contracted to the punctate state while melanophores are well expanded (from Bagnara, 1976).

On white surfaces, these two pigment cells respond in an opposite manner. Adaptation to more particular backgrounds, such as to specific colors or shades or even mottled surfaces, may entail the participation of all three basic pigment cell types. Aside from their role in cryptic coloration, chromatophores function in a variety of other roles. Thermal regulation, protection from solar radiation, nuptial coloration, and warning coloration represent a few of the other more prominent roles of lower vertebrate chromatophores. Because of their roles in so many adaptive activities, the physiology of lower vertebrate chromatophores is by necessity exceedingly complex and is an important component of the integrative biology of poikilotherms. A fundamental feature is the fact that color change results from the translocation of pigmentary organelles within the cell (Figs 2.4 and 2.6). In early times, chromatophores were referred to as being “expanded or contracted” with the implication that the cell boundaries were actually motile. We now know that pigment cells are firmly attached to the extracellular matrix and that contractile motility actually refers to movements of the pigmentcontaining organelles within the chromatophore. Nevertheless, unfettered chromatophores, as in cell culture, do have the capacity to expand and contract, but it is not known whether this is a passive result of intracellular organellar motility or the result of some other contractile event. In any case, we now 26

consider color change to result from the aggregation or dispersion of pigmentary organelles within the cell (Fig. 2.24). This type of chromatophore motility has been the subject of continued scrutiny in recent years, especially as new knowledge about the cytoskeletal system of pigment cells has accumulated. The responses of pigment cells during color change are integrated, and mechanisms for this integration reside in several signaling mechanisms. As color change often results from the visual perception of background change, the nervous system has long been considered to be a key element in chromatophore signaling, and direct innervation of chromatophores was an early focus of investigation. Over the years, we have learned that chromatophore control through direct innervation occurs in some poikilotherms, but is by no means a general phenomenon. While best evidence for such innervation comes from studies of piscine chromatophores (see Bagnara and Hadley, 1973; Fujii, 1993a), not all orders of the class Osteichthyes possess innervated chromatophores. Early reports of possible innervation of amphibian chromatophores have been discounted (Iga and Bagnara, 1975); however, the early reports of the late 1880s describing innervation of reptilian chromatophores (chameleons) certainly remain valid (Whimster, 1971). In other lizards, such as Anolis, direct nervous control of chromatophores does not

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

exist (see Bagnara and Hadley, 1973); thus, we are left with the unanswered question of how pervasive direct innervation of chromatophores exists among reptiles.

Responses of Pigment Cells Physiologic Color Change Physiologic color changes are defined as those rapid changes in color caused by an intracellular movement of pigmentbearing organelles (Parker, 1948). As has been indicated, the response can be elicited by a variety of stimuli such as light or hormone action. The response is rapid, requiring a few minutes or a few hours to occur, and is transitory, i.e. the animal either regains its original coloration or assumes an intermediate state depending on stimulatory cues. Perhaps the most common manifestation of physiologic color change occurs in background adaptation. On a darkcolored background, amphibians that can adapt contain melanophores with dispersed melanosomes, while those on light-colored backgrounds are pale because their melanosomes have aggregated to a perinuclear position within the cell (Figs 2.4, 2.6 and 2.24). Iridophores respond in an opposite manner, being dispersed on light-colored backgrounds and aggregated on dark backgrounds. Apparently, the lateral eyes play a fundamental role in governing these background responses of both melanophores and iridophores. In most amphibian larvae, there is a distinct point in development at which they become able to adapt to background. Before this time, when they are not able to adapt to background, they remain dark colored whether on a light or a dark background, and they are said to be in a primary phase or stage. Older larvae that can adapt to background are said to be in a secondary stage. The secondary stage describes what essentially constitutes adaptation to background based upon the reception of light through the eyes and the control of MSH release from the pituitary. Melanosome aggregation can occur in primary-stage larvae in darkness, but this does not result from alterations in MSH release, but relates to other physiological systems that operate in darkness as mentioned earlier and to be described more fully later. It should be noted that onset of the secondary phase varies with species: whereas melanophores of Xenopus larvae respond to background changes from very young stages onward, larvae of Rana and Hyla do not display the secondary response until they are several weeks old, and some Bufo larvae never seem to acquire the secondary phase. These observations of the ontogeny of physiologic color change have been made primarily on amphibians and, although much is known about the physiology of chromatophores of fishes, the majority of the work has been on adults or at least on post-embryonic stages. Morphologic Color Change Color changes that are evoked slowly and result from alterations in the amount of pigment contained in the integument have long been called morphologic color change (Parker, 1948). Morphologic color change is a relatively slow process that includes the synthesis or destruction of relatively large

amounts of pigment in response to a persistent stimulus. Background adaptation is a common cause of morphologic color change: animals maintained on dark backgrounds develop more melanin whereas those on light backgrounds lose their melanin (Dawes, 1941). The opposite is true of iridophores with respect to their purine pigments (Bagnara and Neidleman, 1958; Fernandez, 1988; Fernandez and Collins, 1988; Taylor, 1969). The total increase or decrease in the amount of pigment can result from several causes, one of which relates to the number of chromatophores present. For example, Xenopus larvae that have been deprived of their pituitary gland (and hence have no melanophore stimulation) contain many fewer dermal melanophores than they do normally (Bagnara, 1957). That the hypophysis exerts its influence on morphologic color change by causing a profound proliferation of dermal melanophores has been elegantly shown (Fig. 2.25) by Pehlemann (1967a, b, 1972). Probably, the increase in chromatophore number following persistent stimulation also results from the synthesis of pigments in hitherto undifferentiated melanoblasts. This phenomenon seems to be a general one and provides a valid interpretation of the fact that iridophores appear in the tail fin of Xenopus larvae that have been deprived of their hypophysis (Fig. 2.26), although such chromatophores are not observed in intact normal larvae (Bagnara, 1957). Amphibian iridophores are markedly inhibited by MSH, both physiologically and morphologically (Bagnara, 1958). In his PhD dissertation, Hadley (1966) described how, in juvenile Xenopus maintained on dark backgrounds for 10 weeks, there is a fivefold increase in the number of web melanophores present in comparison with controls maintained on white backgrounds. The spacing of these new melanophores seems to exclude the presence of doublets as would be expected in cases of rapid increase in melanophore numbers due to proliferation; thus, it is presumed that these new melanophores were derived from latent melanoblasts that underwent melanization in response to the elevated blood levels of MSH present in those animals on black backgrounds. Similar results were obtained by Fernandez and Bagnara (1991, 1993), who studied morphologic color change in leopard frogs (Figs 2.27 and 2.28). They showed that the intense melanization corresponded directly to elevated blood levels of MSH as measured by radioimmunoassay (RIA) (Fig. 2.29). Definitive proof that latent undifferentiated melanoblasts are the source of new melanophores comes from an unpublished study described by Bagnara and Fernandez (1993) in which the pars intermedia of juvenile Xenopus were implanted into the lower unpigmented jaw of larvae (Fig. 2.30). Melanophores differentiated in an area concentric to the implant, implying that they were derived from latent melanoblasts. Manifestations of morphologic color change occur at the organellar level and in some cases can be seen with the electron microscope. For example, after administration of large dosages of MSH to adult frogs, a condensation of the reflecting platelets can be seen in iridophores (Bagnara et al., 1968, 1969; Taylor, 1969). Through action on the adjacent epidermal cells, epidermal 27

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Fig. 2.25. Details of the upper right side of the head of a Xenopus larva, (A) 1, (B) 5, (C) 7 days after transection of the hypophysial stalk leading to greatly increased levels of circulating MSH and melanophore proliferation. A single melanophore (arrow) can be traced through two mitotic divisions in (B) and (C) (courtesy of Dr F. W. Pehlemann; from Pehlemann, 1967b).

melanophores also play an important role in morphologic color change (Hadley and Quevedo, 1967). During profound melanophore stimulation, such as prolonged exposure to a dark background, pronounced cytocrine activity of the dendrites of epidermal melanophores causes melanin to be deposited in adjacent epidermal cells (Figs 2.4 and 2.8). This process in frogs has not been studied with the electron microscope, and the mechanism of melanin deposition is not understood; however, studies made at lower magnification indicate that considerable amounts of melanin are deposited in the epidermis in this manner. In adult frogs, cytocrine deposition of melanin is the primary means of morphologic color change (Hadley and Quevedo, 1967). The importance of epidermal melanophores (-cytes) in homeotherms is well known, and it has been suggested that epidermal melanocytes of all vertebrates are homologous in that they seem to be identical in every way (Bagnara and Ferris, 1971).

The Control of Color Change The Pituitary and the Role of MSH Fig. 2.26. A Xenopus larva hypophysectomized as a tailbud embryo. Note the presence of iridophores and the great reduction in melanophores of the integument, brain case, and other deep areas (from Bagnara, 1957).

28

The ability of lower vertebrates to carry out morphologic and physiologic color change in response to appropriate environmental cues is based upon the existence of precise control mechanisms involving the pars intermedia of the pituitary and the release of MSH. Whereas the existence of this hormone was inferred from experiments on poikilotherms, characteri-

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

α–MSH (pg/ml)

200 175 150 125 100 75 50 25 0

α–MSH (pg/ml)

Fig. 2.27. Dorsal (top) and ventral (bottom) views of adult R. chiricahuensis adapted to dark backgrounds (left individuals) or white backgrounds (right individuals). Note the profound darkening of the dark background-adapted individuals and especially the ventral surface (from Bagnara and Fernandez, 1993).

1800 1600 1400 1200 1000 800 600 400 200 0

Rana pipiens

White

Gray

Fig. 2.28. Dorsal (top) and ventral (bottom) views of adult R. pipiens adapted to dark backgrounds (left individuals) or white background (right individuals). Note the darkening of the dorsal surface of the dark background-adapted individuals and the lack of darkening of the ventral surface of the dark background-adapted individuals (from Bagnara and Fernandez, 1993).

Black

Rana chiricahuensis

Gray Black White Background color

Fig. 2.29. Plasma a-MSH of R. pipiens and R. chiricahuensis on white, gray, and black backgrounds. Each bar represents the mean (±SE) of 1–4 frogs. Plasma a-MSH of frogs on a black background is greater than those on white or gray (P < 005). Note the particularly profound increase in plasma a-MSH of R. chiricahuensis (modified from Fernandez and Bagnara, 1991).

zation of MSH was achieved from mammalian sources. A personal account of the biochemical characterization of the MSHs in the mid-1950s has been graciously written by Professor Aaron B. Lerner (1993). It was not until some 30 years later that poikilothermic MSHs were isolated, purified, and characterized from fishes and amphibians. Thus, some idea of

Fig. 2.30. Lower jaw of a larva of Xenopus laevis that had been implanted subcutaneously with the pars intermedia of a froglet. A spherical blood clot marks the site of the transplant, which releases MSH chronically. Note that the nearby dermis is populated with melanophores distributed over the graft. No melanophores were observed in the dermis of the jaw before the transplantation (from Bagnara and Fernandez, 1993).

the phylogeny of MSH was obtained (Dores et al., 1993). It has been shown that, although the tridecapeptide a-MSH is a ubiquitously secreted peptide derived from the ancestral molecule, proopiomelanocortin, N-acetylation of the hormone is 29

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a key element in the phylogeny of the molecule. Like that of mammals, ranid a-MSH is N-acetylated, a scheme inherited from an ancient tetrapod. During evolution, the scheme was retained except among some of the squamate reptiles, which have lost this capacity. Among the latter is the fence lizard Anolis, which secretes a non-N-acetylated a-MSH that is nevertheless a functional melanotropin.

Melanophore Stimulation The most well-known activity of MSH is to stimulate the dispersion of melanosomes within melanophores of poikilotherms. In the absence of MSH, melanophores assume a punctate (contracted) configuration in which melanosomes are concentrated in the central (perinuclear) portion of the melanophore (Figs 2.4, 2.6 and 2.24). Both dermal and epidermal melanophores respond to stimulation by MSH by demonstrating physiologic color change, whereas prolonged exposure to the hormone, especially at high concentration, is conducive to morphologic color change. It has been thought that morphologic color change is an indirect function of MSH activity that actually results from persistent physiologic color change; however, Pehlemann’s (1972) demonstration of increased melanization resulting from enhanced mitotic activity in melanophores under MSH stimulation indicates that some expressions of morphologic color change are independent of physiologic color change. It is concluded that MSH affects both dermal and epidermal melanophores and is capable of eliciting morphologic color change in both chromatophores. The possible role of MSH on pigment pattern formation through its effects on morphologic color change will be considered later. Iridophore Effects Earlier work on color change centered around melanophore responses. During later years, however, attention was given to the effect of MSH on iridophores, the reflecting pigment cells commonly found in poikilotherms (Figs 2.8, 2.31, and 2.32). In normal larvae and in intact adult frogs as well as in fishes, iridophores are in a punctate state due to a concentration of reflecting platelets toward the center of the cell (see Bagnara and Hadley, 1973; Fujii, 1993a). In the absence of the pituitary, reflecting platelets are dispersed and the iridophore exists in an expanded state. The “silvery” appearance of hypophysectomized ranid larvae observed by Smith (1916) and Allen (1916) results from the “expansion” of iridophores and the “contraction” of melanophores; in intact larvae, the reverse is true. Smith (1920) recognized that the hypophysis exerted an influence on iridophores, which he referred to as xantholeukophores; however, except for Hogben and Winton (1924) and Stoppani et al. (1954), most other workers emphasized melanophore effects, and gradually the prominent role of iridophores in amphibian color change was overlooked and almost forgotten. Interest in amphibian iridophores was renewed when it was discovered that these cells are controlled by MSH just as are melanophores (Bagnara, 1958). Substantial proof for this 30

Fig. 2.31. A mixed primary cell culture of chromatophores, containing mostly iridophores, isolated from R. pipiens tadpoles. Left, attached iridophores in their normal semi-aggregated (expanded) state. Right, a similar culture after 1 h of exposure to [Nle4,D-Phe7]a-MSH (10-5 M). Both photographs were taken under epi-illumination. Note the aggregated (contracted) state of the treated iridophores, which have actually detached from the surface of the culture dish.

Fig. 2.32. Cells similar to those in Fig. 2.31, viewed in thin sections. Upper left, a rounded iridophore before attachment. Bottom, a dispersed iridophore attached to the culture dish. Upper right, detached and rounded iridophores following MSH treatment.

concept was obtained when highly purified a- and b-MSH first became available and were shown to cause iridophore aggregation in both hypophysectomized larvae (Bagnara, 1964b) and in isolated skins of adult frogs (Hadley, 1966; Hadley and Bagnara, 1969). That iridophores are not only sensitive to MSH, but also react by aggregating rather than dispersing under its influence led to the question of whether both melanophore expansion and iridophore contraction require the same sites on the MSH molecule (Fig. 2.24). That this is indeed the case was demonstrated through the utilization of the fact that, in adult frog skins, the entire MSH molecule is not essential for melanophore-expanding activity (Lee et al., 1963) and that some melanophore response can be induced

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

by the centrally located polypeptide sequence, His-Phe-ArgTrp-Gly. When pentapeptide and larger synthetic MSH peptides were injected into hypophysectomized larvae, definite iridophore aggregation was observed, and the iridophore response was always paralleled by melanophore dispersion (Bagnara, 1964b). A similar parallelism of iridophore and melanophore responses was obtained with a thermal polymer of arginine, glutamic acid, histidine, phenylalanine, and tryptophan (Bagnara and Hadley, 1970b). This early work that utilized short amino acid sequences of the MSH molecule to study iridophore responses made little impact because it was very much obscured by the rapid accumulation of new knowledge about MSH chemistry that was mounting up. This work centered on the frog skin bioassay, which was generally considered to involve melanophore responses, although Hadley and Bagnara (1969) clearly described the integrated responses of both melanophores and iridophores in this assay. Early on, Bagnara (1958) had demonstrated that the alkali-enhanced activity of MSH peptides was evidenced in both melanophore and iridophore responses. In later years during the development of superpotent a-MSH analogs (Sawyer et al., 1980), their effects on iridophores were not observed. Recently, such a study has been carried out (J. T. Bagnara et al., unpublished) on tadpole iridophores in culture and, as can be seen in Figure 2.31, iridophores exposed to the superpotent analog [Nle4,D-Phe7]a-MSH indeed become aggregated. This is of special interest not only because it demonstrates iridophore sensitivity to the hormone, but because the response displays both aggregation of reflecting platelets and actual contraction of the iridophore, causing it to round up and detach from its substrate (Fig. 2.32). Early work on the role of MSH in iridophore control was done on amphibians, and only in recent years was it shown that piscine iridophores respond to the hormone in the same way (see Fujii, 1993a). Leukophores, however, may provide a bit of a paradox in that leukophores of the medaka may disperse in the presence of MSH (Negishi and Obika, 1980). Iridophores play a role in both physiologic and morphologic color changes. When high doses of MSH are administered or when hormone treatment is prolonged, larvae of some species such as Rana sylvatica display a morphological response so great that the many iridophores of hypophysectomized larvae may completely lose their pigment (Bagnara, 1958; Bagnara and Neidleman, 1958). This morphological effect of MSH on iridophores has been studied by Taylor (1969), who points out that reflecting platelets in iridophores become thinner in frogs receiving MSH injections. The diminution in reflecting platelet thickness is a morphological manifestation of a quantitative loss of measured purines. Xanthophore Effects Relatively little is known about the physiological responses of amphibian xanthophores or erythrophores to MSH because these chromatophores are difficult to see. They are often pale, especially at their margins, and the presence of large numbers of iridophores and melanophores serves to mask the xan-

thophores. Nevertheless, there are at least some clear cases in which xanthophores are influenced by MSH. In adults of Hyla arenicolor, xanthophores of skins kept in Ringer solution are aggregated; however, upon administration of MSH, these cells expand (Bagnara, 1969; Bagnara et al., 1968). In more recent times, there have been numerous studies on the xanthophores and erythrophores of fish, and it is now well documented that both these chromatophores generally disperse under the influence of MSH (see Fujii, 1993a). The morphological effect of MSH on xanthophores of amphibians is more prominent and is best demonstrated by quantitative changes in pteridine pigments. In the skin of hypophysectomized larvae, the content of pteridines is considerably lower than that of intact larvae, but it returns to normal when such larvae are injected with MSH (Bagnara, 1961, 1969; Bagnara and Neidleman, 1958). Xanthophores in normal larvae of the salamander Pleurodeles waltlii are expanded such that their broad arms form a continuous yellow sheet over the surface. Xanthophores of hypophysectomized larvae are also dispersed; however, individual processes are thin and delicate and individual chromatophores stand out clearly. Thus, the morphological effect of MSH on xanthophores is manifested by the amount of pteridine pigments contained and is reflected in the general appearance of the chromatophore. In Pleurodeles, there is also a marked difference in the total carotenoid content between normal and hypophysectomized larvae (Bagnara, 1969). It is not known, however, whether the diminution of carotenoids from the skin of hypophysectomized larvae is a result of the absence of MSH or an indirect manifestation of the lack of a hypophysis. Despite the profound effect of MSH on the pteridine content of xanthophores, there has been no observation of an MSH effect on the pterinosome. No differences have been noted between the pterinosomes of skins from normal frogs and those from frogs receiving MSH injections. Cyanophore Effects With their discovery of cyanophores in both the mandarin fish, Synchiropus splendidus, and the psychedelic fish, S. picturatus, Goda and Fujii (1995) showed that these chromatophores responded to various stimulatory cues. Whereas their exposure to norepinephrine resulted in slow aggregation, a-MSH induced pigment dispersion.

Melanin-Concentrating Hormone (MCH) MCH is a cyclic heptadecapeptide secreted from the posterior lobe of the pituitary that has the capacity to bring about melanin aggregation in piscine melanophores (Kawauchi et al., 1983). It seems to be present primarily in teleost fishes (Sherbrooke et al., 1988). This neuropeptide has been found in the brain of other lower vertebrates where its neuronal cell bodies seem to be localized in the hypothalamus (Baker, 1993). In most species, only a few of its axons project to the posterior pituitary; however, in teleosts, the neurohypophysial lobe receives many MCH fibers, and thus the neuropeptide has 31

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achieved legitimate hormonal status in this group. It seems to function in the regulation of skin color through its ability to bring about pigment aggregation within melanophores and apparently in xanthophores and erythrophores as well (Baker, 1993). It is possible that the use of MCH in the control of color change in this group of fishes is an evolutionary novelty that arose near the end of the Paleozoic or during the early Mesozoic, just before or in the evolution of the Holostei, a group ancestral to modern teleosts (Sherbrooke and Hadley, 1988). Baker (1993) has summarized the evidence that MCH is used in color change in teleosts. Among the points that she raises is the fact that MCH can induce melanin concentration in isolated fish skin melanophores. The differential effects observed following the use of MCH fragments on different species suggests variation in the MCH receptor. MCH has the capacity to antagonize the melanin-dispersing action of aMSH directly at the melanophore level. MCH may also play a role in pigmentary control by depressing release of MSH from the pituitary. Among the fruits of continuing investigation in this field is the revelation by Oshima and Wannitikul (1996) that cyclic AMP is probably the messenger for MCH action.

The Effects of Other Hormones on Pigment Cells In addition to MSH, other hormones are known to affect chromatophores. Two of these, melatonin and epinephrine, will be discussed in the following sections because they are associated with specific responses. The thyroid has long been implicated in pigmentary changes that occur during amphibian metamorphosis. Woronzowa (1932) reported that thyroid extracts affected the spotting pattern in metamorphosing ambystomids, and Kollros and Kaltenbach (1952) observed pigmentary changes in the vicinity of thyroid implants in Rana pipiens larvae. A profound increase in chromatophore number occurs at metamorphic climax in such larvae (Bagnara and Hadley, 1973). Similarly, specific pteridine changes were induced in localized areas of the skin of Pleurodeles larvae following the implantation of thyroxine cholesterol pellets (Bagnara, 1964c). Among pteridine-associated changes is the abrupt disappearance of “Pleurodeles blue,” long thought to be a pteridine, but now known to be a novel pyridine derivative (Yoshida et al., 1988). The mode of action of thyroxine on the metabolism of this compound is a question that begs an answer. Effects of thyroxine on physiologic color change have been observed. Chang (1957) indicated that the blanching of frogs following administration of thyroxine may be attributable to an inhibition of MSH release from the pars intermedia. The same may hold for salmonids (Bagnara and Hadley, 1973). The direct action of this hormone on pigment cells was found in the in vitro study by Wright and Lerner (1960). Triiodothyronine is more potent than thyroxine or any other known hormonal agent, except for melatonin, in reversing the action of MSH on isolated frog skins (Bagnara and Hadley, 1970a; Hadley and Bagnara, 1969). 32

Studies on other vertebrates have long indicated that steroid hormones influence pigmentation, and such an effect on amphibian pigmentation was reported by Himes and Hadley (1971), who observed that progesterone exerts an MSH-like response on frog skins in vitro. A strong case for a role for sex hormones in in vivo color changes was presented by Richards (1982), who demonstrated a profound alteration of chromatophore expression by sex hormones in the Kenyan reed frog Hyperolius. Adaptive Mechanisms Background Adaptations MSH has long been considered the major hormone involved in background adaptation, and this peptide has provided the basis for the unihumoral theory of chromatophore control (see Parker, 1948). This theory considers that, during adaptation to white backgrounds, little or no MSH is released from the pars intermedia, resulting in low circulating levels of this hormone and a consequent lack of stimulation of chromatophores. Thus, melanophore pigments are aggregated and iridophore pigments are dispersed. During black background adaptation, MSH is released leading to a dispersion of melanophore pigments and an aggregation of reflecting platelets within iridophores; consequently, the animal darkens. This concept is supported by observations of cytological changes in the pars intermedia of frogs in correlation with background adaptation (Cohen, 1967; Imai, 1971; Perryman, 1974; Saland, 1967). The appearance of pars intermedia cells in frogs adapted to black backgrounds is consistent with a state of hormone synthesis and release. This was also the conclusion of Burgers et al. (1963) made on the basis of actual MSH assays. In background adaptation, it is implicit that the animal can perceive differences in background. It seems that the lateral eyes are involved as blinded animals equilibrate to an intermediate state of pigmentation and do not alter coloration in response to changes in background (see Parker, 1948; Bagnara and Hadley, 1973). Moreover, direct electrophysiological studies by Dawson and Ralph (1971) have shown that changes in illumination of the lateral eyes of adult R. pipiens are clearly recorded in the pars intermedia. The mechanism by which the retina discriminates background difference is not understood, although it has long been thought (Butcher, 1938) that dorsal and ventral regions of the retina have different sensitivities to incident and reflected light and thus allow appropriate perception of albedo (the amount of light reflected from the substrate, as explained in Fig. 2.33). Ultimately, background adaptation is regulated by the control of MSH release through the involvement of the hypothalamus. The control is mediated through the inhibition of MSH release, as was suggested by Etkin (1941, 1962a, b) who along with others obtained extremely dark tadpoles after isolating the pituitary by hypophysial transplantation or by hypophysial stalk section (Fig. 2.25). During the past 30 years, an immense literature has accumulated in this area, and the current state of knowledge has been summarized by René et al. (1993) and Roubos et al. (1993). The evidence is over-

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

Light source

Reflecting substrate High albedo

Absorbing substrate Low albedo A

Light source

High albedo environment

Light source

Low albedo environment

Fig. 2.33. Perception of light by Arizona tiger salamanders differs between white and black substrates. Light is reflected from a white substrate and provides a high albedo, whereas a black substrate absorbs light and the albedo is low. Larvae in a turbid pond (lower left) experience a high albedo because of light reflected from suspended particles, while those in a clear pond are subjected to a low albedo because light is absorbed by plants and the dark-colored pond bottom (courtesy of P. Fernandez; from Fernandez and Collins, 1988).

whelming that hypothalamic inhibition of MSH secretion is controlled by catecholamines, notably dopamine. Presumably, the pars intermedia chronically releases MSH, and this release is controlled through neurosecretory fibers that reach the pars intermedia from the hypothalamus. Perception of a light background (high albedo) results in inhibition of MSH release and consequent paling of the animal. On a dark background (low albedo), hypothalamic neurosecretory neuron release of dopamine is reduced and resulting high levels of MSH lead to darkening (Figs 2.27–2.29). Adaptation to Darkness Unlike adults, larval stages of most amphibians display a

B

Fig. 2.34. Xenopus larvae, stage 42. (A) Larva under normal room illumination on a white background; larva does not background adapt because it is in a primary stage. (B) Similar larva in darkness for 1 h; note prominent melanophore contraction in response to melatonin release from the pineal (from Bagnara, 1976).

remarkable ability to blanch when they are maintained in darkness (see Bagnara, 1965, 1966b; Bagnara and Hadley, 1970a). This blanching reaction was first observed long ago (Babak, 1910); however, it was only much later that a basic mechanism controlling this response was described (Bagnara, 1960, 1961). The proposed mechanism describes a role for the pineal in a normal physiological function. It is essentially hypothetical, but it remains unchallenged and is widely accepted (Bagnara and Hadley, 1970a; Bogenschütz, 1965). The hypothesis suggests that, under conditions of darkness, the pineal is stimulated to release melatonin, presumably a pineal hormone, into the general circulation. Melatonin exerts a profound contracting effect on dermal melanophores leading to a rapid blanching (Fig. 2.34). As first described by Bagnara (1960), the involvement of the pineal relates to two aspects of its physiology: light reception and endocrine function. The former has been a role attributed to the pineal since before the classic work of von Frisch (1911) on fishes and has since found substantial support from both ultrastructural and electrophysiological studies (see Eakin, 1973). The role of the pineal as an endocrine organ is more obscure and requires a fuller explanation. The first evidence that the pineal contains a humoral agent is attributable to the studies of McCord and Allen (1917), who discovered that feeding mammalian pineals to tadpoles evoked a profound blanching. Later, Lerner et al. (1958) isolated a potent melanophore-contracting agent from beef pineal glands, which they identified as melatonin (Nacetyl-5-methoxytryptamine). A detailed analysis of the data supporting the concept that the body-blanching reaction is mediated by the pineal has been presented elsewhere (Bagnara, 1965; Bagnara and Hadley, 1970a; Eakin, 1973); however, a few important points need to be made here. First of all, it should be mentioned that 33

CHAPTER 2

blinded larvae become pale when they are placed in darkness (Bagnara, 1960; Laurens, 1915, 1917). Moreover, the blanching reaction is abolished by “pinealectomy” (Bagnara, 1960, 1963; Charlton, 1966). Temporal events in the onset and recovery from the blanching reaction are consistent with the view of an endocrine mediation of the response. Of many indoles tested, only melatonin is a potent melanophorecontracting agent (Quay, 1968; Quay and Bagnara, 1964), and the pigmentary changes induced by the action of melatonin duplicate the responses that occur in darkness. Young tadpoles, not yet able to respond to dark backgrounds by inhibiting MSH release from the pars intermedia, are in the primary stage and thus have dispersed melanophores. Such larvae blanch when placed in darkness due to the direct effects of melatonin on dermal melanophores causing them to aggregate. In larvae that possess epidermal melanophores or iridophores, these chromatophores do not change in darkness because they do not respond directly to melatonin. While these data support the hypothesis that the bodyblanching reaction of amphibian larvae is controlled by the pineal, it must be emphasized that this mechanism is restricted to larvae and does not appear to be generally functional in adults. It is well known that adult amphibians do not blanch in darkness (see Parker, 1948). Moreover, both epidermal and dermal melanophores in skins of adult frogs are generally unresponsive to the administration of melatonin, as has been demonstrated both in vitro (Bagnara and Hadley, 1970a; Hadley and Bagnara, 1969) and in vivo. Whether they lack the melatonin receptor originally deduced by Heward and Hadley (1975) on the basis of structure–function studies and characterized recently by expression cloning from Xenopus (Ebisawa et al., 1994) is not known. Among the various color changes displayed by vertebrates, those resulting from the direct action of light are among the most striking. Perhaps the most remarkable of these is the tail-darkening reaction observed when hypophysectomized Xenopus larvae were placed in the dark (Bagnara, 1957) and subsequently shown to occur in isolated tails maintained in darkness (Fig. 2.35) (Bagnara, 1957, 1966b; Burgers and van Oordt, 1962; van der Lek et al., 1958). Under usual conditions of illumination, dermal melanophores of the fin are punctate, or nearly so, and the tail is essentially transparent. When isolated tails or whole tadpoles are placed in darkness for half an hour or more, a profound dispersion of melanosomes occurs in these melanophores so that the tail becomes black. Upon resumption of illumination, these melanophores quickly revert to the original punctate state and the tail becomes pale. Illumination of given regions of the tail (Bagnara, 1957) or of individual melanophore processes (van der Lek, 1967) can cause aggregation of melanosomes, suggesting that the response is mediated by a photochemical system operating at the level of the chromatophores themselves. The tail-darkening reaction was first considered to be a rather peculiar specialization of Xenopus larvae, but we now know that it occurs in larvae of a Mexican leaf frog, P. dacnicolor (Bagnara, 1974), and in many other hylid tadpoles. 34

Fig. 2.35. In vitro tail-darkening response of isolated tails from Xenopus larvae under normal conditions of illumination (top) and in darkness (bottom). The tail-darkening response was prevented in a central area which was left illuminated (center) (from Bagnara, 1957).

The dynamic light-sensitive tail fin melanophores have been used to address another infrequently considered phenomenon of chromatophore physiology, that of the energy requirements for the aggregation–dispersion phenomenon. Some investigators have considered that pigment aggregation is the principal energy-requiring step (Horowitz, 1958), whereas others have concluded that dispersion is the principal energy-demanding process. Iga and Bagnara (1975) have demonstrated that both aggregation and dispersion of melanosomes are energy requiring and will not occur in the absence of oxygen (Fig. 2.36). Furthermore, they showed that, although darkness-induced darkening of Xenopus tail fin melanophores does not occur in oxygen-free media, restoration of oxygen to such tails even after they have been returned to lighted conditions leads to a dispersion aftereffect. This indicates that the phenomena of light reception and melanosome dispersion are two separate components of the tail-darkening reaction. In addition to the tail-darkening reaction, the direct effect of light on melanophores cultivated in vitro has been reported. Kuhlemann (1960) indicated that embryonic melanophores of Xenopus grown in tissue culture respond to illumination by becoming punctate (confirmed more recently by Daniolos et al., 1990), just as do embryonic melanophores of neural crest explants of several anurans (Fig. 2.37) (Bagnara and Obika, 1967). The significance of these direct effects of light on melanophores grown in culture is not known.

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES A

Fig. 2.37. Neural crest explant of Xenopus. Under normal room illumination, melanophores remain in an aggregated state (left) but, following a 45-min period in darkness, these melanophores become punctate (right) (modified from Bagnara and Obika, 1967). B

C

Fig. 2.36. (A) Response of tail fin melanophores of a Xenopus tadpole to darkness (D) and to light (L) in Ringer solution; the dark period is indicated on the abscissa by a solid black bar. Melanosome dispersion takes place gradually in darkness, and aggregation occurs quickly in light. (B) Inhibition of melanosome dispersion in the tail fin melanophores of a Xenopus tadpole in oxygen-free Ringer solution and the dispersion “aftereffect,” which occurs in oxygen-containing Ringer solution. The dark period is indicated on the abscissa by a solid black bar. (C) A typical example of retardation of melanosome aggregation in the tail fin melanophores of a Xenopus tadpole in oxygen-free Ringer solution; the dark period is indicated on the abscissa by the solid black bar (D). Pigment dispersion of the melanophores was induced by dark treatment of the tails for 45 min in Ringer solution, and then the tails were transferred into oxygen-free Ringer solution in a dark room (from Iga and Bagnara, 1976).

In addition to amphibian melanophores, examples of direct light sensitivity of fish chromatophores are well documented. Iga and Takabatake (1986) have demonstrated that teleost melanophores may respond to local light stimulation by aggregating in darkness and dispersing in the light. Of special interest among fishes is the demonstration of light-sensitive iridophores in the neon tetra (Lythgoe et al., 1984; Nagaishi and Oshima, 1989), such that the distance between reflecting platelets within these cells is changed through the action of the light. In a search for the basis of this photosensitivity of iridophores, Lythgoe et al. (1984) demonstrated the presence of an opsin-based visual pigment in these iridophores, either or both rhodopsin or/and porphyropsin. The presence of visual pigments in a photosensitive chromatophore is both fascinating and significant and asks the question whether similar visual pigments are present in other chromatophores such as the light-sensitive melanophores discussed previously. It seems likely that, as more species are studied with respect to lightinduced color changes, more unusual phenomena will be revealed. Such seems to be the case with P. dacnicolor, in which the skin of brown individuals displays a direct photosensitivity (Iga and Bagnara, 1975). When masks are placed on the brown surface, the skin beneath becomes green in exact conformation with the mask (Fig. 2.11).

Mechanisms of Hormone Action on Pigment Cells The Cyclic AMP as a Second Messenger of MSH Action In accordance with the first messenger–second messenger scheme (Sutherland et al., 1965), MSH acts as a first messenger and brings about its effects by promoting an intracellular increase in a second messenger, 3¢,5¢-cyclic adenosine monophosphate (cyclic AMP), which is in turn responsible for the particular response of the effector cell (Sutherland et al., 1968), in this case pigment cells. Thus, cyclic AMP mimics the action of MSH by darkening frog skin in vitro (Bagnara and Hadley, 1969; Bitensky and Burstein, 1965; Novales and Davis, 1967). The response is not as effective as that of MSH itself or as that of the dibutyryl derivative of cyclic AMP (Goldman and Hadley, 1969); however, the response to either of these cyclic nucleotides is truly MSH-like, for cytological 35

CHAPTER 2

examination of darkened skin reveals that both melanophores and iridophores react. Probably, both iridophores and melanophores contain similar or identical MSH receptor sites, and the different events in the two cells following MSH stimulation could then be attributable to the interaction of the second messenger and the specific distal functional elements of the particular chromatophore. Further support for the involvement of the first messenger–second messenger concept in the regulation of chromatophore control is derived from the fact that methylxanthines, such as caffeine or theophylline, can bring about both iridophore and melanophore responses (Fig. 2.24). Methylxanthines are known to increase cellular levels of cyclic AMP by inhibition of cyclic nucleotide phosphodiesterase (Butcher and Sutherland, 1962; Sutherland and Rall, 1958). Furthermore, it has been reported that MSH stimulates cyclic AMP formation in frog skin in correlation with the degree of darkening (Abe et al., 1969). The parallelism between iridophore and melanophore responses to the various MSH peptides, be they unnatural synthetic partial sequences or entire molecules such as a-MSH or b-MSH (Bagnara, 1958, 1964b), is to be expected from the point of function. The most efficient darkening response involves both melanosome dispersion and reflecting platelet aggregation. Accordingly, it seems logical to conclude that, despite the differing intracellular responses of these two divergent chromatophores, they must each possess similar MSH receptors. In modern times, the quest for characterization of the MSH receptor has led to the discovery of several different MSH receptors that function in accordance with specific roles for MSH (Cone et al., 1993). At present, at least five receptors are known from mammals for the melanocortin [MSH/adrenocorticotropic hormone (ACTH)] peptides. These melanocortins are labeled numerically in order of their discovery, with melanocortin-1 (MCR-1) assigned to the surface of melanocytes. It is known through the use of various specific a-MSH antagonists that frog melanophores possess at least one MCR, but which of the five is not known (Hruby et al., 1995). An interesting question concerns the possible similarities or differences between the mammalian MCR-1 and that of amphibians. An answer to this question may be forthcoming when the amphibian MCR-1 gene is cloned. In the meantime, we assume that the amphibian MCR-1 is present on the iridophore cell surface, but the distribution of this receptor on the surfaces of these differing pigment cells is unknown, and the question of whether these two chromatophores share identical profiles is unanswered. It is known through the use of various specific a-MSH antagonists that frog melanophores possess multiple MSH receptors, but such studies are in their infancy (Hruby et al., 1995). As such studies proceed, problems of species variation will be of concern. For example, an interesting study by Quillan et al. (1995) has revealed that D-Trp-Arg-Leu-NH2 is a potent a-MSH antagonist in Xenopus and that it is capable of causing pallor in adults when injected systemically. Moreover, it has the capacity to cause local lightening when applied top36

ically to such frogs. Xenopus is a rather particular amphibian; thus, the question arises of whether this MSH antagonist is generally active among amphibian species or is uniquely active in Xenopus.

The Role of Adrenergic Receptors While MSH seems to be the major factor regulating amphibian chromatophores, other hormones may also play a role in color change. Among these are catecholamines, such as epinephrine and norepinephrine, that apparently control the rapid color changes associated with “excitement pallor” (see Parker, 1948). Catecholamines are known to mediate certain effects through two types of receptors, a and b (Ahlquist, 1948), each of which controls responses that are antagonistic to the other. Accordingly, the response of a system to catecholamine stimulation depends on the presence or absence of a and b receptors, and it has been shown that both receptors may be present on amphibian chromatophores. With the aid of the receptor concept, it becomes possible to elucidate some previously unexplained paradoxical effects. For example, while both epinephrine and norepinephrine lighten the skins of R. pipiens by overriding the MSH effects (Wright and Lerner, 1960), catecholamines darken the skin of both Xenopus (Burgers et al., 1963) and Scaphiopus (Goldman and Hadley, 1969). Apparently, a receptors are present in R. pipiens (Lerner et al., 1954; Novales and Novales, 1965), accounting for the paling reaction, whereas in Xenopus (Graham, 1961; Novales and Davis, 1969) and Scaphiopus (Goldman and Hadley, 1969), b receptors predominate allowing darkening to occur in the presence of catecholamines. Although later advances in the pharmacology of a and b receptors have elaborated subclasses of these receptors, little has been done with respect to their definition in amphibians and reptiles. In contrast, the adrenoreceptors of fishes have been better characterized in this regard (Fujii, 1993a). The color changes that occur in response to receptor stimulation in R. pipiens are attributable to changes in both iridophores and melanophores (Hadley and Bagnara, 1969). Moreover, the presence or absence of a and b receptors on iridophores and melanophores seems to complement one another. This is shown in the variation of response to catecholamine stimulation by different sibling species of R. pipiens (Hadley and Goldman, 1970). Norepinephrine lightens the MSH-darkened skins of R. pipiens of northern origin, but further darkens the skins of frogs of Mexican origin, probably Rana forreri. Lightening of the northern species is based not only on the presence of a receptors on the melanophores leading to an aggregation of pigment in these cells, but it appears that a receptors on the iridophore lead to dispersion of the reflecting platelets. In skins from southern frogs (R. forreri), b receptors predominate on both melanophores and iridophores so that catecholamine stimulation of skins in which MSH has already stimulated melanophores and iridophores leads to further melanosome dispersion in melanophores and greater reflecting platelet aggregation in iridophores. The simultaneous activation of the same receptor

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

type on both melanophores and iridophores does not always occur. For example, the catecholamine darkening of Scaphiopus skins mentioned above entails the activation of b receptors on only melanophores; although melanosome dispersion takes place in melanophores, iridophores remain unchanged (Goldman and Hadley, 1969). It seems reasonable that, as more studies are made relative to the application of the adrenergic receptor concept to pigment cell biology, it will be revealed that these receptors comprise an important part of the chromatophore system that can be used by a variety of agents known to affect chromatophores. An obvious question in this respect concerns the relationship between adrenergic receptors and cyclic AMP; does stimulation of adrenergic receptors lead to an alteration in the level of cyclic AMP within the chromatophore? It is tempting to speculate on this point in view of the report by Turtle and Kipnis (1967) that stimulation of b adrenergic receptors in tissues leads to an increase in tissue levels of cyclic AMP, whereas a adrenergic stimulation leads to a decrease in this substance. For a review of this topic, especially with respect to amphibians and reptiles, consult Bagnara and Hadley (1973) and Hadley and Bagnara (1975). An even richer literature has accumulated with respect to fishes and has been cogently discussed by Fujii (1993a).

Molecular Mechanisms for Intracellular Translocation of Pigment Granules A variety of techniques and procedures have been utilized in order to comprehend the mechanism for intracellular transport of pigmentary organelles. In pharmaceutical studies of such motile responses, small pieces of skin or scales containing chromatophores were often used after removal of the epidermis by treatment with collagenase and/or chelating agents. For cell manipulation and microinjection of drugs and antibodies, in vitro cultured chromatophores were obtained as emigrants from skin explants or from tissues dissociated with collagenase and trypsin with subsequent centrifugal purification in Percoll or Ficoll and immortalized as cell lines (Akiyama and Matsumoto, 1983; Akiyama et al., 1981, 1987; Aspengren et al., 2003; Daniolos et al., 1990; Fujii 1993a, b; Matsumoto et al., 1978, 1984; Negishi and Obika, 1980; Rogers et al., 1997, 1998). Electron microscopy discloses that fish and amphibian chromatophores contain an abundance of microtubules and actin filaments in a definite arrangement, together with relatively few intermediate filaments, the density and pattern of which are variable among species. When these cells are exposed to microtubule-disassembling agents such as colchicine, lumicolchicine, vinblastine, hexylene glycol, and nocodazole, as utilized in earlier studies, pigment aggregation is totally blocked or severely disrupted, mostly irreversibly and concomitantly with a disarrangement of their cytoplasmic framework (Obika, 1986; Obika and Negishi, 1985; Porter, 1973; Schliwa, 1984). When these cells are treated with drugs affecting actin filaments, viz. cytochalasins, DNase I, phalloidin, the results

are mostly confusing: in Xenopus and Rana melanophores treated with cytochalasin, dispersion is inhibited (Malawista, 1971; McGuire and Moellmann, 1972), whereas in fish melanophores exposed to cytochalasin B, pigment migration is not affected (Visconti and Castrucci, 1985). In goldfish xanthophores, cytochalasin B inhibited heavy meromysin (HMM)-binding dispersion of carotenoid droplets (Lo et al., 1980; Obika et al., 1978). In swordtail erythrophores, microinjection of the anti-actin antibody clearly interferes with both aggregation and dispersion (Fig. 2.38) (Akiyama and Matsumoto, 1983). From these findings, it becomes clear that these two cytoskeletons are implicated in pigment translocation, more decisively for microtubules and with some diversification for actin filaments. A unique aspect of this motility is bidirectionality with repeated centripetal or centrifugal migrations of the organelles occurring synchronously in whole dendrites of the cells. The question arises as to how these directed movements and their reversal occur. Inasmuch as microtubules have a polarity with minus and plus ends in their structure (Euteneuer and McIntosh, 1981; McNiven and Porter, 1986; McNiven et al., 1984), and inasmuch as they exist under a parallel alignment along the migratory tracks, as was indicated from pioneering studies (Bikle et al., 1966; Porter, 1973), these cytoskeletons are considered to be key instruments in this motility. As to motors driving pigment granules along microtubules, it was shown that cytoplasmic dyneins are inseparably implicated in aggregation as a minus end-directed motion whereas kinesins, mechanochemical ATPases, are involved in a plus end-directed dispersion (Asai and Lee, 1995; Haimo, 1996; Horikawa, 1998; Horikawa et al., 1998; Karki and Hozbaur, 1999; Rodionov et al., 1991; Thaler and Haino, 1996). The involvement of these motor proteins is primarily suggested by the requirement of this motility for a nucleotide triphosphatelike adenosine triphosphate (ATP) or guanosine triphosphate (GTP). This seems to be the case because the application of vanadate, which inhibits ATPase activity of dyneins, disrupts pigment aggregation in fish pigment cells (Beckerle and Porter, 1982; Clark and Rosenbaum, 1982; Luby and Porter, 1980; Negishi et al., 1985; Rozdzial and Haimo, 1986). Later, it was shown that cytoplasmic dyneins are colocalized with melanosomes along microtubules in both fish and amphibian melanophores (Nilsson et al., 1996; Rogers et al., 1997). Further decisive evidence supporting cytoplasmic dynein as the motor resides in the fact that purified melanosomes of Xenopus are capable of moving along microtubules towards the minus end in vitro (Rogers et al., 1997), and that microinjection of an anti-dynein antibody blocks pigment aggregation in fish melanophores (Nilsson and Wallin, 1997). In addition to these findings, it was also shown that microinjection of an antibody raised against kinesins (domain) into frog melanophores blocks pigment dispersion without affecting aggregation (Rogers et al., 1998). This suggests the possible participation of different motors in two phases of dispersion and aggregation. Biochemical analyses of purified 37

CHAPTER 2

A

D

B

E

C

F

melanosomes from Xenopus melanophores disclosed that kinesins are tightly associated with these organelles, moving them in vitro in the absence of cytosolic factors (Rogers et al., 1997, 1998). As kinesin II is a heterotrimer composed of two motor subunits of 85 and 95 kDa and a nonmotor of 115 kDa, mutations in these subunits should disrupt movement along microtubules. When a dominant-negative mutant of the 95 kDa motor subunit, which lacks the ATP-binding region of the motor domain, is produced, this headless kinesin II is unable to move organelles along microtubules (Tuma et al., 1998). In actin-based motility, myosin is considered to be the most reasonable candidate motor protein. As early as 1973, it was shown with mammalian melanocytes that myosin V, a nonmuscle myosin, is involved in melanosome transport from the cell center to the cell periphery where these melanosomes are transferred to keratinocytes (Wolff, 1973). In the dilute mutant of mice bearing defects in hair pigmentation, it is known that melanosomes are distributed in the perinuclear region of the melanocytes. Inasmuch as the dilute gene encodes myosin V, it is considered that melanosome transport along actin filaments toward the cell periphery is disordered by these mutated motor proteins, disrupting melanosome transfer from melanocytes to hair keratinocytes (Wu et al., 1997, 1998). 38

Fig. 2.38. The blockade of pigment displacement in cultured swordtail erythrophores by microinjection of an antiactin antibody. (A) Before microinjection. Cells are in a dispersed state in standard culture medium. (B) The same as (A) after administration of 5 ¥ 10-4 M epinephrine. All pigment (pterinosomes) is aggregated to the cell center. (C) The same as (B) after a brief rinsing in phosphate-buffered saline and subsequent administration of 10-3 M theophylline. Pigment is redispersed. (D) The same as (C) immediately after microinjection of the anti-actin antibody into a cell present in the center of the field of view. A liquid paraffin droplet injected following the antibody solution is seen inside the cell body (arrow). (E) The same as (D) after exposure to epinephrine after injection. Note that pigment aggregation is blocked only in the injected cell, whereas uninjected cells nearby display complete pigment aggregation. (F) The same as (E) after exposure to theophylline. Note that no change is seen in the state of pigment dispersion in an injected cell, whereas pigment in the others is redispersed. Scale bar = 50 mm (from Akiyama and Matsumoto, 1983).

In Xenopus, it was shown with the use of isolated melanosomes that myosin V, although coexisting with kinesin II and cytoplasmic dynein, is tightly associated with these organelles, and that such isolated melanosomes are able to move along actin filaments in vitro (Rogers and Gelfand, 1998; Rogers et al., 1997). The localization of myosin V on melanosomes was also revealed in fish (cod) melanophores by immunoelectron microscopy combined with on-grid labeling using an antibody raised against 200 kDa mouse myosin Va heavy chain (Skold et al., 2002). The results of these studies strongly support the view that myosin V is implicated in pigment migration along actin filaments. Judging from the distribution pattern of actin filaments, which are rich at the cell periphery, it appears that motility based on their presence is of short-range nature. Physiological color change is mostly fast in fish and slow in amphibians, although fairly variable among species. In melanophores of the fish Oryzias latipes, the migratory speed of melanosomes is estimated as 0.06~0.3 mm/s as mass movement and 1~2 mm/s at 22∞C in an ideal linear track without a marked difference in the rate between aggregation and dispersion (Obika, 1986). In swordtail erythrophores (Fig. 2.39), pterinosomes migrate on average at 1 mm/s in aggregation and 0.01 mm/s in dispersion at room temperature, over about

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES A

50 µm

Aggregation(%)

B 100

50

Voltage 0.01mV

0 0

20 0 20 40 Time (min)

60

Fig. 2.39. Pigmentary responses of a swordtail erythrophore in an isolated scale. (A) Tracing of one cycle of pigment aggregation (left row) and dispersion (right row). The red pterinosomes are aggregated by administration of 5 ¥ 10-4 M epinephrine and dispersed by 10-3 M theophylline after washing in physiologic salt solution. The frame indicates the dimension of a recording photo cell. Note that there is little change in cell shape after one cycle of pigment responses. (B) Photoelectric recording of pterinosome displacement made on a single cell using a Rose chamber at room temperature (about 25∞C). The effects of yellow carotenoid vesicles are blocked by the use of a filter with a peak at 470 nm (see Matsumoto et al., 1984 for further details on instrumentation).

a 15~35 mm track in the dendrites (Matsumoto et al., 1984). In myosin-based motion, the velocity is estimated as about 0.04 mm/s, which would be appropriate for melanosome migration in amphibian melanophores (Tuma and Gelfand, 1999). The time needed for the completion of aggregation or dispersion would be affected by the proportion of processes using microtubule- or actin filament-based motors as well as the efficiency of their linkage. A proposal for the relationship of these two motility systems is depicted in Figure 2.40. It suggests that a microtubule-based motor works in long-range movement, utilizing cytoplasmic dynein for aggregation and kinesins for dispersion, while an actin filament-based motor, myosin V, acts in short range (Radionov et al., 1998; Wu et al., 1998). In the latter, pigment granules at aggregation are trapped at the cell periphery to be brought to microtubules for transport by cytoplasmic dynein, whereas at dispersion, pigment granules are dispersed evenly throughout the cytoplasm after transport from the cell center through microtubule-based motors (Tuma and Gelfand, 1999). This model is referred to as the “dual filament model of transport” by Langford (1995). Because fish chromatophores, when their actin filaments are drug disrupted, exhibit hyperdispersion possibly through a strong, centrifugal driving force of unaffected microtubule-based motors, and as frog melanophores under similar treatment show uneven distribution of melanosomes, possibly due to the lack of a local, short-range drive by a myosin motor (Radionov et al., 1998), it appears that this model is suitable for the interpretation of available findings. In signaling pathways for this motility, as mentioned earlier, the control of intracellular levels of cAMP plays a crucial role. This naturally suggests the involvement of protein phosphatases and protein kinases such as cAMP-dependent protein kinase A (PKA) or calcium-dependent protein kinase C (PKC) in this cascade (Nery and Castrucci, 1997). It is known that phosphatase 2A, but not 1 and 2B, is required for pigment aggregation of Xenopus melanophores, whereas phosphatase 2B, calcineurin, is necessary in fish (Reilein et al., 1998; Thaler and Haimo, 1990). In melatonin-induced aggregation of Xenopus melanophores, it was shown that mitogen-activated protein kinase (MAPK) is activated to supplement movement operated by the cAMP/PKA pathway (Andersson et al., 2003). On the other hand, it was shown that both PKA and PKC are implicated in pigment dispersion of Xenopus melanophores, despite the manner of their action being different and the role of PKC being supplemental. Activation of these two protein kinases is essential for dispersion of fish melanophores (Daniolos et al., 1990; Graminski et al., 1993; Reilein et al., 1998; Sammak et al., 1992; Sugden and Rowe, 1992). From these findings, it is apparent that phosphorylation and dephosphorylation of a target subunit of motor proteins is associated with regulation of bidirectional pigment transport (Rozdzial and Haimo, 1986). Current studies on this subject are focused on identification of target subunits or phosphorylation sites of the motor proteins associated with the direction of migration. Knowledge of the role of calcium 39

CHAPTER 2 Aggregation

Dispersion Cytoplasmic dynein Kinesin II Myosin V

Melanosome Microtubule Actin filament

in this signaling cascade is still fairly controversial, depending upon the species used.

Cellular Associations in Color Change Alterations in the state of dispersion or aggregation of any single chromatophore type can often lead to profound color changes in an animal. However, color change often results from the integrated responses of the various pigment cells that exist together in well-organized associations. The association of one chromatophore type with another does not necessarily mean that either cell must undergo a physiological color change. Often, the association of two pigment cells is passive and serves to emphasize a permanent color pattern. As an example, the red spots on the adult dorsal surface of the newt, Notophthalmus viridescens, are based upon a precise superimposition of an erythrophore layer upon an iridophore layer (J. T. Bagnara, unpublished; Forbes et al., 1973). The red coloration of the spots is enhanced by reflection of light from the iridophores beneath. A similar situation applies to the bright spots of other species such as the spotted salamander, Ambystoma maculatum. Here, the yellow spots are based upon the exact superimposition of xanthophores upon iridophores in an otherwise homogeneous background of dermal melanophores (J. T. Bagnara, unpublished). There are many similar examples of chromatophore associations; however, the most important are the dermal chromatophore unit (Bagnara et al., 1968) and the epidermal melanin unit (Fitzpatrick and Breathnach, 1963). Neither of these units represents a precise structure in the anatomical and functional sense, but rather are concepts based upon the location of various chromatophores in the dermis and epidermis.

The Dermal Chromatophore Unit The primary function of dermal chromatophores is the pro40

Fig. 2.40. A model illustrating the implication of three motor proteins in melanosome translocation of fish and amphibian melanophores (adapted from Gross et al., 2002; Tuma and Gelfand, 1999; Wu et al., 1998).

duction of physiological color changes through the rapid intracellular mobilization of pigment-containing organelles. Among the adults of many amphibians, color changes are brought about by coordinated responses of the three basic chromatophore types, which are so situated that they comprise an integral, functional unit that has been designated the dermal chromatophore unit (Bagnara et al., 1968) (Figs 2.13, 2.18, and 2.41). More recently, a comparable unit has been described for fishes (Fujii et al., 1989). Uppermost in the unit, just below the basement membrane, is a layer of xanthophores and immediately beneath this layer of yellow pigment is found a layer of iridophores. In frogs, the iridophore layer that forms the reflecting component of the unit is composed of a layer of melanophores that have dendrites extending upward. In frogs, these dendrites terminate in fingerlike processes on the surface of the iridophore, just beneath the xanthophore layer. During adaptation to dark-colored backgrounds, melanosomes fill these processes, thus obscuring the reflecting surface of the iridophore and leading to consequent darkening of the animal (Fig. 2.9). When the animal lightens, melanosomes move from the terminal processes and occupy a perinuclear position. As a result, their dermal melanophores are almost completely obscured by overlying xanthophore and iridophore layers and the animal appears light in color. The pigmentary role of the xanthophore layer relates to the establishment of the green color of many forms (Figs 2.23 and 2.42). In such animals, light waves leaving the iridophore surface appear blue because of Tyndall scattering and, as the light waves pass through overlying yellow pigment cells, the shortest wavelengths are absorbed so that finally the animal appears green (Fig. 2.17). The importance of the xanthophore pigments in imparting green coloration is shown not only by the blue coloration of green frogs from which yellow pigments have been leached (Fig. 2.9), but by the existence of blue mutants of frogs or

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

BL

X

PT

CV F

I RP

Fig. 2.42. Wholemount in Karo syrup of dorsal skin of a green tree frog, Hyla cinerea, photographed with epiillumination. Because of the presence of xanthophore pigments, green color is manifested. When yellow pigments are leached away, the preparation appears blue as in Fig. 2.9. The numerous black spots are openings of skin glands (see also Plate 2.12, pp. 494–495).

as larvae but not as adults when they have acquired the ability to change color rapidly. The loss of epidermal melanophores as metamorphosis approaches is paralleled by the differentiation of dermal chromatophore units.

M C MS

Fig. 2.41. Transverse section of Hyla cinerea skin from the dorsal surface showing dermal chromatophore unit in white backgroundadapted state. M, melanophore; I, iridophore; X, xanthophore. Melanosomes (MS) are uniformly distributed in processes extending around the sides of the iridophore, but fingerlike endings (F) over iridophores are empty. BL, basal lamella; RP, reflecting platelets; CV, carotenoid vesicles; C, collagenous masses; PT, pterinosomes. Magnification ¥ 9700 (from Bagnara et al., 1968).

snakes (Fig. 2.22). In such mutants, the pigment content of the overlying xanthophores or erythrophores is almost completely depleted (Bagnara et al., 1978b). The effectiveness of dermal chromatophores and the dermal chromatophore unit as elements of physiological color change is very much affected by the presence of epidermal melanophores and epidermal melanin units. Because the latter lead to the deposition of melanin in overlying epidermal cells, physiologic color changes in the dermis beneath are obscured, and thus an inverse correlation exists between the degree of development of the two systems. In tree frogs, which undergo rapid and profound color changes, a well-developed system of dermal chromatophore units is present and epidermal melanin units are lacking. These frogs have epidermal melanophores

Subcellular Associations Over the past 25 years, it has been suggested on several occasions that the various kinds of pigment-containing organelles of dermal chromatophores are closely related to one another from the point of view of origin (for full details, see Bagnara, 1972; Bagnara and Ferris, 1971; Bagnara et al., 1979; Taylor and Bagnara, 1972). Essentially, it is believed that specific pigment-containing organelles, melanosomes, reflecting platelets, and pterinosomes may be derived from a common equipotential primordial organelle that may form, depending on specific developmental cues, any of the definitive organellar types. The strongest evidence in support of this conclusion is based upon the existence of chromatophores of one type that contain pigmentary organelles of another type (Fig. 2.43). So many examples of this phenomenon exist among amphibians that new observations are often not reported. It was first noted that dermal melanophores of the canyon tree frog, H. arenicolor, sometimes contain a few reflecting platelets intermingled between melanosomes (Bagnara, 1972; Bagnara and Ferris, 1971; Taylor, 1971). Another example is represented in the skin from the dorsal surface of the red-backed salamander, Plethodon cinereus (Bagnara and Taylor, 1970). Erythrophores of the red form of this species contain pterinosomes for the most part but, in addition, a few melanosomes are found. Similarly, in melanophores of the dark form of this species, all three of these organelles are found. Also observed in erythrophores of this species are electron-dense organelles that may be mosaic intergrades between the melanosome and the pterinosome. The first example of a 41

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C

P

RP

Fig. 2.43. A mosaic dermal chromatophore of the leaf frog (P. dacnicolor). Melanosomes, reflecting platelets (RP), pterinosomes (P), and carotenoid vesicles (C) are all present in the same cell (from Bagnara, 1983).

definitive normal pigmentary organelle containing at least two different unrelated pigments within the same limiting membrane is the earlier mentioned melanosome of adult P. dacnicolor, which contains both eumelanin and a pteridine pigment (Fig. 2.10). This species has also been shown to produce mosaic pigment cells and mosaic organelles of several types (Bagnara et al., 1979). Mosaic pigment cells and mosaic organelles are not limited to amphibians but have also been observed in reptiles and fishes (Matsumoto et al., 1980). The general phenomenon of chromatophore mosaicism speaks to the obvious close developmental relationships among these neural crest-derived cells that are so obviously different in phenotype. It may also provide an explanation for the remarkable existence of chromatophore transdifferentiation, whereby a fully differentiated chromatophore of one type may change its phenotype and convert to a chromatophore of another type (see Ide, 1986). It appears likely that, as chromatophores of more species are examined, more examples of such chromatophore polymorphism will be observed, which may contribute to a fuller understanding of the mechanisms of chromatophore differentiation.

Pigmentation Patterns The striking and varied pigmentation patterns of poikilotherms have long attracted interest, and an understanding of their biological and physical bases has been sought after for 42

Fig. 2.44. Ventral surface of the same froglet which had received a neural fold graft at the neurula stage. Note the clearly circumscribed area of the graft and its pattern of atypical small spots. The original graft included prospective dorsal skin which had been already determined at the time of transplantation (see also Plate 2.13, pp. 494–495).

many years. Despite these efforts, substantial definitive knowledge about the development and maintenance of pigmentation patterns is lacking, although it is obvious that the ultimate expression of specific patterns depends upon the distribution of specific pigment cells. Pigmentation patterns may be general, such as the frequently encountered one in which the dorsum is darkly pigmented while the ventrum is light colored (Fig. 2.44 and see Fig. 2.49). The cellular basis for this pattern usually involves a greater presence of melanophores in the dorsum than in the ventrum and, conversely, a maximal presence of iridophores or leukophores in the ventrum and a minimal expression of these cells in the dorsum. More specific patterns result from the localized expression or lack of expression of selected chromatophores in circumscribed areas that are shaped as spots, stripes, mottlings, etc. These points are illustrated in Fig. 2.44 and see also Figures 2.47–2.49.

The Role of Endogenous Factors of the Integument The concept that patterns of chromatoblast differentiation are

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

Fig. 2.45. Dorsal surface of a newly transformed froglet of Rana pipiens showing the typical large spot pattern (see also Plate 2.14, pp. 494–495).

established early, even at the level of the neural crest before migration starts, has been considered for many years (see Bagnara, 1987); however, much recent experimentation has supported the alternative view that the pattern resides in the embryonic skin and that uncommitted chromatoblasts are influenced by specific local factors that influence the differentiation of these chromatoblasts (Bagnara et al., 1979). The first concerted effort to discover factors of local integumental origin that might regulate chromatoblast differentiation was made by Fukuzawa and Ide (1988), who discovered a putative inhibitor of melanoblast differentiation in ventral but not dorsal skin of young Xenopus froglets. They considered that this agent present in ventral skin inhibited melanization of melanoblasts and thus designated it a melanization-inhibiting factor (MIF). They considered the differential activity of MIF in dorsal and ventral skin to be responsible for dorsal/ventral patterns. Later, Fukuzawa and Bagnara (1989) demonstrated that MIF (ventral conditioned medium) could override the stimulatory effects of trophic factors such as MSH that would normally be present in the living organism. They further showed (Bagnara and Fukuzawa, 1990) that putative MIF had

Fig. 2.46. The dorsal surface of a half-grown adult which had a portion of dorsal skin rotated 180° at a mid-larval stage. Note the exactness of fit between the separated areas, indicating that the spot pattern was determined precisely at the time of rotation, when no indication of the pattern was yet evident (see also Plate 2.15, pp. 494–495).

a stimulatory effect on iridophores, as would be expected if MIF were indeed responsible for dorsal/ventral pattern formation (see Figs 2.47–2.49). The MIF molecule has been partially characterized, and a monoclonal antibody against MIF has been obtained (Samaraweera et al., 1994). Use of this antibody as an immunohistochemical probe has revealed that MIF is specifically localized in ventral skin of leopard frogs (Fukuzawa et al., 1995). The disclosure of an MIF from lower vertebrates coincides with the discovery that the agouti locus of mammals codes for a specific protein that similarly inhibits melanization of both mammalian melanocytes and melanophores of Xenopus and may be responsible for the dorsal and ventral pigmentation pattern of mice (Miller et al., 1993; Vrieling et al., 1994). The possibility that the agouti protein is related to MIF is a distinct one in view of the fact that agouti protein is an antagonist of the MSH receptor (Lu et al., 1994) and MIF blocks the melanogenic effect of MSH on Xenopus neural crest cells (Fukuzawa and Bagnara, 1989). 43

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Fig. 2.47. Iridophores of Pachymedusa dacnicolor in primary culture in control medium. Each iridophore has few processes and little or no branching of attached cells (see also Plate 2.16, pp. 494–495).

There is evidence that MIF has a similar effect on the mammalian system as it has been found that MIF extracted from the ventral skin of the leopard frog, Rana forreri, has an inhibitory effect on the activity levels of tyrosinase and dopachrome tautomerase in B16/F10 and Cloudman S-91 melanoma cell lines (Lopez-Contreras et al., 1996). Moreover, MIF seems to block the stimulatory effects of a-MSH on these enzymes. As MIF does not block the melanogenic effects of theophylline on these melanoma cells, it appears that it acts proximal to the MSH receptor. A similar search for a melanization-stimulating factor (MSF) has revealed the presence of such a putative factor in fishes (Zuasti et al., 1992, 1993), and this factor has been partially characterized from catfish skin (Johnson et al., 1992). What may be a similar factor has been demonstrated in the dorsal skin of the leopard frog where it appears to be particularly manifested in the dark spots (Mangano et al., 1992). Consistent with the view that the integument itself is responsible for the establishment of specific patterns is the demonstration by Bagnara (1982) that, in the leopard frog, dorsal ectoderm at the open neural plate stage has already been deter44

Fig. 2.48. A similar culture of iridophores in a medium conditioned by exposure to ventral skin of a leopard frog and presumably containing MIF. Note the large size of the iridophore mass containing confluent cells and possessing many branching processes. MIF is presumed to stimulate iridophore differentiation (see also Plate 2.17, pp. 494–495).

mined as dorsal and can program immigrating chromatoblasts to differentiate in accordance with a dorsal spot pattern of a general type. He also showed that, at some point in early larval life, a highly specified final spot pattern is put in place, long before it is expressed during metamorphic climax. Very likely, this specification (or determination) takes place at the onset of the feeding stage (Naughten, 1971). It is attractive to consider the possibility that, at this early stage, factors such as MIF and MSF are expressed at a sufficient level to affect chromatophore differentiation and thus to dictate the highly specified pigmentation pattern (see Figs 2.44–2.49 for details). The putative MIF and MSF molecules are presumably produced by cellular elements of the environment in which the pigment cells are found or pass through. In contrast, it is possible that patterns of migration and expression are influenced by molecules produced by the pigment cells themselves. In a study to investigate this possibility, Fukuzawa and Obika

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

(1995) considered that cell adhesion molecules (CAMs) expressed by specific pigment cell types might be important in poikilotherms. They discovered that, in both the medaka (Oryzias latipes) and Xenopus, N-CAM and N-cadherin are expressed specifically in xanthophores, but not in melanophores and iridophores. At present, it is too early to assess the meaning of these findings in the total expression of pigmentation patterns, but continuing studies hold promise of augmenting our knowledge in this area. It would be helpful if numerous pigmentary mutations were available in order to add a genetic component to our understanding of amphibian pigmentation patterns; however, only a few are adequately described, and even these are difficult to utilize as development to sexual maturity in amphibians is relatively slow, and breeding colonies are restricted to only a few species, most notably the Mexican axolotl, Ambystoma mexicanum. Three pigmentary genes in this species, wild type (D), white (d), and albino (a), have been utilized in conjunction with microsurgical manipulations (chimera formation, reciprocal neural crest grafts, and grafts of gonadal primordia) to provide insights into an understanding of pigment pattern formation (Fig. 2.50). Houillon and Bagnara (1996) noted that “in chimeras between white and albino embryos, melanoblasts from the white half crossed the graft interface to differentiate in albino skin. Neural crest grafts from white embryos to albinos provided melanophores of white origin that were capable of differentiation in albino skin. Grafts of gonadal primordia from albino to white embryos provided albino germ cells that formed unpigmented ovocytes together with dark ovocytes: white ovocytes from the albino grafted ovary, and dark ovocytes from the host ovary. The donor albino white ectoderm included in the graft was able to support the differentiation of melanophores, iridophores, and xanthophores that invaded the graft ectoderm from the neural crest of the white host. It was concluded that manifestation of the white or wild phenotypes may be related to the possible presence or absence of inhibiting or stimulating pigmentary factors in the skin.” It seems possible that these respective inhibiting or stimulating factors are the circumscribed agents MIF and MSF that have been discussed.

Color Pattern Formation in Zebrafish Integumental color patterns of fish are amazingly diverse even among mutants or variants of a single species (Kirschbaum, 1975; Yamamoto, 1975). It is no wonder then that biologists and fish fanciers alike are attracted to them: the former to elucidate the mechanisms responsible for the expression of particular patterns and the latter to produce new and interesting color forms or patterns. With the advent of the present age of molecular genetics, the zebrafish, Danio rerio, and related species of this genus have become widely utilized as model organisms in studies on pattern formation. These species offer a wide choice of pigmentary mutants that are readily detectable through visible characteristics that are advantageous for the screening of mutations (Haffter et al., 1996). Integumental pigmentary patterns of adult zebrafish are

Fig. 2.49. The margin of a graft of ventral larval skin of R. pipiens to the dorsal surface. Note that, after metamorphosis, the typical adult skin was expressed and thus a precise margin is manifested between the heavily iridophore-laden ventral skin and the dorsal surface. The former is considered to contain a melanization-inhibiting factor (MIF) that inhibits melanization and stimulates iridophore expression (see also Plate 2.18, pp. 494–495).

characterized primarily by melanophores that form either narrow, horizontal stripes on the body, as seen in the wild type, or dispersed discrete spots as in the panther or leopard mutants (McClure, 1999; Quigley and Parichy, 2002; Rawls et al., 2001) (Fig. 2.51). In other related species of this genus, such as Danio malabaricus, melanophores are also involved in the development of complicated reticulations over the flank (McClure, 1999). All these color patterns of adults are formed after metamorphosis when the basic body structure switches from the larval to the adult form. In contrast to the diverse pigmentation patterns of wild-type or mutant adults of this genus, larvae display an essentially similar pigmentation pattern (McClure, 1999). Pigmentation of the larvae begins with the expression of melanophores in a few loose lines and is followed, with some delay, by the appearance of xanthophores distributed randomly. Based upon the onset of their appearance and cell size and density, 45

CHAPTER 2

A

B

A

B (a)

(b)

(c) C

(d)

(e)

Fig. 2.50. A. Two-year-old adult albino white chimeras. On the left chimera, note the clear line of demarcation separating the white (d/d) anterior half from the albino (a/a) posterior half. Some melanoblasts from the white half have migrated a short distance across the junction to differentiate and provide the black patches seen in the albino half. The same explanation applies to the chimera on the right except that, in this individual, white is posterior and albino anterior. Female on left, male on right (from Houillon and Bagnara, 1996). B. Two aD/aD albinos 2 years after having received neural crest grafts. Note the presence of melanophore patches distributed randomly in the dorsal area corresponding with the level of the neural crest graft. Female on left, male on right (from Houillon and Bagnara, 1996). C. A young adult female white Ad/Ad host 9 months after having received a gonadal primordium graft from an albino aD/aD donor. Note that the grafted albino integument is clearly delineated from that of the white host by its heavy pigmentation identical to that seen in wild-type individuals; both melanophores and iridophores are clearly visible. This female oviposited a majority of pigmented eggs corresponding with her dd genotype and a significant number of white eggs derived from albino (a/a) germ cells contained in the graft (from Houillon and Bagnara, 1996) (see also Plate 2.19, pp. 494–495).

it is considered that there are two types of melanophores in these fish, larval and adult types. The occurrence of larval- and adult-type melanophores during ontogeny is well known for other teleost species (Matsumoto et al., 1960; Matsumoto, 1965b). In wild-type zebrafish, adults are marked by several horizontal and regularly spaced melanophore stripes over the 46

(f)

(g)

Fig. 2.51. Schematic drawings of pigment (melanophore) patterns of zebrafish, Danio rerio, and its related species. (A) A larva of the wild-type zebrafish. (B) Adults: a, wild-type zebrafish; b, panther (fms) mutant; c, sparse (kit) mutant; d, rose (endothelin) mutant; e, nacre (mitf) mutant; f, D. kerri; g, D. malabaricus (adapted from McClure, 1999; Quigley and Parichy, 2002; Rawls et al., 2001).

flank. This pattern is known to form on the basis of two events: (1) intensive proliferation of adult-type melanophores at or after metamorphosis; and (2) expulsion of these melanophores by pre-existing xanthophores (Johnson et al., 1995; McClure, 1999). On the basis of the careful tracing of pigment cell development, McClure indicates clearly that adult-type melanophores appearing after metamorphosis are forced to coalesce, through contact with xanthophores, into lines along loosely aligned larval melanophore stripes, and that two populations of melanophores and xanthophores are gradually seg-

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

regated into two regions, narrow melanophore stripes with distinct margins and interspaces of xanthophores that are free of melanophores. The capacity of xanthophores to expel melanophores is clearly substantiated from observations of pigment pattern formation in the panther (fms), which lacks xanthophores in larval forms and in which stripes fail to form at metamorphosis. Instead, numerous tiny melanophorecontaining spots form over the adult body (Haffter et al., 1996; Johnson et al., 1995; McClure, 1999). It was also shown that the width of the black stripes, when xanthophores are present, depends upon the growth rate of adult-type melanophores appearing after metamorphosis and leading to the formation of wide stripes or large reticulations as a marked increase in their numbers occurs (Johnson et al., 1995; McClure, 1999; Quigley and Parichy, 2002). All these findings indicate that interactions between different pigment cell types, melanophores and xanthophores, and a genetically defined proliferation rate of xanthophores play crucial roles in pigment pattern formation in these fish. In some related species such as Danio albolineatus and Danio sp. cf. aequipinnatus, erythrophores have been shown to play a role in pattern formation similar to that of xanthophores. An apparent “hostile” relationship between adult-type melanophores and xanthophores is considered to function in maintaining stripes in a definitive pattern (McClure, 1999). When melanophore migration along the dorso-ventral axis is disrupted locally in the course of stripe formation, the regularity of stripe alignment is lost, resulting in irregular-shaped reticulations or patches as seen in Danio kerri or D. malabaricus (McClure, 1999). Additional factors leading to such distorted stripes may include the development of neuromasts in the migratory pathways of melanophores and an uneven distribution of extracellular matrix. As to the apparent hostile relationship between adult-type melanophores and xanthophores, the possibility is suggested that it derives from their differing dependency on fms and kit in the course of development from common precursors, as the former requires either kit or fms, depending upon lineage, while the latter definitely requires fms (Mellgren and Johnson, 2002; Parichy and Turner, 2003; Rawls and Johnson, 2000). With respect to cell adhesion molecules of chromatophores, it was shown in the fish medaka (Oryzias latipes) and the amphibian, Xenopus laevis, that N-CAM and N-cadherin are specifically expressed in xanthophores, but not in melanophores and iridophores (Fukuzawa and Obika, 1995). If the same holds true for zebrafish or its closely related species, such differences between melanophores and xanthophores may relate to a hostile relationship in their respective behaviors. It is interesting that this concept of chromatophore hostility, so well documented for zebrafish in modern studies, is a reaffirmation of the views of Twitty and Niu (1954), voiced more than 50 years ago from work on newts. Recent studies on zebrafish mutants with the use of the techniques of molecular genetics have disclosed that a variety of genes, most of which are orthologs of those in mammals, func-

tion in pigment cell development of this species and ultimately determine their pigmentation patterns (Haffter et al., 1996; Rawls et al., 2001). With respect to genes associated with melanophore development, the following mutations are known to be well-established examples (Fig. 2.51). In homozygous nacre (mitf ) mutants, no melanophores develop during embryonic, larval, and adult stages, indicating an absence of larval- and adult-type melanophores. In homozygous panther (fms) and rose (ednrb1) mutants, the proliferation of adulttype melanophores associated with body stripe formation is reduced, as mentioned previously, although fms and enrb1 are ortholog genes encoding, respectively, a type III receptor tyrosine kinase and a G-protein-coupled endothelin receptor B in mammals. In homozygous sparse (kit) mutants, the development of adult-type melanophores to be incorporated into body stripes and dorsal scales is disrupted. In double sparse (kit) and rose (ednrb1) mutants, two types of larval- and adulttype melanophores fail to develop, while a certain number of melanophores appear in the caudal and anal fins. In view of the development of caudal and anal fin melanophores in sparse and rose double mutants, even in the absence of larvaland adult-type melanophores in the flank, and in light of the development of melanophores having different requirements for kit in regenerating fins, it seems that four different classes or lineages exist in zebrafish (Rawls et al., 2001). Some of these genes associated with the fate of melanophores of this species are also implicated in the development of other types of chromatophores as exemplified by fms for adult-type xanthophores and ednrb1 for adult-type iridophores (Rawls et al., 2001). In addition, a group of genes such as pfeffer (pfe) and salz (sal) causes reduction in xanthophore populations, whereas another group including rose (rse), shady (shd), and transparent (tra) does the same for iridophores (Haffter et al., 1996; Rawls et al., 2001). With respect to mutations affecting melanogenesis itself in adults, a number of genes including albino (alb), mustard (mrd), and sandy (sdy) are reported to be incapable of supporting the production of melanin, and a group of brass (brs), fading vision (fdv), and golden (gol) express a phenotype of reduced melanin (Haffter et al., 1996). As studies of the molecular genetics of zebrafish pigmentation are largely facilitated by those on mammals, available information on pigment genes of this species is rather restricted to melanophores. The information so far derived indicates that the functions of pigmentation-associated genes in zebrafish are in essence comparable to those in mammals, except for the occurrence of a variety of melanophore lineages in this species. In view of the fact that Wnt-1 and Sox 10, essential for the development of mammalian neural crest, are expressed in developing melanophores of zebrafish (Rawls et al., 2001), it is evident that a common basis is shared in the development of pigment cells of both groups. Knowledge about pigment cells of lower vertebrates certainly provides a useful key for understanding the mechanisms of pigment pattern formation and its related phenomena in the vertebrate kingdom. 47

CHAPTER 2

Hormonal Influences on the Development of Pigmentation Patterns Because of its importance in morphological color change, MSH would seem to be a likely candidate as a major role player in pigment pattern formation. As was pointed out earlier, MSH exerts a powerful proliferative effect on melanophores (Pehlemann, 1967a, b, 1972) and, as shown in Figure 2.30, apparently stimulates the differentiation of latent melanoblasts (Bagnara and Fernandez, 1993). Its stimulatory effects can be so potent on some frogs that the normally pale ventrum is darkened (Figs 2.27 and 2.28) (Fernandez and Bagnara, 1991, 1993). The morphological effect stemming from the lack of MSH must also be considered when evaluating the expression of pigmentation patterns. In this case, iridophores are maximally expressed in the absence of MSH. As was pointed out earlier, Xenopus larvae deprived of MSH from the earliest larval stages develop iridophores in the tail fin (Fig. 2.26) where they normally never appear (Bagnara, 1957). One of the best examples of the influence of MSH on the expression of normal pigmentation patterns is that in wild isogenic populations of tiger salamanders, in which the environment markedly affects the amount of MSH secreted and thus the pigmentation pattern that is manifested (Fig. 2.33) (Fernandez and Collins, 1988). High-albedo environments result in low MSH levels and the high expression of iridophores that mask melanophore influences on pigmentation patterns. Low-albedo individuals secrete high levels of MSH and thus a pattern dominated by melanophores is expressed. As potent as these effects of MSH are on morphological color change, they nevertheless must not be construed as an indication that MSH is a primary determinant of pattern formation (Bagnara and Fernandez, 1993). In fact, a clearly formed pigmentation pattern of dorsal spots is manifested in leopard frogs hypophysectomized as embryos, but induced to metamorphose (Fig. 2.52). The role of MSH is not a causative one in the development of primary pigmentation patterns; rather, it is a modifier through its actions on the differentiation of latent chromatoblasts or on proliferation. Similarly, thyroxine plays a permissive role in pigment pattern formation through its general action on the skin, and thus on pigment pattern expression, at metamorphic climax (Bagnara, 1982).

Pigmentation Anomalies Obviously, in nature many undiscovered pigmentary abnormalities exist. These range from albinism and other color mutations to serious pathological conditions such as pigment cell malignancies. With the tremendous growth in the popularity of fishes, amphibians, and reptiles as pets, a large industry has grown up in the breeding of these poikilotherms, and color variants have been selected for. Unfortunately, the professional research community has not taken advantage of these variants for research, in large measure because of the lack of financial support for such seemingly esoteric studies. In contrast, pigment cell malignancies have been the focus of inves48

Fig. 2.52. A R. pipiens tadpole in metamorphic stasis due to an anomalous development of the hypophysis such that MSH is lacking and TSH is insufficient to induce metamorphic climax. Note the obvious onset of the formation of a typical spot pattern even in the absence of MSH.

tigation for many years and, in fact, the establishment of pigment cell biology as a recognized discipline (Bagnara, 1991) stems from the discovery of the Xiphophorine GordonKosswig Melanoma System. This subject has been reviewed frequently in recent years by Professor Fritz Anders, and thus the reader is referred to one of his important works (Anders, 1991). A lesser known malignancy is erythrophoroma from goldfish, an erythrophore neoplasm first described by Prince Masahito and his collaborators (Ishikawa et al., 1978), who ultimately established several lines of neoplastic erythrophores (Matsumoto et al., 1980) (Fig. 2.53). Later, uncloned cell lines of iridophores and melanophores were derived from an iridophore–melanophore tumor found on a marine teleost, the nibe croaker (Matsumoto et al., 1981). This neoplasm (iridomelanophoroma) metastasized on both dorsal and ventral regions of the fish with the melanophore component more prevalent in the dorsum and the iridophore phenotype more prevalent in the ventrum. It is tempting to speculate that the two phenotypes appear where they do as a result of the actions of MSF and MIF respectively. A similar neoplasm has been described from the pine snake (Fig. 2.54) (Jacobson et al., 1989). It was referred to as an iridosarcoma, but it appears very much like the irido-melanophoroma and has the capacity to transform from a largely iridophore type to one that is more phenotypically melanophore-like. Although the malignancy is primarily dermal, neoplastic cells seem to traverse the basement membrane with ease to invade the epidermis (Fig. 2.55). Lower vertebrate pigment cell neoplasms (chromatophoromas) have much to offer in basic research; however, little is being done with them at present. Very likely, there are numerous such malignancies that appear both in nature and in the colonies of fish, frog, and reptile breeders.

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

Fig. 2.53. Morphological properties of spontaneous cutaneous erythrophoromas and irido-melanophoromas occurring in teleost fish. Left column, erythrophoroma formed on the dorsal surface of an adult goldfish. Note that the tumor contains several growing nodules (middle). Component cells of the tumor, even in the central portion, are pigmented red to orange as are normal erythrophores present in the skin. Right column, the profiles of cells from the GEM 81 cell line of goldfish erythrophoromas (top), and nibe croaker irido-melanophoroma (middle and bottom) exhibiting either melano(phoro)ma-like pigmentation (middle) or iridophoroma-like cells (bottom) depending upon the site of the lesion. Scale bars = 100 mm (see also Plate 2.20, pp. 494–495).

Unfortunately, these usually escape the attention of pigment cell investigators; however, the case of the aforementioned goldfish erythrophoroma is an exception, for its discovery followed by appropriate investigation has led to valuable contributions to our knowledge of pigment cells. Interesting aspects of goldfish erythrophoroma cells are

their instability or multiplicity of phenotypic expression. The vast majority of erythrophoromas in situ are recognized by their yellow or orange pigmentation as a tumor of erythrophores, although some may contain a few black lumps composed of numerous melanophore-like cells (Ishikawa et al. 1978; Matsumoto et al., 1980). When these erythrophoroma 49

CHAPTER 2

cells are cultured in a standard culture medium in vitro as a permanent cell line derived from a sporadic tumor of orangecolored goldfish, they remain essentially unpigmented, although they may become pigmented following the addition of fish serum to the culture medium (Matsumoto et al., 1980; 1983). When these cells are subjected to factors that induce differentiation, viz. the use of carp serum in combination with

Fig. 2.54. An adult northern pine snake (Piterophia) with irregularly thickened and pitted black and white ventral scales. A large subcutaneous iridosarcoma nodule bulges to overlying skin (arrow) (from Jacobson et al., 1989).

dimethyl sulfoxide (DMSO) or others, they begin to form, mostly on a clonal basis, a variety of products such as melanin, reflecting substance, teeth- or bone-like structures, and lenslike bodies (Matsumoto et al., 1981, 1983, 1989) (Fig. 2.56). Cells expressing an abundance of melanin are essentially similar to melanophores with respect to both their dendritic appearance and their immense deposition of melanosomes and, upon clonal subcultivation, develop a responsiveness to aggregating or dispersing agents such as epinephrine or cAMP respectively (Matsumoto et al., 1982, 1989). Cells that contain a reflecting substance appear similar in profile to iridophores, as evidenced by the presence of bizarre-shaped platelets (Matsumoto et al., 1981, 1989). Within cell mounds that develop in long-sustained cultures of these cells are lentoid bodies that form occasionally and in which crystallins are detectable by immunochemical assay (Akiyama et al., 1986). In other cases, teeth-like structures are recognized within such cell mounds, suggesting their differentiation toward odontoblasts or the like (Matsumoto et al., 1983). In some cultures, neuron-like cells appear, extending thin, long dendrites that lack pigment deposition (Matsumoto et al., 1983). As most of the cell characteristics that are thus expressed are of neural crest origin (Weston, 1970), it is considered that immortalized erythrophoroma cells are neural crest stem cells in nature. It seems likely that previously described differentiations such as schwannomas or neurofibromas, etc. deriving from goldfish tumors (Duncan and Harkin, 1969) are manifestations of the multiplicity of possible phenotypic expression that these tumor cells are capable of. The capacity for multiple differentiation exhibited by both goldfish erythrophoroma cells and nibe iridomelanophoroma cells offers a wide potential for their use in studies of pigment cell differentiation.

Fig. 2.55. Dermal epidermal junction from an abnormal black scale from the affected region shown in the previous figure. Note the many abnormal iridophores (left) and a single iridophore in the corresponding epidermis just above the basement membrane (arrows). Magnification ¥ 3700 (modified from Jacobson et al., 1989).

50

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

Fig. 2.56. Multiple differentiation of goldfish erythrophoroma cells in vitro. Top, clone of melanophore-like cells that are capable of exhibiting pigmentary responses such as melanosome displacement from a dispersed state (left) to an aggregated state (right). Aggregation is induced by the addition of epinephrine at 5 ¥ 10-4 M. Arrows indicate corresponding sites of the clone. Middle, clone of iridophore-like cells that exhibit brownish profiles under a transmitted light (left) and reflectance under reflecting illumination (right). Bottom, formation of tooth-like (left) structures (arrows) and bone-like (right) structures (arrows) within a cell mound. Differentiation is induced by the administration of dimethyl sulfoxide (DMSO) and fish serum (see Matsumoto et al., 1989 for details). Bars indicate 50 mm for top/middle and 25 mm for bottom (see also Plate 2.21, pp. 494–495).

Perspectives Much of the knowledge discussed herein has accumulated during the last 50 years and, given the low number of scien-

tists engaged in the area of pigment cell research on lower vertebrates, there is a wealth of untapped knowledge to be gained. In order properly to take advantage of the opportunities offered by these lower vertebrates, investigators engaged in research on mammalian pigmentary problems must realize 51

CHAPTER 2

that the pigmentary phenomena studied in lower vertebrates are not necessarily unique to these lower forms. Lower vertebrate pigmentary problems should not be studied merely because, by analogy, they offer model systems for relatively distant mammalian or human phenomena. It must be realized that, frequently, homologies exist among the pigmentary phenomena displayed by lower and higher vertebrates alike. After all, the pigment cells of all vertebrates are of neural crest origin, and they migrate in a similar manner to distal sites where they respond to similar factors present in either the immediate area they occupy or in the organism as a whole. An exceedingly important problem about vertebrate pigment cells that needs study is that of chromatophore organellogenesis. The few studies available have indicated that melanosomes, reflecting platelets, and pterinosomes are of endoplasmic reticular origin (Bagnara et al., 1979), but little is known about details. Unlike the situation with respect to melanosome formation, which has been much studied for mammals and can thus be extrapolated to lower vertebrates, there is no such possibility for iridophores and xanthophores. Perhaps the reverse will be true one day when it becomes appreciated that vestiges of xanthophores and iridophores are present in the iris of homeotherms, which may serve as a refuge for these chromatophores that have long been considered to be unique to poikilotherms (Oliphant et al., 1992). A related problem and one ultimately related to the question of chromatophore characterization and identification concerns the mysterious pigmentary organelles that have been found in chromatophores of little-studied species such as the Centrollenid and Dendrobatid frogs mentioned earlier. A better understanding of these organelles can only be derived from studies on species of less recognized taxa. Probably, the existence of some of the unusual pigmentary organelles is related to specific ecological needs of the various taxa. Certainly, background color matching is an important antipredator behavior; however, it is possible that some of the unusual organelles contain pigments that help in other means of protection from predators. For example, Schwalm et al. (1977) have shown that some leaf-sitting frogs reflect infrared, and they suggest that this ability may confer advantages to the frogs with respect to both thermoregulation and infrared cryptic coloration. In this regard, some of the unusual organelles of poison arrow frogs, the Dendrobatids, may contain pigments that provide the bright warning colors typical of this group. Because of the rather favored status of ecological studies during present times, it is not unlikely that the role of chromatophores and their pigmentary organelles in topics such as thermoregulation, osmoregulation, predator avoidance, and reproduction stand a good chance for study. This is in contrast to the possibilities of studying chromatophores, merely for the sake of knowledge, for such investigations are too esoteric for these modern times. One of the intriguing aspects of chromatophore physiology that certainly needs investigation is that of photoreception. The suggestion that we made, almost 50 years ago, that

52

amphibian chromatophores may possess visual pigments (Bagnara, 1957), seems to have been vindicated by the demonstration that piscine chromatophores do, indeed, seem to possess visual pigments (Lythgoe and Thompson, 1984). However, this discovery seems to have escaped the attention of investigators of higher vertebrate pigmentation and of photobiology. Sooner or later, this knowledge will strike home, especially because of possible implications in the photobiology of tanning or in oncology. It would be fascinating, indeed, if it were found that some chromatophores of all types contain visual pigments, either those already characterized from the retina or entirely new types. Moreover, given the absence of the usual photoreceptive membranes typical of photoreceptors, it would be interesting to know whether the limiting membranes of the pigmentary organelles, themselves, serve as the surface repository for such pigments. Of possible relatedness to the question of direct light reception by chromatophores is the extracutaneous distribution of such cells. In one species or another, chromatophores occur in practically every internal organ, on blood vessels, body cavity linings, meninges, etc. It has been suggested that the presence of pigment cells and melanin in internal organs is related to the impact of solar radiation. It was beyond the scope of this chapter to delve into this subject, especially as much of the work is older and speculative; however, in this regard, two possible functions for these internal pigment cells stand out, thermoregulation and protection. As an example of the former, in a fascinating study by Guillete et al. (1983), it was discovered that one testis in several related species of spiny lizards is heavily melanized and is thus warmer. As a consequence, its spermatogenesis is accelerated over that of its partner. From the standpoint of protection, the presence of melanin in the kidney (Zuasti et al., 1989) and the liver (Sichel, 1988) has been viewed as being part of antioxidant systems based upon the reducing action of melanin. There is so little work being done in this area on lower vertebrates that it stands out as an important target for future investigation. Probably the area of research that holds the most promise for investigators of lower vertebrate pigmentation is that of pattern expression. In particular, the extrapolation of results stemming from mammalian molecular genetics to these lower forms has the most to offer. Earlier, it was commented upon that the putative MIF from amphibians is analogous to the agouti protein derived from the cloning of the gene from the mouse agouti locus. As can be seen from later chapters in this volume, several mammalian genes dealing with pigmentation have been characterized, and an array of mutations affecting mouse coat color have been described (see Jackson, 1994). Among the affected genes are those that affect melanocyte development and migration, melanocyte morphology, melanosomal structure and function, the enzymes of melanogenesis, and the MSH receptor. These characters are elements of pigment cell biology common to all vertebrates. Thus, it remains for the lower vertebrate counterparts of these mammalian genes to be found. Analyses of piscine and amphibian

COMPARATIVE ANATOMY AND PHYSIOLOGY OF PIGMENT CELLS IN NONMAMMALIAN TISSUES

tyrosinase genes and their products are well under way (Miura et al., 1995; Morrison et al., 1994; Peng et al., 1994; Takase et al., 1992; Yamamoto et al., 1992); however, genes dealing with pattern-specific products have not been extended to lower vertebrates. As mentioned earlier, attention to the nature of MSH receptors of lower vertebrates is just commencing. Such studies need to move forward with urgency given the important and varied roles of MSH in lower and higher vertebrates alike. The recent discovery by Valverde et al. (1995) that alterations in the MSH receptor markedly affect the pigmentary phenotype of man should provide special impetus to studies of MSH receptors and the genes that govern them in lower vertebrates. It is important to remember that the pigmentary systems of higher vertebrates are homologous to those of lower forms; thus, investigators involved in research at each of these levels should keep open minds and be able to apply knowledge obtained at one level to pigmentary problems of vertebrates at another level.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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The Science of Pigmentation

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

3

General Biology of Mammalian Pigmentation Walter C. Quevedo Jr. and Thomas J. Holstein

Part I: The Melanin Pigmentary System of Postnatal Mammals from Basic Design to Evolutionary Origins Part II: The Development of the Melanin Pigmentary System and the Molecular Genetic Systems that Regulate It

Summary 1 This chapter examines the structure and function of the mammalian pigmentary system in the light of its diversity, evolutionary origin, and contributions to the adaptiveness of mammals to natural environments. 2 A major focus is on the variation in skin color based on the interactions between melanocytes and keratinocytes in the pigmenting of the epidermis. 3 The inborn constitutive melanin pigmentation of human skin is contrasted with facultative melanin pigmentation or tanning in response to solar radiation. 4 As in the epidermis, melanocytes of hair follicles synthesize melanin and transfer it to keratinocytes (epidermal cells) that form the hair proper. 5 With respect to hair growth, follicular melanocytes that pigment each generation of hair appear to die at the end of each cycle and are replaced in the next one by melanocytes recruited from a stem cell population situated in the upper hair follicle. 6 Dermal melanocytes of humans are most frequently found at sites where hair is thin. “Mongolian spots” serve as a general model for the colors generated by dermal melanin. 7 The presence of dermal melanin is highly variable in mammals, suggesting that dermal melanocytes may have functions in addition to providing photoprotection. 8 Although mammals lack the capacity for rapid color changes by shifting pigment within melanocytes, the color interactions between static deposits of dermal melanin and surrounding blood vessels colored red by hemoglobin produce rapid flashes of bright blue color in such primates as the mandrill (Mandrillus sphinx). 9 At least seven adaptive functions have been proposed for melanin in protecting humans from environmental hazards and for promoting effective metabolic functions of skin exposed to solar radiation. 10 The effectiveness of melanin in photoprotection is being debated. The big picture suggests that melanin is photoprotective to the extent that it is in harmony with its other functions.

11 The evolution of very light-skinned humans created the paradox of cutaneous melanin potentiating solar damage rather than providing photoprotection. 12 As an interface, the hair coat provides many signals to observers when used as communication or none at all when used as camouflage. The pigmentation of the hair coat is carefully crafted for these purposes. 13 Among mammals, youth is a time to play and learn. It is a time for protection from aggression from the adults that surround them. Coat colors signal to adults the privileged status of their conspecific juveniles. The coat colors of juveniles also elicit protection by their adults when predators threaten. In some cases, the coat colors of juveniles, rather than calling attention to them, facilitate concealment. 14 Radicalism and conservatism are evident in the human pigmentary system evolved from that of reptiles. Human skin retained certain features of the spatial organization in which melanocyte function is integrated into the functions of skin as a whole. Examples of this integration are melanin’s role in thermoregulation, the paracrine activity of melanocytes in the inflammatory response, and melanin’s neutralization of certain cytotoxic agents. 15 Many of the most basic features of the mammalian pigmentary system have been carried over from reptiles (melanocytes, melanosomes, tyrosinase, melanin, etc.). The major evolutionary change was the evolution of new regulatory mechanisms for orchestrating both genetically and epigenetically the patterns of pigment distribution in the skin and hair to facilitate the survival of mammals in an acutely visionoriented world. The details of the embryonic and postnatal settings in which the melanin pigmentary system is formed and integrated with local surrounding cells and distant glands that regulate it indicate the great importance of melanocytes in the rise of mammals to dominance in the natural world.

Historical Background The evolution of mammals from reptilian ancestors was characterized by the invention of a new system of protection, a 63

CHAPTER 3

coat of hair. This innovation required a revision of strategy for displaying skin color. Unlike their reptilian, amphibian, and fish ancestors, mammals could no longer profit by using dermal chromatophores (xanthophores, melanophores, and iridophores) to produce stable or rapidly changing patterns of skin pigmentation, for their skin was no longer completely visible. In fact, the ability of chromatophores rapidly to disperse or concentrate pigments within their cytoplasm became a liability in mammals. In their case, the strategy for protective coloration had to be shifted from patterning of the skin with a variety of pigments to one of programming the pigmentation of hairs that grew from hair follicles. The pigment used for this purpose was melanin deposited within melanosomes, specialized organelles synthesized by melanocytes. Each hair had to be pigmented precisely from top to bottom to make its appropriate contribution to the pattern expressed by the dead hair coat that invested the skin. There was no place in mammals for disruptive reversals in the movement of melanosomes within melanocytes in response to endocrine or neural stimuli. Although such physiologic color changes were of great importance in lower vertebrates, they were nonadaptive in the hair follicles of mammals. In this location, unscheduled reversible shifts in the distribution of melanosomes within melanocytes would have interrupted the programmed synthesis and transfer of melanosomes from melanocytes into cells (keratinocytes) being incorporated into the developing hair. Any disruption in the program for pigmentation of small populations of individual hairs could produce a serious flaw in the visual impact of the overall coat color pattern. Although melanocytes lost their capacity rapidly to disperse and concentrate their melanosomes in response to hormones, hormones did retain the ability to regulate pigmentation, hence hair color, by influencing the amount and type of melanin [eumelanin (black/brown) vs. pheomelanin (yellow/red)] synthesized by follicular melanocytes. Physiology deals with the functions of living organisms with particular attention to the mechanisms that regulate them at all levels of biological organization, from subcells to social groups and external environmental influences. In many ways, the mammalian pigmentary system is an ideal example for affirming this interpretation of nature. Accordingly, this chapter emphasizes two approaches to exploring the melanin pigmentary system of mammals. Part I, starting from the top down, from complexity to “simplicity,” describes the big picture of the gross and microscopic organization of the melanin pigmentary system, its universal and variable features among mammals, its adaptive significance, evolutionary origin, and the constraints placed on it by its inheritance from reptilian ancestors. Part II begins from the bottom up, from “simplicity” to complexity, in describing the shaping of the melanin pigmentary system during embryonic development. It stresses the roles played by events at the molecular level that regulate development, including the genes and their products, enzymes, hormones, and growth factors that fine-tune the life, differentiation, and death of melanocytes. The interplay (inductions) of the epidermis and dermis prior to the devel64

opment of hair follicles unleashes the appropriate genetic mechanisms that specify and regulate the path that growth will take. Some features of the melanin pigmentary system are important in both mammalian evolution and the molecular biology of development. This chapter examines the links between them. In doing so, it crosses the boundary between physiological genetics and molecular genetics of the mammalian pigmentary system. In general, molecular genetics has focused on the central dogma of DNAÆmRNAÆprotein, whereas physiological genetics has emphasized the path from the gene product, a protein, the initial phenotype, to the derived phenotype, in this case the melanin pigmentary system that emerges during embryonic and postnatal development (Quevedo and Holstein, 1992). In an attempt to give an integrated picture of events at the micro level, some repetition of information about the macro level has been included to expand what was stated earlier in another context. At the end of the chapter, there is a list of other chapters in this book that expand on the topics covered here.

Part I: The Melanin Pigmentary System of Postnatal Mammals from Basic Design to Evolutionary Origins To begin with, the human melanocyte system will be used to illustrate the general principles of mammalian pigmentation. More is known about the melanin pigmentation of humans than for any other mammal, except for the laboratory mouse (Mus musculus). In general structure and function, the human pigmentary system shows little difference from that of other mammals. However, unlike other mammals, the marked reduction of hair that characterized human evolution once again placed the epidermis and not the hair coat as the primary interface between the organism and its environment. In addition, while hemoglobin imparts significant color to human skin, its impact is not as great as in certain other mammals. Therefore, to complete the generalized picture of mammalian pigmentation based on Homo sapiens, (1) mice will be recruited for an analysis of the pigmentary system in skin sprouting dense hair and (2) the mandrill, a baboon-like primate, will be examined for it epitomizes the ability of the circulatory and pigmentary systems to cooperate in producing bright patches of red and blue in glabrous skin.

Current Concepts Human Skin Color In humans, skin color derives mainly from the interplay of two pigments, hemoglobin and melanin. Eumelanin in the epidermis typically imparts a brown to black hue, whereas eumelanin in the dermis may appear blue. However, the epidermis of orange–red-haired, fair-skinned, freckled individuals may

GENERAL BIOLOGY OF MAMMALIAN PIGMENTATION

appear slightly orange–yellow, possibly owing to the presence of pheomelanin in higher proportions than eumelanin than is characteristic of individuals lacking red hair and freckles (Thody et al., 1991). However, recent findings by Hunt et al. (1995) cast doubt on this interpretation. Oxygenated hemoglobin in the superficial dermal arterioles and capillaries appears pink/red and deoxygenated hemoglobin in the venules blue. Dietary carotenoids may contribute a slight yellow tint to the epidermis (Jimbow et al., 1999). For most humans alive today, melanin is the major source of skin color. Although melanocytes are found in hair follicles and occasionally in the dermis, worldwide ethnic variation in heritable (“constitutive”) skin color results mainly from differences in the amount of melanin within the epidermis. Constitutive skin color (CSC) is defined as the level of melanization generated within the epidermis of an individual through the operation of cellular genetic programs in the absence of influences from ultraviolet light. In practical terms, it is taken to be the level of pigmentation in those parts of the human body that are rarely exposed to sunlight. Humans vary in CSC from dark black/brown to almost alabaster white in skin color. CSC can be enhanced by exposure to ultraviolet light (UVR). Facultative skin color (FSC) designates increases in melanin pigmentation of the skin above CSC and is induced by UVR and/or hormones. FSC is reversible in that the hyperpigmentation tends to decline toward CSC when solar and/or endocrine stimulation is discontinued (Quevedo et al., 1974).

The Histological Basis for CSC and FSC: Epidermal Melanocytes Human epidermis is conventionally described as a stratified squamous epithelium consisting of keratinocytes that are modified as they ascend from their origin in the basal layer and ultimately cornify and die as they near the outer surface where they are shed (Bloom and Fawcett, 1962) (Fig. 3.1A). However, the epidermis is a considerably more complex community of cells than this definition implies (Montagna and Parakkal, 1974; Odland, 1991). Although keratinocytes are the principal cells of the epidermis, there are also several types of nonkeratinocytes. Particularly prominent are the melanocytes found in the basal layer of the epidermis (Fig. 3.1A and B). Epidermal melanocytes typically synthesize melanosomes that become ellipsoidal in shape and more or less uniformly pigmented by melanin deposited on an internal matrix of aligned filaments (cf. Chapter 7). The melanin produced by epidermal melanocytes is most often described as eumelanin based on its tendency to appear brown/black in color. The mature melanosomes are moved from the cell body (perikaryon) of the melanocyte to its dendrites that are in contact with neighboring keratinocytes of the Malpighian layer (cf. Chapter 8). The melanosomes move from the dendrites into the keratinocytes. There are three major theories about how melanosomes are acquired by keratinocytes: (1) keratinocytes phagocytize bits of the melanosome-containing dendrites; (2) melanosomes are discharged by the dendrites into the intercellular space and subsequently phagocytized by

SC SS SG

D

A K

MC

MN

G G K

RER B

*

D

Fig. 3.1. Vertical section of skin from the buttocks of a heavily pigmented adult African American male. (A) Note the heavily pigmented keratinocytes of the epidermal basal layer. In some keratinocytes, the arrangement of melanosomes in the form of supranuclear caps is evident (arrowheads). A “clear cell” (arrow), presumably a melanocyte, is present in the basal layer of the epidermis (SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum; D, dermis). (B) Ultrastructure of a basal melanocyte flanked by two keratinocytes. Melanosomes at various stages of development (arrows) are distributed throughout the cytoplasm of the melanocyte. Arrowheads indicate melanosomes that have been transferred to the cytoplasm of keratinocytes (MN, melanocyte nucleus; MC, melanocyte cytoplasm; K, keratinocytes; G, Golgi apparatus; RER, rough endoplasmic reticulum; *basement membrane at the dermal–epidermal junction; D, dermis) (electron micrograph courtesy of Raymond Boissy).

keratinocytes; (3) the dendrite and keratinocyte plasma membranes fuse, opening a cytoplasmic channel through which the melanosomes pass directly from the melanocyte into the keratinocyte (Jimbow et al., 1999; Marks and Seabra, 2001; Yamamoto and Bhawan, 1994). On incorporation into keratinocytes, melanosomes, which are themselves considered to be specialized lysosomes (Orlow, 1995), interact with lysosomes of the keratinocyte and are sequestered within them forming secondary lysosomes (cf. Chapter 7). As a general rule, melanosomes larger than approximately 1 mm in greatest 65

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diameter are packaged singly in secondary lysosomes, whereas those smaller than 1 mm are sequestered in groups of varying size within a secondary lysosome (Jimbow et al., 1999; Quevedo et al., 1986). Lysosomal hydrolases degrade melanosomes as the keratinocytes move to the epidermal surface. The smaller melanosomes packaged in groups appear to be the most susceptible to lysosomal degradation. The degradation of melanosomes appears to reduce the melanin into finer particles, which may increase its effectiveness in shielding the epidermis from penetration and damage by ultraviolet light (Jimbow et al., 1999). The close relationship that exists between a melanocyte and a small population of keratinocytes with which it maintains contact by its dendrites is formalized in the concept of the epidermal melanin unit (EMU) (Fitzpatrick and Breathnach, 1963). An EMU consists of a melanocyte and the population of keratinocytes that acquires, transports, metabolizes, and disposes of the melanin/melanosomes synthesized within the melanocytes (Quevedo et al., 1974) (Fig. 3.1A and B). It is estimated that an EMU contains approximately 36 viable keratinocytes (Quevedo et al., 1974). Although the uninterrupted horizontal distribution of melanin in the outer cornified layer suggests a functional overlap between EMUs, epidermal pigmentation is largely a mosaic of the individual contributions of millions of EMUs. Just as the unaided human eye fails to resolve the large number of individual dots that constitute a newspaper photograph, the same unaided eye fails to detect that individual EMUs vary in their melanin/melanosome content. Consequently, the brain concludes that skin color is essentially homogeneous. However, under the light microscope, it is evident that melanocytes on the ridges of epidermis that press into the dermis are more melanogenically active than those of interridge areas of epidermis that form a flat interface with the dermis. Not only is more melanin found in the basal melanocytes and keratinocytes of the ridges, the stratum corneum above them often appears to be more heavily pigmented than the interridge cornified layer (Quevedo et al., 1989). This condition is partially evident in Becker’s nevus where there is enlargement of the epidermal ridges and an increase in melanocyte numbers (Quevedo et al., 1989). The freckled epidermis of sun-exposed, fair-skinned, red-haired humans is a classic extreme manifestation of the mosaic nature of EMU structure and function (Fig. 3.2A and B). The clusters of EMUs in freckles produce and harbor abundant melanosomes, resulting in islands of dark epidermis surrounded by a sea of light epidermis composed of EMUs containing weakly melanogenic melanocytes (Quevedo et al., 1986). Melanosomes tend to be largest in African Americans, black Africans, and Australian aborigines and are generally arranged singly within secondary lysosomes of keratinocytes (>1 mm). Melanosomes are smaller (<1 mm) in white individuals of European ancestry, Mongoloid Asians (e.g. Chinese and Japanese ancestry), and certain East Indians; they tend to form groups of two or more in secondary lysosomes (Jimbow et al., 1999; Szabo et al., 1972). 66

A

B Fig. 3.2. Black light photographs of freckled subjects with “carrot red” hair. (A) In this portrait, the population of freckles is so dense that freckles lose their individuality as they increase in size and number and finally merge with one another in facial areas most prone to sun exposure. (B) Close-up view of discrete freckles on a sun-exposed forearm (photographs courtesy of Dr Robert D. Fleischmann).

When sections of skin are examined under the light microscope, the distribution of melanin within the epidermis correlates with the darkness of the skin. In fair-skinned, white individuals, melanin is scant in the epidermis including the basal layer where only occasional keratinocytes have melanosomal caps shielding their nuclei (Gates and Zimmerman, 1953). When skin sections are treated with ammoniated silver nitrate to darken melanin, greater amounts of melanin are found in all epidermal layers, suggesting that melanosomes may lighten in color because they are under attack by lysosomal hydrolases or passing from an oxidized state to a more reduced one during their ascent within keratinocytes from the basal layer (Pathak and Stratton, 1969; Quevedo et al., 1989) (W. C. Quevedo et al., unpublished observations). In the skin of the dark African Americans, melanosomes are largely concentrated in the basal layer where they form supranuclear apical caps in the germinal keratinocytes (Fig. 3.1A). There are fainter supranuclear melanin caps (Fig. 3.1A) throughout the spinous layer, and scattered melanosomes are found throughout the fully keratinized cells of the stratum corneum. As in white skin, silver nitrate

GENERAL BIOLOGY OF MAMMALIAN PIGMENTATION

treatment reveals greater amounts of melanin throughout the epidermis of black skin, but its greatest concentration continues to be found in the lower levels of the epidermis (W. C. Quevedo et al., unpublished observations). Along a series of progressively lighter African Americans, the epidermis shows a parallel decrease in melanosomes in all layers as well as an increasingly marked unevenness in the melanin burden of individual keratinocytes (Gates and Zimmerman, 1953). At first, the uneven reduction in melanosomes is more pronounced in the corneum and spinous layers than in the basal layer. In more lightly colored African Americans, there are islands of heavy melanin deposits, scattered among lightly pigmented ones, forming a mosaic pattern somewhat similar to freckles in white skin (Gates and Zimmerman, 1953). Corneocytes stripped from the epidermis of human subjects by Scotch tape and treated with silver nitrate to darken melanosomes give a more accurate impression of the numbers of melanosomes reaching the stratum corneum (Goldschmidt and Raymond, 1972). There are large numbers of melanosomes forming a large central cluster in the corneocytes of a very dark individual of African descent. There are few melanosomes and no central clusters in the corneocytes of fairskinned, white individuals. Among individuals of intermediate colors, there is a gradient of melanosome numbers and frequency of clustering paralleling gradually increasing CSC/FSC among the subjects examined (Goldschmidt and Raymond, 1972). The density of melanogenically active (dopa-positive) epidermal melanocyte populations varies between different parts of the human body. The most dense populations are found on the face and genitals and the least dense on the abdomen. Melanocyte populations are distributed symmetrically in the human integument, so that those on the left side match those on the right side. Regardless of skin color, all humans have approximately the same melanocyte densities at corresponding anatomical sites (Szabo, 1967). Therefore, differences in human skin color result from differences in the numbers of melanosomes within EMUs and not from differences in the numbers of EMUs. In addition, the distribution of melanosomes within keratinocytes (single vs. multiple melanosomes in secondary lysosomes) may have some influence on overall skin color (Minwalla et al., 2001; Szabo et al., 1972; Thong et al., 2003). It is generally considered that regional populations of melanogenically active melanocytes are but one expression of the mosaic nature of the skin established and maintained by a normal developmental program that operates throughout life (Jimbow et al., 1999; Quevedo et al., 1989). Nonetheless, this interpretation has been questioned. It has been pointed out that, with the exception of dopa-positive melanocyte populations in male genitalia (~2000 mm-2), the regional variations in melanocyte numbers parallel the amount of chronic sun exposure that the various parts of the body are likely to have received (Jimbow et al., 1999). It is possible that the high density of dopa-positive melanocyte populations in the male genitalia may be brought about by sex hormones.

In young adults, the habitually exposed face has ~2000 melanocytes mm-2, whereas the generally shielded thigh has ~1000 melanocytes mm-2. As the number of dopa-positive melanocytes increases within skin treated with UVR (Quevedo et al., 1969), it is proposed that repeated exposure to UVR brings about an irreversible elevation of melanogenically active epidermal melanocyte populations (Gilchrest et al., 1979). This would in part explain differences in epidermal melanocyte populations found between individuals. Another reason is that the populations of dopa-positive epidermal melanocytes in human skin decline steadily with advancing age (Quevedo et al., 1969). Therefore, age must be taken into account when comparing melanocyte populations between individuals. Although, at all ages, dopa-positive melanocytes are increased in UVR-treated skin, the populations of melanocytes in irradiated old skin are not as great as in young skin (cf. Chapter 23) (Quevedo et al., 1969). It important to be aware that the sizes of melanocyte populations determined by light microscope cell counts of dopa-incubated skin specimens are substantially smaller than those estimated by electron microscopy (Mishima and Widlan, 1967). As essentially all the studies on melanocyte populations in human skin have involved light microscopy, the regional differences, if any, in the absolute size of melanocyte populations, defined as the total number of dopa-positive and dopanegative melanocytes, remains to be determined. Essentially all estimates of epidermal melanocyte populations have been based on counts of melanocytes in a particular physiological state rather than their absolute numbers. Consequently, the two contrasting interpretations of melanocyte distributions in human skin, developmental programs vs. UVR history, must be given equal status until more is known about the true densities of melanocyte populations.

Pigmentation of the Hair: Follicular Melanocytes In mammals, hair is produced in a hair follicle during a hair growth cycle lasting approximately 21 days in the mouse and as long as several years in humans (see Part II for more details). The hair generated by a hair follicle when properly interpreted is a permanent record of its developmental history. This is particularly valid for its pigmentation. For example, Figure 3.3A is a wholemount of a hair from a uniformly intense black mouse. The eumelanosomes are grouped to form steps of a ladder-like structure when the medullary cells that contain them undergo keratinization. The translucent areas are considered to be “air” spaces that develop in medullary cells when the hair emerges above the skin and dehydrates on exposure to the atmosphere (Roth, 1967). The thin vertical struts supporting the steps of the ladder are melanized cortex cells. The outer cuticle of the hair is rarely pigmented and therefore not evident in this photograph. Figure 3.3B illustrates a dilute black (d/d) phenotype. In this case, the clumping of pigmented melanosomes reduces the amount of pigment available to coat the steps, producing a modest reduction in the intensity of black coat color. Figure 3.3C captures a large mass of melanin pigment in a dilute black (d/d) mouse. The eumelanin mass is 67

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A

A

B

B

C

C

situated near the base of the medullary compartment. Smaller clumps are frequently found in this region near the hair club. Figure 3.4A shows a wholemount preparation of a hair from a Light (Blt/Blt) mouse where the upper region of the hairs displays eumelanin masses that are either whole or large fragments of melanocytes that have been dislodged from the upper hair bulb. As a result, there is an absence of melanin in a long segment of the lower hair shaft. In Figure 3.4B, the hair of a Light mouse contains a single dislodged large melanocyte. Owing to the lack of obscuring melanin in the background, the keratinized framework of the medulla and cortex is clearly evident. In Figure 3.4C, although the hair of mice heterozygous for the Blt gene (not shown) contains pigment clumps, the presence of the pink-eyed gene in the heterozygous state corrects the condition, as revealed by the ladder-like pigment pattern. Figure 3.4D illustrates the failure of the pink-eyed gene to correct pigment clumping caused by the leaden (ln) gene. Each picture tells a different tale about gene action 68

Fig. 3.3. Wholemount preparations of pigmented hairs. Magnification ¥370. (A) Intense black; (B) Dilute black (d/d); (C) Dilute black (d/d). For descriptions, see text.

D

Fig. 3.4. Wholemount preparations of pigmented hairs. Magnification ¥370. (A) Light (Blt/Blt); (B) Light (Blt/Blt); (C) Dark sepia (P/p; Blt/b); (D) Pink-eyed leaden (p/p; ln/ln). For descriptions, see text.

(McGrath and Quevedo, 1965). Each hair records the interactions of melanocytes and keratinocytes of the follicular hair bulb as the latter pass the melanocytes on their way toward forming the hair. (For coverage of hair growth and pigmentation in depth, see Part II.) In human hair, the cortex dominates and the medulla is small, so that the most evident pigment in the hair is the vertical streams of melanosomes, reflecting their entrapment within elongated spindle-shaped cortical cells. This pattern is particularly evident in hair containing eumelanosomes. In some bright red (pheomelanic)-haired humans, the pigment appears for the most part to be a diffuse yellow rather than granular as found in black or brown hair. In human follicular melanocytes producing melanin chemically defined as pure pheomelanin, the melanosomes are spherical membrane-limited vacuoles containing matrices of microvesicles and amorphous proteinaceous filaments or lamellae (cf. Chapter 7) (Jimbow et al., 1984; Marks and

GENERAL BIOLOGY OF MAMMALIAN PIGMENTATION

Seabra, 2001). Pheomelanin is unevenly deposited on the inner matrix at first but accumulates to produce a mature melanosome that is highly electron dense. Pheomelanic melanosomes are degraded following transfer to keratinocytes. In ultrastructure, the pheomelanosomes of human hair containing chemically defined pheomelanin are identical to the pheomelanosomes of mice and guinea pigs (Jimbow et al., 1984, 1999; Marks and Seabra, 2001). Mature follicular melanosomes in human subjects with black (eumelanic) hair are black and ellipsoidal. Electrondense eumelanin is deposited on a filamentous/lamellar matrix of the membrane-limited eumelanosome. As a surface for melanin deposition, small vesiculoglobular bodies also make a contribution to the eumelanosomal matrix (Jimbow et al., 1984; Marks and Seabra, 2001). The majority of red-haired individuals studied by Jimbow et al. (1984) displayed chemically defined “mixed melanogenesis,” a mix of pheomelanin and eumelanin synthesis. Two types of melanocytes were found in their hair bulbs. One produced eumelanosomes, the other pheomelanosomes. The pheomelanocytes also produced a mosaic melanosome incorporating features of both eumelanosomes and pheomelanosomes. The population of eumelanocytes was much smaller than that of pheomelanocytes. On occasion, a keratinocyte was observed to contain both eumelanosomes and pheomelanosomes, indicating that it had been serviced by a eumelanocyte and a pheomelanocyte. Eumelanosomes were singly distributed in keratinocytes, whereas pheomelanosomes were arranged singly or in groups and were highly susceptible to degradation (Jimbow et al., 1984). The vast majority of humans today have black hair. Variation in hair color is largely restricted to white Europeans and their descendants. It is among Europeans that red, blond, brown, and black hair colors are common. Red hair may be found in other ethnic groups but is most frequently associated with a pigmentary disorder such as oculocutaneous albinism (Quevedo et al., 1985). In mice, numerous genes have been shown to influence eye, skin, or coat color. Their action and interaction have produced a bewildering array of color patterns. The complexity of genetic regulation of melanin pigmentation in mice and also in humans underscores the importance of pigments in the adaptation and evolution of mammals (Hearing and King, 1993; Jackson, 1994; Jackson et al., 1994; Jimbow et al., 1999; Ozeki et al., 1995; Prota et al., 1995; Quevedo et al., 1985; Silvers, 1979; Witkop et al., 1989). In contrast to humans, eumelanosomes and pheomelanosomes have been found within the same follicular melanocyte of agouti mice. They were observed during a shift from eumelanogenesis to pheomelanogenesis (Jimbow et al., 1979; Sakurai et al., 1975). Mice have a juvenile coat of hair that is replaced by an adult one that may differ from it in pigmentation. For example, on some genetic backgrounds, the hair coats of juvenile lethal yellow (Ay/–) mice may appear quite dark (“sooty”) but become clear yellow in adults (Silvers, 1979). Apparently, modifier genes stimulate eumelanin synthesis in hairs pro-

grammed to synthesize pheomelanin specified by the a-locus, but their ability to do so is restricted to a brief period early in life. Recessive yellow (e/e) mice also exhibit a sooty pheomelanic juvenile pelage that becomes clear yellow in adults (Silvers, 1979). Although the hair coat of light (Blt/Blt) mice is eumelanic throughout life, light mice parallel yellow mice, in that their juvenile pelage is darker, more melanized, than the adult one (Quevedo and Chase, 1958). It is well known that humans with blond or red hair when young may become brunettes with advancing age.

Pigmentation of the Dermis: Dermal Melanocytes Dermal pigmentation results from the presence of melanized melanocytes within this skin layer. Melanocytes synthesize melanosomes that they retain and, possibly, degrade and replace periodically. Although dermal pigmentation is not uncommon in infants of certain human ethnic groups, it is relatively rare in the adult. The classic expression of normal dermal hyperpigmentation is the Mongolian spot or congenital dermal melanocytosis observed in newborns (cf. Chapter 50) (Mosher et al., 1987). The lesions impart a blue–black tint to the skin and range from the typical size of a few centimeters in diameter to very large ones, in some cases extending over the lower back and buttocks. The macroscopic Mongolian spots typically disappear gradually, frequently within a few years. However, persistent Mongolian spots have been found in 3–4% of adult Japanese. The Mongolian spots consist of dermal nests of melanocytes that have been shown to become melanotic at 3 months’ gestation and to lose their melanogenic capacity (dopa reaction) in children from 2 to 10 years of age. Despite the disappearance of the macroscopic spot as a consequence of melanocyte degeneration, inactive dermal melanin-bearing melanocytes have been found throughout life (Kikuchi and Inoue, 1981). The Mongolian spot is evident at birth in 90% of Asian (Mongoloid) and Native American (Amerindian) infants. It is less common in African Americans, black Africans, and European whites (less than 10% in the latter) (Mosher et al., 1987). There are a number of nevi (noncongenital) such as the Nevus of Ota (cf. Chapter 50) that bear some resemblance to the congenital Mongolian spots (Gonzalez-Campora et al., 1994; Mosher et al., 1987). The blue color imparted to the skin by dermal pigmentation requires explanation (R. R. Anderson, in Mosher et al., 1987). When the epidermis is separated from the dermis of the Nevus of Ota and placed on a neutral-colored background, it appears gray or brown in reflected light. It appears brown or yellow when observed by transmitted light. In contrast, the pigmented dermis, in the absence of the epidermis, appears blue in reflected light. The melanin dispersed in the dermis absorbs incident visible light such that the diffuse reflectance in the longer (red) wavelengths is reduced, giving the pigmented sites a blue appearance. However, epidermal melanin does the opposite, eliciting a reduction in diffuse reflection at the shorter wavelengths, giving the epidermis a yellow to dark brown tint. As reflected radiation from the dermis must reach 69

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the observer by passing through the epidermis, the degree to which dermal melanin appears blue depends on the amount of melanin in the epidermis. Therefore, Mongolian spots with little overlying melanin should appear bluer than those with more epidermal melanin. Possibly, this is why they are so striking in infants before full CSC has been acquired. In addition, based on the contribution that hemoglobin makes to the brightness of blue reflectance from dermal melanin in mandrills (see below), the vascular system may have some influence on the blueness of Mongolian spots in humans. Interference with dermal bluing of the skin by epidermal melanin is suggested by Mosher et al.’s (1987) statement that: “The color [of the dermal melanocytotic nevus of Ota] may be of various hues: black, purple, blue-black, slate-blue, purplish brown and brown . . . Curiously and unexplained, the intensity of the pigmentation may vary daily, and in females during menstruation.” Based on the mandrill, vascular changes bringing more or less blood (hemoglobin) to the spots may produce these rapid color changes (Quevedo et al., 1981).

The Dermal Melanocytes of Other Mammals In wild-type (haired) mice, melanotic dermal melanocytes are commonly found throughout the skin during embryonic development. Melanized dermal melanocytes routinely persist in the relatively glabrous skin (e.g. ear pinnae, tail, and perineum) of the wild-type adult. However, along with epidermal melanocytes, they essentially disappear from the trunk (haircovered) skin during the first month of postnatal life (Quevedo et al., 1966). Occasional free dermal melanocytes and dermal perifollicular networks of melanocytes are found in the trunk skin of adult wild-type mice (Quevedo, 1981a). Their frequency of expression is greater in certain mutant varieties of mice. For example, greater numbers of dermal melanocytes persist sprinkled throughout the dermis of adult hairless (hr/hr) mice, suggesting a regulatory defect in a system that normally orchestrates their demise (Quevedo et al., 1995). Compared with wild-type mice, dermal perifollicular networks of melanocytes are also more evident in mice expressing the gene for “dominant alopecia” (Mann, 1969). Well-developed dermal perifollicular melanocytes are found in the hairy trunk skin of the adult Syrian golden (agouti) hamster (reviewed by Quevedo, 1981a). The perifollicular melanocytic networks are associated with the connective tissue sheaths of hair follicles and are located near the sebaceous glands. Topical applications of croton oil, a tumorpromoting agent in which the active ingredient is TPA (12-Otetradecanophorbol-13-acetate), greatly increase the number of perifollicular melanocytic networks, suggesting that many hair follicles have affiliated amelanotic dermal melanocytes that are activated and possibly proliferate in response to croton oil (Quevedo, 1981b). Pigmented dermal perifollicular melanocytic networks in normal (untreated) mice are most often associated with specialized tylotrich hair follicles. Tylotrich hair follicles are richly innervated, produce coarse hairs, and function as specialized mechanoreceptors (Straile, 1964). (See Part II for a description of Straile’s model of a 70

cutaneous mosaic of hair follicle groups each surrounding a tylotrich hair follicle and possibly regulated by the nervous system.) Although melanotic perifollicular melanocytes are thought to be absent from the tylotrich follicles of primates including man (Mann, 1969), they are present around the sensory whiskers of at least one primate species, the New World squirrel monkey, Saimiri sciurea (Machida and Perkins, 1967). It is worth emphasizing that the existence of amelanotic perifollicular melanocytes accompanying tylotrich follicles has not been ruled out for primates. As melanocytes are known to synthesize cytokines (Nordlund, 1992), perifollicular melanocytes may be more important for this function than for melanogenesis. If this is the case, either amelanotic or melanotic melanocytes might release cytokines. The tylotrich follicle has been found in the skin of a great variety of mammals, suggesting that it is old in its origin and plays an important role in neurophysiology at the levels of the skin and organism. The mammals possessing tylotrich follicles include duckbilled platypus, spiny anteater, American opossum, European hedgehog, starnose mole, little brown bat, domestic cat, ocelot, tiger, lion, puma, domestic dog, pig, domestic sheep, Japanese deer, house mouse, rat, hamster, guinea pig, porcupine, rabbit, baboon, and a broad assortment of monkeys.

The Interplay of Dermal Melanin and Hemoglobin in Skin Color: The Mandrill (Mandrillus sphinx) One of the most dramatic displays of dermal melanin as a secondary sexual characteristic is in the mandrill (Fox, 1976; Hill, 1955, 1970; Napier and Napier, 1967; Zuckerman and Parkes, 1939). In the adult male of this species, a brilliant red band runs down from the center of the enlarged muzzle from the top of the nose to the base of the nostrils and joins the red lips. On either side of the red nose are brilliant electric blue paranasal swellings, which are cleaved by skin folds. There are tufts of white hair on the cheeks and a yellow to orange beard. The anogenital region of the male roughly matches the “red, white, and blue” pattern of the face. The circumanal region is a brilliant scarlet red and the callosities (thickened skin pads) a pale pink. Immediately lateral to each callosity, the skin is sky blue. Still more laterally, the skin is purplish lilac. The skin extending from the callosities to the penis is blue. The scrotum grades from lilac to pink. There are numerous bright orange scrotal hairs. The penis is pink and the foreskin is scarlet. Although there have been few histological studies of Mandrillus sphinx, the areas characterized by blue coloration are stated to contain abundant dermal melanotic melanocytes with an overlying epidermis devoid of melanin (Fox, 1976). It is this combination of a translucent epidermis with a pigmented dermis that provides the conditions of light absorbance and reflectance that produce the blue coloration observed in Mongolian spots. With sexual maturity, major changes in the vascularity occur, resulting in the brilliant blue and scarlet colors of the face and anogenital region. Superficial blood sinusoids develop in the nasal tract of the male

GENERAL BIOLOGY OF MAMMALIAN PIGMENTATION

mandrill after sexual maturity and become engorged with blood producing the bright red color (Zuckerman and Parkes, 1939). At this time, there is increased blood volume in regions with dermal melanin that produce, depending on the specifics of vascularity and pigmentation, colors ranging from blue to lilac. In the adult female Mandrillus sphinx, the bright red color characteristic of the male is absent, in part because of the presence of greater amounts of epidermal melanin, which act to obscure dermal blood vessels (Hill, 1955). Nonetheless, in females, the paranasal swellings are a “livid blue” in color and traces of red may be found on the nose. With regard to the anogenital region of the female, there are dark and light areas, but color differentials are in no way comparable to those expressed in the male (Hill, 1955). In male guenons (Ceropithecus spp.), dermal melanocytes in the scrotum contribute a blue color that is more vivid in some species of the genus than in others (Fox, 1976; Napier and Napier, 1967; Wickler, 1967). In free-ranging male mandrills (Mandrillus sphinx), there are positive correlations between testosterone levels and position in the dominance hierarchy. Positive correlations exist between dominance rank (which male wins in agonistic encounters), “levels of plasma testosterone, testicular volume, body weight, and development of secondary sexual characteristics (red and blue sexual skin on the muzzle and rump areas)” (Wickings and Dixson, 1992). Such coloration may be important in sexual selection, for females in some animal species respond to males with the brightest colors, presumably because they signal good health and general fitness. In brief, hemoglobin in blood vessels within the heavily pigmented dermis is required to produce the electric blue coloration found in the male mandrill and, probably, guenons. The combination of changing blood supply and fixed dermal melanin in specialized areas of their skin makes possible transient flashes of bright blue color, the mammalian equivalent of the physiologic color changes brought about by dispersion and aggregation of chromatophore pigments in the lower vertebrates.

Humans Among the Primates The pongids (gorilla, chimpanzee, and orangutan) of today have an extensive covering of hair. Among the early members of the human family descended from pongid ancestors, this coat of hair was reduced but not discarded (Montagna, 1985). Hair was dramatically reduced on all parts of the body except for the scalp, beard, axillae, and pubis, sites at which it played roles in sex-related ornamentation, protecting the skin from damage, in projecting agonistic social signals and, in the case of the axillae, reduction of friction between interfacing skin surfaces (Quevedo et al., 1985). Middle-aged white males acquire gray hair that is largely restricted to the scalp, beard, and chest (Fig. 3.5) (Quevedo et al., 1985). The display of this pattern in more hairy early men would have been quite striking and may have symbolized strength and experience, reducing overt challenges from young males while attracting females. Guthrie’s (1970) life cycle illustration of changing

Fig. 3.5. Composite drawing of graying patterns observed in six white middle-aged males superimposed on a gorilla for artistic convenience. It is evident that the graying of the beard, scalp, and upper chest is intimidating when the length of the hair is exaggerated. Except for the blaze of white on their chests, the humans matched the gorilla (in this illustration) in having pigmented general body hairs. Among the human subjects studied, there appeared to be a strong tendency for the beard to develop streaks of gray initially that gave way to more uniform graying with advancing age.

patterns of the scalp and facial hair of the male suggests that baldness, if early men lived long enough to bald, would have caused a male’s display to shift from a positive signal of virility to a negative one of implied vulnerability. Thus, the tendency for age-dependent male baldness may have devalued the currency of displays originally designed to project male strength and potency. The ability to project dominance signals via coat color in hominids is documented in the saddle-like pattern of gray found on the backs of mature male gorillas (Napier and Napier, 1967). It suggests that displays involving both hair coat and skin color may have formed part of the behavioral program of ancestors common to humans as well as chimpanzees and gorillas, their closest relatives. Recently, there has been renewed interest in hypertrichosis in humans, a condition that, in the extreme, is characterized by a dense covering of body hair sparing only the palms of the hands and soles of the feet. In some cases, although increased, body hair is less dense, finer, and more limited in its distribution. Most types of hypertrichosis are associated with severe developmental abnormalities. Recently, the gene for a form of hypertrichosis that is essentially free of any associated developmental abnormalities has been assigned to the long arm of the X chromosome (Figuera et al., 1995). Congenital generalized hypertrichosis is inherited as an X-linked dominant trait. It causes generalized hypertrichosis in males but patchy hyper71

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trichosis in females. Females are typically heterozygous for the mutant gene and, owing to random X inactivation early in development, the wild-type and mutant alleles are randomly expressed. As a result, a cutaneous mosaic of scattered patches of normal and mutant skin is produced, thereby reducing the severity of hypertrichosis (Figuera et al., 1995). It is possible that the gene mutation that produces hypertrichosis today may be a reversion to a very ancient (“wild-type”) hypertrichosis allele that might have mutated many millions of years ago to precipitate the loss of the dense hair cover characteristic of other primates (Angier, 1995; Figuera et al., 1995; Hall, 1995). It is speculated that the “hypertrichosis locus” may have some additional major function besides regulating hair density and distribution (Angier, 1995). This would account for the persistence of the current so-called “normal” but historically mutant gene that does not perform its ancient function of inducing a dense coat of hair. Periodically, this impotent gene, as far as hair growth is concerned, may undergo back-mutation, leading to a reactivation of the program for hypertrichosis. This model implies that hair reduction early in the evolution of humans or prehumans may have been a very simple genetic event (i.e. a saltation, a jump, producing a new developmental state without gradual change through intermediate forms). With regard to human pigmentation, knowledge of the graying patterns of individuals manifesting extreme congenital generalized hypertrichosis would be particularly interesting, for it might reveal the impact of aging on the display patterns of members of the human lineage prior to the time that the hair coat was reduced historically. With the reduction in hair cover on the appendages and torso in early man, epidermal melanin took on greater significance. Like humans, the gorilla and chimpanzee, their closest relatives, have abundant melanogenic epidermal and follicular melanocytes and few dermal melanocytes. Nonetheless, the importance of epidermal pigmentation in humans is still debated. Suggestions as to why melanin is or has been important as a human adaptation include: (1) it absorbs UVR, thus increasing resistance of the skin to sunburn, premature aging, and cancer; (2) it camouflages humans in evasion of predators and in hunting; (3) it facilitates thermoregulation by enhancement of absorption of solar radiation or by loss of metabolic heat; (4) it regulates vitamin D synthesis by influencing penetration of UVR into skin; (5) it protects light-sensitive substances from photodestruction; (6) it interacts with scalp and beard in producing aggressive display patterns; (7) it forms part of a system designed to neutralize the action of noxious chemicals reaching the skin, either directly from the external environment or indirectly from those generated when exogenous agents (e.g. UVR) damage the skin (e.g. free radical compounds) (Nordlund, 1995; Pathak, 1995; Quevedo et al., 1974, 1985). Most, if not all, these suggestions have been challenged, and negative attributes of epidermal melanin have been cited (Hill, 1995; Quevedo et al., 1995). For example, it has been suggested that heavily pigmented humans are more sensitive to frostbite than more lightly pigmented ones. This suggestion has also been questioned (Robins, 1991). 72

Melanin and Human Adaptations to Solar Radiation The effectiveness of melanin as a sunscreen in humans has not gone unchallenged (Kligman, 1995; Nordlund, 1995). This debate is not a new one for, in 1961, Harold Blum ranked its photoprotective value below that of other components of human skin (thickness of the epidermis). In fact, Blum went much further: The widely accepted idea that melanin pigment in human skin protects against sunlight, and that this has bearing upon adaptation to life in the tropics and the distribution of races, [when] examined in terms of its physical and physiological aspects . . . appears to have little merit. It is concluded that whereas the pigment may have a slight adaptive value as regards some aspects of the organism–environment relationship, it may be non-adaptive as regards others; and the respective values may depend upon various complicating factors of the environment. Moreover, the distribution of races according to skin color [if it is adaptive] does not appear to conform well to what would be expected from the spectral distribution of sunlight. (Blum, 1961) While there is logic to Blum’s argument, there is also a fatal flaw, for his bottom line is essentially one of perfectionism rather than relativism in evaluating melanin as an adaptive trait (Blum, 1961, 1969). Relativism holds that an adaptation is not necessarily perfect; it might have both good and bad effects. As small advantages appear to have determined the survival of many lineages in history, as long as the virtues outweigh the liability of a trait, it has a chance to be a winner in the sweepstakes of natural selection. Blum recognizes relativism when he constructs a balance sheet for the positive and negative values of melanin. However, he ignores relativism when he concludes that it is highly questionable whether melanin is a significant human adaptation. Failing to find a perfect match between melanin’s properties, the worldwide distribution of human skin color, and the problems facing humans in different ecological settings, he assigns melanin a minor role, if any, in the origin, survival, and territorial expansion of the human species. However, based on the numerous possible functions of melanin in humans (Quevedo et al., 1974, 1986, 1995), more than those mentioned by Blum, it is likely that melanin meets a multiplicity of needs simultaneously. Recently, Gilchrest et al. (1996) have demonstrated that increased melanogenesis is stimulated by DNA dimers excised during repair of UVR-induced DNA damage. This intimate link between photorepair of DNA damage and melanogenesis leading to melanin amplification indicates that melanin forms an important part of the homeostatic photoprotection mechanism of skin. As different functions of melanin may require different amounts, the level of melanin maintained in human skin, although less or more than the optimal amount for any one function, nonetheless is the best compromise amount for meeting all melanin’s duties while sacrificing as little as possible in effectiveness in any one of them. Therefore, there is an adaptive compromise between contrasting functions of

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melanin in skin rather than an impossible perfection in all of them. Jacob (1982) has emphasized that evolutionary change results from the tinkering of existing structures and substances into new forms and functions. Consequently, evolution does not produce optimal designs, for it can only work on the materials at hand. The failure to recognize this reality greatly weakens Blum’s position. Whether melanin is or is not a good sunscreen will be determined by thoroughgoing research. However, whatever the outcome of quantitative research, the results will have to be evaluated in the light of a qualitative question: Where would the human species be if melanin was absent from the skin of the early naked human ape evolving in the tropics? The most reasonable answer seems to be: If the emergence of humans was an evolutionary long shot, the odds would have been even longer for humans lacking cutaneous melanin pigmentation. Apart from not considering adaptive compromise, Blum failed to factor in the human life cycle. Among early hominids evolving in the tropics, dark skin color would have been particularly adaptive during early childhood, a time when susceptibility of the still developing skin to solar damage would have been at a maximum. Childhood is also the time when the risk of death from starvation or infectious disease is greatest. It is possible that the adult levels of constitutive skin color (CSC) are primarily the result of natural selection acting on pigment levels during infancy and childhood (Quevedo et al., 1985). In unclad, depigmented, prehistoric humans living in the tropics, sun damage would have been the straw that broke the camel’s back, particularly in children attempting to cope with parasites, infectious diseases, diarrheal dehydration, and malnutrition, conditions that would have been exacerbated by severe sunburn (Quevedo et al., 1985).

The Evolutionary and Biological Basis for Melanin Photoprotection and Phototoxicity Based on the Melanin Content of Human Skin Based on the principle of adaptive compromise, one might envision a scenario for human evolution as follows (Quevedo et al., 1995): 1 The skin of the earliest members of the human family evolving in the solar-inundated continent of Africa was probably hair covered and lightly or darkly pigmented. When reduction in body hair took place, if the skin was lightly pigmented, there would have been strong natural selection for darker skin color, particularly during childhood. Consequently, today’s black Africans are resistant to sunburn and solar-induced skin cancer. They also have the ability to amplify their CSC substantially by increasing the levels of epidermal melanin following exposure to solar radiation, particularly its UVA and UVB components. 2 Melanin distributed throughout the viable and cornified layers of dark pigmented epidermis, based on biological assays, would have blocked the passage of 90% or more of incident UVB and 80% or more of incident UVA to the dermis. Much of the UVB would have been blocked by the stratum corneum (Kaidbey et al., 1979).

3 At low doses of solar radiation, UVA and UVB reaching the viable layers of the epidermis would have generated free radicals subsequently neutralized by general antioxidants and the free radical scavenging redox actions of melanin. The result would be survival and restored functions of epidermal cells. At higher doses, significant damage wrought by free radicals in excess of the reducing capacity of the antioxidant defense system would have brought a second line of defense into action. In melanocytes, cytotoxic melanin, its cytotoxic monomers, as well as their cytotoxic precursors released from UVR-damaged melanosomes would exacerbate UVR-induced nuclear damage (DNA strand breaks) and cytoplasmic membrane damage resulting in melanocyte death. Similar events would transpire in UVR-damaged keratinocytes where melanin and its breakdown products would be released from secondary lysosomes (phagolysosomes) and would destroy the functionally impaired cell (Daniels and Johnson, 1974; Quevedo et al., 1995; Riley, 1974). Quevedo et al. (1995) emphasize that: “Melanin/melanogenesis-induced death of photodamaged melanocytes and keratinocytes in vivo may be important in rapidly restoring the essential ‘housekeeping’ functions of sunburned skin and in lowering the potential incidence of skin cancer.” In particular, the removal of damaged stem cells (melanocytes and keratinocytes) of the germinal layer of the epidermis would prevent the cloning of defective cells, which would otherwise chronically undermine essential functions of the epidermis (Riley, 1974; Young, 1987). Quevedo et al. (1995) point out that “[in darkly pigmented humans] the melanin-associated deletion of genetically and functionally compromised melanocytes and keratinocytes is an important factor in the restoration of UVR-disrupted epidermal homeostasis in the short term and in the reduction of precancerous and cancerous lesions in the long term.” The scenario thus far gives substance to melanin adaptations 1 (sunscreen), 5 (photoprotection of vital metabolites), and 7 (detoxification of noxious chemicals) listed on p. 72. Melanin adaptations 2 (camouflage), 3 (thermoregulation), 4 (regulation of vitamin D synthesis), 6 (aggressive display patterns), and 7 might have forced a level of melanin pigmentation less than optimal for defense of the skin from UVR damage. If childhood was the critical time when melanin exerted its major influence in depressing mortality in the face of disease and starvation, then photoprotection (melanin adaptations 1, 5, and 7) and possibly photosynthesis of vitamin D (melanin adaptation 4) may have overshadowed all the other postulated functions of melanin. 4 The depigmentation of skin in many human populations following their dispersal from Africa produced a range of colors dominated by individuals with black hair, dark eyes, and moderate levels of constitutive skin color, coupled with marked ability for facultative tanning on exposure to solar radiation. In effect, for most humans, the photoprotection provided by the dark constitutive skin color of Black Africans was retained. Therefore, the same statements made about adaptive compromise in Black Africans holds for most other humans living today. 73

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5 In what is today Western Europe, populations of highly depigmented (fair-skinned) humans evolved with limited ability for facultative darkening in response to solar radiation. The Celtic peoples with red hair, blue eyes, and fair skin are representative of this group. Their melanocytes lack the ability to produce melanin in quantities adequate to provide an effective sunscreen. In addition, melanin/melanogenesis levels are too low to provide effective free radical scavenging or to elicit the death of functionally compromised melanocytes and keratinocytes. As a result, the irradiated skin of such individuals is at greater risk for developing a variety of benign (freckles and keratoses) and malignant (melanoma, basal cell carcinoma, and squamous cell carcinoma) lesions (Quevedo et al., 1995). Considering the negative features of depigmented skin and suggestive evidence that there may have been secondary repigmentation of some depigmented human populations (e.g. Asian Indians), there appears to be no clear adaptive explanation for why reduction in cutaneous melanin should have occurred in the first place. To account for geographic/racial differences in skin color, major emphasis has been placed on (1) the requirement for UVR-induced cutaneous synthesis of vitamin D in humans inhabiting latitudes where sunlight is limited and where dietary sources of the vitamin are scarce and/or (2) although controversial, evidence suggesting that black skin is more susceptible to frostbite than white skin. There is no strong consensus of opinion in favor of either one of them (reviewed by Robins, 1991). Perhaps lightening of skin color in certain human populations dispersing out of Africa reflected persistence of genetic changes permitted by a reduction in the formerly strong selection for cutaneous melanin in the sundrenched tropics (Quevedo et al., 1985). The resolution of the problems surrounding the current role of the melanin pigmentary system in the skin of humans will have to be solved in humans, for the comparative anatomy of melanin pigmentation elsewhere among the primates is more bewildering than informative.

Cutaneous Melanin Pigmentation of Nonhuman Primates Machida and Perkins (1967) estimated the degree of melanotic pigmentation within the general body skin of 49 species of nonhuman primates. They found that, paralleling rodents, dermal melanocytes in primates are most frequently found in specialized areas of skin, such as the eyelids, lips, nose, and anogenital region. Overall, although some primate species have dense populations of dermal melanocytes in their general body skin, the numbers of dermal melanocytes are reduced in the general body skin compared with specialized skin. Taken as a whole, the more primitive primates, prosimians, have little dermal melanin in their general body skin and, in many species, epidermal melanin is absent as well. Dermal melanin increases in frequency among the Old and New World monkeys but is sharply reduced in humans and their closest relatives, the gorilla and chimpanzee. This brief survey suggests that epidermal and hair follicle pigmentation may be primitive traits dating from the origin of primates and, there74

fore, different from dermal pigmentation, which may be a derived character arising independently in some lineages but not others during their evolutionary divergence. It is important to emphasize that the patterns of epidermal and dermal pigmentation are highly variable in the Old and New World monkeys. Perkins (1975) considers the variation in pigmentation within any group of monkeys to be greatest among the New World monkeys. One might speculate that there has been a reduction in evolutionary selection for epidermal and dermal pigmentation in monkeys, possibly because selection acts only on their exposed surfaces, i.e. their hair coat, and it is adequate for photoprotection. Another possibility is that they live mainly in shaded areas where the hair coat, regardless of density, is sufficient to protect the skin from solar damage. As a result, one finds a kaleidoscope of cutaneous pigment patterns that, at least in some cases, appear to have no obvious positive or negative adaptive value. The rhesus monkey illustrates the difficulty in finding adaptive explanations for pigmentation in hair-covered skin, for it is not clear how its presence would improve the ability of the organism to survive and reproduce in its native habitat. In the rhesus monkey, the hair-covered general body skin, but not the hair, is pigmented in a piebald (spotted) fashion. The epidermis lacks melanogenic melanocytes, but they can be induced by treatment with UVR (Erickson, 1976; Montagna et al., 1964; Yun and Montagna, 1966). Patches of dermal melanocytes are scattered throughout the skin. Within the pigmented patches, dermal melanocytes are frequently observed to be wrapped around blood vessels. Although UVR stimulates epidermal pigmentation, it does not influence the activity or distribution of dermal melanocytes (Erickson, 1976; Montagna et al., 1964; Yun and Montagna, 1966). UVR has also been demonstrated to increase the number of melanogenic melanocytes in the epidermis of the potto, Perodicticus potto (Montagna, 1967). In view of the ability of melanocytes to synthesize cytoactive substances, it is possible that melanocytes enveloping blood vessels might play a role in skin physiology unrelated to melanogenesis. The same can be said for melanocytes associated with sebaceous glands and the perifollicular networks of dermal melanocytes. James J. Nordlund (1992) is to be credited for emphasizing the roles played by melanocytes in processes other than those associated with melanin. Mammalian melanocytes are considered to be important modulators of inflammation and have been demonstrated to synthesize interleukin (IL)-1 and transforming growth factor (TGF)-a and other growth factors and cytokines (Nordlund, 1995). It is important to keep in mind the diversity of functions that melanocytes may perform in the hairy skin of primates when attempting to interpret their often bizarre distribution patterns. The cases in which pigmentary explanations for purpose in melanocyte patterns utterly fail may be ones where closer scrutiny reveals heretofore unknown functions of melanocytes, or familiar ones seen in a different context. From this perspective, biologists should not be frustrated by evolutionary paradoxes in pigmentation but rather they should be delighted.

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The Hair Coat as the Interface between the Mammal and its Environment Coat color patterns are considered to be important in protective (concealing) coloration, sexual attraction, warning displays, and thermoregulation. Although the distribution of epidermal and dermal melanocytes shows significant arbitrariness in the skin of many mammals with dense coverings of hair, the color pattern of the hair coat does not. It appears to be produced by developmental genetic mechanisms that have been closely scrutinized by natural selection. However, nongenetic maternal environmental factors operating during gestation may have some influences on coat color patterns (Caro and Durant, 1991). African cheetahs (Acinonyx jubatus) are dark spotted over most of their bodies with the exception of the apex of the tail where there are stripes. The pattern of tail stripes of sibling cheetahs is very similar and distinguishable from the array of tail stripes on their mother. The pigment patterns of siblings are more alike than they are similar to those of nonsiblings of equal age. It has been hypothesized that “environmental factors in utero are responsible for (pattern) differences in cheetahs in combination with maternal or paternal genetic influences,” thus producing the greater phenotypic coat color variation observed in cheetah populations than expected in a species with very high levels of genetic homozygosity. It is possible that coat color patterns may directly influence bonding or are markers of other developmental events that control bonding. Male cheetahs may form coalitions consisting solely or predominantly of brothers. Such coalitions have better odds of holding territories favoring hunting and reproductive success (Caro and Durant, 1991). Individual and geographic variation in pigmentation patterns of the harbor porpoise, Phocoena phocoena, suggests that melanin pigmentation may play a role similar to that in the cheetah in recognition between individual porpoises of a group. However, the variation in color patterns of geographically isolated groups of porpoises is insufficient to establish taxonomic distinctions between them (Koopman and Gaskin, 1994). Unlike the symmetrical pigmentation patterns in the cheetah, those of Phocoena phocoena are asymmetrical (Koopman and Gaskin, 1994). Mammals may escape from constraining developmental rigidity of pigmentation in another way. In adapting to temperate habitats, mammals such as the snowshoe hare have seasonal changes in hair color such that hair grown in the spring is brownish and that grown in the fall is white (Keogh, 1969). In response to changing photoperiods, hormones trigger the appropriate melanocyte responses (Rust, 1965; Rust and Meyer, 1968). However, the changes brought about are economy minded, for the whitening of the coat involves the growth of overhairs that are white (pigmentless) at their exposed tips but dark in their concealed bases. All the concealed undercoat of insulating hairs remains pigmented regardless of season. Such selectivity in the types of hairs participating in seasonal color changes and the mathematical precision with which hairs are partially pigmented suggest a

tightknit relationship between the neuroendocrine system and the skin (Rust, 1965; Rust and Meyer, 1968). The thin winter coating of white suggests that the seasonal color changes in the snowshoe hare are important in concealing it from predators. The permanent white coat of the polar bear is considered to be important in concealing it from prey when hunting on ice and snow. In summer, in some parts of the world (e.g. the Hudson Bay area, Canada), with the melt of sea ice, it is not uncommon for polar bears to be restricted to land temporarily devoid of ice and snow (Nowak, 1991). Therefore, at least for some populations of polar bears, the white coat may prove a seasonal disadvantage for, rather than being concealing, it calls attention to its owner. This disadvantage may be partly reduced by the polar bear’s omnivorous lifestyle. Seals are the major source of food, particularly in winter. Depending on location, when feeding on seals is not an option in summer and fall, polar bears feed on sea birds and their eggs plus berries and other vegetation. In the absence of snow and ice, its coat color may make it more conspicuous to prey and thus prove a disadvantage. On occasion, polar bears may eat the carcasses of stranded marine mammals, small land mammals, reindeer, and fish. Nonetheless, a life without seals is a difficult one. In the James Bay region of Canada from July through December, the polar bear lives off stored fat to a significant extent, hanging on until the sea ice returns. This suggests that the coat color of polar bears has real advantages and disadvantages, with the former greater than the latter when it comes to feeding (Nowak, 1991). A competing hypothesis is that the white coat and darkly pigmented epidermis of the polar bear are associated with thermoregulation (Nowak, 1991). Possibly, the true explanation for the evolution of the polar bear’s coat is that it was selected for both functions. The agouti (A) coat of feral house mice, Mus musculus, appears to be associated with concealment from predators, as do the light and dark coats of the pocket mouse matching light (sandy) and dark (lava) backgrounds in New Mexico (Cott, 1957). The agouti pattern of coat color in house mice arises from a complex array of hairs that differ from each other in shape as well as the color that they display, thus rendering their bearers less visible, safer, when in harmony with the background coloration of their habitat. For further details about the wild-type coloration of mice, see Part II. The lion cub has dark spots and dark rings on its tail; the adult has not. Presumably, the disruptive coloration of the cub favors concealment in the grassy landscapes on which they are reared and/or possibly identifies them as juveniles, protecting them from adult aggression. The traditional explanation of the alternating light and dark stripes (hair color reinforced by skin color) of the zebra is that they produce disruptive coloration, making it particularly difficult to detect zebras on the African savannahs at dusk when risk of attack is greatest (Cott, 1957). Alternatively, when zebras are in herds, their swift movement may cause a blurring of individual outlines, momentarily distracting their predators (MacClintock and Mochi, 1976). However, these 75

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interpretations have been questioned (MacClintock and Mochi, 1976; Owen, 1980). One suggestion is that the color pattern of the zebra disorients biting flies, decreasing their success (Waage, 1981). In addition, it is possible that zebra stripes, because they vary between individuals, may facilitate intraspecific recognition as well as membership in a particular herd, a role comparable to that already suggested for color pattern in cheetahs (MacClintock and Mochi, 1976). A variety of evidence suggests that zebras are black with white stripes produced by the gradual evolutionary enlargement and coalescence of small white flecks in the descendants of their darkcoated ancestors (MacClintock and Mochi, 1976). Based on his experiments, Waage (1981) concludes that black-bodied zebras would be more prone to fly bites than their striped counterparts.

The Coat Colors of Juvenile Mammals: A Time to Learn and Play Among mammals, hair color patterns often change between infancy, adolescence, and adulthood in association with the different roles played and problems faced at different stages of the life cycle. The young chimpanzee has a patch of white hair on its rump that elicits tolerance in its misbehavior toward the powerful adults that surround it (Napier and Napier, 1985). The young of many monkey species are dramatically different in color from the adult. The dusky leaf monkey (Presbytis obscura) as an adult wears a dark coat of hair whereas the infant wears one that is apricot in color (Napier and Napier, 1985). In some human populations, hair frequently darkens with age. In these cases, lighter hair color along with other juvenile traits may have some influence as a releaser of empathy directed by human adults toward their young. Clearly, many social primates emphasize in their coat colors that youth is a time for learning and support by the community. In primates other than man, particularly in the Old and New World monkeys, there is generally a dense covering of body hair, sparing only certain regions such as the face and anogenital region. It is in many of these highly social animals that there is an intricate pattern of glabrous skin pigmentation framed by hairs of various hues. It is clear that these faces are intended to be seen and to transmit information. The associations of tufts of hairs of different colors together with patterned facial skin pigmentation indicates that this complex of features is a defining element of certain species and has been shaped in fine detail by evolutionary mechanisms (Napier and Napier, 1985). In some primates, the color of the hair coat is less important than what is on it. In South American sloths, the coat is covered by algae that impart a green color that camouflages the sloths as they hang from tree limbs (Cott, 1957; Owen, 1980). According to Nowak (1991), “the color [of the polar bear] is pure white following the molt but may become yellowish in the summer, possibly because of oxidation by the sun. The pelage sometimes appears grey or almost brown, depending on season or light conditions.” The mechanisms 76

involved are not stated. It is possible that the variations in hair coloration result from the oxidation of lipids of sebaceous gland origin that coat the hair. In some cases, the off-white color of the polar bear might render it less visible, particularly on snowless landscapes and on snow under certain lighting and weather conditions. Although a start has been made (Vrieling et al., 1994), the developmental mechanisms involved in establishing the complex coat color patterns of mammals have not been identified. A number of different mathematical models have been proposed but have not as yet been attached to real events transpiring within the developing skin (Nagorcka and Mooney, 1992; Pearson, 1993; Quevedo et al., 1986).

Radicalism and Conservatism in the Evolution of the Mammalian Cutaneous Pigmentary System from that of Reptiles The evolution of mammals from reptiles did not occur in one big step. It took place gradually; the traits that define mammals (warm-bloodedness, diaphragm, hair, etc.) accumulated and became more efficient over considerable periods of evolutionary time. These events transpired within a lineage known as the mammal-like (synapsid) reptiles (Kemp, 1982). The innovative adaptations of mammal-like reptiles to changing environments were put to good use by the mammals, their gift to posterity. Following the demise of the dinosaurs, the adaptive traits of mammals facilitated an evolutionary increase not only in the number but also in the diversity of species. More precisely, the ancestral diminutive mammals of limited diversity rapidly produced many divergent lineages that, in turn, generated a great variety of specialized descendants capable not only of filling the habitats vacated by the ruling reptiles but also equipped to enter new habitats as well. The skin and its appendages played an important role in determining the ecological success of mammals. The evolutionary changes in the skin associated with the reptile– mammalian transition were complicated ones that tested the relevance of each skin component to emergent new functions. Some components did not stand the test and disappeared; others became less prominent. In the case of the dermal chromatophores, the instruments of change in vertebrates other than the birds and mammals, the cutaneous xanthophores and iridophores presumably disappeared from advanced mammallike reptiles while their dermal melanophores were replaced by smaller numbers of melanocytes lacking the ability for rapid, reversible movement of melanosomes between their cell bodies and dendrites (Bagnara and Hadley, 1973; Oliphant et al., 1992). Only epidermal melanocytes prospered by the evolutionary test. They became the major source of skin pigment in mammals. They retained their ability to transfer melanosomes to keratinocytes but, like the dermal melanocytes, they lost the ability for rapidly aggregating or dispersing melanosomes intracellularly in response to endocrine and neural stimulation (Klaus and Snell, 1967; Reams et al., 1968). In reptiles, all the chromatophores are thought to be derived

GENERAL BIOLOGY OF MAMMALIAN PIGMENTATION

from pluripotential stem cells of neural crest origin, the specific differentiated fate of which (melanophore, xanthophore, iridophore) is determined by the particular cutaneous site that each stem cell becomes lodged in during embryonic development (Bagnara et al., 1979). Consequently, major changes in regulation of cutaneous development were indispensable for the evolutionary emergence of mammalian pigmentary patterns produced by epidermal and follicular melanocytes (Quevedo and Holstein, 1992). Three major events in the reorganization of the skin among mammal-like reptiles were: (1) the loss of epidermal scales from the body surface; (2) the development of hair; and (3) the modification of the molecular biological pathway for epidermal keratinization, which produced new patterns of molting in those mammal-like reptiles approaching full status as mammals. In the ancestors of mammals, as in their descendants, molting became increasingly identified with periodic shedding of hair rather than the shedding of cornified epidermis. The loss of epidermal scales resulted from modifications in epidermal keratinization. Their disappearance over most of the body erased or respecified a complex mosaic of morphogenetic fields formerly embodied in the epidermal scales, the intervening scale hinges of which had served as the “grout” that held the mosaic together. This change was not a minor one, for the scales had imposed order on the skin, as evidenced by the intricate associations of diverse dermal chromatophores within each scale territory (Whimster, 1965, 1971). In mammalian skin, there is evidence of a mosaic consisting of repeated “vertical units” containing epidermal, neural, and vascular elements arranged around a sensory tylotrich hair follicle (Maderson, 1972; Straile, 1969). These morphogenetic units may reflect in some highly derived way the morphogenetic fields of scale-covered skin in ancestral reptiles. In reptilian lineages other than that of the mammal-like reptiles, species-specific skin color patterns continued to be produced by the programming of groups of adjacent scales to make coordinated site-specific skin displays of coloration. The “scale color display units,” like the colored tiles in ceramic mosaics fashioned by human artisans, collectively gave rise to a grosser distinctive pattern of value in a sighted world. In the case of reptiles, the pattern concealed its bearer, attracted conspecifics, and repelled potential predators including conspecifics (Whimster, 1965, 1971). These behavior-triggering functions of pigment patterns emerged when the observing animal contemplated the whole integument of the object of its attention rather than its individual component parts (scales). The loss of scales in advanced mammal-like reptiles was presumably precipitated by events associated with the origin of hair and almost certainly destabilized the reptilian-style dermal pigmentary system from a developmental point of view. In addition, an increasingly dense covering of hair exacerbated the stress imposed by scale loss on the dermal pigmentary system by making dermal chromatophores largely irrelevant in photoprotection and adaptive color changes. Maderson (1972) has outlined a plausible scenario for the

origin of hair and loss of scales in mammal-like reptiles. It centers on the premise that hair first arose as a mechanoreceptor that provided mammal-like reptiles with information about the position of their scales and about external objects in contact with their skin. Accordingly, the insulating function of hair must have been a secondary rather than a primary adaptation. The first hairs were limited in number and distributed diffusely over the body. The tylotrich hair follicles that serve as mechanoreceptors and their companion hairs show a special distribution in the dorsal skin, which suggests that they arose in the scale hinges and were separated by scales at some time in the remote past. Maderson (1972) suggests that the proliferation of hair follicles to amplify monitoring of the environment (e.g. sensing the air currents passing across the skin) would result in their invasion and destruction of scales. Accompanying the spread of hair follicles would be an altered pattern of epidermal keratinization featuring keratohyalin granules in the superficial epidermis. Maderson hypothesizes that “hair” began as a simple column of epidermal cells but gradually evolved into the multilayered hair follicle of today, a structure significantly governed by interactions of a dermal papilla with germinal keratinocytes of the hair follicles (Cotsarelis et al., 1990). The tylotrich hair follicle, with its encircling networks of nerves, is thought to mirror features of the advanced prototypic hairs of progressive mammal-like reptiles. The broad distribution of the tylotrich hair follicle in mammals from duckbilled platypus to humans suggests that it was an early component of the hair coat and in all probability was present in mammal– reptiles (Maderson, 1972; Mann, 1968, 1969) (see Part II for coverage of the embryology of hair formation). By this stage in the evolution of mammal-like reptiles, a sophisticated behavioral pattern for thermoregulation would have evolved so that shifts made in body position in response to air currents would have facilitated appropriate heat gain or loss. Sustained selection for a thickened hair coat to enhance mechanoreception was eventually co-opted to provide insulation in support of thermoregulation (Maderson, 1972). Of the pigment cells in the skin, epidermal melanocytes would have retained an adaptive melanin-based purpose in animals covered with hair. They were required to pigment the hair as well as any areas of skin epidermis that was devoid of hair cover. In addition, many mammals bask in the sun, accumulating heat by exposing their ventral surfaces where hair density is lowest. Reptiles habitually sunbathe. A pigmented epidermis in an early reptile–mammal may have facilitated heat absorption while preventing sunburn of the skin during basking (Hamilton, 1973). Such behaviors reduced the requirement for feeding (caloric intake) in metabolic support of thermoregulation and may have been very important during the transition from exothermy to endothermy. Therefore, the presence of a pigmented epidermis sometimes accompanied by a pigmented dermis in skin exposed to solar radiation was adaptive and thus a target for natural selection leading to developmental genetic programs that assured appropriate 77

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embryonic deployment of cutaneous melanocytes. In polar bears, for example, the combination of white fur and melanized (black) epidermis is thought to trap and transfer substantial amounts of solar heat into the blood vessels of the skin. The skin of mammals was programmed regionally in order to specify the presence or absence of differentiated melanocytes in the epidermis and/or dermis in keeping with the specialized functions of the particular site. For example, in the trunk skin of mice, differentiated melanocytes are present in the epidermis, dermis, and hair follicles at birth. Many of the epidermal melanocytes enter the hair follicles and survive. However, numerous epidermal melanocytes do not. The epidermal melanocytes that do not enter hair follicles and dermal melanocytes disappear almost completely during the first month of life (Quevedo et al., 1966). In contrast, melanocytes are retained in the epidermis and dermis of essentially glabrous skin such as the perineum, tail, and ear pinnae. The persistence of melanocytes in this case may aid concealment of feral mice as well as play some role in photoprotection and courtship of mice in the wild.

Part II: The Development of the Melanin Pigmentary System and the Molecular Genetic Systems that Regulate It Introduction Thus far, the major focus of this chapter has been threefold: the structure and function of the melanin pigmentary system (MPS) of mammals; the great variety of melanin pigment patterns found in the integument of mammals; and the adaptive significance of the MPS during millions of years of mammalian evolution. On the short scale of time, knowledge of the mechanism by which such patterns are laid out during embryonic development is equally as important as the long-term events that shape them. A substantial number of the specialized chapters in this volume are dedicated to exploring in depth the biochemistry, developmental biology, molecular biology, and cellular biology of the MPS that emerges during embryogenesis and functions throughout the life of the organism. By way of an introduction to these chapters, what follows is a brief integrated account of the genetic program that regulates the development of the MPS in the mouse. It is our hope that it will be of assistance to readers without a background in molecular and developmental biology. For readers desiring additional coverage in greater depth, the links between this introduction to development and the matching more technical chapters are listed at the end of this account. To begin with, the development of the MPS is described to establish a context in which to discuss the mechanisms that regulate its origin and function. Based on the large number of hair color mutants known and preserved in laboratory mice, their developmental genetics of 78

pigmentation is particularly well understood. Historically, the development of the wild-type color pattern of the laboratory mouse has been treated as the “standard pattern” from which all other color mutations in laboratory mice have departed. The account of MPS development that follows adheres to this tradition. It is noteworthy that numerous pigment gene homologies have been found between mice and humans.

The Development of the Hair Coat of Wild-type Mice A brief account of the development of the hair coat in mice is in order to establish a structural context within which to discuss regulatory mechanisms related to pigmentation of the hair. Embryonic development of hair follicles is tightly controlled by numerous genes that encode growth factors. In addition to regulating epidermal and dermal cells directly related to hair growth, growth factors, by chance or necessity, may also benefit the invading melanocytes that are of little use to the hair follicle, but of great importance in the survival of the organism as a whole. The hair coat is complex in its structure. According to Chase and Rauch (1950), the dorsal pelage of the mouse consists of at least four distinct types of hairs divisible into two groups: the long overhairs and the shorter underhairs. The overhairs consist of the monotrichs (1.6% of hair coat), awls (8.7%), and auchenes (7.8%). The underhairs, called zigzags, make up 81.9% of the dorsal pelage. In wild-type (agouti) mice, the overhairs differ in the frequency with which they express the agouti band, a band of yellow pigment that divides the otherwise uniformly eumelanic black hair (except for a short portion of the tip and base that lacks pigment) into a hair shaft that contains a black eumelanic band near the tip followed by a yellow pheomelanic band and a long basal eumelanic segment accounting for much of the remaining hair shaft. Zigzag hairs invariably have an agouti band; auchenes usually, but not invariably, have an agouti band; awls lack agouti bands more frequently than auchenes; and the monotrichs invariably lack them (Galbraith, 1964, 1969). In mice, the mix of black hairs and yellow-banded hairs produces a hair coat in which blackness and yellowness are muted to an indefinite intermediate overall color. Hundreds to thousands of tiny flecks of yellow and black may serve as camouflage and confuse the eyes of potential predators as well as the human eye so that one mouse can appear dark grayish, brownish, or blackish and inconspicuous depending on the background and ambient light.

Embryonic Establishment of the Hair Coat in Mice The initiation of the growth of hair follicles begins on the 14th day post coitus (dpc) in the mouse and continues until slightly before birth (Mann, cited in Claxton, 1966) or slightly after

GENERAL BIOLOGY OF MAMMALIAN PIGMENTATION

birth (Claxton, 1966). Its earliest stages are dependent on exchanges of signals between dermal mesenchyme cells (precursor dermal papilla) and overlying foci of epidermal cells (keratinocytes). In response, epidermal cells crowd together, with the precursor dermal papilla condensing beneath them. They are separated by a basement membrane (basal lamina). The crowded keratinocytes sink slightly into the dermis producing a node (bud), the collective cells of which are designated as the initial hair follicle germ. In later hair generations, as discussed below, the equivalents of the initial hair germ and initial dermal papilla interact to induce the growth of a new hair (Hardy, 1992; Lavker et al., 1999). In the mouse embryo, signals from the initial dermal papilla are considered to induce the cells of the epidermal node (bud) to grow into the dermis and elongate into a peg, a cellular cylindrical rod-like structure. In doing so, the solid cylindrical rod (peg) of epidermal cells (keratinocytes) with its former hair germ leading grows over the dermal papilla producing a hair (follicle) bulb that takes the lead as the peg extends deeply into the dermis (Figs 3.6A and 3.7) (Chase et al., 1951). The follicle is now characterized by the hair bulb and two other bulges,

the presumptive sebaceous gland and, slightly below it, a bulge of the outer root sheath (ORS) to which the arrector pili muscle attaches (Fig. 3.7). The ORS bulge is thought to be a reservoir of stem keratinocytes and possibly melanocytes (Akiyama et al., 1995). The permanent (upper) hair follicle ends slightly below the ORS bulge. The part of the hair follicle below the ORS bulge is temporary and is destroyed during each hair growth cycle only to be recreated by the next one. Within the hair bulb of the lower follicle, the basement membrane separates the dermal papilla not only from the surrounding keratinocytes but also from the migratory melanocytes that have invaded the hair follicle (Fig. 3.6B). The keratinocytes of the bulb form a matrix. Within the matrix, the keratinocytes located in approximately the lower half of the bulbous portion of the lower hair follicle dominate in mitotic activity and create a pressure for upward movement of keratinocytes toward the skin surface. In pigmented mice, keratinocytes destined to form the hair acquire melanosomes when they encounter melanocytes adjacent to the dermal papilla in the upper hair bulb. During their upward movement, keratinocytes are organized into concentric channels

K

G

MC

MN DP

G

A

K

B

Fig. 3.6. Vertical sections of hair follicles plucked from a human scalp. (A) Incubation of a follicle in “dopa-reagent” to enhance melanin synthesis decisively distinguishes dendritic melanocytes (arrows) from keratinocytes in the upper hair bulb. Note that the melanocytes reside at the junction of the dermal papilla (DP) and follicular keratinocytes. (B) Ultrastructure of a follicular dendritic melanocyte in intimate contact with several keratinocytes. Numerous melanosomes at formative stages ranging from unmelanized (Stages I and II) (arrows) to fully melanized (Stage IV) populate the melanocyte cytoplasm. Melanized melanosomes are also present in the keratinocytes (arrowheads). (MN, melanocyte nucleus; MC, melanocyte cytoplasm; G, Golgi apparatus; open arrow, melanocyte dendrite; K, keratinocyte) (both micrographs courtesy of Raymond Boissy).

79

CHAPTER 3 Telogen

Anagen I

Anagen II

Anagen III

Epidermis Dermis

Adipose layer

Permanent follicle

Sebaceous gland

Outer root Arrector pili Sheath bulge muscle Temporary follicle

Anagen IV Anagen V Anagen VI Catagen

Dermal papilla

within which the ascending cells meet substantially different developmental fates (Chase et al., 1951; Lavker et al., 1999). Within the solid column of keratinocytes forming the cellular follicular peg above the hair bulb, central cells die and decompose, providing a space within which the upward movement of two major channels of cells, each divisible into subchannels, can take place. Within the most central channel, there is a transformation of streams of medulla, cortex, and cuticle cells into a hair shaft. It grows through the channel in the follicular rod (peg) and out of the hair canal, an opening in the epidermis. This pattern of development not only permits the hair to display itself to the sighted world beyond the skin, but also aids the organism in keeping warm and capable of sensing features of its environment everywhere in the integument that encloses its body [see Straile and Mann (1972) on touch receptors below]. During hair growth, the upper matrix cells of the hair bulb are the first cells incorporated into the hair shaft as well as the inner root sheath (IRS) that surrounds the hair shaft. The IRS consists of three layers: an outer Henle’s layer, middle Huxley’s layer, and inner cuticle layer. Although the IRS keratinizes and ascends with the hair shaft toward the skin surface, it stops short and sheds itself into the hair canal near the duct of the sebaceous gland. The outer root sheath (ORS), which surrounds the IRS, although of epidermal origin, is maintained by the epidermis with which it is contiguous. Completing the layers of the hair follicle is the connective tissue sheath of dermal origin that surrounds the ORS of the follicle. It con80

Fig. 3.7. Critical stages in the growth cycle of mouse hair follicles, courtesy of Dr William Montagna (slightly modified). Left: an actively growing hair follicle illustrating some of its important features. Right: illustration of the stages of the hair growth cycle: telogen (quiescent period), anagen [period of active follicle and hair growth (six substages characterize anagen)], and catagen characterized by the cessation of hair growth and regression of the hair follicle to the quiescent state.

sists of three layers: the inner basement membrane/basal lamina and an intermediate layer of collagen. The outer component of the connective tissue sheath is cellular, composed of fibroblasts and macrophages, and in contact with the papillary area adjacent to the dermal papilla (Lavker et al., 1999; Parakkal and Alexander, 1972). Once the growth of hair is completed, mitotic activity in the bulb matrix is terminated. The hair follicle tends to “deconstruct” itself in the sense that melanocytes stop synthesizing melanosomes and die by apoptosis, a complicated process by which a cell destroys itself (Lavker et al., 1999). The growth of the hair shaft stops and its base is left unpigmented. However, cells that have started to differentiate continue to do so and migrate upward to form the last part of the hair shaft. The hair shaft forms a brush-like ending taking on the appearance of a club fashioned by modified cortical cells that ascend with it. Once the hair club has been completed and surrounded by transformed ORS keratinocytes (germ cells) that anchor the club hair in the permanent follicle, the lower hair follicle is essentially reabsorbed. The death of matrix keratinocytes is thought to be brought about by terminal differentiation. Other processes such as heterophagy have been suggested as factors in the demolition of the lower hair follicle (Lindner et al., 1997). The dermal papilla moves upward stopping just below the hair germ of the now quiescent hair follicle. During the resting stage of the hair growth cycle, the configuration of the hair germ and dermal papilla is reminiscent of their association at the start of hair follicle formation in the embryo. They are still separated by a basement mem-

GENERAL BIOLOGY OF MAMMALIAN PIGMENTATION

brane (Chase et al., 1951; Hardy, 1992; Lavker et al., 1999; Parakkal and Alexander, 1972). The recurrent hair growth cycle in mammals consists of three major stages: (1) anagen, during which the hair shaft is formed by the hair follicle; (2) catagen, the transitional stage between anagen and telogen, during which the hair follicle regresses to the telogen configuration; and (3) telogen, (“resting stage”), beginning after the growth of the hair has been completed and a basal club is formed that anchors it in the upper hair follicle (Fig. 3.7) (Chase et al., 1951; Montagna and Parakkal, 1974; Parakkal and Alexander, 1972). All hair growth cycles that follow the first one also depend on signaling between the dermal papilla and hair germ. There is some debate as to the source of matrix keratinocytes for each generation. Challenging the concept of the traditional self-perpetuating hair germ as the sole source of matrix keratinocytes is evidence that the matrix keratinocytes originate in the bulge of the ORS, which may also be the source of the cells that form the hair germ for each generation (Chase et al., 1951; Cotsarelis et al., 1990; Parakkal and Alexander, 1972). Like the mouse, the human upper hair follicle is thought to contain a population of stem keratinocytes, which implies that the mouse model for hair growth applies to humans as well (Lavker et al., 1999; Moll, 1995; Yang et al., 1993). A variety of evidence suggests that the melanocytes that pigment hair in one growth cycle are not involved in the pigmentation of the next. First, in light (Blt/-) mice and silver (si/si) mice, melanocytes start to die approximately midway in anagen, resulting in their being uprooted and incorporated into the developing hair shaft (see Figs 3.3 and 3.4) (Quevedo and Chase, 1958; Quevedo et al., 1994). As a consequence, all melanocytes are physically removed from the hair bulb and disposed of in the hair shaft. The hair continues to grow in the absence of melanocytes for approximately a week, so that it is pigmented at its tip and pigment free in much of its base. Nonetheless, the hair bulbs of the next generation have what appears to be a full complement of melanocytes that ultimately meet the same fate as their predecessors. As noted above, during catagen in wild-type mice, both melanocytes and keratinocytes of the temporary region of the hair follicle die, suggesting that melanocytes for the next hair generation come from another source. The presence of amelanotic melanocytes within hair germs of resting follicles was inferred when melanogenic (DOPA-positive) dendritic melanocytes were found following treatment of pigmented mice with multiple small doses of X-rays that stimulate melanogenesis (Quevedo and Isherwood, 1958). Electron microscopy subsequently revealed amelanotic melanocytes containing immature melanosomes (Sugiyama et al., 1979). In addition to their presence in the hair germ, cells in the bulge of the ORS have not only been determined to be stem keratinocytes, but others have been identified as melanocytes by melanocyte-specific antibodies (Akiyama et al., 1995). Therefore, although stem melanocytes and keratinocytes in the traditional hair germ and/or bulge might provide a new complement of matrix keratinocytes and melanocytes for each hair growth cycle, it

seems most likely that the ORS is the sole source for both, particularly as the germ cells for each hair follicle generation are apparently derived from the ORS (Chase et al., 1951; Cotsarelis et al., 1990; Parakkal and Alexander, 1972). Recently, Sharov et al. (2004) published a brief account of their work suggesting that, in TRP2-LaxZ transgenic mice, melanocytes incorporated in the follicle germs of “resting” (telogen) hair follicles originate from more than one source. Not only are they derived from amelanotic stem cells of the ORS bulge but also from other ORS amelanotic melanocytes that escape the widespread cell death that occurs within hair follicles during catagen. The latter ORS melanocytes are thought to do so by retreating upward in the regressing, surviving portion of the hair follicle (Sharov et al., 2004). The technology employed in this study is more sophisticated than previous ones dealing with the same topic. The details of the second generation of hair growth, with some exceptions, are comparable to those of the first generation. Two examples of the exceptions are, first, that the hair germ and dermal papilla are in position to initiate the second hair generation as a legacy of the first hair generation. Second, the hair of the second generation exits the skin via the slightly altered transepidermal channel established for that of the first generation. The first hair shaft may be shed or remain anchored in the follicle so that two or more shafts emerge from one follicular orifice (Chase and Silver, 1969). Embryogenesis of the hair follicle becomes even more complex when viewed populationally. Hair follicles are not distributed randomly in the skin for they are arranged in linear bands in the adult mouse. However, in neonatal mice, they form discrete groups consisting of hair follicles that produce overhairs (monotrichs, awls, and auchenes) and underhairs (zigzags) (Claxton, 1966). With regard to the four types of hairs that characterize the hair coat, monotrichs, awls, auchenes, and zigzags are generated by hair follicles that are initiated in that order in groups (Claxton, 1966). Monotrichs come from the oldest follicles and zigzags from the youngest, with awls and auchenes in between (Claxton, 1966). The mechanism that regulates the production of the various hair types by follicles is largely unknown. It is known that several mutant genes interfere with the embryonic development of a hair follicle despite its initiation by cell signaling, while several other genes regulate the duration of the hair growth cycle in young and adult mice (Hebert et al., 1994; Lindner et al., 2000). Evidence that the embryology of hair growth is under tight genetic regulation is evident in the crinkled mouse. A recessive mutant allele at the crinkled (cr) locus produces a radical change in the hair coat of mice. Guard hairs, auchenes, and zigzags are missing from the thin hair coat of crinkled mice. The only hairs present are modified awls. Their hairs display many wrinkles absent in normal awls. Their hairs are thinner and the pigment deposits in their hair are more irregular than in normal awls. The agouti pattern of crinkled mice is altered. The dorsum tends to be darker and the flanks yellower than in normal wild-type mice, apparently the result of alterations 81

CHAPTER 3

in the amount and distribution of black and yellow hair pigment as well as the disorderly appearance of the crinkled hair coat in general (Gruneberg, 1952). Straile and coworkers (Straile, 1969; Straile and Mann, 1973) have provided a model for the structural and functional organization of mammalian skin. He proposes that their skin is divided into numerous subunits, each containing nerves, blood vessels, hair follicles, and glands organized into a complex coordinated vertical system. A major component of each unit is a tylotrich hair follicle. Tylotrichs are atypical guard hairs/monotrichs. Tylotrich hair follicles are richly innervated, produce coarse hairs, and function as mechanoreceptors. They are thought to have other more complex neurophysiologic functions as well. Straile has noted that the richly innervated tylotrich follicles are typically surrounded by pigmented networks of melanocytes, suggesting that nerve fibers servicing the hair follicle may stimulate melanocyte melanogenesis directly or lower the threshold for melanocyte melanogenesis in response to stimulation by endogenous or exogenous regulatory agents (e.g. hormones and growth factors). In contrast, dermal melanocyte networks associated with small, poorly innervated hair follicles are thought to be amelanotic because of the lack of neurological stimulation that also sets a higher threshold for their proliferating and “melaninizing” in response to intrinsic nonneural stimulation by the agents already mentioned. Only powerful exogenous factors, such as high doses of sex hormones or tumor promoters, can unleash these activities. For example, perifollicular melanocyte networks are more numerous in the trunk skin of female compared with male hamsters, indicating some response to estrogens (Ghadially and Illman, 1964; Illman and Ghadially, 1966). Consistent with Straile’s “threshold theory” is the fact that administration of estrogen to male hamsters increases the number of perifollicular melanocytes considerably beyond that of normal females of comparable age (Ghadially and Illman, 1964; Illman and Ghadially, 1966). Additional support for Straile’s threshold model for melanocyte activation and responsiveness to nonneural regulatory factors comes from the demonstration that the interruption of the adrenergic nerve supply to the irides of rabbits results in reduced tyrosinase activity within stromal melanocytes of the iris (Laties and Lerner, 1975). Within tylotrich units, the perifollicular melanocytes may influence other cellular components by secreting a variety of cytokines and matrix molecules (Chapter 21).

The Developmental Genetics and Regulation of Melanoblast Migration General Embryology: the Neural Crest Origin of Melanoblasts and their Migration to Populate the Skin of the Developing Wild-Type Mouse In mice, embryonic development is timed from copulation (coitus), indicated by the presence of a vaginal plug usually found in the vulva of females. It results from the coagulation of the male ejaculate. The timing of embryonic events below is expressed as the number of days post coitus (dpc). 82

During development, neural crest cells arise on the dorsal ridges of the neural folds that fuse to form the neural tube, precursor of the brain and spinal cord, etc. The neural crest cells give rise to many different types of cells, the melanocytes being one of them (Chapter 5). The precursors of the epidermal melanocytes are known as melanoblasts. They start to migrate dorsolaterally from the trunk region of the neural crest at 8.5 days, by which time the bilateral neural ridges of the embryo have already fused to form the neural tube (Serbedzija et al., 1990). The melanoblasts migrate in contact with the extracellular matrices surrounding the neural tube and “somites” and enter the mesenchymal layer (presumptive dermis) situated below the ectoderm (presumptive epidermis). Within the dermal mesenchyme, they migrate to all regions of the developing skin including that of the appendages (Pavan and Tilghman, 1994). By 10.5 days, neural crest cells are no longer seen leaving the neural crest (Serbedzija et al., 1990). By approximately 12 days, melanoblasts populate essentially all regions of the developing skin, including that of the limb buds (Mayer, 1973). With migration almost completed, the melanoblasts begin to populate the epidermis on day 12, where they proliferate and differentiate into melanocytes (Mayer, 1973). Apparently, many of the melanocytes migrate into the developing hair follicles. Those that do not remain in the basal layer of the epidermis. While the epidermal melanocytes establish intimate associations with keratinocytes of the interfollicular epidermis, the melanocytes of hair follicles do the same with respect to keratinocytes destined to form the hair produced by the follicle. By 16 days, melanocytes with dopa-positive premelanosomes are present within hair follicles (Hirobe, 1995). Throughout the migration of melanoblasts from the neural crest, there is no guarantee that melanoblasts will survive to complete their journey. Even melanocytes that enter the epidermis of the skin of mouse embryos are not immune to death. In the hairy skin of the trunk of mice, most of the interfollicular epidermal melanocytes that did not enter the hair follicles essentially disappear from the epidermis of the trunk skin by the 14th day post partum (Quevedo et al., 1966; Weiss and Zelickson, 1975). Electron microscopy indicates that the interfollicular epidermal melanocytes degenerate and die, questionably by apoptosis, a programmed event resulting in cell death (Quevedo et al., 1976; Weiss and Zelickson, 1975). However, the hair follicle melanocytes persist in the same areas where the epidermal melanocytes perish. In contrast to the trunk skin, the hair cover of the external ear of mice is sparse. The dark color of the outer ear results from melanin produced by epidermal and dermal melanocytes that persist throughout life.

Regulation of the Development of the Pigmentary System Embryogenesis is regulated by interactions at the subcellular, cellular, and tissue levels of organization. Growth factors,

GENERAL BIOLOGY OF MAMMALIAN PIGMENTATION

signal transduction mechanisms, and transcription factors play essential roles orchestrating development. Substantial numbers of growth factors secreted by paracrine cells stimulate target cells to proliferate and often to differentiate. Signal transduction mechanisms acting within the target cell transform the stimulus provided by a growth factor into a new one capable of provoking an appropriate response by the target cell. Transcription factors interact with short sequences of chromosomal DNA situated in the enhancer and promoter regions of a gene in order to solicit its transcription into RNA and, indirectly, its translation into protein. Regulation of the distribution of melanoblasts from the neural crest of the mouse to the appropriate sites required to establish the pigmentary system begins with release of melanoblasts along with other neural crest cells from bondage in the neural crest. Initially, the neural crest cells are bound together by tight junctions that prevent their dispersal. In order to do so, these bonds have to be broken. This is brought about by the expression of BMP4 and BMP7 (bone morphogenetic protein) genes initiating a chain of events that includes the stimulation of the production of Slug gene and RhoB gene proteins. One of them activates factors capable of breaking the bonds of tight junctions (Slug), and the other (RhoB) brings about the enhancement of neural crest cell motility (RhoB). The removal of cell adhesion molecules from the surfaces of neural crest cells completes their preparation for invading regional environments on their way to those where they finally lodge. Depending on their gene expression, these environments may play an essential role in determining the fate of the melanoblasts (Gilbert, 2000; Markert and Silvers, 1956). The hair coat of the wild-type house mouse is pigmented throughout, indicating that the melanoblasts that migrate from the neural crest encounter hospitable environments during their journey. These supportive environments encountered by wild-type melanoblasts include regions of extracellular matrix lacking migration inhibitors but containing proteins such as proteoglycans capable of supporting melanoblast migration toward the presumptive skin. They encounter paracrine factors such as endothelins, notably endothelin-3, that direct the melanoblasts toward the skin and instruct them to differentiate as melanocytes there (Baynash et al., 1994; Chapter 13). On entering the skin, the melanoblasts encounter paracrine Wnt protein, which is secreted by mesenchymal cells and serves to reinforce the commitment of melanoblasts to become melanocytes (Dorsky et al., 1998). The substitution of some mutant alleles for their wild-type allele can drastically alter the generation of pigment in skin and hair follicles by blocking melanin synthesis. Substitution of other mutant alleles for their wild-type counterparts may produce less dramatic absence of melanin. Still other mutant alleles alter the color of melanosomes or their distribution in the hair. Collectively, the mutant alleles reveal the role that individual wildtype genes play in the formation of the pigmentary system and the complexity of their interaction. For example, homozygous Steel (Sl/Sl) mutants are charac-

terized by severe macrocytic anemia, rarely survive to birth, and are completely deficient in germ cells as well as neural crest-derived melanocytes. The early death of Sl/Sl mice makes study of their deficiencies difficult. However, the Dickie mutation (Sld) of the Steel locus, in combination with the Sl mutant allele, yields a Sl/SId mouse that is sterile, anemic, and reduced in life expectancy (may survive up to a year) and, except for its black eyes, unpigmented. Melanocytes are missing from their skin and hair follicles (Silvers, 1979). Such mice are capable of living long enough for study of the consequences of stem cell deficiencies resulting from mutations at the Steel locus. Fujita et al. (1989) have demonstrated that the mutant Sl/Sld fibroblasts fail to produce and secrete a growth factor, Steel factor, better known as stem cell factor (SCF), essential for the proliferation and differentiation of stem cells, which give rise to reproductive cells, blood cells, and melanocytes. It is also important in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture (Hirobe et al., 2004). In wild-type mice, SCF is active and binds to a transcell membrane melanoblast receptor, c-kit tyrosine receptor kinase (c-kitTRK), a product of the Dominant spotting W locus. The binding of SCF protein (ligand) to its receptor c-kitTRK activates the receptor, resulting in the onset of a sequence of reactions that activates the Mitf transcription factor encoded by the microphthalmia gene. As a result, Mitf transcription factor combines with a protein coactivator. The combination of activated Mitf and coactivator is capable of activating the transcriptions of genes central to the production of melanin (Price et al., 1998). In contrast to wild-type SCF of fibroblast origin that exerts an environmental influence on wild-type melanoblasts, the Dominant spotting W locus is expressed within the melanoblast (Silvers, 1979). Like the Steel mutant, Dominant spotting (W/Wv) mice have black eyes and are otherwise unpigmented, sterile, and anemic. The defective proliferation, differentiation, and survival of the W/Wv melanoblasts results from their failure to respond to appropriate cues from their tissue environment, namely by binding and responding to potent SCF. The fault resides in their defective c-kit tyrosine receptor kinase that fails to respond to SCF. As a result, c-kitTRK does not autophosphorylate and activate functions essential for normal development (Nocka et al., 1989). Injection of anti-c-kitTRK antibody (ACK2) into pregnant black (C57BL/6) mice reveals a c-kit-dependent stage of melanoblast development on day 14.5 of gestation, just before c-kitTRK-dependent melanoblasts enter the epidermis from the dermis (Nishikawa et al., 1991). In addition, treatment of adult black mice with ACK2 indicates a requirement for ckitTRK action on follicular melanocytes at the onset of each hair growth cycle. It seems likely that c-kitTRK activity is required to promote melanoblast proliferation within the embryonic dermis and melanocyte proliferation at the onset of each hair growth cycle (Nishikawa et al., 1991). Okura et al. (1995) found that c-kitTRK dependency was not restricted to a brief period in the dermis prior to melanoblast invasion of 83

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the epidermis, but persisted during their differentiation into melanocytes and migration into the hair follicles. As noted above, the absence of neural crest-derived melanocytes in the skin and hair of Steel mutant (Sl/Sld) mice and Dominant spotting (W/Wv) mice results from the failure of fibroblasts of Steel mutant mice to deliver SCF. Alternatively, as in the case of mutant Dominant spotting mice, the fibroblasts’ SCF signal is available to melanoblasts, but they lack the competent c-kit tyrosine receptor kinase required to register and react to it. The impact of mutations at the Steel and Dominant spotting loci on the development of the neural crest-derived melanin pigmentary system has been examined. The goal was not only to determine what went wrong but also to obtain hints as to the role that each wild-type gene plays in establishing the wild-type pigmentary phenotype. A major problem that investigators have encountered when tracing the migration of melanoblasts from the neural crest is the close resemblance between melanoblasts and the population of mesenchymal cells through which they migrate on their way to the epidermis and hair follicles of the skin. A particularly useful method for making this distinction is the construction of probes that bind to specific molecules, often proteins that are unique to melanoblasts and melanocytes. One such protein is tyrosinase-related protein #2 (TYRP-2), an enzyme that plays an important role in the synthesis of melanin (Pavan and Tilghman, 1994; Steel et al., 1992). It is produced relatively early in the migration of melanoblasts even though melanogenesis is a late event that characterizes the transformation of melanoblasts into melanocytes. TYRP2 protein is associated with the limiting membrane of melanosomes, the cell organelles within which melanogenesis takes place. Please remember that, in the research described below, wherever melanoblasts and melanocytes are mentioned, they were judged to be so based on a positive response to a TYRP-2 protein probe, tyrosinase mRNA probe, TYRP1 mRNA probe, or TYRP-2 mRNA probe. With regard to the time of first detection of neural crest melanoblasts, TYRP-2 was first with day 10 dpc, followed by TYRP-1 (day 14.5 dpc), tyrosinase (day 14.5 dpc), and pigment deposition (day 16.5 dpc) (Pavan and Tilghman, 1994; Steel et al., 1992). Steel et al. (1992) emphasized the TYRP-2 mRNA probe in their study because of its early detection of neural crestderived melanoblasts and its ability to distinguish between migrating melanoblasts and mesenchymal cells in their study of Steel mice. They found that SCF characteristic of wild type was absent in Sl/Sld mutant mice, a deficit that led to premature melanoblast/melanocyte death or dedifferentiation beginning around day 11 dpc and continuing thereafter, resulting in a uniformly white coat of hair. Predictably, in mice homozygous for the wild-type allele, the hair coat was pigmented throughout. The study also revealed that, although SCF signaling is essential for the survival of melanoblasts, it does not play a role in their early migration or initial differentiation. Steel et al. (1992) (see also their communication to Pavan and Tilghman published in their 1994 paper) explored melanoblast fates in mutant mice of the Dominant spotting W 84

locus. The genotypes examined were Wv/Wv, Wsh/Wsh, and W41/W41. All were characterized by white hair coats due to melanoblast/melanocyte death by day 12 dpc, a consequence of their lack of a c-kit tyrosinase receptor kinase responsive to SCF signaling. The study also revealed that, although c-kit tyrosinase receptor kinase is essential for the survival of melanoblasts, it does not play a role in their early migration or initial differentiation. Pavan and Tilghman (1994) built on the foundation laid by Steel et al. (1992) when they used an antibody specific for the TYRP-2 protein as a probe to identify the function of the piebald s locus by examining the influence of the piebald lethal mutant allele (sl) on the fate of neural crest-derived melanoblasts. In wild-type mice by day 12 dpc, numerous melanoblasts/melanocytes are localized in the head region back to the forelimbs followed by numerous melanoblasts from the hindlimbs back to the tail. The region between the limbs, the trunk of the body, has significantly fewer melanoblasts than the regions fore and aft until days 13–17 dpc when a wave of mitotic activity sweeps across the trunk increasing melanoblast numbers to levels comparable to those elsewhere along the anterior–posterior axis. This is not the case in piebald lethal sl/sl mice. Typically, variable patches of pigmented hair are found on the head, rump, or base of tail, with the space between them populated by white hair. From day 10.5 dpc, when neural crest-derived melanoblasts are seen, their numbers are extremely low compared with wild type. In piebald lethal mice, melanoblasts were only found in regions of the skin where patches of pigmented hair were frequently observed. As the numbers of melanoblasts found in the skin of piebald lethal mice was low from their first appearance on day 10 dpc, Pavan and Tilghman (1994) concluded that the s locus wild-type gene must express early in melanocyte development either in the neural crest or during melanoblast migration. They suggest that the expression of the piebald locus is upstream from the Steel and Dominant spotting loci. In an effort to integrate the interaction of Steel, Dominant spotting, and Microphthalmia signals, a provisional pathway for the developmental genetic events that culminate in the onset of melanogenesis within differentiating melanoblasts has been assembled: fibroblasts homozygous for the wild-type Steel gene that encodes a protein, SCF, secrete it into the dermis during embryogenesis. Its target is the melanoblasts arriving from the neural crest. SCF becomes bound to the melanoblast cell surface c-kit receptor tyrosine kinase (ckitRTK) encoded by the wild-type allele of the Dominant spotting W locus. The binding of SCF to the c-kitRTK dimerizes the receptor, leading to its phosphorylation. This event is critical in the activation of a pathway resulting in the phosphorylation of the wild-type microphthalmia locus transcription factor (Mitf). The phosphorylated form of Mitf is capable of initiating a reaction that results in the onset of transcription and translation of the wild-type alleles for the enzymes essential in the synthesis of melanin: tyrosinase, DHICA oxidase/TYRP-1, and DOPAchrome tautomerase/TYRP-2, and other gene products relevant to melanogenesis.

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It is reasonable to conclude that a major function of the wild-type alleles at many of the mutant “white-spotting” gene loci, including the Dominant spotting locus, is to orchestrate melanoblast migration, proliferation, and differentiation via influences on the availability of growth factors and by determining the response of melanoblasts to them. The physiological genetics of the Steel locus and Dominant spotting W locus stand as a classic model for such interactions.

The Regulation of the Fully Differentiated Melanocyte in Producing the Wild-type Phenotype The production of hairs with the appropriate pigmentation to produce the agouti pattern requires close timing of the growth of the hair and the function of melanocytes that pigment it. Although some of the regulatory mechanisms involved have been identified, much remains uncertain. In wild-type agouti mice, the melanocytes of hair follicles programmed to produce banded hairs shift their synthesis of pigment from eumelanin to pheomelanin and back to eumelanin during hair growth. As a result, melanin is deposited in viable keratinocytes destined to keratinize and die while forming the hair shaft. In doing so, they also take part in coloring the hair. The regulation of melanocyte function in this process is as follows. The signal for the production of pheomelanin is provided by agouti protein encoded by the wild-type allele of the agouti a locus activated within dermal cells that form the dermal papilla of hair follicles (Jackson, 1994; Chapter 19). Within the hair bulb, melanocytes are arranged so that they are adjacent to the dermal papilla on one side and to keratinocytes on the other. Alpha-melanocyte stimulating hormone (MSH), also known as melanocortin I, binds to a specialized cell surface receptor on the melanocytes, the MSH receptor encoded by the wild-type allele of the extension locus (e) (Jackson, 1994). The receptor-bound MSH stimulates events that result in the activation of adenyl cyclase, which elevates the levels of cyclic adenosine monophosphate (AMP), leading to the synthesis of eumelanin. The eumelanin-containing melanosomes are transferred to the population of keratinocytes destined to form the hair tip (Boissy, 2003; Marks and Seabra, 2001). Subsequently, agouti protein emanating from the dermal papilla cells gains access to the MSH receptor on the melanocytes and blocks the binding of MSH to them. Eumelanin synthesis is halted and pheomelanin synthesis is commenced within melanocytes. The pheomelanosomes are transferred to the population of keratinocytes scheduled to form the agouti band. Finally, MSH replaces agouti protein on the receptors, and eumelanin is synthesized and distributed to keratinocytes scheduled to form the remainder of the hair shaft. Hair growth stops and developmental changes occur so that the hair is elevated and anchored in the now quiescent follicle. While this description accurately relates the expression of the wild-type agouti gene, some agouti alleles regulate the dorsum and

ventrum of the mouse independently, yielding different color patterns than wild type (Millar et al., 1995; Vrieling et al., 1994). Within the follicular melanocytes during the periods of eumelanin synthesis in agouti mice, numerous genes are activated to support the process. The synthesis of eumelanin involves a pathway whereby tyrosine is converted via intermediates into the complex polymer eumelanin. Not surprisingly, the transcription of genes that encode enzymes that catalyze the melanogenic pathway is activated: the wild-type allele at the albino locus (c) encoding tyrosinase, the wild-type allele at the brown locus (b) encoding the protein TYRP-1 (DHICA oxidase), and the wild-type Slaty locus (Slt) allele encoding TYRP-2 (DOPAchrome tautomerase) (Chapter 10). In addition, the wild-type allele at the pink-eyed dilution locus (p), a gene that encodes a protein that is not an enzyme, is also activated. It is believed that all the foregoing proteins are aggregated to form a “melanogenic complex” that is bound to the limiting membrane of the melanosome. The “melanogenic complex” is postulated to generate the eumelanin that is deposited on and between the protein filaments of a well-organized matrix that occupies the interior of the eumelanosome. Possible functions of the wild-type p protein might be that it functions as a structural element of the melanosome, a coordinating molecule of the “melanogenic complex,” a transporter of melanogenic proteins, or is involved in the transport of sulfhydryl compounds out of the melanosome to permit the formation of eumelanin (Lamoreux et al., 1995). During the pheomelanic phase of melanogenesis in the agouti mouse brought on by binding of agouti protein to the MSH receptors on follicular melanocytes, the biochemistry of melanin formation takes a new direction. Pheomelanin synthesis begins with the oxidation of tyrosine catalyzed by tyrosinase, but then diverges to interact with sulfur-containing compounds such as glutathione or cysteine to form the sulfurous compound of cysteinylDOPA. CysteinylDOPA is modified and then polymerized to form pheomelanin (Chapter 10). These events take place within pheomelanosomes that lack the more complex internal organization found in eumelanosomes. Pheomelanosomes are spherical in shape, whereas eumelanosomes are ellipsoidal. The “melanogenic complex” of eumelanosomes is absent in pheomelanosomes with only tyrosinase activity in evidence in the four members of the “melanogenic complex.” Microvesicles dominate the interior of the pheomelanosome, and protein filaments, where present, are scattered rather than arrayed to form an organized matrix. Pheomelanin is deposited on the surfaces of the microvesicles and filaments and fills the space between them. Tyrosinase may be present within the microvesicles where it converts tyrosine to dopaquinone, which diffuses out of the vesicles and interacts with cysteine or glutathione to form cysteinylDOPA that, in modified form, is polymerized into pheomelanin. The pheomelanosomes are discharged into the keratinocytes scheduled to form the agouti band (Chapter 19; Jimbow et al., 1999). 85

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Examples of Influences of Hair Follicle Growth Regulators on the Hair Growth Cycle with Possible Implications for Follicular Melanocytes Fibroblast growth factors (FGFs) form a family, the members of which have been described as possibly the most important “endogenous mediators of hair follicle growth and development” (Danilenko et al., 1995). The family includes acidic FGF (aFGF), basic FGF (bFGF), and FGF-5, the receptors for which are found within hair follicles or very close to them. Another member of the family (KGF or FGF-7) and its receptor, although localized to the skin, influences hair growth (Danilenko et al., 1995). Halaban in Chapter 22 and Hirobe (1992) have also stressed the importance of the members of the FGF family for their role as supporters of melanocyte proliferation and viability in vitro. Halaban notes that, although bFGF is the most potent supporter of melanocyte proliferation/survival in vitro, other members can substitute for it. The question that remains to be answered is whether the commitment of FGFs to regulating hair growth permits their simultaneously supporting follicular melanocytes of the hair bulb that pigment the developing hair shaft while performing largely unknown functions in the ORS. One case study says no, not because the growth factor is necessarily impotent but because, in meeting the hair growth cycle’s requirement, it is expressed too late, that is, just as hair growth is about to stop. Fibroblast growth factor-5 (FGF-5) expresses and is sharply localized in the external root sheaths of hair follicles late in anagen (Hebert et al., 1994). Evidence suggests that FGF-5 is an essential component of the signaling system that elicits the end of anagen and onset of catagen and terminates each hair growth cycle. Supporting this role for FGF-5 is the demonstration that genetically engineered mice homozygous for a null (nonfunctional) allele of FGF-5 (FGF5neo) produced by gene targeting in embryonic stem cells have longer hairs than unaltered mice. In addition, Angora (go) mice are indistinguishable from FGF5neo mice in their coat of elongated hair, compared with that of wild-type mice. The Angora gene is considered to be an allelic mutant of the FGF-5 gene (Hebert et al., 1994). The late expression of the FGF-5 gene and its restriction to the ORS argues against any influence on the hair bulb melanocytes, nor has there been a report of its influence on melanocytes of the ORS. Examination of the timing of gene expression and distribution patterns of other FGFs may reveal one or more that meets the needs of melanocytes and the growth of the hair. Another candidate for a growth factor influencing both hair growth and melanocytes is hepatocyte growth factor (HGF) and its receptor MET. The team is known to be an important mediator of mesenchymal–epithelial interactions and involved in the control of hair growth (Lindner et al., 2000). HGF is restricted to the dermal papilla of hair follicles, and its receptor MET is present nearby in keratinocytes of the hair bulb. 86

HGF has been shown to repress the onset of the catagen stage of the hair growth cycle, thus prolonging it (Jindo et al., 1994; Lindner et al., 2000). Consequently, HGF action on hair growth is just the opposite of that of FGF-5. While HGF acts to delay the onset of catagen, FGF-5 acts to promote its onset. In this case, the duration of hair growth might be a compromise between the antagonistic influences. HGF is known to act either alone (Hirobe et al., 2004) or synergistically with bFGF in supporting melanocyte proliferation in vitro (Chapter 22). The distribution pattern of HGF and its receptor in growing hair follicles, as well as its expression beginning early in anagen, are consistent with a possible role in supporting the proliferation and viability of follicular melanocytes during the hair growth cycle, particularly those of the hair bulb.

Conclusion The wild-type agouti coat color is but one pattern of the collective expression of gene loci involved in pigmenting the skin and hair. Several of the chapters that follow this one explore in greater depth the mutant alleles at these loci that, when present, result in departures from the wild-type color pattern. In doing so, they help to reveal the nature of the steps in the pigmentary process that a particular gene locus influences. Significant numbers of mutant “pigment genes” have been characterized, not only for their gross influences on coat color but also for their actions at the molecular level. Only a sample has been discussed in this chapter. The same is true for the hormones, growth factors, signal transduction mechanisms, and other factors that regulate melanin synthesis by melanocytes. More information on these and other topics can be found in the chapters listed below. Human Skin Color Chapter 20: Human Pigmentation: Its Regulation by Ultraviolet Light and by Endocrine, Paracrine, and Autocrine Factors Chapter 27: The Normal Color of Human Skin Melanin and Human Adaptations to Solar Radiation Chapter 17: Photobiology of Melanins The Embryogenesis of the Melanin Pigmentary System Chapter 5: Regulation of Melanoblast Migration and Differentiation Chapter 6: Melanoblast Development and Associated Disorders Chapter 7: Biogenesis of Melanosomes Genetics of the Pigmentary System Chapter 10: The Regulation of Melanin Formation Chapter 11: The Tyrosinase Gene Family Chapter 19: Regulation of Pigment Type Switching by Agouti, Melanocortin Signaling, Attractin, and Mahoganoid

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The Regulation of the Fully Differentiated Melanocyte Chapter 10: The Regulation of Melanin Formation Chapter 11: The Tyrosinase Gene Family Chapter 12: Molecular Regulation of Melanin Formation: Melanosome Transporter Proteins Chapter 14: Enzymology of Melanin Formation Chapter 15: Chemistry of Melanins Chapter 20: Human Pigmentation: Its Regulation by Ultraviolet Light and by Endocrine, Paracrine, and Autocrine Factors Chapter 22: Growth Factor Receptors and Signal Transduction Regulating the Proliferation and Differentiation of Melanocytes

Perspectives This chapter has surveyed the products of Jacob’s (1982) evolutionary tinkering that transformed the pigmentary system of reptiles into that of mammals. The great diversity of coat color patterns found in mammals represents developmentally subtle manipulations of a pigmentary system that appears to be highly conserved at its core. The basic ingredients for hair and skin color (melanocytes, melanosomes, melanogenic enzymes, and melanin) seem to be pretty much the same throughout the class of mammals, as does the basic ability of melanocytes to transfer melanosomes to keratinocytes, an attribute inherited from the reptilian ancestors of mammals. What is different are the regulatory factors that program the timing of pigment pattern expression, the anatomical locations of hair follicles and the coarseness of the hairs that are produced, and the qualitative and quantitative functions of melanocytes. More is being learned daily about these regulatory systems. The importance of the melanin pigmentary system of mammals cannot be overemphasized, for it is at the interface between the organism and the world in which it must survive and reproduce. What mammals have inside them is insignificant unless it is in harmony with their integument. Mammals communicate their strengths and desires through their color patterns as well as concealing their vulnerability. In the animal kingdom, color patterns form a powerful silent language focused on aggression, attraction, repulsion, submission, and aloofness. Tragically, with all its capacities for doing good things, taken as a whole, the human species has still not been able to penetrate beneath the superficiality of skin color in assessing the true worth of its individual members.

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Silvers, W. K. The Coat Colors of Mice. A Model for Mammalian Gene Action and Interaction. New York: Springer-Verlag, 1979. Steel, K. P., D. R. Davidson, and I. J. Jackson. TRP-2/DT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor. Development 115: 111–119, 1992. Straile, W. E. Carcinogen-induced melanotic tumours of the tylotrich hair follicle. Nature 202:403–404, 1964. Straile, W. E. Vertical cutaneous organization. J. Theor. Biol. 24: 203–215, 1969. Straile, W. E., and S. J. Mann. Characteristics of hairs and related mechanoreceptors in the mouse and rabbit. J. Exp. Zool. 185:189– 208, 1973. Sugiyama, S., T. Uesugi, K. Jimbow, and A. Kukita. Activation and differentiation of melanogenesis in melanocytes of telogen and early anagen hair follicles. In: Pigment Cell, vol. 4, S. Klaus (ed.). Basel: S. Karger, 1979, pp. 185–190. Szabo, G. The regional anatomy of the human integument with special reference to the distribution of hair follicles, sweat glands and melanocytes. Philos. Trans. Roy. Soc. London 252:447–485, 1967. Szabo, G., A. B. Gerald, M. A. Pathak, and T. B. Fitzpatrick. The ultrastructure of racial color differences in man. In: Pigmentation: Its Genesis and Biologic Control, V. Riley (ed.). New York: Appleton Century Crofts, 1972, pp. 23–41. Thody, A. J., E. H. Higgins, K. Wakamatsu, S. A. Burchill, and J. M. Marks. Pheomelanin as well as eumelanin is present in human epidermis. J. Invest. Dermatol. 97:340–344, 1991. Thong, H.-Y., S.-H. Jee, C.-C. See, and R. E. Boissy. The pattern and mechanism of melanosome distribution in keratinocytes of human skin is one determining factor of skin colour. Br. J. Dermatol. 149:498–505, 2003. Vrieling, H., D. M. J. Duhl, S. E. Millar, K. A. Miller, and G. S. Barsh. Differences in dorsal and ventral pigmentation result from regional expression of the mouse agouti gene. Proc. Natl. Acad. Sci. USA 91:5667–5671, 1994. Waage, J. K. How the zebra got its stripes — Biting flies as selective agents in the evolution of zebra coloration. J. Entomol. Soc. S. Afr. 44:351–358, 1981. Weiss, L. W., and A. S. Zelickson. Embryology of the epidermis. III. Maturation and primary appearance of dendritic cells in the mouse with mammalian comparisons. Acta Dermatovener 55:431–442, 1975. Whimster, I. W. An experimental approach to the problem of spottiness. Br. J. Dermatol. 77:397, 1965. Whimster, I. W. Symmetry in dermatology. In: Modern Trends in Dermatology — 4, P. Borrie (ed.). London: Butterworths, 1971, pp. 1–31. Wickings, E. J., and A. F. Dixson. Testicular function, secondary sexual development and social status in male mandrills (Mandrillus sphinx). Physiol. Behav. 52:909–916, 1992. Wickler, W. Socio-sexual signals and their intra-specific imitation among primates. In: Primate Ethology, D. Morris (ed.). London: Weidenfeld and Nicolson, 1967, pp. 69–147. Witkop, C. J., Jr., W. C. Quevedo, Jr., T. B. Fitzpatrick, and R. A. King. Albinism. In: The Metabolic Basis of Inherited Disease, 6th edn, vol. II, C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle (eds). New York: McGraw-Hill, 1989, pp. 2905–2947. Yamamoto, O., and J. Bhawan. Three modes of melanosome transfers in Caucasian facial skin: hypothesis based on an ultrastructural study. Pigment Cell Res. 7:158–169, 1994. Yang, J.-S., R. M. Lavker, and T. T. Sun. Upper human hair follicle contains a subpopulation of keratinocytes with superior in vitro proliferative potential. J. Invest. Dermatol. 101:652–659, 1993. Young, A. R. The sunburn cell. Photodermatology 4:127–134, 1987. Yun, J. S., and W. Montagna. The melanocytes in the epidermis of the rhesus monkey (Macaca maculatta). Anat. Rec. 154:161–174, 1966. Zuckerman, S., and A. S. Parkes. Observations on secondary sexual characters in monkeys. J. Endocrinol. 1:430–439, 1939.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

4

Extracutaneous Melanocytes Raymond E. Boissy and Thomas J. Hornyak

Historical Background Our analysis and knowledge pertaining to the structure/ function correlates of pigment and melanocytes/chromatophores have primarily focused on the various populations of pigment-synthesizing cells in cutaneous tissues. These populations of cells have included the dermal melanophores of lower vertebrates, which exhibit rapid melanosome dispersion or constriction within the cell as an immediate adaptation to the environment. In addition, other chromatophores of lower vertebrates, in association with the melanocyte, interact in the dermis and at the dermal/epidermal junction to provide an array of cutaneous coloration for environmental and social interactions. Follicular melanocytes in the bulb region of the hair follicles of higher vertebrates and primates are responsible for making melanosomes that are transferred to the hair keratinocytes and thus give coloration to the hair shaft, which presumably functions to provide cues to and possibly protection from the environment. Interfollicular epidermal melanocytes are primarily, but not exclusively, found in most primates including humans. This population of melanocytes developed in conjunction with the dramatic decrease in the terminal type of hair follicles distributed throughout the body surface. These interfollicular melanocytes of the epidermis seem to have evolved for additional and/or different functions, including the absorption of ultraviolet and visible light and the scavenging of free radicals. Melanin, eventually transferred to keratinocytes, appears to protect the body in general, and the skin specifically, against damaging environmental assaults. Thus, looking globally at the evolutionary development in the various populations of cutaneous pigment cells, one can see that the functions of melanin change or increase as described above. However, all these various functional roles for cutaneous melanocytes reflect mechanisms responsible for the interaction between an individual and the environment. Less has been known about extracutaneous melanocytes found primarily in the eye, ear, and internal organs such as the leptomeninges. These populations of melanocytes have some morphologic and functional similarities to the various cutaneous melanocytes outlined above. However, there are some significant differences between extracutaneous and cutaneous melanocytes that have yet to be fully assessed. It appears that extracutaneous melanocytes must have unique functional roles for the organism distinct from those of cutaneous melanocytes by virtue of the fact that extracutaneous melanocytes are not positioned immediately at the organ-

ism/environmental interface. This precludes the function of extracutaneous melanocytes as primarily providing environmental protection and/or socially interactive cues. A review of the characteristics of extracutaneous melanocytes is presented in this chapter.

Development and Nature of Ocular Melanocytes Ocular melanocytes have traditionally been categorized into two groups of cell types: the uveal melanocytes and the retinal pigmented epithelial (RPE) cells. It should be mentioned that a controversy exists as to whether the RPE cells are truly melanocytes. It has been proposed that, as these cells express melanocyte-specific proteins for the specialized synthesis of pigment granules and melanin, they are by definition “melanocytes.” Conversely, it has been argued that these cells that form a continuous epithelial tissue in association with the retina proper, thus forming a border or lining between the body (i.e. mesenchyme tissue of the uvea) and a body cavity (i.e. vitreous chamber), are actually epithelial cells. This proposal presumes that the synthesis of melanin by these epithelial cells is an accessory function. The reality is probably somewhere in between, as the epithelial function of the RPE is important for eye development whereas its role in pigment synthesis is critical for proper retinal ganglion cell pathfinding. We will refer to these cells as RPE melanocytes. In any case, the uveal and RPE categories of ocular melanocytes can be distinguished from each other on both a developmental and a morphologic basis. For comprehensive reviews of ocular embryology and structure, we recommend Jakobiec (1982) and Fine and Yanoff (1979). Developmentally, melanocytes that eventually reside in the uvea, comprising the choroid, ciliary bodies, and the iris, are of neural crest origin similar to cutaneous melanocytes (see Chapter 5). Neural crest-derived melanoblasts migrate through the mesenchyme of the developing embryo and are subsequently targeted to this ocular site (Bartelmex and Blount, 1954; LeLievre and LeDouarin, 1975; Noden, 1991). In contrast, cells of the RPE are derived directly from cells in the neuroectoderm of the developing forebrain of the embryo (Mann, 1964). Specifically, after the diencephalon (forebrain) forms the optic cup that invaginates into a bilayered structure, the outermost layer adjacent to the mesenchyme of the developing uveal tract differentiates into the RPE. Therefore, no migratory phase occurs for these RPE melanocytes. Again, 91

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it has traditionally been stated that these two groups of melanocytes originate from different embryonic sources. This is misleading in that the precursors for both groups (the neural crest cells and the posterior surface of the optic neuroectoderm) actually originate from the same source, i.e. the neural tube. Neural crest cells emigrate from the dorsal surface of the neural tube shortly after the neural fold closes (Le Douarin, 1982). The neural ectoderm of the developing forebrain, which gives rise to the RPE, is the cephalic extension of the neural tube. Therefore, the developmental divergence that separates these two groups of ocular melanocytes is not their original embryonic origin per se, but rather their mode of reaching the target site. Melanoblasts that differentiate into uveal melanocytes undergo a migratory phase from neural tube to target site; in contrast, melanoblasts that differentiate into RPE melanocytes do not undergo a migratory stage but evaginate from a specific region of the neural tube. Analysis of murine coat color mutants has provided important insights into how neural crest-derived and RPE melanocytes are differentially regulated during development. The determinants of neural crest-derived melanocyte development are discussed elsewhere (Chapter 5) and should apply to the control of survival, migration, and initial differentiation of uveal melanocytes because of the evidence supporting their origin from the neural crest. Cell surface receptors such as Kit and Ednrb, their respective ligands KitL/SCF and ET-3, as well as the transcription factors Mitf, Pax3, Sox10, and Snai2 have all been shown to be important in governing the development of neural crest-derived melanocytes during embryogenesis. There are distinct differences between some of these determinants and determinants of RPE development. One example is the difference in Mitf isoform expression between neural crestderived melanocytes and RPE cells. Although Mitf is critical to the development of both types of cells, neural crest-derived melanocytes express the Mitf-M isoform exclusively, whereas RPE cells express the Mitf-A, Mitf-H, and Mitf-D isoforms, all expressed from different promoters and utilizing different first exons (Takeda et al., 2002; Udono et al., 2000). As these isoforms are regulated differently, it follows that the transcriptional determinants for Mitf expression in RPE melanocytes are different than those for neural crest-derived melanocytes. In fact, neither Pax3 or Sox10, which regulate the expression of the neural crest-derived melanocyte isoform Mitf-M (Bondurand et al., 2000; Potterf et al., 2000), are expressed in RPE melanocytes. It appears that the major regulators of RPE melanocytes are the factors Mitf, Otx2, and Pax6 linked in a transcriptional network (Martinez-Morales et al., 2004), perhaps regulated by the activity of fibroblast growth factors expressed by the overlying endoderm (Nguyen and Arnheiter, 2000). Additionally, mutations affecting the Kit or Ednrb systems inhibit successful migration of melanoblasts into the skin, the ear, and uveal tract without affecting survival or differentiation of RPE melanocytes (Cable et al., 1994; Deol, 1970a; Mackenzie et al., 1997; Markert and Silvers, 1956).

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It was originally demonstrated in classic studies by Noden (1975) that the uveal melanocytes of the eye originate from neural crest cells derived from the cephalic portion of the neural tube. Two basic possibilities may explain the distinct differentiated phenotype of uveal melanocytes from cutaneous melanocytes. One possibility is that uveal melanocytes, although neural crest derived, are specified different from cutaneous neural crest-derived melanocytes and adopt their differentiated phenotype regardless of local environmental cues. The other possibility is that their specified precursor melanoblast is indistinguishable from those contributing to cutaneous melanocytes and that end-organ environmental signals are critical for conferring the differentiated phenotype. Some evidence exists to support each of these models. For example, uveal-like melanocytes have been demonstrated to emigrate from all areas of the neural tube (Boissy et al., 1990). A unique mutant allele at the albino (c) locus in Recessive white chickens affects the function of tyrosinase in cutaneous and not in uveal melanocytes (Brumbaugh et al., 1983; Oetting et al., 1985). Neural tubes extracted from 3-day-old embryos of this mutant line, trisected into cephalic, medial, and caudal segments, and used to develop separated cultures of melanocytes demonstrated that equal numbers of amelanotic and pigmented melanocytes could be derived from all areas of the neural tube. Therefore, the commitment to differentiate into cutaneous vs. extracutaneous melanocytes may be determined at some preneural crest cell stage. It has also been demonstrated that the normal embryonic development of the RPE and choroidal melanocytes is essential for the developmental establishment of the neural retina and its projection and connection to the visual cortex of the brain. Classic studies in all forms of albinism demonstrate that the associated innate hypopigmentation of the uveal tract and the RPE correlates with a significant decrease in visual acuity associated with strabismus and nystagmus of the eyes (Creel et al., 1990; Fulton et al., 1978; King et al., 1994; Kinnear et al., 1985; Summers et al., 1988). These ocular/vision aberrancies are the consequence of a misrouting of the optic fibers from the neural retina. There is an increase in neurons crossing at the optic chiasma to the contralateral geniculate, resulting in the scrambling of the neuronal input to the visual cortex of the brain (Creel et al., 1978; Guillery, 1974). Most intriguing is the fact that this misrouting of the visual system occurs in patients with OCA1, OCA2, or OCA3. These three forms of albinism result from nucleotide lesions in the gene encoding the tyrosinase enzyme (Chintamaneni et al., 1991; Giebel et al., 1990; King et al., 1991; Tomita et al., 1989), the P protein (Gardner et al., 1992; Ramsay et al., 1992; Rinchik et al., 1993), and tyrosinase related protein-1 (TRP-1) (Boissy et al., 1996) respectively. These three melanocyte-specific proteins regulate pigment synthesis and do not appear to directly affect any developmental process. Therefore, the aberrant visual system occurring in albinism demonstrates that the development of ocular melanin during embryogenesis is critical for, and probably directs, the normal establishment of the

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optic neuronal network (Colello and Jeffery, 1991; Silver and Sapiro, 1981; Strongin and Guillery, 1981) as well as photoreceptor cells directly, specifically rods (Grant et al., 2001). In mice, the number of rod photoreceptor cells that are produced decreases correlatively with extent of pelage hypopigmentation (Donatien and Jeffery, 2002). The mechanism underlying the regulation of the development of both the retina and the visual pathway by ocular melanins is still elusive (Ilia and Jeffery, 2000). Not only is the synthesis of melanin key in the development of the visual system of an organism, but the presence of RPE melanocytes is also responsible for the maintenance as well as the successful embryonic development and organization of the neural retina proper. Experimentally induced (Raymond and Jackson, 1995) or natural mutant (Boissy et al., 1993; Mullen and LaVail, 1976) perturbations in the development of the RPE during embryogenesis have correlated with the aberrant development of photoreceptor cells and other layers of the neural retina. Therefore, melanocytes and pigment development in the eye provide important cues for the development of the ocular and visual system. In addition, cells of the pigmented ciliary margin in mice have been shown to function as putative retinal stem cells (Tropepe et al., 2000), ascribing a potential postnatal role in retinal maintenance to a small subset of ocular pigment cells.

Morphology of Uveal (i.e. Choroidal) and RPE Melanocytes Once the uveal group and RPE group of melanocytes migrate to and/or differentiate in their respective anatomical sites, distinct morphologic criteria distinguish the two. These

differences are reflected in both the shape of the melanocytes (Fig. 4.1) and the morphology of the melanosomes synthesized. Pertaining to the cellular shape, the melanocytes of the uveal tract are dispersed throughout the connective tissue stroma of these areas in a territorial manner without any direct contact with each other (Feeney and Hogan, 1961). These uveal melanocytes are dendritic. They have a centrally placed nucleus in the cell body and peripherally radiating dendrites. Wholemounts of the choroidal component of the uveal tract have demonstrated that a majority of these melanocytes are bipolar; however, some uveal melanocytes develop numerous dendrites (Torczynski, 1982). The choroid is a layer of connective tissue per se of about 80 mm in thickness. The cellular component of the choroid is primarily composed of fibroblasts and melanocytes in a 2:3 ratio and numerous endothelial cells which line the cavernous blood sinus of this optic layer (Fig. 4.2). Occasionally, nerve bundles containing groups of myelinated and nonmyelinated neurons exist (Fig. 4.2) (Bergmanson, 1977; Castro-Correia, 1967). These cellular components are embedded in a fibrous stroma composed primarily of collagen and elastin fibers (Feeney and Hogan, 1961). The choroidal melanocyte is congested with melanosomes, predominantly at the terminal Stage IV state of maturation (Fig. 4.2). Relatively minimal amounts of other cellular organelles, consisting of endoplasmic reticulum, Golgi apparatus, mitochondria, etc., exist in the cytoplasm of the choroidal melanocyte. In contrast, the RPE melanocytes are highly polarized cells approximately 14 mm in height and width. They form a single layer of cuboidal cells at the uveal/retinal junction and are connected to each other on their juxtaposed lateral surfaces by both elaborate terminal bar complexes, consisting of both

S Sinus

*

C *

RPE

R

A

B

P

Fig. 4.1. The back of the eye (A) consists of four major layers: the innermost neural retina (R), the retinal pigmented epithelium (RPE), the choroid (C), and the outermost sclera (S), as shown in the light micrograph cross-section of an avian eye. Melanocytes are confined to the RPE and the choroid (B). The melanocytes of the choroid are heavily pigmented and multidendritic. They reside in the connective tissue component between the extensive sinus (sinus) and blood vessels (*) of the choroid. Melanocytes comprising the retinal epithelium have a relatively amelanotic cell body, with prominent nuclei (arrowheads) adjacent to the choroid, and numerous slender, pigmented dendrites (arrows) extending toward and interdigitating with the photoreceptor cells (P) (see also Plate 4.1A and B, pp. 494–495). Bar in A, 50 mm; bar in B, 15 mm.

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E

bv F

*

* N

M *

bv

RPE

zonula occludens and zonula adherens (Hudspeth and Yee, 1973), and desmosomes (Miki et al., 1975) (Fig. 4.3). Morphologically, these cells have a basal surface facing the uveal tract with numerous regular invaginations of the plasma membrane, called basal infoldings, extending 1 mm in length. Between these basal infoldings and the choroid of the uveal tract there exists: (1) an extremely thickened basement membrane (of approximately 2 mm) adjacent to the RPE melanocytes called Bruch’s membrane; and (2) an extensive anastomosing network of fenestrated blood vessels called the choriocapillaris adjacent to the stroma of the choroid (Fig. 4.3). Both these structures facilitate the transport of nutrients, waste, and presumably cytokines to and from the retina proper (Bill, 1975; Torczynski, 1982). Facing the photoreceptors of the retina, the RPE melanocyte apical surface has extensive slender cellular processes, the apical villi, that interdigitate between the photoreceptor cells (Fig. 4.3). These villi impart two basic functional roles unique to the RPE 94

Fig. 4.2. The choroid is the connective tissue spanning the sclera and the retinal pigmented epithelium (RPE). It contains numerous fibroblasts (F) and dendritic melanocytes (M) that have cytoplasm congested with Stage IV melanosomes. Also distributed throughout the choroid is extracellular ground substance (*), nerve bundles (N), collagen (arrows), elastin, and endothelial cells (E) lining the extensive blood vascular network (bv) of the choroid. Bar = 3.0 mm.

melanocytes. First, the apical processes can lengthen or contract in correlation with the increased or decreased amount of light that enters the orbit, although it has been suggested that the melanosomes within the villi, and not the villi themselves, are translocated in response to light (Cohen, 1992). It is presumed that this translocation functions to coordinate the surface area of the photoreceptor cells available to receive photon activation under differential lighting conditions in the environment. The mechanism regulating the movement of the apical process of the RPE melanocyte or the melanosomes within the villi is unknown but is thought to be mediated through actin filaments (Burnside and Laties, 1976). Second, the RPE melanocytes also somehow facilitate the regeneration of rhodopsin, the photochemical responsible for transducing the visual signals (Dowling, 1960; Hubbard and Colman, 1959). In addition, the apical processes also participate in the phagocytosis of the eventually shed tips of the photoreceptor cells (Hogan et al., 1974) for the subsequent degradation of

EXTRACUTANEOUS MELANOCYTES

cap

*

* G RPE

PC

Fig. 4.3. The retinal pigment epithelium (RPE) is a single layer of polarized cuboidal melanocytes positioned between the choroid and the photoreceptor cells (PC) of the neural retina. Elaborate specializations exist at the RPE/choroid junction, which consist of the choriocapillaris (cap) and Bruch’s membrane (brackets) of the choroid and the basal infoldings (asterisks) of the RPE. At the RPE/neural retina junction, the RPE melanocytes exhibit numerous slender apical processes containing melanosomes (arrows), which interdigitate between the rod and cone photoreceptor cells (PC). The cell body of the RPE melanocyte is also relatively polarized, in that Stage IV melanosomes are predominantly restricted to the apical half of the cell. Also distributed throughout the cytoplasm of the RPE melanocyte are the Golgi apparatus (G), lipofuscin granules (arrowheads), and mitochondria (open arrows). Bar = 1.5 mm.

this material in the cytosol of the RPE melanocyte. The RPE melanocytes thus contain an elaborate lysosomal system for this purpose (Feeney, 1973). Abundant in the RPE melanocyte are residual bodies, the end-stage of the lysosome-based process of phagosomal degradation, which have been called lipofuscin granules (Weiter et al., 1986; Wing et al., 1978). These granules can be distinguished from the melanosomes of the RPE melanocytes in that the former are very irregular in shape and their inclusions predominantly consist of laminated whorls representing degraded lipids (Feeney-Burns, 1980). However, melanosomes can occasionally be incorporated into these residual bodies. The total amount of melanin eventually produced by the choroidal melanocytes and the RPE varies markedly among individuals. However, it has been shown that between races

this variation is not significantly different for RPE melanin. In contrast, choroidal melanin is approximately twice as dense in African Americans as opposed to Caucasians (Geeraets et al., 1962; Hunold and Malessa, 1974; Weiter et al., 1986). The morphology of the melanosomes synthesized by the melanocytes of the uvea and the RPE can also be distinguished from each other. The shape of melanosomes in the uveal melanocyte is oval to spherical and generally 0.3 to 0.4 mm in diameter (Feeney and Hogan, 1961) but can be as large as approximately 1 mm in diameter (Boissy et al., 1988). In contrast, the melanosomes in the RPE melanocyte are extremely elongated and rod shaped ranging from 1.0 to 2.5 mm in length and 0.5 to 1.5 mm in width (Feeney-Burns, 1980; Kaczurowski, 1962). These distinctive RPE melanosomes are primarily localized toward and within the apical villi. However, spherical melanosomes ranging dramatically in diameter from 0.5 to 4.5 mm can also exist in the more perinuclear areas of the RPE (Kaczurowski, 1962). Another characteristic unique to the ocular melanocyte, in contrast to follicular and interfollicular cutaneous melanocytes, is their permanent halt in melanin synthesis and retention of melanosomes that occur shortly after birth of the organism. Melanization in the melanocytes of both the uveal tract and the RPE is transient, occurring only for a short period of time after initial differentiation in the target site (Boissy et al., 1988; Feeney-Burns and Mixon, 1979; Hu and Montagna, 1971). This contrasts markedly with melanocytes of the interfollicular epidermis which continuously synthesize melanin and transfer melanosomes to neighboring keratinocytes throughout the life of the organism (see Chapters 8 and 9). Hair follicle melanocytes also continually synthesize melanin and transfer melanosomes during their differentiated state, which occurs intermittently during the anagen cycles of hair growth (see Chapter 3). The transcription of melanocyte-specific genes responsible for the synthesis of melanin, and the subsequent production of melanin, has been documented in ocular melanoblasts during embryogenesis. Histologically, melanin synthesis (Mund et al., 1972; O’Rahilly, 1975) appears first in the RPE melanocytes at 10–12 weeks of gestation in the human embryo and, as demonstrated in primates, subsequently in the uveal melanocytes at about 28–36 weeks in equivalent human gestation (Endo and Hu, 1973; Hu et al., 1973). In the mouse, transcripts for Dct/Tyrp2 were initially identified in presumed RPE melanocytes at the optic cup at 9.5 days post coitum (dpc) and in presumed uveal melanoblasts occurring in the mesenchyme immediately outside the developing optic vesicle at 11.5 dpc (Steel et al., 1992). By birth, both groups of ocular melanocytes contain numerous Stage IV melanosomes congestively packed within their cytoplasm. However, melanin synthesis is still occurring at this time. The increase in melanin (Matheny and Dolan, 1975; Pierro, 1963), expression of tyrosinase activity (Miyamoto and Fitzpatrick, 1957), DOPApositive reactivity in the trans-Golgi network (Endo and Hu, 1973), and the presence of tyrosinase, Tyrp1, and Dct/Tyrp2 (Chiu et al., 1993) have been demonstrated in neonatal eyes 95

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of various species. These criteria of active melanization disappear permanently within days or weeks of birth leaving both groups of ocular melanocytes melanogenically inert for the rest of the life of the individual (Hearing et al., 1973; John and Fitzpatrick, 1972). However, it has been reported that eye color can darken with age, suggesting that melanogenesis may be reactivated in the melanogenically silent ocular melanocytes (Carino et al., 1994; see also review by Schraermeyer, 1993). It has recently been demonstrated that cultured human ocular melanocytes can be induced by alpha melanocyte-stimulating hormone (a-MSH) under certain conditions to increase DOPA oxidase activity (Smith-Thomas et al., 2001). Certain pathologic and therapeutic conditions also demonstrate that ocular pigmentation can be regulated postnatally. Many human ocular diseases have been linked to ocular pigmentation or directly attributed to the abnormalities of ocular melanogenesis, as exemplified by uveal melanoma, macular degeneration, senile cataract, Fuch’s heterochromic uveitis, and pigmentary glaucoma (Aggarwal et al., 1994; Cruickshanks et al., 1993; Regenbogen and Naveh-Floman, 1987; Scotto et al., 1976; Spector, 1984; Weiter et al., 1985). There is also evidence implicating a physiological association between innervation and pigmentation in the eye. Autonomic nerves innervate the eye in general (Laties, 1974; Laties and Lerner, 1975; Palumbo, 1976). More specifically, both adrenergic and cholinergic nerves frequently juxtaposition against melanocytes of the iridial stroma (Calhoun, 1919; Laties, 1974; Laties and Lerner, 1975; Ringvold, 1975). This iridial innervation appears to have influences on the regulation of ocular pigmentation, especially iris color. Cervical sympathectomy in various animal models has resulted in a marked decrease in iris coloration (Angelucci, 1893; Dryja and Albert, 1980; Giles and Henderson, 1958; Krebs et al., 1989; Laties, 1974). In humans, Horner’s syndrome results from a congenital or acquired unilateral paralysis of the cervical sympathetic nerves. This syndrome can frequently present with hypopigmentation of the iris associated with the lesional side (Calhoun, 1919; Weinstein et al., 1980). A postmortem electron microscopic analysis of the irises from a patient with Horner’s syndrome demonstrated that, in conjunction with a depletion of sympathetic nerve fibers, stromal melanocytes were reduced in numbers with a few morphologically normal melanocytes remaining (McCartney et al., 1992). This suggests that iris stromal melanocytes depend on the sympathetic nerve supply to survive. Furthermore, epidermal melanocytes were also demonstrated electron microscopically to be innervated by neurons (Hara et al., 1996). Cultured uveal melanocytes demonstrate stimulated growth and melanogenesis upon exposure to epinephrine, isoproterenol, salbutamol, and metaproterenol, adrenergic agonists that can activate beta(2)-adrenoceptors (Hu et al., 2000). Prostaglandins have been a successful therapeutic treatment for intraocular pressure in glaucoma (Bito et al., 1987, 1993). Latanoprost, a phenyl-substituted analog of prostaglandin F2a administered as eye drops, has been demonstrated to darken the eye color of monkeys (Selen et al., 1997) and glaucoma 96

patients (Wistrand et al., 1997). Recently, it has been demonstrated that latanoprost can induce tyrosinase gene transcription in cultured iridial melanocytes (Lindsey et al., 2001; Stjernschantz et al., 2000).

Variation in Morphology/Function of Uveal and RPE Melanocytes Uveal Tract The uveal tract consists of the choroid, the ciliary bodies, and the iris. It is predominantly composed of the choroidal component that encases the entire posterior orbit of the eye. As the choroidal layer approaches the anterior, frontal area of the orbit, it transforms first into the stromal component of the ciliary bodies and then into that of the iris, the anterior termination of the uveal tract. In a similar fashion, the RPE melanocyte layer is primarily found in the posterior orbit juxtaposed with the choroid. It also consists of anterior localized components that become the covering layer of the ciliary bodies and the inner or posterior surface of the iris. The bi- and/or multidendritic melanocytes of the choroid exhibit changes in their morphology as they reside in the stromal part of the ciliary bodies and the iris. At the base of the ciliary bodies, which is directly contiguous with the anteriormost area of the choroid, the melanocytes become relatively adendritic, in that they appear as large spherical cells congested with melanosomes. These spherical uveal melanocytes of the ciliary body are densely packed (Fig. 4.4). The ciliary bodies are primarily composed of processes that

C

NPE

PE CB

IRIS

Fig. 4.4. The pigmented tissues of the posterior chamber of the eye, i.e. the choroid and retinal pigmented epithelium (asterisk), are modified apically, first into the ciliary bodies (CB) and then into the iris (I). The ciliary body is a pleated structure with a heavily pigmented uveal stroma (arrow) lined by an epithelial bilayer of proximal pigment cells (PE) and distal nonpigmented cells (NPE). The iris lies beneath the cornea (C) and is composed of an anterior (outer) face containing a heavily pigmented stroma continuous with the uvea of the ciliary bodies, and a posterior (inner) face containing a bilayer of pigmented melanocytes continuous with the RPE layer of the ciliary bodies. Bar = 100 mm.

EXTRACUTANEOUS MELANOCYTES

extend into the vitreous body forming an extensively pleated, cylindrical ring-like structure anchoring the zonular apparatus (i.e. system of 10-nm fibers) responsible for accommodation of the lens during focusing (Coleman, 1970). The melanocytes distributed throughout the stroma of this part of the ciliary body become dendritic and resemble the melanocytes of the stroma of the choroid. The morphology of the melanosomes is identical to those of the choroidal melanocyte. The uveal tract leaves the ciliary bodies and continues on as the anterior stroma of the iris (Fig. 4.4) forming a circular ring of tissue (for review, see Miller, 1985). The iris, which is approximately 12 mm in thickness, marks the most frontal region of the posterior chamber of the eye and forms a diaphragm or wall separating it from the anterior chamber of the orbit. The most anterior surface of the iris is covered by a discontinuous layer of fibroblasts. Immediately beneath this layer of fibroblasts is a population of stromal melanocytes with dendrites orientated parallel to the iris surface. The remainder of the uveal component of the iris, called the iris stroma, contains numerous melanocytes with dendrites that are more extensively branched and more irregularly oriented. The melanosomes occurring in the melanocytes of the iris stroma, like those throughout the rest of the uveal tract, are oval to spherical in shape, measuring between 0.25 and 0.75 mm in diameter (Zinn et al., 1973). However, the size of these melanosomes varies depending on the genetic makeup of the color of the iris (see below). Like the choroid and ciliary body stroma, the iris stroma also contains an extensive amount of fibroblasts, blood vessels, nerves, and collagen fibers.

Clump Cells of Koganei Unique to the iris stroma is a cell type called the clump cell of Koganei (Wobmann and Fine, 1972). This spherical cell, much larger than the melanocyte, has a cytoplasm congested with many membrane-bound vesicles varying dramatically in size and containing primarily melanosomes with some intermittent granular and/or particular material. These giant pigmented cells have been proposed to be resident macrophages that have phagocytized a large number of melanin granules (Wobmann and Fine, 1972). Macrophages that appear to be precursors of clump cells of Koganei can occasionally occur on the anterior surface of the iris. These cells increase both in number and in content of engulfed melanosomes either during aging or in some pathologic conditions (Boissy et al., 1987; Hogan and Zimmerman, 1962; Wobmann and Fine, 1972). The most posterior face of the iris is lined with a double layer of melanocytes derived from the anteriormost continuation of the RPE layer as detailed below.

Retinal Pigment Epithelium Similar to the uveal tract, the RPE melanocyte also exhibits morphologic changes as it progresses anteriorly along and in juxtaposition with the uveal layer. Covering the ciliary bodies, the RPE becomes a bilayer called the ciliary epithelium (i.e.

ciliary RPE) lined on both surfaces by a basement membrane (Fine and Zimmermann, 1963; Straatsma et al., 1968). The cells of the outermost layer are relatively devoid of melanosomes and are called the nonpigmented epithelial cells (NPE) (Fig. 4.4). In contrast, the innermost layer contains numerous melanosomes and is called the pigmented epithelium (PE) (Fig. 4.4). These two cell layers of the ciliary epithelium are interjoined by numerous junctions including gap junctions, desmosomes, and tight junctions (Raviola and Raviola, 1978). The melanosomes of the PE resemble those of the RPE except that a higher percentage of them are more spherical and less elliptical. However, as a group, they are much larger than the melanosomes in the melanocyte of their stromal counterpart (Streeten, 1982). The RPE layer extending anteriorly toward the iris eventually forms the posterior leaf of this tissue and is specifically called the iris pigment epithelium (IPE) (for review, see Miller, 1985). The IPE, like that of the ciliary epithelium, is a double layer of cuboidal cells rather than a single layer. The posterior layer of the IPE is composed of heavily pigmented cuboidal melanocytes with a typical basement membrane on the posterior surface separating the cells from the vitreous chamber of the eye. The lateral surfaces of the posterior IRP cells are extensively connected to each other by lateral infoldings, desmosomes, and tight junctions. The posterior or basal surface manifests areas of infoldings, whereas the anterior or apical surface expresses numerous desmosomes and tight junctions adhering these cells to the anterior layer of the IPE. The anterior layer of the IPE is composed of an extremely unique type of melanocyte that expresses numerous myofilaments and is called the anterior epithelium–dilator muscle layer. The elongated melanocytes exhibit striking polarity and measure approximately 12.5 mm in height. The basal cytoplasm contains the nucleus and most of the heavily pigmented melanosomes. In contrast, the apical portions, which are wavy or spindle-shaped and interwoven with the apical portions of neighboring melanocytes, are relatively devoid of melanosomes and contain the classical arrangement of myofilaments. When these myofilaments induce contraction of the apical processes of these IPE cells, the iris layer is retracted and the pupil dilates (Salzmann, 1912; van Alphen, 1963, 1979). The contractile properties of the apical processes of the IPE cells resemble those of the apical processes of the RPE cells interdigitating with the photoreceptor cells of the retina as described above. Regulation of this contractile property appears to be under direct sympathetic stimulation by either neighboring neurons or humoral adrenergic substances such as norepinephrine and adrenal catecholamines (Miller, 1985). Therefore, it has been proposed that the ocular melanocyte must express functional receptors responsive to various neurokines that influence this contractile property (Bonvallet and Zbrozyna, 1963; Miller, 1985; van Alphen, 1963, 1979) and possibly melanogenesis as well (Laties, 1974; McCartney et al., 1992), as described earlier in this chapter. Finally, the melanosomes in the melanocytes of both layers of the IPE are relatively elongated like those distinctive of the rest of the RPE 97

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and are on average 0.8 mm in diameter and 2.5 mm in length (Zinn et al., 1973).

Eye Color Mammalian The color of human irises exhibits a transitional range from light (blues) to medium (greens/hazels) to dark (browns) and was demonstrated at the turn of this century to be under genetic regulation (Davenport and Davenport, 1907). The ultimate inherited color of an iris appears to be under the influence of both the amount of pigmentation in and the size of melanosomes within the stroma of the iris, and not by the number, density, and/or shape of the melanocytes in the stroma nor the melanosomes or melanocyte components of the IPE (Imesch et al., 1996; Wilkerson et al., 1996). The number, size, and pigmentation of the melanosomes produced in both cuboidal layers of the IPE appear to be constant and maximal regardless of the iris color (Feeney et al., 1965; Fine and Yanoff, 1979). In contrast, initial investigations demonstrated that variation in pigmentation of the anterior stroma layer correlated with eye color (Fuchs, 1913; Wolfrum, 1922). It was originally proposed that the number of melanocytes in the iris stroma increased as the color of the iris darkened (Dietrich, 1972). However, more recent quantitative analyses have demonstrated that the number of stromal melanocytes does not differ with eye color (Eagle, 1988; Wilkerson et al., 1996). Instead, quantitative differences in the density (Eagle, 1988; Imesch et al., 1996) and size (Eagle, 1988) of melanosomes do vary, in that stromal melanocytes of darker colored eyes contain larger and more numerous melanosomes per cytoplasmic area than lighter colored eyes. However, it has not yet been determined whether the quantity or quality of melanin within the melanosomes also varies with iris color. It is possible that the amount of eumelanin vs. pheomelanin or the ratio of brown-soluble vs. black-insoluble forms of eumelanin varies between different colored irises. It has recently been demonstrated that the ratio of eumelanin to pheomelanin in ocular tissue does not differ in blue or brown eyes (Menon et al., 1992) or in bovines of different coat colors (Dryja et al., 1979). The question of variation in melanogenesis correlating with eye color is being readdressed with the indication that both amount and type of melanin varies with different iris coloration (Sarna and Wielgus, 2004). Therefore, it is the function of melanocytes once established in the iridial stroma, and not the embryonic development of the melanocyte population in the eye, that determines iris color. Ultimately, the increased amount of backscattered reflective light occurring as a consequence of less melanosomal density in blue eyes, as opposed to more light absorption and relatively less reflection of light due to the relative higher density of melanosomes in brown eyes, is principally responsible for the color of the iris one observes (Miller, 1978).

Nonmammalian While the color of the iris of most birds and mammals is brown to black, there are notable exceptions of yellow, red, 98

RP

M

M

Fig. 4.5. The iris of the ground dove contains iridophores, congested with spaces representative of reflecting platelets (RP), residing next to myocytes (M) of the stroma. Bar = 1.0 mm (micrograph courtesy of Joseph Bagnara).

or orange irides, especially among avian species. This is not so surprising as the integument of birds and lower vertebrates also exhibits yellow, red, and orange as a result of the involvement of other neural crest-derived chromatophores, i.e. iridophores/leukophores and xanthophores/iridophores, in addition to melanophores (see Chapter 2). A study of the irides of two species of ground doves with yellow/orange and deep red iris color (Ferris and Bagnara, 1972) revealed the presence of true iridophores and their constituent reflecting platelets (Fig. 4.5). It is presumed that these capillary-associated iridophores function to enhance the red color stemming from the capillaries during vasodilation (Chiasson et al., 1968). No evidence of yellow chromatophores with their constituent pterinosomes was seen in the irides of ground doves, although the possible presence of pteridines was suggested by the fluorescence exhibited by iris extracts. A study of the great horned owl iris with yellow irises by Oliphant (1981) revealed the presence of both pterinosome-like organelles and pteridines of the type usually found in xanthophores and erythrophores of cold-blooded vertebrates. It was suggested that crystalline deposits of pteridines were present in the reflecting platelets

EXTRACUTANEOUS MELANOCYTES

of the iris, a fact later shown to be true for the reflecting platelets of frog iridophores (Bagnara et al., 1988). Observations on the irides of other avian species revealed the presence of both xanthophore and iridophore pigments (Oliphant, 1987a, b, 1988) and, later, Hudon (1994) extended the list of avian species possessing these pigments and demonstrated that irides of some birds possess unique pigments. One of these, pterorhodin, is a pteridine dimer that has been found in the chromatophores of only one vertebrate group, a subfamily of tree frogs (see Chapter 2). Thus far, mammalian irides have not been studied from the aspect of xanthophore or iridophore presence, although some regular and elongated organelles described from the cat iris (Tousimis, 1963) may represent the presence of iridophores.

Otic Melanocytes At the outset, we recommend the following excellent literature for more substantial information on the development and anatomy of the ear (Anson and Davies, 1980; Donaldson and Miller, 1980) and its melanocyte component (Cable and Steel, 1991; Conlee et al., 1989; Meyer zum Gottesberge, 1988a; Tachibana, 1999). Melanocytes have been identified in various sites of the ear. Primarily, they are localized throughout the stria vascularis and the modiolus of the cochlea, but they also exist in the vestibular organ composed of the utricle, the ampullae, the semicircular ducts, and the saccule as well as in the endolymphatic sac (Meyer zum Gottesberge, 1988a). In all cases, the melanocytes are extensively dendritic, heavily pigmented, and basically reside beneath the epithelial layer of each respective area. However, in the stria vascularis, the melanocytes become dramatically interdigitated with the extensive basolateral plasma membrane infoldings of the marginal (i.e. epithelial) cells (see below) (Cable and Steel, 1991). Much of the structural/functional analyses of otic melanocytes have almost exclusively focused on the melanocytes residing in the stria vascularis of the murine and/or chinchilla models. Therefore, much of the following discussion of otic melanocytes pertains to this subpopulation. Although the neural crest origin of otic melanocytes has not been as rigorously determined, using labeling and/or transplantation studies, as that of cutaneous and ocular melanocytes, it has nonetheless been proposed, primarily on account of association, that the otic melanocytes are of neural crest origin (Hilding and Ginzberg, 1977). The most convincing data implicating the neural crest as the source from which otic melanoblasts arose come from observations of mice mutant at the W locus. The W gene encodes the c-kit tyrosine kinase receptor that regulates the embryonic development of various cell types, specifically neural crest-derived melanocytes of the skin and uveal tract (Geissler et al., 1988; Hilding and Ginzberg, 1977; Mackenzie et al., 1997). Concomitantly, mutant W mice have a loss of melanocytes in the ear (Deol, 1970a; Schrott and Spoendlin, 1987; Steel et al., 1987). Studies on mice mutant for various alleles of the W locus demonstrated a halt in the expression of Dct/Tyrp2-positive

cells (i.e. presumptive melanoblasts) at a migratory site outside the developing ear (Cable et al., 1995), similar to the defective survival and migration of neural crest-derived melanoblasts observed in KitW-v/KitW-v mice during embryonic development using a different melanoblast marker (Mackenzie et al., 1997). The similarity between the in vivo effects of Kit mutations upon melanoblasts migrating to both the integument and the otic vesicle, together with the lack of an apparent effect of Kit mutations upon RPE melanocyte development, provides important correlative evidence for the neural crest origin of otic melanocytes. Interestingly, Mitf, but not Sox10, is expressed in strial melanocytes in immediately postnatal mice, despite the fact that Sox10 regulates Mitf expression during melanocyte development (Bondurand et al., 2000; Potterf et al., 2000), suggesting that this regulatory event in otic melanocytes may be specific to earlier developmental stages of those cells (Watanabe et al., 2002). Minimal amounts of melanin synthesis have occurred in the stria vascularis by birth (Fig. 4.6). During the subsequent weeks, melanogenesis dramatically increases until the otic melanocytes appear congested with melanosomes (Cable and Steel, 1991), similar to the process occurring in uveal and dermal melanocytes. Both eumelanin and pheomelanin appear to be synthesized by these otic melanocytes (Inoue et al., 1984). However, DOPA-reactive products in the trans-Golgi network, indicating active synthesis of tyrosinase, continue to be detectable well into adult life (Cable and Steel, 1991; Escobar et al., 1995), contrasting with the halt in tyrosinase synthesis postnatally that occurs in uveal and dermal melanocyte populations (Chiu et al., 1993). Although there is no concomitant synthesis of premelanosomes at this time, the target site and ultimate function of the tyrosinase-like enzyme in otic melanocytes of the adult remain unknown. Interestingly, it has also been demonstrated that the amount of otic melanosomes can increase in adult animals after exposure to impulse noise (Gratton and Wright, 1992) and experimentally induced endolymphatic hydrops (Meyer zum Gottesberge, 1986). The stria vascularis of the ear continues to develop after birth with extensive restructuring of the marginal, intermediate (i.e. melanocytes), and basal cell layers. As a result, there is a coordinate development of the endocochlear potential of the ear and the interdigitating of the melanocytes and the elaborate basal processes of the marginal cells (Cable and Steel, 1991). At this time, the melanocytes in the intermediate cell layer are called light cells. Morphologically, they have an extensive amount of organelles involved in protein synthesis (i.e. RER and Golgi apparatus) and numerous melanosomes individually distributed throughout the cytosol. Eventually, dark intermediate cells, which exhibit relatively no cytoplasmic organelles except for complexed, acid phosphatasepositive melanosomal complexes plus a pyknotic nucleus, appear in the same vicinity. These dark cells are presumed to be a terminal stage toward which the light intermediate melanocytes eventually progress (Cable and Steel, 1991). This is very unique, as no other subpopulation of melanocytes 99

CHAPTER 4

* *

M

E IV III

II E

throughout the body ever naturally progresses to such a morphologically necrotic-like state. Many functions for otic melanocytes in the physiology of the ear and hearing have been proposed and debated. These putative roles include: (1) the embryonic development of the ear and hearing; (2) the maintenance of endocochlear potential; and (3) the prevention of noise- and/or toxin-induced hearing loss. In general, if otic-destined, neural crest-derived melanoblasts do not migrate successfully into the ear or fail to survive in that location as a function of mutations in spotting genes such as Kit (Schrott and Spoendlin, 1987; Steel et al., 1987), KitL (Mayer and Green, 1968), or Mitf (Deol, 1970b, 1971; Green, 1989), hearing loss occurs. Similarly, deafness in humans that occurs in the majority of patients with Waardenburg syndrome (Hageman and Delleman, 1977; Read and Newton, 1997) and occasional variants of piebaldism (Giebel and Spritz, 1991; Hulten et al., 1987) has also been associated with the lack of normal otic melanocyte development. In KitW-v/KitW-v murine mutants, absence of strial melanocytes results in reduced interdigitations between marginal cells and basal cells, changes that can be observed as early as 6 days after birth (Steel and Barkway, 1989). More extensive cellular and structural degeneration of the cochlea, apparent after 6 weeks of age, occurs in other coat color mutants that lack cochlear melanocytes (Deol, 1970a). In addition, the morphology of the marginal cells is also compromised in cases where melanocytes are successfully established in the inner ear but remain amelanotic, i.e. in albinism (Conlee et al., 1986; Conlee and Bennett, 1993). Albinism also presents with abnormal brainstem auditory evoked responses associated with a reduction in neuronal size of the superior olivary complex (Baker and Guillery, 1989; Conlee et al., 1984, 1986; Creel et al., 1980, 1983). This is reminiscent of the neural alterations in the visual system that 100

Fig. 4.6. Otic melanocyte in the apical coil of the cochlea in an 18.5-day mouse fetus. The cell body of the melanocyte (M) resides among the epithelial cells (E) of the developing stria vascularis. Developmental stages (II, III, and IV) of melanosome biogenesis are present in the melanocyte. A cytoplasmic process of the melanocyte (arrowhead) appears to extend into the underlying mesenchyme (asterisk) through gaps (arrows) in the basal lamina (open arrows), perhaps indicating melanocyte migration from the mesenchyme into the epithelium. Continuity of this process with the cell body of the melanocyte was confirmed in serial sections (inset). Bar = 0.5 mm (micrographs courtesy of Rick Friedman and Emma Lou Cardell).

also correlate with the absence of melanin in albino models (see previous section in this chapter). In summation, the presence of pigmented melanocytes appears to be essential for the complete development of the marginal cells and the otic centers of the brain. The endocochlear potential, a measurement that reflects the ionic and electrochemical gradients within the fluids of the inner ear responsible for transducing sound waves to the hair cell receptors (Bosher et al., 1973), is also affected in aberrant pigment systems. Evidence supporting the importance of melanocytes to the generation of the endocochlear potential has also resulted from the analysis of murine coat color mutants. In most KitW-v/KitW-v mice, the development of an endocochlear potential, which normally occurs gradually over the course of the first two neonatal weeks, does not occur at all in the large majority of homozygous mutants that contain no strial melanocytes (Steel and Barkway, 1989). In addition, the extent to which the endocochlear potential is suppressed in development correlates with the extent of otic depigmentation associated with the various W alleles (Cable et al., 1994). The endocochlear potential in albinos is also reported to be altered (Conlee and Bennett, 1993; Conlee et al., 1991), although this has been contested (Bock and Steel, 1984). Therefore, hearing loss in these mutant pigmentary systems results directly from the absence of an endolymph mediator system. A combination of electrophysiologic, immunolocalization, and knockout mouse studies has demonstrated that the melanocyte, or intermediate cell, in the cochlea expresses the potassium ion channel directly responsible for EP generation. The depression of the EP by Ba2+ suggested that an inwardly rectifying K+ (Kir) channel located in the stria vascularis was responsible for the EP. A screen conducted of the stria vascularis for expression of various Kir channels revealed that only

EXTRACUTANEOUS MELANOCYTES

one, Kir4.1, was expressed in this tissue (Hibino et al., 1997). Although the cell type expressing Kir4.1 was originally in question, whole-cell patch clamp experiments described Ba2+responsive, inwardly rectifying K+ currents across the cell membranes of dissociated strial intermediate cells (Takeuchi and Ando, 1998), leaving open the possibility that the intermediate cell is responsible for generating the EP because of Kir4.1 expression. A subsequent study using an anti-Kir4.1 antibody in conjunction with immunofluorescence of dissociated strial cells and immunogold electron microscopy localized Kir4.1 to the intermediate cell membrane (Ando and Takeuchi, 1999). Finally, the lack of EP observed in mice lacking the Kir4.1 channel due to a homozygous knockout mutation confirmed the important role of this channel in supplying the electromotive force required for EP generation (Ando and Takeuchi, 1999). Melanin in otic tissues has been proposed also to protect against the loss of hearing resulting from noise- and/or toxininduced trauma. Susceptibility to noise-induced hearing loss has been demonstrated in some animals and humans with albinism (Barrenas and Lindgren, 1990; Conlee et al., 1986, 1988; Hood et al., 1976). However, the baseline hearing capabilities in albinos do not appear to be compromised (Harrison et al., 1984; Versnel et al., 1990; Wasterstrom, 1984). The presence of otic melanin has correlated with susceptibility (Comis and Leng, 1980; Wasterstrom, 1984), as well as resistance (Conlee et al., 1991), to ototoxicity. Therefore, the role of melanin in the ear is complex. It can absorb excess sound waves and toxins to prevent the subsequent damage to hair cells. On the other hand, melanin can promote ototoxicity depending on the circumstances (Dencker et al., 1973; Lindquist, 1973; Lindquist and Ullberg, 1972; McGinness et al., 1974; Meyer zum Gottesberge, 1988b). Currently, it has been questioned whether the maintenance of melanocytes and pigment in the ear of adults is essential. Loss of melanocytes, which occurs in brown mice homozygous for the Blt allele at the gene encoding Tyrp1 (Cable et al., 1993) and human patients with vitiligo (Dereymaeker et al., 1989; Orecchia et al., 1989; Tosti et al., 1987), has been associated with the development of hearing loss. It has also been suggested that pigment loss in the ear in conjunction with aging has a cause and effect relationship with the eventual deterioration of otic activity (Dum, 1982; Rosen et al., 1962).

Melanocytes of Internal Body Sites Melanocytes in the Leptomeninges A focal area of the human brain, specifically the meninges covering the ventrolateral surfaces of the medulla oblongata and the upper cervical cord, appears to be macroscopically pigmented (Baader, 1935; Symmers, 1905). This pigmentation is the consequence of melanocytes within this tissue, as confirmed by ultrastructural analysis (Goldgeier et al., 1984). Melanocytes residing in the meninges of the mouse have also been reported (Barden, 1986). In the adult human, melanocytes of the leptomeninges are multidendritic and congested with predominantly Stage IV melanosomes. The

presence in the cytoplasm of relatively few early-stage melanosomes and premelanosomes, as well as the absence of histochemically induced DOPA-positive reaction product in the trans-Golgi network, suggest that in the adult these cells are melanogenically inactive (Goldgeier et al., 1984), similar to the melanocytes of the dermis, the uveal tract, and the RPE. It should be noted that the pigment (i.e. melanin) of the melanocytes in the leptomeninges differs from the dark pigment found within neurons throughout the brain. The latter is classified as neuromelanin and is thought to be autooxidative product(s) of dopamine generated by the neuronal synthesized tyrosine hydroxylase molecule (see Breathnach, 1988; Enochs et al., 1993, 1994; Odh et al., 1994; Zecca et al., 1992). The function of the melanocytes confined to the leptomeninges is not clear. Of interest, it has been speculated that these melanocytes may be affected in patients with severe vitiligo (i.e. the Vogt–Koyanagi–Harada syndrome) and that their loss may correlate with the aseptic meningitis component of this syndrome (Goldgeier et al., 1984; Nordlund et al., 1980). Additionally, aberrant proliferation of these melanocytes during embryonic development, usually associated with the presence of a large congenital melanocytic nevus, may result in the condition neurocutaneous melanosis (Marghoob et al., 2004).

Melanocytes in the Harderian Gland The harderian gland is a secretory structure located at the medial back of the optic orbit with a duct that connects it to the nictitating membrane of the eyelid (Payne, 1994). It exists in all groups of terrestrial vertebrates but is absent in some types of mammals (i.e. bats, cows, and horses), terrestrial carnivores, and higher primates including man. Structurally, the gland is composed of tubules of simple columnar epithelial cells surrounded by myoepithelial cells, and it primarily synthesizes lipids, porphyrins, and indoles. Many putative and diverse functions have been hypothesized for this secretory gland (Payne, 1994). Melanocytes have been identified in the harderian glands of mice (Shirama et al., 1988), gerbils (Johnston et al., 1983), and frogs (Di Matteo et al., 1989). These melanocytes are primarily localized throughout the connective tissue capsule surrounding the harderian gland and intermittently among the connective tissue septa invaginations into the gland that separates the stroma into lobules (Fig. 4.7). Like the melanocytes of the leptomeninges, the melanocytes in the harderian gland of an adult animal are multidendritic, melanogenically silent, and congested with fully pigmented melanosomes (Fig. 4.7). Harderian glands ultrastructurally observed from the murine hypopigmented mutants ruby eye (Ru/Ru) and dilute (di/di) exhibited relatively fewer melanocytes with a dramatic reduction in melanosomes distributed throughout the cytoplasm (R. E. Boissy and K. Moore, personal observations). The function of melanocytes in the harderian gland is unknown, and no dysfunction in any of the putative roles of the gland has been reported in association with the various murine hypopigmentary mutants. 101

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E

E A

with the connective tissue coverings of cartilage (perichondrium) and bone (periostium). In addition, melanocytes were prevalent in the gonads, the lungs, the thymus gland, the thyroid gland, the spinal ganglia, and the gray matter of the spinal cord, but absent from the liver, the pancreas, and the spleen (Hallet and Ferrand, 1984; Makita and Mochizuki, 1984; Makita and Tsuzuki, 1986; Stolle, 1968). This mesenchymal distribution of melanocytes is primarily the result of an autosomal dominant gene (P) that can be intensified by a sex-linked recessive gene (d) (Stolle, 1968). Current analysis of the migration pathway of neural crest-derived melanoblasts in Silkie embryos demonstrated that these precursors are not restricted to the dorsolateral pathway as in normal development, but also migrate aberrantly through the ventral pathway (Reedy and Erickson, 1995). This genetically controlled diversion of melanoblast migration appears to be under cues extrinsic to this neural crest derivative (Hallet and Ferrand, 1984; see also Chapter 5).

Perspectives

E

B Fig. 4.7. The harderian gland is composed primarily of epithelial cells (E) housing numerous and varied types of secretory granules. Melanocytes congested with Stage IV melanosomes exist occasionally within the connective tissue septa separating the lobes of the harderian gland (arrow in A) and prominently within the connective tissue capsule encasing the harderian gland (arrow in B). Bar = 5.0 mm.

Extracutaneous melanocytes found in the eye, ear, and various internal body structures are unique as a whole from epidermal melanocytes in that: (1) they synthesize melanosomes and melanin only during a short interval after arriving in their target tissue; and (2) they retain their melanosomes throughout the life of the organism as opposed to transferring them to neighboring cells. The general morphology and ultrastructure of these extracutaneous melanocytes and the specific variations that exist among them have been well documented in the literature. However, the role of these morphologically dormant melanocytes in their respective tissues has primarily been ascertained anecdotally to date. Fortunately, experimental analysis investigating the function of extracutaneous melanocytes is beginning to make a reappearance in the scientific literature. The advent of molecular genetic technology, and its combination with cellular and physiological approaches, will help to resolve the elusive function of these relatively ignored populations of melanocytes that have been retained through evolution.

Melanocytes in Mesenchymal Tissues There have been a few reports of melanocytes distributed throughout internal body sites, most notably related to the PET mouse (Reams, 1963) and the Silkie chicken (Stolle, 1968). In the PET mouse, melanocytes appearing multidendritic, heavily pigmented, and inactive are distributed throughout the connective tissues of multiple sites in the body cavity, including cartilage, bone, serosae, lungs, heart, gonads, etc. (Nichols and Reams, 1960; Rovee and Reams, 1964). The specific genetics of this undefined strain of mouse was not clearly described; however, the inheritance of the mesenchymal pigmentation in the PET mouse appears to have been autosomal recessive in nature (Mayer and Reams, 1962; Reams, 1967). In the Silkie chicken, melanocytes were identified in the loose connective tissue throughout the body and associated 102

Acknowledgments We wish to thank Joseph Bagnara for providing information and illustrations in the section on nonmammalian eye color and Emma Lou Cardell for providing information and the illustration on otic melanocytes.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

5

Regulation of Melanoblast Migration and Differentiation David M. Parichy, Mark V. Reedy, and Carol A. Erickson*

Summary 1 The discovery of the relationship between the neural crest and pigment cells is briefly reviewed. 2 In all animals studied thus far, neural crest cells are known to migrate along specified pathways and to differentiate into a variety of cell types. These phenomena are directed by a variety of determining factors. 3 With regard to understanding pigment cell migration and differentiation, fish have recently contributed in a significant way primarily because of the large number of mutants that have been isolated in zebrafish and other species. Moreover, the clear embryos are amenable to experimental manipulation and visualization. 4 Amphibians have provided extensive opportunities to examine the movement and differentiation of a variety of pigment cell types, and in this case a number of mutants have been studied in some detail. However, this system has not been exploited to its full potential. 5 Although many reports of pigment variants exist in reptiles, little work has been done on developmental aspects of reptilian chromatophores. 6 Our understanding of the cellular and biochemical controls of pigment cell development has been greatly enhanced by work done in avian systems. Recently the development of methods to up- and downregulate gene expression in the avian embryo has contributed importantly to the understanding of melanogenesis in this system. 7 The many coat color mutants of mice provide extensive opportunities for better understanding the motive forces in pigment cell development. 8 A comparative evolutionary approach to studye pigment cell development will continue to be essential for understanding how such discrete cells (pigment cells) from a well-defined progenitor cell population (the neural crest) have differentiated and evolved.

Historical Background Embryologists have long been interested in the mechanisms that produce the correct arrangement of cells and tissues in *Kenneth A. Mason and Sally K. Frost-Mason were also authors of the previous version of this chapter.

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the developing embryo. Two of the processes that drive this event, called pattern formation, are morphogenesis and differentiation. Morphogenesis refers to the generation of biologic structure by the rearrangement of cells or tissues. In many cases this is achieved by the active migration of cells from one location in the embryo to another. Differentiation, on the other hand, is the process by which cells acquire morphologic and biochemical characteristics distinct from other cells. Although morphogenesis and differentiation usually are studied as independent developmental events, in fact these two processes are highly interconnected. The development of the neural crest exemplifies this complex interplay between morphogenesis and differentiation. Neural crest cells are a population of cells found in all vertebrate embryos. Excellent introductions to the older literature on neural crest development can be found in Hörstadius (1950) and Le Douarin and Kalcheim (1999). The early studies demonstrated three important aspects of neural crest development. First, neural crest cells detach from the neuroepithelium shortly before or soon after fusion of the neural folds. Second, neural crest cells migrate extensively throughout the developing embryo. Third, the neural crest is not a terminally differentiated population of cells, but instead can give rise to a wide range of cell and tissue types. These include neurons and glial cells of the peripheral nervous system, the secretory cells of the adrenal medulla, certain cardiac structures, enteric ganglia, and connective tissues of the craniofacial skeleton. Pigment cells are another derivative of the neural crest. There are several types of pigment cell, also called chromatophores, in vertebrate ectotherms (fishes, amphibians, reptiles) including: black, melanin-containing melanophores; yellow, pteridine-containing xanthophores; and silvery, purine-containing iridophores. In contrast, endotherms (birds and mammals) have a single type of skin pigment cell, the melanin-containing melanocyte. The study of pigment cell development in vertebrates has a rich historical tradition, and many older reviews provide access to these classical studies (e.g., Bagnara and Hadley, 1973; Le Douarin, 1982). The purpose of this chapter is to present an overview of the current state of knowledge on the development of neural crestderived pigment cells in vertebrate embryos. We begin with a discussion of themes in neural crest and pigment cell development common to all vertebrates. Because this textbook is organized around the concept of taxonomy, we have similarly organized this chapter by taxon. Thus, we summarize what is

REGULATION OF MELANOBLAST MIGRATION AND DIFFERENTIATION

known about pigment cell development specifically within the different classes of vertebrates: fishes, amphibians, reptiles, birds and mammals. The reader is encouraged to note the similarities and differences in the control of pigment cell development across these major phylogenetic groups. Although mathematical modeling is one technique that has been employed to study the generation of pigment patterns in vertebrates (reviewed in Murray, 1990), we approach the topic from a molecular and cellular perspective, and thus do not discuss any of these models specifically. Instead, our discussion is framed around the subtopics of morphogenesis and differentiation. Morphogenesis and differentiation are treated separately because most empirical studies to date have focused principally on one process or the other. Nevertheless, there is clear evidence that these two events are closely linked during pigment cell development (reviewed by Erickson and Reedy, 1998). Additionally, we discuss the role that pigmentation mutants have played in elucidating the molecular control of pigment cell development. In some animal models, such as birds and amphibians, pigment mutants have been useful but not the primary means of understanding melanogenesis. In the case of fishes and to a lesser extent mammals, most of what we know about pigment patterns has been derived from mutants and these are discussed extensively throughout the topics of morphogenesis and differentiation. Finally, we discuss major unresolved questions and suggest future research directions in the field.

Current Concepts: Overview of Neural Crest and Pigment Cell Development An understanding of the mechanisms that control neural crest and pigment cell morphogenesis is predicated on knowledge of the precise pathways these cells follow as they migrate. Several methods have been used to trace the migratory pathways of neural crest cells. One approach is to employ vital labeling of premigratory neural crest cells, for example with lysinated rhodamine dextran (LRD) or the lipophilic dye, DiI, and subsequently following migrating cells in living embryos over time. Alternatively, immunohistochemical markers and molecular probes specific for neural crest cells often are used to assess the distributions of cells in fixed tissues. In all species studied thus far, neural crest cells migrate along stereotyped routes. For reasons of space and simplicity, we focus on the migration of neural crest cells in the trunk. Discussions of cranial neural crest migration are available elsewhere (e.g., Köntges and Lumsden, 1996; Kulesa, 2004; Kulesa et al., 2004; Lumsden et al., 1991; Noden, 1991; Trainor et al., 2003). There are two principal migratory pathways for neural crest cells in the trunk (reviewed in Erickson, 2003; Erickson and Perris, 1993). One subpopulation of neural crest cells follows a ventromedial, or medial, pathway between the somite and neural tube. Glial and neurogenic precursors of the peripheral nervous system are among the neural crest cells that differentiate along this path, although in fishes

and amphibians some of the neural crest cells in this path can also differentiate as pigment cells. A second subpopulation of neural crest cells migrates along a dorsolateral, or lateral, path between the somites and the overlying epidermis. Neural crest cells in this path give rise to pigment cells (reviewed in Erickson, 1993). In fishes and amphibians, some neural crest cells take a third pathway and migrate dorsally into the developing fin. In birds and mammals, neural crest cells in the dorsolateral path differentiate exclusively as pigment cells. Similarly in ectotherms, there is no definitive evidence that neural crest cells in the dorsolateral path contribute to any neural crest derivative other than pigment cells. Conversely, there is a bias toward nonpigment cell phenotypes in ventromedially migrating neural crest. This correlation between migratory path and differentiated phenotype is well documented, but its developmental basis is controversial. There is evidence not only to support the notion that environmental factors encountered within a particular migratory path dictate neural crest fate, but also conversely, that prior specification dictates the choice of migratory path (discussed in Erickson, 2003; Le Douarin et al., 1993). The developmental potential of neural crest cells has been studied by a variety of means, including clonal analysis of neural crest differentiation and grafting neural crest cells or neural tube segments to ectopic axial levels and determining the differentiated phenotypes of the transplanted cells. Taken together, these studies suggest that most early neural crest cells are multipotent but become fate-restricted gradually over time (reviewed in Weston, 1991; Stemple and Anderson, 1993; Le Douarin et al., 1993, 2004; see below). Defining the precise lineage segregation of pigment cells has been difficult for two reasons. First, pigment cells differentiate from neural crest cells at most, if not all, axial levels of the trunk. As a result, heterotopic transplantation studies of the sort used to study the differentiation of other crest subpopulations in ectopic locations are not useful for studying the commitment of neural crest cells to a pigment cell fate. Second, markers for pigment cell precursors at the earliest stages of differentiation were not available until relatively recently. Consequently, early studies inferred that a particular cell was a pigment cell precursor simply by virtue of it being in the dorsolateral path but could not further evaluate the cell’s phenotype. Now, however, there is a substantial arsenal of lineage-specific markers for pigment cell precursors in most taxa. As detailed below, these markers have proven critical in studying the factors involved in pigment cell development.

Fishes Fishes provide three advantages for studies of pigmentation. First, the embryos of many species are oviparous and transparent so that neural crest cells and chromatophores can be observed throughout early development. Second, some fishes have short generation times and can be bred and reared in the laboratory, permitting genetic approaches to studies of pigment cells and pigment pattern development. Third, 109

CHAPTER 5 Early larva

Adult

Mel

Mel

Xan Irid

Mel Irid

Mel

pigment patterns of fishes are exceptionally diverse across taxa (e.g., Orton, 1953; Quigley et al., 2005; Riehl, 1991), allowing for comparative analyses of pigment cell development. To date, the advantages of fish are being exploited most fully in the tropical, freshwater zebrafish Danio rerio, as well as the freshwater medaka Oryzias latipes. Several sources provide information on staging, husbandry, experimental techniques, as well as the histories of these species as research models [zebrafish: Grunwald and Eisen (2002), Kimmel et al. (1995), Westerfield (2000), Zebrafish Information Network (http://zfin.org); medaka: Iwamatsu (2004), Shima and Mitani (2004), Medaka Homepage (http://biol1.bio.nagoya-u.ac. jp:8000/)]. Studies of these species have revealed numerous similarities in the genetic and cellular bases for neural crest and pigment cell development between teleosts and amniotes (see below). Recently several more extensive reviews of teleost pigment pattern formation have appeared (Kelsh, 2004; Parichy, 2003; Quigley and Parichy, 2002; Rawls et al., 2001). Like other vertebrates, a neural crest origin for fish pigment cells was long suspected (Borcea 1909), though not verified until considerably later (Humm and Young, 1956; Kajishima, 1958; Lopashov, 1944; for experiments on lampreys, see Newth, 1951, 1956). Early light microscopic studies of neural crest cells and chromatophores in teleosts identified these cells by location, shape, or externally visible pigmentation (e.g., Milos and Dingle, 1978a; Orton, 1953; Shephard, 1961). Grafting of [3H]thymidine-labeled cells also has been employed to examine neural crest migration (Lamers et al., 1981). Similarly, the HNK-1 antibody, which recognizes migrating neural crest cells of avian, rat, and turtle embryos (see below), has been used to identify neural crest cells in some 110

Fig. 5.1. Different pigment patterns of early larval and adult zebrafish (see also Plate 5.1, pp. 494–495). Mel, melanophore stripes; Xan, xanthophores; Irid, iridophores.

species (Sadaghiani and Vielkind, 1989, 1990a; Sadaghiani et al., 1994). Finally, vital dyes have been used to label neural crest cells and their derivatives in zebrafish (e.g., Dutton et al., 2001; Raible and Eisen, 1994; Smith et al., 1994; Vaglia and Hall, 2000). Teleost chromatophores retain their pigments intracellularly and pigment patterns thus reflect the numbers and arrangements of the various chromatophore classes (black melanophores, yellow xanthophores, iridescent iridophores, white leukophores, and red erythrophores). The precise complements of these chromatophores differ among species. For example, erythrophores are present in several Danio species, though not in zebrafish (McClure, 1999; Quigley et al., 2004, 2005). Adding to the complexity of teleost pigment cell development, pigment patterns of many species differ between early larval and adult stages (Fig. 5.1). Early larval patterns of many species (including zebrafish and medaka) comprise stripes of melanophores at the dorsal and ventral edges of the myotomes, along the horizontal myoseptum, and over the dorsal and ventral surfaces of the yolk (Kimmel et al., 1995; Kirschbaum, 1975; Lamoreux et al., 2005; Milos and Dingle, 1978a, b; Milos et al., 1983; Orton, 1953; Quigley et al., 2004). Iridophores or leukophores are found within the melanophore stripes, whereas xanthophores are widely dispersed outside of the stripes. This early larval pigment pattern arises during embryogenesis and is largely completed by hatching. After several days to weeks depending on the species, early larval pigment patterns are transformed into adult pigment patterns that are exceptionally diverse among species (Booth,

REGULATION OF MELANOBLAST MIGRATION AND DIFFERENTIATION

1990; Kirschbaum, 1975; Johnson et al., 1995; Mabee, 1995; Parichy and Johnson, 2001). For example, even within the genus Danio, different species exhibit horizontal stripes of varying number, width, and distinctiveness, vertical bars, uniform patterns, and more ornate patterns (Quigley et al., 2004, 2005). Pigment patterns are even more diverse among other groups including cichlids and coral reef fishes (Allender et al., 2003; Lorenz, 1962; Streelman et al., 2003).

Morphogenesis of Fish Chromatophores Prospective neural crest cells in most vertebrates are found within the neural folds, which fuse to form a neural tube. In fishes, the neural tube arises instead from a ventral thickening of the dorsomedial ectoderm termed the “neural keel.” Subsequently, the neural keel delaminates from surrounding epidermal ectoderm and a central canal develops to generate the definitive neural tube (Lowery and Sive, 2004; Schmitz et al., 1993). Neural crest cells first become apparent along the dorsal surface of the developing neural tube as the neural keel separates from the epidermis (Lamers et al., 1981; Lowery and Sive, 2004; Raible et al., 1992; Sadaghiani and Vielkind, 1989, 1990a; Sadaghiani et al., 1994; Schmitz et al., 1993). The details of neural crest migration in fishes are best documented in zebrafish. After segregating from the neural keel, zebrafish neural crest cells migrate along the two major pathways discussed previously: ventromedially between the somites and neural tube, and dorsolaterally between the somites and epidermis (Raible and Eisen, 1994; Raible et al., 1992; Vaglia and Hall 2000; Wood et al., 1992). In addition, some neural crest cells migrate dorsally and localize in the fins. Zebrafish neural crest cells in all pathways give rise to pigment cells (Raible and Eisen, 1994). The mechanisms underlying early neural crest cell dispersal and migration remain unknown in teleosts, though transient roles for somitic mesoderm in blocking early migration are likely (Jesuthasan, 1996). Some insights into early larval pigment cell morphogenesis have come from analyses of sparse mutant zebrafish. In contrast to wild-type, sparse mutants have fewer melanophores and these cells are largely absent from the anterior head as well as ventral regions of the flank. Molecular cloning identified sparse as a teleost ortholog of Kit (formerly c-kit; Parichy et al., 1999), long-studied for its role in mouse melanocyte development (see Mammals, below). kit encodes a type III receptor tyrosine kinase containing five extracellular immunoglobulin repeats and a split kinase domain. The ligand of Kit is Steel factor (SLF; also known as stem cell factor, mast cell growth factor, and kit ligand). Like other receptor tyrosine kinases, dimerization of Kit upon ligand binding is necessary for activation of the kinase domain (Blume-Jensen et al., 1991). Downstream signal transduction events initiated by Kit signaling in melanoblasts are largely unknown, but evidence suggests that the phospholipase C and phosphatidylinositol-3¢-kinase pathways are activated (reviewed in Galli et al., 1994). Zebrafish kit is expressed by melanophores and their precursors. In null alleles, melanophores undergo apoptosis and are extruded from the

skin (Parichy et al., 1999). More recent analyses of an allelic series including conditional, temperature-sensitive alleles reveal genetically and temporally separable roles for kit in melanocyte migration (at early stages) and melanocyte survival (at later stages) (Rawls and Johnson, 2003; see also, Rawls and Johnson, 2000, 2001). Interestingly, the kit-related gene colony stimulating factor-1 receptor (csf1r; formerly, fms) is required for the survival and migration of zebrafish xanthophores (Parichy et al., 2000b), suggesting that similar requirements for receptor tyrosine kinase activity are fulfilled by kit and csf1r in melanophore and xanthophore lineages, respectively. Recent studies have identified a second locus required for melanophore survival during early larval pigment pattern formation. Danio rerio touchtone mutants exhibit normal migration and specification of melanophores, but melanoblasts and melanophores subsequently undergo apoptosis. Genetic mosaic analyses reveal that touchtone acts autonomously to the melanophore lineage (Arduini and Henion, 2004; Cornell et al., 2004). The touchtone mutant phenotype results from lesions in trpm7, which encodes a member of the transient receptor potential (TRP) family (Elizondo et al., 2005). The transmembrane trpm7 protein serves as both a cation channel and as a kinase (Wolf, 2004), and mutants have additional defects in kidney function and skeletal development. A role for trpm7 channel activity during melanophore development is demonstrated by the partial rescue of trpm7 mutant melanophores by simple calcium or magnesium supplementation to the medium. While trpm7 appears to act independently of the kit pathway during melanophore development (Cornell et al., 2004), the upstream regulators and downstream targets of trpm7 remain unknown. Beyond permissive roles for cell migration, survival, and proliferation, of particular interest are the instructive factors that generate specific arrangements of chromatophores. From several recent studies we now have a clearer notion of what these factors are both in early larval and adult pigment pattern formation. The arrangement of early larval melanophores depends in part on the stromal-derived factor 1a (sdf1a) chemokine. sdf1a is a chemoattractant that contributes to migration of primordial germ cells (PGCs) as well as the lateral lines, a bilateral sensory system that develops by the directed migration of a bullet-shaped “primordium” of cells within the epidermis. Both PGCs and lateral line primordia express cxcr4b, a Gprotein-coupled receptor for sdf1a, and migrate towards sources of sdf1a in the prospective gonads and along the horizontal myoseptum, respectively (David et al., 2002; Doitsidou et al., 2002; Li et al., 2004). A role for sdf1a in melanophore patterning is revealed by choker mutant zebrafish (Kelsh et al., 1996). By early larval stages, these mutants have a “collar” of melanophores that covers the normally melanophore-free regions of the lateral myotomes. Myotome development is severely disrupted in choker mutants and sdf1a is expressed ectopically along the myotomes where melanophores accumulate. Morpholino knockdown of sdf1a 111

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in the choker mutant background reduces the numbers of collar melanophores, whereas ectopic sources of human sdf1a attract melanophores in wild-type embryos (V. Svetic and R. N. Kelsh, personal communication). During normal development then, sdf1a plausibly contributes to melanophore localization. Whether sdf1a acts directly on melanophores or through an intermediary signaling pathway remains unclear, particularly since melanophores do not express detectable levels of cxcr4b by in situ hybridization. Intriguingly, choker mutants lack xanthophores in the collar region. Genetic analyses show this defect is independent of the defect in melanophore patterning, whereas the defect in melanophore patterning is independent of the defect in xanthophore distribution. Development of the zebrafish adult pigment pattern depends on a complex interplay between directed chromatophore migration and death, as well as differentiation (see below; Parichy and Turner, 2003b; Parichy et al., 2000b; Quigley et al., 2004, 2005). During metamorphosis, new xanthophores arise just ventral to the horizontal myoseptum and at the dorsal and ventral margins of the flank, whereas new melanophores arise widely dispersed over the flank and outside of the early larval stripes. Some of these “metamorphic” melanophores migrate short distances to join the first two adult melanophore stripes, immediately dorsal and ventral to the horizontal myoseptum. Simultaneously, additional metamorphic melanophores develop already within the adult stripes. Many early larval melanophores initially within the lateral stripe along the horizontal myoseptum die, though some migrate to join the developing adult stripes, thereby producing a melanophore-free “interstripe region” that is, however, populated by xanthophores. As metamorphosis proceeds, the adult stripe borders become increasingly distinct. During juvenile and subsequent adult development, additional melanophore stripes are added dorsally and ventrally as the fish grows. In the mature adult pigment pattern of zebrafish, dark stripes comprise melanophores and iridophores with occasional xanthophores, whereas light stripes comprise xanthophores and iridophores (Hirata et al., 2003; Parichy and Turner, 2003a). Dermal and epidermal chromatophores also are found outside of stripes; for example, scale melanophores contribute an overall dark cast to the dorsum of the fish. The morphogenetic bases for the zebrafish adult striped pattern have been examined in several recent studies that have identified a critical role for interactions within and among chromatophore classes. The notion that chromatophore interactions could be important comes initially from the csf1r mutant phenotype (Parichy et al., 2000b; Fig. 5.2). As compared to wild-type, csf1r mutants have fewer adult melanophores and these cells fail to organize into normal stripes. Beyond the melanophore defect, csf1r mutants lack embryonic xanthophores (as mentioned above) as well as adult xanthophores. The correlated adult melanophore and xanthophore defects raised the possibility that interactions between these lineages might underlie melanophore stripe development. Support for this notion comes from two experimental paradigms (Parichy and Turner, 2003a). First, orga112

nized melanophore stripes are recovered in genetic mosaics in which melanophores and xanthophores are experimentally juxtaposed: when cells are transplanted between csf1r mutants (which lack xanthophores but retain melanophores) and nacre (mitfa) mutants [which lack melanophores but retain xanthophores (Fig. 5.2 and see below)] the resulting chimeras develop stripes. Second, if csf1r is conditionally activated or inactivated using a temperature-sensitive allele, xanthophores are gained or lost, respectively, and the number of xanthophores present correlates with the degree of melanophore stripe organization. Indeed, xanthophores and melanophore stripes can be gained or lost in a csf1r-dependent manner throughout development, revealing roles for csf1r in both initiating and maintaining stripe formation. Since csf1r is not detectably expressed by melanophores but is expressed by the xanthophore lineage, these results suggest that interactions between melanophores and csf1r-dependent cells of the xanthophore lineage are required for stripe organization. Chromatophore interactions probably also contribute to development of stripes on the fins (Goodrich and Greene, 1959; Goodrich and Nichols, 1931; Goodrich et al., 1954; Parichy and Turner, 2003a). Analyses of other Danio species suggest that evolutionary changes in chromatophore interactions may underlie interspecific variation in adult pigment patterns (Parichy and Johnson, 2001; Quigley et al., 2005). Further indications that chromatophore interactions underlie stripe development come from studies of obelix (jaguar) and leopard mutant zebrafish (Haffter et al., 1996; Johnson et al., 1995; Fig. 5.2). obelix mutants exhibit fewer and broader stripes than wild-type and xanthophores are intermingled with melanophores in the melanophore stripes (Maderspacher and Nüsslein-Volhard, 2003). leopard mutants often develop spots, but alleles of differing severity range in phenotype from irregular stripes to nearly uniformly distributed, intermingled melanophores and xanthophores (Asai et al., 1999; Maderspacher and Nüsslein-Volhard, 2003). Thus, mutant phenotypes may be interpretable in the context of defective boundary formation between chromatophore classes. Cell transplantation and genetic analyses indicate that obelix acts within melanophores to promote homotypic interactions between these cells, whereas leopard acts within melanophores and xanthophores to promote both homotypic and heterotypic interactions (Maderspacher and NüssleinVolhard, 2003). The obelix mutant phenotype results from mutations in kir7.1, encoding an inwardly rectifying potassium channel (S. L. Johnson and S. Kondo, personal communication); the cellular basis for translating channel activity into cellular pattern remains to be elucidated. leopard mutant phenotypes result from mutations in the gap junction gene, connexin-40, suggesting that direct cell–cell contacts and cytoplasmic communication may underlie leopard-dependent chromatophore interactions and boundary formation (S.L. Johnson, personal communication). Interestingly, N-CAM and N-cadherin had previously been associated with xanthophores but not other chromatophore classes in O. latipes (Fukuzawa and Obika, 1995), consistent with roles for dif-

REGULATION OF MELANOBLAST MIGRATION AND DIFFERENTIATION

Wild-type

picasso (erbb3)

puma

panther (csf1r)

sparse (kit)

panther (csf1r); sparse (kit)

rose (ednrb1)

panther (csf1r); rose (ednrb1)

sparse (kit); rose (ednrb1)

nacre (mitfa)

leopard (cx40)

jaguar (kir7.1)

Fig. 5.2. Mutational analyses allow a genetic dissection of adult pigment pattern development in zebrafish. Shown are wild-type zebrafish as well as single and double mutant zebrafish with corresponding pigment pattern defects (see also Plate 5.2, pp. 494–495). See text for details.

ferential adhesion in the sorting-out of specific chromatophore classes, although this model has yet to be tested directly. Besides the spatial arrangements of chromatophore classes across the flank, different chromatophores types occupy different strata within the skin itself (Hawkes, 1974a, b). In zebrafish, light stripes comprise xanthophores superficial to iridophores, whereas dark stripes comprise occasional xanthophores superficially, followed by iridophores, then melanophores, and additional iridophores more internally. Moreover, the organization of iridophore reflecting platelets differs depending on whether or not these cells are associated with xanthophores or melanophores (Hirata et al., 2003). The molecular signals associated with these arrangements are not known.

Differentiation of Fish Chromatophores Factors controlling the specification and differentiation of teleost chromatophores are beginning to be elucidated. An early step in neural crest specification towards chromatophore fates requires the transcription factor sox10, as revealed by analyses of colourless mutant zebrafish in which the sox10 gene is disrupted (Dutton et al., 2001; Kelsh and Eisen, 2000). sox10 (colourless) mutants have severe defects in chromatophores, glia, and neurons: precursors to these lineages fail

to leave the neural crest and instead undergo apoptosis. In contrast, ectomesenchymal neural crest derivatives (e.g., craniofacial skeleton and fin mesenchyme) are unaffected. Nonectomesenchymal sox10-dependent neural crest cells often are fate-restricted even prior to leaving the neural crest, as revealed by elegant single cell lineage analyses (Dutton et al., 2001; Raible and Eisen, 1994). Such analyses do not yet provide a clear picture of lineage relationships between chromatophores and other nonectomesenchymal lineages, or even among chromatophore classes themselves. (For a fuller discussion of neural crest specification, see Birds, below.) Nevertheless considerable progress has been made in defining the mechanisms of fate specification and differentiation in the melanophore lineage. An important advance came from the isolation of nacre mutant zebrafish that lack melanophores. This mutant phenotype results from mutations in microphthalmia-a (mitfa) (Lister et al., 1999), encoding a member of the basic-loophelix-leucine-zipper (bLH-ZIP) family of transcription factors (Hodgkinson et al., 1993; see Steingrimsson et al., 2004 for a recent review). Mammalian orthologs of mitfa have long been studied and are known to directly upregulate genes required for melanin synthesis (see Mammals, below). Additional studies of zebrafish have provided insights into how 113

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mitfa is itself regulated, with important roles identified for both intrinsic and extrinsic factors. An intrinsic factor relative to the melanophore lineage is the sox10 transcription factor. mitfa expression is curtailed in sox10 (colourless) mutants (Dutton et al., 2001). The sox10-dependence of melanophore development lies entirely in sox10-dependent regulation of mitfa: sox10 binds and activates the mitfa promoter, sox10 overexpression upregulates mitfa expression in vivo, and forced mitfa expression rescues melanophore development in sox10 mutant backgrounds (Elworthy et al., 2003). An extrinsic factor relative to the melanophore lineage lies in the Wnt signaling pathway. Wnt1 and Wnt3a are expressed in the dorsal neural keel. This expression domain has functional significance in promoting chromatophore over nonchromatophore fates as revealed by injections of single premigratory neural crest cells with activating or inhibitory mRNAs for the Wnt signal transduction pathway (Dorsky et al., 1998). This effect is mediated, at least in part, by the binding of Wnt-responsive Lef1/T-cell factor (TCF) transcription factors within the mitfa promoter, which upregulate mitfa transcription and thus melanophore fate specification (Dorsky et al., 2000a, b). More recent studies using a heat shock inducible transgene for a dominant negative form of TCF demonstrate that Wnt signals are required at several distinct steps in the neural crest–melanophore lineage (Lewis et al., 2004; see also Birds, below). Beyond the critical roles for mitfa, sox10, and Wnts in melanophore specification, a large number of mutants have been isolated in zebrafish and medaka that identify genes likely to function primarily in melanophore differentiation (Kelsh et al., 1996, 2000, 2004). Many of these probably affect melanin synthesis or melanosome biogenesis (e.g., Fukamachi et al., 2001; Schonthaler et al., 2005). One gene likely to regulate these processes encodes transcription factor activating protein-2a (tfap2a). The zebrafish lockjaw and mont blanc mutant phenotypes result from mutations in tfap2a and have severe defects in melanophore as well as iridophore development, in addition to defects in craniofacial, glial, and neural derivatives of the neural crest (Barrallo-Gimeno et al., 2004; Knight et al., 2003, 2004). The failure of melanophore development results at least in part from a tfap2a-dependence of kit expression, and epistasis analyses combined with morpholino knockdown support a model in which tfap2a and kit act in a common pathway (O’Brien et al., 2004). As melanoblasts express early markers of the melanophore lineage, and some melanophores develop but are lightly pigmented, it appears tfap2a allows the terminal differentiation of this cell type. While considerable effort is directed towards understanding specification and differentiation of chromatophores during embryonic stages, mutational analyses have also started to reveal genetic requirements of metamorphic melanophores that contribute to adult pigment patterns. For example, puma mutant zebrafish have a severe deficit of metamorphic melanophores despite having a normal complement of early larval melanophores (Parichy and Turner, 2003b; Parichy 114

et al., 2003; Fig. 5.2). puma is required during metamorphosis for promoting melanoblast and melanophore development and acts autonomously to the melanophore lineage. A superficially similar phenotype is observed for picasso mutants (Quigley et al., 2004; Fig. 5.2), which result from mutations in the receptor tyrosine kinase gene erbb3 (Lyons et al., 2005; E. Budi and D. Parichy, unpublished data). Nevertheless, it has yet to be determined whether puma and picasso (erbb3) function primarily to promote metamorphic melanophore specification and differentiation, or survival and proliferation. Regardless of the precise mechanism, comparative analyses demonstrate that most adult pigment patterns among danios arise from metamorphic melanophores that differentiate from latent precursors during the larval-to-adult transformation (Quigley et al., 2004); understanding the factors that promote the establishment, maintenance, and recruitment of such latent precursors is thus critical to elucidating the mechanisms underlying naturally occurring pigment pattern variation in this group. As an extreme example of this notion, the zebrafish sister species, D. nigrofasciatus, mostly lacks metamorphic melanophores and their precursors owing to evolutionary changes that are extrinsic to this melanophore lineage and likely to involve the puma pathway (Quigley et al., 2004). Despite their apparent uniformity, metamorphic melanophores actually comprise two genetically and temporally distinct populations, as revealed by analyses of kit, csf1r, and endothelin receptor b1 (ednrb1) mutants (Johnson et al., 1995; Parichy et al., 1999, 2000a, b). kit mutants not only lose their early larval melanophores, but lack early-appearing, dispersed metamorphic melanophores; nevertheless, these mutants ultimately recover metamorphic melanophores, already situated in stripes, during late metamorphosis and juvenile development. In contrast, csf1r and ednrb1 mutants have normal numbers of early-appearing, initially dispersed metamorphic melanophores and instead are deficient for late-appearing metamorphic melanophores at sites of stripe formation. That early kit-dependent and late csf1r- and ednrb1-dependent melanophores are distinct populations is supported by epistasis analyses: in contrast to single mutants, fish doubly mutant for kit and csf1r, or kit and ednrb1, lack nearly all body melanophores (Johnson et al., 1995; Parichy et al., 2000b; Fig. 5.2). Additional discussion of endothelin receptors is provided in sections on Birds and Mammals, below. In contrast to melanophores, relatively less is known about the specification and differentiation of xanthophores, iridophores, or other chromatophore classes. Early larval iridophores are reduced in tfap2 mutants whereas adult iridophores are absent in ednrb1 mutants. Molecular identification of additional mutants affecting iridophore development [e.g., shady and parade (Kelsh et al., 1996)] will surely provide valuable new insights. Virtually nothing is known about xanthophore fate specification, though precursors of these cells and xanthophore numbers are somewhat reduced in mitfa mutants (Lister et al., 1999; Parichy et al., 2000b). Xanthophore pigments are

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derived, in part, from the pteridine pathway (Ziegler, 2003; Ziegler et al., 2000) and enzymes within this pathway are expressed by xanthophore precursors, although also in some other lineages, including melanophores (Parichy et al., 2000b; Parichy, unpublished data), suggesting caution in their use as lineage markers. Mutational analyses reveal that xanthophore differentiation requires PAM (protein associated with cmyc), which regulates pteridine synthesis enzymes in a cell autonomous manner (Le Guyader et al., 2005), as well as somatolactin, which acts as an endocrine factor (Fukamachi et al., 2004).

Mutants Besides those discussed above, a very large number of other mutants affecting chromatophore development have been isolated, particularly in zebrafish (Golling et al., 2002; Haffter et al., 1996; Kelsh et al., 1996; Odenthal et al., 1996; Rawls et al., 1993) and O. latipes (Kelsh et al., 2004). These mutants, high-throughput screens for chemically-induced phenotypes (Peterson et al., 2000), and genomic resources (Pickart et al., 2004) offer outstanding opportunities for dissecting morphogenesis and differentiation, as well as the evolution of these processes. Descriptions of mutant screens and on-line resources for these mutants can be found at: ∑ http://zfin.org ∑ http://biol1.bio.nagoya-u.ac.jp:8000/ ∑ http://protist.biology.washington.edu/dparichy/ ImagesMutants.htm

Amphibians Amphibians offer several advantages for studies of pigment cells and pigment pattern formation. For example, embryos of many species are quite large and develop oviparously. Moreover, development rate often can be controlled by incubating embryos at a wide range of temperatures. This makes amphibian embryos highly accessible to both observation and experimentation. Finally, amphibians offer a wide range of patterns to study: not only do different taxa exhibit an extraordinary diversity of larval and postmetamorphic patterns [e.g., urodeles (Bishop, 1941; Epperlein and Löfberg, 1990; Olsson, 1993, 1994; Pfingsten and Downs, 1989; Twitty, 1936, 1966), reviewed in Parichy, 1996b; anurans (Altig and Johnston, 1989; Andres, 1963; Bagnara, 1982; Elias, 1941; Ellinger, 1979; Frost and Robinson, 1984; Gosner and Black, 1957; Smith-Gill, 1974), reviewed in Frost-Mason et al. (1995)], but mutations have been identified in some species that specifically affect chromatophores or pigment pattern formation (see below). Salamander embryos were used by DuShane (1934, 1935, 1938) in his pioneering studies that first revealed a neural crest origin for chromatophores. These findings were later extended to anurans (Stevens, 1954) and inspired numerous analyses of pigment pattern development in amphibians. Several reviews provide an introduction to these classical studies (Bagnara, 1983; DuShane, 1943; Epperlein and Löfberg, 1993; Erickson, 1993; Frost and Malacinski, 1980; Hall and

Hörstadius, 1988; Lehman and Youngs, 1959; Parichy, 1996b; Rawles, 1948; Twitty, 1949, 1953, 1966). Amphibian chromatophores in the integument can localize in both the dermis and the epidermis, though dermal chromatophores are primarily responsible for distinctive pigment patterns in most taxa (e.g., Bagnara et al., 1968; DuShane, 1943; Stearner, 1946).

Morphogenesis of Amphibian Chromatophores In salamanders, rounded, presumptive neural crest cells are first detectable within the neural folds (Hirano and Shirai, 1984; Raven, 1931; Spieth and Keller, 1984). As the neural folds fuse at the midline, prospective neural crest cells form a wedge within the neuroepithelium and finally emerge as a distinctive but transient cord or “crest” running anteroposteriorly along the dorsal aspect of the completed neural tube (Fig. 5.3). Presumptive neural crest cells also have been identified in the neural folds of the anuran Xenopus laevis (Mayor et al., 1995; Sadaghiani and Thiébaud, 1987; Schroeder, 1970). Many techniques have been used to identify neural crest cells in amphibian embryos, including light and electron microscopy (Hirano and Shirai, 1984; Löfberg and Ahlfors, 1978; Löfberg et al., 1980, 1985; Spieth and Keller, 1984), vital labeling (Collazo et al., 1993; Detwiler, 1937), and intrinsic characteristics or labeling of transplanted cells in chimeric embryos (Chibon, 1967; Krotoski et al., 1988; MacMillan, 1976; Moury and Jacobson, 1989, 1990; Sadaghiani and Thiébaud, 1987; Raven, 1931, 1936). Finally, amphibian — like teleost — chromatophores often produce pigment prior to reaching their final destination and can be observed directly (Epperlein and Claviez, 1982; Epperlein and Löfberg, 1990; Keller and Spieth, 1984; Tucker and Erickson, 1986a; Twitty, 1945). Amphibian neural crest cells disperse along dorsolateral and ventromedial pathways and also are found within the expanding fin. In salamanders, most or all neural crest cells that contribute to pigment patterns on the dorsal flank of young larvae migrate dorsolaterally between the somites and epidermis (Epperlein and Löfberg, 1990; Olsson and Löfberg, 1992). Neural crest cells traveling dorsolaterally are probably the major contributors to externally visible pigment patterns in most frog tadpoles as well (Andres, 1963; Tucker, 1986). In contrast, most X. laevis neural crest cells and melanophores fail to migrate subepidermally in the anterior trunk and instead remain near the dorsal apex of the myotomes or first travel medially and only secondarily migrate to the epidermis (Andres, 1963; Collazo et al., 1993; Krotoski et al., 1988; MacMillan, 1976; Tucker, 1986). Medial migration of melanophores or their precursors in other amphibians is suggested by the observation that many amphibians exhibit chromatophores lining internal structures (DuShane, 1943; Tucker and Erickson, 1986a). The factors controlling amphibian neural crest and chromatophore morphogenesis are not well understood, but results from many studies suggest roles for the extracellular matrix (ECM). Work in Ambystoma mexicanum suggests that changes in the composition of the ECM may influence the 115

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Fig. 5.3. Neural crest cells (arrows) emerging from the neural tube of a salamander embryo. Bar = 200 mm.

initial dispersal and migration of neural crest cells (Löfberg and Ahlfors, 1978; Löfberg et al., 1980, 1985). A variety of ECM components have been identified in the vicinity of early or later migrating amphibian neural crest cells and chromatophores, including fibronectin, laminin, collagens type I and III, chondroitin and keratan sulfate proteoglycans, tenascin, hyaluronan, and molecules that bind the lectin peanut agglutinin (PNA) (Epperlein and Löfberg, 1990; Epperlein et al., 1988; Olsson et al., 1996; Parichy, 1996a, 2001a; Perris et al., 1990; Tucker, 1986; Tucker and Erickson, 1986a, b). Several studies have examined the behavior of salamander neural crest cells on ECM substrata in vitro (Epperlein et al., 1988; Perris and Johansson, 1987, 1990; Tucker and Erickson, 1986a, b). These investigations have shown that salamander neural crest cells can disperse and migrate on a variety of ECM components, including fibronectin, vitronectin, laminin, and collagens type I, IV, and VI. In contrast, purified tenascin inhibits the adhesion of neural crest cells, and motility of these cells can be modulated in a concentrationdependent manner by addition of chondroitin sulfate proteoglycans and hyaluronan. In the early larvae of several salamander taxa, melanophores first are uniformly distributed over the flank but subsequently concentrate near the dorsal apex of the myotomes and along the dorsal margin of the yolk mass. Because xanthophores typically are present over the lateral face of the myotomes, the resulting pattern consists of dark, horizontal dorsal and lateral stripes bordering a pale “melanophore-free region” along the middle of the flank (Parichy, 1996b, 2001a, b). In one of these taxa, Taricha torosa, fibronectin is abundant at sites of melanophore localization, whereas hyaluronan is abundant within the dorsal fin matrix where melanophores are not found (Tucker and Erickson, 1986a, b). In Triturus alpestris, the local appearance of tenascin and chondroitin sulfate proteoglycans over the lateral face of the myotomes correlates with the evacuation of 116

melanophores from this region (Epperlein and Löfberg, 1990). Moreover, interspecific differences in tenascin and laminin correlate with species-specific melanophore distributions (Parichy, 2001a). In several other taxa within the families Ambystomatidae and Salamandridae, the lateral line sensory system (Northcutt, 1992; Northcutt et al., 1994) is directly responsible for excluding melanophores from the middle of the flank (Parichy, 1996a, c). Lateral line-dependent perturbations of the subepidermal basement membrane and increased deposition of tenascin correlate with the evacuation of melanophores from this region (Parichy 1996a, 2001a). Changes in the ECM also may influence chromatophores and chromatoblasts during later development. In A. t. tigrinum for example, staining for laminin, tenascin, and PNA-binding activity increase dramatically during terminal formation of the early larval pigment pattern, probably reflecting the elaboration of a largely acellular dermis that is hypothesized to stabilize melanophore positions (Parichy, 1996c; Stearner, 1946). Likewise, breaks in the collagenous basement membrane and the appearance of fibronectin “tracts” correlate with changes in melanophore positions at metamorphosis in the frog Rana esculenta (Denèfle and Lechaire, 1990, 1991, 1992; Denèfle et al., 1987). Another class of factors that probably influences the morphogenetic behavior of chromatophores is interactions among the cells themselves, as in teleosts. Based on a series of studies in vitro, Twitty (1944) and Twitty and Niu (1948) suggested that the directed dispersal of neural crest cells and melanophores results from the production of diffusible substances that repel neighboring cells. Although such negative chemotaxis has not been ruled out in urodeles, attempts to verify these conclusions using quail neural crest cells have not been successful (Erickson and Olivier, 1983). More recently, melanophore and neural crest cell dispersal has been suggested to result from contact inhibition of movement (Abercrombie, 1970) coupled with high cell densities in dorsal regions of the

REGULATION OF MELANOBLAST MIGRATION AND DIFFERENTIATION

flank [Tucker and Erickson (1986a); but see Keller and Spieth (1984)], concordant with similar proposals in avian embryos (Rovasio et al., 1983; Erickson, 1985). In contrast to repellent interactions that appear to mediate pigment cell dispersion, adhesive interactions among melanophores have been suggested to contribute to the condensation of these cells during stripe formation in T. torosa (Parichy, 1996a; Twitty, 1945). Cell–cell interactions also may be involved in the development of vertical barring patterns in several salamanders within the family Ambystomatidae (Epperlein and Löfberg, 1990; Lehman, 1954, 1957; Olsson, 1994; Olsson and Löfberg, 1992; Parichy, 1996a, b, 2001b); similar patterns also are present in some frog tadpoles (Altig and Johnston, 1989; Gosner and Black, 1957). In these taxa, melanophores scatter widely over the somites during early stages while xanthophores are retained in aggregates at variable intervals dorsal to the neural tube. Xanthophores subsequently disperse from these aggregates, and melanophores on the flank recede short distances from the advancing xanthophores. This results in alternating vertical bars of melanophores and xanthophores in young larvae. Epperlein and Löfberg (1990) hypothesize that aggregates arise at the level of the premigratory neural crest by xanthophores “sorting out” from surrounding melanophores via differential adhesion (Steinberg, 1970). Observations from experimentally manipulated and mutant embryos are consistent with this idea. Nevertheless, the events of aggregate formation have yet to be described in vivo. During later stages, repulsive interactions between melanophores and xanthophores appear to drive morphogenesis of the definitive vertical barring pattern (Epperlein and Löfberg, 1990) though the underlying mechanisms have yet to be established. In addition to interactions among chromatophores and between chromatophores and the ECM, gross physical features of the extracellular environment probably influence the migration and localization of amphibian chromatophores. For example, a physical barrier near the dorsal margin of the yolk mass may contribute to the failure of T. torosa melanophores to migrate ventrally over the lateral plate mesoderm (Twitty, 1936). Melanophores of this species also localize along the lateral line nerve perhaps due to contact guidance (Dunn, 1982; Tucker and Erickson, 1986a). Finally, the lateral line nerve in A. tigrinum is hypothesized to act as a physical barrier that prevents dorsal melanophores from migrating further ventrally (Parichy, 1996c). A variety of hormones and growth factors also could influence the behavior of embryonic amphibian neural crest and pigment cells. Candidate molecules have been identified in teleost and amniote embryos, but the distributions and functions of these molecules and their receptors are only beginning to be investigated in amphibians at stages relevant to early pigment pattern formation (e.g., Baker et al., 1995). Finally, externally visible pigment patterns of most amphibians change markedly during later larval stages and especially during and after metamorphosis (Bagnara, 1982; DuShane,

1943; Fernandez and Collins, 1988; Lehman, 1953; Lehman and Youngs, 1959; Niu and Twitty, 1950; Parichy 1998, 2001b; Smith-Gill, 1974; Stearner, 1946; Twitty, 1936). These changes presumably reflect persistent or renewed migration of existing pigment cells as well as neural crest cells or chromatoblasts that have not yet overtly differentiated. Nevertheless, it seems likely that these chromatophore movements are influenced by endocrine-derived hormones (reviewed in FrostMason et al., 1995). Hormones and localized growth factors probably also influence the massive restructuring of skin that occurs during metamorphosis (Heatwole and Barthalmus, 1995), which may indirectly impact the establishment of a new pigment pattern. For example, thyroid hormones trigger a cascade of events during metamorphosis, including extensive remodeling of the ECM, cell death, proliferation, and differentiation (e.g., Patterton et al., 1995). Intriguingly, thyroxine stimulates the migration of melanophores from the dermis to the epidermis in cultured frog integument, possibly reflecting a direct effect of the hormone on melanophores (Yasutomi, 1987).

Differentiation of Amphibian Chromatophores At least some amphibian neural crest cells begin to differentiate into melanophores and xanthophores shortly after dispersing, or even while in the premigratory position dorsal to the neural tube (Epperlein and Löfberg, 1984, 1990; Olsson and Löfberg, 1992; Tucker and Erickson, 1986a). In contrast, iridophores typically are detectable only after the appearance of melanophores and xanthophores. It seems likely that additional populations of chromatoblasts remain covert within the integument and differentiate at later larval stages, during metamorphosis, or during adult life (Bagnara et al., 1979; Brown, 1997; DuShane, 1943; Fernandez and Collins, 1988; Frost et al., 1984a; Ohsugi and Ide, 1983; Parichy, 1998, 2001b; Stearner, 1946). In A. mexicanum, differentiation into melanophores and xanthophores is stimulated by serum (Dean and Frost-Mason, 1991), or alternatively if the cells are co-cultured with neural tube, epidermis, or ECM isolated from the dorsolateral (but not medial) pathway (Perris and Löfberg, 1986; Perris et al., 1988). Similar requirements for serum are observed in cultures of neural crest cells isolated from T. torosa (Tucker and Erickson, 1986b; Twitty, 1944, 1945) and X. laevis (Fukuzawa and Ide, 1988). One molecule known to stimulate melanogenesis specifically is a-melanocyte stimulating hormone (a-MSH), which is produced in the pituitary gland of adult animals. a-MSH originally was identified as a circulating hormone that causes the dispersion of melanin-containing granules (melanosomes) within melanocytes (e.g., Bagnara and Hadley, 1969; Fernandez and Bagnara, 1991; Sawyer et al., 1983). In addition to these short-term physiological responses, a-MSH probably also mediates long-term morphologic changes in pigmentation (reviewed in Bagnara and Hadley, 1969). In vitro, a-MSH stimulates both proliferation and melanization of anuran neural crest cells (Fukuzawa and Bagnara, 1989; 117

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Wilson and Milos, 1987) and melanophores (Fukuzawa and Bagnara, 1989). A possible role for a-MSH or related peptides during early development also has been suggested in a preliminary report demonstrating a-MSH immunoreactivity in the epidermis of A. mexicanum embryos and larvae (FrostMason et al., 1992). It will be interesting to see if a-MSH activity contributes to the early differentiation of neural crest cells into chromatophores, particularly in light of taxon- and stage-specific differences in the ability of epidermis to promote melanophore differentiation (DeLanney, 1941; Thibaudeau and Frost-Mason, 1992; Twitty, 1936). In addition to a-MSH, presently unidentified factors in the dorsal integument and serum of Rana pipiens and X. laevis stimulate both melanization and outgrowth in cultured X. laevis neural crest cells (Fukuzawa and Bagnara, 1989; Mangano et al., 1992). Conversely, a partially purified factor from ventral integument of Rana and X. laevis inhibits outgrowth and melanization of these cells (Fukuzawa and Bagnara, 1989; Fukuzawa and Ide, 1988), whereas medium containing factors derived from ventral integument stimulates adhesion and proliferation of iridophores in vitro (Bagnara and Fukuzawa, 1990). An antibody raised against a putative “melanization-inhibiting factor” has been shown to block inhibitory effects in vitro, and this antibody stains the ventral but not dorsal integument of R. pipiens (Fukuzawa et al., 1995). Further characterization of this factor could provide important insights into the control of regional differences in pigmentation (e.g., the dark dorsal and white bellies seen in many amphibians and other vertebrates).

Mutants Numerous pigment mutants have been found across several amphibian taxa, including R. pipiens (Davison, 1964; SmithGill, 1973, 1974, 1975; Volpe and Dasgupta, 1962), Bombina orientalis (Ellinger, 1980; Frost et al., 1982), X. laevis (Droin, 1992), and P. waltl (Collenot et al., 1989). Perhaps the most studied are those of A. mexicanum: melanoid, albino, axanthic, and white (reviewed in Frost and Malacinski, 1980; Frost et al., 1984b). Early larval “wild-type” A. mexicanum display abundant melanophores and xanthophores, whereas older larvae and paedomorphic adults exhibit melanophores, xanthophores, and iridophores in an irregular mottled green and black pattern that covers the integument. melanoid (m) In contrast to wild-type A. mexicanum, m/m animals exhibit few xanthophores as larvae or adults and completely lack iridophores at all stages of development (Frost et al., 1984c). Thus, melanoid adults are a velvety black. Genetic mapping places the melanoid locus on linkage group 14 (Smith et al., 2005). The melanoid defect is believed to act autonomously within the neural crest lineage (Sawada and Dalton, 1979). Total pigment cell numbers do not differ between melanoid and wild-type animals (Hoerter, 1977). Biochemically, the deficit of xanthophores and iridophores correlates with a decrease in overall pteridine levels during 118

development (Frost et al., 1984c) as well as lower levels of pteridines compared to wild-type animals (Benjamin, 1970; Frost et al., 1984b; Thorteinsdottir and Frost, 1986). Treatment of wild-type A. mexicanum with an inhibitor of the pteridine synthesis enzyme xanthine dehydrogenase results in a phenocopy of the melanoid defect (Frost et al., 1989; Thorteinsdottir and Frost, 1986). albino (a) The a gene was originally introduced into A. mexicanum from the closely related A. tigrinum [an undertaking that required truly heroic efforts; see Humphrey (1967)]. Homozygotes for the albino gene completely lack melanin but do have unmelanized “melanophores” containing organelles typical of early stages in melanogenesis (Frost et al., 1986a). Because a/a individuals possess both xanthophores and iridophores, their overall coloration is a golden yellow. Albino A. mexicanum also exhibit differences in pteridine profiles within the integument compared to wild-type A. mexicanum (Frost et al., 1986a). The biochemical defect underlying the albino phenotype remains unknown. axanthic (ax) Little is known about the axanthic defect. The ax/ax A. mexicanum completely lacks iridophores whereas “xanthophores” are identifiable but remain unpigmented; melanophores are apparently unaffected (Frost et al., 1984a, 1986b). white (d) Homozygous d/d individuals lack all three types of chromatophores in the integument during most or all of their lives (Frost et al., 1984a), resulting in a white phenotype. Notably, eye pigmentation is unaffected, suggesting that the defect is specific for neural crest-derived pigment cells. Deficient cutaneous pigmentation is thought to result from a failure of neural crest cells and chromatophores to disperse into the dorsolateral migratory pathway (Dalton, 1950; Keller et al., 1982), though lineage tracers have yet to be employed to test explicitly the relative roles of inhibited dispersal versus inhibited differentiation or survival [see also DuShane (1939)]. Indeed, some chromatophore precursors must disperse, because some d/d animals repigment spontaneously in haphazard patches after reaching sexual maturity (Frost and Malacinski, 1980; Frost et al., 1984b). The defect associated with the d gene traditionally has been attributed to a change within the epidermis, since wild-type epidermis grafted to white embryos permits melanophore dispersal whereas the converse does not (Keller et al., 1982). Löfberg et al. (1989) obtained analogous results when microcarriers coated with only white or wild-type subepidermal ECM were grafted between embryos. In contrast, microcarriers coated with subepidermal ECM from older white embryos permitted neural crest dispersal in both white and wild-type hosts. This led to the hypothesis that changes in the subepidermal ECM normally required for neural crest cell migration are transiently retarded in white embryos. Unfortunately, com-

REGULATION OF MELANOBLAST MIGRATION AND DIFFERENTIATION

parisons of white and wild-type ECM have yielded conflicting results (Epperlein and Löfberg, 1993; Olsson et al., 1996; Perris et al., 1990) and do not adequately explain what the white defect might be. Genetic mapping places d on linkage group 1, allowing the exclusion of several candidate genes, including steel, encoding the kit ligand, SLF (Parichy et al., 1999; Smith et al. 2005; Voss et al., 2001).

Reptiles Little is known concerning neural crest and pigment cell development in reptilian embryos. Hou and Takeuchi (1994) used the HNK-1 antibody to show that neural crest cells of the softshell turtle Trionyx sinensis migrate along both ventromedial and dorsolateral pathways, as in other vertebrates. Melanophores in this species are found cutaneously and internally (Hou and Takeuchi, 1991), suggesting that both ventromedially and dorsolaterally migrating neural crest cells can differentiate as melanophores. Intriguingly, early migrating neural crest cells also enter the myotome (Hou and Takeuchi, 1994). This could reflect enhanced invasiveness of a melanogenic subpopulation of neural crest cells traveling along the medial pathway, as has been suggested in the Silkie chicken (Faraco et al., 2001; Reedy et al., 1998b).

Birds Avian embryos are amenable to experimental manipulation at virtually all stages of embryogenesis and thus have been widely exploited as a model system for studying neural crest and pigment cell development. By far the two most commonly used avian species for these studies are the domestic chicken, Gallus gallus, and the Japanese quail, Coturnix japonica. Many studies have taken advantage of the fact that quail neural crest cells, when grafted into a chick embryo host, can be distin-

guished by their characteristic nuclear staining pattern with Feulgen dye (Le Douarin, 1982) or the quail-specific antibody QCPN. In addition, avian neural crest cells can be identified on the basis of their immunoreactivity with the HNK-1 antibody (Tucker et al., 1988) and by their expression of specific cadherins (Nakagawa and Takeichi, 1995) or transcription factors, such as Sox 10 (Cheng et al., 2000). The neural crest origin of avian melanocytes, the sole skin pigment cell type found in birds, was demonstrated over 50 years ago using experimental embryological techniques (Dorris, 1936, 1938, 1939; Ris, 1941; Watterson, 1942; Willier and Rawles, 1940). In adult birds, melanocytes are distributed in both the dermis and the epidermis (Hulley et al., 1991). In addition, melanocytes are found internally in some birds, for example the Silkie chicken (e.g., Faraco et al., 2001; Makita and Mochizuki, 1984; Makita and Tsuzuki, 1986).

Morphogenesis of Avian Melanocytes The dynamics of neural crest migration in the avian trunk have been well documented. In chickens, neural crest cells first detach from the neuroepithelium in the trunk at stage 11 (Tosney, 1978) and invade the ventromedial pathway shortly thereafter (Bronner-Fraser, 1986; Loring and Erickson, 1987). These neural crest cells differentiate into the neurons and glial cells of the peripheral nervous system. The entry of avian neural crest cells into the dorsolateral path is delayed by about 24 hours with respect to ventromedial migration and begins at stage 20 at the level of the forelimb (Erickson et al., 1992; Serbedzija et al., 1989). Neural crest cells destined for the dorsolateral path initially pause between the lateral neural tube and the tip of the dermamyotome (Fig. 5.4). This region has been termed the migration “staging area” (Weston, 1991). Cells that enter this pathway will differentiate into pigment

Regulation of Melanoblast Migration and Differentiation +24 hrs

+24 hrs

Fig. 5.4. Schematic diagram of the major migratory pathways followed by neural crest cells in the trunk of amniote embryos. Anterior is to the left. (Bottom) Sagittal section through somites -6 to -12 indicating the simultaneous development of the myotome and neural crest invasion into the somite from the ventromedial path. (Top) Cross-sections at different axial levels show the ventromedial and dorsolateral pathways taken by neural crest cells. Diagram by Martha Spence and used with permission from Academic Press.

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cells of the skin. Melanoblasts begin entering the ectoderm approximately two stages after entering the dorsolateral path (Erickson et al., 1992). Eventually, they invade the feather follicle and complete their differentiation into melanocytes (Hulley et al., 1991). A major question about neural crest morphogenesis is what determines the positioning of neural and glial cells in the ventral pathway and pigment cells in the dorsolateral path. The generally accepted model for patterning neural crest derivatives proposes that neural crest cells are a pluripotent population of cells that migrate haphazardly into the ventral or dorsolateral paths, and differentiate according to the cues encountered in these paths (Le Douarin, 1982, 1986; Le Douarin et al., 1993; Selleck et al., 1993; Stemple and Anderson, 1993). This model is supported by experimental evidence from heterotopic grafting experiments (Le Douarin and Teillet, 1974; Le Douarin et al., 1975), as well as backtransplantation of crest-derived structures into the early migratory crest pathways (Ayer-Le Lievre and Le Douarin, 1982; Dupin, 1984; Le Douarin et al., 1978; Le Lievre et al., 1980), which show that, as a population, neural crest cells migrate and differentiate according to their new environment and not their origin. However, an alternative mechanism directs the migration of chicken melanoblasts into the dorsolateral path at the trunk level. Specifically, melanoblasts are developmentally biased at the time that they depart from the neural tube: only neural crest cells that are specified as melanocytes enter the dorsolateral path (Reedy et al., 1998a), and melanoblasts are the only neural crest cells that can exploit the dorsolateral path under experimental conditions (Erickson and Goins, 1995). Thus, the final distribution of pigment cells is directed by cellautonomous migratory properties unique to that subpopulation. The evidence that melanoblasts are specified prior to entering the dorsolateral pathway will be discussed in the following section. Here we will first consider what controls the choice of pathway. Why are neural and glial precursors unable to invade the dorsolateral path, whereas the later-migrating melanoblasts can exploit this pathway? The dorsolateral path at the trunk level is initially refractory to neural crest cell migration (Erickson and Goins, 1995; Erickson et al. 1992; Serbedzija et al., 1989). By stages 19–20, beginning at the wing-bud level, dorsolateral migration is initiated as ventral migration gradually ceases (Kitamura et al., 1992; Oakley et al., 1994; Serbedzija et al., 1989). The molecular basis for this switch in pathways, initially suggested by Oakley et al. (1994), was that inhibitory molecules are gradually lost, so that the environment in the dorsolateral path is now permissive for migration. However, experimental studies do not support this proposal. They instead suggest that there are changes in those neural crest cells specified as pigment cells that allow them to overcome the inhibitory cues. When nonmelanogenic neural crest cells are grafted into stage-19 embryos at the wing-bud level, which is the time when the host neural crest cells would be embarking on the dorsolateral path, the grafted cells only 120

migrate ventrally (Erickson and Goins, 1995). However, when melanoblasts are grafted into the early neural crest migratory pathway (stage-15 host), the grafted cells invade the dorsolateral space immediately, well in advance of the time when dorsolateral migration usually takes place (Erickson et al., 1992). These experimental studies indicate that molecular changes occurring during melanoblast specification, rather than changes in the environment, permit dorsolateral migration. One possible molecular change in melanoblasts is that they develop unique sensitivity to positive guidance cues from the dermamyotome, allowing them to migrate dorsolaterally. In the mouse, melanoblasts depend upon SLF produced by the dermamyotome for their initial dispersal onto the dorsolateral path (Wehrle-Haller and Weston, 1995, 1997; Wehrle-Haller et al., 1996; and see Mammals below), and they express the receptor for SLF, kit, prior to embarking on the dorsolateral path. However in the avian embryo, kit is first expressed by melanoblasts long after they have migrated dorsolaterally (stage 25), and SLF is not produced by the dermamyotome, but rather by the ectoderm and only after stage 25 (Lecoin et al., 1995; Reedy et al., 2003). Furthermore, morpholino knockdown of kit does not affect either melanoblast migration or specification (Harris and Erickson, unpublished data). Thus, acquiring responsiveness to SLF owing to kit upregulation is unlikely to control the timing of dorsolateral migration in the chicken. In the past few years a number of guidance molecules have been identified that cause the collapse or repulsion of growth cones, filopodia or lamellipodia (Tessier-Lavigne and Goodman, 1996). These include the netrins, the collapsin/ semaphorins, Slits and ephrins (Gale and Yancopoulos, 1997). Three laboratories have shown that ventrally migrating neural crest cells are constrained to migrate in the anterior half of somites because inhibitory ephrins are expressed in the posterior somite (Flenniken et al., 1996; Krull et al., 1997; Wang and Anderson, 1997). Ephrins are also distributed in the dorsolateral space at the time of early crest migration and they have also been shown to be responsible for preventing neural and glial precursors from entering this space (Santiago and Erickson, 2002). In contrast, ephrins stimulate melanoblast migration, and this positive guidance cue permits melanoblasts to invade the dorsolateral space. This is not unlike the netrins and semaphorins, which can be inhibitory under some circumstances and stimulatory under others. A major task will be to understand how Eph receptors, the receptors for ephrins, can differentially signal in melanoblasts and nonmelanoblasts. Further, we do not yet understand how ephrin signaling results in enhanced melanoblast migration, although integrin activation is likely to be a factor (Beauvais et al., 1995; Santiago and Erickson, 2002). Ephrins are likely not the only signaling molecules that control the directional migration of melanoblasts into the dorsolateral space. Recently, Jia et al. (2005) have demonstrated that Slits, which are secreted chemorepellants, expressed in the dermamyotome also prevent the migration of early migratory neural crest cells into the dorsolateral space. When the Slit

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receptor, Robo 1, is inactivated by expressing a dominant negative form in the premigratory neural crest, ventrally migrating crest cells now precociously invade the dorsolateral pathway. Still unanswered is how melanoblasts can overcome the chemorepellant action of Slit. Potentially the Robo receptors could be downregulated on neural crest cells during melanoblast specification, or melanoblasts may be attracted to Slits rather than being repelled. Yet another signaling molecule/receptor pair that potentially controls directed migration into the dorsolateral space is endothelins/endothelin receptors (reviewed by Pla and Larue, 2003). Endothelins are 21-amino acid peptides originally identified for roles in vasoconstriction (Yanigasawa et al., 1988) and their receptors are G-protein-coupled, heptahelical, cellsurface receptors. In the chick, ventrally migrating neural crest cells express Ednrb, whereas Ednrb2 is upregulated just prior to neural crest cells migrating in the dorsolateral pathway. Endothelin-3, the presumptive ligand for avian Ednrb and Ednrb2, is expressed by the ectoderm (Nataf et al., 1998). When mouse embryonic stem (ES) cells are transfected with Ednrb2 and grafted into an early chick embryo, they take the dorsolateral pathway precociously, unlike the wild-type ES cells or ES cells expressing Ednrb, which migrate ventrally (Pla et al., 2005). This study suggests that endothelin signaling may positively attract melanoblasts into the dorsolateral space. It would be interesting to manipulate the composition of endothelin receptors on neural crest cells in situ to test this possibility directly. Beyond effects on migration, several studies of cultured quail neural crest cells show that endothelin-3 clearly affect proliferation of melanoblasts (Lahav et al., 1996). Additionally clonal analysis reveals that endothelin signaling enhances the survival of clonogenic cells that give rise to melanocytes only, glial cells only, and the melanocyte/glial bipotent precursors (Lahav et al., 1998). During later morphogenesis, melanoblasts enter the ectoderm, which is separated from the dermis by a basal lamina. Although previous studies have shown that basal laminae are impenetrable by early-migrating neural crest cells, melanoblasts clearly have enhanced invasive properties (Cuddenec, 1977; Erickson, 1987; Erickson and Goins, 1995; Sears and Ciment, 1988; Teillet, 1971). Localized discontinuities in the basal lamina adjacent to neural crest cells in the dorsolateral pathway suggest that melanoblasts selectively degrade the basal lamina prior to invading the ectoderm (Erickson et al., 1992). The mechanisms of melanoblast invasiveness are not clear, but one possibility is that they produce matrix-degrading proteases that allow them access to sites where other crest cells normally cannot enter (Duong and Erickson, 2004; Erickson and Isseroff, 1986; Valinsky and Le Douarin, 1985).

Differentiation of Avian Melanocytes The melanoblast lineage was believed to be specified only after neural crest cells had migrated from the neural tube, because a number of cloning or culture studies using avian neural crest showed that pigment cells could arise from a pluripotent pre-

cursor in 24-hour outgrowths (Baroffio et al., 1991; Dupin and Le Douarin, 2003; Le Douarin et al., 1993; Sieber-Blum et al., 1993; Stocker et al., 1991; Weston, 1991). However, when quail trunk-level neural tubes are cultured and the fate of those neural crest cells that emigrated during the first six hours is assessed, no melanocytes differentiate, even after six days in culture under conditions that are permissive for melanogenesis (Reedy et al., 1998a). Thus there is heterogeneity in the melanogenic potential of different temporal subpopulations of neural crest cells from the same axial level. Corroborating studies by Henion and Weston (1997) showed that many of the neural crest cells are fate-restricted (that is, the progeny from a single neural crest cell all have the same phenotype) at the time that they detach from the neural tube, and that neurogenic and some gliogenic precursors migrate from the neural tube before any melanogenic precursors. They did note that there are some clones that are bipotent and could give rise to glial cells and pigment cells, which are likely to be the intermediate precursor population identified in previous cultures studies (Sherman et al., 1993; Sieber-Blum and Cohen, 1980). Together these two studies show that many neural crest cells are developmentally restricted when they leave the neural tube, and that neurogenic, gliogenic, and melanogenic neural crest cells are specified at different times prior to their migration. Similar observations have been made in the zebrafish (Raible and Eisen, 1994; Dorsky et al., 1998, 2000a) and in axolotls (Epperlein and Löfberg, 1984). See also reviews by Anderson (2000), Dorsky et al. (2000b), and Erickson (2003). We know a great deal about melanogenesis, including the biochemical pathways that generate pigment, the regulation of expression of the enzymes in that pathway, and the role of numerous growth factors that play a role in melanoblast maintenance, proliferation, and terminal differentiation (discussed in detail in many chapters in this textbook). Nevertheless, very little is known about the molecular events that specify the lineage as distinct from the other neural crest derivatives. The studies cited above suggest that there is a lineage switch from the nonmelanogenic lineages to the melanogenic lineage; that is, the premigratory neural crest cells may be specified first as neuro/gliogenic cells [or bipotent neural/glial and glial/melanogenic precursors (Le Douarin et al., 2004)] and subsequently some switch to the melanogenic fate. Further, this lineage switch occurs while the neural crest precursors are still resident in the neural tube or shortly after they detach from it. Studies in the chick (Jin et al., 2001) and in zebrafish (Dorsky et al., 1998, and see Fishes, above) show that this lineage switch is controlled, at a minimum, by two competing classes of signaling molecules: the Wnts and the BMPs. Specifically, Wnt 1/3a signaling specifies melanocytes whereas BMP 2/4 signaling specifies neural and glial cells. Despite the evidence that Wnt and BMP signaling is involved in fate specification, we do not know the cellular mechanisms by which this specification is achieved, or how Wnt- and BMPdependent signals compete with each other in this context. There are undoubtedly many genes involved in the specification of the melanogenic lineage downstream of Wnt 121

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signaling, including transcription factor cascades (Anderson, 1994; Groves and Anderson, 1996; Groves et al., 1995). Only a few transcription factors have been identified to date that regulate the melanogenic fate. Microphthalmia (Mitf), a basichelix-loop-helix-zipper transcription factor, has been shown in human, mouse, zebrafish (nacre, mitfa), and chickens to bind to the so-called M-box, an 11-bp sequence containing the core element CATGTG (Hodgkinson et al., 1993), in the upstream regulatory region of the tyrosinase and tyrosine-related protein 1 (TRP-1) genes and activate their transcription (review by Goding, 2000; see Mammals, below, for more detail). Downregulation of Mitf in chick neural crest cells using morpholinos eliminates melanoblasts and misexpression of Mitf by electroporation gives rise to excess pigments cells (Thomas and Erickson, unpublished). Mitf appears to be the primary transcription factor that drives melanoblast specification (Goding, 2000). Specification of different neural crest subpopulations at the trunk level occurs in sequential temporal waves, and yet many transcription factors that control neural, glial, and melanogenic lineages are expressed at the same time in the dorsal neural tube. For example, Sox10 and Pax3 are both required for the development of all three lineages in mouse, chick, and probably zebrafish (Britsch et al., 1998; Cheung et al., 2005; Dutton et al., 2001; McKeown et al., 2005; Lacosta et al., 2005; Potterf et al., 2001), so what prevents neuroblasts, glioblasts, and melanoblasts from developing simultaneously? One possibility is that a transcriptional repressor for melanogenesis is expressed early, which would prevent the early migratory crest from having melanogenic potential, whereas Wnt signaling may cause the downregulation of this putative repressor, which would then permit melanogenesis. We predict that the lineage switch from neural/glial to melanoblast occurs as a result of the downregulation of this repressor. A good candidate for such a transcriptional repressor is FoxD3. FoxD3 is a member of the winged-helix family of transcription factors (Kaestner et al., 2000). FoxD3 represses melanogenesis but is required for gliogenesis in the chick (Kos et al., 2001). The zebrafish homolog (formerly, fkh-6) similarly stimulates glial cell differentiation (Kelsh et al., 2000) and foxd3 mutants exhibit defects in neurons, glia, and ectomesenchymal lineages, though not chromatophore lineages (Stewart et al., 2005). In the neural epithelium, FoxD3 controls another lineage switch from neural crest to interneuron (Dottori et al., 2001), and in the early mouse embryo it is critical for maintaining embryonic stem cells (Hanna et al., 2002). Since FoxD3 in most experimental contexts acts as a transcriptional repressor (Freyaldenhoven et al., 1997b; Hromas et al., 1999), the most parsimonious hypothesis that accounts for the current data is that FoxD3 represses Mitf expression directly by binding to the Mitf promoter. Using a luciferase assay, chick FoxD3 expressed in B16 melanoma cells will reduce luciferase activity that is being driven by a chicken Mitf enhancer (Thomas and Erickson, unpublished results). This model further predicts that when FoxD3 is downregulated under the influence of Wnt signaling, Mitf is no longer repressed 122

by FoxD3, and is therefore transcribed by the transcriptional activators Tcf/Lef-1, Sox10, and Pax3 (Goding, 2000).

Mutants There is considerable variation in plumage and skin pigmentation among domestic fowl (reviewed in Smyth, 1990; Staples, 2001) and a variety of mutants with melanocyte defects have been isolated (e.g., Niwa et al., 2002) including a mutation in Mitf (Mochii et al., 1998). Congenital hypopigmentation resulting from inefficient melanoblast migration, differentiation, or both also has been shown in several instances (Jimbow et al., 1974). Some chicken strains have been used as model systems for studying pigment defects such as vitiligo (Boissy and Lamoreux, 1988). Most of these defects are characterized by the spontaneous depigmentation of normally pigmented birds and probably represent mutants in melanocyte physiology rather than true developmental mutants. One exception is the Silkie strain of chickens, which is characterized by extensive dermal and internal pigmentation caused by a prevalence of melanocytes in ectopic locations (Faraco et al., 2001; Hallet and Ferrand, 1984; Makita and Mochizuki, 1984; Makita and Tsuzuki, 1986). Unlike White Leghorn and quail melanoblasts, Silkie melanoblasts migrate ventrally, as well as dorsolaterally, during neural crest morphogenesis (Faraco et al., 2001; Reedy et al., 1998b). The abnormal migration of melanocytes in Silkie chickens does not reflect a defect in the neural crest, because Silkie neural tubes grafted into White Leghorn hosts never exhibit internal pigmentation, whereas quail neural tubes placed in Silkie embryos do generate internal melanocytes (Hallet and Ferrand, 1984). A molecular marker for barrier molecules, PNA (Oakley et al., 1991), indicates that inhibitory molecules are normally distributed beneath the dermamyotome in regions through which melanoblasts do not migrate. These PNA barriers are absent in the Silkie embryo (Faraco et al., 2001; de Freitas et al., 2003), suggesting that barriers develop during the late migration of melanoblasts that keep them in the dorsolateral path and prevent them from dispersing ventrally. Thus this mutant has identified a process late in melanoblast migration that constrains melanoblasts to the skin. Identification of the factors expressed in the Silkie environment responsible for melanocyte morphogenesis will be of considerable interest.

Mammals As in birds, the only pigment cell found in mammalian skin is the melanocyte. The neural crest origin of mammalian epidermal melanocytes was demonstrated by Rawles (1947, 1948) in an elegant study in which she grafted isolated mouse neural tube, somite, or skin (ectoderm plus dermis) of varying ages into the chick coelom and then scored for the presence of pigmented cells. Studies of mammalian development are hampered by several factors, including the inaccessibility of the embryo to experimental manipulation and higher maintenance costs compared with other vertebrate systems. Nevertheless, the mouse has proved to be a popular system for

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studying pigment cell development because of the ability to make and analyze transgenic embryos and the availability of many coat color mutants. In this section we discuss the development of pigmentation in mammals, focusing primarily on mouse and to a lesser extent on humans.

Morphogenesis of Murine Melanocytes Several studies have followed the early stages of trunk neural crest migration in mouse embryos (e.g., Erickson and Weston, 1983; Serbedzija et al., 1990). At the level of the forelimb, neural crest cells first emerge from the neural tube at day E8.5 (i.e., 8.5 days postcoitus). As in avian embryos, mammalian trunk neural crest cells take one of two migratory pathways: ventrally between the somite and neural tube, or dorsolaterally in the space between the dermamyotome and overlying somite (Rawles, 1947; Serbedzija et al., 1990). Unlike the situation in avians, however, the entry of mouse neural crest cells into the dorsolateral path does not appear to be delayed significantly with respect to ventral migration (Rawles, 1947; Serbedzija et al., 1990, 1994). By E10.5, the emigration of neural crest cells from the dorsal neural tube has ceased (Serbedzija et al., 1990, 1994). Mayer (1973) recombined flank dermis and ectoderm from albino or normally pigmented mouse embryos to demonstrate that melanoblasts begin entering the epidermis at E12. This was subsequently confirmed using probes against the melanogenic enzyme dct to identify melanoblasts (Cable et al., 1995; Pavan and Tilghman, 1994; Steel et al., 1992). Interestingly, the first melanoblasts to enter the ectoderm are located lateral, as opposed to dorsal, to the neural tube (Pavan and Tilghman, 1994). This corresponds to the spatial pattern of the onset of dct immunoreactivity and suggests that entry into the epidermis depends on the cells reaching a particular point in melanogenesis. Once in the epidermis, melanoblasts migrate into the developing hair follicles, and by E16 premelanosomes are detectable by the dopa reaction (reviewed in Hirobe, 1995). More recently, several studies have used mice that express the lacZ reporter under the control of the Dct promoter/ enhancer elements to track murine melanoblasts more carefully. For example, Wilkie and coworkers (2002) used Dct-lacZ transgenic mice to study the spatial and temporal patterns of melanoblast appearance and migration. They noted that melanoblasts do not arise uniformly along the length of the neural tube. Rather, a large pool of melanoblasts is present in the cervical region by E11.5, and significantly fewer melanoblasts are found in the trunk. The cervical pool appears to be the source of craniofacial melanocytes, as no melanoblasts were found to originate in rostral regions. Also using Dct-lacZ reporter mice, Jordan and Jackson (2000b) found evidence that there is a kit-independent population of melanoblasts present in the trunk between E15.5 and E16.5, and that these cells are capable of colonizing areas of the flank that are otherwise devoid of melanoblasts in the Ph mutants. The ontogeny of these cells is as yet unclear, but if confirmed this is a very interesting observation because of the

obvious parallels reported in zebrafish (discussed in Fish, above). Distribution and patterning of pigment cells is controlled not only by guidance molecules but also by signaling systems that control cell proliferation and cell survival. In the mouse there is, as yet, little information about the molecular nature of directed migration of melanoblasts. However, two general classes of mouse mutants have identified critical regulators of melanoblast proliferation and survival that in turn affect patterning: piebald/lethal spotting and Steel/Dominant spotting. piebald mutants display a range of white spotting depending on the allele. Only neural crest-derived melanocytes are affected, however, as retinal pigmentation is normal (Silvers, 1979). In addition to white spotting, piebald mutants exhibit dramatically reduced numbers of two other neural crest derivatives: the cochlear melanocytes of the inner ear, and the enteric ganglia of the myenteric plexus (Deol, 1967, 1970; Lane, 1966; Silvers, 1979; Webster, 1973). In homozygotes of the most severe allele, piebald-lethal (sl), this results in deformities of the cochlear duct and in megacolon, the latter of which causes neonatal death. The product of the s locus was identified as the endothelinB receptor (Ednrb) (Greenstein et al., 1994; Hosoda et al., 1994). Analysis of the Ednrb gene in piebald mutants provides an explanation for the variation in allelic severity; whereas the entire Ednrb gene is deleted in the sl allele, the less severe s allele is structurally normal but expressed at much lower levels compared to wild type (Hosoda et al., 1994). Interestingly, early tissue recombination experiments (Mayer 1965, 1967a, b, 1977) suggested that although the s phenotype is largely cell autonomous, there were non-cell autonomous effects as well. More recent tissue recombination experiments by Lou and coworkers (2004) support this conclusion. Pavan and Tilghman (1994) demonstrated that the white spotting phenotype in sl mutants is manifested very early in development by an almost complete lack of melanoblasts in the trunk. Experiments by Shin and coworkers (1999) using a conditional allele confirmed that melanocytes are dependent upon Ednrb very early in their development, between E10 and E12, consistent with Ednrb expression in the head fold and future dorsal neuroepithelium prior to neural crest dispersal (Tilghman et al., 1995). What happens to melanocyte precursors in the absence of Ednrb function? Recent studies by Lee et al. (2003) have shown that melanoblasts form and even begin to disperse from the neural tube in Ednrb-null embryos. However, Ednrb-null melanoblasts do not migrate very far along the dorsolateral pathway and eventually disappear. Whether these cells ultimately die or simply fail to complete melanogenesis remains unclear. It should also be noted that in the chicken embryo (see above), melanoblasts chemotact toward endothelin-3. Whether endothelins provide directional cues to murine melanoblasts has not been explored. The identification of Ednrb as the gene at the piebald locus suggested that one or more of the endothelins might play a role 123

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in melanocyte development. The phenotype of mutants at the lethal spotting (ls) locus is virtually identical to that of piebald mutants, including white spotting and megacolon (Silvers, 1979). Baynash et al. (1994) demonstrated that the product of the ls locus is EDN-3. In addition, they showed that ls mutants carry a point mutation that prevents normal proteolytic processing of the inactive polypeptide precursor of EDN-3. The other mouse mutants that have provided important insights into morphogenesis are Dominant spotting (W) and Steel (Sl). W and Sl mutants are phenocopies of one another and are characterized by being sterile, anemic, lacking in mature mast cells, and devoid of epidermal pigmentation (Silvers, 1979). These deficiencies are caused by the abnormal development of three stem cell populations: the germ cells, hematopoietic precursor cells, and the neural crest [Silvers (1979) and references therein; reviewed in Fleischman (1993), Morrison-Graham and Takahashi (1993)]. Early work demonstrated that the Sl mutation, but not the W mutation, could be rescued by a “normal” environment, implying that the W mutation was cell autonomous whereas the Sl mutation was not (Mayer, 1970; Mayer and Green, 1968). These results, together with the complementary mutant phenotypes, suggested that the W and Sl gene products act in a common pathway, possibly as a receptor–ligand complex. The cloning and characterization of these genes proved that this is, in fact, the case, with W encoding Kit and Sl encoding the Kit ligand, SLF (Anderson et al., 1990; Flanagan and Leder, 1990; Geissler et al., 1988; Huang et al., 1990; Martin et al., 1990; Williams et al., 1990; Zsebo et al., 1990a, b). Several studies have attempted to define exactly when melanoblasts require Kit and SLF activity (see Wehrle-Haller, 2003 for a comprehensive review). The patterns of expression of both Kit and SLF mRNAs suggest an early role in melanocyte development, with Kit+ melanoblasts detectable in the trunk as early as E10.5 (Keshet et al., 1991; Manova and Bacharova, 1991; Wehrle-Haller and Weston, 1995). Likewise, in utero injection of ACK2, a Kit function-blocking antibody, revealed that Kit is required at this time (Nishikawa et al., 1991; Yoshida et al., 1993). These results are consistent with several studies that examined the dependence of melanogenesis on SLF in vitro (Morrison-Graham and Weston, 1993; Murphy et al., 1992; Reid et al., 1995). In vitro and in vivo studies support the idea that mammalian Kit signaling is required for the survival of melanoblasts and their proper migration (Cable et al., 1995; Ito et al., 1999; Morrison-Graham and Weston, 1993; Murphy et al., 1992; Reid et al., 1995; Steel et al., 1992; Wehrle-Haller and Weston, 1995; Wehrle-Haller et al., 2001; reviewed in Yoshida et al., 2001) as in fish (Parichy et al., 1999; Rawls and Johnson, 2003). Later in development, Kit/SLF signaling is also required for the entry of melanoblasts into the developing hair follicle, having largely chemokinetic effects on melanoblasts (Jordan and Jackson, 2000a; Peters et al., 2002); the precise factors that guide these precursors to hair follicles remain unknown. Remaining unresolved is the importance of two forms of 124

SLF. Alternative splicing of the primary Sl transcript results in two isoforms, soluble and membrane bound, both of which are equally capable of binding to and activating Kit (Flanagan et al., 1991; Majumdar et al., 1994). In Sl dickey (Sld) mutants, the entire transmembrane and cytoplasmic domains of the gene are deleted. As a result, Sld mutants produce functional, soluble SLF but no transmembrane SLF (Brannan et al., 1991; Flanagan et al., 1991). Sld/Sld homozygotes are phenotypically identical to homozygotes for Sl null alleles (Silvers, 1979), indicating that soluble SLF alone is not sufficient for normal melanogenesis. Wehrle-Haller and Weston (1995) demonstrated that soluble SLF is, however, sufficient for the initial dispersal of melanoblasts along the dorsolateral pathway, while membrane-bound SLF is required for later survival, differentiation, or both. One explanation for the importance of membrane-bound SLF is that it would be expected to result in a higher local concentration of SLF because it cannot diffuse away from the cells producing it. An additional possibility, however, is that the membrane-bound form of SLF functions as a cell adhesion partner for Kit expressing melanoblasts. The ability of SLF/Kit binding to promote cell adhesion directly has been demonstrated in cell lines (Flanagan et al., 1991), primordial germ cells (Marziali et al., 1993), mast cells (Adachi et al., 1992; Kaneko et al., 1991), and megakaryocytes (Avraham et al., 1992).

Morphogenesis of Human Melanocytes Despite the evidence for a neural crest origin for pigment cells in other vertebrate species, many early studies of human pigmentation were prefaced on the theory that human melanocytes are derived from the basal cells of the epidermis, rather than the neural crest [Bloch (1917, 1921), as reported in Zimmerman and Becker (1959a)]. Information on human melanocyte development is reviewed in Holbrook et al. (1988) and Boissy and Nordlund (1995) and is summarized briefly here. The migration of neural crest through the lateral pathway brings melanoblasts first to the dermis. From the dermis, melanoblasts migrate into the epidermis to their eventual location on the basal lamina of the basal epithelial layer of the epidermis. Early work on the timing of appearance of fetal melanoblasts and melanocytes was limited to indirect approaches that utilized the reactivity of premelanosomes with reduced silver and subsequent identification of reactive cells by electron microscopy. Mature melanocytes have also been identified using the dopa reaction and morphologic criteria. More recently, the discovery of a cytoplasmic epitope shared by melanoma cells and fetal melanocytes, the HMB-45 monoclonal antibody (Gown et al., 1986), has allowed a reanalysis of data on the timing of appearance of human melanocytes (Holbrook et al., 1988). Early studies indicated that in human embryos neural crest migration is complete by seven weeks and that melanoblasts appear in the dermis by 11 weeks (Zimmerman and Becker, 1959a, b). The timing of appearance of melanoblasts in the epidermis has since been refined to earlier than seven weeks using HMB-45 staining (Holbrook et al., 1988). The density

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of this initial population of melanocytes observed by seven weeks is about 50% of the density observed at birth. By 10 weeks, melanocytes in the epidermis are observed to have premelanosomes based on both the reaction with reduced silver and direct electron microscopic observation (Fujita et al., 1970; Zimmerman and Becker, 1959b). It was thought that a large influx of melanoblasts occurred at the 12–14-week stage of development based on counts derived by reduced silver staining, but this has been reinterpreted by the HMB-45 staining data, which seem to imply that the levels of melanocytes thought to appear at the twelfth week are already in place by the eighth week and thus there is no new influx. There is a rough doubling of the number of melanocytes between weeks 10 and 14, with the numbers observed by HMB-45 staining much greater than the numbers observed by reduced silver staining. This may be due to mitosis of melanocytes that are already in the epidermis. The final number and distribution of melanocytes in the adult is relatively constant, although it can be affected by such stimuli as ultraviolet (UV) light. The distribution of melanocytes appears to be nonrandom (Holbrook et al., 1988), and each melanocyte is found with 36 keratinocytes in an epidermal melanin unit. Melanocytes that are associated with hair follicles arrive at their final location by following the downgrowth of epidermal cells in the developing hair germ (Mishima and Widlan, 1966). The production of melanin pigment developmentally begins at around 12 weeks based on both electron microscopic studies and dopa staining (Zimmerman and Becker, 1959a; Zimmerman and Cornbleet, 1948). The transfer of pigment to keratinocytes occurs late in the second trimester of development (Zimmerman and Cornbleet, 1948). The human piebald trait, characterized by a white forelock and patchy pigmentation, is likely due to a defect in melanocyte morphogenesis resulting in reduction or absence of these cells in discrete areas of the skin. Like white spotting in mice, piebaldism is congenital (Halaban and Moellmann, 1993) and many piebald patients have been shown to have mutations in the human homolog of Kit (e.g., Fleischman, 1992, 1993; Fleischman et al., 1991; Giebel and Spritz, 1991; Spritz et al., 1992, 1993; reviewed in Baxter et al., 2004). Defects in melanocyte and melanoblast morphogenesis resulting from disruptions in endothelin-3 and Ednrl signaling are likewise implicated in Hirschsprung disease in humans, which is also characterized by aganglionic megacolon (McCallion and Chakravarti, 2001).

Differentiation of Mammalian Melanocytes As discussed previously, it is well documented in both teleost and avian embryos that melanocyte lineage segregation begins very early in neural crest development, and that melanoblasts are specified prior to or at least coincident with their emigration from the neural tube. A recent study by Wilson et al. (2004) demonstrates that the same is true in murine embryos. Using mice that express lacZ under the control of the kit regulatory elements (Wlacz), they identified distinct melanogenic (kit+) and neurogenic (p75+) subpopulations of

premigratory neural crest cells in the midline of the dorsal neural tube prior to neural crest emigration. What signaling pathways are responsible for the initial segregation of the melanocyte lineage? There is strong evidence to suggest that Wnt/b-catenin signaling plays a critical role in this process. Wnt signaling promotes melanocyte formation in both birds and fish (see previous sections), and targeted disruption of the b-catenin gene in the dorsal neural tube of mouse embryos results in specific loss of the melanocyte and sensory ganglia neural crest subpopulations (Hari et al., 2002). The most likely activators of b-catenin in vivo are Wnt1 and Wnt3a, both of which are expressed in the dorsal neural tube and required for melanocyte formation (Ikeya et al., 1997). Dunn et al. (2005) have recently shown that forced overexpression of b-catenin, Wnt1, or Wnt3a in vitro results in an endothelin-dependent increase in melanocyte differentiation as well. Several transcription factors have been identified that are required for neural crest development overall (e.g., slug/snail, foxd3, and Pax 3) and are therefore important for melanocyte formation as well. The first transcription factor that is relatively specific for melanocyte differentiation was identified in the microphthalmia (mi) mutant. Both neural crest-derived and non-neural crest-derived melanocytes are affected in mi mutants. As a result, homozygotes of many alleles display white spotting, small, unpigmented eyes, and a lack of melanocytes in the inner ear (Silvers, 1979). Mice with certain mi alleles are also deficient in osteoclasts and mast cells (Silvers, 1979). mi encodes the transcription factor Mitf (Hodgkinson et al., 1993). Members of this family bind DNA as homo- or heterodimers (Hemesath et al., 1994), which explains why the original mi allele is semidominant and may also explain the high degree of intrallelic complementation and interallelic interactions that characterize mi mutants (Hodgkinson et al., 1993; Silvers, 1979; Steingrimsson et al., 1994). As previously discussed, Mitf regulates the expression of several melanocytespecific genes, including dopachrome tautomerase, tyrosinase, tyrosinase-related protein 1, Pmel17, and Melan-a (Baxter and Pavan, 2003; Bentley et al., 1994; Du et al., 2003; Yasumoto et al., 1995; Yokoyama et al., 1994). In transgenic mice carrying mutations in mi, melanoblasts form initially but fail to survive and reach the migration staging area (Hornyak et al., 2001). It is unlikely that this phenotype is related to Mitf’s role regulating expression of the melanin biosynthesis genes mentioned above, as mutations at the brown (TRP-1), slaty (dct), and albino (tyrosinase) loci do not result in melanocyte death but rather prevent the formation of normal melanin (see Silvers, 1979; Bennett, 1993; and references therein). However, McGill et al. (2002) have shown that Mitf is a critical transcriptional regulator of the antiapoptotic gene Bcl2 in melanocytes, suggesting that Mideficient melanoblasts die as a result of Bcl2 downregulation. Mitf can activate transcription in vitro through the M box found in the promoters of several melanocyte-specific genes (Bentley et al., 1994; Lowings et al., 1992; Shibahara et al., 1991; Yasumoto et al., 1994; Yavuzer and Goding, 1994). 125

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Moreover, misexpression of Mitf can convert nonmelanocytes into melanin-producing cells and rescue melanogenesis in mutant mice (Opdecamp et al., 1997). As in other vertebrates, Mitf appears to be one of the major links between Wnt signaling and melanogenesis in two ways. First, MITF is a direct target of Wnt3a-mediated LEF-1 activation (Takeda et al., 2000; see also Fishes). Second, the Mitf protein interacts in trans with LEF-1 to regulate expression of melanocyte-specific genes (Yasumoto et al., 2002). In humans, mutations in Mi are associated with Waardenburg’s syndrome type 2A (WS2A), an autosomal dominant condition exhibiting deafness and patchy, abnormal pigmentation (reviewed in Baxter et al., 2004; Hughes et al., 1994; Tassabehji et al., 1994b). The importance of Mitf in WS may be even more far reaching because two other genes linked to WS discussed below, sox10 and Pax3, regulate Mitf expression (Lee et al., 2000; Potterf et al., 2000; Verastegui et al., 2000). Thus, it is possible that the common etiologies between the various forms of WS ultimately result from a lack of Mitf expression. Sox10 is another transcription factor that is critical for melanocyte development (reviewed in Mollaaghababa and Pavan, 2003). Sox10 directly regulates the expression of MITF (Watanabe et al., 2002), and the sox10 protein also cooperates with the Mitf protein to regulate expression of Dct (Potterf et al., 2001; Ludwig et al., 2004). Sox10 is also associated with WS2A in humans. A third transcription factor in the melanogenesis regulatory network was identified in the Splotch mutant. The pigment defect in Splotch heterozygotes is characterized by white spotting on the feet, tail, and belly (Silvers, 1979). For most alleles, the homozygous phenotype is lethal due to defects in the neural crest and to the failure of the neural tube to close properly (Jackson, 1994; Silvers, 1979 and references therein). Other neural crest derivatives affected in homozygous splotch mutants include the spinal root ganglia and Schwann cells (Silvers, 1979). Interestingly, these derivatives are not noticeably affected in heterozygotes. The splotch locus corresponds to Pax3, encoding a member of the paired-box transcription factor family (reviewed in Chalepakis et al., 1993) that is expressed in the dorsal neural tube and early neural crest cells (Goulding et al., 1991). Melanoblasts do develop in splotch mice, but they are present in far lower numbers than normal (Hornyak et al., 2001). This may be due at least in part to the role of Pax3 in regulating expression of Mitf. Some patients with WS1 have been shown to have mutations in the human homolog of Pax3 (e.g., Tassabehji et al., 1992, 1994a). A recent report shows that Pax3 activates Mitf, while simultaneously competing with Mitf for sites in the dct promoter. Wnt signaling and b-catenin activation relieve this repression, allowing melanogenesis to proceed. Thus, Pax3 appears to prime cells for the differentiation whereas Wnt signaling allows the cells to proceed down this pathway (Lang et al., 2005).

Mutants Much of what we know about melanocyte development in mammals has come from studies of the over 70 mouse pigment 126

mutants. A detailed discussion of the development and initial characterization of these lines can be found in Silver’s classical 1979 treatise, The Coat Colors of Mice. In general, mouse coat color mutants can be assigned to one of three categories: those that affect the subcellular structure of melanocytes, those that perturb the biochemical pathways of melanin synthesis, and those that disrupt normal melanocyte development. Mutants in the latter category typically display various degrees of white spotting, i.e., an absence of melanocytes from distinct regions of the head, trunk, or rump. Comprehensive discussions of these coat color mutants can be found elsewhere (e.g., Baxter et al., 2004; Bennett, 1993; Jackson, 1994; Silvers, 1979). While many have been identified at the molecular level, much work remains to be done. For example, the patchwork mouse, which exhibits a salt-andpepper phenotype, may be especially interesting for understanding melanoblast colonization and survival in the hair follicle (Aubin-Houzelstein and Panthier, 1999; AubinHouzelstein et al., 1998). A final mutant worth mentioning for historical reasons is Patch, a homozygous-lethal dominant mutation. Heterozygotes have a sharply demarcated white belly spot that is variable in size, sometimes encircling the entire trunk (Silvers, 1979). The Patch mutation was mapped to a deletion that encompasses the gene for platelet-derived growth factor receptor-a (Pdgfra) (Stephenson et al., 1991). The Pdgfra gene is linked to Kit on chromosome 5 (Nagle et al., 1994). Interestingly, the Patch phenotype (MorrisonGraham et al., 1992) is similar to that of KitW–sh mutants. The KitW–sh mutation is a gross rearrangement of the 5¢ upstream region of the Kit gene that results in the ectopic misexpression of Kit in a variety of tissues, including the dermatome and developing limb (Duttlinger et al., 1993). It is hypothesized that this misexpression results in a reduction of the amount of SLF available to migrating melanoblasts (Besmer et al., 1993; Duttlinger et al., 1993). The phenotypic similarity of KitW–sh and Patch, together with the fact that the two genes are closely linked, led to the hypothesis that the Patch phenotype is due to the misexpression of Kit, rather than a reflection of melanoblast or neural crest dependence on Pdgfra activity in vivo. This hypothesis is supported by the observation that Kit mRNA is expressed ectopically in the dorsal neural tube and dermatome of Patch heterozygotes, despite the fact that Kit is not affected structurally by the Patch deletion (Duttlinger et al., 1995). The absence of pigment defects in targeted Pdgrfa knockout mice is consistent with a primary role for Kit misexpression in explaining the Patch phenotype (Soriano, 1997).

Perspectives The study of pigment cell development has been an area of active research in developmental biology for over a century because pigment cells are so readily identifiable and pigment patterns vary strikingly both within and among vertebrate

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taxa. Until recently, embryologists studying pigment cell development were limited to careful descriptive analyses or physical perturbations of developing embryos. Now, these techniques are complemented by a growing understanding of the factors at the molecular and cellular level that regulate the morphogenetic behavior and differentiation of pigment cells. A comparative approach to studying pigment cell development has been, and will continue to be, absolutely critical for a thorough understanding of the subject. Although each system has it own unique advantages, no single system is ideal for studying both the molecular and cellular aspects of neural crest and pigment cell development. For example, identification of the molecular basis for some of the mouse coat color mutants has provided definitive evidence for specific factors involved in melanocyte development. Nevertheless, murine embryos typically are not an experimentally tractable system, so characterizing the cellular effects of these factors is difficult. Conversely, amphibian and avian embryos are highly accessible to experimental manipulation, but are only beginning to emerge as useful systems for genetic or molecular analyses. For this reason zebrafish has become a powerful model system to study pigment patterns since they are both genetically and experimentally amenable. Comparative studies of pigment cell development also are useful for studying questions of developmental biology in an evolutionary context. Pigment cells in all vertebrates are derived from the same progenitor cell population, the neural crest, and at least one type of pigment cell, the melanophore/cyte, utilizes a common pigment biosynthesis pathway across species. These commonalities, and considerable research over the last decade, suggest that specific molecules will play similar roles in the morphogenesis or differentiation of pigment cells in different taxa. Equally interesting, however, are those instances where this is not true. For example, the importance of Kit signaling in the early migration and survival of zebrafish melanophores and murine melanocytes is well established. However, the expression patterns of kit in amphibians and birds suggest this gene is not involved in the same events as in other vertebrates (Baker et al., 1995; Lecoin et al., 1995; Reedy et al., 2003). Discrete cells (pigment cells) from a well-defined progenitor cell population (the neural crest) will thus continue to provide intriguing experimental materials for cell and developmental biologists in their quest to understand how eukaryotic cells differentiate and evolve.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

6

Melanoblast Development and Associated Disorders Richard A. Spritz

As discussed in Chapter 29, genetic disorders of melanocyte development, such as piebaldism and Waardenburg syndrome, are associated with heterogeneous distribution of pigmentation resulting from abnormal distribution of melanocytes during embryogenesis. These disorders result from mutations in a complex network of interacting genes that regulate differentiation and development of the neural crest, and the melanoblast/melanocyte lineage in particular, in both man and the laboratory mouse. Indeed, elucidation of the molecular basis of these disorders has provided a paradigm for the use of human and mouse genetics in combination to dissect the molecular etiology of genetic diseases. In the following discussion of these human genetic abnormalities of melanocyte development, MIM numbers for each disorder are from Online Mendelian Inheritance in Man (2004).

Piebaldism (MIM #172800) As discussed in Chapter 29, piebaldism is an autosomal dominant disorder characterized clinically by congenital patches of white skin and white hair, principally located on the frontal scalp, forehead, ventral chest and abdomen, and extremities (cf. Campbell and Swift, 1962; Comings and Odland, 1966; Cooke, 1952; Froggat, 1951; Geerts-Marchandise and Lachapelle, 1977; Grupper et al., 1970; Jahr and McIntire, 1954; Keeler, 1934; Küster, 1987; Pearson, 1913; Smith and Schulz, 1955; Storkan and Hosmer, 1954; Sundfor, 1939; Taylor and Robinson, 1976; Winship et al., 1991), which typically lack most or all melanocytes (Breathnach et al., 1965; Comings and Odland, 1966; Fukai et al., 1989; Jimbow et al., 1975). Piebaldism is now known to result from mutations of the KIT gene, which encodes the cell surface receptor for a growth factor ligand that plays a key role in melanogenesis, gametogenesis, and the early stages of hematopoiesis. The critical clues to the molecular basis of human piebaldism came from studies of what ultimately proved to be the homologous disorder of mice (reviewed by Spritz, 1992, 1993; Spritz and Hearing, 1994). W mutant mice, with so-called dominant white spotting, exhibit patches of white skin and fur very similar to the patches of leukoderma and poliosis of human piebaldism, as well as the features of hypoplastic anemia and sterility (reviewed by Bennett and Lamoreux, 2003; Lyon and Searle, 1989; Silvers, 1979). Molecular genetic studies demonstrated that mouse dominant white spot140

ting results from deletions or point mutations of the c-Kit (officially, Kit) protooncogene (Chabot et al., 1988; Geissler et al., 1988; Hayashi et al., 1991; Larue et al., 1992; Nocka et al., 1990; Reith et al., 1990; Tan et al., 1990; reviewed by Morrison-Graham and Takahashi, 1993). The Kit gene, originally identified in the genome of the HZ4-feline sarcoma virus (Besmer et al., 1986), encodes the cell surface receptor for an embryonic growth factor, usually called “stem cell factor” (SCF), although also variously referred to as “Steel factor” (SLF), “mast cell growth factor” (MCGF), and “Kit ligand” (KL) (Copeland et al., 1990; Flanagan and Leder, 1990; Huang et al., 1990; Murphy et al., 1992; Zsebo et al., 1990; reviewed by Morrison-Graham and Takahashi, 1993). SCF is the product of the Steel (Sl) locus of mice, and both Sl and W mutant mice display similar piebald-like phenotypes. The KIT receptor is expressed on the surface of melanocytes and, whereas Sl mutant melanocytes develop normally when transplanted to normal skin, because normal ligand is available, W mutant melanocytes do not, as they have an intrinsic defect of receptor function. The KIT protein is a member of the tyrosine kinase family of transmembrane receptors. As illustrated in Fig. 6.1, the KIT polypeptide consists of an amino-terminal, extracellular, ligand-binding, receptor domain composed of five immunoglobulin-type domain repeats, a short transmembrane domain, and an intracellular domain consisting of a bipartite tyrosine kinase domain followed by a carboxyl-terminal tail. Within the extracellular domain, the first three immunoglobulin-type repeats bind the SCF ligand (Blechman et al., 1993; Jiang et al., 2000; Lev et al., 1993), the fourth may (or may not) be involved in receptor dimerization (Blechman et al., 1995; Lemmon et al., 1997), and the fifth is required for proteolytic release of the KIT receptor from the cell surface (Broudy et al., 2001). The stoichiometry of interaction between SCF and the extracellular domain of the KIT receptor is apparently monovalent (Lev et al., 1992), each subunit of the SCF dimer binding to one KIT chain on the cell surface (Jiang et al., 2000; Lemmon et al., 1997; Philo et al., 1996) and thereby inducing dimerization of the KIT receptor within the cell membrane and consequent activation of its intracellular tyrosine kinase (Lev et al., 1992). This, in turn, results in autophosphorylation of specific tyrosine residues within the KIT kinase domain, enhancing the binding of various proteins, including phosphatidylinositol 3¢ kinase and phospholipase Cg1, which act as downstream mediators of the mitogenic signal in the KIT-dependent pathway of signal transduction

MELANOBLAST DEVELOPMENT AND ASSOCIATED DISORDERS

Fig. 6.1. Schematic diagram of the human KIT polypeptide and locations of mutations that are associated with human diseases. The amino-terminal extracellular leader sequence (LS) and penta-repetitive ligandbinding domain, the transmembrane domain (TM), and the intracellular bipartite tyrosine kinase domain are indicated. Missense amino acid substitutions are indicated by filled circles, frameshifts by filled diamonds, nonsense mutations by filled triangles, splice junction mutations by zig-zags, and in-frame deletions by brackets. AML, acute myelogenous leukemia; GIST, gastrointestinal stromal tumor.

(reviewed by Morrison-Graham and Takahashi, 1993), Src family members, the JAK/STAT pathway, and the Ras–Raf–MAP kinase cascade (reviewed by Ronnstrand, 2004; Taylor and Metcalfe, 2000). The specific mechanism by which reduced KIT function results in the piebald phenotype is not known exactly. Injection of anti-kit antibodies into developing mouse embryos resulted in W-like pigmentary anomalies (Nishikawa et al., 1991), and incubation of cultured human melanocytes with antisense KIT oligonucleotide significantly reduced melanocyte proliferation but not cell survival (Spritz et al., 1994a). It is clear that, in the mouse and zebrafish, and presumably also the human, KIT function is required for normal proliferation and survival of both melanoblasts and melanocytes both before and after migration from the neural crest (Mackenzie et al., 1997; Nishikawa et al., 1991; Parichy et al., 1999; Rawls and Johnson, 2003; Spritz et al., 1994a; reviewed by Yoshida et al., 2001). Reduced KIT-dependent signal transduction results in a greatly reduced number of dermal melanocytes in the regions of involved skin. Nevertheless, the precise basis for the aberrant regional distribution of skin melanocytes, and particularly the typical pattern seen in human piebaldism, remains unknown. The first clue to the molecular genetic basis of human piebaldism came from the observation of several patients with sporadic, nonfamilial piebaldism associated with de novo chromosomal deletions involving region 4q12 (Funderburk and Crandall, 1974; Hoo et al., 1986; Lacassie et al., 1977; Yamamoto et al., 1989). The human KIT gene was subsequently mapped to approximately the same chromosomal location (d’Auriol et al., 1988; Yarden et al., 1987), and this

coincidence suggested that human piebaldism might, like mouse “dominant white spotting,” also result from abnormalities of the KIT gene. In fact, the human KIT gene consists of 21 exons spanning almost 83 kb at chromosome segment 4q12 (Giebel et al., 1992; Vandenbark et al., 1992) and is part of a gene cluster encoding a family of closely related “type III” receptor tyrosine kinases, organized PDGFRAKIT-KDR (Spritz et al., 1994b). PDGFRA encodes the platelet-derived growth factor receptor a chain, and KDR the so-called “vascular endothelial growth factor receptor 2” (VEGFR2). The first direct evidence for the involvement of KIT mutations in human piebaldism was provided by Giebel and Spritz (1991), who studied KIT as a candidate gene in an extended kindred with piebaldism and identified a missense mutation, Gly664Arg, within the highly conserved intracellular tyrosine kinase domain. This mutation showed complete genetic linkage with the piebald phenotype in this family. Further support came from analyses of two patients with sporadic piebaldism associated with de novo cytogenetic deletions, one of 4q12–q21.1 (Yamamoto et al., 1989) and the other submicroscopic; in both patients, the KIT gene and the adjacent PDGFRA gene were both deleted (Fleischman et al., 1991; Spritz et al., 1992a). Subsequent work has led to the identification of at least 37 different point mutations and a number of deletions of the KIT gene in patients with piebaldism and their families (Ezoe et al., 1995; Fleischman, 1992; Fleischman et al., 1996; Giebel and Spritz, 1991; Murakami et al., 2004; Nomura et al., 1998; Richards et al., 2001; Riva et al. 1995; Shears et al., 2004; Spritz et al., 1992b, c, 1993a, b; Syrris et al., 2000, 2002) (Fig. 6.1). About two-thirds of the patients 141

CHAPTER 6

studied have identifiable point mutations, and many of the rest appear to have KIT gene deletions (Ezoe et al., 1995; Fleischman et al., 1991). It is now clear that piebaldism is caused by reduced function of the KIT receptor, resulting in decreased KIT-dependent signaling and consequent abnormal proliferation, survival, or distribution of melanoblasts and melanocytes during embryologic development. Correlation of point mutations of the human KIT gene with the associated clinical phenotypes has proved highly instructive in ascertaining the molecular pathogenic mechanisms underlying piebaldism, as there appears to be a clear and relatively robust genotype–phenotype relationship (Spritz et al. 1992c). As shown in Figure 6.1, KIT point mutations associated with piebaldism can be grouped into four general classes, each associated with phenotypes of differing severity. Thus, a hierarchical paradigm of pathologic human KIT mutations appears to account for a graded series of dominant phenotypes in human piebaldism, correlating reasonably well with predicted effects on KIT-dependent function. The first class of KIT gene mutations, all associated with relatively severe piebald phenotypes, consists of missense substitutions located within the highly conserved tyrosine kinase domain. Most of these involve amino acid residues that have been particularly conserved during evolution, and several correspond closely to similar mutations in W mutant mice (reviewed by Morrison-Graham and Takahashi, 1993; Spritz, 1992, 1993; Spritz and Hearing, 1994), underscoring the importance of these sites to the function of the KIT receptor. All these KIT missense substitutions are associated with relatively severe piebald phenotypes. This is a consequence of the fact that the KIT kinase is only activated on dimerization of the receptor, and KIT receptor heterodimers consisting of one normal KIT polypeptide and one abnormal KIT polypeptide are inactive. Thus, patients heterozygous for these “dominantnegative” KIT missense mutant alleles have only one-quarter of the normal amount of KIT receptor dimer and, accordingly, they exhibit relatively severe piebald phenotypes. Even these patients exhibit something of a spectrum of clinical severity, with rare mutations tending to result in extreme phenotypes in which depigmentation may extend to the dorsal aspects of the trunk and head (Fleischman, 1992). The second class of KIT mutations is associated with a relatively wide range of piebald phenotypes, from severe to mild even within an individual family. This group consists of a variety of different nonsense, frameshift, and splice junction mutations, all located distally, within the part of the gene encoding the intracellular portions of the KIT protein that constitutes the tyrosine kinase domain. All these mutations would result in premature termination of translation, truncating the nascent KIT polypeptide distally, within the intracellular tyrosine kinase domain. Clearly, these mutations would abolish the expression of normal KIT polypeptide from this allele. However, such truncated KIT receptors can still bind SLF and even form dimers, dominant negatively inhibiting function of the normal KIT polypeptide (Lev et al., 1992). As both the 142

incompletely translated KIT mRNA and the truncated KIT polypeptide are most likely relatively unstable, even though these truncated KIT polypeptides act dominant negatively, they are present at less than a full dose. Accordingly, this class of mutations presumably reduces KIT function to between onequarter and one-half of normal, accounting for the generally intermediate but highly variable piebald phenotype. The third class of KIT mutations is a group of frameshifts and other “loss of function” mutations that completely eliminate the production of KIT protein by the defective allele. Patients heterozygous for these “loss of function” mutant alleles thus express half the normal amount of KIT receptor, resulting in haploinsufficiency for KIT-dependent signal transduction. These KIT mutations are thus associated with relatively mild piebald phenotypes, in which the depigmented patches are usually small, and poliosis is often absent, although affected individuals frequently experience early graying of the hair. In fact, some members of families with KIT mutations of this type have been so mildly affected that the clinical diagnosis was only made subsequent to DNA diagnosis. Interestingly, one of this group of mutations, a four-base deletion (delCAGT) involving codons 250–251, has been observed in at least three unrelated families (Spritz, 1993) and appears to be a recurrent deletion resulting from a direct sequence repetition in this region. The fourth class of KIT mutations constitutes a series of missense substitutions located in the extracellular ligandbinding domain (Fig. 6.1). All these mutations are associated with mild clinical phenotypes (in fact, at least one, Met318Gly, is not pathologic at all). The extracellular domain of the KIT polypeptide consists of five immunoglobulin-like repeats (Yarden et al., 1987), the first three of which bind the SCF ligand (Blechman et al., 1993; Jiang et al., 2000; Lev et al., 1993), and structural redundancy in this region may result in some functional redundancy, thus rendering some amino acid substitutions in this region phenotypically silent. The identification of mutations in the KIT gene in human piebaldism opened the door to relatively simple and accurate DNA-based diagnosis and prenatal diagnosis in selected cases. DNA-based molecular diagnosis and even prenatal diagnosis of piebaldism are now routinely accomplished. Sanchez-Martin et al. (2003) have reported heterozygous de novo deletions of the SNAI2 gene (MIM #602150) on chromosome 8q11 in some patients with sporadic piebaldism. SNAI2 encodes Slug, a zinc finger transcription factor expressed in migratory neural crest cells (Perez-Losada et al., 2002). Although SNAI2 is thus an attractive candidate for a human piebaldism locus, it is puzzling that these same authors have also reported de novo homozygous SNAI2 deletions in patients with sporadic Waardenburg syndrome type 2 (Sanchez-Martin et al., 2002).

Related Disorders KIT was first identified as the active oncogene of the HZ-4 feline sarcoma virus, suggesting that, in contrast to piebaldism, in which KIT expression is reduced, in certain forms of

MELANOBLAST DEVELOPMENT AND ASSOCIATED DISORDERS

human neoplasia or hyperproliferative states, KIT expression might be elevated or dysregulated. Furitsu and coworkers (1993) identified two KIT gene mutations, Val560Gly and Asp816Val, in the human mast cell leukemia line HMC-1; the Asp816Val substitution constitutively activates the KIT receptor in these cells. Subsequent studies (Buttner et al., 1998; Longley et al., 1996; Nagata et al., 1995) showed that de novo activating mutations of KIT account for most cases of acquired adult mastocytosis and mastocytoma (reviewed by Tsujimura et al., 1997). Activating mutations of KIT have also been found in a small fraction of patients with various types of myeloid leukemias (Beghini et al., 1998; Kimura et al., 1997; Nakata et al., 1995), including about half of adults with core binding factor acute myeloid leukemia (CBFL) (Beghini et al., 2004). Activating mutations of KIT have also been found in a small fraction of gastrointestinal stromal tumors (GIST; MIM #606764) (Hirota et al., 1998; Lasota et al., 1999, 2000; Metaxa-Mariatou et al., 2004; Nakahara et al., 1998; Nishida et al., 1998), but have not been found in most other solid tumors (Chilton-Macneill et al., 2004; Lasota et al., 1999; Lassus et al., 2004; Miettinen et al., 2000; Sihto et al., 2005). In the mouse, a white spotting disorder similar to W is caused by mutations at the Steel (Sl) locus, which encodes SCF, the ligand for the KIT receptor (Lyon and Searle, 1989; Silvers, 1979). The human homolog of the mouse Sl locus gene is called KITLG (for “Kit ligand”). Although aberrant expression of KITLG has been observed in cutaneous mastocytosis (Longley et al., 1993), no abnormalities of the MGF gene have been found in human patients with the piebald phenotype (Ezoe et al., 1995). There may thus be no human homolog to the Steel mutations of mice. Perhaps the function of SCF is redundant in humans, and human KITLG gene mutations thus result in no clinical phenotype, in a clinical phenotype that does not include piebald-like pigmentary anomalies or, alternatively, in humans, KITLG gene mutations might be lethal. Another mouse locus closely related to the Kit/W complex is Patch (Ph), characterized by a dominant white spotting phenotype similar to that of W (Lyon and Searle, 1989; Silvers, 1979). The original Ph mutation was found to comprise a 400kb chromosomal deletion that does not involve the Kit gene at all, but instead deletes the adjacent Pdgfra gene (Nagle et al., 1994; Smith et al., 1991; Stephenson et al., 1991). Although it was therefore initially suggested that white spotting in Ph mice might result from deletion of the Pdgfra gene itself, it was subsequently found that the Ph deletion alters the normal developmental pattern of expression of the cis-linked Kit allele (Duttlinger et al., 1995). Similarly, the mouse rumpwhite (Rw) locus, which is also associated with dominant white spotting (Lyon and Searle, 1989; Silvers, 1979), constitutes a chromosomal inversion that includes Kit (Nagle et al., 1994; Stephenson et al., 1994) and likewise alters the expression of the cis-linked Kit allele during mouse development. Nevertheless, some human GISTs have recently been found to have activating mutations of PDGFRA (Chompret et al.,

2004; Hirota et al., 2003; Lasota et al., 2004; Sakurai et al., 2004; Yamamoto et al., 2004), raising the possibility that the functions of PDGFRA and KIT may overlap to some extent, thus resurrecting the possibility that deletion of Pdgfra might contribute directly to the murine Ph mutant phenotype.

Waardenburg Syndrome Type I (WS1; MIM #193500), Waardenburg Syndrome Type III (WS3; Klein–Waardenburg Syndrome; MIM #148820), and Craniofacial–Deafness–Hand Syndrome (MIM #122880) As discussed in Chapter 29, WS1 is an autosomal dominant disorder characterized clinically by piebald-like hypopigmented patches, heterochromia irides, dystopia canthorum, and sensorineural deafness. WS3 is similar to WS1, but is additionally associated with musculoskeletal abnormalities. Craniofacial–deafness–hand syndrome is similar to WS1, but involves more severe abnormalities of craniofacial development and includes developmental anomalies of the hand. All three of these disorders are now know to be allelic, resulting from mutations of a single gene, PAX3. Elucidation of the molecular bases of WS1 and WS3 was facilitated by studies of a similar disorder of mouse, Splotch (Sp) (reviewed by Pierpont and Erickson, 1993; Spritz, 1993; Spritz and Hearing, 1994). Splotch mice exhibit piebald-like patches of nonpigmented skin and hair (Auerbach, 1954; Epstein et al., 1991, 1993; Goulding et al., 1991, 1993; Moase and Trasler, 1992; Russell, 1947; Vogan et al., 1993), probably due to a reduction in the number of primordial neural crest melanoblasts or to their delayed migration so that melanoblasts fail to reach the affected regions before hair follicles develop (reviewed by Tachibana et al., 2003). The gene for human WS1 was first mapped to distal chromosome 2q on the basis of a patient with a chromosomal inversion of 2q35–q37.3 (Ishikiriyama et al., 1989). Subsequent genetic linkage analyses confirmed localization of the WS1 gene to this chromosomal region (Asher et al., 1991; Foy et al., 1990), which corresponds to the location of the murine Splotch locus on mouse chromosome 1, suggesting that human WS1 and mouse Splotch might be homologous. Analysis of the mouse Sp2H mutation showed it to consist of a deletion in the Pax3 gene (Epstein et al., 1991), one of a family of socalled “paired box” (PAX) genes involved in embryologic development (Burri et al., 1989). Two reports refined the localization of the human PAX3 gene to 2q35–q36.2 (Lu-Kuo et al., 1993; Tsukamoto et al., 1992), and Tassabehji et al. (1992) and Baldwin et al. (1992) quickly identified pathologic PAX3 mutations in patients with WS1. More than 50 different point mutations of the PAX3 gene have since been identified in patients with Waardenburg syndrome (Baldwin et al., 1994, 1995; Butt et al., 1994; Fortin et al., 1997; Hoth et al., 1993; Morell et al., 1992, 1993; Pardono et al., 2003; Tassabehji et al., 1993, 1994a, 1995; Wildhardt et al., 1996; 143

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Wollnik et al., 2003; Zlotogora et al., 1995), including missense substitutions, nonsense mutations, frameshifts and small in-frame deletions, and splice junction mutations. WS1 is one of the autosomal dominant disorders for which there is evidence of paternal age effect on the occurrence of new mutations (Jones et al., 1975). The PAX3 gene product is a transcription factor that is critical for activating melanoblasts and other cellular elements to proliferate or to begin migration from the neural crest (Stuart and Gruss, 1995). The human PAX3 polypeptide contains four principal structural motifs: a “paired box” domain, a homeobox domain, a conserved octapeptide, and a serinethreonine-proline-rich carboxyl segment. The 128-amino-acid paired box domain, from which the name of the gene is derived, and the 60-amino-acid homeobox domain are both highly conserved amino acid sequence motifs present in a number of mammalian and Drosophila genes involved in controlling segmentation. These motifs form a helix–turn–helix structure and are both thought to mediate DNA binding. Expression of PAX3 is spatially and temporally regulated during development, with localized expression during formation of the embryonic primitive streak (Goulding et al., 1993), during differentiation of the dorsal neuroepithelium (Goulding et al., 1991), during migration of dermomyotomal cells from somites to limb buds (Bober et al., 1994; Franz et al., 1993), and in adult brain (Stoykova and Gruss, 1994). PAX3 acts in concert with SOX10 to trans-activate the promoter of the MITF gene (Bondurand et al., 2000; Watanabe et al., 1998), which encodes one of the key regulators of melanocyte development and melanogenesis (Hornyak et al., 2001; reviewed by Steingrimsson et al., 2004). PAX3 also controls a transcriptional cascade that is involved in regulating skeletal myogenesis (Ridgeway and Skerjanc, 2001). There has been considerable effort to correlate specific types of mutations of the PAX3 gene with the different clinical subtypes of Waardenburg syndrome, but with only limited success. Clearly, most mutations in the PAX3 gene result in the phenotype of WS1 (Baldwin et al., 1995; Tassabehji et al., 1995). It is apparent that simple loss of function of one allele will result in the WS1 phenotype due to haploinsufficiency for the PAX3 gene product. The importance of gene dosage in WS1 is further supported by the observation of several patients with WS1 who have cytogenetic rearrangements that involve PAX3 (Lu-Kuo et al., 1993; Pasteris et al., 1993; Tassabehji et al., 1994a; Tsukamoto et al., 1992; Wu et al., 1993). However, a small fraction of PAX3 mutations do not result in WS1, but instead result in the WS3 (Klein–Waardenburg) phenotype in which features of WS1 are associated with limb anomalies (Hoth et al., 1993; Milunsky et al., 1992; Pasteris et al., 1993; Tassabehji et al., 1995; Tekin et al., 2001). Some patients with WS3 have heterozygous PAX3 mutations, whereas other have homozygous PAX3 mutations (Ayme and Philip, 1995; Wollnik et al., 2003; Zlotogora et al., 1995). Yet other mutations in PAX3 result in “craniofacial–deafness– hand syndrome” (Asher et al., 1996). Thus, WS1, WS3, and craniofacial–deafness–hand syndrome are allelic, and the 144

interplay among functional domains in the PAX3 polypeptide is exceedingly complex (Chalepakis et al., 1994; DeStefano et al., 1998; Fortin et al., 1997), accounting for a remarkable diversity of clinical and developmental phenotypes and patterns of inheritance associated with PAX3 mutations in man. One patient with WS2 was reported to have a mutation in PAX3 (Tassabehji et al., 1993); however, it now seems likely that this patient was misclassified. No patients with WS2 have mutations in PAX3, and WS2 was found not to be genetically linked to markers from chromosome 2q (Farrer et al., 1992, 1994), indicating that genes responsible for WS2 are not located in this region of the genome. Indeed, it is now clear that at least some patients with WS2 have mutations of the MITF gene, located on chromosome 3p, and others have mutations in the WS2B, WS2C, and WS2D loci, located on chromosomes 1p, 8p, and 8q11 respectively (see below).

Related Disorders Surprisingly, the PAX3 gene also turns out to be rearranged in the t(2;13)(q35;q14) chromosomal translocation frequently observed in cases of alveolar rhabdomyosarcoma (MIM #268220), an aggressive solid tumor of childhood (Barr et al., 1993; Galili et al., 1993). In several such tumors, the chromosome 2 breakpoints were located within the fourth intervening sequence of the PAX3 gene, juxtaposing the upstream PAX3 DNA-binding motifs, including the paired box domain, conserved octapeptide, and homeobox domain, with a specific “forkhead domain” gene, FOXO1A (FKHR), located on chromosome 13q14.1, which is involved in regulating the growth of a number of different cell types (Medema et al., 2000; Modur et al., 2002; Nakamura et al., 2000). As a result, an abundant novel mRNA is transcribed that encodes a PAX3/FOXO1A fusion protein, which interferes with the normal control of striated muscle cell proliferation (Fredericks et al., 1995; Khan et al., 1999; Lagutine et al., 2002; Relaix et al., 2003; Scheidler et al., 1996; Sublett et al., 1995). Thus, the PAX3 gene appears to lead a double life: inherited loss of function mutations result in Waardenburg syndrome, and acquired gain of function mutations result in alveolar rhabdomyosarcoma, consistent with a role for PAX3 in the developmental regulation of skeletal myogenesis (Ridgeway and Skerjanc, 2001).

Waardenburg Syndrome Type II (WS2; MIM #193500) As discussed in Chapter 36, the phenotype of WS1 is very similar to that of WS1 except that patients with WS2 lack the clinical feature of dystopia canthorum (Arias, 1971; Farrer et al., 1992). Human WS2 can be caused by mutations in at least four different genes, only two of which have thus far been identified. WS2A (MIM #193510) results from mutations in the MITF gene on chromosome 3p. WS2B (MIM #600193) maps to chromosome 1p and WS2C (MIM #606662) to chro-

MELANOBLAST DEVELOPMENT AND ASSOCIATED DISORDERS

mosome 8p. WS2D (MIM #608890) results from mutations in SNAI2 on chromosome 8q11. Genetic linkage analysis first demonstrated that WS2 did not result from mutations in the PAX3 gene (Farrer et al., 1992, 1994), and subsequent studies demonstrated genetic linkage between some (but not all) cases of WS2 (WS2A) and the MITF gene (Hughes et al., 1994), the human homolog of the mouse microphthalmia (mi) locus (Tachibana et al., 1994), located in human chromosome segment 3p12. Pathologic mutations of the human MITF gene have since been identified in a number of patients with WS2A (Lalwani et al., 1998; Morell et al., 1997; Nobukuni et al., 1996; Smith et al., 2000; Tassabehji et al., 1994b, 1995). Moreover, re-evaluation of a family with “Tietz albinism–deafness syndrome” (Tietz, 1963; MIM #103500) demonstrated that this family segregates a pathologic MITF mutation (Smith et al., 2000). The MITF gene product is a homodimeric transcription factor of the basic helix–loop–helix leucine zipper class (Hemesath et al., 1994; Hodgkinson et al., 1993). In addition to its involvement in regulating melanocyte development (reviewed by Steingrimsson et al., 2004), the MITF protein also appears to play a role in regulating melanocyte function, as it is critical for transcriptional activation of the genes encoding tyrosinase (TYR; Bentley et al., 1994; Tachibana et al., 1996; Yasumoto et al., 1994) and TRP-1 (TYRP1; Yavuzer et al., 1995). Indeed, Morell et al. (1997) showed that the phenotype of WS2A plus ocular albinism (OA) apparently results from “digenic” inheritance, the coincidence of a MITF gene mutation and the R402Q allele of tyrosinase that is associated with reduced enzymatic activity (Tripathi et al., 1991), with resultant downregulation of already reduced tyrosinase catalytic function. The phenotype of the microphthalmia mutant mouse includes hypopigmentation, reduced eye size and cataracts, osteopetrosis, and early onset of deafness, and various different alleles can be either dominant or recessive (Lyon and Searle, 1989; Moore, 1995; Silvers, 1979). Interestingly, although only a modest number of human MITF mutations have been identified in patients with WS2, one of these is precisely homologous to the mouse mi mutation itself, an inframe deletion of one of a run of four arginines in the basic domain (Tassabehji et al., 1995), and is associated with a somewhat atypical clinical phenotype. Dominant mouse mi alleles operate via dominant negative effects (Hemesath et al., 1994; Steingrimsson et al., 1994), whereas mi mutations that prevent dimerization are recessive. Nevertheless, it is clear that, in humans, WS2A can result from loss-of-function mutations of MITF, which presumably result in haploinsufficiency for MITF function. It is intriguing that patients with WS1 resulting from PAX3 mutations and patients with WS2A due to MITF mutations exhibit virtually identical clinical phenotypes except for the absence of dystopia canthorum in WS2. It may be that the embryologic processes responsible for dystopia canthorum are more sensitive to dosage of the PAX3 protein than of the MITF protein. Even within WS1 families with known PAX3

mutations, not all affected individuals exhibit dystopia canthorum, and it has been suggested that variation at some other genetic locus may be responsible for much of this clinical variation in WS1 (Reynolds et al., 1996). Overall, only a relatively small fraction of families with WS2 appears to have mutations in the MITF gene (Hughes et al., 1994; Tassabehji et al., 1995). Lalwani et al. (1994) mapped a second WS2 locus, designated WS2B, to chromosome 1p21–p13.3. Selicorni et al. (2002) assigned WS2C on the basis of a family with a translocation breakpoint involving 8p23. WS2D corresponds to the SNAI2 locus on 8q11 (Sanchez-Martin et al., 2002), with these authors detecting homozygous SNAI2 deletions in two of 38 unrelated WS2 patients who lacked MITF mutations. SNAI2 encodes Slug, a zinc finger transcription factor expressed in migratory neural crest cells (Perez-Losada et al., 2002). Whereas SNAI2 is thus an attractive Waardenburg syndrome candidate locus, this result is puzzling as these same authors have reported de novo heterozygous SNAI2 deletions in patients with sporadic piebaldism (Sanchez-Martin et al., 2003). Yet another WS locus may reside on chromosome 13q, as van Camp et al. (1995) reported a patient with both WS2 and Hirschsprung disease with a de novo interstitial cytogenetic deletion of chromosome 13q. These same authors also reported a family in which two brothers both exhibited WS2 and Hirschsprung disease, and in which linkage to PAX3 and MITF could be excluded, but linkage to 13q markers could not (see also below).

Waardenburg Syndrome Type IV (WS4; Waardenburg–Shah Syndrome; MIM #277580) As discussed in Chapter 36, WS4 is an autosomal recessive disorder phenotypically similar to WS3, although sometimes with more extreme hypopigmentation (Gross et al., 1995), but without deafness, and involving the additional feature of Hirschsprung disease (HSCR; MIM #142623), the congenital absence of intrinsic ganglion cells of the myenteric and submucosal plexi of the gastrointestinal tract (aganglionic megacolon) (Gross et al., 1995; Shah et al., 1981). Like melanocytes, gut enteric plexus ganglion cells are derived from the neural crest, and thus it is not surprising that there is an association between these phenotypic features. In fact, HSCR has been reported in rare cases of piebaldism, WS1, and WS2; similarly, Hirschsprung disease itself has occasionally been associated with pigmentary anomalies, in both humans and mice. Evidence has begun to emerge for a complex network of interacting genes and proteins that regulate the development of melanocytes and enteric plexus neurons during embryogenesis (Barsh, 1995). Indeed, it is now clear that the WS4 phenotype can result from mutations in genes encoding at least three of these regulatory factors: endothelin-3 (EDN3), the endothelin-B receptor (EDNRB), and the transcription factor SOX10. 145

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WS4 Associated with Mutations of the Endothelin-3 Gene (EDN3; MIM #131242) Targeted disruption of the mouse Edn3 gene, encoding endothelin-3, resulted in a recessive phenotype of Hirschsprung disease and spotted coat color (Baynash et al., 1994), similar to the phenotype of human WS4. This laboratory induced mutation is not complemented by the natural mouse mutation, lethal spotting (ls), which was found to result from a point mutation in the Edn3 gene (Baynash et al., 1994). Hood et al. (1989) described a child with sporadic Hirschsprung disease, deafness, and piebald-like white spotting associated with a de novo translocation t(7;20)(q22.1;q13.13); the location of the EDN3 gene at 20q13.2–q13.3 (Arinami et al., 1991; Gopal Rao et al., 1991) suggested that EDN3 may have been disrupted by this translocation. These observations, as well as earlier findings involving the EDNRB gene, encoding the endothelin-3 receptor (discussed below), made the human EDN3 gene a likely candidate for WS4. Subsequently, two groups identified homozygous or heterozygous mutations of the EDN3 gene in patients with WS4 (Edery et al., 1996; Hofstra et al., 1996), confirming the homology between human WS4 and murine ls. Mutations of EDN3 may also result in isolated Hirschsprung disease without pigmentary anomalies (Bidaud et al., 1997; Svensson et al., 1999), as well as congenital central hypoventilation syndrome associated with Hirschsprung disease (Bolk et al., 1996).

WS4 Associated with Mutations of the Endothelin Receptor B Gene (EDNRB; MIM #131244); Hirschsprung Disease/Waardenburg Syndrome (HSCR2; MIM #600155); ABCD Syndrome (MIM #600501) Puffenberger and coworkers (1994a) initially mapped a locus associated with recessive “multigenic Hirschsprung’s disease,” frequently associated with white forelock, to chromosome segment 13q22 (Puffenberger et al., 1994a), and van Camp et al. (1995) reported a patient with clinical features of WS2A and a de novo interstitial deletion of chromosome 13q, as well as a family with two siblings with WS2 and Hirschsprung disease. The localization of the EDNRB gene to this same chromosome segment (Puffenberger et al., 1994b), and the fact that both targeted and natural (piebald-lethal; sl) mutations of the mouse Ednrb gene result in a similar recessive phenotype of Hirschsprung disease and spotted coat color (Hosoda et al., 1994) due to a profound deficiency of neural crest-derived melanocytes (Pavan and Tilghman, 1994), made EDNRB an obvious candidate for WS4. Two groups (Attie et al., 1995a; Puffenberger et al., 1994b) then identified mutations of the EDNRB gene, encoding the endothelin B (endothelin-3) receptor, in patients with HSCR2, an autoso146

mal dominant disorder in which Hirschsprung disease is associated with pigmentary anomalies, overlapping the clinical definition of WS4. Additional dominant EDNRB mutations have since been identified in other WS4 patients (Syrris et al., 1999). Recessive EDNRB mutations can also result in isolated Hirschsprung disease (Amiel et al., 1996; Kusafuka et al., 1996; Puffenberger et al., 1994a), as well as ABCD syndrome (MIM #600501), characterized by bilateral deafness, albinism, black scalp lock, and retinal depigmentation (Gross et al., 1995; Verheij et al., 2002).

WS4 Associated with Mutations of the SOX10 Transcription Factor Gene (SOX10; MIM #602229); Yemenite Deaf–Blind Hypopigmentation Syndrome (MIM #601706) SOX10 is mutated in the spontaneous dominant megacolon (Dom) mouse (Herbarth et al., 1998; Southard-Smith et al., 1998) and encodes a transcriptional activator related to that encoded by the testis-determining gene SRY. Heterozygous SOX10 mutations have been identified in a number of patients with WS4 (Kuhlbrodt et al., 1998; Pingault et al., 1998; Southard-Smith et al, 1999), with the so-called WS4 “neurologic variant” (Inoue et al., 1999, 2002; Touraine et al., 2000), and one patient with the “Yemenite deaf–blind hypopigmentation syndrome” (MIM #601706; Bondurand et al., 1999). It is thought that SOX10 mutations that cause pure loss of function may result in WS4, whereas mutations associated with neurological dysfunction may act via dominant-negative mechanisms (Inoue et al., 2002, 2004). SOX10 strongly transactivates the MITF promoter, acting in synergy with PAX3 (Bondurand et al., 2000; Lee et al., 2000; Potterf et al., 2000).

Related Disorders An autosomal dominant form of Hirschsprung disease (MIM #142623) is associated with mutations in the RET protooncogene (Angrist et al., 1995), located at chromosome 10q11.1 (Edery et al., 1994; Romeo et al., 1994), which appears to be a major locus for sporadic Hirschsprung disease (Angrist et al., 1995; Attie et al., 1995b; Bolk Gabriel et al., 2002; Seri et al., 1997). RET encodes a receptor tyrosine kinase related to the torso gene of Drosophila, although its natural ligand is not yet known. RET gene mutations have most frequently been associated with multiple endocrine neoplasia type 2 (MEN2; MIM #171400 and 162300; Eng et al., 1994; Mulligan et al., 1993, 1994), a disorder involving tumors of multiple neural crest-derived endocrinologic tissues and occasional Hirschsprung disease (Eng, 1996; Lyonnet et al., 1993; Mahaffey et al., 1990; Verdy et al., 1982), and with medullary thyroid carcinoma (MTC; MIM #155240; Marsh et al., 1996). However, RET mutations have not yet been associated with developmental pigmentary anomalies in humans.

MELANOBLAST DEVELOPMENT AND ASSOCIATED DISORDERS

Aganglionic megacolon is a relatively frequent occurrence in patients with Down syndrome (Garver et al., 1985; Passarge, 1967), and evidence has been presented for a “modifier locus” on human chromosome 21q22 that affects expression of Hirschsprung disease in patients with EDNRB gene mutations (Puffenberger et al., 1994a). Up to six genetic modifier loci may modulate the severity of white spotting associated with homozygosity for Ednrb mutations in piebald (s) mice (Pavan et al., 1995). Whereas some of these modifiers may be known genes, such as W and Sl, others may well be novel genes that may themselves be associated with developmental mutations that affect pigmentation. It is of some interest, for example, that the human homolog of the Drosophila white gene is located at 21q22.3 (Chen et al., 1996), although this gene has not thus far been associated with pigmentary abnormalities in humans.

X-linked Albinism–Deafness Syndrome (ADFN; MIM #300700) ADFN is an X-linked recessive disorder characterized by extreme piebald-like hypopigmentation and deafness affecting only males in a large Israeli kindred (Margolis, 1962; Ziprkowski et al., 1962). Genetic linkage and somatic cell hybridization studies have localized the gene to Xq24–q26 (Shiloh et al., 1990); however, as of this writing, the gene for this disorder had not yet been identified.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

7

Biogenesis of Melanosomes Raymond E. Boissy, Marjan Huizing, and William A. Gahl

Introduction The major product of the melanocyte is the specialized organelle called a pigment granule, or melanosome. As discussed in the preceding chapters, the cutaneous melanocyte synthesizes this organelle for eventual transfer to neighboring keratinocytes. In contrast, noncutaneous melanocytes retain their quota of pigmented melanosomes. This chapter addresses the origin of this specialized organelle, the processing and transport of gene products destined to occupy this organelle, and the melanosome’s maturation to a specialized microenvironment.

Origin of the Stage I Premelanosome During the early part of the twentieth century, pigmented tissue such as skin and eyes was shown to have a dark granular morphology when examined by light microscopy. This prompted biologists to speculate that pigment granules could originate from: (1) nuclei via extrusion; (2) mitochondria via conversion; or (3) the Golgi apparatus via production (Barnicot and Birbeck, 1958; Guttes, 1953). When investigators began exploring pigmented tissue using electron microscopy in the latter half of the twentieth century, it was initially concluded that pigmented organelles formed from colorless, thick-walled, hollow ellipsoids in which a fibrous matrix was laid down and on to which melanin was subsequently deposited (Barnicot et al., 1955; Birbeck et al., 1956). These observations resulted in discarding the hypothesis that pigment granules originate from a mitochondrial or nuclear origin. At this time, the various ultrastructural profiles of pigment granules were collaterally termed “melanosomes” and staged into morphological categories suggesting a developmental process (Birbeck, 1963; Moyer, 1961; Seiji et al., 1961, 1963). To this day, these stages are routinely used. They consist of: Stage I (also termed “premelanosome”) — a relatively spherical organelle with matrix material (i.e. filaments) beginning to assemble; Stage II — an oval-shaped organelle containing an organized internal matrix without the appearance of melanin; Stage III — an organelle exhibiting deposition of melanin on the matrix; and Stage IV — an organelle completely filled with melanin (Fig. 7.1). During the initial ultrastructural analyses of melanosomes, Moyer (1961) identified Stage I melanosomes associated with membranes of the endoplasmic reticulum (ER). Specifically, Moyer stated that, in

embryonic retinal pigmented epithelium, “one often finds vesicular dilations in the ER that are approximately the same size as stage I granules and they usually appear empty but occasionally show traces of very thin fibers and small vesicles in the lumen” (Moyer, 1961). This suggested that the premelanosome originated from the ER. Many subsequent authors agreed with this idea (Eppig and Dumont, 1972; Ide, 1972; Maul, 1969; Maul and Brumbaugh, 1971; Stanka, 1971; Stanka et al., 1981). With the advent of DOPA histochemistry, evidence for a non-Golgi origin of melanosomes was strengthened. DOPA incubation is a technique in which this exogenously supplied tyrosinase substrate is converted into melanin at cellular sites where functional tyrosinase resides. As a consequence of this experimental procedure, many trans-Golgi elements and perinuclear melanosomes increased in reaction product (Fig. 7.2). However, many premelanosome organelles in the vicinity of the Golgi remained negative for the reaction product, suggesting that melanosomes did not originate directly from the Golgi (Maul, 1969; Maul and Brumbaugh, 1971). In addition, many 50-nm coated vesicles appeared to fuse with the DOPA-negative premelanosomes, suggesting that the tyrosinase enzyme was trafficked to the preformed, recipient premelanosome. This “bipartite” hypothesis of melanosome biogenesis was substantiated by Turner et al. (1975), who demonstrated that premelanosome structures were derived from the ER in the goldfish. In addition, Turner et al. (1975) demonstrated that DOPA-positive vesicles fused with the premelanosome, inverted, and reformed within the lumen. Vesicles within premelanosomes were originally described by Tousimis (1963), and these vesicles sometimes “opened directly into the cytoplasm.” This was subsequently reported by Durrer and Villiger (1967). These vesicles were further explored by Jimbow et al. (Jimbow and Fitzpatrick, 1974; Jimbow and Kukita, 1970; Jimbow et al., 1979) and proposed to be inverted vesicles trafficking tyrosinase. By the end of the 1990s, conventional dogma held that premelanosomes formed from the ER and that enzymes and regulatory proteins were subsequently trafficked to them from the trans-Golgi in 50-nm clathrin-coated vesicles. However, this concept is currently being challenged in light of recent research focusing on a molecule called Silver/Pmel17. Silver/Pmel17 (also known as Pmel17, gp100, ME20, RPE1, and the silver locus protein) is a type 1 transmembrane glycoprotein that localizes to the matrix of Stage I and II melanosomes (Kobayashi et al., 1994; Lee et al., 1996; Raposo 155

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IV

II III

Fig. 7.1. Perinuclear area of normal human melanocytes from a lightly pigmented Caucasian skin demonstrating melanosomes in various stages of maturation. Melanosomes in the vicinity of the Golgi apparatus (G) exist as Stage I premelanosomes (I) that are more oval in shape containing floccular material and short irregular melanofilaments, Stage II melanosomes (II) that are more elongated containing an organized, scaffold-like matrix of melanofilaments, Stage III melanosomes (III) with melanin being sequentially deposited on the melanofilament matrix, and Stage IV melanosomes (IV) with melanin completely filling the organelle and obscuring the matrix. This melanocyte is from a lightly pigmented Caucasian individual and consists of Stage II and III melanosomes predominantly with relatively few Stage IV melanosomes. CE, coated endosome; N, nucleus; M, mitochondria. Bar = 1.0 mm.

et al., 2001). Silver/Pmel17 appears to be the initial melanocyte-specific molecular marker for melanosomes. Consistent with the hypothesis that the pre-/stage I melanosome originates from the ER, it was demonstrated in mouse melanocytes that Silver/Pmel17 lacks Golgi-modified N-linked glycans (Kobayashi et al., 1994) and coexists with ER-resident proteins (e.g. calreticulin, calnexin, ribophorin) in purified premelanosomes (Basrur et al., 2003; Kobayashi et al., 1994; Kushimoto et al., 2001). However, other data suggest that premelanosomes arise from compartments of the late secretory (post-Golgi) and/or endocytic pathways. Golgi-processed, glycosylated forms of Silver/Pmel17 have been identified in human premelanosomes (Berson et al., 2001; Raposo et al., 2001), suggesting that the delivery of this molecule to a structure designating it as a melanosome occurs post Golgi. Detailed ultrastructural studies (Raposo et al., 2001) showed that Silver/Pmel17 was enriched in coated, multivesicular, endosomal compartments containing protruding

156

I

Fig. 7.2. Perinuclear area of normal human melanocytes from a lightly pigmented Caucasian skin demonstrating localization of tyrosinase by l-DOPA histochemistry. DOPA reaction product indicating cellular sites of catalytically functional tyrosinase appears in the trans-Golgi network (G) including the trans-most cisternae (arrows), coated vesicles budding from these cisternae (arrowheads), and vesicles migrating from the Golgi apparatus (arrowheads with asterisk). Minimal reaction product exists in some Stage I premelanosomes (I), indicating incorporation of tyrosinase into these organelles. After DOPA histochemistry, this lightly pigmented, Caucasian melanocyte consists of Stage IV melanosomes predominantly with relatively few Stage II and III melanosomes. CE, coated endosome; N, nucleus; M, mitochondria. Bar = 1.0 mm.

smooth tubules. This compartment was shown to evolve into a multivesicular endosomal intermediate, the coated endosome, proposed to represent the premelanosome (Fig. 7.3) (Raposo et al., 2001; Raposo and Marks, 2002). The coated endosome contains large clathrin lattices on the cytoplasmic side (Fig. 7.3), which are thought to function in sorting premelanosome-bound cargo, including Silver/Pmel17, away from other endocytic cargo bound for lysosomes. Currently, unidentified chaperones likely facilitate this sorting, perhaps by recognizing specific domains of Silver/Pmel17 and other premelanosomal components. One candidate chaperone in this sorting process is adaptor protein-3 (AP3). Coated endosomes are also found in nonpigmented cells and are endosomal precursors of multivesicular bodies (Sachse et al., 2002). Proteomic analysis revealed the presence of Silver/Pmel17 in the premelanosome, but tyrosinase and other melanosomal proteins were also detectable (Kushimoto et al., 2001). However, the highly proteolytic environment in these vesicles appears to degrade or partially cleave these proteins and impair their enzymatic activities. Silver/Pmel17 also undergoes

BIOGENESIS OF MELANOSOMES

CE I

CE

? A

CE CE I I

B

C

Fig. 7.3. Profiles of coated endosomes/premelanosomes within the perinuclear area of normal human melanocytes. Perinuclear areas of normal human melanocytes without (A and B) or with (C) DOPA histochemistry demonstrate coated, multivesicular endosomal compartments (CE) proposed by Marks and Raposo (Raposo et al., 2001; Raposo and Marks, 2002) as precursor organelles developing into premelanosomes (I). These structures are characterized by focal, clathrin lattices (arrowheads) present on the cytoplasmic face. Profiles ultrastructurally resembling intermediates between the coated, multivesicular endosome and the premelanosome with melanofilaments undergoing assembly can be observed occasionally (?). Bar = 1.0 mm.

cleavage in this environment by a furin-like proprotein convertase (Berson et al., 2003). Properly cleaved Silver/Pmel17 has a key function in restructuring the premelanosomal matrix into fibrous striations, leading to a Stage II melanosome (Berson et al., 2003). In conclusion, it remains unsubstantiated whether the organelle that becomes a premelanosome originates from the ER and/or cis-Golgi or as a specialization of the endosomal compartment. In addition, the discrepancy regarding the presence of tyrosinase and Tyrp1 in early Stage I melanosomes is not completely resolved.

Development of the Melanosome Regardless of the actual origin of the premelanosome, it eventually becomes a distinct organelle with a specialized microenvironment to which a battery of enzymes and regulatory proteins are trafficked to facilitate the conversion of tyrosine to melanins. The major group of enzymes and regulatory proteins that eventually reach the melanosome are members of the tyrosinase gene family [tyrosinase-related proteins

(TRPs)], i.e. tyrosinase, tyrosinase-related protein-1 (Tyrp1), and dopachrome tautomerase/tyrosinase-related protein-2 (Dct/Tyrp2). These Type 1 membrane glycoproteins are transcribed at the rough endoplasmic reticulum (RER), shuttled through the Golgi apparatus for carbohydrate maturation, and ultimately transported to premelanosomes in the vicinity of the trans-Golgi network (TGN) via 50-nm coated vesicles. The initial processing of TRPs has been thoroughly explored in recent years. All three TRPs exhibit similar initial processing events, but distinct differences exist. Tyrosinase is initially translated as a 60-kDa polypeptide that is heavily glycosylated in the RER to a 70- to 74-kDa molecular weight form (Muller et al., 1988; Ujvari et al., 2001). Tyrosinase contains up to seven Asn-X-Ser/Thr sequons, and Glc3Man9GlcNAc2 glycans are added to six or seven of them (Branza-Nichita et al., 2000a; Ujvari et al., 2001). The proper amount of glycosylation, the appropriate folding into a tertiary structure, and the acquisition of copper are regulated by glycosylation enzymes and RER chaperone molecules such as calnexin (Branza-Nichita et al., 1999; Toyofuku et al., 1999; Wang and Androlewicz, 2000). Calnexin is a lectin involved in ER quality control that acts by driving the incompletely folded nascent chains into glucosylation/deglycosylation cycles (Ellgaard and Helenius, 2003; Kowarik et al., 2002; Zapun et al., 1997). Mutations in tyrosinase, either experimentally induced (Branza-Nichita et al., 2000b) or occurring naturally in patients with oculocutaneous albinism type 1 (Halaban et al., 2000; Park et al., 1993; Spritz et al., 1990), indicate that aberrations in N-glycosylation or conformational structure result in the degradation of the abnormal molecule via an ER-associated and/or proteasome mechanism (Berson et al., 2000; Halaban et al., 1997; Mosse et al., 1998). The mechanism of ER-associated protein degradation has been reviewed recently (Jarosch et al., 2002). Copper binding, specifically at the CuB site (Olivares et al., 2003), also appears to facilitate the initial processing of tyrosinase into a form capable of being transported out of the RER. In addition, DOPA, tyrosine (Halaban et al., 2001), and Tyrp1 (Kobayashi et al., 1998) apparently facilitate the maturation of tyrosinase in the RER. The initial synthesis of Tyrp1 and Dct (Tyrp2) in the RER is similar to that of tyrosinase. Tyrp1 is also a glycoprotein containing six sites with glycosylation sequons (BranzaNichita et al., 2000a; Xu et al., 2001). It is initially translated as a 55-kDa polypeptide, ultimately matures to a 75-kDa glycoprotein (Vijayasaradhi et al., 1991), and constitutes the predominant tyrosinase-related protein synthesized by the melanocyte (Vijayasaradhi et al., 1990). Tyrp1 undergoes a more rapid carbohydrate processing than tyrosinase (Negroiu et al., 1999a; Vijayasaradhi et al., 1991), i.e. ranging in duration from 2–3 hours vs. 3–6 hours respectively (Halaban et al., 1997; Negroiu et al., 1999a; Vijayasaradhi et al., 1995). This range varies with cell line but is primarily related to ER residency time. Treatment of melanoma cells with glycosylation inhibitors does result in a modified conformation

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(Negroiu et al., 2000). However, Tyrp1 is less sensitive to inhibitors of N-glycan processing than is tyrosinase (Negroiu et al., 1999b). Structural analyses of N-linked glycans for tyrosinase and Tyrp1 demonstrate significant differences in the extent and complexity of carbohydrate processing, indicating that the two molecules have different accessibility to the same processing milieu, probably because of differences in their three-dimensional structures (Negroiu et al., 1999b). Alterations in Tyrp1 that affect either glycosylation (Toyofuku et al., 2001) or amino acid sequence at the cytoplasmic tail (Xu et al., 1998) result in retention/degradation in the ER. Dct (Tyrp2) is also glycosylated in the ER and shares conserved potential N-glycosylation sites with tyrosinase and Tyrp1 (Xu et al., 2001). Processing of N-glycan in the ER is mediated by calnexin, which also facilitates the formation of stabilizing disulfide bridges (Negroiu et al., 2003). Upon proper initial synthesis, the precursor TRPs are trafficked out of the ER to the Golgi, primarily via vesicles directed by COPII coat complexes (Antonny and Schekman, 2001; Duden, 2003; Murshid and Presley, 2004). TRPs are ultimately shuttled through the Golgi apparatus where sequential modifications of the sugar residues occur. This shuttling, at least for tyrosinase, appears to be regulated in part by glycosphingolipids. A mouse melanoma cell line deficient in the synthesis of glycosphingolipid demonstrates retention of tyrosinase, but not Tyrp1, in the cisternae of the Golgi apparatus (Sprong et al., 2001). Upon reaching the trans-most cisternae of the Golgi, the mature tyrosinase is a catalytically functional enzyme, as demonstrated by DOPA histochemistry (Fig. 7.2) (Maul, 1969; Maul and Brumbaugh, 1971). Interestingly, this catalytically active enzyme is sequestered from its substrate until deposited into the premelanosome. Targeting sequences on the carboxyl ends of the TRP molecules are essential for accurate targeting to the melanosome (Table 7.1). Tyrosinase contains a dileucine-based motif with flanking regulatory amino acids (two leucine or isoleucine residues with an acidic residue four or five positions upstream) that interacts with adaptin-3 (AP3)

to facilitate its shipment to the melanosome (Calvo et al., 1999; Honing et al., 1998; Simmen et al., 1999). In the absence of this interaction, as in AP3 mutants (human HPS2, pearl, and mocha mice), tyrosinase does not reach the premelanosome efficiently (Huizing et al., 2001a). Tyrp1 also contains carboxy-terminal dileucine sorting signals that mediate its trafficking. Truncated Tyrp1 molecules are erroneously diverted to the plasma membrane (Vijayasaradhi et al., 1995). There is a requisite dileucine motif that exists in the carboxy region, but it is not utilized by AP3 for trafficking (Huizing et al., 2001a) because it lacks the correct adjoining/regulatory amino acid sequence. Instead, Tyrp1 may exit the trans-Golgi network (TGN) via an adaptor complex-1 (AP1)-mediated mechanism (Raposo et al., 2001). Recently, Vijayasaradhi (Liu et al., 2001a) identified by yeast twohybrid screening of a melanocyte cDNA library the interaction of Tyrp1 with GIPC, a member of a large family of PDZ domain proteins that recognize the sequence motif Ser/ThrXaa-Val on putative interactive/cargo proteins (Songyang et al., 1997). Tyrp1 expresses carboxyl-terminal Ser-Val-Val residues that interact transiently with GIPC after synthesis (Liu et al., 2001a). Interestingly, GIPC cofractionates with adaptin 1 (De Vries et al., 1998), suggesting that Tyrp1 utilizes AP1 as opposed to AP3 in its trafficking. While the specific adaptor complexes utilized for tyrosinase and Tryp1 trafficking differ, a dileucine motif functions in melanosomal sorting of both these melanogenic proteins. Tyrosine-based sorting signals (YXX F, Y = tyrosine, X = any amino acid, F = bulky hydrophobic amino acid), present in tyrosinase, Tyrp1 and Dct/Tyrp2, may also play a role in the sorting process (Simmen et al., 1999). This raises the possibility that melanosomal proteins follow alternative paths to the melanosome by utilizing tyrosine-based sorting signals and AP1 recognition for sorting at the TGN, and AP2-mediated sorting for internalization from the plasma membrane. Another possibility is that tyrosine-based signals are utilized to retrieve mistargeted melanosomal proteins. Melanosomal proteins may also utilize signals different from tyrosine and

Table 7.1. Putative sorting signals in the cytoplasmic domains of proteins targeted to the melanosome. Protein

Human (murine) disorder

Targeting motif

Tyrosinase Tyrp1 DCT/Tyrp2 Pmel17 p MART1/MLANA OA1

OCA1 (albino, platinum) OCA3 (brown) Unknown (slaty) Unknown (silver) OCA2 (pink-eye dilute) Unknown (unknown) OA1 (Moa1)

TMD-X9-EEKQPLLMEKEDYHSLYQSHL-cooh TMD-X8-DEANQPLLT-X8-EYEKLQNPNQSVV-cooh TMD-RRLRKGYTPLMETHLSSKRYTEEA-cooh TMD-X25-PRIFCSCPIGENSPLLSGQQV-cooh nh2-X90-KEDTPLLW-X77-TMD7-VV-cooh TMD-X6-GYRALMD-X43-AYEKLSAEQSPPPYSP-cooh TMD-X114-cooh

Bold highlighted gray, dileucine-based sorting signal (two leucine or isoleucine residues with an acidic residue 4 or 5 positions upstream). Highlighted black, tyrosine-based sorting signals (YXXF, Y = tyrosine, X = any amino acid, F, bulky amino acid) (Calvo et al., 1999; Honing et al., 1998; Simmen et al., 1999). Not all the signals shown in this table have been experimentally demonstrated to be important for sorting; -cooh, carboxyl-terminus; nh2-, aminoterminus; TMD = transmembrane domain, OCA = oculocutaneous albinism, OA = ocular albinism. See text for details.

158

BIOGENESIS OF MELANOSOMES

dileucine signals, as indicated by the absence of dileucine motifs from Dct/Tyrp2 and the OA1 protein. Dct/Tyrp2 processing exhibits post-translational modifications that suggest transcription in the RER followed by maturation in the Golgi (Tsukamoto et al., 1992). Some data indicate that Dct/Tyrp2 is predominantly maintained in the TGN (Negroiu et al., 2003) and is less prevalent in late melanosomes. Unlike tyrosinase and Tyrp1, Dct/Tyrp2 does not have a dileucine motif, but it does have a tyrosine-based signal (Table 7.1) (Jackson et al., 1992), resembling LAMP-1 and MART-1 (De Maziere et al., 2002; Dell’Angelica et al., 2000a). This suggests that Dct’s trafficking route/mechanism is distinct from that of tyrosinase and Tyrp1. It is uncertain whether TRPs traffic directly from the Golgi to the melanosome. Tyrosinase and Tyrp1, for example, may transit through a late endosome/lysosome compartment en route to the melanosome (Huizing et al., 2001a; Jimbow et al., 1997; Orlow et al., 1993). Another melanocyte-specific protein related to melanosome biogenesis is the product of the ocular albinism type 1 (OA1) gene. OA1 is an X-linked recessive disorder in which giant macromelanosomes exist predominantly within ocular but also within cutaneous melanocytes (Garner and Jay, 1980; O’Donnell et al., 1976). The OA1 gene encodes a novel integral membrane protein of seven transmembrane domains with homologies to G protein-coupled receptors (Bassi et al., 1995; Schiaffino et al., 1999). Initial localization studies using immunoelectron microscopy suggested that the OA1 protein localizes in part to melanosomes (Schiaffino et al., 1999). However, recent studies utilizing immunocytochemistry, cellular fractionation, and transfection of GFP-OA1 analysis has demonstrated that the OA1 protein is predominately localized to a late endosome/lysosome compartment and is largely absent from mature melanosomes (Samaraweer et al., 2001; Shen et al., 2001). This suggests that the OA1 protein is positioned to facilitate in part the trafficking of melanosomedistinct proteins through the intermediate compartment en route to the melanosome or to direct the endolysomal compartment into a melanosome. Loss of function of the OA1 protein may result in accumulation of melanogenic enzymes in the late endosome/lysosome and divert this organelle into the unique macromelanosome, as proposed by Shen et al. (2001). Chemically, melanins have been divided into the black/ brown eumelanins and the red/yellow, sulfur-containing pheomelanins. Correlative morphologic features distinguish melanosomes that exist in tissues predominantly eumelanotic vs. pheomelanotic (Hearing et al., 1973; Jimbow et al., 1979). Eumelanosomes are relatively oval organelles, in which the melanofilaments organize into a regularly registered matrix. In contrast, pheomelanosomes are relatively spherical organelles, in which the melanofilaments exist in short, irregularly arranged profiles. As melanin synthesis progresses, eumelanosomes acquire a uniform pigmentary pattern whereas pheomelanosomes exhibit an uneven deposition of pigment. It is currently unknown what regulates the unique features of these two types of melanosomes. Interestingly,

expression of Tyrp1, Dct/Tyrp2, and the silver protein (Pmel17) is not present in tissue undergoing pheomelanogenesis (Kobayashi et al., 1995), suggesting that the presence of these melanosome-residing, transmembrane proteins may impart some structural integrity to the eumelanosome.

Chaperoning of Tyrosinase Family Members to the Premelanosome Numerous chaperone proteins, responsible for the accurate trafficking and sorting of the tyrosinase family member proteins from the Golgi apparatus to the premelanosomes, are being identified. Many of these chaperones are encoded by genes that result in human and murine pigmentation disorders, such as Hermansky–Pudlak syndrome (HPS), Chediak–Higashi syndrome (CHS), and Griscelli syndrome (GS) (Huizing et al., 2000). These disorders of lysosomerelated organelle biogenesis typically affect melanosomes, platelet dense granules, and lysosomes; patients consequently show symptoms of hypopigmentation, prolonged bleeding, and other abnormalities of the lung, gut, and immune system (Clark and Griffiths, 2003; Dell’Angelica et al., 2000b; Huizing et al., 2000; Shiflett et al., 2002). HPS patients have oculocutaneous albinism, absent platelet dense bodies, and, sometimes, pulmonary fibrosis and granulomatous colitis (Huizing et al., 2000, 2002a; Huizing and Gahl, 2002). CHS patients manifest giant intracellular inclusions in leukocytes and platelets; without a bone marrow transplant, they die of infections in the first decade of life (Barak and Nir, 1987; Huizing et al., 2001b; Introne et al., 1999; Shiflett et al., 2002; Ward et al., 2002). GS patients also have immunological problems and may manifest neurological disorders as well (Anikster et al., 2002; Griscelli et al., 1978; Sanal et al., 2002). Most CHS and GS patients who do not undergo a bone marrow transplant eventually experience a fatal accelerated phase of lymphocytic proliferation. Identification of the chaperone-encoding genes involved in the above disorders can help us to unravel the intertwined biochemical machinery active in melanosome biogenesis. The best understood chaperone in this set is adaptor protein-3 (AP3). This ubiquitously expressed heterotetrameric complex binds clathrin to provide structure to newly forming vesicles and bind dileucine sorting motifs in the cytoplasmic tails of cargo proteins destined to be part of the nascent organelles (Dell’Angelica et al., 1997a, b; Simpson et al., 1997). Defects in a specific AP3 subunit, b3A (AP3B1), have been identified in human HP syndrome type 2 or HPS-2 (Clark et al., 2003; Dell’Angelica et al., 1999; Huizing et al., 2002b; Shotelersuk et al., 2000). In addition, AP3 subunit defects are responsible for the pearl (Ap3b1) (Feng et al., 1999) and mocha (Ap3d) (Kantheti et al., 1998) mouse coat color mutants and for pigmentary eye defects in the Drosophila melanogaster mutants ruby (Kretzschmar et al., 2000) and garnet (Ooi et al., 1997). Current dogma suggests that AP3 mediates melanosomal cargo sorting and vesicle budding at the TGN, but AP3 may 159

CHAPTER 7

also operate at other intracellular locations. AP3 could function in the retrieval of tyrosinase, and possibly other melanosomal proteins, from endosomal membranes, with subsequent placement in vesicles destined to become mature melanosomes. AP3 could also retrieve tyrosinase from melanosomal membranes in order to maintain homeostatic levels of the enzyme (Peden et al., 2004; Raposo and Marks, 2002). In AP3-deficient melanocytes, tyrosinase accumulates in multivesicular structures similar in appearance to the coated endosome (Huizing et al., 2001a). This could be due to missorting at the TGN or to failure to retrieve tyrosinase from endosomes. In contrast to tyrosinase, Tyrp1 localization does not appear to be affected by AP3 deficiency (Huizing et al., 2001a). In fact, colocalization of Tyrp1 with AP1 in melanocytes suggests a role for AP1 in Tyrp1 transport to melanosomes (Raposo et al., 2001). A novel PDZ domain protein, GIPC, might also have a role in Tyrp1 sorting to melanosomes (Liu et al., 2001b). HP syndrome exhibits locus heterogeneity, and genes associated with human HPS subtypes 1, 3, 4, 5, 6, and 7 (and their respective mouse orthologs pale ear, cocoa, light ear, ruby-eye 2, ruby eye, and sandy), all code for proteins that contain no recognizable motifs or homologies that could predict their biochemical function (Huizing et al., 2002a; Huizing and Gahl, 2002). However, some of these proteins can associate with each other into distinct multisubunit complexes, namely BLOC-1, BLOC-2, and BLOC-3 (biogenesis of lysosomerelated organelles complexes), which function to form and traffic intracellular vesicles (Di Pietro et al., 2004; FalconPerez et al., 2002; Gautam et al., 2004; Li et al., 2003; Martina et al., 2003; Nazarian et al., 2003; Starcevic and Dell’Angelica, 2004) (Fig. 7.4).

Snapin

indin Dysb

AP3

BLOC-2

Table 7.2. Human and mouse genes causing HPS and related disorders. Complex

Human gene

Human disorder

Mouse mutant

AP3

AP3B1 (b3A) AP3D1 (d) AP3S1 (s3A) AP3M1 (m3A)

HPS-2 – – –

pearl mocha – –

BLOC-1

HPS7/Dysbindin Pallidin Muted Cappuccino BLOS1 BLOS2 BLOS3 Snapin

HPS-7 – – – – – – –

sandy pallid muted cappuccino

BLOC-2

HPS3 HPS5 HPS6

HPS-3 HPS-5 HPS-6

cocoa ruby-eye 2 ruby eye

BLOC-3

HPS1 HPS4

HPS-1 HPS-4

pale ear light ear

Others

RGGTA Rab38 VPS33A MYO5A RAB27A Melanophilin

– – – GS1 GS2 GS3

gunmetal chocolate buff dilute ashen leaden

LYST

CHS

beige

Pallidin

Muted

BLOC-1

BLOC-3

Fig. 7.4. Assembly of tyrosinase-related protein chaperone complexes. Proposed assembly structures of adaptor complex 3 (AP3) and biogenesis of lysosome-related organelles complexes (BLOCs). ?, undefined subunit(s).

160

BLOC-1 consists of the palladin, muted, cappuccino, HPS7/dysbindin proteins, and the recently identified novel subunits BLOS1, BLOS2, BLOS3, and Snapin (Falcon-Perez et al., 2002; Li et al., 2003; Martina et al., 2003; Starcevic and Dell’Angelica, 2004). The genes encoding pallidin, muted, cappuccino, dysbindin, and BLOS3 are mutated in the HPS mouse strains pallid, muted, cappuccino, sandy, and reduced pigmentation respectively (Table 7.2) (Ciciotte et al., 2003; Huang et al., 1999; Li et al., 2003; Starcevic and Dell’Angelica, 2004; Zhang et al., 2002). These mouse strains exhibit the most severe coat color dilution phenotypes among all mouse HPS models (Li et al., 2004). Except for one patient with a dysbindin/HPS7 defect (Li et al., 2003), no mutations in any of the other BLOC-1 subunits have been identified in human HPS patients. The molecular function of BLOC-1 remains unknown, but indications from its potential binding partners suggest that BLOC-1 operates in the regulation of SNARE-mediated membrane fusion. SNAREs are soluble N-ethylmaleimide-sensitive factor attachment protein receptors involved in vesicle membrane fusion. Palladin is able to interact with syntaxin 13, a SNARE family member localized to early endosomes and involved in membrane fusion at early stages of the endocytic pathway (Huang et al., 1999; Prekeris et al., 1998), and

reduced pigment

BIOGENESIS OF MELANOSOMES

Snapin interacts with SNAP-25/23, two closely related SNARE proteins implicated in membrane fusion events (Buxton et al., 2003; Ilardi et al., 1999; Starcevic and Dell’Angelica, 2004). Dysbindin is a binding partner of alpha and beta dystrobrevins, i.e. dystrophin-related proteins that are part of large dystrophin-associated, membrane-spanning complexes functioning in membrane stability and signaling events (Benson et al., 2001). BLOC-1 associates with F-actin (FalconPerez et al., 2002), supporting a role for the complex in promoting vesicle movement in the periphery of cells. BLOC-2, a stable complex containing the HPS3, HPS5, and HPS6 proteins, exists in both human and mouse (Table 7.2) (Di Pietro et al., 2004; Gautam et al., 2004). Clinically, HPS3, HPS-5, and HPS-6 are relatively mild forms of the disease (Anikster et al., 2001; Huizing et al., 2001c, 2004; Tsilou et al., 2004), with no reported involvement of pulmonary fibrosis, suggesting a common biological basis underlying the pathogenesis of these three HPS subtypes. A regulatory role for BLOC-2 in the secretion of lysosomes and related organelles has been suggested (Di Pietro et al., 2004), based on reduced secretion of lysosomal enzymes by kidney and platelets from both ruby eye (HPS-6) and ruby-eye 2 (HPS-5) mice (Swank et al., 1998). In addition, an increase in the frequency and duration of transient fusion events in ruby-eye mast cells during stimulation implicates a function for the ruby-eye gene product in regulating closure of the cell fusion pores (Oberhauser and Fernandez, 1996). These pores connect the lumen of a secretory vesicle with the extracellular environment during exocytosis. However, fibroblasts from cocoa (HPS-3) and ruby-eye (HPS-6) mice showed normal secretory levels of the enzyme b-hexosaminidase, indicating that BLOC2 is not critical for secretion of this lysosomal enzyme (Di Pietro et al., 2004). Recent studies in fibroblasts have shown that HPS3 localizes to clathrin-coated vesicles in the perinuclear area and that HPS3 contains a clathrin-binding domain at residues 172–176 (LLDFE), which is essential for the accurate localization of LAMP1 (Helip-Wooley et al., 2004). Studies on human HPS-3 melanocytes indicate that melanosome-targeted cargos (tyrosinase, Tyrp1, and Tyrp2) do not become incorporated efficiently into melanosomes in the perinuclear region but are retained in 50-nm vesicles that extend into the periphery of the HPS-3 melanocyte. Therefore, BLOC-2 and its association with clathrin may be necessary for the efficient incorporation of melanogenic cargo targeted to the melanosome (Boissy et al., 2004). BLOC-3 contains HPS1 and HPS4 (Table 7.2) (Chiang et al., 2003; Martina et al., 2003; Nazarian et al., 2003), which explains the phenotypic similarity of HPS-1 and HPS4 patients. In addition to oculocutaneous albinism and prolonged bleeding, present in all HPS subtypes, HPS-1 and HPS-4 patients are at high risk of developing progressive pulmonary fibrosis and granulomatous colitis (Anderson et al., 2003; Hermos et al., 2002; Huizing et al., 2002a), which might result from accumulation of ceroid lipofuscin (an autofluorescent compound) in various tissues (Huizing et al., 2000). The mouse mutants pale ear (HPS1 deficient), light ear

(HPS4 deficient), and double-homozygous pale ear/light ear have identical coat colors and a similar unique hypopigmentation of ears, tail, and feet (Lane and Green, 1967; Li et al., 2004; Suzuki et al., 2002), strongly suggesting a physical and/or functional interaction between HPS1 and HPS4. In addition, tissues of the light ear mouse (HPS4 deficient) show no HPS1 protein expression (Suzuki et al., 2002). A similar phenomenon is seen in cells deficient in subunits of AP3 (Dell’Angelica et al., 1999; Huizing et al., 2002b) or BLOC1 (Ciciotte et al., 2003; Falcon-Perez et al., 2002; Starcevic and Dell’Angelica, 2004), in which the absence of one subunit leads to the loss of other subunits of the corresponding complex. Even though HPS1 and HPS4 are part of a stable complex, no direct interactions between the two proteins have been shown, indicating that other unidentified BLOC-3 subunits might exist (Martina et al., 2003; Nazarian et al., 2003). In addition, the possible existence of BLOC-4 and BLOC-5 complexes, sharing some BLOC-3 subunits, has been suggested (Chiang et al., 2003). The biochemical pathway in which BLOC-3 functions remains speculative. The HPS1 and HPS4 proteins contain no recognizable homology or structural motifs that can help to predict BLOC-3 function. In human melanotic cells, the HPS1 protein resides in uncoated vesicles and early-stage melanosomes, and the lack of HPS1 protein expression in these cells results in mislocalization of tyrosinase and Tyrp1 to large membranous structures rather than melanosomes, thereby affecting melanin synthesis (Sarangarajan et al., 2001). Subcellular fractionation studies have revealed that BLOC-3 expression is mostly cytosolic, with only a small fraction associated with membranes (Martina et al., 2003). Light ear and pale ear mice have enlarged kidney lysosomes containing increased amounts of lysosomal hydrolases and decreased secretion of lysosomal hydrolases into the urine (Meisler et al., 1980; Swank et al., 1998), suggesting BLOC3 involvement in lysosomal biogenesis. Pale ear fibroblasts exhibited a reduced perinuclear localization of lysosomal and late endosomal markers compared with control fibroblasts, indicating a role for BLOC-3 in the movement and/or localization of these organelles (Nazarian et al., 2003). Finally, ultrastructural studies of eye melanosomes have shown abnormal macromelanosomes within the choroids of pale ear and light ear mice, presumably a result of abnormal melanosome fusion events (Li et al., 2004). The LYST protein, defective in human CHS and in the beige mouse, might also serve as a chaperone in the trafficking of tyrosinase family members to the premelanosome (Barbosa et al., 1996; Huizing et al., 2001b; Nagle et al., 1996; Perou et al., 1997; Ward et al., 2002). LYST is a large cytoplasmic protein (425 kDa) (Nagle et al., 1996), thought to function as an adaptor that may juxtapose proteins that mediate intracellular membrane fusion reactions (Tchernev et al., 2002). LYST contains a BEACH motif, which is highly conserved in a large family of eukaryotic proteins and is crucial for their functions in vesicle trafficking, membrane dynamics, and receptor signaling (Huizing et al., 2001b; Nagle et al., 1996; Ward et al., 161

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2002). LYST also contains a series of hydrophobic helices resembling HEAT and ARM domains (Huizing et al., 2001b; Nagle et al., 1996; Ward et al., 2002). HEAT repeat proteins are involved in vesicle transport, and ARM domains are thought to mediate membrane associations (Huizing et al., 2001b; Peifer et al., 1994; Ward et al., 2002). The carboxylterminus of LYST contains several WD40 repeat motifs, which can function in protein–protein interactions (Huizing et al., 2001b; Neer et al., 1994; Ward et al., 2002). Melanocytes in CHS frequently produce large premelanosomes that can reach 10 times the size of a normal melanosome and express an aberrant trafficking of tyrosinase (Zhao et al., 1994). Griscelli syndrome (GS) is associated with defects in three distinct proteins, myosin 5A (GS1), rab27A (GS2), and melanophilin (GS3), the orthologs of the genes mutated in the dilute, ashen, and leaden mice respectively (Anikster et al., 2002; Griscelli et al., 1978; Menasche et al., 2000, 2003; Pastural et al., 1997; Sanal et al., 2002). Myosin 5A, GTP-rab27A, and melanophilin are the main components of a complex that regulates actin-based melanosome transport in melanocytes (Fukuda et al., 2002; Menasche et al., 2003; Wu et al., 2001), as presented in the following Chapter 8. Melanophilin can bind to actin filaments through its actinbinding domain and serves as the linker protein between myosin 5A and GTP-rab27A (Fukuda et al., 2002). A defect in any component of this complex results in failure of melanosomes to localize to the dendritic tips of melanocytes, thereby preventing melanosome transfer to adjacent keratinocytes (Boissy, 2003; Fukuda et al., 2002; Wu et al., 2001). Several other protein complexes are beginning to emerge as chaperones in melanosome biogenesis. All eukaryotes are known to utilize a combination of coat-associated proteins, SNAREs, syntaxins, rabs (GTPases of the ras superfamily), cytoskeletal proteins, and membrane lipids for vesicle targeting, transport, and fusion events (Karcher et al., 2002; McMaster, 2001; Pelham, 2001; Seabra et al., 2002; Teng et al., 2001). Apart from rab27A, several other rabs also appear to function in melanosome-specific trafficking, budding, and fusion events (Seabra et al., 2002; Whyte and Munro, 2002). For example, the chocolate coat color mutant mouse, defective in rab38, has impaired targeting of Tyrp1 (Loftus et al., 2002). Rab7, which is localized to late endosomes, is also thought to traffic tyrosinase and Tyrp1 from the TGN to melanosomes (Gomez et al., 2001; Hirosaki et al., 2002), and the HPS mutant mouse gunmetal is mutated in rabggta, an enzyme that attaches geranylgeranyl groups to rabs (Detter et al., 2000). SNARE complexes, which can be either in a vesicle (vSNARE) or in the vesicle’s fusion target (tSNARE), consist of a syntaxin, a vesicle-associated membrane protein (VAMP), and synaptosome-associated proteins (SNAPs) (Pelham, 2001; Teng et al., 2001). The role of the SNARE complex in melanosome biogenesis is slowly becoming apparent. The pallid mutant mouse protein pallidin, a component of BLOC1, interacts with the tSNARE syntaxin 13 (Huang et al., 162

1999). This complex may function as a premelanosomal tSNARE that assists specific Tyrp1- and Tyrp2-containing vesicles to fuse with the premelanosome (Falcon-Perez et al., 2002; Marks and Seabra, 2001). Other melanosomeassociated SNAREs include syntaxin 4, SNAP-23, and SNAP25 (Scott and Zhao, 2001). Interestingly, the recently described BLOC-1 component Snapin (Starcevic and Dell’Angelica, 2004) can interact with both SNAP-23 and SNAP-25 (Buxton et al., 2003; Ilardi et al., 1999). The so-called ‘granule group’ of Drosophila melanogaster eye color mutants encodes proteins that function in the delivery of proteins to specialized eye pigment granules (Lloyd et al., 1998). Drosophila homologs of all four subunits of AP3 correspond to granule group mutants (garnet/AP3-d, carmine/AP3-m, orange/AP3-s, ruby/AP3-b) (Kretzschmar et al., 2000; Mullins et al., 2000; Ooi et al., 1997), supporting the concept that fly pigment granule defects are related to melanosomal defects in higher organisms. Other proteins defective in the granule group mutants are VPS41 (light), VPS33 (carnation), and VPS18 (deep orange) (Sevrioukov et al., 1999; Shestopal et al., 1997; Warner et al., 1998). Mutations of any of these proteins in yeast result in the accumulation of prevacuolar multiple vesicular bodies (Peterson and Emr, 2001), as in deep orange/VPS18 (Shestopal et al., 1997) and carnation/VPS33 pigment granules (Sevrioukov et al., 1999). VPS41 interacts with the d subunit of AP3 and is required for the formation of AP3-coated carrier vesicles (Rehling et al., 1999). Despite the impressive progress already made in understanding intracellular vesicle formation, a great deal remains unknown. Mutant animal and human pigmentation models provide an invaluable resource for elucidating the pathways involved in the formation and precise trafficking of melanosomal components. These pathways may have broader applicability to cell types other than the melanocyte.

The Melanosome as a Specialized Microenvironment Within the pigment-producing melanosome, TRPs ultimately form into a high-molecular-weight, multimeric complex that acts as a functional, stable, enzymatic unit (Halaban and Moellmann, 1990; Hearing et al., 1982; Jimenez-Cervantes et al., 1998; Orlow et al., 1994). Because of the different mechanisms responsible for the trafficking of TRPs to the melanosome, as described above, this complex presumably does not form until all TRPs are inserted into the limiting membrane of the melanosome. In addition to the tyrosinase gene family members, many other gene products are putatively part of the melanosome and contribute to its specialized microenvironment, allowing melanin synthesis. The luminal acidity of the melanosome appears to regulate melanization. The catalytic activity of tyrosinase exhibits a lag at neutral pH that is lost under acidic conditions (Devi et al., 1987; Lerner et al., 1949; Tripathi et al., 1988), and acidity

BIOGENESIS OF MELANOSOMES

has been shown to control the rate of melanization and tyrosinase activity (Ancans et al., 2001; Saeki and Oikawa, 1983). Other investigators have demonstrated that tyrosinase activity is optimal at neutral pH and reduced in acidic conditions (Hearing and Ekel, 1976; Saeki and Oikawa, 1985; Townsend et al., 1984). Regardless, fluorometric (Bhatnagar et al., 1993) and histocytochemical (Moellmann et al., 1989, personal communication) methods indicate that melanosomes have an acidic lumen in both melanoma cells and normal human melanocytes. Experimental manipulation of light vs. dark skin-derived melanocytes, using inhibitors of the vacuolar proton pump V-ATPase, cause a dramatic increase in tyrosinase activity in light melanocytes and no change in dark melanocytes, without a concomitant alteration in tyrosinase amount (Fuller et al., 2001). This demonstrates that acidity in the melanocyte regulates the efficiency of tyrosinase activity. Translocation of tyrosinase out of the RER is impeded in amelanotic melanoma cells exhibiting aberrant vacuolar ATPasemediated proton transport; the defect was corrected by treatment with V-ATPase inhibitors (Halaban et al., 2002). Sodium–hydrogen exchangers (NHEs) are known to regulate intracellular pH (Counillon and Pouyssegur, 2000; Orlowski and Grinstein, 1997). Melanocytes treated with a chemical inhibitor of NHEs result in a rapid dose-dependent inhibition of tyrosinase activity (Smith et al., 2004). In addition, melanocytes express five of the seven isoforms of NHE (Sarangarajan et al., 2001; Smith et al., 2004), several of which, along with the vesicular proton pump V-ATPase, colocalize with Tyrp1 (Smith et al., 2004). It remains unknown whether regulation of acidity occurs in the melanosome, directly affecting tyrosinase, or within the RER and/or translocational compartments, influencing the maturation of tyrosinase and/or its efficient delivery to the melanosome. The gene product of OCA2 is the P protein, i.e. the pinkeye dilute protein (Lee et al., 1994), a limiting membrane protein with 12 transmembrane domains (Gardner et al., 1992; Rinchik et al., 1993). Brilliant (2001) demonstrated that melanocytes mutant for P exhibit a loss of colocalization of acidic organelles and Tyrp1, suggesting that the P protein acidifies melanosomes (Puri et al., 2000). Verification of this finding is pending. The P protein also regulates processing in the RER. Specifically, melan-p1 cells, null at the p locus, exhibit an elevation in tyrosinase activity and protein with concomitant secretion of the enzyme after intracellular cleavage (Manga et al., 2001; Potterf et al., 1998). In addition, the P protein has been shown to exhibit a prominent ER localization and to contribute to the correct processing of tyrosinase in the ER (Chen et al., 2002). It is noteworthy that the P protein, as well as membrane-associated transport protein (MATP), was not identified by protein analysis of Stage II melanosomes (Basrur et al., 2003), suggesting that these proteins function upstream of the melanosome and are not localized to the organelle. Therefore, it appears that the transmembrane, pore-like P protein is not a prominent component of melanosomes. Its contribution in regulating the environment of the RER and/or Golgi cisternae to facilitate folding

and/or trafficking of tyrosinase, and thus its requirement for stabilization of the TRP complex (Lamoreux et al., 1995), have yet to be elucidated. The P protein was originally hypothesized to be the tyrosine transporter essential to import this substrate into the melanosome for induction of melanization. However, poor sequence homologies between P and known tyrosine transporters (Rosemblat et al., 1994) and the lack of a deficiency in tyrosine transport in P null cells (Gahl et al., 1995) refute this. Regardless, the melanosomal membrane does indeed contain a transport protein with a high affinity for tyrosine (Gahl et al., 1995; Potterf et al., 1996). Melan-A/MART-1 is a melanocyte-specific protein currently of unknown function. It was originally identified in melanoma patients who expressed T lymphocytes reactive to this melanocyte-specific protein (Coulie et al., 1994; Kawakami et al., 1994). Melan-A/MART-1 is a 22- to 24-kDa transmembrane glycoprotein oriented with the carboxy terminus exposed to the cytosol (Rimoldi et al., 2001). Melan-A/ MART-1 does not contain consensus signals for N-glycosylation, but it is post-translationally acylated (De Maziere et al., 2002), a common finding among proteins involved in signaling and trafficking mechanisms (Dunphy and Linder, 1998; Mumby, 1997). Localization studies have demonstrated that Melan-A/MART-1 is an integral membrane protein of melanosomes but decreases in amount as the melanosome matures (De Maziere et al., 2002; Kawakami et al., 1997). However, most of Melan-A/MART-1 (i.e. 80%) is present in the TGN area associated with trans-Golgi cisternae, 50-nm vesicles, and coated early endosomes (De Maziere et al., 2002). These latter structures also import Silver/Pmel17, which subsequently helps to direct their development toward a melanosome lineage as opposed to a lysosome lineage (Raposo et al., 2001). The predominance of Melan-A/ MART-1 in the coated early endosome suggests that it may also participate in directing the early biogenesis of melanosomes. Recently, MATP has been identified as the product of the gene responsible for oculocutaneous albinism type 4 (OCA4) (Newton et al., 2001). MATP has 12 predicted membranespanning regions lacking significant similarities to transporters of amino acids or monosaccharides but with one loop exhibiting similarities to a sucrose/proton symporter in plants (Newton et al., 2001). Melanocytes cultured from the murine model for OCA4, underwhite (uw), express normal levels of tyrosinase synthesis but exhibit dramatically reduced levels of tyrosinase activity (Lehman et al., 2000). In addition, uw melanocytes secrete into the medium dark vesicles that contain tyrosinase, Tyrp1 and Dct (Costin et al., 2003). Similar to the P protein, proteomic analysis of Stage II melanosomes did not identify MATP as a component of mature melanosomes (Basrur et al., 2003). Therefore, it appears that MATP may regulate the trafficking of melanocyte proteins from the Golgi to the premelanosome via its influence on the luminal milieu of trafficking components, in a fashion similar to that described above for the P and Melan-A/MART-1 protein. 163

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Endoplasmic reticulum Golgi

Tyrosinase

Silver/Pmell7

lti

v

ic u

lar/s o r ti n g

bo

no

ed

Mu es

so me

t Coa

en d

a osome/premel

dy

o Melanos

me –

e II stag

Clathrins -

Adaptins Molecules for recognition, docking and fusion -

Fig. 7.5. Schematic diagram of melanosome biogenesis. To date, the initial defining event in the biogenesis of a melanosome is the incorporation and processing of Silver/Pmel17 in a cytoplasmic organelle, defining it as a premelanosome. This cytoplasmic organelle was originally thought to be derived from the endoplasmic reticulum. However, current observations suggest that it may be derived from a coated, early endosomal component. Vesicles containing the precursor form of Silver/Pmel17 may be derived from the endoplasmic reticulum, cis-Golgi and/or trans-Golgi network. Once Silver/Pmel17 is processed in the premelanosome (i.e. Stage I melanosome), matrix assembly progresses, and the organelle develops into a Stage II melanosome that is capable of receiving tyrosinase-related protein (TRP) cargo. After their biosynthesis is complete, TRPs exit the trans face of the Golgi via clathrin-coated vesicles, presumably with the aid of adaptins. Cargo vesicles containing TRPs travel along cytoskeletal elements, possibly being diverted through an intermediate compartment (i.e. multivesicular/sorting body) en route to the Stage II melanosome. At the target Stage II melanosome, recognition, docking, and fusion molecules facilitate incorporation of the TRPs (see also Plate 7.1, pp. 494–495).

Summary Defining all cellular and molecular events regulating the biogenesis of the melanosome is still a work in progress (Fig. 7.5). Original observations intimated that precursor structures for premelanosomes are derived from the ER. However, current observations suggest that premelanosomes originate from the 164

endosomal network of components. A defining event in the commitment of cellular structures to premelanosomes is the incorporation of Silver/Pmel17 into vesicles and its subsequent assembly into a matrix scaffold that defines the Stage II melanosomes. Concomitant with the formation of Silver/ Pmel17-positive organelles must be the incorporation of pumps and transporters essential for the specialized environment within the melanosomes. Upon progression to Stage II melanosomes, this organelle becomes receptive to enzymes and regulatory molecules that coordinate the synthesis of melanins within. The tyrosinase gene family of proteins are trafficked from the Golgi apparatus to Stage II melanosomes, where molecular steps regulate recognition, docking, and fusing that facilitate their incorporation into melanosomes. The pathways and mechanisms of transit for each tyrosinase gene family member are currently being defined. Interestingly, it appears that differential pathways as well as alternate routes may exist for each TRP. Eventually, these cargo proteins are incorporated into melanosomes, putatively as a complex, and pigment is synthesized. Ultimately, the pigmented melanosomes must be translocated down the melanocyte dendrites for transfer to keratinocytes or to confer color adaptation. Many specialized molecules exist on the melanosome to facilitate this process, as described in the following chapter.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

8

Melanosome Trafficking and Transfer Glynis A. Scott

Introduction In prior chapters, the elegant and complex process of melanosome biogenesis and the biochemistry and regulation of pigmentation have been described. However, there would be little benefit in creating this amazing organelle if the machinery to transport it to the dendrite tip, and transfer it to a keratinocyte, was lacking. Indeed, adequate pigmentation of the skin is as dependent upon successful transport and transfer of melanosomes as it is on the formation of the organelle itself. It has long been recognized that melanocytes and keratinocytes interact both in culture and in skin in vivo (Fig. 8.1). This interaction occurs through long arborizing dendrites that result in the transfer of melanosomes to keratinocytes. Whole organelle donation from one cell to a heterologous cell is unique to the melanocyte–keratinocyte unit. Effective melanosome transfer requires a confluence of successful steps that starts with the formation and maintenance of the dendrite, transport of the melanized melanosome from the cell body to the periphery of the dendrite, followed by transfer of the organelle to a keratinocyte. A virtual explosion of knowledge has occurred in the area of melanosome trafficking and transport in the last 10 years. In this chapter, I will describe the current understanding of these two processes and will review the data that led to these conclusions.

Melanocyte Dendrite Formation and Transport on Microtubules: the First Step in the Process For melanosome trafficking and transfer to occur, effective dendrite formation is a prerequisite. Recent studies have shown that dendrite formation is dependent upon actin polymerization, which is controlled by the small guanosine triphosphate (GTP)-binding proteins of the Rho family (Busca et al., 1998; Scott, 2002; Scott and Cassidy, 1998; Scott and Leopardi, 2003). The Rho subfamily of GTP-binding proteins orchestrates a variety of cellular shape changes, including lamellipodia formation (Rac), filopodia formation (Cdc42), and stress fiber formation (Rho), through their ability to regulate actin assembly in response to growth factors (Etienne-Manneville and Hall, 2002). Similar to other small GTP-binding proteins, Rho proteins alternate between active (GTP-bound) and inactive (GDP-bound) forms, a process that is controlled by regulatory associated proteins (Fig. 8.2). Several studies have

demonstrated the crucial importance of two members of the Rho family of proteins, Rac and Rho, in melanocyte dendrite formation. Our studies have shown that the introduction of constitutively active Rac mutants into human melanocytes and B16F1 melanoma cells stimulates dendrite formation, whereas the introduction of C3 botulinum exoenzyme, which inactivates Rho, inhibits dendrite formation (Fig. 8.3; Scott and Leopardi, 2003). We also showed that ultraviolet irradiation (UVR) and melanocyte-stimulating hormone (MSH), known to stimulate dendrite formation, activate Rac (Scott and Cassidy, 1998). Busca et al. (1998) reported that Rho inhibition is responsible for dendrite formation in B16F10 cells. They showed that treatment of B16F10 cells with C3 botulinum exoenzyme stimulated dendrite formation and that constitutively active forms of Rho inhibited the ability of cAMPelevating agents to stimulate dendricity. We also demonstrated a link between the cAMP second messenger system, known to stimulate dendrite formation, and Rac activation and Rho inhibition (Scott and Leopardi, 2003). Treatment of melanocytes with agents known to stimulate cAMP, such as forskolin and dibutyryl-cAMP, resulted in the rapid activation of Rac and inhibition of Rho (Scott, 2002; Scott and Leopardi, 2003). Therefore, melanocyte dendrite formation is dependent upon actin polymerization, which is in turn controlled by the activity of Rac and Rho, which are regulated by the cAMP second messenger pathway. Whether other second messenger pathways regulate Rac or Rho activity in melanocytes has not been determined. Although the Rho family of proteins is clearly important for melanocyte dendrite formation, other proteins have been shown to influence dendrite formation, including the Rab protein Rab27B (Chen et al., 2002). Rab27B shares 72% identity with Rab27A, and the introduction of dominant-negative mutants of Rab27B into melanocytes resulted in a decrease in the length and number of dendrites. Although the mechanisms of Rab27B-dependent dendrite formation were not investigated, it is likely due to the role of Rabs in facilitating vesicle transport containing proteins necessary for dendrite formation and/or maintenance. Myosin Va, an unconventional myosin motor (see below), has also been implicated in dendrite formation in melanocytes (Edgar and Bennett, 1999) because the introduction of antisense oligonucleotides to myosin Va inhibits de novo dendrite formation in murine melanocytes. Historically, it had long been known that melanocyte dendrites were composed of both actin and microtubules (Moellmann and McGuire, 1975; Moellmann et al., 1973). Treatment of melanocytes with cytochalasin D, which disrupts the actin cytoskeleton, inhibits melanosome trafficking. 171

CHAPTER 8

Fig. 8.1. Melanocytes contact multiple keratinocytes in vivo. Hematoxylin and eosin-stained section of skin showing a melanocyte at the basement membrane with multiple arborizing dendrites in contact with keratinocytes (see also Plate 8.1, pp. 494–495).

Basement membrane

1µm

Hormones GDP

GEF

GDP GTP Rho

GTP

Rho

Pi

GAP Downstream effectors

Cdc42

Filopodia

Rac

Rho

Lamellipodia

Stress fibers

Fig. 8.2. Schematic of regulation and function of Rho GTP-binding proteins. Rho GTP-binding proteins alternate between active (GTP) and inactive (GDP) bound forms that are controlled by regulatory factors including guanine nucleotide exchange factors (GEF) and GTPase activating proteins (GAP).

However, whether this was due to specific effects on melanosome transport proteins or to disruption of the integrity of the dendrite was not determined (McGuire and Moellmann, 1972). More recent studies have definitively established that melanosomes are transported along the length of the dendrite on microtubules. Direct visualization of melanosome trafficking by video microscopy reveals a saltatory back and forth movement with a pathlength and speed characteristic of microtubule-based transport. The addition of nocodizole, which inhibits microtubule formation, results in the rapid redistribution of melanosomes to the cell body, but leaves the integrity of the dendrite intact (Scott et al., 2002; Wu et al., 1997). In many ways, melanosome transport in mammalian cells parallels that seen in amphibians, in which pigment granules are transported on microtubules (Rogers 172

et al., 1997). Several studies have identified the molecular motors involved in melanosome transport on microtubules. Co-localization studies and Western blotting have shown that melanosomes co-localize with dynein (a minus end microtubule motor) and kinesin (a plus end microtubule motor) (Byers et al., 2000; Hara et al., 2000; Vancoillie et al., 2000). Treatment of human melanocytes with antisense oligonucleotides to a dynein results in enhanced anterograde movement of melanosomes from the cell body to the periphery, providing evidence that dynein transports melanosomes centripetally (Fig. 8.4; Byers et al., 2000). Similarly, treatment of melanocytes with antisense oligonucleotides to kinesin heavy chain (Hara et al., 2000) results in the rapid translocation of melanosomes from the periphery to the cell body, providing evidence that kinesin is the motor that transports melanosomes centrifugally on microtubules. Dynein heavy chains bind microtubules and adenosine triphosphate (ATP) and are responsible for producing forces that move the motor and cargo along microtubules. The intermediate chains link dynein to dynactin, which is responsible for cargo binding. The light intermediate chains, three forms of which have been identified, are responsible for targeting dynein to different organelles. Analysis of dynein on melanosomes suggests that melanosomes are associated with a distinct light chain (Vancoillie et al., 2000). Kinesin is unrelated in sequence to the other known class of microtubule motor proteins, the dyneins, and is thought to perform functions in the cell distinct from the dyneins. Conventional kinesin is a dimer (Hirose and Amos, 1999) consisting of two identical 120- to 130-kDa chains, commonly known as heavy chains. When kinesin is purified from brain homogenates, two light chains (60–70 kDa) are associated with the dimers (Kull, 2000). It is thought that both dynein and kinesin remain on melanosomes and that motor activity is

MELANOSOME TRAFFICKING AND TRANSFER A

B

C

Fig. 8.3. Human melanocytes become dendritic in response to Rac activation and Rho inhibition. Human melanocytes in culture were microinjected with either C3 exoenzyme (B, which inhibits Rho; see also Plate 8.2B, pp. 494–495) or constitutively active Rac mutants (C, V12Rac; see also Plate 8.2C, pp. 494–495). Melanocytes become markedly dendritic in response to Rho inhibition and Rac activation compared with cells microinjected with dye alone (A; see also Plate 8.2A, pp. 494–495).

regulated to modify the direction of melanosome movement (Gross et al., 2002). This would account for the observed rapid ability of melanosomes to change direction.

Three Mouse Mutants Prove Critical for Understanding Melanosome Trafficking As has been the case for melanosome biogenesis, mouse mutants proved to be critical in furthering our understanding of melanosome transport. The dilute, leaden, and ashen mice all share a similar phenotype: pigment dilution with a pale or silver coat color, and the microscopic identification of clumped melanosomes within the hair follicles. These three mice turned out to be the living embodiment of specific genetic defects in three individual proteins that cooperate in the final stages of melanosome trafficking (Table 8.1).

The first major breakthrough came with the identification of the mouse dilute gene as the unconventional myosin, myosin Va (Mercer et al., 1991). Unconventional myosins are actin-based motor proteins and are widely expressed (Hasson and Mooseker, 1995). Myosin Va has a globular head domain containing the ATP- and actin-binding sites, a “neck” domain, which is the site of calmodulin (or light chain) binding, and a tail domain, which interacts with cargo. The dilute mouse shows pigment dilution characterized by irregular clumping of pigment in the hair, as well as opisthotonic seizures and death, and both melanocytes and neural cells have been shown to express myosin Va. The tail domain of myosin V serves as an anchor for a variety of cargoes, including melanosomes and synaptic vesicles in mammals to vacuoles and messenger RNA in yeast (Provance and Mercer, 1999). Clues to the function of myosin Va came through immunofluorescence colocalization 173

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studies in which myosin Va was shown to be localized to melanosomes (Provance et al., 1996). Immunoelectron microscopy further confirmed the co-localization of myosin Va and melanosomes (Wu et al., 1997). Melanocyte dendrites were normal in dilute mice, indicating that the primary defect caused by myosin Va mutation was in melanosome distribution, rather than in dendrite formation. Similarly, melanosome biogenesis was also shown to be normal in dilute mice (Provance et al., 1996). Because myosin Va, like other myosins, is an actin-based motor, the question of how myosin Va functioned in the transport of melanosomes became one of intense interest. One possibility was that myosin Va transported melanosomes along actin filaments that extend the length of the dendrite. The other possibility was that the myosin Va–melanosome unit was “captured” at the actin-rich tip of the dendrite, thus positioning the melanosome for transfer to keratinocytes. By taking advantage of the ability to observe melanosomes in vivo through their high reflectivity under Nomarsky optics, Wu and colleagues used melanocytes cultured from dilute and wild-type melanocytes in combination with agents that disrupt microtubules to determine that myosin Va functions to facilitate the “capture” of melanosomes at the actin-rich tip of the dendrite (Wu et al., 1997). Evidence to support this model included the demonstraSense DNA

Antisense DNA

12h A

B

C

D

12h

Fig. 8.4. Antisense DNA complementary to cytoplasmic dynein sequence disperses perinuclear melanosomes. Sense DNA-treated melanocytes show normal perinuclear distribution of melanosomes at 12 h (A) and 24 h (C). In contrast, antisense DNA-treated cells show a perinuclear melanosome-free zone (arrow) with increased peripheral membrane pigment distribution at 12 h (B) and loss of perinuclear melanosomes and marked net anterograde dispersion of melanosomes at 24 h (D). Photograph used with kind permission of Dr R. Byers (Byers et al., 2000).

tion that actin filaments are concentrated at the dendrite tip, and that their orientation is essentially random. Melanosome movement in melanocytes cultured from dilute mice, viewed in real time, demonstrated bidirectional movement with an average speed of 1.04 mm/s for centrifugal movement and 1.13 mm/s for centripetal movement. In the dilute mice, which lack myosin Va, melanosome transport along the dendrite was similar to that in wild-type mice; however, peripheral accumulation of melanosomes did not occur. Introduction of the myosin Va tail domain into wild-type mice resulted in a dilute phenotype, with redistribution of melanosomes to the cell body suggesting that the tail domain acted in a dominant-negative fashion. Further analysis of the myosin Va gene revealed that alternative splice variants contain different combinations of exons B, D, and F. Further, it was shown that cargo selection depends upon the splice variant expressed in a cell (Huang et al., 1998). Analysis of mutations in dilute mice have shown that both the tail and the head regions of myosin Va may be mutated and that exon F is responsible for binding of the tail region of myosin Va to melanosomes (Au and Huang, 2002).

Rab27A and Melanophilin: the Next Pieces of the Puzzle Subsequent to the discovery that myosin Va was the product of the dilute locus, and that it functions to allow capture of melanosomes at the tip of the dendrite, the defect in the ashen mouse was identified as the Rab protein Rab27A (Wilson et al., 2000). Similar to dilute mice, dendrites in ashen mice are normal; however, melanosome transfer to follicular keratinocytes is abnormal. Rab proteins are a large and diverse family of small GTP-binding proteins that are critical for targeting membrane fusion of vesicles from different cellular compartments. How Rabs work is still unknown; however, it is known that they recruit a large group of different proteins (“effector molecules”) to membranes when they are activated by GTP binding. Over 60 Rab proteins have been identified in humans. Ashen mice also have bleeding abnormalities, suggesting that Rab27A directs the transport of both melanosomes and vesicles in platelets. Based on genetic evidence, many Rab27A effector proteins have been identified that share the conserved N-terminal Rab27A binding domain and show Rab27A binding activity in vitro, which suggests that the Rab27A subfamily regulates exocytotic pathways in a variety of cell types. At this point, it was clear that myosin Va and Rab27A both functioned in directing melanosome transport to the dendrite tip, but whether they worked in parallel yet independent

Table 8.1. Mouse mutants, their corresponding human homolog, and protein affected in melanosome trafficking. Mouse mutant

Human homolog

Phenotype

Protein affected

Dilute mouse Ashen mouse Leaden mouse

Griscelli syndrome I Griscelli syndrome II Griscelli syndrome III

Hypopigmentation and neurologic disorder Hypopigmentation and immunologic abnormality Hypopigmentation

Myosin Va Rab27A Melanophilin

174

MELANOSOME TRAFFICKING AND TRANSFER

pathways or as a complex was unknown. When the genetic defect in the leaden mouse was identified as melanophilin, the product of the MLPH gene, genetic and biochemical techniques were utilized to show that these three proteins, myosin Va, Rab27A, and melanophilin, formed a complex that functioned to capture melanosomes at the dendrite tip. A series of papers demonstrated that Rab27A associates with the membrane of melanosomes, which then recruits melanophilin, which in turn recruits myosin Va (Fig. 8.5; Barral et al., 2002; Fukuda et al., 2002; Strom et al., 2002; Wu et al., 2002a). Rab27A associates with myosin Va and melanophilin (also known as Slac2-a) through distinct regions of the protein, and melanophilin acts as a linker to bridge the interactions between activated Rab27A and myosin Va. The middle region of melanophilin directly binds the globular tail of myosin Va and adjacent exon F (Fukuda et al., 2002; Provance et al., 2002; Strom et al., 2002; Wu et al., 2002b). Melanophilin binds to Rab27A via its N-terminal Slp homology domain. Melanophilin has also been shown to bind actin, and the absence of the actin binding domain of melanophilin results in accumulation of melanosomes in the cell center, adding further support for the “capture model” of melanosome transport originally proposed by Wu and colleagues (Fukuda et al., 2002; Wu et al., 1997). The ability of melanophilin to bind actin, in addition to its function as a Rab27A and myosin Va linker protein, is postulated to transfer melanosomes from microtubules to actin filaments and may help melanosomes bind to peripheral actin near the tip of the dendrite (Fukuda et al., 2002). In retinal pigment epithelial cells, a similar tripartite complex of Rab27A, MyRIP, and myosin VIIA has been described (El-Amraoui et al., 2002). Rab27A has also been shown to bind to Gnph (involved in insulin secretion) and several other novel proteins of which the function is still unknown (JFC1/Slp1, Slp2-a, Slp3-a, and Slac2-b) (Strom et al., 2002).

Griscelli Syndrome Griscelli syndrome (GS) is an autosomal recessive genodermatosis characterized by hypopigmentation of the skin and hair, clusters of pigment in the hair shaft, and either a primary neurologic impairment or severe immunodeficiency. Griscelli syndrome and three defined variants (I–III) are caused by mutations in genes that regulate vesicle transport and are the human homologs of the dilute, ashen, and leaden phenotypes. While all patients with Griscelli syndrome share pigment dilution, they are differentiated by secondary manifestations including mental retardation, developmental delay, and neurologic abnormalities in GS type I and severe immune abnormalities including T cell and macrophage activation, leading to organ infiltration and damage in GS type II. The phenotype of patients with GS type III is restricted to hypopigmentation. Griscelli syndrome type II, the largest group, is the human homolog of the ashen phenotype and is due to mutations in Rab27A. Patients exhibit partial albinism, hemophagocytic syndrome, and immune abnormalities. Griscelli syndrome type I is due to mutations in myosin Va

Myosin Va

Actin Head

Proximal tail

Neck Globular tail NH2

Melanophilin

COOH

Rab27A GTP

Melanosome

Fig. 8.5. Schematic representation of the tripartite protein complex responsible for melanosome transport. GTP-bound Rab27A is associated with the melanosome and binds to melanophilin at the amino-terminal domain of melanophilin. Melanophilin binds the globular tail of myosin Va through the C-terminal domain. The head (motor) domain of myosin Va interacts with actin filaments.

and manifests with partial albinism and neurologic abnormalities. While these two subtypes are generally accepted, there is some evidence that GS types II and I may both represent defects in Rab27A. A report identified a Muslim Arab kindred with mutations in Rab27A, but not myosin Va, who exhibited variable neurologic abnormalities as well as hemophagocytic syndrome (Anikster et al., 2002). The authors propose that previously reported patients with defects in myosin Va who exhibited neurologic defects but no immune abnormalities may instead have Elejalde syndrome, a condition characterized by mild hypopigmentation and severe, primary neurologic abnormalities, rather than Griscelli syndrome (Anikster et al., 2002). Griscelli syndrome type III is due to mutations in melanophilin (Anikster et al., 2002; Menasche et al., 2000, 2003a), but may also be seen in patients with deletion mutations in exon F of myosin Va (Menasche et al., 2003b).

Melanosome Transfer to Keratinocytes Having produced a melanosome complete with enzymes necessary for melanin synthesis, and having successfully transported it to the tip of the dendrite, the final duty of the melanocyte is to transfer the melanosome to the keratinocyte. It is generally agreed that UVR and the hormone MSH stimulate melanosome transfer, although the proteins and 175

CHAPTER 8

signaling intermediates involved in UVR- and MSH-activated melanosome transfer are still unknown (Virador et al., 2002). Other agents, such as niacinamide, the physiologically active amide of niacin, have been shown to suppress melanosome transfer (Hakozaki et al., 2002). A major hurdle that has severely limited progress in understanding the molecular basis of melanosome transfer has been the lack of a model system. The majority of studies of melanosome transfer to keratinocytes have been based on cocultures of nonhuman cells observed by electron microscopy. Studies utilizing time-lapse video microscopy have been limited by the relatively poor resolution achieved (Cohen and Szabo, 1968; Mottaz and Zelickson, 1967; Wolff, 1973). Other more recent studies have utilized gold particle uptake by keratinocytes, melanin uptake, or transfer of cytoplasmic dyes from melanocytes to keratinocytes to measure transfer (Minwalla et al., 2001; Seiberg et al., 2000; Sharlow et al., 2000). In toto, these prior studies led to observations that suggested phagocytosis of melanocyte dendrites by keratinocytes as the major mode of melanosome transfer, although exocytosis of melanosomes into the extracellular space with uptake by keratinocytes and insertion of melanocyte dendrites and transfer of melanosomes to keratinocytes have also been proposed (Yamamoto and Bhawan, 1994). In this chapter, I will first review current information about melanosome transfer at the structural level and then discuss data in which molecular mediators of melanosome transfer have been identified.

Melanosome Transfer Occurs Along Filopodial Extensions of the Melanocyte Dendrite and Cell Body We used high-resolution movies made from digital images of human melanocyte–keratinocyte cocultures to observe directly melanosome transfer to keratinocytes in vitro (Scott et al., 2002). These movies, along with electron microscopy of cells in vitro and skin in vivo, provided evidence showing that melanosome delivery to keratinocytes occurs along filopodia (Figs 8.6 and 8.7). Time-lapse digital microscopy showed the presence of long (up to 16 mm) dynamically active filopodia arising from melanocyte dendrite tips and the melanocyte cell body, many of which contained melanosomes that were easily visualized under Nomarsky optics (Fig. 8.6). Close analysis of the movies showed that melanosomes moved along the filopodia and were transferred to the keratinocytes (Fig. 8.7). We also observed filopodia arising from melanocytes in human skin biopsies by electron microscopy. These novel observations contradicted the long-held belief that whole tips of dendrites, relatively large structures, are engulfed by keratinocytes. Another important observation was that melanosome transfer is a relatively uncommon event, occurring perhaps once over the course of 45 min of observation. In many instances, melanosomes within filopodia were observed to travel bidirectionally along the length of the filopodia, and transfer was often aborted. The filopodia were observed to attach and detach from the keratinocyte membrane and, in most instances, melanosome transfer did not occur. In a report by Zhang et al. (2004), atomic 176

fp

fp fp

fp A

fp 2µ

2µ

fp

fp

fp

KM

KM

KM

B

C KM

KM

KM

D 2µ Fig. 8.6. Melanocytes exhibit filopodia. Melanocytes extend long filopodia from dendrite tips that transport melanosomes to keratinocytes. (A) Two melanocyte dendrites with prominent filopodia (fp; arrowhead) are shown in images taken at 8 s. The arrowheads point to thin structures consistent with filopodia. (B) A melanosome (circle) is present in a filopodium (fp) and moves towards the keratinocyte membrane (KM; outlined by hatched line). Over the course of 16 s, the melanosome has moved along a filopodium toward the keratinocyte membrane. (C) The tip of a melanocyte dendrite is shown with multiple connections with a keratinocyte membrane (KM; outlined by hatched line). A melanosome (circle) moves toward the KM over the course of 40 s. (D) Two sequential images captured 5 min after treatment of cells with nocodazole are shown. Melanosomes have redistributed toward the melanocyte cell body, leaving a dendrite that appears empty. Filopodia (arrowhead) were not affected by nocodazole treatment.

force microscopy was used to image human epidermal melanocytes. They also noted filopodia arising from melanocyte dendrites and cell body and the presence of melanosomes within the filopodia, further establishing filopodia as a conduit for melanosome transfer. In neural cells, filopodia primarily function as a guide to the neural growth cone through receptors on its membrane, which responds to exogenous hormones that stimulate growth cone turning and directionality. It is likely that melanocyte filopodia also function as pathfinding structures and that they express receptors that respond to keratinocytederived factors that guide them to the keratinocyte membrane where they attach. While the receptors used for attachment of filopodia to the keratinocyte membrane are still unknown, work by several groups (Cerdan et al., 1991; Condaminet et al., 1997; Min-

MELANOSOME TRAFFICKING AND TRANSFER A 1

3

2

4

5

6

KC

MD 10µ

2µ

2µ

B

KC MD

10µ

*

Fig. 8.7. Melanosomes are transported to the keratinocyte along filopodia. (A) A scanning view of a melanocyte dendrite (MD) contacting a keratinocyte (KC) is shown. The boxed area is shown in detail in sequential images taken every 8 s from movies made from this area. A filopodium arising from the lateral aspect of the dendrite is either attached or inserted into the keratinocyte membrane. A string of melanosomes (approximately six of them) moves in single file toward the keratinocyte (2–5). (B) A scanning view of a melanocyte dendrite (MD) adjacent to a keratinocyte (KC) is shown. The hatched line delineates the KC membrane in the upper right-hand corner. The boxed area is shown in detail in images that span 152 s. A string of melanosomes (approximately four of them; arrowhead) is present within a projection arising from the side of the body of the melanocyte dendrite. These projections were frequently observed in melanocytes and were shorter and thicker than filopodia. Other similar projections are present (asterisks).

walla et al., 2001) has shown that lectins and their glycoconjugate ligands may function as receptor–ligand pairs in keratinocyte–melanocyte interactions. Selective lectin binding sites have been demonstrated on melanocytes in normal skin (Nau and Schaumburg-Lever, 1990), and Minwalla et al. (2001) performed functional studies to show that the addition of selected lectins and neoglycoconjugates inhibited melanosome transfer in melanocyte–keratinocyte cocultures.

Molecular Mediators of Melanosome Transfer Direct examination of melanosome transfer shows that melanosomes are trafficked along filopodia; however, the mechanism by which the melanosome is finally donated to the keratinocyte is still unclear. One potential mechanism may be exocytosis of melanosomes from filopodia tips with uptake by keratinocytes through endocytosis, analogous to synaptic vesicle release and uptake in neural cells. Although we did not directly observe exocytosis in digital movies, it is still possible that exocytosis occurs. Virador and colleagues (2002) examined murine melanocyte–keratinocyte cocultures and concluded that exocytosis of melanosomes occurred following UVR or the addition of MSH through determination of melanin content in culture supernatants. However, whether changes in melanin reflected an increased number of melanosomes through exocytosis, increased tyrosinase activ-

ity, or sloughing of dendritic tips was not determined. In support of a role for exocytosis in melanosome transfer is the identification of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Rab3a on melanosomes. SNARE and Rab family of proteins are involved in vesicle transport and docking (Rab) and membrane fusion (SNAREs). The Rab family of GTP-binding proteins regulates vesicle transport as well as exocytosis (for a review, see Jahn and Sudhof, 1999) and localizes to the surface of organelles involved in vectorial transport. Rab3a has been shown to be critical for exocytosis of synaptic vesicles in neurons, secretion from pancreatic b cells, granule secretion in mast cells, and zymogen secretion from pancreatic acinar cells (Takai et al., 1996). Studies have shown that Rab3a, VAMP2, SNAP-23, SNAP-25, and syntaxin-4 are expressed by melanocytes and that Rab3a and SNARE proteins are present on melanosomes (Araki et al., 2000; Scott and Zhao, 2001). These data suggest that SNARE proteins and members of the Rab protein family may be involved in exocytosis of melanosomes. Interestingly, DNA array analysis of genes upregulated by MSH in murine melanocytes showed that several SNARE proteins, including syntaxin-4 and a synaptobrevin-like protein, are upregulated (Virador et al., 2002). Therefore, the presence of SNARE proteins on melanosomes, in conjunction with reports of exocytosis observed ultrastruc177

CHAPTER 8 Serine protease cleavage site

NRSSKGR

Tethered ligand

NH2

NH2 TRYPSIN

COOH COOH

turally or in video capture movies, raises the possibility that exocytosis is a mechanism of melanosome transfer. Another likely mechanism for melanosome transfer is phagocytosis of melanosome-containing filopodia tips. This hypothesis has been strengthened by work showing that a receptor expressed by keratinocytes, the proteinase activated receptor-2 (PAR-2) is involved in melanosome transfer and skin pigmentation (Seiberg, 2001; Seiberg et al., 2000; Sharlow et al., 2000). The PAR family are G-coupled transmembrane receptors that are activated by serine proteinases, which cleave the extracellular amino-terminal domain of the PAR and expose a tethered ligand that undergoes a conformational change with subsequent activation of the receptor (Fig. 8.8; for a review, see Macfarlane et al., 2001). In HaCat, PAR-2 is activated by trypsin, mast cell tryptase, factor VIIa, or factor Xa, and by synthetic peptides that mimic the amino-terminal portion of the receptor (Camerer et al., 2000; Santulli et al., 1995; Steinhoff et al., 1999). Like all Gcoupled protein receptors, PARs have seven transmembrane domains and associate with heterotrimeric G proteins. PAR-2 is involved in a broad spectrum of physiological processes, including induction of cytokine and prostaglandin expression, mitogenesis, and inflammatory responses (Asokananthan et al., 2002; Mirza et al., 1997). Seiberg et al. (2000) and Sharlow et al. (2000) demonstrated that, in keratinocyte–melanocyte cocultures, PAR-2 is activated by UVR, and activation induces melanosome transfer through increased keratinocytic phagocytosis of melanosomes. Electron micrographs of PAR-2 activated cells demonstrated complex membrane ruffling and plasma membrane extensions compared with control cells, and PAR-2 activation induced increased actin polymerization and a-actinin filament organization beneath the plasma membrane (Sharlow et al., 2000). The importance of the actin cytoskeleton in PAR2-mediated melanosome uptake was shown by experiments in which activation of PAR-2 resulted in activation of Rho, which has been implicated in phagocytosis in other cell types (Qualmann and Mellor, 2003; Scott et al., 2003). Activation of PAR-2 has also been demonstrated to alter pigmentation via modulation of melanosome uptake in vivo. Serine protease inhibitors that interfere with PAR-2 activation, such as soybean 178

Fig. 8.8. Schematic representation of PAR-2 activation. PAR-2 is a seven transmembrane receptor that is activated by serine proteases such as trypsin. These act through cleavage of a specific sequence at the amino-terminal domain of the PAR, which results in unmasking of a new N-terminus that then folds back onto the receptor and activates it. Activation may also be achieved through the use of synthetic peptides (such as SLIGRL-NH2) that mimic the tethered ligand sequence.

trypsin inhibitor, induced a dose-dependent depigmentation of the skin of dark-skinned Yucatan swine. The inhibitors work by inhibiting melanosome transfer and distribution, which leads to skin lightening in vivo (Seiberg et al., 2000a). Moreover, inhibition of PAR-2 activation prevented UVB-induced pigmentation both in vitro and in vivo (Seiberg et al., 2000a). We showed recently that PAR-2 receptor expression is increased in human skin and in cultured keratinocytes following UVR (Scott et al., 2001). Levels of PAR-2 expression, as well as trypsin, which activates PAR-2, are higher in dark-skinned compared with light-skinned individuals (Seiberg et al., 2003). We also showed that UVR-induced PAR-2 expression is attenuated in people who tan poorly (skin type I) compared with people who tan more easily (skin types II and III). Together, these studies suggest that PAR-2 is a critical receptor involved in keratinocyte uptake of melanosomes. Activation of PAR-2 has also been shown to stimulate the release of prostaglandins from human keratinocytes (Scott et al., 2004). Because prostaglandins stimulate dendricity in human melanocytes (Scott et al., in press), as well as melanin synthesis and potentially other aspects of melanocyte biology (Morelli et al., 1989; Nordlund et al., 1986; Parsad et al., 2002; Sauk et al., 1975; Tomita et al., 1992), PAR-2 may control pigmentation through diverse mechanisms.

Conclusion The ability of melanosomes to be trafficked along dendrites and then transferred to keratinocytes is of critical importance for cutaneous pigmentation. It is clear that melanosomes are moved along microtubules, are captured at the dendrite tips through the molecular complex of myosin Va, rab27A, and melanophilin, and are then transferred to keratinocytes along filopodia. Of the two processes, trafficking and transfer, much more is known about the former than the latter. Future studies will be directed at defining in more precise detail the adhesion molecules that function to regulate melanocyte dendrite and filopodia attachment to the keratinocyte membrane, the molecules involved in the regulation of phagocytosis of

MELANOSOME TRAFFICKING AND TRANSFER

melanosomes by keratinocytes, and possibly the proteins involved in exocytosis of melanosomes. Still to be determined is the relationship between signaling intermediates, such as the cAMP/PKA pathway, known to stimulate melanosome transfer, and their role in regulating proteins involved in the process of melanosome trafficking and transfer.

References Anikster, Y., Huizing, M., Anderson, P. D., Fitzpatrick, D. L., Klar, A., Gross-Kieselstein, E., Berkun, Y., Shazberg, G., Gahl, W. A., and Hurvitz, H. Evidence that Griscelli syndrome with neurological involvement is caused by mutations in RAB27A, not MYO5A. Am. J. Hum. Genet. 71:407–414, 2002. Araki, K., Horikawa, T., Chakraborty, A. K., Nakagawa, K., Itoh, H., Oka, M., Funasaka, Y., Pawelek, J., and Ichihashi, M. Small Gtpase rab3A is associated with melanosomes in melanoma cells. Pigment Cell Res. 13:332–336, 2000. Asokananthan, N., Graham, P. T., Fink, J., Knight, D. A., Bakker, A. J., McWilliam, A. S., Thompson, P. J., and Stewart G. A. Activation of protease-activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells. J. Immunol. 168:3577–3585, 2002. Au, J. S., and Huang, J. D. A tissue-specific exon of myosin Va is responsible for selective cargo binding in melanocytes. Cell Motil. Cytoskeleton 53:89–102, 2002. Babiarz-Magee, L., Chen, N., Seiberg, M., and Lin, C. B. The expression and activation of protease-activated receptor-2 correlate with skin color. Pigment Cell Res. 17:241–251, 2004. Barral, D. C., Ramalho, J. S., Anders, R., Hume, A. N., Knapton, H. J., Tolmachova, T., Collinson, L. M., Goulding, D., Authi, K. S., and Seabra, M. C. Functional redundancy of Rab27 proteins and the pathogenesis of Griscelli syndrome. J. Clin. Invest. 110:247–257, 2002. Busca, R., Bertolotto, C., Abbe, P., Englaro, W., Ishizaki, T., Narumiya, S., Boquet, P., Ortonne, J. P., and Ballotti, R. Inhibition of Rho is required for cAMP-induced melanoma cell differentiation. Mol. Biol. Cell 9:1367–1378, 1998. Byers, H. R., Yaar, M., Eller, M. S., Jalbert, N. L., and Gilchrest, B. A. Role of cytoplasmic dynein in melanosome transport in human melanocytes. J. Invest. Dermatol. 114:990–997, 2000. Camerer, E., Huang, W., and Coughlin, S. R. Tissue factor and factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proc. Natl. Acad. Sci. USA 97:5255–5260, 2000. Cerdan, D., Grillon, C., Monsigny, M., Redziniak, G., and Kieda, C. Human keratinocyte membrane lectins: characterization and modulation of their expression by cytokines. Biol. Cell 73:35–42, 1991. Chen, Y., Samaraweera, P., Sun, T. T., Kreibich, G., and Orlow, S. J. Rab27b association with melanosomes: dominant negative mutants disrupt melanosomal movement. J. Invest. Dermatol. 118:933–940, 2002. Cohen, J., and Szabo, G. Study of pigment donation in vitro. Exp. Cell Res. 50:418–433, 1968. Condaminet, B., Redziniak, G., Monsigny, M., and Kieda, C. Ultraviolet rays induced expression of lectins on the surface of a squamous carcinoma keratinocyte cell line. Exp. Cell Res. 232:216–224, 1997. Edgar, A. J., and Bennett, J. P. Inhibition of dendrite formation in mouse melanocytes transiently transfected with antisense DNA to myosin Va. J. Anat. 195 (Pt 2):173–184, 1999. El-Amraoui, A., Schonn, J. S., Kussel-Andermann, P., Blanchard, S., Desnos, C., Henry, J. P., Wolfrum, U., Darchen, F., and Petit, C. MyRIP, a novel Rab effector, enables myosin VIIa recruitment to retinal melanosomes. EMBO Rep. 3:463–470, 2002. Etienne-Manneville, S., and Hall, A. Rho GTPases in cell biology. Nature 420:629–635, 2002. Fukuda, M., Kuroda, T. S., and Mikoshiba, K. Slac2-a/melanophilin,

the missing link between Rab27 and myosin Va: implications of a tripartite protein complex for melanosome transport. J. Biol. Chem. 277:12432–12436, 2002. Gross, S. P., Tuma, M. C., Deacon, S. W., Serpinskaya, A. S., Reilein, A. R., and Gelfand, V. I. Interactions and regulation of molecular motors in Xenopus melanophores. J. Cell Biol. 156:855–865, 2002. Hakozaki, T., Minwalla, L., Zhuang, J., Chhoa, M., Matsubara, A., Miyamoto, K., Greatens, A., Hillebrand, G. G., Bissett, D. L., and Boissy, R. E. The effect of niacinamide on reducing cutaneous pigmentation and suppression of melanosome transfer. Br. J. Dermatol. 147:20–231, 2002. Hara, M., Yaar, M., Byers, H. R., Goukassian, D., Fine, R. E., Gonsalves, J., and Gilchrest, B. A. Kinesin participates in melanosomal movement along melanocyte dendrites. J. Invest. Dermatol. 114:438–443, 2000. Hasson, T., and Mooseker, M. S. Molecular motors, membrane movements and physiology: emerging roles for myosins. Curr. Opin. Cell Biol. 7:587–594, 1995. Hirose, K., and Amos, L. A. Three-dimensional structure of motor molecules. Cell Mol. Life Sci. 56:184–199, 1999. Huang, J. D., Mermall, V., Strobel, M. C., Russell, L. B., Mooseker, M. S., Copeland, N. G., and Jenkins, N. A. Molecular genetic dissection of mouse unconventional myosin-VA: tail region mutations. Genetics 148:1963–1972, 1998. Jahn, R., and Sudhof, T. C. Membrane fusion and exocytosis. Annu. Rev. Biochem. 68:863–911, 1999. Kull, F. J. Motor proteins of the kinesin superfamily: structure and mechanism. Essays Biochem. 35:61–73, 2000. Macfarlane, S. R., Seatter, M. J., Kanke, T., Hunter, G. D., and Plevin, R. Proteinase-activated receptors. Pharmacol. Rev. 53:245–282, 2001. McGuire, J., and Moellmann, G. Cytochalasin B: effects on microfilaments and movement of melanin granules within melanocytes. Science 175:642–644, 1972. Menasche, G., Pastural, E., Feldmann, J., Certain, S., Ersoy, F., Dupuis, S., Wulffraat, N., Bianchi, D., Fischer, A., Le Deist, F., and de Saint Basile, G. et al. Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nature Genet. 25:173–176, 2000. Menasche, G., Feldmann, J., Houdusse, A., Desaymard, C., Fischer, A., Goud, B., and de Saint Basile, G. Biochemical and functional characterization of Rab27a mutations occurring in Griscelli syndrome patients. Blood 101:2736–2742, 2003a. Menasche, G., Ho, C. H., Sanal, O., Feldmann, J., Tezcan, I., Ersoy, F., Houdusse, A., Fischer, A., and de Saint Basile, G. Griscelli syndrome restricted to hypopigmentation results from a melanophilin defect (GS3) or a MYO5A F-exon deletion (GS1). J. Clin. Invest. 112:450–456, 2003b. Mercer, J. A., Seperack, P. K., Strobel, M. C., Copeland, N. G., and Jenkins, N. A. Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature 349:709–713, 1991. Minwalla, L., Zhao, Y., Cornelius, J., Babcock, G. F., Wickett, R. R., Le Poole, I. C., and Boissy, R. E. Inhibition of melanosome transfer from melanocytes to keratinocytes by lectins and neoglycoproteins in an in vitro model system. Pigment Cell Res. 14:185–194, 2001. Mirza, H., Schmidt, V. A., Derian, C. K., Jesty, J., and Bahou W. F. Mitogenic responses mediated through the proteinase-activated receptor-2 are induced by expressed forms of mast cell alpha- or beta-tryptases. Blood 90:3914–3922, 1997. Moellmann, G., and McGuire, J. Correlation of cytoplasmic microtubules and 10-nm filaments with the movement of pigment granules in cutaneous melanocytes of Rana pipiens. Ann. N. Y. Acad. Sci. 253:711–722, 1975. Moellmann, G., McGuire, J., and Lerner, A. B. Ultrastructure and cell biology of pigment cells. Intracellular dynamics and the fine structure of melanocytes with special reference to the effects of MSH

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CHAPTER 8 and cyclic AMP on microtubules and 10-nm filaments. Yale J. Biol. Med. 46:337–360, 1973. Morelli, J. G., Yohn, J. J., Lyons, M. B., Murphy, R. C., and Norris, D. A. Leukotrienes C4 and D4 as potent mitogens for cultured human neonatal melanocytes. J. Invest. Dermatol. 93:719–722, 1989. Mottaz, J. H., and Zelickson, A. S. Melanin transfer: a possible phagocytic process. J. Invest. Dermatol. 49:605–610, 1967. Nau, P., and Schaumburg-Lever, G. Ultrastructural localization of lectin binding sites in human melanocytes. Arch. Dermatol. Res. 282:520–525, 1990. Nordlund, J. J., Collins, C. E., and Rheins, L. A. Prostaglandin E2 and D2 but not MSH stimulate the proliferation of pigment cells in the pinnal epidermis of the DBA/2 mouse. J. Invest. Dermatol. 86:433–437, 1986. Parsad, D., Pandhi, R., Dogra, S., and Kumar, B. Topical prostaglandin analog (PGE2) in vitiligo — a preliminary study. Int. J. Dermatol. 41:942–945, 2002. Provance, D. W., and Mercer, J. A. Myosin-V: head to tail. Cell. Mol. Life Sci. 56:233–242, 1999. Provance, D. W., Jr., Wei, M., Ipe, V., and Mercer, J. A. Cultured melanocytes from dilute mutant mice exhibit dendritic morphology and altered melanosome distribution. Proc. Natl. Acad. Sci. USA 93:14554–14558, 1996. Provance, D. W., James, T. L., and Mercer, J. A. Melanophilin, the product of the leaden locus, is required for targeting of myosin-Va to melanosomes. Traffic 3:124–132, 2002. Qualmann, B., and Mellor, H. Regulation of endocytic traffic by Rho GTPases. Biochem. J. 371:233–241, 2003. Rogers, S. L., Tint, I. S., Fanapour, P. C., and Gelfand, V. I. Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro. Proc. Natl. Acad. Sci. USA 94:3720–3725, 1997. Santulli, R. J., Derian, C. K., Darrow, A. L., Tomko, K. A., Eckardt, A. J., Seiberg, M., Scarborough, R. M., and Andrade-Gordon, P. Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes. Proc. Natl. Acad. Sci. USA 92:9151–9155, 1995. Sauk, J. J., Jr., White, J. G., and Witkop, C. J., Jr. Influence of prostaglandins E1, E2, and arachidonate on melanosomes in melanocytes and keratinocytes of anagen hair bulbs in vitro. J. Invest. Dermatol. 64:332–337, 1975. Scott, G. Rac and rho: the story behind melanocyte dendrite formation. Pigment Cell Res. 15:322–330, 2002. Scott, G. A., and Cassidy, L. Rac1 mediates dendrite formation in response to melanocyte stimulating hormone and ultraviolet light in a murine melanoma model. J. Invest. Dermatol. 111:243–250, 1998. Scott, G., and Leopardi, S. The cAMP signaling pathway has opposing effects on Rac and Rho in B16F10 cells: implications for dendrite formation in melanocytic cells. Pigment Cell Res. 16:139–148, 2003. Scott, G., and Zhao, Q. Rab3a and SNARE proteins: potential regulators of melanosome movement. J. Invest. Dermatol. 116:296–304, 2001. Scott, G., Deng, A., Rodriguez-Burford, C., Seiberg, M., Han, R., Babiarz, L., Grizzle, W., Bell, W., and Pentland, A. Proteaseactivated receptor 2, a receptor involved in melanosome transfer, is upregulated in human skin by ultraviolet irradiation. J. Invest. Dermatol. 117:1412–1420, 2001. Scott, G., Leopardi, S., Printup, S., and Madden, B. C. Filopodia are conduits for melanosome transfer to keratinocytes. J. Cell Sci. 115:1441–151, 2002. Scott, G., Leopardi, S., Parker, L., Babiarz, L., Seiberg, M., and Han, R. The proteinase-activated receptor-2 mediates phagocytosis in a

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Rho-dependent manner in human keratinocytes. J. Invest. Dermatol. 121:529–541, 2003. Scott, G., Leopardi, S., Printup, S., Malhi, N., Seiberg, M., and Lapoint, R., Proteinase-activated receptor-2 stimulates prostaglandin production in keratinocytes: analysis of prostaglandin receptors on human melanocytes and effects of PGE and PGF on melanocyte dendricity. J. Invest. Dermatol. 122:1214–1224, 2004. Seiberg, M. Keratinocyte-melanocyte interactions during melanosome transfer. Pigment Cell Res. 14:236–242, 2001. Seiberg, M., Paine, C., Sharlow, E., Andrade-Gordon, P., Costanzo, M., Eisinger, M., and Shapiro, S. S. The protease-activated receptor 2 regulates pigmentation via keratinocyte–melanocyte interactions. Exp. Cell Res. 254:25–32, 2000. Sharlow, E. R., Paine, C. S., Babiarz, L., Eisinger, M., Shapiro, S., and Seiberg, M. The protease-activated receptor-2 upregulates keratinocyte phagocytosis. J. Cell Sci. 113 (Pt 17):3093–3101, 2000. Steinhoff, M., Corvera, C. U., Thoma, M. S., Kong, W., McAlpine, B. E., Caughey, G. H., Ansel, J. C., and Bunnett. N. W. Proteinaseactivated receptor-2 in human skin: tissue distribution and activation of keratinocytes by mast cell tryptase. Exp. Dermatol. 8:282–294, 1999. Strom, M., Hume, A. N., Tarafder, A. K., Barkagianni, E., and Seabra, M. C. A family of Rab27-binding proteins. Melanophilin links Rab27a and myosin Va function in melanosome transport. J. Biol. Chem. 277:25423–25430, 2002. Takai, Y., Sasaki, T., Shirataki, H., and Nakanishi, H. Rab3A small GTP-binding protein in Ca(2+)-dependent exocytosis. Genes Cells 1:615–632, 1996. Tomita, Y., Maeda, K., and Tagami, H. Melanocyte-stimulating properties of arachidonic acid metabolites: possible role in postinflammatory pigmentation. Pigment Cell Res. 5:357–561, 1992. Vancoillie, G., Lambert, J., Mulder, A., Koerten, H. K., Mommaas, A. M., Van Oostveldt, P., and Naeyaert, J. M. Cytoplasmic dynein colocalizes with melanosomes in normal human melanocytes. Br. J. Dermatol. 143:298–306, 2000. Virador, V. M., Muller, J., Wu, X., Abdel-Malek, Z. A., Yu, Z. X., Ferrans, V. J., Kobayashi, N., Wakamatsu, K., Ito, S., Hammer, J. A., and Hearing, V. J. Influence of alpha-melanocyte-stimulating hormone and ultraviolet radiation on the transfer of melanosomes to keratinocytes. FASEB J. 16:105–107, 2002. Wilson, S. M., Yip, R., Swing, D. A., O’Sullivan, T. N., Zhang, Y., Novak, E. K., Swank, R. T., Russell, L. B., Copeland, N. G., and Jenkins, N. A. A mutation in Rab27a causes the vesicle transport defects observed in ashen mice. Proc. Natl. Acad. Sci. USA 97:7933–7938, 2000. Wolff, K. Melanocyte-keratinocyte interactions in vivo: the fate of melanosomes. Yale J. Biol. Med. 46:384–396, 1973. Wu, X., Bowers, B., Wei, Q., Kocher, B., and Hammer, J. A., 3rd. Myosin V associates with melanosomes in mouse melanocytes: evidence that myosin V is an organelle motor. J. Cell Sci. 110 (Pt 7):847–859, 1997. Wu, X., Wang, F., Rao, K., Sellers, J. R., and Hammer, J. A., 3rd. Rab27a is an essential component of melanosome receptor for myosin Va. Mol. Biol. Cell 13:1735–1749, 2002a. Wu, X. S., Rao, K., Zhang, H., Wang, F., Sellers, J. R., Matesic, L. E., Copeland, N. G., Jenkins, N. A., and Hammer, J. A., 3rd. Identification of an organelle receptor for myosin-Va. Nature Cell Biol. 4:271–278, 2002b. Yamamoto, O., and Bhawan, J. Three modes of melanosome transfers in Caucasian facial skin: hypothesis based on an ultrastructural study. Pigment Cell Res. 7:158–169, 1994. Zhang, R. Z., Zhu, W. Y., Xia, M. Y., and Feng, Y. Morphology of cultured human epidermal melanocytes observed by atomic force microscopy. Pigment Cell Res. 17:62–65, 2004.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

9

Melanosome Processing in Keratinocytes H. Randolph Byers

Summary 1 Melano-phagosomes are transported along microtubule arrays toward the cell center. 2 The motive force of transport of the melano-phagosomes is the microtubule-based motor cytoplasmic dynein. 3 Melano-phagosome fusion with primary lysosomes near the centrosome gives rise to melano-phagolysosomes. 4 Melano-phagolysosomes occasionally form cisternae where melanosomes and dendrite cytoplasm are digested. 5 Melano-phagolysosomes are sometimes distorted into cisternae and stretched into fingerlike projections until individual or small aggregates of melanosomes are pulled off the distorted membrane by dynactin complex/cytoplasmic dynein step motoring along microtubules. 6 Cytoplasmic dynein/dynactin complex maintains the phagocytosed melanosomes and melano-phagolysosomes into perinuclear aggregates in the basal layer and supranuclear melanin caps in the suprabasal keratinocytes. 7 The perinuclear and supranuclear melanin caps (microparasols) protect the nucleus from UV irradiation-induced DNA damage.

Introduction Skin color and protection of the skin from ultraviolet (UV) radiation is primarily dependent on the amount and distribution of melanin in the epidermis. Melanin is the major component of melanosomes, which are produced by melanocytes and transferred into adjacent basal keratinocytes. The suprabasal keratinocytes move upward through progressive keratinocyte layers of the epidermis where melanocyte dendrites continue to transfer melanosomes to the maturing keratinocytes. Melanin is eventually shed with the dead, cornified keratinocytes from the top layers of the stratum corneum. Melanosome transfer is a complex process whereby melanosomes are released or pinched off from the melanocyte dendrite and phagocytosed by adjacent keratinocytes singly or in aggregates. This chapter on melanosome processing in keratinocytes will exclude the rapid advances in our understanding of melanocyte–keratinocyte melanosome transfer mechanisms as they are presented in another chapter (Chapter

8). Therefore, post-phagocytic processing of the phagocytosed melanosome will be the focus of this chapter. The golden age of electron microscopy led to insights into the cellular and ultrastructural relationship of melanocytes, keratinocytes, transference of melanosomes, and the process whereby melano-phagosomes eventually fuse with other primary lysosomes and mature into melano-phagolysosomes (Cohen and Szabo, 1968; Jimbow et al., 1976; Klaus, 1969; Mottaz and Zelickson, 1967; Wolff et al., 1974). More recent immunologic, biochemical, and molecular techniques have accelerated our understanding of melanosome processing in keratinocytes. The close embryologic relationship of cellular derivatives of the neuroectoderm, including neurons, melanocytes, and keratinocytes, has allowed the cellular and molecular insight gained from one cell type to be translated into the other cell type. For example, our understanding of axoplasmic transport has parallel mechanisms in melanosome transport and melano-phagosome transport in keratinocytes. This chapter is divided into two sections, one on the mechanism of keratinocyte melano-phagolysosome distribution and the other on melano-phagolysosome maturation and degradation. The first section reviews how elucidating the molecular motors of pigment transport in lower vertebrate pigment cells (chromatophores; including melanophores and erythrophores) led to a deeper understanding of the mechanisms of melano-phagolysosome distribution in human keratinocytes. The second section reviews how our recent understanding of phagosome processing and maturation into phagolysosomes in all eukaryotes and melanocytes has led to insight into the maturation of melano-phagolysosomes in human keratinocytes.

The Mechanism of Melano-Phagosome and Melano-Phagolysosome Distribution in Keratinocytes Early investigators appreciated that the distribution of melanin in the epidermis and keratinocytes is nonrandom (Fig. 9.1). The basal keratinocytes exhibit more melanophagolysosomes than the spinous, granular, and cornified cell layers. The basal keratinocytes show perinuclear aggregation of melano-phagolysosomes around the nucleus. In the suprabasal keratinocytes of tanned individuals or darkly pigmented individuals, melanin is concentrated into “supranuclear 181

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Fig. 9.1. Melano-phagolysosomes are organized into supranuclear “caps” within keratinocytes. Note melanized dendritic melanocytes and adjacent keratinocytes with supranuclear “caps.” Melanin silver-stained (Fontana Masson) section of heavily melanized human epidermis. Bar = 50 mm. (Reprinted by permission of Elsevier Science, Inc., from Byers et al., 2003, copyright 2003 by The Society for Investigative Dermatology.)

caps” (Gates and Zimmermann, 1953). This melanin cap is precisely oriented toward the outer skin surface, enhances the protective effect of the nucleus from UV irradiation, and acts as an internal sunscreen. Investigators suspected that, similar to mechanisms responsible for movement of melanosomes observed in melanocytes, a force-generating factor would be found associated with the keratinocyte melano-phagosome. Electron microscopy provided insight into the mechanism of transport of melanosomes in melanocytes but was less helpful in elucidating the mechanism of the melano-phagolysosome distribution in keratinocyte cytoplasm. Only one ultrastructural study specifically addressed the “forces responsible for the establishment and maintenance of the supranuclear melanin cap” (Findlay and Liebenberg, 1976). These investigators subjected human epidermis to ultracentrifugation and found the melanin cap was relatively shear prone compared with the surrounding intermediate filaments or tonofilaments (cytokeratin filaments). This was somewhat of a paradox, as it suggested perhaps a passive displacement or exclusion from the periphery by the abundant intermediate filaments (cytokeratin filaments). Still, how would the cytoplasm become organized if it did not involve force generation? Clearly, there is a centripetal force that collects the peripheral phagocytosed melanosomes and transports them to the centrosomes in the perinuclear region. The mechanism of centripetal melanosome movement following phagocytosis in keratinocytes may be traced to our understanding of centripetal aggregation or inward migration of pigment granules in pigment cells of fish and amphibians. Electron microscopy identified microtubules oriented in the direction of pigment granule translocation of fish melanophores, suggesting a role for microtubules in mediating pigment migration (Bikle et al., 1966). Indeed, the dramatic light microscopic radial orientation of pigment granules 182

in pigment cells on fish scales drawn so beautifully near the end of 1800s by Ballowitz (1893) correlated perfectly with the elegant radial organization of microtubules seen with the electron microscope (Green, 1968; Wikswo and Novales, 1969) or by immunofluorescent localization in vitro (Schliwa et al., 1978) and in situ (Byers et al., 1980) with the fluorescent microscope. Microtubules were functionally shown to be involved in the mechanism when investigators found that the microtubule inhibitors colchicine or vinblastine inhibited pigment granule migration (Green, 1968; Malawista, 1971; Wikswo and Novales, 1969), and that inhibition correlated with microtubule disappearance (Wikswo and Novales, 1972). These investigators occasionally identified cytoplasmic bridges connecting the pigment granule with the microtubule. However, these bridges were difficult to visualize because of the ultrathin sectioning by conventional electron microscopy. Wholemount cultured pigment cells prepared for the highvoltage electron microscope provided three-dimensional visualization of microtubule to pigment and pigment to pigment fine cytoplasmic bridges (Byers and Porter, 1977). These fine bridges were called the microtrabeculae (Byers and Porter, 1977; Wolosewick and Porter, 1976). This was simply a descriptive term; the motive force appeared to reside within them and, although their molecular nature was completely unknown, speculation centered on acto-myosin-based systems, microtubule-associated proteins, and other uncharacterized cytoskeletal-associated proteins (Byers and Porter, 1977; Luby and Porter, 1980; Schliwa et al., 1981). The first microtubule-associated molecular motor characterized was the force-generating dynein arm that produces shear forces in the beating of cilia and flagella (Brokaw, 1994). The hypothesis was put forth that perhaps dynein and microtubules generated the shear forces necessary for translocation of pigment granules (Fujii and Novales, 1969). This mechanism was supported by elegant observations including statistical morphometric analysis of electron micrographs (Murphy, 1975; Murphy and Tilney, 1974) and ingenious use of inhibitors of dynein-like ATPases on fish pigment cells (Beckerle and Porter, 1982; Clark and Rosenbaum, 1982). Furthermore, dynein hooklet assays on intact and severed dendrites of fish pigment cells determined that the polarity of microtubules was maintained or repositioned such that their plus ends are oriented peripherally and their minus ends are positioned centrally (McNiven and Porter, 1986; McNiven et al., 1984). The discovery of cytoplasmic dyneins in all eukaryotes (Holzbaur and Vallee, 1994; Milisav, 1998; Schroer, 1994) and the early characterization of cytoplasmic dynein in fish melanoma cells (Ogawa et al., 1987) implied a role for this microtubule-based motor in pigment transport. Finally, the self-centering of the centrosome in pigment cells and all interphase cells appears to involve microtubule polymerization and formation of the radial microtubule array by cytoplasmic dynein (Burakov et al., 2003; Rodionov and Borisy, 1997; Vorobjev et al., 2001). Cytoplasmic dynein is a double-headed ATPase mechanochemical motor that steps along the microtubule

MELANOSOME PROCESSING IN KERATINOCYTES

toward the minus end of the microtubule at the perinuclear cell center. It functions as a molecular gear mechanism in response to a load by decreasing step distance and increasing force generation (Mallik et al., 2004). The dynein ATPase “nanomotor” heads are composed of two cytoplasmic dynein heavy chains of about 530 kDa each (Holzbaur and Vallee, 1994; Schroer, 1994). Light intermediate cytoplasmic dynein chains of 55 kDa and intermediate chains of 74 kDa bind the motor portion to dynactin, which attaches to membranebound organelles (Gill et al., 1991; Karki and Holzbaur, 1995; Schafer et al., 1994; Vaughan and Vallee, 1995). Heterogeneity of dynein chain subunits with multiple isoforms of the three different chains is hypothesized to participate in coordinated organelle migration or positioning in different cell types (Brill and Pfister, 2000; Criswell and Asai, 1998; King et al., 1998; Koonce et al., 1992; Maeda et al., 1998; Mikami et al., 1993; Nurminsky et al., 1998; Paschal et al., 1992; Pfister et al., 1996a, b; Tai et al., 2001; Tanaka et al., 1995; Tynan et al., 2000; Vaisberg et al., 1996; Zhang et al., 1993). Indeed, recent evidence suggests that only one of three forms of dynein light intermediate chains fractionates with melanosomes (Reilein et al., 2003). Immunofluorescent and functional blocking studies also point to the role of cytoplasmic dynein in minus-directed melanosome transport. Cytoplasmic dynein co-localizes to melanosomes that are previously stimulated to aggregate in fish melanophores (Nilsson et al., 1996). Furthermore, microinjection of anti-dynein antibody results in release of aggregated pigment granules and inhibits minus-end microtubule-directed melanosome transport (Nilsson and Wallin, 1997). Similarly, immunolocalization of cytoplasmic dynein shows concentration in the perinuclear region in melanocytes where most of the pigment is positioned (Byers et al., 2000) and localizes precisely with melanosomes (Vancoillie et al., 2000a). Treatment of pigment cells with antisense directed toward the cytoplasmic heavy chain released many of the perinuclear melanosomes (Byers et al., 2000). These studies indicate a role for cytoplasmic dynein in directing transport of melanosomes toward the perinuclear microtubule-organizing centers. Melanosomes, similar to all organelles, are displaced by at least three families of ATPase molecular motors stepping along actin filaments or microtubule arrays (Lambert et al., 1999; Smith and Simmons, 2001; Vancoillie et al., 2000a). Melanosome transport within human melanocytes is the result of the forces generated by the balance of cell-centered directed cytoplasmic dynein–dynactin microtubule-based transport (Byers et al., 2000; Vancoillie et al., 2000a, b), the outwarddirected myosin V actin-based transport (Provance et al., 1996; Wu et al., 1998), and the peripherally directed kinesin microtubule-based transport (Hara et al., 2000; Vancoillie et al., 2000c). In contrast, in keratinocytes, phagocytosed melanosomes or melanosomal aggregates are rapidly transported to the perinuclear center and tightly maintained there similar to lysosomes (Fig. 9.2) (Byers et al., 2003). Indeed, cytoplasmic dynein knockout results in lysosome dis-

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Fig. 9.2. Time-lapse analysis of cocultures indicates that transferred melanosomes accumulate as perinuclear melano-phagolysosomes in cultured human keratinocytes. (A) Phase-contrast photomicrograph of human melanocyte/keratinocyte coculture. Heavily melanized dendritic melanocytes and keratinocytes with perinuclear melanophagolysosomes are seen. Bar = 50 mm. (B–E) Time-lapse series with 10-s intervals showing distal fragmentation of melanocyte dendrite (see arrows), phagocytosis, melano-phagosome formation, and perinuclear aggregation of melano-phagolysosomes within keratinocytes. Bar = 20 mm. (Reprinted by permission of Elsevier Science, Inc., from Byers et al., 2003, copyright 2003 by The Society for Investigative Dermatology.)

persion in cultured mouse blastocyst cells (Harada et al., 1998). Organelles undergoing transport are linked to the dynactin complex, which in turn is linked to three intermediate chains of cytoplasmic dynein that associate with the two ATPase heavy chains (Hirokawa, 1998). The 74-kDa intermediate chain (Pfister et al., 1996a, b) of cytoplasmic dynein is a central link between the organelle and the motor heads stepping along the microtubule. It binds to the p150Glued subunit of the dynactin complex anchored to membrane-bound organelles (Karki and Holzbaur, 1995, 1999; Steffen et al., 1997; Vaughan and Vallee, 1995). Not surprisingly, the 74kDa chain is also present in human melanocytes where it concentrates or co-localizes with pigment granules (Byers et al., 2000; Vancoillie et al., 2000a). The dynactin complex subunits p150Glued and p50 dynamitin also co-localize to melanosomes (Vancoillie et al., 2000b). Similar to human melanocytes, keratinocytes also express the 74-kDa chain and, 183

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Fig. 9.3. Intermediate chain of cytoplasmic dynein co-localizes with perinuclear melano-phagolysosomes and perinuclear microtubules in cultured human keratinocytes. (A) Differential interference contrast image showing perinuclear aggregation of melano-phagolysosomes and other large vesicular organelles compared with small peripheral cytoplasmic vesicular organelles. (B) Anti-tubulin staining showing radial arrangement of microtubules with convergence into perinuclear microtubule-organizing centers. (C) Distribution of the intermediate chain of cytoplasmic dynein is concentrated in the perinuclear region corresponding to the melano-phagolysosomes and other large vesicular organelles with only scant, scattered, small aggregates of peripheral cytoplasmic staining. (D) Overlap image of microtubules and cytoplasmic dynein. Bar = 10 mm. (Reprinted by permission of Elsevier Science, Inc., from Byers et al., 2003, copyright 2003 by The Society for Investigative Dermatology.)

in support of its functional role in the maintenance of perinuclear melano-phagolysosomes, the intermediate chain co-localizes with the perinuclear distribution of melanophagolysosomes and concentrates at the microtubuleorganizing centers of the radial microtubule framework (Fig. 9.3) (Byers et al., 2003). Cytoplasmic dynein is thus in position as the motive force directing the “inward” pull on melano-phagolysosomes to the keratinocyte cell center. In contrast to melanocytes, the inward force on melanosomes in keratinocytes must override the outward “pull” of kinesin (the plus end-directed motor protein family) and myosin V (the actin-based motor protein). Nonetheless, the presence of incomplete overlap of cytoplasmic dynein and microtubules in the perinuclear region (Byers et al., 2003, p. 169) may indicate a role for other proteins in targeting melano-phagolysosomes to the perinuclear aggregation. For example, cytoplasmic dynein light intermediate chain 1 binds to pericentrin, a molecule associated with centrosomes 184

Fig. 9.4. Antisense DNA complementary to cytoplasmic dynein sequence disperses perinuclear melano-phagolysosomes in keratinocytes at 24 and 48 h compared with sense DNA-treated controls. Cocultures of human neonatal melanocytes and keratinocytes (A–C). (A) Sense DNA-treated cells at 24 h show tight perinuclear aggregates of melano-phagolysosomes, whereas antisense DNA treated cells at 24 h (B) reveal perinuclear and numerous dispersed melano-phagolysosomes. (C) Sense DNAtreated cells at 48 h still show tight perinuclear aggregates, whereas antisense DNA-treated cells at 48 h (D) exhibit numerous dispersed melano-phagolysosomes. Bar = 10 mm. (Reprinted by permission of Elsevier Science, Inc., from Byers et al., 2003, copyright 2003 by The Society for Investigative Dermatology.)

at the center of the microtubular array (microtubuleorganizing center). Cytoplasmic dynein also appears to take part in the assembly of pericentrin and g-tubulin-containing microtubule initiation sites (Tynan et al., 2000; Young et al., 2000). Functional evidence for the role of cytoplasmic dynein in retrograde (minus-directed) microtubular melanophagolysosome transport in keratinocytes came from timelapse microscopy of cocultured melanocytes and keratinocytes treated with diluent (control), sense (control), or two different antisense cytoplasmic dynein heavy chain oligonucleotides (Byers et al., 2003). High-resolution time-lapse microscopy allowed visualization of the movement of individual melanosomes or small aggregates of melano-phagolysosomes. Diluent-treated and sense-treated cells showed normal perinuclear aggregation of melanosomes/melano-phagolysosomes, whereas antisense-treated cells exhibited progressive dispersion of the keratinocyte perinuclear aggregates throughout the cytoplasm (Fig. 9.4). Indirect evidence suggests that the microtubule polarities in keratinocytes resemble melanocytes. The antisense experiments on human melanocytes (Byers et al., 2000) infer that the majority of the melanocyte dendrite microtubules are oriented similar to nerve axons (Baas et al., 1988) and fish melanophores (McNiven and Porter, 1986) with the plus ends directed toward the periphery as opposed to nerve dendrites with mixed polarity (Baas et al., 1988). Similarly, the antisense

MELANOSOME PROCESSING IN KERATINOCYTES

experiments on keratinocytes suggest that the microtubules are oriented with the plus ends in the periphery and the minus ends in the perinuclear centrosome (Byers et al., 2003). Keratinocytes do not show the bidirectional motility of melanosomes that is observed in melanocytes; therefore, it is apparent that the force acting on keratinocyte melanophagolysosomes by the cytoplasmic dynein–dynactin complex overrides any outward-directed forces by myosin V or kinesin motors acting on these organelles. In addition to cytoplasmic dynein’s perinuclear concentration, it is also scattered throughout the cytoplasm, although to a lesser degree, with peripheral punctate staining (Byers et al., 2003). This finding agrees well with the bidirectional movement of cytoplasmic dynein-associated structures and switching activity of both plus- and minus-directed motors associated with different membrane-bound cargoes (Ma and Chisholm, 2002). Indeed, in order for the melano-phagosome to be pulled from the peripheral cytoplasm, there must be dynactin complexes and cytoplasmic dynein available in the periphery. In fact, cytoplasmic dynein is found at the peripheral membrane in association with adherens junctions containing b-catenin and E-cadherin in epithelia (Ligon et al., 2001). However, the peripheral membrane-associated cytoplasmic dynein that is ready to shuttle cargo toward the cell center is less concentrated than that found in the perinuclear region holding already transported melano-phagolysosomes in place (Byers et al., 2003). Cytoplasmic dynein also has several other functions: the breakdown of the nuclear envelope during prophase (Salina et al., 2002); reorganization of the microtubule array into the mitotic spindle during prophase and metaphase; and possibly chromosomal segregation during anaphase (Banks and Heald, 2001; Barton and Goldstein, 1996; Karki and Holzbaur, 1999; Rusan et al., 2002). Differential expression of members of the dynein superfamily and assignment of specific isoforms of heavy, intermediate, and/or light intermediate chains may specify cargo trafficking within different cellular compartments and among different cell types. There are at least four cytoplasmic dyneins and 12 members of the axonemal dyneins in the dynein superfamily (Brokaw, 1994; Hirokawa, 1998). Cytoplasmic dynein heavy chain Dyh1 (MAPlc, DHcl, and DHC1a; Koonce et al., 1992; Mikami et al., 1993; Vaisberg et al., 1993; Zhang et al., 1993) is ubiquitous throughout phylogeny from amoeba to vertebrate neurons. In the latter cells, it is responsible for microtubuledependent fast axonal retrograde (minus end-directed) organelle transport (Muresan et al., 1996; Schroer et al., 1989). At least two cytoplasmic dynein family members are present in brain tissue (Tanaka et al., 1995); three have been identified in fibroblasts and HeLa cells (Vaisberg et al., 1996) and possibly four in rat testis (Criswell and Asai, 1998). Isoform specificity to cell type or cellular compartment may be involved in different organelle transport, secretion, mitotic spindle organization, chromosome or nuclear migration (Holzbaur and Vallee, 1994; Schroer, 1994; Tanaka et al., 1995; Vaisberg et al., 1996). The cytoplasmic dynein antisense oligonucleotides that induced dispersion of melanosomes from the cell center in melanocytes and

keratinocytes were made to a short Dyh1 sequence cloned from melanocytes (Byers et al., 2000). The lack of complete peripheral dispersion of melano-phagolysosomes in keratinocytes with Dyh1 antisense studies (Byers et al., 2003) suggests functional redundancy of cytoplasmic dynein heavy chain isoforms or functioning of other minus-directed microtubule motors including kinesin-related motors (Case et al., 1997; Henningsen and Schliwa, 1997; Hirokawa, 1998; Matuliene et al., 1999; Noda et al., 2001; Sablin et al., 1998; Smirnova et al., 1998; Wade and Kozielski, 2000). The human epidermis is a polarized epithelium. In tanned or pigmented skin, the supranuclear melanin cap is another feature of polarization. This concentrated aggregate of melano-phagolysosomes over the nucleus of the keratinocyte acts as an internal sunscreen against UV-induced nuclear DNA damage. To emphasize the functional–structural photoprotective effect of aggregation of pigment on a subcellular radial microtubular array over the nucleus, the “microparasol” has been coined (Byers et al., 2003; Scott, 2003). Animal models have demonstrated that the “protective caps” also reside on the side of the nucleus closest to the skin surface and, similar to the tanning response, are increased with aliphatic and alicyclic diols (Brown et al., 1998). The melanin caps of keratinocytes are also seen in vitro with keratinocytes of cultured skin equivalents (Archambault et al., 1995) and increase following UV irradiation or isobutyl methylxanthine (IBMX) treatment (Gibbs et al., 2000). Interestingly, increased supranuclear caps also occur in hyperpigmentation of fullthickness skin grafts (Li et al., 1997), suggesting that UV exposure is not necessary for their formation. In situ immunofluorescent studies also support a role for cytoplasmic dynein as the force-generating motor involved in the formation of the melanin cap. Frozen sections of human skin from a moderately pigmented individual incubated with monoclonal antibodies against the cytoplasmic dynein intermediate chain reveal that cytoplasmic dynein co-localizes with the supranuclear melanin cap (Fig. 9.5) (Byers et al., 2003). Thus, melanin is not randomly distributed throughout the keratinocyte cytoplasm; cytoplasmic dynein forms the supranuclear melanin cap or microparasol to protect the nucleus from UV-induced DNA damage. Earlier studies calculating total melanin content in the skin and testing its protective effect using solutions of melanin are analogous to shredding all the parasols at the beach and spreading them like confetti over the sunbathers and the beach, only to find that areas on the bather’s skin would burn, similar to nuclear mutational hits in a basal layer nucleus if the microparasol melano-phagolysosomes were dispersed. The most vulnerable germinative layer, the stratum basale, has the greatest number of melano-phagolysosomes surrounding the nucleus. It may have more pigment surrounding the nucleus as a result of the polarized nature of cell division of daughter cells (one remains on the basal layer and the other moves into the suprabasal layer), and asymmetric segregation of pigment granules may reflect a functional anisometry in the melanocyte and keratinocyte cytoskeletal organization. It may also relate to the 185

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A

B

C

D

Fig. 9.5. Intermediate chain of cytoplasmic dynein in keratinocytes is concentrated in a supranuclear pattern in human epidermis. (A) Phase-contrast image of frozen section of human epidermis showing focal supranuclear aggregates or “caps” of melano-phagolysosomes (arrows). (B) Confocal fluorescence image showing focal increased cytoplasmic staining of the intermediate chain of cytoplasmic dynein in keratinocytes. (C) Double fluorescent confocal image with nuclear staining shows increased supranuclear localization of intermediate chain of labeled cytoplasmic dynein over labeled nuclei (arrows) corresponding to “caps” in (A). (D) Co-localization of melano-phagolysosomes and cytoplasmic dynein intermediate chain by computer-generated pseudo-bright overlay. Bar = 50 mm. (Reprinted by permission of Elsevier Science, Inc., from Byers et al., 2003, copyright 2003 by The Society for Investigative Dermatology.)

temporal sequence of phagocytosis, melano-phagosome transport, processing, and elongation of melano-phagolysosomes into cisterns along the sides of the nuclei prior to maturation of the microparasol. Morphologic and molecular evidence for this is discussed in part below.

Maturation and Degradation of MelanoPhagolysosomes in Keratinocytes Electron microscopic and time-lapse photomicrography studies provided early insights into the processing of melanophagosomes into melano-phagolysosomes. Melanosome size, uptake, and retention as aggregates or complexes were studied as well as the digestion of melanosomes, membranes, and residual melanocyte cytoplasm during maturation into melano-phagolysosomes. Individuals with minimal to lightly pigmented epidermis (phototypes I or II) exhibit small melanosomes packaged within membrane-bound melanosome complexes in the keratinocyte cytoplasm. This contrasts with 186

the single large melanosomes packaged singly in the keratinocyte cytoplasm in individuals with moderately to heavily pigmented epidermis (phototype III or IV) (Hori et al., 1968; Konrad and Wolff, 1973; Szabo et al., 1969). This difference is due to the size of melanosomes produced in the melanocyte; smaller melanosomes are found in phototype I or II and larger melanosomes are seen in phototype III or IV individuals. The size difference influences the type of transfer and phagocytosis by the keratinocyte (Toda et al., 1972; Wolff et al., 1974; Wolff and Konrad, 1971, 1972). However, recent work indicates a more complex size gradient of melanosomes found in the spectrum of global skin coloration (Thong et al., 2003). In addition, mix and matched co-culture experiments of melanocytes and keratinocytes from dark and light skin have shown that the keratinocytes determine the type of melanosome packaging as either predominantly single melanosomes or predominantly membrane-bound clusters of melanosomes (Minwalla et al., 2001). Nonetheless, depending on the mechanism of transfer and phagocytosis (discussed in Chapter 8), melanosomes are identified within keratinocytes as: (1) membrane-bound melanosomes as complexes within a pinched-off portion of a membrane-bound melanocyte dendrite and enfolded by keratinocyte membrane; (2) melanosomes enveloped by melanosomal membrane, melanocyte membrane, and keratinocyte membrane to form a trilaminate organelle; and (3) single membrane-bound melanosomes as coated vesicles (Okazaki et al., 1976; Yamamoto and Bhawan, 1994). The general maturation sequence that follows is that keratinocyte primary lysosomes fuse with the melanophagosomes, and the dendrite and/or melanosomes begin to undergo lysosomal digestion. The presence of acid phosphatase in these secondary lysosomes (melano-phagolysosomes) in the keratinocyte supports this pathway (Hori et al., 1968). Nonetheless, melanosomes entering the keratinocyte appear to exhibit a heterogeneous pathway of entrance. In addition, it has become apparent that melanosomes themselves are related to lysosomes in the melanocyte (Orlow, 1995; Zhou et al., 1993). To complicate matters further, melanocytes also exhibit phagocytosis, at least in vitro, of previously released melanosomes (Le Poole et al., 1993). It is thus quite probable that at least a subset of transferred melanosomes may exhibit lysosomal-associated proteins prior to fusion with keratinocyte lysosomes. On the other hand, recent evidence indicates that lysosomes and melanosomes are largely segregated during endosome processing in melanocytes (Fujita et al., 2001; Raposo et al., 2001, 2002; Setaluri, 2003). In addition, the Golgi, lysosomes, and melanosomes are centrally aggregated by similar inward-directed, microtubulebased motor mechanisms involving microtubules and cytoplasmic dynein (Allan et al., 2002; Corthesy-Theulaz et al., 1992; Harada et al., 1998; Karki and Holzbaur, 1999; Valetti et al., 1999). Therefore, caution in the interpretation of mutations or targeted knockout experiments of elements of this mechanism may be in order if a general disruption in the membrane trafficking leads to aberrant membrane fusion and

MELANOSOME PROCESSING IN KERATINOCYTES

mixing of various vesicle compartments that are usually kept relatively separate. The molecular complexity of newly formed phagosomes and mature phagolysosomes is highlighted by the finding that at least 200 polypeptides associate with these organelles (Desjardins et al., 1994). More recent work has identified many of the proteins involved in endosome/ lysosome fusion mechanisms and vesicle trafficking, including multiple SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein families including syntaxins and VAMPs that bind to SNAPs (SNARE receptors) (Abeliovich et al., 1999; Advani et al., 1999; Atlashkin et al., 2003; Burd et al., 1998; Gerst, 2003; Hay and Scheller, 1997; Houle and Marceau, 2003; Mullins and Bonifacino, 2001; Mullock et al., 2000; Ward et al., 2000). These advances were derived from yeast endosome, lysosome fusion to neuronal synaptic vesicle fusion studies and led to evidence of a role for the SNARE complexes and Rab3a in melanosome transfer (Scott and Zhao, 2001). Undoubtedly, investigators will soon elucidate similar SNARE or Rab GTPase-mediated mechanisms of keratinocyte phagocytosis of melanosomes, melano-phagosome maturation, and transport. In fact, the dynein–dynactin bridging to phagosomes is mediated by Rab GTPase; specifically, active Rab7 on the phagosomal membrane binds with the effector protein RILP (Rab7-interacting lysosomal protein), which links to the dynein–dynactin complex (Harrison et al., 2003). These investigators observed that phagosomes are transported centripetally, and the dynactin–cytoplasmic dynein microtubule-associated motor complex distorts the membrane into phagosomal tubules toward late endocytic compartments. When RILP was truncated, the tubules did not form, and fusion with late endosomes or lysosomes could not occur. These findings shed light on the cisternal and tubular membrane structures of late melano-phagolysosomes observed in keratinocytes in earlier electron microscopic investigations (Yamamoto and Bhawan, 1994). Mature melano-phagolysosomes derived from enfolded or sac–dendrite complexes appear to demonstrate two different morphologies: (1) the digestion of the dendrite organelles with fragmentation of the complex and dispersion of individual, pairs, or small aggregates of melanosomes (Okazaki et al., 1976); or (2) digestion of the dendrite organelles with formation of cisternal structures within the keratinocyte (Yamamoto and Bhawan, 1994). These latter cisterns contain electrondense amorphous material and fragments of cytoplasmic components or melanosomes or both. The cisterns become elongate with fingerlike projections containing melanosomes in the tips of the cisterns. The cisterns are often seen running parallel to the nuclear membrane below and to the side of the nucleus, and melanosomes appear to pinch off from the distal cistern. Ultimately, an “apical cap” of aggregated melanosomes may be identified above the nucleus (Yamamoto and Bhawan, 1994). It is highly probable that the cisterns, as distorted phagosomes and similar to Golgi cisterns, are tubulovesicular components that are pulled and formed by associated cytoplasmic motor proteins acting on microtubules

(Harrison et al., 2003; McIlvain et al., 1993). The exact mechanisms of melano-phagolysosomal maturation and intrakeratinocytic melanosomal membrane trafficking need further clarification; nonetheless, the elucidation of many basic structural and molecular features provides a framework for the testing of additional hypotheses.

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phorylation of the 74-kDa intermediate chains of cytoplasmic dynein in cultured neurons and glia. J. Biol. Chem. 271:1687–1694, 1996b. Provance, D. W., Jr., M. Wei, V. Ipe, and J. A. Mercer. Cultured melanocytes from dilute mutant mice exhibit dendritic morphology and altered melanosome distribution. Proc. Natl. Acad. Sci. USA 93:14554–14558, 1996. Raposo, G., B. Fevrier, W. Stoorvogel, and M. S. Marks. Lysosomerelated organelles: a view from immunity and pigmentation. Cell. Struct. Funct. 27:443–456, 2002. Raposo, G., D. Tenza, D.M. Murphy, J.F. Berson, and M.S. Marks. Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells. J. Cell Biol. 152:809–824, 2001. Reilein, A. R., A. S. Serpinskaya, R. L. Karcher, D. L. Dujardin, R. B. Vallee, and V. I. Gelfand. Differential regulation of dynein-driven melanosome movement. Biochem. Biophys. Res. Commun. 309:652–658, 2003. Rodionov, V. I., and G. G. Borisy. Self-centring activity of cytoplasm. Nature 386:170–173, 1997. Rusan, N. M., U. S. Tulu, C. Fagerstrom, and P. Wadsworth. Reorganization of the microtubule array in prophase/prometaphase requires cytoplasmic dynein-dependent microtubule transport. J. Cell Biol. 158:997–1003, 2002. Sablin, E. P., R. B. Case, S. C. Dai, C. L. Hart, A. Ruby, R. D. Vale, and R. J. Fletterick. Direction determination in the minus-enddirected kinesin motor ncd. Nature 395:813–816, 1998. Salina, D., K. Bodoor, D. M. Eckley, T. A. Schroer, J. B. Rattner, and B. Burke. Cytoplasmic dynein as a facilitator of nuclear envelope breakdown. Cell 108:97–107, 2002. Schafer, D. A., S. R. Gill, J. A. Cooper, J. E. Heuser, and T. A. Schroer. Ultrastructural analysis of the dynactin complex: an actin-related protein is a component of a filament that resembles F-actin. J. Cell Biol. 126:403–412, 1994. Schliwa, M., M. Osborn, and K. Weber. Microtubule system of isolated fish melanophores as revealed by immunofluorescence microscopy. J. Cell Biol. 76:229–236, 1978. Schliwa, M., K. Weber, and K. R. Porter. Localization and organization of actin in melanophores. J. Cell Biol. 89:267–275, 1981. Schroer, T. A. Structure, function and regulation of cytoplasmic dynein. Curr. Opin. Cell Biol. 6:69–73, 1994. Schroer, T. A., E. R. Steuer, and M. P. Sheetz. Cytoplasmic dynein is a minus end-directed motor for membranous organelles. Cell 56:937–946, 1989. Scott, G. Photo protection begins at the cellular level: microparasols on the job. J. Invest. Dermatol. 121:viii, 2003. Scott, G., and Q. Zhao. Rab3a and SNARE proteins: potential regulators of melanosome movement. J. Invest. Dermatol. 116:296–304, 2001. Setaluri, V. The melanosome: dark pigment granule shines bright light on vesicle biogenesis and more. J. Invest. Dermatol. 121:650–660, 2003. Smirnova, E. A., A. S. Reddy, J. Bowser, and A. S. Bajer. Minus enddirected kinesin-like motor protein, Kcbp, localizes to anaphase spindle poles in Haemanthus endosperm. Cell. Motil. Cytoskel. 41:271–280, 1998. Smith, D. A., and R. M. Simmons. Models of motor-assisted transport of intracellular particles. Biophys. J. 80:45–68, 2001. Steffen, W., S. Karki, K.T. Vaughan, R.B. Vallee, E.L. Holzbaur, D.G. Weiss, and S.A. Kuznetsov. The involvement of the intermediate chain of cytoplasmic dynein in binding the motor complex to membranous organelles of Xenopus oocytes. Mol. Biol. Cell 8:2077–2088, 1997. Szabo, G., A. B. Gerald, M. A. Pathak, and T. B. Fitzpatrick. Racial differences in the fate of melanosomes in human epidermis. Nature 222:1081–1082, 1969.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

10

The Regulation of Melanin Formation Vincent J. Hearing

Summary 1 This chapter addresses important regulatory controls of melanogenesis that operate at the subcellular level in melanocytes to affect the structural and/or the functional nature of the melanins and melanin granules produced. 2 Pigment-producing cells in lower vertebrates (usually known as melanophores) are regulated by a number of distinct factors, including hormonal agents such as melanocytestimulating hormone (melanotropin), melanin-concentrating hormone, and melatonin; these generally work by affecting the distribution of existing melanin granules within melanophores. 3 Many of these same factors also affect pigmentation in mammalian melanocytes, but function to regulate the quantity or quality of the melanins synthesized; in addition to melanotropin, the sex hormones (estrogen and progesterone) also affect melanocyte function, and yet another hormone termed agouti signal protein also plays an important physiologic role in regulating melanogenesis in the hair follicle. 4 Melanotropin and agouti signal protein have antagonistic roles and possibly antagonistic mechanisms of action in melanocytes; melanotropin stimulates melanogenic enzyme function and elicits increases in the amount of eumelanins produced; in contrast, agouti signal protein reduces melanin production and elicits the synthesis of pheomelanin rather than eumelanin. 5 There are other levels of controls over pigmentation that are regulated at the melanosomal level, including substrate availability, various cytokines and growth factors, intracellular pH, trafficking and sorting pathways, proteasomes, and ultraviolet radiation, all of which can dramatically affect melanin production and/or the maturation and distribution of melanin granules. 6 The quality and quantity of melanins produced by melanocytes have important physiological consequences for melanocyte function, and undoubtedly play important roles in the various functions of the melanins per se, including hair and skin coloration and photoprotection.

Historical Background Visible pigmentation in the skin, hair, and eyes depends on a

wide variety of factors that influence melanocyte function at various levels. For correct pigmentation, a number of distinct steps in development, proliferation, and differentiation must occur with high fidelity; these include factors that affect melanoblast development and migration in the developing embryo, affect melanocyte survival and proliferation once in place in situ, affect melanocyte function in response to environmental stimuli, and affect melanin granule distribution and subsequent processing by neighboring keratinocytes. There are a plethora of genes that affect these processes at each of the levels noted, either directly or indirectly. Early on, more than 60 distinct genes were identified in mice that affect pigmentation (Silvers, 1979), but the number has recently increased to more than 120 (Bennett and Lamoreux, 2003); no doubt that number will increase further in the future. At this time, approximately 50% of those genes have been cloned and characterized, and their general, often their specific, function(s) in affecting melanogenesis in mammals have been identified. Several of those genes encode proteins that are localized in melanin granules and play active roles in the structure and function of that organelle, either as catalytic entities involved in melanin synthesis or as structural proteins important to the integrity of the melanosome. Many of those genes, perhaps most of them, have been implicated in various inherited pigmentary disorders in humans, as detailed elsewhere in this book. An excellent resource summarizing the current state of pigment genes, mutations, and associated diseases is maintained online (http://www.cbc.umn.edu/ifpcs/micemut.htm). Most of the genes and factors that modulate melanogenesis at the tissue and cellular levels have been discussed in other chapters of this book and will not be discussed in detail here. This chapter focuses on regulatory controls of melanogenesis that operate at the subcellular level, particularly those that affect the structural or functional nature of melanins and melanin granules produced and/or their processing and distribution within melanocytes. The critical requirement for an enzyme activity to produce melanin from the amino acid tyrosine has been known for some time. The role of tyrosinase [EC 1.14.18.1] was initially described in mushrooms (Bourquelot and Bertrand, 1895; Bertrand, 1896) and, several decades later, tyrosinase was shown also to play a regulatory role in the production of melanin in mammalian melanocytes (Bloch, 1916; Bloch and Schaaf, 1925; Lerner et al., 1949; Lerner and Fitzpatrick, 1950). There was initially some controversy over the identity 191

CHAPTER 10

of the substrate(s) that mammalian tyrosinase uses in melanin production, as the specificity of the mammalian enzyme was highly restricted compared with tyrosinases present in lower animals and plants. For example, the rate of the initial reaction (tyrosine hydroxylase) proceeds very slowly in the absence of a suitable cofactor, and there was some doubt that tyrosine was the appropriate biologic substrate, but it has now been shown conclusively that tyrosine, via a series of complex reactions, gives rise to the biopolymer melanin. This reaction cascade (discussed in more detail in Chapters 14, 15, and 18) is generally referred to as the Raper–Mason scheme (Raper, 1927; Mason, 1959, 1967); it involves a complex series of oxidations and rearrangements, which results in the formation of indole–quinone ring structures that readily polymerize to high-molecular-weight pigmented biopolymers. Owing to the rate-limiting nature of tyrosinase in this pathway and to the fact that other accessory enzymes were only identified very recently, early studies assumed that the regulation of melanin production per se was accomplished solely via regulation of tyrosinase activity, and that this in turn was essentially regulated by rates of transcription of its encoding gene. This concept, although in part correct, has been modified in light of more recent studies. In the past decade, a number of other regulatory points in the melanogenic cascade have been described; these involve inhibitors (which modulate tyrosinase activity), other posttyrosinase enzymes (which regulate the nature of the melanins produced), intracellular pH, proteolytic processing, and other intracellular components that modulate the processing and sorting of melanogenic enzymes, including tyrosinase. As the different types of melanins that can be produced vary greatly in their phenotypic characteristics, it is reasonable to assume that these regulatory factors have great importance to the functional properties of melanins produced. It is important to note that the early concept of direct tyrosinase regulation of melanin formation remains essentially correct, as it is certainly true that, in the absence of active tyrosinase, there will be no melanin formation. However, we now know that there are many ways in which tyrosinase activity is regulated in the melanocyte and that it is not solely at the level of gene transcription; there are many instances in which active tyrosinase is present yet no melanin is produced. We also now know that there are many other switchpoints in the melanin biosynthetic pathway that modulate what type of melanin is produced and how much of it is produced. It should be noted from the outset that we are only now starting to unravel the highly complex series of interactions that is involved in the biogenesis and function of melanin granules, as underscored by the complexity of their proteomic analyses (Kushimoto et al., 2001; Basrur et al., 2003). This chapter can only be considered as a starting point for understanding this field, which is an incredibly active area of scientific research in which important new findings are being published on a regular basis. 192

Current Concepts Overview It has become increasingly apparent that the pigmentation of mammals and lower animals is under diverse and multifaceted controls. As noted above, there are now a number of factors known to affect pigmentation at the tissue, the cellular, and the subcellular levels, and many of these have been noted and discussed in other chapters of this book. Although the balance of this discussion will focus on the regulation of melanogenesis at the subcellular level, the reader must always keep in mind that regulation at the tissue and cellular levels is equally relevant to the control of pigmentation. As examples, factors that affect the distribution, proliferation, and survival of melanocytes in various tissues are of paramount importance (reviewed by Hearing and King, 1993; King et al., 1997; Spritz and Hearing, 1994); a number of hypo- and hyperpigmentary disorders have been described that result from lesions at this level, including piebaldism and vitiligo. Obviously, if environmental stimuli, e.g. ultraviolet radiation or melanocytestimulating hormone (melanotropin), are operating normally in situ, but functional melanocytes are absent, there will be no pigmentation in that tissue. Similarly, important regulatory effects occur at the cellular level, both within and without melanocytes. Some examples of these types of parameters have also been detailed more completely in other chapters of this book, but they include such cellular determinants as melanocyte structure (e.g. dendrite formation), interactions with keratinocytes (both as a source of regulatory factors and as a repository of donated melanosomes), and other such cellular processes that affect the ultimate distribution and processing of melanosomes in the tissues. Finally, there is active regulation of melanogenesis that occurs at the subcellular level; parameters that fall into this group are those that affect the production and processing of melanin and the melanosomes in which melanin is deposited, at the transcriptional, translational, and/or post-translational levels. Some of these factors are produced within the melanocyte per se, while the vast majority of them derive from outside the melanocyte (e.g. ultraviolet radiation, melanotropin, and agouti signal protein); many of them have pleiotropic effects at different levels, stimulating or inhibiting proliferation of melanocytes in concert with their action in increasing or decreasing pigment production by those same melanocytes. Several of those agents are known not only to modulate the amount of melanin synthesized but also to regulate the type of melanin that is produced (at least in mammals); between those two effects there can be astounding differences in visible phenotype. Chapter 15 discusses in detail the various types of melanin that can be produced, at least in mammals, and the effects on their structure and function. In brief, the two major types of melanins produced are termed pheomelanins and eumelanins, which are yellow/red or black/brown respectively (Fig. 10.1). Interestingly, pheomelanins have not yet been identified in lower vertebrates and

THE REGULATION OF MELANIN FORMATION

Tyrosinase COOH NH2

HO

Tyrosinase COOH

HO

NH2

HO

Tyrosine

DHI

-O

N H

+

N COOH H DOPAchrome

DOPAchrome tautomerase / DCT O

HO

O

HO

N H Indole-5,6-quinone

NH2

NH2

Glutathione HO or DOPAquinone cysteine N2 H

S

COOH CysteinyIDOPA

HO

COOH N H LeucoDOPAchrome

DHICA oxidase / TYRP1 O N COOH H DHICA

COOH

HO

HO

O

HO

O

DOPA

HO

COOH

O

O

N COOH H Indole-5,6-quinonecarboxylic acid

Eumelanin

COOH

HO

NH2

N S

1,4-Benzothiazinylalanine

Pheomelanin

Fig. 10.1. Schematic of the melanin biosynthetic pathway. DHI, 5,6-dihydroxyindole; DHICA, 5,6-dihydroxyindole-2-carboxylic acid; TRP, tyrosinase-related protein; DOPA, 3,4-dihydroxyphenylalanine.

may represent a more recent and mammalian-specific group of pigments. Melanotropin and agouti signal protein are factors produced extrinsic to melanocytes that play dramatic roles not only in regulating levels of melanin synthesis, but also in switching melanocytes between production of those two types of melanins, resulting in profound effects on visible pigmentation. Finally, a number of basic housekeeping processes involved in the biogenesis of cellular organelles also regulate the formation and maturation of melanosomes, particularly through the processing and sorting of tyrosinase; such parameters include intracellular pH, proteasome function, and trafficking pathways by which proteins are processed and sorted to their target organelles.

Lower Vertebrates and Invertebrates Pigment-producing cells in lower vertebrates and invertebrates are usually termed melanophores. Similar in morphology and function to mammalian melanocytes, these cells typically appear as highly dendritic cells that are primarily found in the dermis (Bagnara, 1966, 1972; Bagnara and Ferris, 1971; Bagnara and Hadley, 1973; Chavin, 1971; Fujii, 1971; Novales and Novales, 1966). Melanophores are usually present at a higher density on the dorsal surface than on the ventral surface, which in part explains the lighter pigmentation of the latter. However, it has been found more recently that there are also regulatory factors in these tissues, tentatively termed melanization-inhibiting factor and melanizationstimulating factor, which may play important roles in reducing

or promoting pigmentation in these dorsal and ventral surfaces (Bagnara and Fernandez, 1993; Frost, 1988; Fukuzawa and Bagnara, 1989; Fukuzawa and Ide, 1988; Johnson et al., 1992; Wang et al., 2001). These factors have also been shown to have comparable effects on mammalian melanocytes (Kreutzfeld et al., 1989) and may thus play a role in determining the pigmentary patterns in higher vertebrates as well; it is of course possible that some of the melanogenic inhibitors identified (cf. below and Chapter 18) represent the mammalian counterparts of these stimulating or inhibiting factors. An interesting aspect of melanophore pigmentation is the facility rapidly to translocate melanosomes within the melanophore dendrites, which can lead to relatively quick changes in visible pigmentation based primarily on the distribution of melanin particles within the cell rather than the synthesis of de novo melanin (Bagnara and Hadley, 1973; Fernandez and Bagnara, 1991; Fujii, 2000; Fujii and Novales, 1972; Potter and Hadley, 1970); comparable rapid transit mechanisms of melanosomes in higher vertebrates and mammals have not yet been described. The actual processes and control mechanisms involved in the dispersion and aggregation of melanosomes, and concurrent rates of melanogenesis, in melanophores is discussed extensively in Chapters 2, 3, 7, and 8.

Hormonal Control Pigmentation in lower vertebrates is also under active control by hormones, among which the most commonly known are 193

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a – Melanotropin b – Melanotropin g – Melanotropin ACTH (residues 4–10)

MCH (salmon) (rat)

Melatonin

Agouti signal protein

Human–1 Mouse–1

Human–61 Mouse–61

Human–121 Mouse–121 Fig. 10.2. Structure of melanotropic hormones and factors. ACTH, adrenocorticotropic hormone; MCH, melanocyte-concentrating hormone.

melanotropin, melanin-concentrating hormone, and melatonin (Fig. 10.2). In fact, the action of melanotropin was initially defined in amphibians, and early studies in this field provided interesting insights into its mechanisms of action (McCord and Allen, 1917; Zondik and Krohn, 1932). Surprisingly, however, more is now known about the signaling pathways and subcellular mechanisms involved in melanotropin action in mammals than in lower vertebrates, primarily because of early studies on the effects of melanotropin in man (Lerner, 1961; Lerner and McGuire, 1961; Lerner et al., 1954; McGuire and Lerner, 1963) and the recent cloning of the family of receptors for melanotropin (Desarnaud et al., 1994; Gantz et al., 1993a, b; Mountjoy et al., 1992). The effect of melanotropin on amphibian melanophores results not only from its effects on the distribution of existing melanin and melanosomes, but also in part from the induction of proliferation of melanophores and/or stimulation of their melanogenic activities, as occurs with mammalian melanocytes (this topic is discussed in more detail in Chapter 2). Melanotropin is a relatively short oligopeptide; it contains 13 residues and is derived from a relatively large precursor termed proopiomelanocortin; this proopiomelanocortin precursor is cleaved proteolytically into a number of hormones, including adrenocorticotropic hormone (ACTH), amelanotropin, b-melanotropin, and g-melanotropin. Proopiomelanocortin (and therefore melanotropin) derives principally from the pituitary gland, but recent evidence has suggested that other cells in the skin (e.g. keratinocytes), as well as melanocytes themselves, can produce proopiomel194

anocortin and may thus act in an autocrine fashion (Chakraborty et al., 1995; Farooqui et al., 1993; Ghanem et al., 1992; Pawelek, 1993; Slominski, 1991a; Slominski et al., 1992, 1995; Wintzen and Gilchrest, 1996). In mammals, a family of melanotropin receptor genes is required for the binding of melanotropin to specific types of cells and subsequent intracellular signaling (cf. below); receptors for melanotropin have been identified (de Graan et al., 1981; Eberle, 1981; Fujii and Miyashita, 1976; Graminski et al., 1993; Sherbrooke and Hadley, 1988) but have not yet been cloned in lower vertebrates. In addition to its role in regulating pigmentation in lower vertebrates, melanotropin also plays other roles as a potential neural regulatory agent and growth factor, among other possible functions (cf. below). The mechanism(s) whereby melanotropin effects melanosome dispersion within melanophores has not yet been identified or characterized; nevertheless, ionic concentrations are important to these responses, especially calcium (Hadley, 1987; McNiven and Ward, 1988; Negishi and Obika, 1985; Veerdonk et al., 1979; Visconti et al., 1989; Visconti and Castrucci, 1990), and no doubt this plays a critical role in the intracellular signaling pathway. The hypothalamus also plays a significant role in regulating pigmentation in lower vertebrates (described primarily in teleost fishes) via another hormone known as melaninconcentrating hormone. This hormone essentially has the reverse effect on melanophores that is elicited by melanotropin, i.e. it results in pigment granule aggregation rather than dispersal (Baker, 1988, 1993; Castrucci et al., 1988;

THE REGULATION OF MELANIN FORMATION

Enami, 1955; Hogben and Slome, 1931; Kawauchi et al., 1993; Visconti et al., 1989), and thus lightens rather than darkens visible pigmentation of the skin. Melanin-concentrating hormone is also a short oligopeptide (Fig. 10.2) that consists of 17 residues; the two cysteines present are involved in a disulfide bond that cyclizes the molecule (Eberle et al., 1986; Nahon et al., 1993; Okamoto et al., 1984; Wilkes et al., 1984). The effects of melanin-concentrating hormone on the aggregation of pigment granules is reversible, and melanosome dispersion will occur if melanin-concentrating hormone is removed from the milieu or if melanotropin is added (Castrucci et al., 1987; Hadley et al., 1987). The gene encoding melanin-concentrating hormone has been cloned in several different species of salmon (Nahon et al., 1991, 1993; Ono et al., 1988; Takayama et al., 1989) as well as in higher species. As found for melanotropin, melanin-concentrating hormone has a number of alternative functions in amphibians and fishes in addition to its role in eliciting color changes (Baker, 1993; Nahon et al., 1993). Melatonin is a cyclic molecule synthesized from serotonin following acetylation and methylation (Lerner et al., 1958, 1959; Quay and Bagnara, 1964); this synthesis is regulated in the pineal gland and is responsive to light (Bagnara and Hadley, 1973). Melatonin also plays a role in melanosome aggregation in melanophores (Cozzi and Rollag, 1992; Kastin et al., 1972; Messenger and Warner, 1977; Novales, 1963; Rollag and Adelman, 1993), and the responses of pigmentation to variations in light via melatonin are discussed in detail in Chapter 2. The action of melatonin on amphibian melanophores is dependent on the expression of highly specific melatonin receptors (Heward and Hadley, 1975; Sugden, 2004); indirect evidence suggests that melatonin-specific receptors also function in mammalian responses to melatonin, although the effect is most likely not on pigmentation per se (cf. below and Chapter 2). Melatonin generally acts in opposition to melanotropin, both directly at the level of the melanophore and indirectly via the hypothalamus, and has been shown to inhibit the effects of melanotropin in amphibians (Abe et al., 1969).

Mammalian More is known about extrinsic regulators of mammalian melanogenesis than for the lower vertebrates, probably because of the added interest in this area due to the cosmetic and pharmacologic implications of the research. This section only briefly discusses hormonal controls and other known extrinsic factors that modify melanogenesis, including ultraviolet radiation and other biologic factors that act in autocrine and/or paracrine manners to influence the biosynthesis of melanins (processes detailed in Chapters 19, 20, and 21).

Hormonal Control As discussed above for lower vertebrates, hormones such as melanotropin, melanin-concentrating hormone, and melatonin also affect mammalian melanocytes, although often in

distinct manners, yet their physiologic role has often not been resolved. Melanin-concentrating hormone has been cloned and sequenced in mammals and has been shown to be expressed in a tissue-specific manner, both in rats and in humans (Nahon et al., 1992; Takahashi et al., 1993; Thompson and Watson, 1990). Although the proteins encoded by the mammalian melanin-concentrating hormone genes have been demonstrated to be biologically active (Drozdz et al., 1993), there is still some difference of opinion as to their actual physiologic role in regulating mammalian pigmentation, and as yet there is no direct evidence for such a role. The evidence for possible function(s) of melatonin in the regulation of mammalian melanogenesis is also controversial at best, both for epidermal (McElhinney et al., 1994; Slominski et al., 1994; Snell, 1965; Voulot, 1969) and for follicular (Logan and Weatherhead, 1980; Weatherhead and Logan, 1981a) melanocytes; again, the specific mechanism(s) of melatonin action, both direct and indirect, in mammalian pigmentation are not yet established. Mammalian melanocytes express specific surface receptors that bind melatonin, and the encoding gene for that receptor has been cloned and mapped (Garratt et al., 1995). That receptor is expressed by mammalian pigment cells (Roberts et al., 2000), and the specificity of expression of those receptors has been a source of interest in targeting neoplastic melanocytes (i.e. melanoma); a number of studies have examined this approach (El-Domeini and DasGupta, 1973; Gonzalez et al., 1991; Kane et al., 1994; Weatherhead and Blackburn, 1985). The sex hormones estrogen and progesterone have also been shown to interact with melanocytes, although the mechanism whereby the proliferation or differentiation of melanocytes might be influenced has yet to be resolved. A number of studies have established that melanocytes express receptors for these hormones (Avril et al., 1985; Chaudhuri et al., 1985; Jee et al., 1994; Walker et al., 1987; Zava and Goldhirsch, 1983), but it is not known whether these hormones have their primary effects on the growth of melanocytes (Beattie et al., 1991; McLeod et al., 1994; Schleicher et al., 1987), activation of the melanogenic pathway (Delacretaz, 1966), and/or specific binding to the melanins directly (Jacobsohn and Jacobsohn, 1992; Jacobsohn et al., 1993; Kalyanaraman et al., 1986, 1989). It is thought that increases in pigmentation often observed during pregnancy are but one phenotypic effect of estrogen production. Melanotropin, and its precursor proopiomelanocortin, are by far the best characterized of the hormones involved in regulating mammalian pigmentation. Although melanotropin in lower vertebrates regulates pigment dispersion in melanophores (as noted above), in mammals the effect of melanotropin is at least at two other levels, i.e. eliciting an increase in the proliferation of melanocytes (Abdel-Malek et al., 1986, 1987, 1995; Abdel-Malek, 2001; Chavin et al., 1963; Geschwind and Huseby, 1972; Halaban et al., 1993; Halaban and Lerner, 1977; Hill et al., 1989a; Pawelek et al., 1975; Scott et al., 2002a, b; Snell, 1967, 1972; Swope et al., 195

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1995; Virador et al., 2002) and/or a stimulation in their production of melanins (Abdel-Malek et al., 2001; Aroca et al., 1993; Burchill and Thody, 1986; Burchill et al., 1986, 1988, 1989; Fuller et al., 1987, 1993; Hadley and Levine, 1993; Hoganson et al., 1989; Weatherhead and Logan, 1981b). The origin of melanotropin that modulates melanocyte function is also an area of active interest — originally proopiomelanocortin was thought to derive only from the pituitary gland, but recent evidence has shown that local production and secretion of proopiomelanocortin by epidermal cells in the skin (Bhardwaj and Luger, 1994; Farooqui et al., 1993; Mazurkiewicz et al., 2000; Pawelek, 1993; Slominski et al., 1992, 1993a, 1993b; Slominski and Wortsman, 2000), particularly keratinocytes (Schauer et al., 1994), may facilitate this paracrine influence. Perhaps even more interestingly, proopiomelanocortin seems to be expressed by melanocytes themselves and thus acts in an autocrine fashion (Farooqui et al., 1993; Slominski, 1991a; Slominski et al., 1995). There are actually three forms of melanotropin (a, b, and g) that are derived by proteolytic cleavage from the proopiomelanocortin precursor, along with other hormones such as ACTH. Binding of melanotropin to the melanocyte depends upon the surface expression of G protein-coupled melanotropin receptors (Ahmed et al., 1992; Ghanem et al., 1988; Hadley et al., 1981; Siegrist et al., 1989; Varga et al., 1974, 1976). A family of melanotropin receptors (termed melanocortin receptors, MC1R, MC2R, etc.) has been identified and cloned (Chhajlani and Wikberg, 1992; Chhajlani et al., 1993; Cone and Mountjoy, 1993; Desarnaud et al., 1994; Gantz et al., 1993b, c; Griffon et al., 1994; HaskellLuevano et al., 1994; Konda et al., 1994; Magenis et al., 1994; Mountjoy, 1994; Mountjoy et al., 1992; Siegrist et al., 1994), but only one of these, termed MC1R, is specifically expressed by melanocytes. MC1R binds the a-melanotropin form of melanotropin preferentially, whereas the distribution patterns and binding characteristics of other members of the melanocortin receptor family differ in other tissues. Presumably, these other receptors have different binding affinities to other members of the proopiomelanocortin derivatives, thus accounting for their varied functions in nonmelanocytic tissues such as the brain. Interestingly, although the MC1R (i.e. the melanocyte-specific receptor) of most mammals responds only to a-melanotropin, the MC1R expressed by human melanocytes responds equally well to ACTH (Abdel-Malek et al., 2000; Cone and Mountjoy, 1993; Hunt et al., 1994a, b; McGuire and Lerner, 1963), thus explaining the phenotypic effects on pigmentation associated with many conditions in which ACTH metabolism is disturbed. These topics are covered in more detail in Chapters 19, 20, and 21. The intracellular mechanism of melanotropin action is quite complex and not yet completely understood. The melanocytespecific melanotropin receptor is a G-coupled protein which, upon activation by melanotropin, stimulates the protein kinase A pathway (Abdel-Malek et al., 1992; Busca and Ballotti, 2000; Eberle et al., 1985, 1987; Friedmann et al., 1990a; Haskell-Luevano et al., 1994; Hol et al., 1993; Körner 196

and Pawelek, 1977; Lambert et al., 1985; Mountjoy et al., 1992; Pawelek, 1979, 1987; Varga, 1979). The intracellular events that lead from there to activation of the melanogenic pathway are not fully characterized, but one level of response may involve the stimulation of expression of the transcription factor AP-1, which in turn upregulates the transcription factor Mitf (cf. Chapters 5 and 13) and in turn stimulates the expression and function of melanogenic enzymes, including tyrosinase, and other differentiation factors. Mutations of the melanotropin receptor are associated with pheomelanin production in humans (i.e. red hair and fair skin) (Rees, 2000; Valverde et al., 1995) and can elicit dramatic changes in eumelanin or pheomelanin production depending on whether they lead to hypo- or hyperfunction of the melanotropin receptor (Abdel-Malek, 2001; Abdel-Malek et al., 1999, 2000, 2001; Cone and Mountjoy, 1993; Flanagan et al., 2000; Healy et al., 2000; Magenis et al., 1994; McGuire and Lerner, 1963; Scott et al., 2002a, b). A number of studies have established a correlation between MC1R genotype and melanoma risk (Cone, 2000; Flanagan et al., 2000, 2001; Healy et al., 1999; Rees, 2000; Scott et al., 2002b), although whether that is due to the pheomelanin or via other implications of MC1R signaling is not known. There is no doubt that other responses are also involved in melanocortin receptor signaling and, among those, it seems clear that the protein kinase C pathway is activated in some manner during the melanotropin response and that this also plays a role in the complex series of events that typically leads to stimulation of differentiation and proliferation of melanocytes (Buffey et al., 1992; Englaro et al., 1995; Park et al., 1993; Siegrist et al., 1995). The protein kinase C pathway is yet another route to regulation of melanocyte function and can be the mechanism whereby different growth factors, retinoic acid, and even ultraviolet radiation affect melanogenesis (Ando et al., 1990; Arita et al., 1992; Brooks et al., 1991; Carsberg et al., 1994; Gruber et al., 1992, 1995; Halaban et al., 1992; Loeffler et al., 1989; Niles and Loewy, 1989; Oka et al., 1995, 1996; Powell and Rosenberg, 1991; Rosenbaum and Niles, 1992). The last major hormone known to regulate mammalian pigmentation is the latest to be cloned and is in many ways perhaps the most intriguing, i.e. the agouti signal protein, the product of the agouti locus (Fig. 10.2). It has been known for some time that, in mice, melanotropin and agouti signal protein interact to regulate the switch of follicular melanocytes to produce eumelanin or pheomelanin (Fig. 10.3) (Barsh, 1995; Duhl et al., 1994a, b; Granholm and Van Amerongen, 1991; Granholm et al., 1990a, b, 1995; Kucera et al., 1996; Miller et al., 1993; Yen et al., 1994). While melanotropin is produced by a number of cell types in the skin and elsewhere (including melanocytes), agouti signal protein is produced by dermal papillae cells (Millar et al., 1995) and is a paracrine factor that modulates pigment production (Bultman et al., 1992; Miller et al., 1993). Agouti signal protein controls whether black/brown eumelanin or yellow/red pheomelanin is produced by follicular melanocytes, although the precise

THE REGULATION OF MELANIN FORMATION

MC1R

Fig. 10.3. Regulation of melanosomal biosynthesis. MSH, melanocyte-stimulating hormone; ASP, agouti signaling protein; DCT, DOPAchrome tautomerase; gp100, pmel17; MART-1, melanoma antigen recognized by T cells-1; MC1R, melanocortin 1 receptor; MITF, microphthalmia-associated transcription factor; OA1, ocular albinism 1; OCA, oculocutaneous albinism; PKA, protein kinase A; TYR, tyrosinase; TYRP1, tyrosine-related protein 1.

OCA2 OCA4

OCA1 OCA3

Adenylate cyclase G protein

PKA activity

MITF

DCT MART1 OA1

mechanism(s) by which this switch is effected is as yet unknown. In wild-type mice, eumelanin is produced by all follicular melanocytes at the beginning of the hair growth cycle (i.e. from 0–4 days). Transient expression of agouti signal protein from 4–6 days of the hair cycle causes melanocytes to produce pheomelanin instead of eumelanin, after which agouti gene expression is turned off and eumelanin is produced again. This pattern of pigment synthesis results in a yellow striped band near the tip of each hair shaft against a black background (Silvers, 1979; Silvers and Russell, 1955). Eumelanin and pheomelanin differ not only in their gross appearance, but also in their chemical composition and physical properties (as detailed in Chapters 15 and 16) and the ultrastructure of melanosomes in which they are synthesized and deposited. Follicular melanocytes of 1- to 2-day-old agouti mice (i.e. in their eumelanic phase) contain ellipsoid and fibrillar eumelanosomes, while follicular melanocytes of 4- to 6-day-old agouti mice contain ovoid and particulate pheomelanosomes. Similar changes in the ultrastructure of pheomelanosomes and eumelanosomes have been confirmed in a number of mouse mutants that produce one or the other type of melanin (Ozeki et al., 1995; Prota et al., 1995). Mutations at the agouti locus can cause the production of all yellow or all black hair, depending on whether the mutation leads to overexpression/hyperfunction or nonexpression/nonfunction of agouti signal protein respectively (Perry et al., 1994; Siracusa, 1994; Vrieling et al., 1994). Consequently, the agouti locus has been recognized as having an important role in regulating the switch between the production of eumelanin or pheomelanin by melanocytes. The biochemical action of agouti signal protein is a bit controversial (Conklin and Bourne, 1993; Jackson, 1993). Several laboratories have shown (Abdel-Malek, 2001; Abdel-Malek et al., 2001; Blanchard et al., 1995; Lu et al., 1994; Rouzaud et al., 2003; Siegrist et al., 1996; Tota et al., 1999; Willard et al., 1995) that agouti signal protein blocks the ability of melanotropin to activate its receptor, which suggests that the effect of the agouti locus on pigment switching is mediated by

reduced signaling through the melanocortin receptor, whereas its extrapigmentary effects may be mediated by reduced signaling through other melanocortin receptors. However, other groups have shown (Hunt and Thody, 1995; Manne et al., 1995; Zemel et al., 1995) that some effects of agouti signal protein might be mediated by an alteration in calcium channels. Therefore, in addition to working as an antagonist of melanotropin function through the melanotropin receptor, agouti signal protein may also function through other as yet undefined receptors to elicit its pleiotropic effects. Whatever the mechanism, the expression and enzyme activity of tyrosinase in vivo has been shown to be reduced during the pheomelanogenic phase of follicular melanocytes in various genotypes of mice, and there was little or no expression or enzyme activity of tyrosinase-related proteins 1 and 2 during pheomelanogenesis (Furumura et al., 1998, 2001; Granholm et al., 1990b; Kobayashi et al., 1995). This pattern of expression is consistent with the fact that tyrosinase is required for both types of pigment synthesis, but expression of the two tyrosinase-related proteins 1 and 2 is required only for eumelanin synthesis (Kameyama et al., 1992, 1995; Sakai et al., 1997). More recently, a number of studies have shown that melanocytes, which normally produce eumelanin exclusively in vivo, begin to produce significant amounts of pheomelanin when cultured in vitro (Hunt et al., 1995a, b; Sakai et al., 1997; Sato et al., 1987), presumably as a result of factors present in the medium. Further, pheomelanin production can be stimulated by in vitro treatment with purified recombinant agouti signal protein (Hunt and Thody, 1995; Sakai et al., 1997). Following treatment with agouti signal protein, cultured melanocytes exhibit physiologic features characteristic of pheomelanogenesis in vivo, thus providing an in vitro model for future characterization of the mechanisms and genes involved in this switch. Studies using such models should be able to resolve which melanogenic proteins are responsible for the eumelanin:pheomelanin switch and may finally elucidate the mechanism(s) involved in this important regulatory point. 197

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The function of agouti signal protein in human pigmentation remains speculative at this time (Kanetsky et al., 2002; Krude and Gruters, 2000; Krude et al., 1998; Voisey and van Daal, 2002; Voisey et al., 2001). The homology between the human and mouse proteins is striking and, in fact, the murine and human agouti signal protein elicits pleiotropic effects on murine and human melanocytes in culture (Abdel-Malek et al., 2001; Suzuki et al., 1997; Thirumoorthy et al., 2001). It remains an interesting topic for future study to define more clearly the role of agouti signal protein in human pigmentation, as well as to characterize its mechanism of action.

demonstrated by several groups (Gahl et al., 1995; Jara et al., 1990a, b, 1991; Pankovich and Jimbow, 1991; Potterf et al., 1996; Saga and Shimojo, 1984). Whether this transport system is constitutively expressed or is actively regulated as a mechanism for controlling melanin synthesis is not yet known, but this seems a reasonable possibility. It also seems quite likely that a distinct membrane transporter will be found to be involved in the control of the pheomelanogenic substrate cysteine through the melanosomal membrane (Potterf et al., 1999) and, again, it will prove interesting to see what role that transporter plays in regulating the switch to pheomelanogenesis.

Substrate Availability

Ultraviolet Radiation

l-Tyrosine, and its hydroxylated derivative l-3,4dihydroxyphenylalanine, are two key substrates of tyrosinase. Increasing the concentrations of these substrates would be expected to increase melanogenesis and, in fact, that does happen. However, such substrate activation of melanogenesis may be biphasic, going beyond the simple substrate activation of enzyme, in that tyrosine and 3,4-dihydroxyphenylalanine can stimulate melanogenesis by stimulating tyrosinase synthesis and activity in the cells (Slominski and Paus, 1990; Slominski et al., 1988). Melanocytes may express receptors that bind the substrates tyrosine and/or 3,4-dihydroxyphenylalanine (Howe et al., 1991; Slominski, 1991b; Slominski and Paus, 1994; Slominski and Pruski, 1992); upon such binding, there is a stimulation of the expression of melanotropin receptors by melanocytes that leads to further increases in proliferation and enzyme activity via melanotropin activation (McLane et al., 1987; Pawelek et al., 1988; Slominski, 1989; Slominski and Costantino, 1991; Slominski et al., 1989a, b). Phosphorylated substrates and other derivatives have been used as an approach specifically to target melanoma cells (Agin et al., 1987; Pawelek and Murray, 1986). In addition to the possible role of melanogenic substrate receptors on melanocyte differentiation and function, other factors that regulate substrate availability have critical importance to melanin formation. Synthesis of tyrosine itself is dependent upon the action of phenylalanine hydroxylase (Breakefield et al., 1978; Katz et al., 1976; Nagatsu et al., 1978; Schallreuter et al., 1998; Treiman and Tourian, 1973), yet little is known about the regulation of melanogenesis at this level. In addition, there are substrate transporters that function to regulate melanogenesis at the level of the melanosomal membrane (Gahl et al., 1995; Jara et al., 1990a, b, 1991; Pankovich and Jimbow, 1991; Potterf et al., 1996; Saga and Shimojo, 1984; Tietze et al., 1989). Tyrosine, the principle substrate of the melanogenic pathway, is not freely permeable through biologic membranes and, thus, a tyrosine transporter in the melanosomal membrane seems an obvious requirement. Early studies indicated that the permeability of the melanosomal membrane might play an active role in regulating melanogenic activity (VanWoert, 1970, 1972, 1973; VanWoert et al., 1971) and, in fact, the presence of a specific tyrosine transporter in the melanosome membrane has been

Ultraviolet radiation is a well-known stimulus of melanogenesis in the skin of mammals, particularly humans, and is commonly known as the tanning response (as detailed in Chapter 17). To summarize the effects with respect to modulation of the melanogenic pathway, the effects noted following ultraviolet radiation are comparable to those elicited by melanotropin, i.e. there is an upregulation in melanocyte proliferation and a stimulation of melanin production (Aberdam et al., 1993; Erickson, 1976; Erickson and Montagna, 1975; Friedmann et al., 1990b; Giacometti et al., 1972; Hunter et al., 1970; Iyengar, 1994; Libow et al., 1988; Morpurgo et al., 1980; Pathak, 1963; Rosdahl and Szabo, 1976; Uesugi et al., 1979; Zelickson et al., 1972). In fact, it has been reported that one mechanism by which ultraviolet radiation elicits its effects on melanocytes is via the induction of expression of melanotropin receptors (Birchall et al., 1991; Chakraborty et al., 1995; Oka et al., 1996; Pawelek et al., 1992), and it has often been noted that melanotropin and ultraviolet radiation produce synergistic effects (Halaban et al., 1993; Seechurn and Thody, 1990). Again, although no direct measurements on quantities and types of melanins produced following ultraviolet radiation of melanocytes have been made (Friedmann and Gilchrest, 1987; Johnson et al., 1972), the upregulation of melanogenic proteins, including tyrosinase and the tyrosinase-related proteins (Allan et al., 1995; Eller et al., 1996; Sharma and Ali, 1976; Sharma et al., 1979), would predict a dramatic increase in eumelanin production. This scenario would be consistent with expectations due to the beneficial ultraviolet-photoprotective properties of eumelanins compared with the more phototoxic properties of pheomelanins (Ahene et al., 1995; Barker et al., 1995; Cesarini and Msika, 1995; Chedekel, 1995; Chedekel et al., 1979; Hill, 1995; Hill et al., 1987, 1989b; Hubbard-Smith et al., 1992; Ishikawa et al., 1984; Jimbow et al., 1995; Menon et al., 1985; Schmitz et al., 1995); this topic is discussed more thoroughly in Chapter 17. Ultraviolet radiation of course can induce different types of photodamage in melanocytes, as well as in other types of cells, which leads to phototoxicity and ultimately to malignant transformation (Donawho et al., 1994; Fenske and Koo, 1994; Kripke, 1974; Nordlund et al., 1981; Setlow et al., 1993; Szabo et al., 1982; Tadokoro et al., 2003; Wolf et al., 1994).

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Other Factors Regulating Pigmentation A number of growth factors are important to melanocyte survival and proliferation; however, as many of these work by stimulating the protein kinase A and/or protein kinase C intracellular signaling pathways, it is not unexpected that they also often have “indirect” effects on rates of melanin production within melanocytes, typically via increased synthesis and activity of tyrosinase (Buffey et al., 1993; Eisinger et al., 1985; Gordon et al., 1989; Halaban et al., 1983). Many of these factors are expressed by neighboring keratinocytes, including basic fibroblast growth factor and endothelin-1 (Ando et al., 1995; Imayama et al., 1994; Imokawa et al., 1995; Nakazawa et al., 1995; Shishido et al., 2001), and their regulation is in turn affected by other environmental stimulants, including ultraviolet radiation (detailed in Chapters 20 and 21). Retinoic acid has been known for some time to upregulate the differentiation (i.e. melanogenesis) and proliferation of mammalian melanocytes (Boskovic et al., 2002; Dupin and LeDouarin, 1995; Edward et al., 1988; Hosoi et al., 1985; Huang et al., 2002; Lotan, 1979; Lotan et al., 1981; Niles and Loewy, 1989; Orlow et al., 1990; Talwar et al., 1993). As with ultraviolet stimulation, this effect seems to be mediated through an increase in expression of melanocortin receptors (Chakraborty et al., 1990, 1991; Gruber et al., 1995; Ho et al., 1992; Niles and Loewy, 1991; Rosenbaum and Niles, 1992). Thus, one might reasonably expect a similar stimulatory pattern of expression as seen with melanotropin itself, i.e. an increase in tyrosinase expression and activity (accompanied by moderate changes in the catalytic functions of tyrosinaserelated proteins 1 and 2) and thus a stimulation of eumelanin production. In addition to factors that upregulate pigment production in melanocytes, there are several physiologically important ones that downregulate melanogenesis. Agouti signal protein is one such notable factor that is produced extrinsic to melanocytes and is discussed above. It is known that such inhibitors play important roles in regulating ventral/dorsal pigmentation patterns in amphibians, and the relationship of agouti signal protein to those factors, as well as the role that differential expression of agouti signal protein plays in regulating mammalian pigmentation patterns, is another interesting question that remains to be examined. The role of melanogenic inhibitors is covered in detail in Chapter 18. Yet another physiologically important inhibitory factor is an intrinsic melanin inhibitor found within murine and human melanocytes, the level of which seems to play an active role in regulating melanin production by existing melanogenic enzymes (Hearing et al., 1995; Kameyama and Hearing, 1990; Kameyama et al., 1989, 1990, 1992, 1995). While tyrosinase is a critical requirement for melanin production, the presence of tyrosinase within melanocytes is no guarantee of melanin synthesis (Aroca et al., 1993; Eller et al., 1990; Hearing and Jiménez, 1989; Jara et al., 1988; Jiménez et al., 1988; Kameyama et al., 1989; Naeyaert et al., 1991), and many amelanotic, but tyrosinase-positive cell lines have been described both in vivo and in vitro. As discussed in Chapters

11 and 12, there are a number of tyrosinase-positive forms of oculocutaneous albinism in which normal levels of active tyrosinase are present, yet little or no melanin is produced. Full chemical characterization of these endogenous inhibitors has remained elusive, and future work will be necessary not only to identify these, but to elucidate their role in the metabolic regulation of melanin production in melanocytes.

Intracellular Trafficking and Melanosome Biogenesis The known melanosomal proteins are all involved in melanogenesis as catalytic and/or structural components of that organelle, and are currently composed of tyrosinase, TYRP1, DCT, MART 1, and gp100 (Kobayashi et al., 1994; Kwon et al., 1991). Although the processing and sorting of these proteins is not completely understood, they are known to be synthesized and translocated into the endoplasmic reticulum and eventually into the Golgi, where their post-translational processing and glycosylation takes place (Francis et al., 2003; Olivares et al., 2003). Following that, they seem to take distinct routes to traffic to melanosomes, the majority of melanosomal proteins predominantly going through the endosomal system to Stage II melanosomes, although that distribution can be disrupted in amelanotic cells. Despite their high structural similarity and conserved primary sequences, the three tyrosinase-related proteins seem to use distinct routes to move from the trans-Golgi network to early melanosomes: tyrosinase uses the AP3 system, TYRP1 uses the AP1 system, and DCT uses yet another unknown sorting vesicle system (Huizing et al., 2002; Negroiu et al., 2003; Spritz et al., 2003). The sorting system for MART 1 is not yet known, but its distribution patterns are highly similar to tyrosinase. gp100 is perhaps the most uniquely processed of the melanosomal proteins; after processing through the endoplasmic reticulum and Golgi, it is normally delivered to Stage I melanosomes without going through the endosomal system (Basrur et al., 2003; Kushimoto et al., 2001; Yasumoto et al., 2004). Following the maturation of Stage I to Stage II melanosomes, which is coincident with the cleavage and refolding of gp100, the enzymatic components are delivered and the synthesis of melanin usually ensues (Kushimoto et al., 2003). Interestingly, the subcellular distribution of melanosomal proteins in amelanotic melanocytic cells that produce normal levels of melanosomal proteins (presumably with intact functional activities) and thus should be pigmented is quite distinct. In pigmented cells, the majority of tyrosinase is correctly processed through the endoplasmic reticulum, Golgi, and endosomes and is delivered to Stage II melanosomes (Kushimoto et al., 2001; Watabe et al., 2004). In contrast, the majority of tyrosinase in amelanotic cells is retained in the endoplasmic reticulum, although a minor amount of tyrosinase can be glycosylated correctly and found in the Golgi, endosomes, and early melanosome fraction (primarily in Stage I melanosomes). Melanosomes are lysosome-related organelles, but their exact biogenesis is still poorly defined (Marks and Seabra, 2001; Raposo et al., 2001). For instance, 199

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melanosomes and lysosomes contain many of the same structural proteins [e.g. lysosome-associated membrane proteins (LAMP), acidic hydrolases, vacuolar-type proton pumps] (Ancans and Thody, 2000; Bhatnagar and Ramaiah, 1998; Diment et al., 1995), and both are affected in several genetic disorders, such as the Chediak–Higashi and Hermansky– Pudlak syndromes (Boissy et al., 1998; Zhao et al., 1994). The correct processing and sorting of tyrosinase to melanosomes is the key to pigmentation and a number of factors regulate it. In addition to pH (as discussed below), other chaperonelike proteins are involved (e.g. TYRP1), as are vesicular components such as the P protein and MATP, and disruptions in the functions of these proteins are associated with oculocutaneous albinism types III, II, and IV respectively (Costin et al., 2003; Toyofuku et al., 2001a, b, 2002) (detailed in Chapters 11 and 12).

Proteasome Function and the Regulation of Tyrosinase Activity In contrast to the distribution and stability patterns of tyrosinase in pigmented cells, the majority of tyrosinase in amelanotic cells is not correctly glycosylated, is trapped in the endoplasmic reticulum, and is quickly degraded by proteasomes. This mechanism is also involved with the hypopigmentation seen in various forms of inherited pigmentary disorders such as oculocutaneous albinism, as noted above. Thus, tyrosinase, which is an extremely stable protein with a half-life of 16–24 h in pigmented melanocytes, can have an extremely short half-life in amelanotic cells, and that stability can be markedly enhanced by treatment with proteasome inhibitors, showing that proteasomes are involved in the degradation of tyrosinase. It remains an interesting hypothesis that proteasomes (and the degradation of tyrosinase) may actually be a physiologically important point of regulating tyrosinase activity and pigmentation under normal circumstances. A recent series of studies has found that free fatty acids can dramatically stimulate or inhibit pigmentation (Ando et al., 1999). Analysis of the mechanism involved in those effects has shown that they specifically affect tyrosinase and, in fact, they reflect the effects of those free fatty acids on proteasome function (Ando et al., 2004; Kageyama et al., 2004). At least one pigment-related gene has been associated with a ubiquitin ligase function in regulating pigmentation (He et al., 2003) and, thus, the role of proteasomes in modulating melanogenesis, perhaps providing rapid responses to environmental stimuli that require no changes in gene expression, is an exciting avenue for future study.

has been some controversy regarding the optimal pH for tyrosinase activity. Melanosomes, like other lysosomal organelles, can be quite acidic, and it has been assumed that this low melanosomal pH facilitates melanogenesis (Devi et al., 1987). It has been suggested that TYR may be activated at an acidic pH and that the enzyme is inactive at neutral pH (Tripathi et al., 1988). However, several other groups have maintained that mammalian TYR has an optimal enzymatic activity near neutral pH and that its activity is gradually lost with decreasing pH (Hearing and Ekel, 1976; Saeki and Oikawa, 1985; Townsend et al., 1984). However, the pH at which melanin is produced and the pH at which the vesicular trafficking system works may function quite independently. TYR levels in amelanotic cells can be dramatically increased following treatment with protonophore or proton pump inhibitors (Fuller et al., 2001; Halaban et al., 2002; Manga and Orlow, 2001; Watabe et al., 2004). Surprisingly, the retention of TYR in the endoplasmic reticulum in amelanotic cells is corrected by these agents (i.e. by increasing intracellular pH), which results in the enhanced transport of tyrosinase from the endoplasmic reticulum to the Golgi. Thus, the abnormal acidification of intracellular organelles also plays an important role in the pathogenesis of hypopigmentation in amelanotic cells, and may even play an important role in regulating the constitutive level of pigmentation in the skin. Although tyrosinase is dramatically redistributed to Stage II melanosomes in amelanotic cells following the neutralization of intracellular pH, tyrosinase levels in endosomes are reduced in the presence of proton pump inhibitors but are increased in the presence of protonophores. This suggests that the processing and trafficking of tyrosinase in amelanotic melanocytes is related to the dysfunction of vATPases. vATPase was recently identified as a melanosomal protein (Basrur et al., 2003) and, thus, it may play an important role in regulating pigmentation. Transport of tyrosinase from the endoplasmic reticulum and its subsequent processing depends on the neutralization of pH in the Golgi, although the translocation of tyrosinase to endosomes then requires the activation of vATPase. Neutralization of the pH within early melanosomes results in the accumulation of tyrosinase in those organelles. Therefore, the activity of vATPases within intracellular organelles plays an important role in the sorting and function of tyrosinase and in modulating pigmentation; dysfunction of this pH regulatory system may be responsible for the depigmented phenotype and pathogenesis of amelanotic melanocytes.

Perspectives Regulation of Melanin Production via Intracellular pH The catalytic domains of tyrosinase and other enzymes involved in melanogenesis are located within the lumen of the melanosome, and it follows that their activity is likely to be dependent upon the intramelanosomal environment, including the pH (Ancans et al., 2001; Puri et al., 2000). However, there 200

The regulation of pigmentation has important consequences for all the varied functions of melanin, including the social implications of skin pigmentation, the cosmetic variations in skin, hair, and eye color, and of course its photoprotective function. It has become progressively obvious in recent years that there are numerous levels of control over pigmentation, which

THE REGULATION OF MELANIN FORMATION I

III

II

IV

Dct Tyr

Fig. 10.4. Melanosomal architecture. Dct, DOPAchrome tautomerase; DOPA, 3,4-dihydroxyphenylalanine; DHI, 5,6-dihydroxyindole; DHICA, 5,6dihydroxyindole-2-carboxylic acid; Tyr, tyrosinase; Tyrp1, tyrosine-related protein 1.

Tyrosine

are exceedingly complex, and that it will be some time before we can fully appreciate the interactions that are involved. Regulatory stimuli for melanocyte function originate from the environment (e.g. ultraviolet radiation), endocrine sources (e.g. melanotropin from the pituitary), paracrine sources (e.g. growth factors, endothelins, or agouti signal protein from keratinocytes or other neighboring cells), or can be autocrine (e.g. proopiomelanocortin from the melanocyte itself). In addition, the pigment distribution depends upon the initial distribution and survival of melanoblasts from the developing embryo and, of course, ultimately in mammals on the transfer and processing of melanosomes to keratinocytes in the skin or hair. Each of these steps is under complex genetic and molecular controls, and it is no wonder that there is such a variety of visible phenotypes in humans. In addition to studies targeted at understanding the regulation of processes involved in pigmentation, the melanosome (Fig. 10.4) is now also being used as a model for study of organelle biogenesis and function as well as for targeting of malignant melanoma. The complexity of controls regulating pigmentation is an impediment to easy understanding of the entire process, but promises a wealth of potential targets for the eventual manipulation of the system, for either cosmetic or pharmacologic purposes.

Acknowledgments The author wishes to thank numerous investigators in this field who have been so cooperative in countless discussions on these topics in recent years. Among them are Drs Zalfa AbdelMalek, Gregory Barsh, Dorothy Bennett, Raymond Boissy, Murray Brilliant, Shosuke Ito, Richard King, Lynn Lamoreux, William Oetting, Seth Orlow, John Pawelek, Giuseppe Prota, Francisco Solano, Richard Spritz, and all former/current members of the Hearing Laboratory — I apologize to many others whose names have been omitted for the sake of brevity.

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DOPAquinone

DOPAchrome + cysteine

Tyrp1

DHICA DHI

DHICA-melanins

DHI-melanins

Pheomelanins

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V. J. Hearing. Influence of a-melanocyte stimulating hormone and ultraviolet radiation on the transfer of melanosomes to keratinocytes. FASEB J. 16:105–107, 2002. Visconti, M. A., and A. M. Castrucci. Ionic requirements for catecholamine aggregating actions on the teleost Poecilia reticulata melanophores. Pigment Cell Res. 3:132–140, 1990. Visconti, M. A., A. M. Castrucci, M. E. Hadley, and V. J. Hruby. Ionic requirements for melanin concentrating hormone (MCH) actions on teleost Poecilia reticulata melanophores. Pigment Cell Res. 2:213–217, 1989. Voisey, J., and A. van Daal. Agouti: from mouse to man, from skin to fat. Pigment Cell Res. 15:10–18, 2002. Voisey, J., N. F. Box, and A. van Daal. A polymorphism study of the human agouti gene and its association with MC1R. Pigment Cell Res. 14:264–267, 2001. Voulot, C. Effect of melatonin and various indoles on the guinea pig melanocyte. C. R. Acad. Sci. 269:2247–2250, 1969. Vrieling, H., D. M. J. Duhl, S. E. Millar, K. A. Miller, and G. S. Barsh. Differences in dorsal and ventral pigmentation result from regional expression of the mouse agouti gene. Proc. Natl. Acad. Sci. USA 91:5667–5671, 1994. Walker, M. J., C. W. Beattie, M. K. Patel, S. M. Roman, and T. K. DasGupta. Estrogen receptor in malignant melanoma. J. Clin. Oncol. 5:1256–1261, 1987. Wang, S. Q., R. B. Setlow, M. Berwick, D. Polsky, A. A. Marghoob, A. W. Kopf, and R. S. Bart. Ultraviolet A and melanoma: a review. J. Am. Acad. Dermatol. 44:837–846, 2001. Watabe, H., J. C. Valencia, K. Yasumoto, T. Kushimoto, H. Ando, J. Muller, W. D. Vieira, M. Mizoguchi, E. Appella, and V. J. Hearing. Regulation of tyrosinase processing and trafficking by organellar pH and by proteasome activity. J. Biol. Chem. 279:7971–7981, 2004. Weatherhead, B., and M. E. C. Blackburn. Growth and melanization of a hamster melanoma cell line, RPMI 3460, in response to melatonin and cyclic nucleotides. In: Biological, Molecular and Clinical Aspects of Pigmentation, J. T. Bagnara, S. N. Klaus, E. Paul, and M. Schartl (eds). Tokyo: University of Tokyo Press, 1985, pp. 471–476. Weatherhead, B., and A. Logan. Interaction of a-melanocytestimulating hormone, melatonin, cyclic AMP and cyclic GMP in the control of melanogenesis in hair follicle melanocytes in vitro. J. Endocrinol. 90:89–96, 1981a. Wilkes, B. C., V. J. Hruby, W. C. Sherbrooke, A. M. Castrucci, and M. E. Hadley. Synthesis and biological actions of melanin concentrating hormone. Biochem. Biophys. Res. Commun. 122:613–619, 1984. Willard, D. H., W. Bodnar, C. Harris, L. Kiefer, J. S. Nichols, S. Blanchard, C. Hoffman, M. Moyer, W. Burkhart, J. Weiel, M. A. Luther, W. O. Wilkison, and W. J. Rocque. Agouti structure and function: characterization of a potent a-melanocyte stimulating hormone receptor antagonist. Biochemistry 34:12341–12346, 1995. Wintzen, M., and B. A. Gilchrest. Proopiomelanocortin, its derived peptides, and the skin. J. Invest. Dermatol. 106:3–10, 1996. Wolf, P., C. K. Donawho, and M. L. Kripke. Effect of sunscreens on UV radiation-induced enhancement of melanoma growth in mice. J. Natl. Cancer Inst. 86:99–105, 1994. Yasumoto, K., H. Watabe, J. C. Valencia, T. Kushimoto, T. Kobayashi, E. Appella, and V. J. Hearing. Epitope mapping of the melanosomal matrix protein gp100 (PMEL17): Rapid processing in the endoplasmic reticulum and glycosylation in the early Golgi compartment. J. Biol. Chem. 279:28330–28338, 2004. Yen, T. T., A. M. Gill, L. G. Frigeri, G. S. Barsh, and G. L. Wolff. Obesity, diabetes, and neoplasia in yellow Avy/- mice: Ectopic expression of the agouti gene. FASEB J. 8:479–488, 1994.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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The Tyrosinase Gene Family William S. Oetting and Vijayasaradhi Setaluri

Summary 1 The focus of this chapter is the molecular biology of the members of the tyrosinase gene family. Genes of this family encode enzymes involved in the melanin biosynthetic pathway. Members of this family include tyrosinase (the rate-limiting enzyme in melanin biosynthesis), 5,6-dihydroxyindole-2carboxylic acid (DHICA) oxidase, and DOPAchrome tautomerase (also known as TRP1 and TRP2 respectively). 2 All three members of this gene family exhibit high nucleotide sequence homology. This homology extends throughout the animal kingdom. 3 The protein products of these three genes are also highly homologous and share common structural domains including a transmembrane domain, two putative copper binding sites, and a cysteine-rich domain containing an epidermal growth factor (EGF)-like protein-binding motif. 4 The transcriptional regulation of the tyrosinase family genes is rather complex, involving multiple cis-regulatory elements and transactivating factors. A 5¢ regulatory element, termed M-box, that is common to all three genes, is necessary for their regulation by the microphthalmia transcription factor, MITF. However, melanocyte-specific expression of tyrosinase genes during development and differentiation may involve both positive and negative regulatory mechanisms.

Historical Background The tyrosinase gene family consists of three genes that code for enzymes active in the melanin biosynthetic pathway, and truncated pseudogenes in some species. Tyrosinase is the most well-known member of this gene family and is responsible for the rate-limiting step in melanin biosynthesis — the hydroxylation of tyrosine to dopaquinone (Cooksey et al., 1997). The first report of tyrosinase was in 1895, when tyrosinase activity in mushrooms was described as a heat-sensitive, watersoluble ferment that turns black upon exposure to oxygen (Bourquelot and Bertrand, 1895). The first descriptions of tyrosinase activity in mammalian tissue were by Gessard (1903), who reported that extracts of horse melanoma had the ability to convert tyrosine to melanin, and by Durham (1904), who described the presence of tyrosinase activity in mammalian skin. Hogeboom and Adams (1942) separated melanin biosynthesis into two enzymatic activities, the oxidation of tyrosine and the oxidation of dopa. Lerner et al. (1949) provided evidence that tyrosinase was responsible for both these

activities. Further details of the melanin biosynthetic pathway were reported by Mason et al. (1955). For many years, it was thought that tyrosinase was the sole enzyme involved in the biosynthetic pathway of melanin, which begins with the substrate tyrosine and produces either the characteristic brown/black eumelanin or red/yellow pheomelanin in the presence of sulfhydryl groups. Tyrosinase was known to be involved in the catalysis of the first two steps, the oxidation of tyrosine to dopa and the subsequent formation of dopaquinone. All subsequent steps were thought to occur spontaneously without the need for other enzymatic catalysts. It was through the work of Logan and Weatherhead (1978), using Siberian hamsters, that other regulatory factors in the melanin biosynthetic pathway were shown to exist. They found that, during autumn molt, when the coat hair was unpigmented, tyrosinase activity was still present as well as normal serum tyrosine levels. They hypothesized that another factor must exist to prevent melanin from being formed. Previous to this, there had been suggestions of other regulatory factors in the melanin biosynthetic pathway, but this was the first experimental evidence that such factors existed and affected coat color. Körner and Pawelek (1980) identified a factor, termed DOPAchrome conversion factor (DCF), in extracts of Cloudman S91 melanoma cells that could rapidly convert red/orange DOPAchrome to a colorless compound. Pawelek and coworkers also reported two other factors: 5,6-dihydroxyindole (DHI) conversion factor, which, when cells are exposed to melanocyte-stimulating hormone (MSH), converts DHI to indole-5,6-quinone, and DHI blocking factor, which stops melanin biosynthesis at 5,6dihydroxyindole (Pawelek et al., 1980). The DOPAchrome conversion factor was subsequently shown to be a new enzyme in the melanin biosynthetic pathway, and was termed DOPAchrome oxidoreductase and finally named DOPAchrome tautomerase (Aroca et al., 1990; Barber et al., 1984; Leonard et al., 1988). A third enzyme, 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA oxidase), has also been included in the menagerie of enzymes involved in the melanin biosynthetic pathway. At present, no enzymes have been shown to be involved specifically in the pheomelanin pathway. As the complex enzymology of the melanin pathway was being worked out, attempts were being made to isolate gene sequences coding for these enzymes. Understandably, efforts to isolate pigment-specific genes began initially with the cDNA for tyrosinase. Instead of isolating the tyrosinase cDNA, a family of cDNAs coding for proteins related to tyrosinase was discovered. The first “tyrosinase” cDNA clone isolated, 213

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termed pMT4, was later shown to code not for tyrosinase, but for a related protein termed tyrosinase-related protein (TRP1), the gene now known to code for DHICA oxidase (JiménezCervantes et al., 1994; Shibahara et al., 1986). This cDNA was found to be homologous to the brown (b) locus of the mouse (Jackson, 1988; Vijayasaradhi et al., 1990). Eventually, both the mouse and the human tyrosinase cDNA were cloned (Kwon et al., 1987; Yamamoto et al., 1987). The tyrosinase sequence was authenticated by comparison of the encoded amino acid sequence with results of direct amino acid sequencing of the isolated enzyme (Wittbjer et al., 1989) and by expression of the isolated tyrosinase cDNA in heterologous cells and observing tyrosinase enzymatic activity (Beermann et al., 1990; Bouchard et al., 1989; Müller et al., 1988). Two other related loci were identified and called the tyrosinaserelated protein-2 (TRP2), a product of the mouse slaty (slt) locus, shown to be the gene for DOPAchrome tautomerase (DT) (Jackson et al., 1992; Tsukamoto et al., 1992a), and a tyrosinase pseudogene (tyrosinase-related sequences; TYRL), which contains only exons 4 and 5 of the tyrosinase gene (Takeda et al., 1989a). In her analysis of tyrosinase in pigmented vertebrate skin, Durham (1904) also reported that tyrosinase activity was absent in albino animals, but she did not draw any conclusions from this observation. A hypothesized association between albinism in man and a lack of enzyme activity was first made by Sir Archibald Garrod in his seminal paper entitled “Inborn Errors of Metabolism” (Garrod, 1908). In his report for the Croonian lectures, Garrod stated that “. . . an intracellular enzyme is probably wanting in the subjects of this anomaly . . . .” Garrod mentions tyrosinase as being the activity responsible for melanin pigment formation, but did not make a clear connection between a lack of tyrosinase activity and albinism. For many decades afterwards, it was not clear whether the lack of tyrosinase activity resulted from a mutation in the gene responsible for tyrosinase or the absence of melanocytes in the skin (Lerner and Fitzpatrick, 1950). Using gold impregnation to visualize melanocytes by light microscopy (Becker et al., 1952) and electron microscopy (Barnicot et al., 1955), melanocytes were identified in human albino skin and hair bulbs. Fitzpatrick et al. (1958), using the dopa reaction of Bloch (1917), later showed that albino hair follicles in both mouse and human skin contained melanocytes but lacked tyrosinase activity. The first mutation within the tyrosinase gene associated with human oculocutaneous albinism (OCA) was reported in 1989 (OCA1; MIM #203199) (Tomita et al., 1989). This type of albinism, termed OCA1A, classically presents with a complete absence of pigment in the skin, eye, and hair, but some mutations produce a residual activity enzyme producing a phenotype with varying amounts of pigment (OCA1B) (Giebel et al., 1991a; King et al., 2000). Mutations of the tyrosinase-related protein-1 (TRP1) gene were shown to result in another type of albinism, OCA type 3 (OCA3, MIM #203290), also termed rufous or red OCA

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(MIM *278400; Boissy et al., 1996; Manga et al., 1997). Individuals with OCA3 are born with minimal pigmentation but develop an increase in pigment with time. The pigmentation changes in the skin, hair, and eyes, as well as the severity of the ocular involvement with OCA3, have not been as well documented as in other types of albinism, but the visual problems do not appear to be as severe in OCA3 as those observed in OCA1 (Kromberg et al., 1990). To date, there have been no reported human pigment phenotypes associated with mutations of TRP2.

Current Concepts Overview The formation of pigment in the melanocyte is a complex process requiring the action and interaction of many genes. In the mouse, there are over 100 loci that affect coat color (Bennett and Lamoreux, 2003; Silvers, 1979), and the majority of these loci are involved in melanocyte differentiation and migration. A subset of these loci is concerned with the formation of the premelanosome and biosynthesis and deposition of melanin in the premelanosome forming the mature melanosome. Three of these loci are members of a gene family that code for the enzymes, tyrosinase, TRP-1, and TRP-2, involved in the eumelanin biosynthetic pathway. Although these three proteins have different enzymatic activities, they share extensive homology in both nucleotide and amino acid sequences. The comparison of the promoter regions of these genes has provided an opportunity to locate common regulatory elements that may control the expression of pigmentspecific genes in a coordinated fashion. Detailed analysis of the 5¢ promoter region of these genes has indicated several possible cis-acting regulatory regions, some common to all three genes. Analysis of the proteins has extended our understanding of protein transport and processing, including the impact of missense mutations on this process.

Lower Vertebrates and Invertebrates Although the greater part of our understanding of the proteins and genes involved in pigmentation has come from studies involving the mouse and human pigment systems, other species have provided important insight into the evolution of the tyrosinase gene family. The earliest work with nonmammalian tyrosinase genes has been with mushroom tyrosinase. Other reports have included work with tyrosinase from the Streptomyces family and Neurospora crassa (Bernan et al., 1985; Huber et al., 1985; Katz et al., 1983). These systems have been instrumental in helping us understand the chemistry occurring in the active site of the enzyme. Lerch and colleagues have used site-directed mutagenesis to alter amino acids in the copper binding sites of Streptomyces tyrosinase to determine the amino acid residues critical to tyrosinase activity and their role in the catalytic site of the enzyme (Huber and Lerch, 1988; Jackman et al., 1991, 1992). These studies have shown

THE TYROSINASE GENE FAMILY

that histidine residues are the ligands involved in copper binding (Huber and Lerch, 1988; Jackman et al., 1991). Other amino acid residues such as the asparagine at codon 190 in Streptomyces glaucescens, adjacent to one of the histidine ligands in the copper B binding site, are thought to play a critical role in the hydrogen-bonding interaction that bridges the two copper atoms (Jackman et al., 1991). It was not until the mouse cDNA for tyrosinase was isolated that tyrosinase cDNAs from other vertebrates were cloned and their sequences determined. The isolation and sequence analyses of all three members of the tyrosinase gene family from the fugu fish (Takifugu rubripes) indicate the high degree of conservation of the pigmentary system in vertebrates (Camacho-Hubner et al., 2002). Partial sequences for all three members in Xenopus laevis have also been reported (Kumasaka et al., 2003). Other nonmammalian vertebrates in which tyrosinase sequences have been identified include the Japanese quail (Coturnix coturnix japonica), the snapping turtle (Trionyx sinensis japonicus), the Japanese pond frog (Rana nigromaculata), and goldfish (Miura et al., 1995, Peng et al., 1995; Yamamoto et al., 1992). The high degree of nucleotide sequence homology between members of the tyrosinase gene family (see below) allowed Jackson and coworkers to create a series of degenerate oligonucleotide primers that amplified the cDNA of all three members of the tyrosinase gene family (Jackson et al., 1994). Using these amplification primers, TRP1 cDNA from the Mexican axolotl (Ambystoma mexicanum) was isolated (Mason and Mason, 1995). These degenerate polymerase chain reaction (PCR) primer pairs should make it much easier to isolate the cDNA sequences of members of the tyrosinase gene family from other species. Numerous tyrosinase gene sequences are now available for all members of the tyrosinase gene family. Partial sequences for all three tyrosinase gene family members for many species can be found in the National Center for Biotechnology Information (Pruitt and Maglott, 2001). With the exception of the signal peptide sequence at the amino-terminal end, the amino acid sequences of these proteins are highly homologous.

The Tyrosinase Gene Family The tyrosinase gene family in humans consists of four loci, three of which produce protein products that are active in the melanin biosynthetic pathway. These include the enzymes tyrosinase (TYR; EC 1.14.18.1), DHICA oxidase (tyrosinaserelated protein-1; TRP-1), and DOPAchrome tautomerase (tyrosinase-related protein-2; TRP-2; EC 5.3.3.12). The fourth locus, the tyrosinase pseudogene (TYRL), is not expressed in melanocytes (Giebel et al., 1991b; Jackson et al., 1994; Takeda et al., 1991). Sequences from TYRL are found in humans and the gorilla, but are not found in other higher primates including the chimpanzee and Barbary ape (Oetting et al., 1993a), and are thought to be absent in lower vertebrates. No additional members are thought to exist in this gene family in humans. In the mouse genome, in addition to the Tyrp1 gene located on chromosome 4, there is also a

TYR TRP1 TRP2 Fig. 11.1. Comparison of the gene structures for tyrosinase, TRP1, and TRP2. The numbered boxes represent the exons of the gene. Vertical lines represent the location of intron/exon junctions. Vertical lines between genes represent identical or in-phase intron/exon junctions between those two genes.

tyrosinase-related protein-1-related sequence 1 (Tyrp1-rs1) on chromosome 8. Little is known about this locus, or whether it is an active gene or a pseudogene. Comparison of the genomic structure of the mouse and human homologs of tyrosinase, TRP1, and TRP2 genes shows that they have identical gene structure and splice junctions. Comparison of tyrosinase, TRP1, and TRP2 with each other, on the other hand, reveals numerous differences in the number of exons and the location of the splice junctions (Fig. 11.1). There are only two splice site junctions that are identical and one location that is similar. Comparisons between the splice sites for intron 2 of TYR and intron 5 of TRP1 and between the splice sites of intron 3 of TRP1 and TRP2 are identical (Budd and Jackson, 1995; Sturm et al., 1995). Intron 4 of TYR and intron 7 of TRP1 and TRP2 are not identical but have the same phasing. The intron/exon boundary and nucleotide sequences between TYR and TYRP are almost identical for exons 4 and 5 (Giebel et al., 1991b; Takeda et al., 1991). Both TRP1 and TRP2 are thought to have arisen from the TYR gene by means of gene duplication and subsequent divergence (Jackson, 1995a; Sturm et al., 1995). This event appears to have at least predated mammalian evolution, in that all mammals studied so far appear to have all three members of this gene family. Furthermore, all three genes have been found in Takifugu rubripes and Xenopus laevis, showing that all vertebrates may contain all three members (Camacho-Hubner et al., 2002; Kumasaka et al., 2003). As gene sequences of the tyrosinase gene family are isolated from more diverse species, direct comparisons of the gene structure should provide valuable insight into the evolution of this gene family. Sequence analysis of the cDNAs has shown that these genes have similar nucleotide sequences (30–50% similarity). Comparison of the amino acid sequences between TRP1 and TRP2 shows a closer relationship to each other (50% identify) than to TYR (40% identify) (Bouchard et al., 1994; Cassady and Sturm, 1994). Figure 11.2 shows the high degree of homology in the amino acid sequence between these three proteins. There are two consensus amino acid patterns that identify members of this protein family, as well as the hemocyanins, but no other proteins in the SWISSPROT database. These sequences are H-x(4,5)-F-[LIVMFTP]-x-[FW]-H-Rx(2)-[LVM]-x(3)-E associated with the copper A binding

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Fig. 11.2. Comparison of the amino acid sequence for tyrosinase, TRP1, and TRP2. The cysteine residues are shown in bold letters, and an asterisk has been placed over the histidine residues involved in copper binding. Vertical lines represent identical amino acids. Dashes represent gaps to aid in alignment of the sequences. The sequence for the EGF-like motif has been underlined.

216

THE TYROSINASE GENE FAMILY

Fig. 11.3. This represents similar structural domains between members of the tyrosinase family of proteins. The shaded box labeled “SP” is the signal peptide sequence. The dots represent cysteine residues. EGF is the location of the EGF-like domain. The hatched boxes Cu(A) and Cu(B) represent the copper-binding domains. The checkered box labeled TM represents the transmembrane domain.

domain and D-P-x-F-[LIVMFYW]-x(2)-H-x(3)-D associated with the copper B binding domain. In both cases, the “H” represents the histidine copper-binding ligands. Analyses of the amino acid sequences of these proteins reveal several regions of similarity that are potential functional domains. All three enzymes contain an amino-terminal signal sequence, 15 conserved cysteine residues, two putative copper-binding regions, a transmembrane region, and a cytoplasmic tail at the carboxy-terminal of the protein (Fig. 11.3) (Jackson et al., 1992; Sturm et al., 1995). These similarities, especially the conservation of the cysteine residues, present a strong indication that all three proteins have a common three-dimensional structure. The amino-terminal signal sequence has been shown to be important for correct processing of the tyrosinase polypeptide and its intracellular trafficking (Oetting et al., 1995). Mutations that eliminate this portion of the protein for tyrosinase result in the enzyme being translated by cytoplasmic ribosomes instead of membrane-bound ribosomes. This results in an aberrantly folded protein that is quickly degraded (Oetting et al., 1995). The proteins also have two cysteine-rich domains (Figs 11.2 and 11.3). The first cysteine-rich region also contains an EGF motif similar to that found in the EGF family, a motif that may be involved in protein–protein interactions (Jackson et al., 1992). This may be important in the formation of the melanin biosynthetic complex discussed later in the chapter. Although the copper(A) and copper(B) binding regions found in tyrosinase, including the copperbinding histidine ligands, can be identified in the other two proteins, copper has not been shown to be present in either TRP-1 or TRP-2. It has been hypothesized that TRP-1 and TRP-2 may have other metal ions as cofactors, but this has yet to be unambiguously established (Chakraborty et al., 1992; Halaban and Moellmann, 1990; Solano et al., 1994). The strongest evidence to date for binding of other metal cofactors has been binding of zinc by TRP-2 (Solano et al., 1994). Work by Furumura et al. (1998) showed that copper is bound most strongly by tyrosinase and less so by TRP-1. TRP-2 binds zinc and could also bind small amounts of iron, but TRP-1 did not bind zinc or iron. This difference in metal ligand binding is due, in part, to differences in the sequence in the metal-binding regions. It is expected that TRP-1 also has a metal cofactor, but the identity of this ligand has yet to be determined. The transmembrane region, found in all three proteins, is a highly hydrophobic region, as shown by hydrophobicity

mapping. Accordingly, all three proteins have been shown to be integral membrane proteins, anchored in the melanosomal membrane by the transmembrane domain (Orlow et al., 1994; Winder et al., 1994a). The carboxy-terminal cytoplasmic region has been shown to be important for the correct intracellular sorting and targeting of TRP-1 to the premelanosome and most likely plays an important role in the targeting of all three proteins. A series of experiments by Vijayasaradhi et al. (1995a) has shown the importance of the cytoplasmic tail region in intracellular sorting. A hexapeptide sequence, AsnGln-Pro-Leu-Leu-Thr, containing the dileucine motif ProLeu-Leu, is conserved among other melanosomal proteins including tyrosinase, Pmel-17, and the P gene product, but not TRP-2 or the melanocyte-stimulating hormone receptor (MSHR; Jimbow et al., 1995). This sequence has been identified as a sorting signal necessary for correct intracellular trafficking of TRP-1 in melanocytic cells. This motif is also observed in some lysosomal membrane proteins, providing evidence for a similar sorting mechanism and pathways for both melanosomes and lysosomes, which will also include additional signals necessary to distinguish the movement of these proteins between the two organelles. The importance of the transmembrane region for correct protein function has also been shown in vivo by two frameshift mutations in the human tyrosinase gene. A frameshift mutation at codon 490 eliminates one half of the transmembrane region and the entire cytoplasmic tail, and a second frameshift mutation at codon 501, located at the carboxy end of the transmembrane region, effectively eliminates the cytoplasmic tail (Chintamaneni et al., 1991a; Giebel et al., 1991a; King and Oetting, 1992; Tripathi et al., 1992a). In both cases, these mutations result in the complete absence of enzymatic activity and are associated with tyrosinasenegative oculocutaneous albinism (OCA1). A mutation in the mouse tyrosinase gene responsible for the platinum phenotype also causes a truncated protein that lacks the cytoplasmic tail (Beermann et al., 1995; Orlow, 1995; Orlow et al., 1993a). The lack of an intact cytoplasmic tail due to the platinum mutation results in the tyrosinase protein being routed to the plasma membrane rather than to the melanosome, causing a severe reduction in tyrosinase activity. The transmembrane region and the cytoplasmic tail appear only to be involved in the correct targeting of the enzyme and do not play a role in the catalytic function of the enzyme. Tryptic cleavage of the mature enzyme, which removes the transmembrane region and the cytoplasmic tail, leaves the truncated enzyme catalytically active.

Tyrosinase Gene/Tyrosinase Tyrosinase is a copper-containing protein and is the rate-limiting enzyme in the melanin biosynthetic pathway. Tyrosinase catalyzes two major steps in melanin formation, the hydroxylation of tyrosine to DOPA, which is the rate-limiting step, and the oxidation of DOPA to DOPAquinone. The catalytic site of the enzyme contains two copper atoms ligated to six 217

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histidine residues. The hydroxylation of tyrosine apparently begins with the binding of dioxygen to the Cu1+ Cu1+ center of tyrosinase to yield a peroxide-bound Cu2+ Cu2+ center termed oxytyrosinase (Lerch, 1988; Mason et al., 1955; Wilcox et al., 1995). It has been proposed that a tyrosine hydroxylate complex forms with one copper of the cluster placing the 3 carbon of the substrate in position to accept the terminal oxygen of the peroxide, which is bound to the second copper to form DOPA. This o-diphenol is then oxidized to the quinone (DOPAquinone) by the enzyme and, upon release, regenerates the Cu1+ Cu1+ center. It is thought that tyrosinase folds in the presence of the protein chaperone calnexin, allowing trimming of sugar chains by a-glucosidase and copper binding (Branza-Nichita et al., 1999; Jimbow et al., 1994). Proper folding of tyrosinase also appears to require other proteins, such as TRP1 and the substrate tyrosine (Francis et al., 2003). Tyrosinase is then transferred from the Golgi network to the premelanosome via interaction between the dileucine motif and a tyrosine-based signal and the AP3 adaptor-like complex (Blagoveshchenskaya et al., 1999; Höning et al., 1998; Le Borgne et al., 1998; Simmen et al., 1999). The tyrosinase gene (TYR) maps to human chromosome 11q14 (Barton et al., 1988). The tyrosinase cDNA codes for a 529-amino-acid polypeptide with a predicted molecular weight of 55 kDa and a mature processed glycoprotein protein of 75 kDa. TYR contains five exons spanning 118 kb (Giebel et al., 1991b; Ponnazhagan et al., 1994). The sizes of the introns are 12.4 kb for intron 1, 36.4 kb for intron 2, 56.8 kb for intron 3, and 10.2 kb for intron 4 (Pruitt and Maglott, 2001). Over 50% of the coding region is found in exon 1 (819 bp) with exons 2 to 5 ranging from 148 to 225 bp. The tyrosinase gene has been shown to produce many alternatively spliced transcripts, but their function, if any, is unknown (Fryer et al., 2001). The tyrosinase gene is the albino (c) locus of the mouse. Mutations that completely eliminate tyrosinase enzymatic activity produce a mouse with a complete absence of melanin pigment in the coat and eye. Several mouse tyrosinase alleles have been isolated that result in a spectrum of pigment loss from a complete absence of pigment (the null mutations) to moderate amounts of pigment including a temperaturesensitive mutation (ch) (Beermann et al., 1990; Jackson, 1995b; Jackson and Bennett, 1990; Kwon et al., 1989; Orlow et al., 1993a; Porter et al., 1991; Schmidt and Beermann, 1994; Shibahara et al., 1990; Silvers, 1979; Yokoyama et al., 1990). Of all the gene products that participate in pigmentation at the subcellular level of the melanocyte, mutations at the c locus have the greatest effect on the reduction in pigmentation. Numerous mutations of the human tyrosinase gene associated with tyrosinase-related OCA have been reported (Oetting and King, 1999). The 5¢ upstream promoter region of the human TYR gene contains four transcriptional start sites with the major site –79 bp from the ATG start codon and minor sites at –78, –42, and –34 bp (Ponnazhagan et al., 1994). Other transcriptional 218

start sites have been reported and may represent differences in the cell lines used (Giebel et al., 1991b; Kikuchi et al., 1989; Takeda et al., 1989b). There are also several potential proximal cis-regulatory elements including a putative TATA box (–106 bp) and CAATT box (–199 bp). Another potential regulatory element is a 230-bp repeat sequence of (GA:TC)n located 713 bp upstream of the translational start site, which can assume a hinged-DNA structure and may play a role in regulation of tyrosinase expression (Kikuchi et al., 1989). This repeat sequence in humans has been found to be polymorphic in several different populations (Morris et al., 1991; Oetting et al., 1993b). There are also numerous AP-1, AP-2, and Oct1 binding cis-acting elements (Ponnazhagan et al., 1994). In an effort to understand the transcriptional control of tyrosinase, there have been several studies to determine the number and location of cis-acting regulatory elements and the trans-acting factors that bind to sequences necessary for both temporal and tissue-specific expression. In expression studies with the mouse tyrosinase gene, as little as 270 bp of the 5¢ promoter has produced cell-specific and temporal-specific expression in transgenic mice, although wild-type levels of pigmentation were not achieved (Beermann et al., 1992; Kluppel et al., 1991). In an effort to mimic wild-type expression, transgenic mice were produced using a 250-kb yeast artificial chromosome (YAC) containing 155 kb of the 5¢ flanking sequence (Schedl et al., 1993). In these experiments, wild-type levels and distribution of pigmentation were observed in a copy numberdependent and position-independent manner, suggesting that in vivo tyrosinase gene expression is regulated by cisregulatory elements located as far upstream as 155 kb. Individual elements involved in the regulation of the tyrosinase gene have now been identified. An element –183 bp upstream of the start codon, TATCAATTAG, was found to bind the transcription factor HMG-1 (high mobility group protein) and its isoform HMG-Y (Sato et al., 1994). HMG1(Y) is thought to be involved in nucleosome phasing, a mechanism for maintaining the undifferentiated state of chromatin. Possibly, this protein alters the structure of the promoter or facilitates the binding and activity of other trans-acting factors at the tyrosinase promoter. At present, the binding of HMG1(Y) has only been shown by in vitro studies and further work is needed. For the human TYR locus, a region between –2020 and –1739 bp from the transcriptional start site was found to contain an important enhancer (Ponnazhagan et al., 1994; Shibata et al., 1992a). The enhancer sequences were localized to a 39-bp core element that was confirmed to direct the melanoma cell-specific expression of a reporter gene under the control of this element (Shibata et al., 1992a). This region was later termed the tyrosinase distal element (TDE) (Yasumoto et al., 1994). The TDE contains one of four copies of a 6-bp motif, CATGTG, found in the 5¢ promoter of the tyrosinase gene, which is also present in the mouse tyrosinase promoter. This same 6-bp motif is found at –104 bp to –99 bp and has been labeled as the “M”-box or the tyrosinase proximal element (TPE) (Lowings et al., 1992; Yasumoto et al., 1994).

THE TYROSINASE GENE FAMILY

The M-box element has also been found in the 5¢ promoter region in the mouse and human TRP1 and TRP2 genes and is considered to be an important cis-regulatory element for melanocyte-specific expression (Hemesath et al., 1994; Jackson et al., 1991; Lowings et al., 1992; Shibahara et al., 1991; Sturm et al., 1995). The third location of this element at –12 bp to –7 bp has been termed the “E”-box (Bentley et al., 1994). Expression studies have shown that the TDE is the strongest of these three elements, with the M-box element being much weaker (Yasumoto et al., 1994). Other elements, both positive and negative, have been reported for the tyrosinase gene (Bentley et al., 1994). The CANNTG motif binds a family of transcription factors, characterized by the presence of a conserved helix–loop–helix structure required for DNA binding and activation (Hodgkinson et al., 1993). It is now established that the protein product of the human homolog of the mouse microphthalmia (mi) gene, a melanocyte-specific factor called the microphthalmiaassociated transcription factor (MITF), binds to both the TDE and the M-box regions (Hemesath et al., 1994; Yasumoto et al., 1994). The murine mi gene product has been shown to increase tyrosinase and TRP1 transcription as well as reporter genes with the M-box in the promoter. The human MITF gene was found to direct transcription of both the mouse tyrosinase and TRP1 genes preferentially in melanocytic cells (Yasumoto et al., 1995). Yasumoto et al. (1995) showed that an 82-bp cis-acting region, which contains the CATGTG motif (position –12 to –7) along with the M-box (positions –107 to –97), was involved in transactivation of the tyrosinase gene in vitro, and concluded that the trans-acting factor MITF is a common factor regulating transcription of the pigment cell-specific genes. A second factor, termed a “ubiquitous transcription factor,” was also found to bind the M-box, but its role in activating melanocyte-specific transcription is unknown (Lowings et al., 1992). Other transcription factors, N-Oct-3 and NOct-5, are expressed at high levels in melanoma cells and may be involved in mechanisms controlling the transcription of pigment-specific genes (Sturm et al., 1994a). Another cis-acting regulatory region has been found –15 kb from the initiation codon (Porter et al., 1991; Porter and Meyer, 1994). In melanoma cells, but not in other nonpigmenting cell types, this region was shown to contain two DNase I hypersensitive sites (HS-site) within a 200-bp region (Ganss et al., 1994). These two DNase I hypersensitive sites were found to be rearranged in the chinchilla-mottled (cm) mouse, resulting in hypopigmentation, showing the importance of these sites for the proper transcription of tyrosinase (Porter et al., 1991). The first site, HS-site (hs1), contained the palindrome TGACTTTGTCA, which resembles the binding motif to both a CREB (cAMP-responsive element binding) transcription factor and the AP-1 (activator protein-1) binding site (Ganss et al., 1994). The inclusion of this region into transgenic mice enhanced tyrosinase expression (Porter and Meyer, 1994). Interestingly, tyrosinase expression was enhanced in neural crest-derived melanocytes but not in neuroectoderm-derived melanocytes, indicating differential

regulation of tyrosinase expression in these two cell lineages (Porter and Meyer, 1994). A similar region has been identified in humans –9 kb upstream of the transcriptional start site (Fryer et al., 2003; Regales et al., 2003). The homologous human sequence was initially found using DNA sequence comparisons with the upstream region of the mouse tyrosinase gene. DNase analysis showed that these sequences exhibited DNase hypersensitivity. This region was cloned and placed upstream of the proximal tyrosinase promoter and a luciferase reporter gene. The core sequences of this region were shown to specifically increase reporter expression in melanocytes in conjunction with the tyrosinase proximal promoter (Fryer et al., 2003; Regales et al., 2003). Preliminary evidence has shown that this region may also contain a negative regulatory region. The trans-acting factors that interact with these sequences still need to be determined. These reports show that the regulation of tyrosinase gene expression is complex and requires the coordinated participation of several negative and positive regulatory elements and factors. As shown above, these regulatory elements are both proximal (e.g. the E-box; –6 bp) and distal (e.g. hs-site; 12–15 kb). Melanocyte-specific and temporal regulation of tyrosinase gene expression is thus achieved by a combination of trans-acting factors that bind to these regulatory elements.

TRP1 Gene/DHICA Oxidase The tyrosinase-related protein-1 (TRP1), the second member of the tyrosinase gene family, remains an enigmatic protein. Accordingly, there have been several different enzymatic functions attributed to DHICA oxidase, including tyrosine hydroxylation (an enzymatic activity of tyrosinase) (Jiménez et al., 1991; Tsukamoto et al., 1992b), DOPAchrome tautomerase (Winder et al., 1994b), and catalase activity (Halaban and Moellmann, 1990). The last activity was used to ascribe the locus name of CAS2 to this gene. Current information shows that the purified protein has DHICA oxidase activity and places it distal to tyrosinase and DOPAchrome tautomerase in the melanin biosynthetic pathway (Jiménez-Cervantes et al., 1994). Interestingly, despite the high degree of homology with the mouse homolog, human TRP-1 protein does not appear to have DHICA oxidase activity (Boissy et al., 1998). The gene (tyrosinase-related protein-1; TRP1) maps to human chromosome 9p23 (Abbott et al., 1991; Chintamaneni et al., 1991b; Murty et al., 1992). The TRP1 gene consists of eight exons within a 25-kb region, with exons 2 through 8 containing the coding sequences (Jackson et al., 1991; Shibahara et al., 1991; Sturm et al., 1995). The TRP1 gene encodes a polypeptide of 537 amino acids with predicted molecular weight of 60 kDa (Cohen et al., 1990; Urquhart, 1993). The mature glycosylated protein has, like tyrosinase, an apparent molecular weight of 75 kDa. In the mouse genome, there is an additional DNA segment that cross-hybridizes to TRP1 probes (Jackson, 1988; Shibahara et al., 1991). This segment contains sequences homologous only to exon 8 of TRP1 (Shibahara et al., 1991). The sequences between the crosshybridizing segment and exon 8 or TRP-1 are 95% identical, 219

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but the segment contains a single base deletion, resulting in a premature stop codon, and is most likely a nonfunctional pseudogene. This is most likely the Tyrp1-rs1 locus. At present, there are no reports of a TRP1 pseudogene in any other species. The brown (b) locus of the mouse encodes the murine TRP1 gene. Mutations at this locus result in the production of brown eumelanin instead of black eumelanin. There are several known alleles of the b locus (Jackson, 1995b; Jackson et al., 1990; Johnson and Jackson, 1992; Silvers, 1979; Zdarsky et al., 1990) as well as several artificially generated deletions that include the b locus (Bell et al., 1995). The recessive mutations result in loss of function in the homozygous state, varying the degree of brown melanin pigment produced in an otherwise normal melanocyte. Two mutations, Light (Blt) and White-based brown (Bw) are dominant mutations and result in melanocyte death (Jackson, 1995b; Johnson and Jackson, 1992). The Light mutation produces a protein with aberrant function that is thought to disrupt the melanosome, allowing toxic intermediates to enter the cytoplasm of the cell, causing cell death. The White-based brown mutation has a chromosomal inversion that places the TRP1 promoter in another chromosomal region. This results in the inactivation of the TRP1 gene and most probably the activation of another protein that is responsible for the observed cell death. Mutations of the human TRP1 gene have been shown to be responsible for brown OCA (OCA3) (Boissy et al., 1996, Manga et al., 1997). Sequences in the first exon of the TRP1 gene appear to be important in determining the efficiency of its expression, but are not sufficient to confer pigment cell-specific expression (Shibata et al., 1992b). The first intron was shown to be necessary for TRP1 transcription in transient transfections. Removal of the first intron, even in the presence of 3 kb of the upstream TRP1 promoter region, reduced expression to background levels (Sturm et al., 1994a). Other reports have shown that the first intron is not necessary for TRP1 expression in cultured cells (Jackson et al., 1991; Lowings et al., 1992). The transcriptional initiation site is 20 bp downstream of the TATAAA box (Sturm et al., 1994a). The minimum promoter in the mouse gene is between –44 bp and +107 bp from the start codon. Within this minimal promoter, both positive and negative regulatory elements have been identified (Lowings et al., 1992; Yamamoto et al., 1992). The M-box regulatory element is one of these elements, which is also found in the tyrosinase gene. The 5¢ upstream region of TRP1 was found to bind to regulatory factors of 50 kDa and 43 kDa (Yamamoto et al., 1992). One of these factors binds to the Mbox element and acts as a positive regulatory factor.

TRP2 Gene/DOPAchrome Tautomerase The tyrosinase-related protein-2 (TRP2) is the third member of the tyrosinase gene family and codes for an enzyme with DOPAchrome tautomerase (DT) activity. TRP-2 maps to human chromosome 13q32 (Sturm et al., 1994b; Bouchard et al., 1994). This gene was initially isolated from an expressed 220

mouse melanoma cDNA library using rabbit antimouse tyrosinase antibody and rabbit antihamster tyrosinase antibody and designated as clone 5A (Jackson, 1988; Jackson et al., 1992). This clone was found to map to the slaty (slt) locus of the mouse and shown to code for DT using an expression vector containing the DT cDNA and expression in COS cells (Kroumpouzos et al., 1994; Tsukamoto et al., 1992a; Yokoyama et al., 1994). The human cDNA for DT was also isolated and sequenced (Bouchard et al., 1994; Cassady and Sturm, 1994; Yokoyama et al., 1994). The coding sequences of the mouse and human TRP2 genes are divided into eight exons spanning a 55-kb region in humans with the translational start site in exon 1 (Budd and Jackson, 1995; Sturm et al., 1995). The increased size of the gene, compared with TRP1, is due to an increased number of repetitive elements. In the mouse, several alleles of the slaty locus have been identified (Budd and Jackson, 1995; Green, 1972; Jackson, 1995b; Jackson et al., 1992). Mutations at this locus result in a dilution of the coat color and slightly yellowish ears (Green, 1972). One mutation, slaty light, acts as a semi-dominant mutation. None of these alleles is thought to be a null allele. To determine the phenotype of a known DT null mouse, a TRP-2 knockout mouse was produced (Guyonneau et al. 2004). The knockout mice were found to be viable and exhibited a diluted coat color phenotype with no detectable abnormalities beyond hypopigmentation in DT-expressing sites such as skin, retinal epithelium, or brain. The phenotype was identical to the slaty mutation. Moreover, unlike slaty mutant melanocytes, DT knockout primary melanocytes were much easier to grow in culture (Guyonneau et al. 2004). At present, no mutations in the human TRP2 gene have been reported. The human DT amino acid sequence has 83% identity and 90% similarity to the mouse sequence (Cassady and Sturm, 1994). Northern blot analysis has shown DT to be expressed as a 4.5-kb and 2.3-kb transcript, whereas the mouse only produces a 2.3-kb transcript (Bouchard et al., 1994; Yokoyama et al., 1994). HeLa cells, which have no endogenous DT activity, exhibited DT activity when transfected with the human DT cDNA, similar to COS cells transfected with the mouse DT cDNA, showing that the TRP2 gene codes for dopachrome tautomerase. Analysis of several different human melanoma cell lines revealed different levels of expression, ranging from no expression through low to moderate to high levels of expression (Bouchard et al., 1994; Cassady and Sturm, 1994). Normal human melanocytes were found to express DT at moderate levels but, in some cell lines, levels of enzymatic activity did not correspond to transcript levels, suggesting that there may be a post-transcriptional regulatory mechanism. There was no direct correlation between the expression of TRP2 and the presence of visible melanin and/or activity of tyrosinase (Bouchard et al., 1994). Like tyrosinase, TRP2 contains the M-box element that binds the MITF transcription factor. It has been shown that SOX10 also binds promoter elements in TRP2 (Jiao et al., 2004). Sox10 binding was demonstrated using gel shifts of

THE TYROSINASE GENE FAMILY

oligonucleotide probes derived from promoter sequences within the region required for Sox10-dependent induction. It was shown that Sox10 acts directly upon the Dct promoter to activate gene expression (Jiao, et al., 2004). It was further hypothesized that Sox10 and Mitf synergistically activate TRP2 expression.

Transcriptional Regulation It has been known for many years that there is a direct response of the pigment system in melanocytes to specific factors such as melanocyte-stimulating hormone (MSH) and UV irradiation. For this general increase in pigment production, it has been assumed that there is an increase in the expression of all pigment genes involved in melanin biosynthesis and premelanosomal formation. Several common regulatory elements for tyrosinase, TRP1, and TRP2 have been identified, and these may be involved in a coordinated regulation of these genes (Shibahara et al., 1991; Sturm et al., 1995; Yamamoto et al., 1992). One regulatory element found to be shared by all three members of the tyrosinase gene family is the M-box (Jackson, 1994). This element is conserved between mouse and human upstream regions in all three members of the tyrosinase gene family, as well as in regulatory elements much more distal (Shibata et al., 1992a; Sturm et al., 1995). One binding factor, termed the M-box binding factor, binds to the M-box element and acts as a positive regulatory factor (Lowings et al., 1992). This is most likely the microphthalmia-associated transcription factor (MITF; Tassabehji et al., 1994). Mutations of MITF result in microphthalmia in the mouse and Waardenburg syndrome type II in humans, which includes alteration in pigmentation due to defects in melanocyte migration. It should be noted, however, that transcriptional regulation of tyrosinase, TRP1, and TRP2 is not always as coordinated as one might expect. Molecular analysis has revealed a high degree of complexity in the transcriptional regulation of these genes. Using mouse B16-F1 cells, it was found that transcription of tyrosinase and TRP1 is coordinately regulated by melanotropic reagents via cAMP-dependent protein kinase and protein kinase C (Kuzumaki et al., 1993). Melanotropic reagents that increased tyrosinase transcription (i.e. cholera toxin, a-MSH, and dibutyryl cAMP) also increased TRP1 transcription. Reagents that decreased eumelanin synthesis, such as 12-O-tetradecaylphorbol 13-acetate (TPA), decreased mRNA levels of both tyrosinase and TRP1. This increase in tyrosinase and TRP1 mRNA resulted in a parallel increase in eumelanin production. Another study showed that this connection between TRP1 protein levels and pigment production occurs only in cells that are producing eumelanin and not in pheomelanin-synthesizing cells (del Marmol et al., 1993). In cells that are producing only pheomelanin, TRP1 mRNA is absent, whereas tyrosinase mRNA is present in all cells producing pigment regardless of the type of melanin being produced. This is not unexpected because TRP1 is thought to be involved only in the eumelanin biosynthetic pathway and not in the pheomelanin pathway. This is also shown by the

fact that murine TRP1 (brown locus) mutations do not affect the coat color of agouti lethal yellow (Ay) or extension (e) mice, which produce only pheomelanin (del Marmol et al., 1993), revealing that there must be different cis- and transacting transcriptional regulators for tyrosinase and TRP1. In other reports, the addition of 2% dimethyl sulfoxide (DMSO) or 5 mM hexamethylenebisacetamide (HMBA) on melanoma cell lines inhibited the expression of TRP1 but had no effect on tyrosinase expression (Sturm et al., 1994a). Vijayasaradhi et al. (1995a, b) found that HMBA reduced the expression of TRP1 to 88% of control but, for several cell lines, tyrosinase expression increased. Sturm et al. (1994a) also found that the addition of differentiating chemicals to melanoma cell lines exhibited opposing effects on tyrosinase and TRP1 expression (Martínez-Liarte et al., 1992). Selective regulation of TRP1 appears to be mediated by protein factors that modulate MITF-mediated transcription (Fang et al., 2002). TRP2 (DOPAchrome tautomerase) also exhibits expression patterns that differ from those of tyrosinase. Addition of a-MSH or other melanogenic activators has opposite effects on tyrosinase and DOPAchrome tautomerase activities (Martínez-Liarte et al., 1992; Sturm et al., 1994a). The addition of a-MSH on B16/F10 melanoma cells caused an increase in tyrosinase activity but a decrease in DOPAchrome tautomerase activity. In another study, Bouchard et al. (1994) found no direct correlation between the expression of TRP2 and pigment production in 10 different human melanoma cell lines. In some cell lines, TRP2 transcripts correlate with DT activity, but there were variations in others. Furthermore, there was no direct relationship between the presence of eumelanin and DOPAchrome tautomerase activity. Moreover, no consistent direct relationship between levels of tyrosinase activity and the amount of visible pigment have been found, suggesting that post-transcriptional and/or -translational regulatory mechanisms play an important role in the control of pigment production (Bouchard et al., 1989, 1994). One possible element interacting at the post-translational level is an inhibitor of these enzymes (Kameyama et al., 1993). One inhibitor isolated from the human amelanotic melanoma cell line SK-MEL-24 was found to suppress the oxidation of DHI in the presence or absence of tyrosinase, but had no effect on DHICA oxidation (Kameyama et al., 1995). Many of these reports analyzing the expression of tyrosinase, TRP1, and TRP2 show conflicting results. This is in part due to laboratories using different cell lines that produce different amounts and types of melanin pigment (eumelanin, pheomelanin, or both) (Bouchard et al., 1994; del Marmol et al., 1993; Taniguchi et al., 1992). It is important that the amount and type of melanin being produced be taken into account when the expression and activity levels of these genes are being determined. Although melanoma cells are convenient model systems to study pigmentation, it must be borne in mind that malignant transformation itself may disrupt the fine balance of the activities of pigmentation machinery. Primary cultures of melanocytes may represent a system closer 221

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to the physiological system, although primary cell lines may still not be an accurate representation of what is happening in the melanocyte in situ. There are also differences in gene expression during embryonic development. Steel et al. (1992) found that DT is expressed very early in developing mouse embryos, 10 days post coitum, whereas TRP1 is expressed at 11.5 days post coitum and tyrosinase transcripts are observed 13.5 days post conception.

Protein Interactions Tyrosinase, TRP-1, and TRP-2, along with the lysosome-associated membrane protein-1 (LAMP-1), have been shown to interact as a high-molecular-weight multimeric enzymatic complex that ranges in size from 200 to >700 kDa (Hearing et al., 1982; Orlow et al., 1993b, 1994; Zhou et al., 1993). This complex is responsible for the conversion of tyrosine to eumelanin but does not appear to be involved in pheomelanin biosynthesis. Copurification of these proteins with antityrosinase antibodies suggests a stable interaction between these proteins in which the EGF motif may be important in the formation and stabilization of this complex (Jackson et al., 1992). Recent studies have shown that tyrosinase activity is stabilized in the presence of TRP-1 and TRP-2, providing further evidence of a functional melanogenic complex (Pawelek, 1991; Winder et al., 1994a). Cotransfection of fibroblast with both tyrosinase and TRP-1 showed an increase in both the stability and the activity of tyrosinase with time. This complex can interact with other factors as well, including melanogenic inhibitors (Kameyama et al., 1989) and products of other pigment loci that determine the quantity and quality of melanin pigment synthesized, including the silver (si), pink-eye (p), and MART-1 proteins (Kameyama et al., 1993). Analysis of mice with mutations at different pigment loci can provide information on the effects of protein interaction in vivo. By assaying melanin production in mouse skin, it was shown that mutations at the pink-eye loci will reduce tyrosinase activity (Coleman, 1962) as well as reduce the levels of tyrosinase transcript in mouse melanocytes compared with normal controls (Tamate et al., 1989). More recently, it has been shown that the pink-eye unstable (Pun) mutation, a mutation that contains an integral duplication inside the P gene resulting in altered P-gene transcription, actually disrupts the tyrosinase enzyme complex (Chiu et al., 1993). In the ocular melanocytes of mice that contained the Pun mutation, the highmolecular-weight enzyme complex could not be detected, and protein levels of tyrosinase, TRP1, and TRP2 were drastically reduced. Another example of the disruptive effect of a mutation in one of the members of this complex is found in the platinum (cp) mouse (Orlow et al., 1993a). This mutation of the mouse tyrosinase gene results in a truncated protein and a coat almost completely devoid of pigment. Enzymatic activity of platinum tyrosinase is higher than that of other tyrosinase mutations that result in a darker coat color, but the tyrosinase enzymatic complex cannot be detected, suggesting 222

that the absence of the complex causes a more severe form of albinism than would be expected by the tyrosinase gene mutation alone. More importantly, alteration of one of these protein products by means of mutations can have a drastic effect on other proteins involved in this complex. It is possible that mutations that result in hypopigmentation (or hyperpigmentation) may not act by specifically altering the function of the protein (i.e. reduced catalytic activity), but by disruption of the protein complex, producing a reduction in pigment biosynthesis. In addition to the potential interactions among family members that affect pigmentation, individual tyrosinase family proteins, specifically their cytoplasmic domains, also interact with components of the intracellular protein sorting machinery (reviewed by Setaluri, 2000). Although all tyrosinase family proteins follow a similar biosynthetic route to melanosomes and are expected to share interactions with the same components of trafficking machinery, it is of interest to note that differences exist in their binding specificities. For example, whereas interaction of tyrosinase with the adaptor protein AP-3 is required for its trafficking, interaction with AP-3 is not necessary for TRP-1 trafficking (Huizing et al., 2001). Similarly, TRP-1, but not tyrosinase, interacts specifically with a multifunctional PDZ-domain protein, GIPC, during its transport through the Golgi (Liu et al., 2001).

Human Pigmentation Disorders Oculocutaneous albinism (OCA) is the most common human genetic disease associated with mutations of pigment genes that act at the subcellular level of the melanocyte. OCA is a clinically heterogeneous genetic disease. Mutations in numerous genes including tyrosinase (OCA1), P (OCA2), TRP-1 (OCA3), membrane-associated transporter protein (MATP; OCA4), Chediak–Higashi syndrome (CHS), and Hermansky–Pudlak syndrome (HPS1–7) are thought to be responsible for different forms of OCA in humans. Although, in certain strains of mice, mutations in TRP-1 have also been found to be responsible for a form of pigmentary glaucoma involving iris stromal dystrophy, no TRP-1 mutations were found to be associated with this disease in humans (Anderson et al., 2002; Lynch et al., 2002). Based on the observation that administration of L-DOPA to cytochrome P450 and TYR null mice alleviates developmental ocular abnormalities found in this model for human primary congenital glaucoma (PCG), it is proposed that the tyrosinase/L-DOPA pathway modifies human PCG (Libby et al., 2003). Because of the effect of ocular albinism gene 1 (OA1) on cutaneous melanosome development, this locus may also be considered to be associated with a type of OCA. Besides their role in melanin pigmentation and pigmentary disorders, tyrosinase family proteins are also involved in the biology of melanoma. For example, levels of expression of TRP-2 in melanoma cells appear to correlate positively with their resistance to certain cancer chemotherapeutic drugs (Chu et al., 2000). The exact mechanism by which TRP-2 confers drug resistance is not known. Paradoxically, elevated

THE TYROSINASE GENE FAMILY

expression of TRP-2, and also tyrosinase, in advanced-stage metastatic melanoma lesions appears to correlate with improved overall survival of patients (Takeuchi et al., 2003). One possible reason for this may be the fact that, in patients with metastatic melanoma, all three proteins are recognized by the immune system (Brichard et al., 1993; Vijayasaradhi et al., 1990; Wang et al., 1995, 1996). Accordingly, a number of strategies are being evaluated, in clinical trials, to elicit immune response to these antigens for immunotherapy of melanoma (Rosenberg, 2001). Much of our understanding about human albinism has come from the work done on mutations of the tyrosinase gene and their relationship to tyrosinase-related OCA (OCA1). This relationship has been shown by both formal linkage of the tyrosinase gene to OCA1 and the finding of mutations in the tyrosinase gene that eliminate tyrosinase enzymatic activity (Giebel et al., 1990). Using a combination of PCR amplification of individual exons and single-strand conformational polymorphism (PCR-SSCP) analysis, a number of mutations have been found (Oetting et al., 1994; Oetting and King, 1999). Mutations that produce a tyrosinase protein completely devoid of enzymatic activity are associated with the severest form of albinism, OCA1A. The missense mutations, on the other hand, result in a spectrum of phenotypes ranging from a complete absence of pigment production to near normal amounts of pigment. These latter mutations have residual amounts of enzymatic activity that allow a small amount of pigment to be produced. The missense mutations of tyrosinase have provided a tool to help us understand the biology of the enzyme and pigment production. At present, the three-dimensional structure of tyrosinase family proteins is unknown, and the effects of different mutations cannot be analyzed at the level of protein structure. However, analysis of mutations and their effect on enzymatic activity have provided some insight into the structure of tyrosinase. Mapping of the missense mutations associated with OCA1 reveals several clusters within the tyrosinase polypeptide. This clustering is thought to define functional domains of the enzyme (King et al., 1991; Oetting and King, 1992a, b; Tripathi et al., 1992b). Two of these domains are within the putative copper(A) and copper(B) binding sites respectively. A third cluster is at the 5¢ end (amino-terminal end of the polypeptide) of the coding region. This latter domain may define a putative substrate and/or cofactor binding site, or may be involved in enzyme stability. Missense mutations within the signal peptide of tyrosinase have allowed us to analyze the importance of this region in the enzymatic activity (Breimer et al., 1994; Fukai et al., 1995). Tyrosinase molecules that lack the signal peptide were found to be translated on free ribosomes instead of membranebound ribosomes. This resulted in a peptide that is incorrectly folded and quickly eliminated by the cell (Oetting et al., 1995). Frameshift mutations in the carboxy-terminal end of the protein also result in an enzyme without enzymatic activity. This region contains the transmembrane region and the cytoplasmic tail, which is important for proper sorting and

melanosomal targeting of these proteins (Vijayasaradhi et al., 1995a). The copper binding site of hemocyanin has many similarities to that of tyrosinase (Beltramini et al., 1990). Determining how different mutations affect tyrosinase activity can be aided using the structure of the copper-containing protein hemocyanin in which the three-dimensional structure has been determined (Gaykema et al., 1984, 1986). Based on amino acid sequence alignment between the copper-containing protein hemocyanin and tyrosinase, and extensive spectroscopic studies of tyrosinase, it is evident that the ligation of the copper atoms involves histidine side-chains from the protein. Site-directed mutagenesis of Streptomyces glaucescens tyrosinase has shown that the predicted histidine ligands are indeed involved in copper binding (Lerch, 1988). The presence and placement of these histidine ligands is likely to be critical for the binding of the copper atoms, which is necessary for both the formation of the reactive peroxide species and the complex with the substrate. Thus, changes in the amino acids that form the copper ligands or that change their position in the protein structure are likely to alter the rate and/or specificity of tyrosine hydroxylation. By using hemocyanin as a model for the copper binding sites of tyrosinase, we can begin to determine possible mechanisms of how some of these tyrosinase mutations cause inactivation of or a reduction in enzymatic activity (Oetting and King, 1994). The predicted secondary structure at both the copper binding sites of tyrosinase indicates that the histidine ligands responsible for copper binding are within two a-helices connected by a loop region (Oetting and King, 1992b). This same type of structure exists in the copper binding sites of hemocyanin (Gaykema et al., 1984, 1986). The amino acid sequences of the copper(B) binding region for hemocyanin and tyrosinase are very similar, but the copper(A) binding regions for both proteins are not, and the locations of the copper(A) tyrosinase mutations should be considered less precise. Two tyrosinase mutations in the copper(A) binding site of human tyrosinase are within conserved amino acid motifs found in most tyrosinases and hemocyanin copper binding sites (Müller et al., 1988). The mutation A206T is found in the motif Pro-X-Phe-X-X-X-His, between the proline and the phenylalanine, causing a change from an alanine to a threonine, and the mutation F176I is within the motif Phe-X-X-XHis, changing the highly conserved phenylalanine to an isoleucine. Both these motifs are thought to be involved in the correct placement of the histidine ligands responsible for copper binding. The other mutations are found to be either within the a-helix or at the junction of the a-helix and the connecting loop structure. Our current hypothesis is that these mutations disrupt correct copper atom orientation by displacing the a-helices relative to each other (Oetting and King, 1992b). Spritz et al. (1997) reported that some missense mutations disrupted copper binding, resulting in an inactive protein. There were two missense mutations, P406L and R402Q, that did exhibit copper binding but still had no detectable enzymatic activity. The action of these mutations 223

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may either prevent one of the copper atoms from binding or displace the copper atoms by altering the copper to copper distance. Either case would prevent the formation of oxytyrosinase and render the enzyme inactive. The limited number of mutations identified to date in TRP-1 and TRP-2 preclude a similar prediction of structure–function relationship for these proteins. There is also evidence that tyrosinase mutations result in retention of the enzyme in the endoplasmic reticulum and subsequent degradation, leading to speculation that albinism is, in part, an ER retention disease (Halaban et al., 1997, 2000; Toyofuku et al., 2001a, b).

Perspectives The tyrosinase gene family provides an excellent model for understanding a number of biological phenomena. These include gene evolution, the biology of a complex biochemical pathway, protein sorting, and protein–protein interaction. Two of these genes, TYR and TRP1, are involved in albinism. Analysis of tyrosinase mutations associated with oculocutaneous albinism type I has provided insight into the spectrum of phenotypes associated with a single gene disorder.

Acknowledgments V. S. acknowledges the support of NIH grant R01 AR048913.

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CHAPTER 11 Shibahara, S., Y. Tomita, T. Sakakura, C. Nager, B. Chaudhuri, and R. Muller. Cloning and expression of cDNA encoding mouse tyrosinase. Nucleic Acids Res. 14:2413–2427, 1986. Shibahara, S., S. Okinaga, Y. Tomita, A. Takeda, H. Yamamoto, M. Sato, and T. Takeuchi. A point mutation in the tyrosinase gene of BALB/c albino mouse causing the cysteine to serine substitution at position 85. Eur. J. Biochem. 189:455–461, 1990. Shibahara, S., H. Taguchi, R. M. Muller, K. Shibata, T. Cohen, Y. Tomita, and H. Tagami. Structural organization of the pigment cellspecific gene located at the brown locus in mouse. Its promoter activity and alternatively spliced transcript. J. Biol. Chem. 266: 15895–15901, 1991. Shibata, K., Y. Muraosa, Y. Tomita, H. Tagami, and S. Shibahara. Identification of a cis-acting element that enhances the pigment cellspecific expression of the human tyrosinase gene. J. Biol. Chem. 267:20584–20588, 1992a. Shibata, K., K. Takeda, Y. Tomita, H. Tagami, and S. Shibahara. Downstream region of the human tyrosinase-related protein gene enhances its promoter activity. Biochem. Biophys. Res. Commun. 184:568–575, 1992b. Silvers, W. K. The Coat Colors of Mice. A Model for Mammalian Gene Action and Interaction. New York: Springer-Verlag, 1979. Simmen, T., A. Schmidt, W. Hunzilker, and F. Beermann. The tyrosinase tail mediates sorting to the lysosomal compartment in MDCK cells via a di-leucine and a tyrosine-based signal. J. Cell Sci. 112:45–53, 1999. Solano, F., J. H. Martinez-Liarte, C. Jimenez-Cervantes, J. C. GarciaBorron, and J. A. Lozano. Dopachrome tautomerase is a zinccontaining enzyme. Biochem. Biophys. Res. Commun. 204:1243– 1250, 1994. Spritz, R. A., L. Ho, M. Furumura, and V. J. Hearing. Mutational analysis of copper binding by human tyrosinase. J. Invest. Dermatol. 109:207–212, 1997. Steel, K. P., D. R. Davidson, and I. J. Jackson. TRP2/DT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor. Development 115:1111–1119, 1992. Sturm, R. A., B. J. O’Sullivan, J. A. F. Thomson, N. Jamshidi, J. Pedley, and P. Parsons. Expression studies of pigmentation and POUdomain genes in human melanoma cells. Pigment Cell Res. 7:235–240, 1994a. Sturm, R. A., E. Baker, and G. R. Sutherland. Assignment of the tyrosinase-related protein-2 gene (TYRP2) to human chromosome 13q31–q32 by fluorescence in situ hybridization: extended synteny with mouse chromosome 14. Genomics 21:293–296, 1994b. Sturm, R. A., B. J. O’Sullivan, N. F. Box, A. G. Smith, S. E. Smit, E. R. J. Puttick, P. G. Parsons, and I. S. Dunn. Chromosomal structure of the human TYRP1 and TYRP2 loci and comparison of the tyrosinase-related protein gene family. Genomics 29:24–34, 1995. Takeda, A., J. Matsunaga, Y. Tomita, H. Tagami, and S. Shibahara. Molecular analysis of the DNA segments cross-hybridizable to the tyrosinase gene in patients afflicted with oculocutaneous albinism. Tohoku J. Exp. Med. 159:333–340, 1989a. Takeda, A., Y. Tomita, S. Okinaga, H. Tagami, and S. Shibahara. Functional analysis of the cDNA encoding human tyrosinase precursor. Biochem. Biophys. Res. Commun. 162:984–990, 1989b. Takeda, A., J. Matsunaga, Y. Tomita, H. Tagami, and S. Shibahara. Nucleotide sequence of the putative human tyrosinase pseudogene. Tohoku J. Exp. Med. 163:295–297, 1991. Takeuchi, H., C. Kuo, D. L. Morton, H. J. Wang, and D. S. Hoon. Expression of differentiation melanoma-associated antigen genes is associated with favorable disease outcome in advanced-stage melanomas. Cancer Res. 63:441–448, 2003. Tamate, H. B., T. Hirobe, K. Wakamatsu, S. Ito, S. Shibahara, and K. Ishikawa. Levels of tyrosinase and its mRNA in coat-color mutants of C57BL/10J congenic mice: effects of genic substitution at the agouti, brown, albino, dilute and pink-eyed dilution loci. J. Exp. Zool. 250:304–311, 1989.

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Taniguchi, M., T. Iwamoto, I. Nakashima, A. Nakayama, O. Masaharu, M. Matsuyama, and M. Takahashi. Establishment and characterization of a malignant melanocytic tumor cell line expressing the ret oncogene. Oncogene 7:1491–1496, 1992. Tassabehji, M., V. E. Newton, and A. P. Read. Waardenburg syndrome type 2 caused by mutations in the human microphthalmia (MITF) gene. Nature Genet. 8:251–255, 1994. Tomita, Y., A. Takeda, S. Okinaga, H. Tagami, and S. Shibahara. Human oculocutaneous albinism caused by a single base insertion in the tyrosinase gene. Biochem. Biophys. Res. Commun. 164:990– 996, 1989. Toyofuku, K., I. Wada, J. C. Valencia, T. Kushimoto, V. J. Ferrans, and V. J. Hearing. Oculocutaneous albinism type 1 and 3 are ER retention diseases: mutation of tyrosinase or Tyrp1 can affect the processing of both mutant and wildtype proteins. FASEB J. 15:2149–2161, 2001a. Toyofuku, K., I. Wada, R. A. Spritz, and V. J. Hearing. The molecular basis of oculocutaneous albinism type 1 (OCA1): sorting failure and degradation of mutant tyrosinases results in a lack of pigmentation. Biochem. J. 355:259–269, 2001b. Tripathi, R. K., V. J. Hearing, K. Urabe, P. Aroca, and R. A. Spritz. Mutational mapping of the catalytic activities of human tyrosinase. J. Biol. Chem. 267:23707–23712, 1992a. Tripathi, R. K., K. M. Strunk, L. B. Giebel, R. G. Weleber, and R. A. Spritz. Tyrosinase gene mutations in Type I (tyrosinase-deficient) oculocutaneous albinism define two clusters of missense substitutions. Am. J. Med. Genet. 43:865–871, 1992b. Tsukamoto, K., I. J. Jackson, K. Urabe, P. M. Montague, and V. J. Hearing. A second tyrosinase-related protein, TRP2, is a melanogenic enzyme termed DOPAchrome tautomerase. EMBO J. 11:519–526, 1992a. Tsukamoto, K., M. Jiménez, and V. J. Hearing. The nature of tyrosinase isozymes. In: The Pigment Cell: From the Molecular to the Clinical Level, Y. Mishima (ed.). Copenhagen: Munksgaard Press, 1992b, pp. 84–89. Urquhart, A. Human tyrosinase-like protein (TYRL) carboxy terminus: closer homology with the mouse protein than previously reported. Nucleic Acids Res. 19:5803, 1993. Vijayasaradhi,S., B. Bouchard, and A. N. Houghton. The melanoma antigen gp75 is the human homologue of the mouse b (brown) locus gene product. J. Exp. Med. 171:1375–1380, 1990. Vijayasaradhi, S., Y. X. Brigitte, B. Bouchard, and A. N. Houghton. Intracellular sorting and targeting of melanosomal membrane proteins: Identification of signals for sorting of the human brown locus protein, GP75. J. Cell Biol. 130:807–820,1995a. Vijayasaradhi, S., P. M. Doskoch, J. Wolchok, and A. N. Houghton. Melanocyte differentiation marker gp75, the brown locus protein, can be regulated independently of tyrosinase and pigmentation. J. Invest. Dermatol. 105:113–119, 1995b. Wang, R. F., P. F. Robbins, Y. Kawakami, X. Q. Kang, and S. A. Rosenberg. Identification of a gene encoding a melanoma tumor antigen recognized by HLA-A31-restricted tumor-infiltrating lymphocytes. J. Exp. Med. 181:799–804, 1995. Wang, R. F., E. Appella, Y. Kawakami, X. Kang, and S. A. Rosenberg. Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes. J. Exp. Med. 184:2207–2216, 1996. Wilcox, D. E., A. G. Porras, Y. T. Hwang, K. Lerch, M. E. Winkler, and E. I. Solomon. Substrate analogue binding to the coupled binuclear copper active site in tyrosinase. J. Am. Chem. Soc. 107:4015–4027, 1995. Winder, A. J., T. Kobayashi, K. Tsukamoto, K. Urabe, P. Aroca, K. Kameyama, and V. J. Hearing. The tyrosinase gene family: Interactions of melanogenic proteins to regulate melanogenesis. Cell. Mol. Biol. Res. 40:613–626, 1994a. Winder, A. J., A. Wittbjer, G. Odh, E. Rosengren, and H. Rorsman. The mouse brown (b) locus protein functions as a dopachrome tautomerase. Pigment Cell Res. 7:305–310, 1994b.

THE TYROSINASE GENE FAMILY Wittbjer, A., B. Dahlback, G. Odh, A. M. Rosengren, E. Rosengren, and H. Rorsman. Isolation of human tyrosinase from cultured melanoma cells. Acta Derm. Venereol. Suppl. 69:125–131, 1989. Yamamoto, H., S. Takeuchi, T. Kudo, K. Makino, A. Nakata, T. Shinoda, and T. Takeuchi. Cloning and sequencing of mouse tyrosinase cDNA. Jpn. J. Genet. 62:271–274, 1987. Yamamoto, H., T. Kudo, N. Masuko, H. Miura, S. Sato, M. Tanaka, S. Tanaka, S. Takeuchi, S. Shibahara, and T. Takeuchi. Phylogeny of regulatory regions of vertebrate tyrosinase genes. Pigment Cell Res. 5:284–294, 1992. Yasumoto, K.-I., K. Yokoyama, K. Shibata, Y. Tomita, and S. Shibahara. Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene. Mol. Cell. Biol. 14:8058–8070, 1994. Yasumoto, K.-I., H. Mahalingam, H. Suzuki, M. Yoshizawa, K. Yokoyama, and S. Shibahara. Transcriptional activation of the melanocyte-specific genes by the human homolog of

the mouse microphthalmia protein. J. Biochem. 118:874–881, 1995. Yokoyama, T., D. W. Silversides, K. G. Waymire, B. S. Kwon, T. Takeuchi, and P. A. Overbeek. Conserved cysteine to serine mutation in tyrosinase is responsible for the classical albino mutation in laboratory mice. Nucleic Acids Res. 18:7293–7298, 1990. Yokoyama, K., H. Suzuki, K.-I. Yasumoto, Y. Tomita, and S. Shibahara. Molecular cloning and functional analysis of a cDNA coding for human DOPAchrome tautomerase/tyrosinase-related protein-2. Biochim. Biophys. Acta 1217:317–321, 1994. Zdarsky, E., J. Favor, and I. J. Jackson. The molecular basis of brown, an old mouse mutation, and of an induced revertant to wild-type. Genetics 126:443–449, 1990. Zhou, B. K., R. E. Boissy, S. Pifko-Hirst, D. J. Moran, and S. J. Orlow. Lysosome-associated membrane protein-1 (LAMP-1) is the melanocyte vesicular membrane glycoprotein band II. J. Invest. Dermatol. 100:110–114, 1993.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

12

Molecular Regulation of Melanin Formation: Melanosome Transporter Proteins Murray H. Brilliant

Summary 1 To make melanin, tyrosinase must be correctly targeted to the melanosome, its substrate, tyrosine, must be transported into the melanosome, and conditions that favor the enzymatic activity of tyrosinase must exist inside the melanosome. All these processes are affected by pH and ionic conditions. Therefore, ion transport across the melanosome membrane is critical for the biosynthesis of melanin. Two proteins, the P protein [encoded by the mouse pink-eyed dilution (p) locus and the human P locus] and MATP (membrane-associated transport protein, previously known as AIM-1, encoded by the formerly named mouse underwhite locus) are critical for normal pigmentation and are likely to be involved in ion flux across the melanosomal membrane. 2 The mouse pink-eyed dilution locus, p, and its human homolog, P, encode a protein of 110 kDa with 12 membranespanning domains that is localized to the melanosome membrane, although a significant fraction is thought to be associated with the endoplasmic reticulum. The p protein is required for normal melanin (primarily eumelanin) biosynthesis. Although the exact function of the p protein is currently unknown, it has a predicted structure in common with anion transporters and Na+/H+ antiporters. Melanocytes lacking a functional p protein have immature melanosomes with aberrant pH that only accumulate minute amounts of primarily eumelanin. In these p mutant melanocytes, tyrosinase is incorrectly processed and targeted. 3 Mutations of the human P gene on chromosome 15q11.2–q12 lead to tyrosinase-positive oculocutaneous albinism, defined as OCA2, a recessive genetic disorder present in the general population at 1:38 000. OCA2 is more common among individuals of African origin, where a predominant haplotype is associated with a 2.7-kb deletion that includes exon 7 of the P gene. In some African populations, this deletion allele comprises up to 80% of the P gene mutations. A larger deletion of different origin is commonly associated with OCA2 among the Navajo people, an independent population that exhibits a high frequency of albinism. 4 The only known function of the P gene is in pigmentation. However, deletions of the human chromosomal interval 15q11q13 that contains the P gene lead to distinct phenotypes known as Prader–Willi syndrome or Angelman syndrome, 230

depending on whether the deletion is paternal or maternal in origin respectively. Although the critical regions and/or genes associated with these imprinting syndromes are distinct from each other and from the P gene, one homolog of the P gene is often deleted in PWS and AS patients. Those AS and PWS patients who are hemizygous for the P gene (in the context of a large deletion of 15q) often exhibit hypopigmentation relative to other family members, a puzzling outcome as carriers of null alleles of the P gene (in the context of a normal chromosome 15) are normally pigmented. Many radiation-induced alleles of the mouse p locus on chromosome 7 are associated with nonpigmentation phenotypes. All these other phenotypes are associated with deletions extending into neighboring genes. 5 The Matp (membrane-associated transporter protein) gene encodes a protein of ~58 kDa with 12 membrane-spanning domains that is very likely associated with the melanosome membrane. The encoded protein, Matp, likely functions as a transporter. One of its closest orthologs encodes a plant sucrose/proton transporter, and the Matp protein may play a role in pH and/or osmotic regulation of the melanosome. In Matp mutant melanocytes (as in p mutant melanocytes), tyrosinase is incorrectly processed and targeted. Matp previously shared the name of AIM-1 with at least two other distinct and unrelated proteins. The mouse locus encoding Matp was formerly called the underwhite (uw) locus. 6 Various mouse mutant alleles of Matp (underwhite) lead to a spectrum of hypopigmented phenotypes, most of which are recessive, but one (MatpUw-dbr) is semi-dominant. 7 Mutations in the human MATP gene are associated with a form of oculocutaneous albinism, defined as OCA4. While rare in the worldwide population, OCA4 is more common in the Japanese population, and cases have been described in the Turkish and German populations. 8 Variations in both the MATP and the P genes are associated with normal variations in human pigmentation.

Historical Background Mouse coat color genes have played an important role in understanding basic aspects of mammalian genetics. For example, the original p mutation and a mutation of the tyrosinase gene (c locus) were used to define the first genetic linkage

MOLECULAR REGULATION OF MELANIN FORMATION: MELANOSOME TRANSPORTER PROTEINS

group in the mouse (Haldane et al., 1915), now assigned to mouse chromosome 7 (Brilliant et al., 1996). The p locus was also used as a marker in transplantation studies to define histocompatability antigens (Snell and Stevens, 1961). The original Matp mutant allele, Matpuw, was used as a coat color marker in many gene linkage and mapping studies (Davisson et al., 1990). The existence of genes that are associated with tyrosinasepositive albinism was hypothesized long ago (Witkop et al., 1970). The P and MATP genes are now known to be the most common genes mutated in tyrosinase-positive albinism, causing OCA2 (Gardner et al., 1992; Rinchik et al., 1993) and OCA4 (Newton et al., 2001) respectively.

Current Concepts Overview Melanin biosynthesis occurs in melanosomes, thought to be derived from the fusion of at least two vesicle compartments, including endosome-derived vesicles that share some features with lysosomes (Dell’Angelica et al., 2000; Orlow, 1995). The initial and rate-limiting step in melanin biosynthesis is catalyzed by tyrosinase, converting tyrosine to DOPAquinone (Cooksey et al., 1997; Hearing and Tsukamoto, 1991). Mature (highly pigmented) “Stage IV” melanosomes derive from earlier stages. The first recognizable stage is the premelanosome, which is distinguished by a disorganized luminal matrix and no melanin. Stage I melanosomes also lack melanin, but are defined by a parallel array of the luminal matrix. Stages II and III are characterized by increased melanin deposition, and Stage IV melanosomes contain the most melanin. Although mouse skin melanosomes are acidic (Bhatnagar et al., 1993; Devi et al., 1987; Ramaiah, 1996), the luminal pH is probably not static as melanosomes develop, and differences in melanosome pH in human melanocytes have been reported between those of Caucasian and black origin (Fuller et al., 2001; Smith et al., 2004). To make melanin: (1) tyrosinase must be correctly processed; (2) tyrosine must be targeted to the melanosome; and (3) conditions that favor the enzymatic activity of tyrosinase must exist inside the melanosome. All these processes are affected by pH (Ancans and Thody, 2000; Ancans et al., 2001; Halaban et al., 2002; Manga and Orlow, 2001; Potterf et al., 1998; Ramaiah, 1996; Watabe et al., 2004). The product of the p (pink-eyed dilution) gene is likely an ion transporter (Gardner et al., 1992; Rinchik et al., 1993) that plays a critical role in melanin biosynthesis through the regulation of melanosome pH (Puri et al., 2000). Matp (membraneassociated transport protein, encoded by the mouse locus previously known as underwhite, uw) also plays a key role in melanin biosynthesis (Lehman et al., 2000; Sweet et al., 1998). Matp likely mediates the transport of an unknown, but critical substance across the melanosome membrane and has high homology at the amino acid level to plant proton/ sucrose transporters (Newton et al., 2001).

The Mouse Pink-eyed Dilution (p) Gene and Its Alleles The original mutant p allele probably arose in Manchuria or Japan in Mus molosinus (Brilliant et al., 1994a) and was incorporated into several common laboratory strains of mice (e.g. SJL/J, 129/J, P/J, and FS/Ei) during the early twentieth century. The first mutant allele of the p locus to be characterized molecularly was the pun (pink-eyed unstable) allele, which exhibits high-frequency (somatic) reversion to wild type (Melvold, 1971). Using the technique of genome scanning, the pun allele was found to result from a duplication of 75 kb of genomic DNA (Brilliant et al., 1991; Gondo et al., 1993) that includes exons 6–18 (Oakey et al., 1996) of the 24-exon p gene. A fragment of genomic DNA that cross-hybridized to various mammalian species (now know to include exon 19) was used to screen a melanoma cDNA library to obtain the p gene cDNA and its human homolog (Gardner et al., 1992). Parallel studies using a candidate gene approach found that a previously unknown human cDNA fragment (DN10) identified an alternative exon of the human P gene (Lee et al., 1995; Rinchik et al., 1993). Many mouse mutant p alleles have been characterized at the molecular level (Culiat et al., 1993, 1994; Gardner et al., 1992; Johnson et al., 1995; Nakatsu et al., 1993; Oakey et al., 1996; Rinchik et al., 1993). The discovery of the human P cDNA has led to an understanding of the molecular basis for tyrosinase-positive oculocutaneous albinism (OCA2). More than 100 p alleles have been identified, some of which were de novo in origin and some of which were induced by X-rays or chemical mutagens (Lyon et al., 1992; Russell et al., 1995; M. F. Lyon, personal communication). In the homozygous state, each mutant p allele causes hypopigmentation ranging from a minor reduction in coat color to a dramatic reduction in both coat and eye color characteristic of the original p mutation. In addition to affecting pigmentation, several mutant alleles are associated with other abnormalities, including decreased neonatal viability, neurologic disorders, cleft palate, male sterility, female semi-fertility, viability, and prenatal lethality (Brilliant, 1992; Culiat et al., 1993, 1994; Hagiwara et al., 2000; Lyon et al., 1992; Nakatsu et al., 1993; Russell et al., 1995). All the mutations with these additional phenotypes were induced by radiation, and all affect surrounding genes (Johnson et al., 1995; Lyon et al., 1992; Russell et al., 1995) or are the result of a chromosomal inversion (Hagiwara et al., 2000). Mutations of the p gene alone cause effects on pigmentation only (Gardner et al., 1992; Johnson et al., 1995; Lyon et al., 1992; Russell et al., 1995). The p mutations result in a dramatic reduction in melanin (primarily eumelanin). In the hair, p pigment granules have been described as shred-like (Russell, 1949). The same description has been applied to the small irregular shaped melanosomes in the harderian gland of p mice (Markert and Silvers, 1956). In p mice, premelanosomes from embryonic choroid and retina are fibrillar in appearance, as are those 231

CHAPTER 12

CHOROID

RPE

CHOROID

RPE

Fig. 12.1. Electron micrograph of wild-type and p/p mutant eyes. A wild-type eye is shown on the left; a p/p mutant eye is shown on the right. Note the aberrant sizes and minimal pigment content of the melanosomes (arrows) in the choroid and retinal epithelium (RPE) of the p/p eye, a feature shared with other forms of albinism.

from the adult choroid. However, most melanosomes from the adult retina are more particulate in appearance. Most p melanosomes show some pigment, indicated as an increase in electron density and diameter of melanofilaments, but none is fully melanized (Hearing et al., 1973). The melanosomes within p/p melanocytes appear as Stage 1 and Stage 2 melanosomes, as described in the above references (Fig. 12.1). Transplantation studies in the mouse have demonstrated that the p defect is intrinsic to melanocytes (Stephenson and Hornby, 1985).

The p Gene Protein and Its Function Molecular analysis of the p transcript and protein have corroborated much of the past phenotypic data and have extended our knowledge about the p protein’s role in pigmentation, even if its precise function remains unknown at this time. Northern blot analyses have confirmed that melanocytes are the predominant p gene-expressing cell type, with lowlevel expression of the p gene in brain, testes, and ovaries (Gardner et al., 1992; Rinchik et al., 1993). The expression of the p gene in testes and ovaries is also conserved in medaka fish (Fukamachi et al., 2004). However, the significance of p gene expression in nonpigmented tissues is unknown as animals and humans lacking P gene expression are normally fertile, and any neurologic problems are limited to the visual system, the same problems as those commonly associated with other forms of albinism. The size of the mouse p gene transcript is 3.3 kb, encoding a predicted protein of 833 amino acids (Gardner et al., 1992; Rinchik et al., 1993). The mouse p gene product, like its 232

human homolog P, encodes a protein with 12 membranespanning domains. Using antisera against a synthetic peptide from the first luminal loop (amino acids 285–298), Rosemblat et al. (1994) were able to characterize the p protein as an integral, melanosomal membrane protein of 110 kDa that does not appear to be glycosylated (tunicamycin treatment does not alter its gel mobility). Subsequent studies (Chen et al., 2002) suggested that a significant fraction of the p protein is associated with the endoplasmic reticulum (ER). Whether this fraction of the p protein functions in the ER or is in transit is unknown. The p protein may be specifically targeted to the melanosome or one of its precursors. In normal pigmented melanocytes, a fraction of the p protein is present in an intracellular compartment distinct from that containing tyrosinase and Trp-1. It has also been noted that melanosomes lacking p protein are lacking a high-molecular-weight complex of the p protein, tyrosinase, and Trp-1, and possess characteristics of immature premelanosomes (Rosemblat et al., 1994). Thus, in addition to a potential transport function, the p protein may also play a critical role in the biogenesis of normal melanosomes, perhaps by providing conditions (e.g. pH) favorable for their proper structure and maturation. The melanosome, as a specialized endosome, may derive its protein components by means of signals and sorting mechanisms that distinguish it from a lysosome. In fact, a specific protein sequence motif, capable of targeting Trp-1 to the melanosomal membrane, exists within its carboxyl-terminus (Vijayasaradhi et al., 1995). When this Trp-1 amino acid sequence was incorporated in chimeric proteins, they were correctly targeted. The sequence capable of targeting Trp-1 is conserved across species, and a related sequence is found in several other melanosome proteins, perhaps including the p protein. However, it remains to be shown whether the sequence in the p protein related to the Trp-1 sorting sequence actually functions in the same way. If so, it would confound the observation that a subset of the p protein is in a different intracellular compartment from tyrosinase and Trp-1 (Chen et al., 2002; Rosemblat et al., 1994). The p protein may interact with melanin. The p protein (along with the silver protein) is far less extractable from melanized melanosomes (in melanocytes or in a cell-free assay system) than it is from poorly melanized melanosomes (Donatien and Orlow, 1995). It may be that some of the loops of the p protein that protrude inside the melanosome are somehow trapped within the melanin polymer. If this close association with melanin impedes p gene function, then perhaps this is one way to limit the melanin content of the developing melanosome. An interesting observation is that the p transcript is expressed in the black dorsal skin, but not the yellow ventral skin in agouti at/at mice (Rinchik et al., 1993), corroborating earlier notions that the p mutations affect eumelanin but not pheomelanin biosynthesis. Eumelanin biosynthesis is favored when the melanocortin 1 receptor (Mc1r) is stimulated by aMSH and upregulates cAMP; pheomelanin is favored when

MOLECULAR REGULATION OF MELANIN FORMATION: MELANOSOME TRANSPORTER PROTEINS

this signaling is antagonized by the agouti protein. Expression of the p gene is upregulated by a-MSH and db-cAMP (a synthetic analog of cAMP), implying that the p protein plays a major role in the eumelanin/pheomelanin switch (Ancans et al., 2003). From its predicted protein structure, the p protein is likely to be a transporter critical to melanocyte function (Gardner et al., 1992; Rinchik et al., 1993). The p protein was proposed to be a tyrosine transporter (Rinchik et al., 1993), based on sequence homology that has subsequently been shown not to be significant (Lee et al., 1995; Rosemblat et al., 1994). Supporting the hypothesis, however, are the observations that both mouse and human melanocytes can be induced to pigment in vitro with high tyrosine (Sidman and Pearlstein, 1965; Witkop et al., 1973). This observation was also confirmed using cultured mouse skin melanocytes (Potterf et al., 1998; Rosemblat et al., 1998). It is possible that the increase in pigmentation detected in mutant p cells when exposed to very high concentrations of tyrosine results from driving the reaction by substantially increasing the substrate. Moreover, the melanin produced in these cells does not appear to be synthesized in melanosomes, and may result from the activity of mislocalized tyrosinase (Manga and Orlow, 1999; Potterf et al., 1998). Most importantly, direct biochemical assays show no difference in tyrosine transport between normal and p melanocytes at the level of the melanocyte or the melanosome. Plasma membrane tyrosine transport was found to be normal in p (pcp/pcp) melanocytes (Km 89 mM; Vmax 302 pmol/min/mg cell protein), and the melanosome-rich granular fractions of normal (melan-a) and p melanocytes (pcp/pcp) were essentially the same, taking up 10 mM [3H] tyrosine at about 21 pmol/min/mg protein (Gahl et al., 1995). Thus, although the p protein may be a transporter, it does not contribute significantly to tyrosine transport. The most homologous bacterial proteins to p include the Staphylococcus aureus and Escherichia coli ArsB proteins (the anion-conducting pathway of a group of proteins that together confer resistance to arsenic), the E. coli Na+/H+ antiporter, and protein 38L from Mycobacterium leprae (Lee et al., 1995; Rosemblat et al., 1994). It may be that the p protein is similarly involved in ion transport, potentially modulating the pH of the melanosome. Indeed, it was found that the mouse p gene can function in yeast and affect cellular sensitivity to arsenic and other metalloids and modulate intracellular glutathione metabolism (Staleva et al., 2002). Although distinct, melanosomes and lysosomes share at least partial endosomal origins, and both types of organelles are characterized by an acidic lumen (Dell’Angelica et al., 2000; Orlow, 1995). Mouse melanosomes have been reported to be very acidic, i.e. pH 3.5 (Bhatnagar et al., 1993; Devi et al., 1987; Moellmann et al., 1988; Tripathi et al., 1988) and, as such, may more closely resemble skin melanosomes from human Caucasians, whereas melanosomes from more darkly pigmented individuals (i.e. black Africans) may be more neutral in pH (Fuller et al., 2001; Smith et al., 2004). Additionally, the pH of melanosomes may be dynamic, with

early stage melanosomes more acidic and later stage melanosomes more neutral in pH allowing for higher tyrosinase activity. In general, acidification of various intracellular compartments is important for a number of processes including receptor-mediated endocytosis, receptor recycling, and membrane trafficking within the cell (Dautry-Varsat et al., 1983). Acidification appears to be required for normal melanosome biogenesis and protein trafficking (reviewed by Brilliant, 2001; Raposo and Marks, 2002), whereas a more neutral pH results in higher tyrosinase activity (Ancans and Thody, 2000; Ancans et al., 2001; Fuller et al., 2001; Manga and Orlow, 2001; Smith et al., 2004). The identity of all the molecular components mediating the pH of endosome-derived organelles is not known. However, it is thought that an anion channel is essential for the acidification of vacuole compartments by ATP-driven proton pumps present in endosomes, Golgi-derived vesicles, and lysosomes (Al-Awqati, 1995). Anion (Cl–, SO4=, or HCO3–) conductance provides the compensating charge balance to electrogenic proton transport. It remains to be determined whether or not there is a melanosome-specific, ATP-driven proton pump. Nevertheless, the melanosome has a unique membrane protein, p, that may be an anion transporter or a Na+/H+ antiporter that may play a role in the pH regulation of melanosomes (Puri et al., 2000). The regulation of pH in melanosomes is likely to be complex and to involve additional proteins, such as Matp (discussed below). Alternative hypotheses for the function of the p protein have been proposed. The p protein may function to transport sulfhydryl compounds out of the melanosome to permit the formation of eumelanin (Lamoreux et al., 1995). The p protein has also been hypothesized to affect the rate of an existing melanin-synthesizing enzyme system to achieve normal pigmentation (Coleman, 1962; Sidman and Pearlstein, 1965). Other observations suggest that the p, tyrosinase, and Trp-1 (tyrosinase-related protein-1) proteins exist in a highmolecular-weight complex that is not formed in p mutant melanocytes (Chiu et al., 1993; Lamoreux et al., 1995). More recently, it has been shown in several studies that, in the absence of a normal p protein, tyrosinase is mistargeted and accumulates in the ER (Manga and Orlow, 1999; Potterf et al., 1998). Other studies imply that a significant portion of the p protein is found in the ER, and their authors have put forward the hypothesis that the P protein is involved in tyrosinase maturation and folding in the ER (Chen et al., 2002; Toyofuku et al., 2002), which may also involve glutathione (Staleva et al., 2002). Melanocytes have a specific factor that is critical for tyrosinase maturation; however, as p-null melanocytes also appear to have this factor, the p protein itself appears to be this factor (Francis et al., 2003). Moreover, the absence of the Matp protein has a similar phenotype (Costin et al., 2003), suggesting that both these proteins may play an indirect role in the folding and targeting of tyrosinase, perhaps by modulating pH (Halaban et al., 2002; Watabe et al., 2004). In any case, the p (and Matp) protein plays a critical coordinating role in melanogenesis. 233

CHAPTER 12

OCA2 and the Human P Gene The human ortholog (P) of the pink-eyed dilution gene has been identified and characterized. Mutations of the human P gene are responsible for type II oculocutaneous albinism (OCA2). In addition, polymorphic variation in the human P gene is responsible, in part, for variations in human pigmentation. Oculocutaneous albinism (OCA) is characterized by abnormally low amounts of melanin in the eyes and skin (see Chapter 31). Abnormally low amounts of melanin in the developing eye lead to abnormal routing of optic nerve fibers, resulting in strabismus and loss of binocular vision. Other ocular effects of albinism include photophobia, nystagmus, and foveal hypoplasia with reduced visual acuity. The reduction in skin pigmentation in individuals with OCA is associated with an increased sensitivity to ultraviolet radiation and a predisposition to skin cancer. Before molecular diagnostics, only two major types of OCA were recognized, tyrosinaserelated OCA and tyrosinase-positive OCA. Mutations of the human tyrosinase gene on chromosome 11 lead to tyrosinaserelated albinism, defined as OCA1 (Tomita et al., 1989; reviewed by King et al., 2003a), whereas tyrosinase-positive albinism is genetically complex (see Chapter 31). In most populations, the major cause of tyrosinase-positive albinism is mutation of the P gene (reviewed by Oetting et al., 1998; see Chapter 31), although mutations of the Matp gene cause a similar phenotype (Newton et al., 2001). The human P gene is on chromosome 15q in a region demonstrated to be linked to OCA2 in native South Africans (Kedda et al., 1994; Ramsay et al., 1992). Mutations in the human P gene lead to tyrosinase-positive OCA, defined as OCA2 (Durham-Pierre et al., 1994; Lee et al., 1994a, b; Rinchik et al., 1993). The phenotype of OCA2 is broad, ranging from minimal to moderate pigmentation of the hair, skin, and iris. The skin pigment tends to be localized in freckles, lentigines, or nevi rather than generalized, and the ability to tan is not well defined (Oetting and King, 1999; see Chapter 31). In contrast to OCA1, individuals with OCA2 usually have pigmented hair at birth that tends to darken somewhat with age. A subtype of albinism known as brown oculocutaneous albinism (BOCA), described in African populations, has been shown to be associated with heterozygosity of a partially functioning allele and a null allele (Kerr et al., 2000; Manga et al., 2001). Additionally, at least one case of autosomal recessive ocular albinism (AROA) is the result of P gene mutations (Lee et al., 1994a). Thus, the phenotypic range of P gene mutations is broad. Moreover, normal MC1R (melanocortin receptor 1) variants dramatically influence the OCA2 phenotype (King et al., 2003b). In the general population of the United States, tyrosinasepositive OCA occurs in 1:30 000 Caucasians and in 1:17 000 blacks (Witkop, 1985). P gene mutations (OCA2) have been detected in these and other racial groups. There are several

234

genetic isolates with a very high frequency of tyrosinasepositive OCA, e.g. the Brandywine, Maryland isolate (1:85) and several native North American Pueblo Indian groups: the Zuni, Hopi, and Jemez people (approximately 1:240; Witkop, 1985; Witkop et al., 1972; Woolf and Dukepoo, 1969), as well as the Navajo people (approximately 1:2000; Yi et al., 2003) and several native South American Indian groups (reviewed by Jeambrun and Sergent, 1991). Presumably, because these represent small restricted populations, individuals within these populations are homozygous for the same recessive mutation of a gene required for normal pigmentation. The P gene is a likely candidate for the OCA seen in all these groups. Indeed, individuals with tyrosinase-positive OCA from the Brandywine, Maryland isolate are homozygous for a deletion allele of the P gene (a 2.7-kb deletion that includes exon 7; DurhamPierre et al., 1994), and Navajos with OCA are typically homozygous for an even larger deletion (122.5 kb) of the P gene. However, it is formally possible that mutations in another gene (e.g. MATP) lead to a similar phenotype of tyrosinase-positive OCA in individuals and groups for which no molecular data are yet available. The tyrosinase-positive OCA phenotype in Africans and African-Americans is characterized by yellow hair, white skin (sometimes with localized pigmented ephelides; Stevens et al., 1995), and irises that are partially or completely pigmented (Oetting and King, 1999; see Chapter 31). This is the most common albinism phenotype because of the high frequency in these populations (ranging from 1:2000 to 1:5000 in large parts of subSaharan Africa). The most common mutation in this group (so far found exclusively in Africans and individuals of African ancestry) is a 2.7-kb deletion that removes exon 7 along with flanking intron sequences, first identified in the Brandywine, Maryland isolate (Durham-Pierre et al., 1994). It is estimated that this single mutation is associated with 25–50% of all mutant P alleles in African-Americans (Durham-Pierre et al., 1994, 1996), although other diverse mutant alleles have been described in this population (Lee et al., 1994b). The 2.7-kb deletion allele accounts for close to 80% of mutant P alleles in South Africa, Zimbabwe, Tanzania, and other parts of subSaharan Africa (Durham-Pierre et al., 1994; Lund et al., 1997; Puri et al., 1997; Spritz et al., 1995; Stevens et al., 1995, 1997). The phenotypic range of OCA2 is now being defined through the molecular characterization of the gene in different individuals with albinism, and it is expected that P gene mutations in OCA2 will be diverse. The missense mutations described to date do not seem to cluster in any specific region of the peptide, as observed for tyrosinase, but most mutations described so far are in the carboxy half of the polypeptide that contains the majority of the 12 membrane-spanning domains (Oetting et al., 1998). A significant portion of the P missense mutations is found at amino acids conserved between the mouse and human P genes and a group of bacterial transport proteins with 12 membrane-spanning domains (Lee et al., 1995). The size of the human P gene transcript is 3.4 kb, encoding

MOLECULAR REGULATION OF MELANIN FORMATION: MELANOSOME TRANSPORTER PROTEINS

a predicted protein of 838 amino acids (Rinchik et al., 1993). Both the human and the mouse proteins encode a 12 membrane-spanning domain protein of unknown function, but related to a group of transport proteins (Gardner et al., 1992; Lee et al., 1995; Rinchik et al., 1993; Rosemblat et al., 1994). The P gene is encoded by 24 exons (plus one alternate exon that contains an in-frame stop codon, corresponding to IR10-1, an anonymous genomic clone) that spans approximately 250 to 650 kb of genomic DNA (Lee et al., 1995). The human proximal promoter region contains sequences that might be binding sites for transcription factors including the following 12 motifs: one AP4, four discrete and one complex AP2, one CF1, one GCF, three SP1, and one TFIID. No TATA or CCAAT motifs or melanocyte-specific motifs (i.e. M-box) have been described (Lee et al., 1995).

In addition to observing hypopigmentation in PWS and AS patients, several cases of PWS and AS are associated with OCA2 (Creel et al., 1986; Fridman et al., 2003; Fryburg et al., 1991; Wallis and Beighton, 1989), with deletion of one homolog of the P gene in the context of PWS or AS and inheritance of a mutation on the other homolog (Brilliant et al., 1994b; Rinchik et al., 1993). Another pigmentation disorder, hypomelanosis of Ito (HI), is genetically complex, with a subset of patients being mosaic for 15q anomalies (reviewed by Pellegrino et al., 1995). These HI patients have hypopigmented whorls, streaks, or patches in addition to some phenotypic similarities to PWS or AS. Just as in PWS and AS, the P gene is likely to underlie at least part of the hypopigmentation phenotype in those HI patients with 15q anomalies.

The P Gene in Prader–Willi Syndrome, Angelman Syndrome, Hypomelanosis of Ito, and Hypopigmentation.

Evolution and the p Gene

Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are genetic diseases associated with chromosome 15q aberrations. PWS and AS both localize to the same chromosomal region, 15q11q13, although the critical regions for the two syndromes are distinct (reviewed by Knoll et al., 1993; Nicholls, 1993). Two common types of deletions are seen in PWS and AS patients (Christian et al., 1995), and both types disrupt the marker D15S12 (IR10) within the human P gene. PWS involves the loss of a paternal component of 15q11q13, with or without maternal disomy for 15q. The opposite inheritance pattern is seen in AS, as it is associated with a deletion of the maternal component of 15q11q13, with or without paternal disomy for 15q. Among the common clinical features of PWS are neonatal hypotonia and failure to thrive, hyperphagia leading to obesity (starting at 1–2 years), mental retardation, behavior problems, craniofacial abnormalities, and hypogonadism (especially in males) leading to infertility (Bray et al., 1983). The clinical features of AS include severe mental retardation, microcephaly, seizures, hypotonia, ataxia, and craniofacial abnormalities (Clayton-Smith, 1993; Magenis et al., 1990). Both syndromes are also associated with hypopigmentation, as many of these patients have much lighter skin, eye, and hair color than other family members (Butler, 1989; ClaytonSmith, 1993; Hittner et al., 1982; King et al., 1993; Saitoh et al., 1994; Weisner et al., 1987). The hypopigmentation is observed in most PWS and AS patients with deletions of 15q11q13, and more specifically among those with a disruption of D15S12, identified by the IR10 probe (Hamabe et al., 1991; Spritz et al., 1997). Thus, patients who are hemizygous for the P gene in the context of a large deletion of 15q are hypopigmented. This observation is difficult to resolve with the recessive nature of both human P and mouse p mutations. Perhaps other genetic determinants of pigmentation are in the 15q11q13 interval. In contrast, people with extra copies of the P gene are hyperpigmented (Akahoshi et al., 2001, 2004).

There is substantial evidence for conservation of the pink-eyed dilution and uw genes in different classes of mammals including ungulates, lagomorphs, canines, marsupials, and primates (see below). For pink-eyed dilution, mice, deermice, rats, rabbits, and cats all have an equivalent locus, defined by a tyrosinase-positive, OCA2-like phenotype, which is part of a conserved linkage group in these species (Heim et al., 1988; reviewed by Little, 1958). The porcine ortholog has been identified (Fernandez et al., 2002). In many mammalian species, the conserved linkage group includes the b-globin gene cluster. However, in humans, the b-globin locus (on human chromosome 11) is not linked to OCA2 (Heim et al., 1988), now known to be associated with mutations of the P gene on chromosome 15 (Durham-Pierre et al., 1994; Lee et al., 1994a, b; Rinchik et al., 1993). Like the mouse p gene, the chicken locus “pinkeye,” pk, causes a severe reduction in retinal pigmentation, produces gray feathers in strains in which wild-type birds produce black feathers, and shows histologic similarities to the melanocytes of p/p mice (Brumbaugh and Lee, 1975). The Medaka fish has a p gene ortholog (Fukamachi et al., 2004), and the p gene shares significant homology with genes from Drosophila, yeast, and bacteria (Gardner et al., 1992; Rinchik et al., 1993).

The Mouse Matp Gene and Its Mutant Alleles Another gene likely to function in transport across the melanosome membrane is Matp (membrane-associated transport protein), previously known as AIM-1 (antigen in melanoma 1), a gene expressed exclusively in melanocytes and melanoma (Harada and Robbins, 1999; Harada et al., 2001; GenBank #AF172849). Note there are two other distinct genes called AIM-1 in the literature (Katayama et al., 1998; Teichmann et al., 1998). Among the mouse mutations that lead to severe hypopigmentation, without other phenotypes

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CHAPTER 12

+

Amino acid transporter

+

Na H exchanger

Tyr (OCA1) Tyrp1 (OCA3)

H+

Na+ Tyrosine Melanin H+ H+ OR Anions Osmolyte Na+ Na+

H+/ATPase Fig. 12.2. Electron micrograph of choroid melanosomes of an adult mouse eye. Left: C57BL/6 (wild type) showing typically well-pigmented and round melanosomes. Right: mutant Matpuw-d/ Matpuw-d showing irregularly shaped melanosomes with uneven distribution of melanin. (Adult Matpuw-d/Matpuw-d mice have dark eyes.) Similar changes are seen in the RPE. Figures of eye melanocytes from these and other mutant Matp alleles are presented in Sweet et al. (1998).

(i.e. effects restricted to melanocytes), mutations in the Matp gene are distinguished by an unusual series of alleles (underwhite) with a spectrum of pigmentation phenotypes. Mutant Matp alleles are associated with small, crenated melanosomes with a range of pigment (Sweet et al., 1998). The original Matpuw allele arose spontaneously in the C57BL/6 strain and is recessive. The outer fur of Matpuw/Matpuw mice is a light beige with very white underfur, and the eyes are pink at birth, but darken with age (Dickie, 1964). Two other recessive alleles, Matpuw-i and Matpuw-d, have also been described (Sweet et al., 1998); homozygous Matpuw-i are slightly more pigmented than homozygous Matpuw; homozygous Matpuw-d are even more pigmented (brown/slate; Sweet et al., 1998). A fourth allele, MatpUw-dbr (dominant brown) is semi-dominant over the wild-type allele, and heterozygous MatpUw-dbr mice have a dark brown coat, while homozygous MatpUw-dbr mice are only slightly darker than uw/uw (Sweet et al., 1998). The phenotype of the eyes of uw allelic series was reported by Sweet et al. (1998), who noted normal numbers of melanocytes or melanosomes in the choroid or retinal pigmented epithelia in mice homozygous for mutant uw alleles. However, the melanosomes of both layers are generally smaller, irregularly shaped (raisin-like or crenated), and contain less pigment than those in wild-type eyes (Fig. 12.2). The action of the Matp gene is autonomous to melanocytes, as shown by transplantation assays (Lehman et al., 2000); its expression is an early marker of pigment cell precursors (Baxter and Pavan, 2002). Because Matp mutations lead to alterations in melanosome shape and pigmentation, it is likely that Matp is associated with the melanosome. The most severe mutant mouse allele, Matpuw/Matpuw is associated with a 7-bp deletion in the coding sequence of exon 3 (Du and Fisher, 2002) leading to a frameshift and resulting in an unstable transcript. Matp transcripts are expressed from the two other alleles that have intermediate pigmentation, but each mutant allele has a unique point mutation that leads to a nonconservative amino acid substitution (Matpuw-d: serine to proline at amino acid 435; MatpUw-dbr: aspartic acid to asparagine at amino acid 153), consistent with their partial function (Du and Fisher, 2002, Newton et al., 2001). 236

OR

P transporter (OCA2)

H+

Matp/uw transporter (OCA4)

Fig. 12.3. A simplified melanosome with key proteins. The melanosome organelle is the site of melanin biosynthesis and storage. The vacuolar H+/ATPase brings in protons (Dell’Angelica et al., 2000; Orlow, 1995; Smith et al., 2004). Two possible functions of the P transporter protein are presented [based on the function of homologous proteins: the P protein may facilitate anion transport needed to maintain electrogenic neutrality or it may function as an Na+/H+ antiporter (exchanger) (Puri et al., 2000)]. One or both Na+/H+ exchangers (NHE3 and/or NHE7) may also modulate the cations present in the melanosome (Smith et al., 2004). Consistent with its high homology to proton- and sodiumsugar symporters, the MATP protein may bring in a sugar (osmolyte) coupled with a proton or sodium (Newton et al., 2001). The model is consistent with changes in tyrosinase activity in melanosomes seen after treatment with agents that disrupt the pH gradient (Ancans et al., 2001; Halaban et al., 2002; Watabe et al., 2004), and differential timing of expression in melanogenesis allows for a dynamic change in the luminal pH. Thus, the P and Matp proteins play a major role in the normal regulation of melanosome biogenesis and melanin content. NB. The melanosome membrane location for Matp is based solely on the phenotype of mutant uw melanocytes. We also note that P protein has been reported in the ER and that both the P and the Matp proteins may primarily function in the vesicles that give rise to melanosomes, if not in the mature organelle.

The Matp Protein and Its Function Matp is transcriptionally modulated by MITF, the melanocytespecific transcription factor essential for pigmentation and a clinical diagnostic marker in human melanoma (Du and Fisher, 2002). Matp shares homology with known transporters, especially with plant H+/sucrose symporters (Newton et al., 2001). Indeed, the five amino acids that are conserved among all known plant H+/sucrose symporters are exactly conserved in mouse and human Matp. Plant H+/sucrose symporters couple the transport of a sucrose molecule with a proton along a proton gradient and often mediate changes in osmolarity (Stadler et al., 1999). Although there are no known mammalian sucrose transporters, it is possible that Matp transports a different sugar (or other related molecule) coupled with proton movement. Along with a reduction in pigment content,

MOLECULAR REGULATION OF MELANIN FORMATION: MELANOSOME TRANSPORTER PROTEINS

melanosomes from Matp mutant mice are small and irregularly shaped (raisin-like, crenated structures; Sweet et al., 1998; Fig. 12.2), consistent with a disruption in osmotic regulation. Moreover, drug- and sugar-mediated disruptions of the osmotic balance of endosomes and lysosomes have effects on protein turnover and luminal pH (Isaac et al., 2000; Schreiber and Haussinger, 1995; Schreiber et al. 1996), and the transport of amino acids across membranes is affected by osmotic imbalance (Gomez-Angelats et al., 1997) and pH (Potterf et al., 1996). Therefore, the Matp transporter is potentially an osmotic and/or pH regulator.

OCA4 and the Human Matp Gene Based on the mouse phenotype, MATP was evaluated as a candidate gene for a form of OCA with a phenotype similar to OCA2 (P gene albinism). The MATP gene was sequenced in several OCA individuals who had minimal pigment, a few of whom had previously been screened for mutations on the P gene. One Turkish OCA individual (with distantly related parents) was found to be homozygous for a splice site mutation of the MATP gene (G to A in the splice acceptor sequence of exon 2) and did not have a P gene mutation. This patient defined a new form of OCA, OCA4 (Newton et al., 2001). Subsequently, MATP mutations leading to OCA4 have been described in additional Turkish individuals (264delC; Ikinciogullari et al., 2004) and several German individuals (L361P, P58A, Y401X, F221del, T329fsX68, Y317C, A477T, W202C, F525fsX15; IVS3+46C4T, IVS3+59+61dup; Rundshagen et al., 2004). In addition, OCA4 is one of the most common forms of albinism in the Japanese population, where 24% of OCA is OCA4, associated with seven mutant alleles [four missense mutations (P58S, D157N, G188V, and V507L) and three frameshift mutations (S90CGGCCAÆGC, V144insAAGT, and V469delG); Inagaki et al., 2004].

Evolution of the Matp Gene The cream color in horses has been associated with the same mutation as the MatpUw-dbr mouse (D153N; Mariat et al., 2003). Mutations of the Medaka ortholog (the B gene) have been identified in the Medaka fish (Fukamachi et al., 2001). The Matp gene shares significant homology with genes from Drosophila and, as mentioned earlier, genes as distant as plant proton/sucrose symporters.

Other Proteins Involved in Transport Across the Melanosome Membrane In addition to the p protein and Matp, other proteins may also play a role in ion transport across melanosome membranes. Recently, two Na+/H+ exchangers, NHE3 and NHE7, have been found associated with melanosomes, and the expression of these Na+/H+ exchangers may vary between Caucasian and black melanocytes (Smith et al., 2004), suggesting that other proteins in addition to p and Matp may regulate ion flux and/or pH, playing a role in the regulation of melanin synthesis in melanosomes. These researchers also confirmed that the melanosome has an ATP-driven proton pump.

Combining what we know about the phenotypes of mutant p and uw melanocytes, the functions of proteins sharing structural similarity with the p and Matp proteins, and the function of other transport proteins known to reside on the melanosome membrane, a model of melanosome transport is presented (Fig. 12.3).

Normal Human Pigmentation Variation and the P and MATP Genes Humans vary considerably in the range of normal pigmentation of the hair, eyes, and skin. It is reasonable to believe that at least part of this variation results from sequence variation in key pigmentation genes, such as the P and MATP genes that underlie forms of albinism. This notion is supported by the range in pigmentation phenotypes seen among mutations of the mouse and human orthologs. Mouse phenotypes vary among different p alleles (Lyon et al., 1992; Russell et al., 1995), and a wide range in OCA2 phenotypes has been noted for various P alleles (Oetting and King, 1999; see Chapter 31). There is also a wide range of phenotypes among mice with various mutant Matpuw alleles (Sweet et al., 1998), and among the limited number OCA4 patients examined to date (Inagaki et al., 2004; Newton et al., 2001; Rundshagen et al., 2004). A gene associated with brown eyes (total brown iris pigment) and a gene associated with brown hair map to 15q, with the P gene as the prime candidate (Eiberg and Mohr, 1996). Population studies looking at the association of specific polymorphisms of the P and MATP genes demonstrate that (together with polymorphisms of the MC1R gene) they play a major role in determining the normal range of pigmentation of the hair (Eiberg and Mohr, 1996, Sturm et al., 2001), skin (Akey et al., 2001; Duffy et al. 2004; Nakayama et al., 2002; Shriver et al., 2003), and eyes (Frudakis et al., 2003; Rebbeck et al., 2002; Sturm and Frudakis, 2004; Zhu et al., 2004). Indeed, a polymorphism of MATP (L374F) may be a useful marker of population origin (Yuasa et al., 2004).

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MOLECULAR REGULATION OF MELANIN FORMATION: MELANOSOME TRANSPORTER PROTEINS Sidman, R. L., and R. Pearlstein. Pink-eyed dilution (p) gene in rodents, increased pigmentation in tissue culture. Dev. Biol. 12:93–116, 1965. Smith, D. R., D. T. Spaulding, H. M. Glenn, and B. B. Fuller. The relationship between Na(+)/H(+) exchanger expression and tyrosinase activity in human melanocytes. Exp. Cell Res. 298:521–534, 2004. Snell, G. D., and L. C. Stevens. Histocompatibility genes of mice, III. H-1 and H4, two histocompatibility loci in the first linkage group. Immunology 4:366–379, 1961. Spritz, R. A., K. Fukai, S. A. Holmes, and J. Luande. Frequent intragenic deletion of the P gene in Tanzanian patients with type II oculocutaneous albinism (OCA2). Am. J. Hum. Genet. 5675: 1320–1323, 1995. Spritz, R. A., T. Bailin, R. D. Nicholls, S. T. Lee, S. K. Park, M. J. Mascari, and M. G. Butler. Hypopigmentation in the Prader– Willi syndrome correlates with P gene deletion but not with haplotype of the hemizygous P allele. Am. J. Med. Genet. 71:57–62, 1997. Stadler, R., E. Truernit, M. Gahrtz, and N. Sauer. The AtSUC1 sucrose carrier may represent the osmotic driving force for anther dehiscence and pollen tube growth in Arabidopsis. Plant J. 19:269–278, 1999. Staleva, L., P. Manga, and S. J. Orlow. Pink-eyed dilution protein modulates arsenic sensitivity and intracellular glutathione metabolism. Mol. Biol Cell. 13:4206–4220, 2002. Stephenson, D. A., and J. E. Hornby. Gene expression at the pinkeyed dilution (p) locus in the mouse is confirmed to be pigment cell autonomous using recombinant embryonic skin grafts. J. Embryol. Exp. Morphol. 87:65–73, 1985. Stevens, G., J. van Beukering, T. Jenkins, and M. Ramsay. An intragenic deletion of the P gene is the common mutation causing tyrosinase-positive oculocutaneous albinism in southern African Negroids. Am. J. Hum. Genet. 56:586–591, 1995. Stevens, G., M. Ramsay, and T. Jenkins. Oculocutaneous albinism (OCA2) in sub-Saharan Africa: distribution of the common 2.7-kb P gene deletion mutation. Hum. Genet. 99:523– 527, 1997. Sturm, R. A., and T. N. Frudakis. Eye colour: portals into pigmentation genes and ancestry. Trends Genet. 20:327–332, 2004. Sturm, R. A., R. D. Teasdale, and N. F. Box. Human pigmentation genes: identification, structure and consequences of polymorphic variation. Gene 277: 49–62, 2001. Sweet, H. O., M. H. Brilliant, S. A. Cook, K. R. Johnson, and M. T. Davisson. A new allelic series for the underwhite gene on mouse chromosome 15. J. Hered. 89:546–51, 1998. Teichmann, U., M. E. Ray, J. Ellison, C. Graham, G. Wistow, P. S. Meltzer, J. M. Trent, and W. J. Pavan. Cloning and tissue expression of the mouse ortholog of AIM1, a betagamma-crystallin superfamily member. Mamm. Genome 9:715–720, 1998. Tomita, Y., A. Takeda, S. Okinaga, H. Tagami, and S. Shibahara. Human oculocutaneous albinism caused by single base insertion in the tyrosinase gene. Biochem. Biophys. Res. Commun. 164:990– 996, 1989.

Toyofuku, K., J. C. Valencia, T. Kushimoto, G. E. Costin, V. M. Virador, W. D. Vieira, V. J. Ferrans, and V. J. Hearing. The etiology of oculocutaneous albinism (OCA) type II: the pink protein modulates the processing and transport of tyrosinase. Pigment Cell Res. 15:217–224, 2002. Tripathi, R. K., C. Chaya-Devi, and A. Ramaiah. pH-dependent interconversion of two forms of tyrosinase in human skin. Biochem. J. 252:481–487, 1988. Vijayasaradhi, S., Y. Xu, B. Bouchard, and A. N. Houghton. Intracellular sorting and targeting of melanosomal membrane proteins: identification of signals for sorting of the human brown locus protein, gp75. J. Cell Biol. 130:807–820, 1995. Wallis, C. E., and P. H. Beighton. Synchrony of oculocutaneous albinism, the Prader–Willi syndrome, and a normal karyotope. J. Med. Genet. 26:337–339, 1989. Watabe, H., J. C. Valencia, K. Yasumoto, T. Kushimoto, H. Ando, J. Muller, W. D. Vieira, M. Mizoguchi, E. Appella, and V. J. Hearing. Regulation of tyrosinase processing and trafficking by organellar pH and by proteasome activity. J. Biol. Chem. 279:7971–7981, 2004. Weisner, G. L., C. M. Bendel, D. P. Olds, J. G. White, D. C. Arthur, D. W. Ball, and R. A. King. Hypopigmentation in the Prader–Willi syndrome. Am. J. Hum. Genet. 40:431–442, 1987. Witkop, C. J., Jr. Inherited disorders of pigmentation. Clin. Dermatol. 3:70–134, 1985. Witkop, C. J., Jr., W. E. Nance, R. F. Rawls, and J. G. White. Autosomal recessive oculocutaneous albinism in man. Evidence for genetic heterogeneity. Am. J. Hum. Genet. 22:55–74, 1970. Witkop, C. J., Jr., J. D. Niswander, D. R. Bergsma, P. L. Workman, and J. G. White. Tyrosinase positive oculocutaneous albinism among the Zuni and the Brandywine triracial isolate: biochemical and clinical characteristics and fertility. Am. J. Phys. Anthropol. 36:397–406, 1972. Witkop, C. J., Jr., C. W. Hill, S. Desnick, J. K. Thies, H. L. Thorn, M. Jenkins, and J. G. White. Ophthalmologic, biochemical, platelet, and ultrastructural defects in the various types of oculocutaneous albinism. J. Invest. Dermatol. 60:443–456, 1973. Woolf, C. M., and F. C. Dukepoo. Hopi indians, inbreeding, and albinism. Science 164:30–37, 1969. Yi, Z., N. Garrison, O. Cohen-Barak, T. M. Karafet, R. A. King, R. P. Erickson, M. F. Hammer, and M. H. Brilliant. A 122.5kilobase deletion of the P gene underlies the high prevalence of oculocutaneous albinism type 2 in the Navajo population. Am. J. Hum. Genet. 72:62–72, 2003. Yuasa, I., K. Umetsu, G. Watanabe, H. Nakamura, M. Endoh, and Y. Irizawa. MATP polymorphisms in Germans and Japanese: the L374F mutation as a population marker for Caucasoids. Int. J. Legal Med. 118:364–366, 2004. Zhu, G., D. M. Evans, D. L. Duffy, G. W. Montgomery, S. E. Medland, N. A. Gillespie, K. R. Ewen, M. Jewell, Y. W. Liew, N. K. Hayward, R. A. Sturm, J. M. Trent, and N. G. Martin. A genome scan for eye color in 502 twin families: most variation is due to a QTL on chromosome 15q. Twin Res. 7:197–210, 2004.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

13

Transcriptional Regulation of Melanocyte Function Kazuhisa Takeda and Shigeki Shibahara

Summary 1 Microphthalmia-associated transcription factor (Mitf) encoded at the mouse microphthalmia locus or its human homolog MITF is a cell type-specific regulator that is required for development and/or survival of pigment cells. Heterozygous mutations in the MITF gene cause auditory–pigmentary syndromes, known as Waardenburg syndrome type 2. 2 MITF consists of at least seven isoforms, such as MITF-A, MITF-B, MITF-C, MITF-H, and MITF-M, differing at their N-termini and in their expression profiles. MITF-M is a founder member of multiple MITF isoforms and represents a melanocyte-specific isoform. Each N-terminal region is encoded by a separate exon 1 of the MITF gene. 3 The melanocyte-specific (M) promoter of the MITF gene is upregulated by several transcription factors, PAX3, SOX10, LEF-1, and CREB. The activity and degradation of MITF-M are also regulated by extracellular signals via protein phosphorylation, such as c-Kit signaling. Together, multiple signals appear to converge on the M promoter as well as on the MITF-M protein. 4 The tyrosinase family consists of tyrosinase, tyrosinaserelated protein 1 (TRP-1), and TRP-2/DOPAchrome tautomerase. We have summarized the short history of identification and characterization of these proteins, which are specifically or preferentially expressed in pigment cells. The cis-regulatory elements of the tyrosinase family genes have been identified in the 5¢ flanking region of each gene. Many of these regulatory elements contain the CATGTG motif, a consensus sequence recognized by a large family of transcription factors with a basic helix–loop–helix structure, such as Mitf. 5 Retinal pigment epithelium (RPE) is derived from the optic cup of embryonic brain and shares the capacity of melanin production with melanocytes. RPE forms a single layer of cells interposed between the neural retina and the vascular-rich choroids, thereby constituting the blood–retinal barrier. RPE is responsible for correct development of the eye and for survival of photoreceptors in adult retinas.

Historical Background Pigmentation is one of the most demonstrable phenotypes in the animal kingdom, and melanin is a principal pigment found 242

in all living organisms, including fungi and plants. In the mouse, at least 90 different loci have been known to affect coat color. Thus, mice coat color mutations provide an excellent model for studying gene action and its regulation. Melanin production is specifically seen in the differentiated melanocytes that originate from the neural crest and in the RPE derived from the optic cup of the brain. Like other cell lineages of neural crest origin, melanoblasts, a precursor to melanocytes, migrate throughout the dermis during development. The RPE, however, does not need to migrate such a long distance during development. The RPE is a monolayer epithelium interposed between the vascular-rich choroid and the neural retina, and such an anatomical organization of RPE is entirely different from that of melanocytes. In spite of these differences, both cell types share the unique capacity of melanin production. Tyrosinase (EC 1.14.18.1) is a rate-limiting enzyme in melanin biosynthesis and has been a symbolic enzyme in pigment cell research. Thus, using various approaches, many investigators have attempted to purify and characterize tyrosinase. The first pigment cell-specific cDNA was isolated by differential hybridization from B16 mouse melanoma cells and was initially suggested to encode tyrosinase (Shibahara et al., 1986). This report definitely facilitated the research related to pigment cells, although the cDNA has been shown not to encode tyrosinase but what is now known as tyrosinaserelated protein 1 (TRP-1) (Jackson, 1988). Since then, many genes related to pigmentation, including tyrosinase, have been cloned. In particular, the second enzyme structurally related to tyrosinase was discovered and termed tyrosinase-related protein 2 (TRP-2) (Jackson et al., 1992; Tsukamoto et al., 1992). There is about 40% amino acid identity among tyrosinase, TRP-1, and TRP-2, all of which constitute the tyrosinase family and are specifically expressed in melaninproducing cells. TRP-2 possesses the activity of DOPAchrome tautomerase (EC 5.3.2.3), catalyzing the conversion of DOPAchrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (Kroumpouzos et al., 1994; Yokoyama et al., 1994a). TRP-2 is now known as DOPAchrome tautomerase (DCT). TRP-1 was shown to catalyze the conversion of DHICA to indole–quinone–carboxylic acid (JiménezCervantes et al., 1994; Kobayashi et al., 1994). Thus, both TRP-1 and DCT/TRP-2 are the enzymes that determine the quality of melanin. The tyrosinase gene family has been characterized as a model to study the regulation of pigment cellspecific and developmental stage-specific gene transcription.

TRANSCRIPTIONAL REGULATION OF MELANOCYTE FUNCTION ∆R217 A→C in splice acceptor (Tietz) R259Stop G→A in splice donor R214Stop N278D S250P S298P

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Fig. 13.1. Schematic illustration of Mitf/MITF and the characterized mutations. A melanocyte-specific isoform, Mitf-M/MITF-M, is shown. The nine exons are indicated by alternate open and closed boxes over the protein structure. The mutations found in individuals affected by WS2 syndrome (Nobukuni et al., 1996; Tassabehji et al., 1994, 1995) are shown above the functional domains. The delR217 is responsible for Tietz syndrome (Amiel et al., 1998). Asterisks and triangles indicate the splicing mutations and the nonsense mutations respectively. The mutations at the 10 Mitf alleles are indicated below (Hodgkinson et al., 1993; Steingrímsson et al., 1994, 1996), and the mutant alleles shown within boxes are unable to bind to DNA in vitro (Hemesath et al., 1994). Region AE indicates the activating exon (exon 4).

Here, we summarize recent advances concerning mechanisms for pigment cell-specific transcription of the tyrosinase family genes. Elucidation of the mechanisms regulating transcription of the tyrosinase family genes is an essential requirement for understanding the molecular events underlying differentiation of pigment cells. A comprehensive text (Silvers, 1979) and good reviews on mouse coat color mutations have been published (Jackson, 1994; Jackson et al., 1994; Urabe et al., 1993).

Genes Regulating the Development of Pigment Cells The genes described in this section are required for development and migration of melanoblasts and melanocytes, and the mutations of each gene cause white spotting or entirely white coat color, characterized by the lack of melanocytes. Most of these mutations do not affect the development of RPE, except for the microphthalmia-associated transcription factor (Mitf); Mitf is required for normal development of neural crestderived melanocytes and RPE.

Microphthalmia-Associated Transcription Factor Overview Mice with mutations at the microphthalmia (mi) locus, now termed Microphthalmia-associated transcription factor (Mitf), have some or all of the following defects: loss of pigmentation, reduced eye size, failure of secondary bone resorption, reduced numbers of mast cells, and early onset of deafness (Silvers, 1979). The most severe manifestations of Mitf alleles, the original Mitf mi and Mitf or (microphthalmia Oak Ridge), are inherited semi-dominantly and include microphthalmia, white coat, deafness (due to lack of inner ear melanocytes),

and osteopetrosis. Less severe alleles tend to be recessive. These phenotypic variabilities have been shown to represent allelic differences. All Mitf alleles are characterized by defects in neural crest-derived melanocytes, leading to coat color dilution, white spotting or complete loss of pigmentation, and deafness. It is clear that the Mitf protein encoded at the Mitf locus plays an essential role in development and/or survival of several cell lineages, including RPE, mast cells, and osteoclasts. Mitf protein has been shown to play a role in promoting the transition of precursor cells to melanoblasts and melanoblast survival (Opdecamp et al., 1997). Mitf as a Member of a Subfamily of Transcription Factors with a bHLH-LZ Independent transgenic insertions disrupted the Mitf locus on mouse chromosome 6 (Krakowsky et al., 1993; Tachibana et al., 1992), which allowed two groups to clone a novel factor encoded at the Mitf locus (Hodgkinson et al., 1993; Hughes et al., 1993). Mitf consists of 419 amino acid residues and is predicted to contain a basic helix–loop–helix and a leucine-zipper (bHLH-LZ) structure (Fig. 13.1). This type of Mitf is now known as Mitf-M representing a melanocyte-specific isoform (Amae et al., 1998). The bHLH-LZ structure is required for DNA binding and dimerization (reviewed by Kadesch, 1993). The predicted Mitf protein shows the highest degree of amino acid similarity to TFE3 and TFEB, both of which are ubiquitously expressed and bind the palindromic sequence (CACGTG), either as homodimers or as TFE3–TFEB heterodimers (Fisher et al., 1991). TFE3 was originally identified as a human protein that binds the E3 box of the m chain immunoglobulin enhancer (Beckmann et al., 1990), and TFEB as the human protein that binds to the adenovirus major late transcription factor (Carr and Sharp, 1990). Hemesath et al. (1994) have shown that Mitf binds in vitro to DNA 243

CHAPTER 13

containing the CACGTG motif derived from adenovirus major late promoter as a homo- or heterodimer with TFEB, TFE3, or TFEC. TFEC was originally identified as a bHLH-LZ protein, forms heterodimers with TFE3, and inhibits TFE3dependent transcription activation (Zhao et al., 1993). TFEC also shows a high degree of amino acid sequence similarity to MITF (Yasumoto and Shibahara, 1997). In contrast, no heterodimers were observed upon mixing Mitf with Max, Myc, or USF. Mitf also bound the CATGTG motif, a core element of the M-box, and was shown to trans-activate the Mbox-driven reporter gene in fibroblasts (Hemesath et al., 1994). Nakayama et al. (1998) reported that expression of TFEB or TFE3 is undetectable in the neural crest at stages when the Mitf mutant phenotype becomes manifest. Furthermore, the phenotypes of Mitf mutant mice in melanocytes and RPE were not affected in double and triple mutant mice lacking Mitf, TFE3, and/or TFEC (Steingrímsson et al., 2002). These results suggest that Mitf may function as a homodimer during the development of melanocytes and RPE. The human homolog of the mouse Mitf gene, MITF, was cloned and mapped to chromosome 3p12.3–p14.1 (Tachibana et al., 1994). MITF-M, a melanocyte-specific isoform, is predicted to consist of 419 amino acid residues and share 94.4% identity with mouse Mitf-M. The predominant species of MITF mRNA is of about 5.5 kb in human melanoma cells (Tachibana et al., 1994). Ectopic expression of MITF-M converts fibroblasts to cells with melanocyte characteristics (Tachibana et al., 1996). Multiple Isoforms of Mitf with Distinct Amino-Termini The MITF gene is expressed as multiple isoforms in humans and mice, termed MITF-M, MITF-A, MITF-B, MITF-C, MITF-D, MITF-E, MITF-H, and MITF-mc (Amae et al., 1998; Fuse et al., 1999; Oboki et al., 2002; Steingrímsson et al., 1994; Takeda et al., 2002; Takemoto et al., 2002; Yasumoto et al., 1994; for a review, see Shibahara et al., 2001). These isoforms share common bHLH-LZ and transcription activation domains, but differ in the amino-terminal regions (Fig. 13.2). MITF-M is a founder member of multiple MITF isoforms, and its amino-terminus, domain M, consists of 11 amino acid residues and is encoded by the exon 1M. All other isoforms with the extended amino-termini share the entire carboxyl portion with MITF-M. The unique aminoterminus of MITF-A, MITF-B, MITF-C, MITF-MC, or MITFH shares the common region of 83 amino acid residues (domain B1b), which is significantly similar to the equivalent portion of TFEB (Amae et al., 1998) and TFE3 (Rehli et al., 1999; Yasumoto et al., 1998). The initiating methionine of MITF-D or MITF-E is located in the B1b domain, as each exon 1 is a noncoding exon. The MITF gene consists of many promoters, their consecutive first exons, such as exon 1A, 1H, 1D, 1B, and 1M (Fig. 13.2), and eight downstream exons that are common to all isoforms (Hallson et al., 2000; Udono et al., 2000). The unique amino-termini of the MITF isoforms are encoded by separate first exons, except for exon 1D, which codes for the 244

Deletion in Mitf mi-rw 1A

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Fig. 13.2. Structural organization of the MITF gene encoding multiple isoforms. The protein-coding regions are indicated by closed boxes, and the 5¢- and 3¢-untranslated regions are indicated by open boxes. Each arrow indicates the transcription start site of separate exon 1. MDE represents the MITF-M distal enhancer. The Mitf mi-rw gene lacks the DNA segment containing exon 1H and MDE (Hallsson et al., 2000; Watanabe et al., 2002). The Mitf mi-bw gene contains the L1 element insertion in intron 3 (Yajima et al., 1999). The bottom panel shows the representative MITF isoforms, MITF-M, MITF-A, MITF-H, and MITF-D. Region A indicates the transcription activation domain. Regions Thr and Ser represent the threonine-rich domain and the serine-rich domain respectively.

5¢ untranslated region of the mRNA. Among the first exons, exon 1B is unique because it is also used as a second exon (B1b exon), when the primary transcripts, initiated from exon 1A, exon 1H, or exon 1D, are subjected to splicing (Udono et al., 2000). Exon 1B therefore encodes the 5¢ untranslated region of MITF-B mRNA, the amino-terminal domain of MITF-B (domain B1a), and domain B1b (Fig. 13.2). Dysfunction of Mitf impairs the differentiation and/or survival of neural crest-derived melanocytes in skin and ear, and optic cup-derived RPE in the eye (Hodgkinson et al., 1993). Among the MITF isoforms, MITF-M is exclusively expressed in melanocytes and pigmented melanoma cells, but is not detectable in any other cell types, including human RPE cell lines (Amae et al., 1998; Fuse et al., 1999). In contrast to MITF-M, other MITF isoforms are widely expressed in many cell types including human RPE cell lines (Amae et al., 1998; Fuse et al., 1999; Takeda et al., 2002; Udono et al., 2000). The Mitf gene is also expressed in heart (Steingrímsson et al., 1994) and male germ cells (Saito et al., 2003a), but no abnormalities in these tissues have been described in Mitf mutants. Informative Mitf Alleles The Mitf gene is altered in two independent Mitf alleles, Mitf mi and Mitf ws (Hodgkinson et al., 1993). Both alleles are semidominant, but the original Mitf m allele shows a more severe phenotype. The Mitf mi allele contains a three-base deletion, resulting in loss of an arginine residue from a series of four consecutive conserved arginines (residues 214–217), located at the carboxyl end of the DNA-binding basic region, whereas the Mitf ws mutation results from an intragenic deletion

TRANSCRIPTIONAL REGULATION OF MELANOCYTE FUNCTION

(Fig. 13.1). Subsequently, it was shown that a protein encoded at the Mitf mi allele is unable to bind in vitro to the CACGTG motif (Hemesath et al., 1994) and to the CATGTG motif (Hemesath et al., 1994; Morii et al., 1994). The deletion of the Mtfi gene was also found in genomic DNA of two Mitf alleles, Mitf ws and Mitf rw (Hughes et al., 1993). Molecular defects associated with eight murine Mitf mutations were characterized (Steingrímsson et al., 1994), and the DNA-binding abilities of mutant proteins were examined in vitro (Hemesath et al., 1994), clearly indicating that the phenotypic variation associated with various Mitf alleles can be explained in large part by the underlying nature of the mutation. For example, a mutation associated with a semi-dominant allele, Mitf or, is an Arg216Lys substitution in the basic region (Steingrímsson et al., 1994), where a deletion of one arginine residue of the four consecutive arginine residues (214–217) is present in the Mitf mi mutation (Hodgkinson et al., 1993) (Fig. 13.1). Indeed, both Mitf mi and Mitf or produce nearly identical phenotypes, including osteopetrosis, which represents the defect of bone resorption and is caused by the osteoclast dysfunction. It was then confirmed that the mutation associated with Mitf mi or Mitf or alleles completely abolishes DNA-binding ability as homodimers or heterodimers with TFE3 in vitro (Hemesath et al., 1994). In addition, the presence of either Mitf mi or Mitf or protein ablated TFE3 homodimeric DNA-binding activity. Thus, the semi-dominant Mitf mutations may represent dominant-negative mutations, as each mutation is predicted to inhibit Mitf function. It has also been reported that the nuclear translocation potential of Mitf mi and Mitf ew proteins is impaired and thus, unlike the nuclear-located Mitf protein, both mutant proteins are predominantly located in the cytoplasm (Takebayashi et al., 1996). Essential requirement of Mitf-M for melanocyte development was verified by the molecular lesion of black-eyed white Mitf mi-bw mice (Yajima et al., 1999), which are characterized by the complete white coat color, deafness, and normally pigmented RPE without any ocular abnormalities. In Mitf mi-bw mice, the insertion of an L1 retrotransposable element in the intron 3 between exon 3 and exon 4 leads to complete repression of Mitf-M mRNA expression and to reduction in Mitf-A and Mitf-H mRNAs expression (Yajima et al., 1999). In this context, the M promoter represents the most downstream promoter of the MITF/Mitf gene (Fuse et al., 1996; Hallsson et al., 2000; Tassabehji et al., 1994; Udono et al., 2000), and may be most susceptible to the transcriptional repression caused by the insertion of the L1 element (Fig. 13.2). These results indicate that MITF-M/Mitf-M is a key regulator of melanocyte development but is dispensable for RPE development. The homozygous red-eyed white Mitf mi-rw mice exhibit small red eyes and white coat with some pigmented spots around the head and/or tail (Steingrímsson et al., 1994), and its molecular defect is a deletion of the genomic DNA segment containing exon 1H and exon 1B (Hallsson et al., 2000) (Fig. 13.2). It is therefore conceivable that the phenotype of Mitf mi-rw mice represents the loss of function of all Mitf isoforms with extended amino-termini. Moreover, these mutant

mice are deficient in melanocytes, probably due to the loss of Mitf-M expression, except for melanocytes located in the head and tail regions, suggesting that the deleted genomic DNA segment may contain the enhancer for the M promoter. In fact, an upstream enhancer has been identified, termed MDE (Fig. 13.2) (Watanabe et al., 2002). Mutations in the MITF Gene, Causing Auditory–Pigmentary Syndrome Waardenburg syndrome type 2 (WS2) is an autosomal dominant disorder of sensorineural hearing loss and pigmentary disturbances, and its responsible gene has been mapped to chromosome 3p12.3–p14.1, close to the position of the MITF gene (Hughes et al., 1994). Subsequently, mutations affecting splice sites in the MITF gene were identified in two families with WS2 (Tassabehji et al., 1994). MITF gene dosage may be more critical in the developing human than in the mouse. A mutation responsible for Tietz syndrome (albinism– deafness syndrome) has been identified as an in-frame deletion of the MITF gene, removing one of four consecutive Arg residues (delR217) in the basic region of MITF-M (Amiel et al., 1998) (Fig. 13.1). Tietz syndrome is characterized by profound deafness, generalized albinism with blue eyes, and hypoplasia of the eyebrows. Unlike WS2, Tietz syndrome shows dominant inheritance with complete penetrance and does not involve the eyes. Thus, the delR217 mutation causes more severe dysfunction of melanocytes than do other MITF mutations. Notably, the delR217 mutation found in Tietz syndrome is equivalent to the mouse semi-dominant Mitf mi mutation (Hodgkinson et al., 1993). The functional consequence of the delR217 mutation was shown to be a loss of DNA-binding activity and to function as a dominant-negative form of MITF-M (Hemesath et al., 1994). In contrast to the haploinsufficiency of MITF in WS2 (Saito et al., 2002), a dominant-negative function of the delR217 MITF-M may be responsible for Tietz syndrome. Regulation of the MITF-M Promoter by Multiple Transcription Factors MITF-M expression is upregulated by Wnt signaling (Dorsky et al., 2000; Takeda et al., 2000a). Wnt proteins, secreted cysteine-rich glycoproteins, have been established as developmentally important signaling molecules (reviewed by Cadigan and Nusse, 1997). Wnt signaling induces intracellular accumulation of b-catenin, which functions as a coactivator for LEF-1/TCF transcription factors (reviewed by Cadigan and Nusse, 1997; Clevers and van de Wetering, 1997; Eastman and Grosschedl, 1999). A double knockout mouse of Wnt-1 and Wnt-3a genes exhibits deficiency of neural crest derivatives, including melanocytes (Ikeya et al., 1997). Wnt signaling promotes pigment cell formation from neural crest in zebrafish (Dorsky et al., 1998). Moreover, the onset of Wnt3a expression is detected at 7.5 embryonic days (Takada et. al., 1994), which precedes the onset of Mitf expression in neural crest cells (9.5–10.5 embryonic days) (Nakayama et al., 1998). Thus, MITF-M is a good candidate for the target genes of Wnt signaling. Indeed, the MITF-M promoter is bound by 245

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LEF-1 and is activated by Wnt-3a (Takeda et al., 2000a). Wnt3a protein induces endogenous Mitf-M mRNA in cultured melanocytes (Takeda et al., 2000a). Interestingly, the MITFM by itself interacts with LEF-1, which efficiently activates its own transcription (Saito et al., 2002; for a review, see Saito et al., 2003b). Recruitment of MITF-M on the M promoter is an essential step for the self-activation of MITF-M expression and depends on the binding of LEF-1 to the three adjacent binding sites (Saito et al., 2002). It is therefore likely that transcription from the M promoter is relatively sensitive to the concentrations of LEF-1 and MITF-M. Importantly, MITF-M is able to transactivate the M promoter by interacting with LEF-1 but without binding to the M promoter, because the M promoter does not contain the CATGTG motif and is not bound by MITF-M in vitro. In addition, the mutant MITF-M protein lacking the DNA-binding activity also enhances the LEF-1mediated transactivation of the M promoter (Saito et al., 2002). We used the Gly244Glu substitution found in the semidominant Mitf brownish (Mitf b) mouse (Steingrímsson et al., 1996). Likewise, Mitf-M has been shown to interact with c-jun to transactivate the mouse mast cell protease 7 gene promoter that lacks a typical MITF-binding element (Ogihara et al., 2001). It is therefore conceivable that MITF-M functions as a nonDNA-binding cofactor for LEF-1 on the M promoter. This notion is of physiological significance in understanding the phenotypic consequences of various MITF and Mitf mutations that alter the DNA-binding activity. MITF-M expression is also upregulated by a-melanocytestimulating hormone (a-MSH), which stimulates cAMP signaling and activates the transcription factor CREB by phosphorylation. CREB binds the cAMP-responsive element in the MITF-M promoter, resulting in transcriptional activation of the MITF-M gene (Price et al., 1998). The cAMP signalingmediated activation of the MITF-M gene is melanocyte specific (Price et al., 1998). This activation requires SOX10, a transcription factor containing a high-mobility-group domain (Huber et al., 2003). The Function of MITF-M Protein is Regulated by Phosphorylation The Steel/c-kit signaling triggers phosphorylation of MITF-M via the MAP kinase ERK2 at Ser-73, and upregulates the function of MITF-M (Hemesath et al., 1998). The function of MITF-M protein is regulated by many signals, which induce phosphorylation of MITF-M (see Fig. 13.4). Consequently, MITF-M could interact with CBP/p300 transcriptional coactivator efficiently (Price et al., 1998; Sato et al., 1997). Moreover, c-kit signaling results in phosphorylation of MITF-M at Ser-409 via p90 Rsk, a member of the serine/threonine kinases, and causes short-lived activation and destruction of MITF-M (Wu et al., 2000). In this context, the ubiquitin-conjugating enzyme hUBC9 was identified as a potential interacting partner for MITF-M (Xu et al., 2000). It was shown that phosphorylation of MITF-M at Ser-73 is a prerequisite for the hUBC9-mediated degradation of MITF-M. 246

Glycogen synthase kinase 3b (GSK3b) phosphorylates MITF-M at Ser-298, enhancing the DNA-binding ability of MITF-M (Takeda et al., 2000b). In fact, a Ser298Pro substitution was found in the individuals affected by WS2 (Tassabehji et al., 1995). It has been reported that GSK3b is activated by cAMP, which stimulates melanogenesis (Khaled et al., 2002). Mitf Regulates the Genes Involved in Melanin Formation and Melanocyte Survival Mitf protein transactivated the mouse tyrosinase (Ganss et al., 1994a) and TRP-1 promoters probably by binding to the Mbox (Yavuzer et al., 1995). It was also shown that Mitf protein activates the human tyrosinase promoter mainly through the initiator E-box (Bentley et al., 1994). Likewise, MITF noticeably increased the expression of a reporter gene under the control of the human tyrosinase promoter, mainly through the CATGTG motif of the tyrosinase distal element, TDE (positions –1861 to –1842) (Yasumoto et al., 1994). Subsequently, MITF was shown to bind in vitro to TDE and was also shown to transactivate the mouse tyrosinase and TRP-1 promoters, probably by binding to the M-box (Yasumoto et al., 1995). The M-box was originally identified as a positive regulatory element in the mouse TRP-1 promoter (Lowings et al., 1992) and is well conserved in the tyrosinase family genes. In contrast, although DOPAchrome tautomerase (DCT) promoter contains the M-box, its promoter is not activated by MITFM alone (Yasumoto et al., 1997). Subsequently, it has been reported that the DCT promoter is activated by cooperation of MITF with LEF-1 (Yasumoto et al., 2002). These results indicate that MITF is a factor required for pigment cellspecific transcription of the tyrosinase family genes. It is noteworthy that MITF may form a dimer with a cell-specific or ubiquitous DNA-binding protein, which in turn functions to direct the pigment cell-specific transcription of the tyrosinase family genes. Other pigment cell-specific target genes of MITF are Tbx2, a member of the T-box family (Carreira et al., 2000), melanosomal proteins Pmel17 (silver/gp100) and MART1/ MELANA (Du et al., 2003), and MELASTATIN (MLSN1/ TRPM1), an ion channel of the transient receptor potential (TRP) superfamily (Miller et al., 2004). MITF is also responsible for melanocyte survival by regulating the antiapoptotic gene Bcl2 (McGill et al., 2002), disruption of which causes depigmentation in second hair follicle change (Yamamura et al., 1996). Moreover, it has been shown that MITF interacts with the retinoblastoma product in vitro (Yavuzer et al., 1995). Thus, MITF may regulate the cell cycle, but it is unknown whether MITF transactivates the cell cycle-related genes.

Pax-3 or Splotch Homozygous splotch (Sp) mice usually die at 13 days of gestation with multiple abnormalities, including neural tube defects and dysgenesis of neural crest-derived cells (Auerbach, 1954; Silvers, 1979), whereas heterozygous splotch mice display white spotting on the belly and occasionally on the back, feet,

TRANSCRIPTIONAL REGULATION OF MELANOCYTE FUNCTION

and tail. It has been shown that the splotch mutation disrupts the development of functioning melanocytes but does not affect RPE (Auerbach, 1954; Pavan and Tilghman, 1994). The splotch locus encodes the Pax-3 protein, a member of a family of putative transcription factors related to the paired box family of Drosophila segmentation genes (Epstein et al., 1991). In the mouse, the Pax gene family encodes a group of eight related proteins, which share a domain homologous to that encoded by the Drosophila paired box (reviewed by Chalepakis et al., 1993). One mutant splotch allele, Sp2H, contains a deletion of 32 bp in the exon sequence coding for the paired homeodomain of Pax-3 protein (Epstein et al., 1991). Human PAX3 gene encodes a paired domain transcription factor located at chromosome 2q35, and its mutations are associated with Waardenburg syndrome type 1 (WS1) and type 3 (WS3) (Baldwin et al., 1992; Tassabehji et al., 1992, 1993, 1995). WS1 and WS3 are dominantly inherited syndromes of hearing loss, pigmentary disturbances, and heterochromia iridis. Unlike WS2, WS1 and WS3 are characterized by dystopia canthorum and limb abnormality, respectively. Watanabe et al. (1998) has shown that the MITF-M promoter is a direct target gene for PAX3, which accounts for the fact that mutations of either transcription factor PAX3 or MITF could cause Waardenburg syndrome. TRP-1 gene is also activated by PAX3 (Galibert et al., 1999).

SOX10 SOX10 is a transcription factor containing a high-mobilitygroup box as a DNA-binding motif, and is responsible for Waardenburg–Hirschsprung syndrome, also known as WS4 (Pingault et al., 1998), which is characterized by aganglionic colon, sensorineural deafness, and pigmentation abnormalities. In the spontaneous mouse mutant Dominant megacolon (Dom), Sox10Dom is functionally inactive due to a frameshift mutation (Herbarth et al., 1998; Southard-Smith et al., 1998). The Dom mice suffer from several neural crest defects including the enteric nervous system and melanoblasts. At E11.5, mouse embryos homozygous for the Sox10Dom mutation entirely lack neural crest-derived cells expressing the lineage marker DOPAchrome tautomerase (DCT) or Mitf. Moreover, neural crest cell cultures derived from homozygous embryos do not give rise to pigmented cells (Potterf et al., 2001). Thus, Sox10 may regulate transcription of the DCT and MITF genes. In the heterozygous Dom mice, c-kit-expressing melanoblasts are observed at E11.5, but lack Dct expression, suggesting that Sox10 may serve as a transcriptional activator of Dct. Indeed, Sox10 and Dct colocalized in early melanoblasts, and Sox10 is capable of transactivating the Dct promoter in a transient expression assay (Potterf et al., 2001). Moreover, Ludwig et al. (2004) have shown the synergistic activation of the Dct gene by Sox10 and Mitf. By in situ hybridization analysis, we have shown the coexpression of Sox10 and Mitf (most likely Mitf-M) mRNAs in migrating melanoblasts at E11.5 (Watanabe et al., 2000). By E13.5, Sox10 mRNA expression became undetectable in

migrating melanoblasts, whereas Mitf expression continues to be detectable. In postnatal day 8 and adult cochleas, Sox10 expression was detected only in the supporting cells of the organ of Corti (Watanabe et al., 2000). Taken together, these results support the notion that Sox10 is required for transcription from the M promoter of the MITF/Mitf gene during the early stage of melanoblast development. MITF-M gene is also activated by SOX10 via at least three SOX10 binding sites of the proximal promoter (Bondurand et al., 2000; Lee et al., 2000; Potterf et al., 2000; Verastegui et al., 2000). SOX10 synergistically activates the MITF-M promoter by cooperating with PAX3 (Bondurand et al., 2000; Potterf et al., 2000). SOX10 also binds the MITF-M distal enhancer (MDE) located at about the –15-kb region upstream of exon 1M (Watanabe et al., 2002). SOX10 was shown to function as an architectural transcription factor by bending the DNA (Peirano and Wegner, 2000). It is therefore conceivable that the DNA bending, induced by SOX10 bound to MDE, may facilitate the interaction with the transcription factors on the proximal MITF-M promoter region (Fig. 13.3). These data support a model in which deafness and hypopigmentation common to all types of WS are caused by a functional disruption of a key transcription factor, MITF-M.

CITED1 CITED1 was identified as a melanocyte-specific gene 1 (msg1), which is a nuclear protein expressed in pigmented melanocytes and melanoma cells, but not in amelanotic melanoma cells (Shioda et al., 1996). The normal tissue distribution of CITED1 in adult mice was confined to melanocytes and testis. Murine Cited1 and human CITED1 genes encode a predicted protein of 27 kDa with 75% overall Wnt 5’ (MDE) –15 kb SOX10

Frizzled

α-MSH MC1R

GSK3β β-Catenin

cAMP Cascade

MITF-M promoter β-Catenin SOX10 PAX3 (WS4) (WS1 and 3)

LEF1 MITF-M

CREB Exon 1M

3’

+1

Fig. 13.3. Transcriptional regulation of the MITF-M promoter. The proximal MITF-M promoter is bound by PAX3, which is responsible for WS1 and WS3, and by SOX10, which is responsible for WS4. SOX10 also binds the MITF-M distal enhancer (MDE) to activate the MITF-M gene. Wnt signaling induces intracellular accumulation of b-catenin, which functions as a coactivator for LEF-1, leading to activation of the MITF-M gene. MITF-M also interacts with LEF-1 and activates its own transcription. a-Melanocyte-stimulating hormone (a-MSH) stimulates cAMP signaling and activates the transcription factor CREB by phosphorylation. CREB induces MITF-M mRNA expression via a cAMP-responsive element in the MITF-M promoter (Price et al., 1998).

247

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also detected in a mouse melanoblast cell line (Eisen et al., 1995; Thomson et al., 1993). Expression of BRN2, in melanoma cells in cotransfection assays, repressed the activity of the melanocyte-specific tyrosinase promoter (Eisen et al., 1995). Additionally, BRN2 ablated melanoma cells revert to a less mature cell type lacking many markers of differentiated melanocytes such as MITF (Thomson et al., 1995). These results suggest that BRN2 maintains the undifferentiated melanocytic phenotype.

Upstream Stimulatory Factor 1 (USF1)

Fig. 13.4. Post-translational regulation of MITF-M by multiple signals. Steel/c-kit signaling triggers phosphorylation of MITF-M via the MAP kinase ERK2 at Ser-73, and increases interaction of MITFM with CBP/p300 transcriptional coactivators. The c-kit signaling phosphorylates MITF-M at Ser-409 via a serine/threonine kinase p90 Rsk, and causes short-lived activation and destruction of MITF-M. For the degradation of MITF-M, the ubiquitinconjugating enzyme hUBC9 interacts with MITF-M. Endothelin 3, which is responsible for WS4, stimulates MAP kinase, and probably phosphorylates MITF-M. Glycogen synthase kinase 3b (GSK3b) is activated by cAMP signaling, and phosphorylates MITF-M at Ser-298, enhancing the DNA-binding ability of MITF-M. MITF-M activates transcription of the target genes, which are related to differentiation of melanocytes, such as tyrosinase family genes, and cell survival, such as Bcl2.

amino acid identity and 96% identity within the C-terminal acidic domain of 54 amino acids. CITED1 is a nonDNAbinding coactivator, and its C-terminal domain can function as a transcription activation domain through interacting with p300/CBP transcriptional coactivators (Fig. 13.4) (Yahata et al., 2000). CITED1 is a member of the CBP/p300-interacting transactivator with an ED-rich tail (CITED). CITED1 also interacts with Smad4 (Shioda et al., 1998) or estrogen receptors a and b (Yahata et al., 2001). Cited1 null mutant mice show growth restriction at 18.5 days post coitum, and most of them die shortly after birth, which suggest that Cited1 is required in trophoblasts for normal placental development and subsequently for embryo viability (Rodriguez et al., 2004). It has been shown that overexpression of CITED1 increases melanin in B16 melanoma (Nair et al., 2001).

BRN2 BRN2 (also called N-Oct3 or POU3F2) is a POU domaincontaining transcription factor, implicated in the development of the brain (reviewed by Nakai et al., 1995; Treacy and Rosenfeld, 1992). BRN2 mRNA was expressed in a range of human melanoma cell lines and was elevated compared with normal human melanocytes, whereas mRNA for Brn-2 was 248

A CANNTG motif is the consensus sequence of the binding site for a large family of transcription factors with a bHLH structure (Murre et al., 1989a, b). The bHLH structure is required for DNA binding and dimerization of transcription factors. A ubiquitous transcription factor, USF1, is one of such factors, containing a bHLH and a leucine zipper (LZ) structure (Gregor et al., 1990; Pognonec and Roeder, 1991). USF1 was initially identified as a nuclear protein that binds to the upstream element of the adenovirus major late promoter (Sawadogo and Roeder, 1985), and was then shown to be involved in the expression of various cellular genes (Carthew et al., 1987; Chodosh et al., 1987; Sato et al., 1990). USF1 was shown to bind in vitro to the mouse TRP-1 gene promoter (Yavuzer and Goding, 1994) and to TDE and TPE of the human tyrosinase gene (Yasumoto et al., 1994) using the antiserum against USF1. The binding of USF1 to TDE was also confirmed using the USF1 produced by in vitro translation. In addition, many properties of the TDE-binding proteins are consistent with those of USF1, such as the elution profile on ion-exchange chromatography and the binding patterns in the gel mobility shift assay (Yasumoto et al., 1994). Furthermore, coexpression of a USF1 cDNA remarkably increased the reporter gene expression under the control of the human tyrosinase promoter. USF1 has been identified as a target of the stress-responsive p38 kinase and could mediate UVinduced tyrosinase expression (Galibert et al., 2001). These results suggest that USF may be involved in efficient transcription of the human tyrosinase gene. However, it should be noted that MITF protein does not form heterodimers with USF in vitro (Hemesath et al., 1994).

Tyrosinase Family Genes The tyrosinase family is directly involved in melanin pigment production and is specifically or preferentially expressed in pigment cells. Each family member has been extensively reviewed in other chapters; this chapter will focus on the transcriptional regulation of these genes. The tyrosinase family genes provide a good system to study the mechanisms of cell type-specific gene transcription. Such understanding is invaluable in studying how pigment cell differentiation is regulated. It is clear that multiple factors, including ubiquitous and cellspecific factors, must cooperate to direct pigment cell-specific transcription of the tyrosinase family genes. Here, we sum-

TRANSCRIPTIONAL REGULATION OF MELANOCYTE FUNCTION

marize some of the transacting factors acting on the tyrosinase family genes.

Tyrosinase or Albino (c) Locus Protein Tyrosinase (EC 1.14.18.1) is a rate-limiting enzyme in melanin biosynthesis that catalyzes the conversion of tyrosine to 3,4dihydroxyphenylalanine (DOPA), DOPA to DOPAquinone, and 5,6-dihydroxyindole to indole-5,6-quinone (Tripathi et al., 1992). The amino acid sequences of tyrosinase were deduced from chicken (Mochii et al., 1992), Japanese pond frog (Takase et al., 1992), Japanese quail, and snapping turtle (Yamamoto et al., 1992). They share significant homology with mammalian tyrosinase. The 5¢ flanking regions of the quail and turtle tyrosinase genes were cloned and compared with mammalian tyrosinase genes (Yamamoto et al., 1992). Through retrovirally mediated gene transfer, it was demonstrated that the quail and mouse promoters of about 500 bp are sufficient to direct efficient expression of a reporter mouse tyrosinase gene in cultured albino chick melanocytes but not in fibroblasts and hepatocytes (Akiyama et al., 1994). This indicates that the quail promoter region used contains the cisacting element responsible for melanocyte-specific expression and that the mouse promoter is also functional in avian melanocytes. The quail promoter contains two copies of the element, containing the CANNTG motif, similar to the proximal melanocyte-specific element (p-MSE) of the mouse tyrosinase and TRP-1 genes (Yamamoto et al., 1992). These results suggest that a molecular mechanism for pigment cell-specific transcription of the tyrosinase gene is well conserved between birds and mammals. Mouse cDNA clones for tyrosinase have been isolated by several groups (Kwon et al., 1988; Müller et al., 1988; Terao et al., 1989; Yamamoto et al., 1987, 1989). The mouse tyrosinase gene, encoded at the albino (c) locus, was characterized and organized into five exons (Müller et al., 1988; Ruppert et al., 1988; Shibahara et al., 1990). The molecular basis of the BALB/c albinism was found as a point mutation, causing a Cys-to-Ser substitution at position 85 (codon 103) (Jackson et al., 1990; Shibahara et al., 1990; Yokoyama et al., 1990). Introduction of functional tyrosinase minigene constructs containing the 5¢ flanking region into the albino mouse can rescue the albino phenotype (Beermann et al., 1990; Tanaka et al., 1992; Yokoyama et al., 1990). The promoter function of the mouse tyrosinase gene was analyzed in transgenic mice, showing that its 5¢ flanking region of only 270 bp is sufficient to provide cell type specificity (Klüppel et al., 1991) and developmental regulation in skin melanocytes and retinal pigment epithelium (Beermann et al., 1992). Within this 270-bp region, there are two copies of a CATGTG motif (positions –104 to –99 and –12 to –7). A high level of position-independent expression of a tyrosinase transgene has been achieved using a yeast artificial chromosome containing the 80-kb mouse tyrosinase gene and the 155-kb 5¢ flanking region (Schedl et al., 1993), indicating that all cis-acting elements required for tyrosinase gene control are located in these DNA segments.

A DNase I hypersensitive site was identified about 15 kb upstream from the first exon of the mouse tyrosinase gene (Porter et al., 1991). This hypersensitive site, located 12 kb upstream, was detected in mouse melanoma cells but not in mouse fibroblasts (Ganss et al., 1994b). Functional analysis of the sequences corresponding to the DNase I hypersensitive site showed the property of a strong cell-specific enhancer in transgenic mice as well as in cultured cells (Ganss et al., 1994b). It was also shown that full enhancer activity in transient expression assays is detected with a minimal sequence of 200 bp located at –12.1 kb. Two protein-binding regions, hs1 and hs2, were identified within the 200-bp enhancer region by DNase I footprinting analysis. Interestingly, either the hs1 or the hs2 region contains a copy of the CANNTG motif. However, by gel retardation assays, the protein-binding activity was detected only with hs1 oligonucleotide, but not with hs2 oligonucleotide, suggesting that protein binding to hs1 is a prerequisite for the binding factors to hs2 (Ganss et al., 1994b). A palindromic sequence 5¢-TGACTTTGTCA-3¢ is present in the hs1 region and is similar to the binding motif of CREB (cAMP-responsive element binding) transcription factor and an AP1 (activator protein 1) binding site, which is recognized by members of the fos/jun family of transcription factors. The enhancer function of the DNase I hypersensitive site of the mouse tyrosinase gene was also confirmed in transgenic mice by other investigators (Porter and Meyer, 1994). They also suggested that the distinct regulatory elements are required for the expression of tyrosinase gene in neural crestderived melanocytes and RPE. The 270-bp upstream region of the mouse tyrosinase gene was analyzed in detail (Ganss et al., 1994a), because this region is sufficient to confer cell-specific and developmentally regulated expression of the tyrosinase gene in the transgenic mice (Beermann et al., 1992). Within this 270-bp region, two positive regulatory elements (positions –245 to –230 and –104 to –93) and one negative regulatory element (position –195 to –125) were identified by transient expression assays (Ganss et al., 1994a). The positive regulatory element, located between –104 and –93, coincides with the M-box, the 11-bp cis-acting element containing the CATGTG motif of the mouse TRP-1 gene (Lowings et al., 1992). It was also shown that MITF activates the mouse tyrosinase gene promoter (Yasumoto et al., 1995). However, the functional analysis of the two positive elements in transgenic mice indicates that these two elements do not determine pigment production in vivo but rather modulate transcription of the mouse tyrosinase gene (Ganss et al., 1994c). Kwon et al. (1987a) isolated a portion of human tyrosinase cDNA by screening a melanocyte expression cDNA library with antityrosinase polyclonal antibodies. Subsequently, the entire structure of human tyrosinase was deduced from the full-length cDNAs (Bouchard et al., 1989; Shibahara et al., 1988; Takeda et al., 1989). The catalytic function of a cloned tyrosinase cDNA was assessed by transient transfection assays, revealing that tyrosinase possesses the two catalytic activities: tyrosine hydroxylase and dopa oxidase (Takeda 249

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et al., 1989). The human tyrosinase gene was mapped to chromosome 11q14–q21, and a second “tyrosinase-related” sequence mapped to 11p11.2–cen (Barton et al., 1988). Tomita et al. (1989) clarified the molecular basis of oculocutaneous albinism (type I) by finding a single base insertion in the exon sequence of the patient’s tyrosinase gene. This insertion causes a frameshift and introduces a premature termination codon. The patient has the mutant gene at both alleles, and the truncated protein encoded by the mutant gene is not functional. The human tyrosinase gene is organized into five exons (Tomita et al., 1989), and its size is greater than 70 kb (Takeda et al., 1990). The nucleotide sequence of the 5¢ flanking region of the human tyrosinase gene was also reported (Kikuchi et al., 1989). The promoter function of the mutant gene is confirmed by in vitro transcription using melanoma whole cell extracts (Takeda et al., 1990). The structural organization and the nucleotide sequence of the 5¢ flanking region of the human tyrosinase gene were also reported (Giebel et al., 1991; Ponnazhagan et al., 1994). It was found that the second site, mapped to chromosome 11p11.2–cen by Barton et al. (1988), represents the truncated human tyrosinase pseudogene, which consists of the nucleotide sequence almost identical to that of exons 4 and 5 (Giebel et al., 1991; Takeda et al., 1991). Shibata et al. (1992a) identified the 39-bp enhancer element of the human tyrosinase gene about 1.8 kb upstream from the transcription initiation site, which is responsible for pigment cell-specific expression of a reporter gene. The enhancer element was narrowed to the 20-bp sequence, termed tyrosinase distal element (TDE) (positions –1861 to –1842) (Yasumoto et al., 1994). TDE, containing a CATGTG motif in its center, was specifically bound by MITF-M (Yasumoto et al., 1994, 1997). There are two additional copies of the CATGTG motif in the human tyrosinase promoter region (positions –104 to –99 and –12 to –7) (Yasumoto et al., 1994). The 20-bp region (positions –112 to –93), containing the CATGTG motif (positions –104 to –99), was identified as a weak pigment cell-specific promoter, termed tyrosinase proximal element (TPE) (Yasumoto et al., 1994). It is noteworthy that the 1.0-kb 5¢ flanking region of the human tyrosinase gene containing TPE but lacking TDE was sufficient to confer cellspecific expression of the tyrosinase gene in transgenic mice (Tanaka et al., 1992). However, no pigmented melanocytes were detected in the epidermis of these mice. It is therefore conceivable that TDE is required for efficient and correct expression of the human tyrosinase gene in a pigment cellspecific manner. Suzuki et al. (1994) have reported that overexpression of neurofibromin cDNA in MeWo melanoma cells, which are deficient in neurofibromin, enhanced the expression of a reporter gene under the control of the tyrosinase promoter. Neurofibromin is responsible for neurofibromatosis type 1 (NF1) or von Recklinghausen’s disease, which is a common autosomal dominant disorder characterized by the presence of neurofibromas, café-au-lait spots, Lisch nodules (hamartomas in the iris), bone deformity, and an increased frequency of 250

malignancies (Cawthon et al., 1990; Viskochil et al., 1990; Wallace et al., 1990). Neurofibromin has a domain related to mammalian ras GTPase-activating protein (GAP) (Xu et al., 1990). Notably, the GAP-related domain of neurofibromin is mainly responsible for the activation of the tyrosinase promoter (Suzuki et al., 1994). Likewise, neurofibromin has been shown to activate the promoter activity of the human DCT gene, as judged by transient cotransfection assays (Suzuki et al., 1998). Neurofibromin may modulate intracellular signaling, which in turn stimulates the activity of a factor required for melanocyte-specific transcription of the tyrosinase and DCT genes.

Tyrosinase-related Protein 1 A pigment cell-specific cDNA, pMT4, was cloned from a B16 mouse melanoma cDNA library by differential hybridization (Shibahara et al., 1986). The expression of pMT4 mRNA is restricted to the skin and pigmented melanoma cells. The deduced structure of the pMT4-encoded protein shares significant homology with Neurospora tyrosinase and contains the several domains thought to be characteristic of tyrosinase, such as a signal sequence, two potential Cu-binding regions, a transmembrane domain, and potential glycosylation sites. Furthermore, transient expression of pMT4 revealed that its protein product is reactive with monoclonal antibodies TMH1 and TMH-2, which have been reported as antityrosinase monoclonal antibodies (Tomita et al., 1985). The pMT4 protein was therefore reported as tyrosinase (Shibahara et al., 1986). However, when its gene was mapped to the brown (b) locus on mouse chromosome 4, which determines the type of melanin produced, it was shown to be different from tyrosinase (Jackson, 1988). The pMT4 protein is now known as TRP-1, and monoclonal antibodies TMH-1 and TMH-2 have been established as anti-TRP-1 antibodies (Tomita et al., 1991). The function of TRP-1 was identified as DHICAoxidase, catalyzing the conversion of DHICA to indole– quinone–carboxylic acid (Jiménez-Cervantes et al., 1994; Kobayashi et al., 1994). Thus, TRP-1 is the first gene of the tyrosinase gene family to be cloned, but its exact function was established last. TRP-1 or brown (b) locus protein is composed of 537 amino acids, including a signal peptide with a molecular mass of 58 kDa (Shibahara et al., 1986). Sequence analysis of the BALB/c mouse TRP1 gene revealed five base differences in the exon regions compared with the sequence of the C57BL/6 gene (Shibahara et al., 1991). One is a deletion of three nucleotides in the 5¢-untranslated region, and the other four are point mutations within the protein coding region: two missense mutations and two silent mutations (Zdarsky et al., 1990). These two missense mutations are also found in another b-mutant mouse DBA/2 (Shibahara et al., 1992). The original b mutation has been identified as a Cys-to-Tyr substitution at position 86 (codon 110) (Jackson et al., 1990; Zdarsky et al., 1990). The mutant TRP-1 containing Tyr-110 is not reactive with an anti-TRP-1 monoclonal antibody TMH-1 (Shibahara et al., 1992).

TRANSCRIPTIONAL REGULATION OF MELANOCYTE FUNCTION

The mouse TRP1 gene is about 15~18 kb long and organized into eight exons (Jackson et al., 1991; Shibahara et al., 1991). Jackson et al. (1991) assigned several potential transcriptional initiation sites, and one of them is the same as that assigned by Shibahara et al. (1991). In this review, we have used the numbering that starts with the upstream initiation site (position 1) (Jackson et al., 1991); thus, the initiation site assigned by Shibahara et al. (1991) is the nucleotide at position 5. The promoter function of the mouse TRP1 gene was analyzed using an in vitro transcription system, suggesting that the DNA segment (position –34 to +158) is sufficient to direct pigment cell-specific transcription in melanoma whole cell extracts but not in HeLa whole cell extracts (Shibahara et al., 1991). On the other hand, through transient transfection assays, Lowings et al. (1992) have identified the regulatory region consisting of both positive and negative elements, which confers efficient expression on a reporter gene in pigmented melanoma cells but not in nonmelanoma cells. The positive element contains the nonconsensus octamer motif, 5¢ATTTGAAT-3¢, and has been shown to be bound in vitro by the ubiquitous OCT-1 transcription factor. In addition, a minimal promoter extending between –44 and +107 is sufficient for cell type-specific expression, and it contains the 11bp cis-acting element, termed the M-box. The M-box contains the CATGTG motif and is required for a basal level of expression of a reporter gene in B16 mouse melanoma cells (Lowings et al., 1992). Yasumoto et al. (1995) have found through transient expression analysis that MITF transactivates the mouse TRP-1 promoter, mainly via the M-box. The structure of human TRP-1 has been deduced from its cDNA sequence (Chintamaneni et al., 1991; Cohen et al., 1990), sharing about 93% amino acid identity with a mouse counterpart. Human TRP-1 is composed of 527 amino acids and is shorter than mouse TRP-1 by 10 amino acids at the carboxyl-terminus (Cohen et al., 1990). Human TRP-1 is also known as a melanoma antigen gp75, which is potentially autoimmunogenic, because immunoglobulin G (IgG) antibodies against gp75 are detected in a patient with metastatic melanoma (Vijayasaradhi et al., 1990). Subsequently, an informative single base change was found in the full-length human TRP-1 cDNA, pHT2a, that was isolated from a MeWo human melanoma cDNA library (Takimoto et al., 1995); namely, the HindIII site (AAGCTT) present in the reported sequence (Cohen et al., 1990) is disrupted in pHT2a because of the Cto-T transition at position 750. The human TRP-1 gene contains the C residue at position 750, favoring the sequence previously reported, which was derived from a different human melanoma cell line (Cohen et al., 1990). This base change is not associated with an amino acid substitution and may represent polymorphism. The human TRP1 gene has been mapped to chromosome 9p22–pter (Chintamaneni et al., 1991), and the 5¢ flanking region and exon 1 of the TRP1 gene have been characterized and the promoter function assessed by transient expression assays (Shibata et al., 1992b). The structural organization of

the human TRP1 gene has been characterized (Sturm et al., 1995). Unlike the mouse TRP1 gene (Yasumoto et al., 1995), the pigment cell-specific promoter function of the human TRP1 gene was not detected by transient expression assays in cultured cells with the 5¢ flanking region of up to 3.5 kb (Shibata et al., 1992b; Yasumoto et al., 1997; Yokoyama et al., 1994b).

DOPAchrome Tautomerase/Tyrosinase-related Protein 2 The phenotype of homozygous slaty mice includes the dilution of coat color and premature hair loss. It was proposed that DCT functions to prevent the accumulation of dihydroxyindole, a more toxic product of DOPAchrome generated spontaneously (Jackson et al., 1992). The DCT gene was mapped to the slaty locus on mouse chromosome 14 and is mutated at the slaty locus, which is an arginine to glutamine change at the first copper binding site (Jackson et al., 1992). It was then confirmed by transient expression assays of cDNA that mouse TRP-2 possesses the catalytic activity of DOPAchrome tautomerase and the slaty mutation dramatically decreases the catalytic function (Kroumpouzos et al., 1994). The mouse DCT gene is expressed in migratory melanoblasts shortly after they left the neural crest (Steel et al., 1992); expression of the DCT gene precedes that of the tyrosinase and TRP1 genes. Likewise, DCT expression is detected in the developing telencephalon and the endolymphatic duct, in which expression of the tyrosinase and TRP1 genes was undetectable (Steel et al., 1992). Furthermore, DCT mRNA is expressed in a glioblastoma (Suzuki et al., 1998) and the retinoblastoma (Udono et al., 2001). Thus, the regulation of DCT gene expression is different from that of tyrosinase and TRP1. The structure of the mouse DCT gene has been reported (Budd and Jackson, 1995), and its promoter region contains the M-box. Human DCT shares about 40% amino acid identity with human tyrosinase or TRP-1 (Bouchard et al., 1994; Cassady and Sturm, 1994; Yokoyama et al., 1994a). It has also been confirmed that DCT possesses the catalytic activity of DOPAchrome tautomerase by transient expression assays of a human cDNA (Yokoyama et al., 1994a). The human DCT gene has been mapped to chromosome 13q32 (Bouchard et al., 1994). The 5¢ flanking region and the portion of the human DCT gene have been cloned and characterized (Sturm et al., 1995; Yokoyama et al., 1994b). Comparison of the nucleotide sequence with that of the other tyrosinase gene family members reveals two putative cis-acting elements (–138/–128 and –34/–21), which are similar to those of the M-box (Lowings et al., 1992) and a pigment cell-specific promoter (Shibahara et al., 1991) respectively. The M-box of the human DCT gene (–138 to –128) is identical to the equivalent element of the mouse DCT gene. Transient expression assays revealed that the 5¢ flanking region of the human DCT gene upstream from a luciferase 251

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reporter gene is more efficiently expressed in MeWo melanoma cells than in HeLa cells (Yokoyama et al., 1994b), indicating the presence of the pigment cell-specific regulatory element(s). It was then found that the 32-bp regulatory element responsible for pigment cell-specific expression is located between –447 and –415, which was termed the DCT distal enhancer (Amae et al., 2000) (Fig. 13.3), although this enhancer alone is not sufficient to confer cell type-specific expression. An additional element, located between –268 and –56, is required for basal transcription, but again this element by itself is not sufficient to confer pigment cell-specific expression. The latter DNA segment (–268/–56) was tentatively termed the proximal region. Notably, it has been shown that the 71-bp region (–415/–345), immediately downstream from the DCT distal enhancer, is required for activation of the DCT promoter by neurofibromin (Suzuki et al., 1998). Thus, multiple regulatory elements are required for the pigment cell-specific transcription of the DCT gene. Yasumoto et al. (1997) have reported that MITF is unable to trans-activate the DCT promoter in transient transfection assays, despite the fact that MITF binds in vitro to the DCT M-box. Subsequently, it has been confirmed that the M-box of the DCT promoter is bound by MITF-M in vivo (Takeda et al., 2003). Furthermore, an experimentally truncated MITF protein lacking the carboxyl-terminal 125 amino acids activated the tyrosinase promoter less efficiently than did MITF but, surprisingly, the truncated MITF activated the DCT promoter (Yasumoto et al., 1997). These results suggest that the carboxyl-terminus of MITF contains a transcriptional activation domain that is also involved in defining the binding sites for MITF. The synergism between LEF-1 and MITF-M is responsible for the transcriptional regulation of the DCT gene but through a different mechanism from that of the M promoter (Saito et al., 2002; Yasumoto et al., 2002). DCT may be important for the survival of melanocytes. The finding that the vit mutation impairs the synergism with LEF-1 on the DCT promoter but not on the M promoter is of particular interest in view of the phenotype of homozygous Mitf vitiligo (Mitf vit) mice that appear normal as young with uniformly lighter color but show aging-dependent melanocyte loss (Lerner et al., 1986). The Asp222Asn substitution in the helix 1 of Mitf-M represents a molecular lesion of the recessive Mitf vit (Steingrímsson et al., 1994), and the Mitf-Mvit protein is able to bind in vitro to DNA (Hemesath et al., 1994). Thus, the Mitf vit mutation does not profoundly alter the fetal development of melanocytes but impairs the postnatal maintenance of follicular melanocytes, which could be explained by the differential effects of the Mitf vit mutation on the synergism with LEF-1 on the M promoter and the DCT promoter (Saito et al., 2002).

Pmel-17 Protein or Silver Locus Protein A portion of cDNA coding for Pmel-17 protein was initially cloned by screening a human melanocyte cDNA expression library with antityrosinase polyclonal antibodies (Kwon et al., 1987b). A full-length Pmel-17 cDNA was subsequently iso252

lated, and Pmel-17 protein is composed of 645 amino acids with a molecular mass of 68.6 kDa (Kwon et al., 1991). Pmel17 protein contains a putative leader sequence, a potential membrane anchor segment, and relatively high numbers of serine and threonine residues. There are five restricted regions of amino acid similarity among tyrosinase, TRP-1, and Pmel17. The Pmel-17 gene has been mapped to human chromosome 12pter–q21, and its mouse homolog has been mapped to the distal region of mouse chromosome 10, a region known to carry the coat color locus silver (si) (Kwon et al., 1991). The recessive silver mutation causes progressive graying of hair due to the loss of functional follicular melanocytes. The silver gene, a mouse homolog of Pmel-17, is thought to act at the subcellular level and is preferentially expressed in melanocytes (Kwon et al., 1987b). Kwon et al. (1995) found that the mouse silver mutation is caused by a single base insertion in the putative cytoplasmic domain of the mouse homolog of Pmel-17 protein. Pmel-17 is expressed in RPE, because the two cDNA clones derived from chicken and bovine RPE turned out to be a homolog of Pmel-17. The former cDNA encodes a melanosomal matrix protein MMP115 of chicken RPE (Mochii et al., 1991), and the latter encodes the bovine retinal pigment epithelial protein RPE-1 (Kim and Wistow, 1992). In particular, MMP115 was shown to be a useful marker for studying the molecular mechanisms for the differentiation of RPE, because the MMP115 gene was transcriptionally inactivated during the process of dedifferentiation of RPE to a bipotent cell, which can be committed to either the lens cell or RPE (Mochii et al., 1988). Furthermore, expression of Pmel-17 mRNA was remarkably increased following the treatment of murine or human melanoma cells with b-MSH or 3-isobutyl1-methylxanthine (Kwon et al., 1987b), and thus the regulation of its gene expression is of interest.

Transcription Factors Acting at Retinal Pigment Epithelium Correct specification and differentiation of RPE underlie eye morphogenesis, and the impairment of RPE development could lead to severe malformation of the eye, such as anophthalmia (Hero et al., 1990). RPE is derived from the optic cup of embryonic brain and differentiates into a single layer of cells interposed between the neural retina and the vascular-rich choroids, thereby constituting the blood–retinal barrier. At the apical side of RPE, many villi interdigitate with the outer segments of photoreceptors, by which RPE is responsible for phagocytosis of outer segments of photoreceptors (Bok, 1988) and for uptake, processing, and transport of retinoids that are essential for visual function (Bok, 1993). RPE therefore plays important roles in the survival and function of photoreceptors in the adult eye. The mutant homozygous mice for Mitf exhibit depigmentation and hyperproliferation of the RPE and the formation of an ectopic neuroretina at the expense of the dorsal RPE, resulting in microphthalmia (Silver, 1979). In the homozygous Mitf VGA-9 mice, where Mitf is not expressed, expression of

TRANSCRIPTIONAL REGULATION OF MELANOCYTE FUNCTION

tyrosinase and TRP1 genes is reduced in RPE, suggesting that MITF is required for efficient transcription of the tyrosinase and TRP1 genes in RPE as well as in melanocytes (Nakayama et al., 1998). But Mitf is dispensable for Dct expression, as expression of Dct is not affected in Mitf VGA-9 mice (Nakayama et al., 1998). MITF-A is preferentially expressed in a human RPE line of fetal origin and is also expressed in many cell types (Amae et al., 1998; Fuse et al., 1999; Udono et al., 2000). Domain A of MITF-A shares significant amino acid identity with the N-terminus of TFE3 (Rehli et al., 1999; Yasumoto et al., 1998). In addition, three consecutive portions, covering the entire domain A, are aligned to the equivalent portions of cytoplasmic retinoic acid-binding protein (CRABP) (Shibahara et al., 2001). Such similarity is of interest, in view of the phenotype of a recessive Mitf mutant, Mitf vit (Lerner et al., 1986; Steingrímsson et al., 1994), which shows late-onset retinal degeneration and abnormalities in retinoid metabolism (Smith et al., 1994). It is therefore conceivable that the Asp-to-Asn substitution in the helix 1 of MITF-A or other MITF isoforms may impair the interaction with a hitherto unidentified protein in RPE. Transcription of the DCT gene is activated by OTX2 (Takeda et al., 2003). OTX2 is a homolog of the Drosophila homeobox-containing gene orthodenticle (otd), a member of a highly conserved family of homeodomain-containing transcription factors, and is expressed not only at the earliest stage of eye morphogenesis including prospective RPE (MartinezMorales et al., 2001; Simeone et al., 1992, 1993), but also in RPE of the postnatal and adult mouse eye (Baas et al., 2000). In addition, Otx2 null homozygous mice exhibit the absence of the forebrain and embryonic lethality (Acampora et al., 1995), whereas the heterozygotes survive until birth but exhibit anomalies in RPE and retinal development (Matsuo et al., 1995). OTX2 interacts with MITF and synergistically activates tyrosinase and TRP1 genes in RPE (MartinezMorales et al., 2003). It has also been reported that MITF interacts with PAX6 in RPE (Planque et al., 2001). PAX6 is a member of the pairedhomeodomain family of transcription factors (Walther and Gruss, 1991), mutations in which cause the aniridia syndrome in humans (Glaser et al., 1992) and the small eye phenotype in mice (Hill et al., 1991). The interaction with PAX6 and MITF abolishes the DNA binding of MITF, leading to a reduction in MITF-mediated activation in RPE (Planque et al., 2001). Several RPE-specific proteins were identified, including tyrosinase family genes, and cDNAs coding for some of them were cloned. A typical example of such proteins is Pmel-17, also known as MMP115 or RPE-1, which was initially cloned as an RPE-specific gene (Kim and Wistow, 1992; Mochii et al., 1991). Another example is RPE65, which was cloned as a novel RPE-specific microsomal protein with a tissue-specific monoclonal antibody RPE9 (Hamel et al., 1993). Bovine RPE65 consists of 533 amino acid residues with no predicted transmembrane domains. RPE65 mRNA was detected in RPE

but not in other ocular tissues, suggesting that RPE65 is an RPE-specific protein. The human RPE65 gene has been mapped to chromosome 1p31 (Hamel et al., 1994). The gene for ocular albinism type 1 (OA1), located at chromosome Xp22.3–22.2, was identified by positional cloning (Bassi et al., 1995). OA1 is an X-linked disorder characterized by severe impairment of visual acuity, retinal hypopigmentation, and the presence of macromelanosomes. The OA1 gene encodes a protein of 424 amino acids with several putative transmembrane domains and is preferentially expressed in retina as well as in melanoma. Five intragenic deletions and a CG dinucleotide insertion causing a premature stop codon were identified in unrelated OA1 patients. It is of interest to study the pathogenesis of OA1, because the OA1 gene is not RPE specific and is also expressed in melanoma, like many other pigment-related genes. The tyrosinase family genes therefore provide a good system to study the mechanism responsible for RPE-specific gene expression, which in turn allows us to compare regulation in melanocytes.

Perspectives The MITF gene consists of widely spaced multiple promoters, which generate not only the diversity in the transcriptional regulation of these promoters but also the structurally different isoforms. The MITF gene provides a good model for studying the mechanism of promoter selectivity during development or under certain metabolic conditions. The isoform multiplicity of MITF also provides functional diversity and redundancy. Future analysis of MITF isoforms in vivo is required to assess the role of each MITF isoform in differentiation and development of the pigment cell and other cell types. It is also important to identify the interacting partners with the unique N-terminal domains as well as the bHLH-LZ domain that modulate the function of MITF isoforms. Such studies will increase our understanding of the regulation of development and differentiation of the pigment cell. Development of gene therapy and chemotherapy toward melanoma are definitely major goals of pigment cell research. Retinal degeneration of diverse etiologies, either acquired or inherited, accounts for a major cause of aging-dependent visual impairment and blindness in the developed world. It is therefore of significance to understand the mechanism by which various transcription factors maintain the survival or function of RPE in the adult retina. Furthermore, the elucidation of the difference in transcriptional regulation of the tyrosinase family genes between RPE and melanocytes is of particular significance. It is also challenging to explore the possibility of cell therapy targeted for RPE in order to deliver a healthy RPE into an impaired retina.

Acknowledgments We thank Dr Hiroaki Yamamoto (Tohoku University) for 253

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helpful discussions. We also thank Ken-ichi Yasumoto, Kazuhiro Takahashi, and other colleagues who contributed to the work on pigment cells.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

14

Enzymology of Melanin Formation Francisco Solano and José C. García-Borrón

Summary 1 Pigmentation is a widely occurring phenomenon. In microorganisms, plants, and lower animals, carotenoids, anthocyanins, tetrapyrroles, and betalains are pigments designed to afford several features, but other pigments are needed for chemo- and photoprotection. These pigments are generically called melanins, and most of them are formed from phenols. In higher animals and mankind, these pigments are found in the integument regions, retina, and inner ear. 2 The key enzyme of the melanogenic pathway is tyrosinase. This enzyme catalyzes the first rate-limiting steps of melanogenesis, the hydroxylation of l-tyrosine, and the subsequent oxidation of the intermediate l-dopa to yield l-dopaquinone. 3 Tyrosinase contains a bicupric active site. The two copper atoms bind to two regions called CuA and CuB, which contain three highly conserved histidines responsible for metal binding. Copper atoms can undergo redox reactions, and tyrosinase catalysis can be explained by the redox cycling of copper between the Cu(II) and Cu(I) states, with the participation of molecular oxygen. 4 The two types of reactions catalyzed by tyrosinases, namely hydroxylation of monophenols and oxidation of diphenols, display different kinetic properties and likely involve a differential interaction of the substrates with the active site. l-Dopa bound directly to tyrosinase is not equivalent to l-dopa as an intermediate of the tyrosine hydroxylation. 5 Tyrosinases from different sources may display different substrate specificities. In particular, mouse tyrosinase does not recognize 5,6-dihydroxyindole-2-carboxylic acid (DHICA) as a substrate, but the human enzyme shows DHICA oxidase activity. 6 In higher animals, there are two other melanosomal proteins displaying extensive sequence similarity to tyrosinase. They are called tyrosinase-related proteins (Tyrps) 1 and 2. 7 Although a number of enzymatic functions have been proposed for Tyrp1, the mouse protein is most likely a low specific activity pseudotyrosinase recognizing DHICA as substrate. However, as human tyrosinase has a similar DHICA oxidase activity, the actual role of TYRP1 remains uncertain. 8 Tyrp2/Dct is a Zn-containing protein. It catalyzes the nondecarboxylative tautomerization of dopachrome to DHICA. As the product of the spontaneous evolution of dopachrome is 5,6-dihydroxyindole (DHI), Tyrp2/Dct is essential for the incorporation of carboxylated indolic units to the melanin. 9 Within melanosomes, tyrosinase and Tyrps may interact in

a melanogenic complex. The functional consequences of their interactions within this metabolon are not well understood, although Tyrp1 and probably also Tyrp2/Dct seem to stabilize tyrosinase, thus increasing its in vivo half-life. 10 Other proteins, such as silver/Pmel-17, P, and the MATP/AIM-1/underwhite protein, are involved in melanogenesis, although their role is probably not enzymatic and may be either structural or related to the regulation of melanosomal pH and transport. 11 Radiometric methods are best suited for the determination of the rate-limiting tyrosine hydroxylase activity (using l-[3,53 H]-tyrosine as substrate) and of the overall melanogenic potential (using l-[U14C]-tyrosine as substrate). 12 Fluorometric assays for tyrosine hydroxylase activity, designed as sensitive alternatives to the radiometric methods, have not been used as frequently. 13 Spectrophotometric assays, especially those based on the trapping of o-quinones by 3-methyl-2-benzothiazolinone hydrazone (MBTH), are sensitive and simple, and therefore the methods of choice for the determination of the oxidation rate of diphenols and dihydroxyindoles. However, at least in mouse melanocyte extracts, the dopa oxidase activity is associated with tyrosinase and Tyrp1, and the relative contribution of each enzyme cannot be directly determined by spectrophotometric assays. This can be only performed by nonreducing gel electrophoresis, followed by activity stains with radioactive substrates or l-dopa plus MBTH. 14 DHICA oxidase, dopachrome tautomerase, and related activities are best determined by high-performance liquid chromatography (HPLC) assays, allowing for a simultaneous determination of substrate consumption and product formation. 15 In spite of the limitations of the currently available methods for the determination of melanogenic enzymatic activities, it is clear that the type, amount, size, and functional properties of melanins are determined not only by tyrosinase, but also by the Tyrps and probably by other above-mentioned proteins with enzymatic activity that is still doubtful. Moreover, the existence of other uncharacterized melanogenic enzymes and/or effectors cannot be ruled out, particularly in the pheomelanogenic pathway.

Introduction to Pigments, Melanin, and Phenol Oxidases: a Historical Background Pigmentation is a widely occurring phenomenon, which has aroused the attention of mankind since the beginning of 261

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history because of the importance of appearance for all living organisms. There are a great variety of natural pigments with different composition and structure. In microorganisms, plants, and lower animals, carotenoids, anthocyanins, tetrapyrroles, and betalains are responsible for most of the colors that we observe (Delgado-Vargas et al., 2000). Most of them are pigments designed to afford environmental advantages, such as camouflage or thermal regulation. Color ornaments are undoubtedly important for sexual processes (from flowers/insects to tropical birds or even mammals). Other pigments are especially designed for chemo- and photoprotection. These pigments are generically called melanins. Their range of colors is not as wide, but melanins are the most abundant and ubiquitous natural pigments throughout the phylogenetic scale. In lower organisms, several types of precursors lead to different types of chemically distinct melanins, but most of them are phenols. Only fungi can form a melanin from acetyl groups through the pentaketide pathway to yield dihydroxynaphthalene-melanin (Tsai et al., 1999). In vertebrates, melanins are formed from the amino acid l-tyrosine by the Raper–Mason pathway. These pigments are found in the integument regions, retina, and inner ear. Although the amount of pigment is genetically determined, in the epidermis, this parameter is also affected by environmental factors, such as exposure to sunlight, as well as by hormonal levels. Tyrosinase, the key enzyme in melanogenesis, is a remarkable protein able to catalyze two types of reactions, hydroxylation of monophenols and oxidations of diphenols. This “ferment” was one of the first enzymes to be reported at the end of the nineteenth century (Bourquelot and Bertrand, 1895). Bourquelot studied up to 281 species of phanerogams for a classification based on pigmentation. He found a large number of pigments, most of them glycosides (some of them still important in pharmacology). Looking for the agent responsible for the massive formation of dark pigments, and using mushrooms as a convenient model, he described one of the first oxidizing ferments in the early age of enzymology, and his report was the first one on the enzyme generically called phenol oxidase and, more specifically, tyrosinase. During the first quarter of the twentieth century, tyrosinase activity was demonstrated in the animal kingdom, and it was also found in cutaneous melanoma from several mammals (horse, hamster, mouse, and others; for a comprehensive review of the state of knowledge at that stage, see Lerner and Fitzpatrick, 1950). The existence of dopa oxidase activity in human skin was first reported by Bloch (1927) by histochemical staining of pigmented human skin specimens immersed in dopa solutions. Because of a very special feature of the tyrosine hydroxylase activity of tyrosinase (a lag period of highly variable length before reaching maximal catalytic rate), this activity was initially very difficult to detect. For a long time, this led to some controversy concerning the double catalytic activity of tyrosinase (tyrosine hydroxylase and dopa oxidase) and the putative existence of two different enzymes to account for those activities. The hypothesis of the occurrence of two inde-

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pendent enzymes subsisted for many years (Patel et al., 1971; Shapiro et al., 1979). However, Mason (1948), Lerner et al. (1949), and others showed that mammalian tyrosinase was a bifunctional enzyme and proved that tyrosine hydroxylase and dopa oxidase activities reside on the same protein. Even in those years, the same conclusion could be drawn from genetic data. Tyrosinase is encoded by the albino c-locus (chromosome 7 in mouse and 11 in human), and it was perfectly demonstrated that albino mice were negative not only for tyrosine hydroxylation but also for dopa oxidation (Russell and Russell, 1948). Nowadays, the single-enzyme theory for tyrosine hydroxylation and dopa oxidation is totally accepted. Owing to the highly reactive nature of l-dopaquinone, the product of tyrosinase action on l-tyrosine, melanins are formed in vitro from l-tyrosine in the presence of tyrosinase alone. This led to the simplistic view that the melanogenic pathway involved only one enzymatic protein, a model that applies to melanogenesis in bacteria and lower organisms. However, the situation is far more complex in higher organisms and particularly in mammals, where several other proteins residing in a specialized melanin-synthesizing organelle, the melanosome, are involved. Therefore, the melanogenic pathway can be considered at the same time simple and complex. It is simpler in lower organisms and more sophisticated in higher organisms where the regulation of melanosynthesis (in terms of amount, type, and location) is an important point. Thus, the study of melanogenesis is a journey from the simplicity of a pathway involving just tyrosinase as the only melanogenic enzyme (in prokaryotes) to the complexity of a multienzymatic complex confined in a specific subcellular compartment where melanogenesis is subject to tight regulation of its rate and of the type of melanin formed (in mammalian melanocytes). The present chapter will describe different aspects of the enzymes involved in the formation of melanin through the modified Raper–Mason pathway, including those “distal” factors whose action takes place after the reactions catalyzed by tyrosinase. We also refer to Chapters 3, 7, 10, 11, 12, and 15 of the present edition for details on the ultrastructure of the melanocyte, the melanosome, the tyrosinase family, and the chemical structure of the melanin pigment. All this information will offer an integrated view of mammalian melanogenesis.

A Global View of the Mammalian Melanogenesis Pathway The key enzyme of the melanogenic pathway is tyrosinase. This enzyme catalyzes the first two rate-limiting steps of melanogenesis, the hydroxylation of l-tyrosine and the subsequent oxidation of the intermediate o-diphenol (l-dopa) to yield l-dopaquinone (Fig. 14.1). The mechanism of these reactions has been controversial and will be discussed below. l-

ENZYMOLOGY OF MELANIN FORMATION

dopaquinone is the first branch point of melanogenesis leading to the two main types of melanin in animals: light (from red to yellow) pheomelanin or dark (from brown to black) eumelanin (Hearing and Tsukamoto, 1991). In the presence of compounds with free thiol groups, such as reduced glutathione or the amino acid l-cysteine, there is a rapid conjugation between l-dopaquinone and the thiol group to yield a family of sulfur-containing compounds related to pheomelanin (Jara et al., 1988a; Rorsman et al., 1973; Prota, 1988). Whenever free thiol compounds are present in sufficient concentration, this pheomelanogenic route is favored over eumelanogenesis. The pheomelanogenic intermediates and the factors that control thiol availability within melanosomes are still poorly known. The preferred position for the addition of the thiol group to l-dopaquinone is 5 (Prota, 1992), but other free positions in the L-dopaquinone ring such as 2 and 6 are also reactive, so that a complex mixture of isomers is in fact obtained. These reactions occur spontaneously. Huge efforts have been made to isolate from melanocytes an enzyme able to catalyze these reactions in a more regulated and ordered way. In this sense, g-glutamyl transpeptidase and some glutathione transferases were proposed (Mojamdar et al., 1983), but so far their role in pheomelanogenesis is, at best, unclear. Recently, it has been proposed that g-glutamyl transpeptidase does not contribute

significantly to pheomelanosynthesis, but it may stimulate the production of hydrogen peroxide and regulate the expression or activity of some transcription factors, ultimately leading to decreased tyrosinase activity (Chaubal et al., 2002). To give a simplified model of pheomelanogenesis (Fig. 14.1, right), it is usually presumed that l-cysteine reacts with l-dopaquinone to yield mainly 5-cysteinyldopa. This compound may undergo some structural rearrangements and dehydration to form alanyl-hydroxy-benzothiazine, the proposed subunit for pheomelanin. It is nevertheless possible that pheomelanin biosynthesis actually proceeds through a more complex pathway. For instance, it is known that glutathione is much more abundant inside cells than l-cysteine, so that 5-glutathionyldopa is likely the first thiol-conjugated species formed. The release of 5-cysteinyldopa would then be catalyzed by a dipeptidase (Agrup et al., 1975; route not shown in Fig. 14.1 to avoid further complexity). This view of the melanogenic pathway including a glutathione-dependent branch may still be an oversimplification of the in vivo situation, because the involvement of still uncharacterized enzymatic activities is likely. Returning to the branch point after l-dopaquinone formation, this compound can undergo a cyclation reaction by Michael addition of the amino group on position 6 of the ring to form l-cyclodopa (also named leukodopachrome), and

Fig. 14.1. The mammalian melanogenic pathway. This updated version of the pathway was originally proposed by Raper (1928) and Mason (1948) taking into account only tyrosinase, but several enzymatic activities in addition to tyrosinase are involved. Concerning the evolution of l-dopachrome in the distal phase, note that, at neutral pH, it undergoes a decarboxylative reaction to dihydroxyindole. This is a spontaneous reaction, but it can be catalyzed by “dopachrome isomerase” (an enzyme related to dopachrome tautomerase from insects and cuttlefish). In mammalian melanocytes, l-dopachrome can undergo a nondecarboxylative rearrangement catalyzed by authentic Tyrp2/Dct. The pathways for pheomelanin synthesis are less well understood than those for eumelanin synthesis, mostly through the contribution of Prota (1988, 1992).

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finally dark eumelanins. This reaction is favored as the pH is increased, because the amino group should be nonprotonated for the nucleophilic attack on position 6. Therefore, at the acidic intramelanosomal pH, cyclation may be rather slow, and other side-reactions such as formation of topaquinone are also possible (Rodríguez-López et al., 1992; not shown in Fig. 14.1). It should be clearly stated that l-dopaquinone cyclation is slower than its conjugation with thiols and other reactive groups. Therefore, l-cyclodopa is only formed in the absence of other chemical alternatives for l-dopaquinone, but this is probably the situation inside the eumelanosome. Accordingly, eumelanin is the main pigment formed in many organisms. l-Cyclodopa is a rather unstable reducing species that reacts rapidly with its precursor l-dopaquinone to yield ldopachrome plus l-dopa (Garcia-Cánovas et al., 1982; Lerner and Fitzpatrick, 1950). Note that half the l-dopa oxidized by tyrosinase to l-dopaquinone is reduced back to l-dopa (Fig. 14.1). This is very important as l-dopa is not just a tyrosinase substrate but also a fine regulator of the tyrosine hydroxylase activity of the enzyme (see below). The chemical generation of l-dopa in this redox reaction can account for the in vivo activation of l-tyrosine hydroxylation. Both ldopaquinone cyclation to l-cyclodopa and the redox reaction to l-dopachrome seem to be very fast reactions proceeding without enzymatic control. l-Dopachrome can be considered the second branch point in melanogenesis leading to different eumelanins. This branch is regulated by the tyrosinase-related proteins (Tyrps, discussed below), and it controls the final content of carboxyl groups in the melanin polymer, which is very important for the size, chelating, and absorption properties of the eumelanin formed. Within a pH range from 3 to 9, including the physiological pH values in melanosomes, l-dopachrome undergoes a spontaneous decarboxylation to form 5,6-dihydroxyindole (DHI). However, Tyrp2/dopachrome tautomerase (Dct) catalyzes the nondecarboxylative rearrangement of ldopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). It is worth noting that traces of metal ions can yield mixtures of DHI and DHICA but, inside the eumelanosome, the level of Dct activity controls the DHICA/DHI ratio (Aroca et al., 1992). This ratio increases at higher Dct and lower tyrosinase activities, but it should be stated that, within melanosomes, there is always a mixture of both indolic species, owing to the spontaneous nature of l-dopachrome decarboxylative rearrangement to DHI at physiological pH. The next step after the formation of 5,6-dihydroxyindoles is their oxidation to 5,6-indolequinones. Oxidation of DHI in aerobic conditions occurs spontaneously at a significant rate, although DHI is a substrate of tyrosinase, so that this enzyme accelerates the reaction. The oxidation of DHICA is more complex. First, spontaneous oxidation is much slower for DHICA than for DHI because of the effect of the carboxyl group at position 2, so that DHICA oxidation in vivo is most likely an enzymatic reaction. Second, this group has 264

important effects on the affinity of the melanogenic enzymes from different mammalian species for the carboxylated indole. Mouse Tyrp1 shows DHICA oxidase activity, but mouse tyrosinase does not (Jiménez-Cervantes et al., 1994; Kobayashi et al., 1994). However, human tyrosinase shows DHICA oxidase activity (Olivares et al., 2001). Therefore, it is possible that the oxidation of DHICA in vivo is carried out by different enzymatic proteins, Tyrp1 or Tyr, depending on the particular mammalian species considered. This opens the question of the physiological role of Tyrp1 in some species, especially in human melanocytes (see section dedicated to this protein), which has been and still is a controversial point. The next steps in eumelanogenesis are poorly characterized. 5,6-Indolequinones (decarboxylated or not) can react with dihydroxyindoles to form semiquinones. These species can spontaneously polymerize in an unordered and intermixed way to form eumelanin. The involvement of the silver protein as an accelerating factor in these last steps was proposed (Chakraborty et al., 1996; Lee et al., 1996), but a catalytic activity has never been definitively proven for this protein. Although this point will be also discussed later, it is clear that the silver protein undergoes a proteolytic process within the eumelanosome that provokes its aggregation and contributes to the formation of the lamellar network of the organelle. These lamellae may trigger melanin deposition (Solano et al., 2000). However, the mechanisms that regulate these processes, from silver protein aggregation to the promotion and binding of indolic units to presumably form a final melanoprotein product containing the silver protein and chains of the melanin polymer, are so far unknown. The size and order of these chains should depend on the initial DHI/DHICA ratio (Aroca et al., 1992; Pawelek, 1991). There is no doubt that l-tyrosine is the main amino acid precursor of melanin in higher organisms, but small amounts of l-tryptophan can also be incorporated into the pigment under special conditions such as an oxidative environment (Blagoeva, 1984; Chakraborty et al., 1986) or in specialized tissues in goldfish or butterflies (Chen and Chavin, 1965; Ubemachi, 1985), yielding pseudomelanin pigments such as papiliochrome and others. This role of l-Trp as a melanin precursor should be considered a highly specialized and rare aspect of melanogenesis, even less frequent than other unusual melanogenic pathways such as, for instance, the fungal pentaketide pathway leading to the dihydroxynaphthalene melanins mentioned above (Tsai et al., 1999). The study of these minor melanins and the enzymes involved in the corresponding pathways are beyond the scope of the present chapter.

The Enzymes Involved in Mammalian Melanogenesis According to Figure 14.1, to date, there are only three enzymes with well-established involvement in mammalian melanogenesis. Tyrosinase is undoubtedly the most important one.

ENZYMOLOGY OF MELANIN FORMATION

Sequence comparison of Tyr, Tyrp1 and Tyrp2/Dct reveals that the three proteins share many key structural features, due to their common origin from a single ancestral gene (Jackson, 1994). They are integral membrane proteins displaying one single membrane-spanning fragment near their C-terminus that anchors them to the melanosomal membrane. More importantly, they share two very similar metal ion binding sites essential for their catalytic action, although the nature of the metal cofactor is not the same. They undergo similar but not identical post-translational processing, including several glycosylation steps in the endoplasmic reticulum (ER) and trimming of the carbohydrate chains in the Golgi network. Glycosylation appears to be crucial for acquisition of full enzymatic activity (Halaban et al., 2000). However, in spite of their extensive sequence similarity, Tyr, Tyrp1, and Dct show remarkable differences in their catalytic abilities. For this reason, the Tyr family has been the subject of intense investigation, not only per se as the mammalian melanogenic machinery but also as an excellent model to study structure–function relationships as well as the molecular basis of divergent functional evolution. Next, we summarize the main properties of the three enzymes and their role(s) in melanogenesis.

Tyrosinase Tyrosinase (monophenol l-dopa: oxygen oxidoreductase, EC 1.14.18.1) is the key enzyme in melanogenesis. It is a glycocopperprotein that catalyzes the first two steps of the pathway, hydroxylation of l-tyrosine (cresolase activity) and oxidation of the o-diphenolic intermediate l-dopa to l-dopaquinone (catecholase activity). Tyr contains a pair of antiferromagnetically coupled copper ions at the active site (Lerch, 1983; Lerner et al., 1949). This coupling is the reason why tyrosinase is not a blue copper electron paramagnetic resonance (EPR)-silent protein in comparison with other copper enzymes such as laccases. So far, mammalian tyrosinase has not been crystallized, probably because it is a microheterogeneous transmembrane glycosylated protein. However, a variety of data based on the damage of H residues after photoinactivation of fungal tyrosinases, the sequence similarity among tyrosinases and hemocyanins (Himmelwright et al., 1980; Huber and Lerch, 1988), and the available crystallographic data on a plant catechol oxidase (Klabunde, 1998) indicate that two copper ions are bound to the protein in a hydrophobic pocket formed by two H-rich peptidic stretches named CuA and CuB. Both CuA and CuB sites contain three conserved H residues that are adjacent and form the binuclear enzyme’s active site due to the folding of the protein (GarcíaBorrón and Solano, 2002). Moreover, this site is very similar in Tyr and Tyrps, although the amino acidic conservation is higher in the CuB peptidic motif. However, it is also in CuB where the most important difference between the Tyr and Tyrps metal binding sites is found. This is related to the third H ligand of the metal ion, with tyrosinases from all sources always displaying two consecutive HH, whereas Tyrps have an LH sequence and, therefore, a single putative ligand at this

position (Martínez-Esparza et al., 1997; Olivares et al., 2002). It is usually accepted that the copper ions are bound only by three H, although some other alternatives with four ligands (one Cys and three His) are still proposed occasionally (Nakamura et al., 2000). It has long been debated whether the hydroxylase and oxidase activities of Tyr share a common catalytic site. The related issue of whether tyrosine hydroxylation and dopa oxidation are obligatorily coupled, or whether they are two independent steps, has also been discussed recently. A direct transformation of l-tyrosine into l-dopaquinone in the catalytic cycle has been proposed (Cooksey et al., 1997), whereas others claim that l-dopa is an intermediate in the oxidation of l-tyrosine that can be released from the tyrosinase active site (Fenoll et al., 2000). Whatever the case, it is clear that ldopa is formed in the pathway and is an alternative substrate for the enzyme, which is oxidized by tyrosinase to yield ldopaquinone. Therefore, the reactions underlying tyrosine hydroxylation and dopa oxidation should differ even if they share the same active site. Some differences between both reactions suggested different requirements at the reaction site (summarized by Lerch, 1983). For instance, the hydroxylase activity, but not the oxidase activity, shows a characteristic lag period before the reaction reaches maximal rate. This lag phase increases with the concentration of the substrate, l-tyrosine, and can be shortened or abrogated by catalytic amounts of l-dopa (Pomerantz, 1966). Therefore, l-dopa is the product of tyrosine hydroxylation, a cofactor for this reaction, and a substrate for the dopa oxidase reaction. In addition, the affinity of mammalian Tyr for l-dopa acting as cofactor for tyrosine hydroxylase is about two orders of magnitude higher than for the same compound acting as substrate for dopa oxidase activity (Pomerantz and Warner, 1967). Lerch (1983) proposed a mechanism to reconcile a common active site for l-tyrosine and l-dopa with the above-mentioned differential features of catalysis. This mechanism has been refined recently by Olivares et al. (2001) using mutant forms of mouse tyrosinase obtained by site-directed mutagenesis of selected active site residues. The main new contributions are related to the role of the HH pair located at the end of the CuB region and the inequivalence of both coppers. It was proposed that l-tyrosine and l-dopa dock to the tyrosinase active site in different orientations (Fig. 14.2). But the key point of the mechanism is the existence of at least three different forms of tyrosinase, called met, oxy, and deoxy, according to the absence/presence of oxygen and the oxidation state of the copper ions (Cu+2/Cu+1) in the active site. Intracellular tyrosinase exists in either the met or the oxy states, with the majority form in the absence of substrates being met-tyrosinase. Only oxy-tyrosinase is able to catalyze tyrosine hydroxylation and dopa oxidation. Met-tyrosinase readily oxidizes l-dopa, but it is inactive for l-tyrosine hydroxylation because a deadend complex is formed (Fig. 14.2). This would account for the low initial tyrosine hydroxylase activity of crude enzymatic extracts in the absence of added cofactors, characteristic of the 265

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Fig. 14.2. Mechanism of action for tyrosinase. Starting in the oxy form of tyrosinase, two catalytic cycles are depicted, dopa oxidase (DO) at the top and tyrosine hydroxylase (TH) at the bottom. Note that met-tyrosinase is involved in the DO cycle but not in the TH cycle. When ltyrosine binds to met-tyrosinase, a dead-end complex is formed, accounting for the lag period of the TH activity and the inhibition by excess of this substrate. Dopa acts as cofactor when bound to the above-mentioned met-tyrosinase. The His-389 residue is essential for l-dopa recognition and orientation to CuB (for more details, see Olivares et al., 2002). Im, imidazole.

lag phase of this activity. A progressive accumulation of ldopa would occur, mainly due to the chemical generation of this compound in the redox reaction between l-dopaquinone and l-cyclodopa during the melanogenic pathway (see Fig. 14.1). This accumulation is responsible for the acceleration of tyrosine hydroxylation. Indeed, the increase in the l-dopa/l-tyrosine ratio as the reaction proceeds leads to a concomitant decrease in the fraction of tyrosinase captured in the dead-end complex and raises the proportion of met-tyrosinase molecules recruited to the productive catalytic cycle and, therefore, the hydroxylase reaction rate. In this hydroxylation cycle, l-tyrosine bound to CuA labilizes the oxygen molecule bound in the catalytic center of oxy-tyrosinase in a side-on manner (Solomon and Lowery, 1993). The resulting polarized molecule ortho-hydroxylates the monophenolic ring of ltyrosine, and the subsequent oxidation of the o-diphenolic product to o-quinone would leave the active site in a reduced bicuprous state (deoxy-tyrosinase), allowing the entrance of a new oxygen molecule and further turnover. In the dopa oxidase cycle, l-dopa bound to CuB of oxytyrosinase would also weaken the oxygen molecule, resulting in oxidation of the organic substrate and release of l266

dopaquinone and water. The enzyme is left in a bicupric state, met-tyrosinase, that can next bind l-tyrosine or l-dopa with higher affinity than the oxy-form as the active center is not occupied by oxygen. l-Dopa may dock to the two copper ions in this form through both hydroxyl groups, so that its affinity would be higher than the affinity for oxy-tyrosinase when the binding proceeds only through CuB. This higher affinity would account for the old observation that the Ka measured for l-dopa as cofactor is in the mmolar range (Pomerantz and Warner, 1967). Thus, oxy-tyrosinase would display a higher affinity for l-tyrosine than for l-dopa, whereas the opposite should hold for met-tyrosinase. This different affinity ratio of the met- and oxy-enzymatic forms also accounts for the fact that d-dopa is a relatively efficient cofactor but a poor substrate (Olivares et al., 2001; Winder and Harris, 1991). The disposition of both hydroxyl groups bound to the copper ions would allow for an easy transfer of two electrons from the odiphenol to the binuclear site, leading to the oxidized quinone and the reduced deoxy-tyrosinase form that is again oxidized upon oxygen binding. Other interesting aspects of tyrosinase aside from the abovedescribed mechanism of catalysis, such as hormonal regula-

ENZYMOLOGY OF MELANIN FORMATION

tion, post-translational modifications including glycosylation, processing of the protein, and correlations between activity and mutations in different parts of the polypeptidic chain, are treated in other chapters in this book, so they are omitted here to avoid overlapping. This omission of crucial aspects of protein maturation and processing also applies to Tyrps, the existence and enzymatic roles of which in mammalian melanogenesis are presented next.

Tyrosinase-related Proteins Two melanosomal proteins displaying extensive sequence similarity to tyrosinase have been found in animals from fishes to mammals. They are called tyrosinase-related proteins 1 and 2 (Hearing and Tsukamoto, 1991). Together with tyrosinase, they constitute the tyrosinase family. This family likely evolved from a single ancestral gene and, in spite of a common general origin, structural similarity is higher between Tyrp1 and Tyrp2 than between these and tyrosinase.

Tyrp1 Tyrp1 was in fact the first member of the Tyr family to be cloned (Shibahara et al., 1986), in an attempt to clone the tyrosinase gene. Soon thereafter, the Tyrp1 gene was mapped to the brown locus known to affect pigmentation in mice. After the cloning of the c-locus tyrosinase gene, the homology of both genes was demonstrated, with an identity score higher than 40%, which increases substantially in the metal ion binding sites. This homology strongly suggests that Tyrp1 may be a metalloenzyme, although the nature of the metal cofactor remains unknown (Furumura et al., 1998). Tyrp1 is important for normal melanogenesis, according to a wealth of converging evidence. For instance, mice homozygous for the brown mutation in Tyrp1 display a lighter coat color than wild-type mice. Mutations in the TYRP1 gene are responsible for a subtype of human albinism, brown OCA3 (Boissy et al., 1996; Sarangarajan et al., 2000) and, in cultured human melanocytes and melanoma cells, expression of TYRP1 positively correlates with eumelanogenesis (Del Marmol et al., 1993). However, in spite of its early cloning, the specific function of Tyrp1 in the Raper–Mason pathway underlying these phenotypic effects has been very elusive and, in fact, it is still controversial. The possible enzymatic activity of mammalian Tyrp1 has been extensively investigated by several independent laboratories. Initially, mouse Tyrp1 was considered to be a second tyrosinase encoded by a different locus and showing a poor dopa oxidase activity compared with the c-locus tyrosinase (Jiménez et al., 1991). The same conclusions were obtained by our group, which for some time used the terminology HEMT and LEMT (for higher or lower electrophoretic mobility tyrosinases) to designate two proteins that could be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions and were positive for an in-gel dopa oxidase activity stain (Jiménez-Cervantes et al., 1993a). Thereafter, specific antibodies developed by V. Hearing allowed the identification of HEMT as tyrosinase and

LEMT as Tyrp1. After purification of Tyrp1 from B16 mouse melanoma, a new enzymatic activity, DHICA oxidase, was demonstrated (Jiménez-Cervantes et al., 1994). This was confirmed by transient expression of the gene in nonmelanocytic cells, as well as by the study of mutant mice (Kobayashi et al., 1994). However, the DHICA oxidase activity of Tyrp1 is quite small, and a similar activity has also been attributed to another melanosomal protein, Pmel-17 (see below). Moreover, in a recent study of DHICA oxidation by extracts from human melanoma cells and by transiently expressed human tyrosinase, it was clearly shown that this enzyme displays a DHICA oxidase activity comparable to mouse Tyrp1 (Olivares et al., 2001). Differences between human and murine Tyrp1 were also observed by Boissy et al. (1998a). This suggests the intriguing possibility that DHICA metabolism could follow different enzymatic pathways in mouse and human melanocytes and raises the question of the actual role of Tyrp1 in human melanocytes. To complicate the situation further, it was reported that transfection of the Tyrp1 gene into fibroblasts conferred dopachrome tautomerase activity to these cells (Winder et al., 1993a). Moreover, other possible catalytic activities were proposed, including a catalase (Halaban and Moellmann, 1990) or even a tyrosine hydroxylase activity devoid of dopa oxidase and responsible for the first l-dopa formed in vivo, and needed to abrogate the lag period in authentic tyrosinase (Zhao et al., 1994). Finally, and in contrast to these different enzymatic activities, it has also been suggested that Tyrp1 could be mostly devoid of enzymatic activity but show a strong stabilizing effect on tyrosinase (Kobayashi et al., 1998). In keeping with the occurrence of a stabilizing effect on tyrosinase, a direct interaction of the two proteins has been clearly demonstrated in vitro (Jiménez-Cervantes et al., 1998). In any case, a stabilizing role is not incompatible with an enzymatic activity for Tyrp1. Indeed, the key to these apparent discrepancies in the role of Tyrp1 could be related to subtle differences among species and its possible double role, on the one hand a catalytic function and on the other hand a stabilizing effect on tyrosinase. In summary, the elusive role of Tyrp1 remains unclear. However, the fact that melanogenesis proceeds in lower organisms in the absence of Tyrp1, and the phenotype associated with the brown mutation in mice and with type 3 human OCA, strongly suggests that this role should be ancillary and related to the determination of the type of pigment formed, rather than to its presence or absence.

Tyrp2 As opposed to Tyrp1, the catalytic activity of Tyrp2 is well established and clearly different from tyrosinase. The protein catalyzes the nondecarboxylative rearrangement of ldopachrome to DHICA and is therefore called dopachrome tautomerase (Dct). Before the cloning of this gene and the characterization of the enzymatic activity of the transiently 267

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Fig. 14.3. Mechanism of action for Dct/Tyrp2. The metal cofactor of this enzyme is zinc. This metal has no redox properties, excluding the participation of oxygen in the catalytic cycle. l-Dopachrome binds to the enzyme through the semiquinonic part of the molecule, and an electronic rearrangement with subsequent hydrogen migration from position 3 to position 2 leads to the tautomerization to DHICA, the product of the reaction.

expressed protein, some enzymatic activity acting on ldopachrome was detected by several laboratories. This activity was called dopachrome conversion factor (Aroca et al., 1990a; Pawelek et al., 1980), dopachrome oxidoreductase (Barber et al., 1984), and dopachrome isomerase (Jackson et al., 1992; Pawelek, 1991). Once the product of the enzyme’s action on l-dopachrome was firmly established as DHICA instead of DHI (Fig. 14.1), the more precise name tautomerase was first proposed (Aroca et al., 1990b) and soon after widely accepted. The definitive assigned EC number is 5.3.3.12, as the reaction catalyzed by Dct is a specific type of isomerization (Fig. 14.3) resulting from hydrogen migration in an intramolecular keto-enol tautomerization (International Union of Biochemistry, 1999). Initially, the actual role of Dct in melanogenesis was questioned based on the observation that metal ions are also competent in catalyzing dopachrome conversion. However, metal ions always lead to mixtures of DHI and DHICA, whereas the latter is the exclusive product of the enzymatic reaction (Palumbo et al., 1991). Moreover, metal ion-catalyzed dopachrome rearrangement is not dependent on the substrate stereospecificity, whereas Dct shows a good specificity, acting only on the natural l-dopachrome. Neither the d-stereoisomer nor the decarboxylated dopaminochrome is a substrate for mouse Dct (Aroca et al., 1991). This indicates that the carboxyl group, in an appropriate spatial orientation, should be an essential requirement for the docking of the substrate at the enzyme active site, probably through its interaction with an unidentified electrophilic residue. In this regard, it is interesting to mention that other mammalian (Matsunaga et al., 1999; Odh et al., 1993) and nonmammalian Dct-related enzymes (Palumbo et al., 1994; Sugumaran and Semensi, 1991) are able to act on d-dopachrome or dopaminechrome, and their product is DHI instead of DHICA. These enzymes should not be considered tautomerases as they catalyze a decarboxylation. Obviously, the mechanism of action should 268

be different, and this difference emphasizes the importance of the carboxyl group in the formation and final structure of the mammalian eumelanin polymer. As for Tyrp1, Tyrp2/Dct shows remarkable homology to tyrosinase. It displays several glycosylation sequons, whose occupancy is important for enzyme function, as treatment of cultured melanoma cells with tunicamycin decreases Dct activity. Moreover, treatment of the enzyme with glycosidases increases the specific activity of the enzyme but decreases its stability (Aroca et al., 1992). Dct is also associated with the melanosomal membrane by a single membrane-spanning helix, but the sorting and trafficking signals in its short cytoplasmic C-terminal extension are different from those in tyrosinase, suggesting a differential intracellular processing (Raposo et al., 2001). The sequences of the metal ion binding sites in Dct share with tyrosinase the position and number of the histidine residues likely to be involved in chelation of the metal cofactor. Yet, the nature of the metal cofactor is different. Purified Dct contains two Zn atoms per protein molecule, as measured by atomic absorption spectroscopy, and enzyme activity can be reconstituted from apoenzymatic preparations by addition of Zn ions, but not Cu or Fe ions (Solano et al., 1994, 1996). Therefore, although direct binding of Zn to Dct could not be demonstrated in melanocytic cells cultured in the presence of a radioactive isotope (Furumura et al., 1998), and in spite of reports suggesting the presence of Fe at the active site (Chakraborty et al., 1992), it is widely accepted that Dct is a Zn protein. In keeping with this, and from the chemical point of view, Zn2+ is preferable to other cations to catalyze a tautomerization, because it has no redox properties and is unable to catalyze oxidative reactions. In Dct, each Zn2+ is probably bound to the polypeptidic chain by three histidine residues in a distorted tetrahedron, and the fourth position is occupied by a water molecule (Fig. 14.3). This water molecule would be displaced by the substrate, l-dopachrome, and a rearrange-

ENZYMOLOGY OF MELANIN FORMATION

ment in the distribution of p-electrons on the indolic ring would follow, thus achieving the tautomerization to DHICA (Solano et al., 1996). The physiological consequences of Dct activity on the type and amount of pigment formed are only partially understood. Tyrp2/Dct may contribute to the formation of DHICAenriched melanins with a higher proportion of carboxylated vs. noncarboxylated indolic monomers. These melanins are lighter in color than DHI-rich melanins (Aroca et al., 1992; Orlow et al., 1992). Moreover, the presence of Dct has been shown to decrease the binding of melanin monomers to proteins and to protect enzymatic activities (Salinas et al., 1994). Therefore, Dct may protect the melanocytes from the inherent cytotoxicity of melanogenesis by promoting a less cytotoxic branch of the pathway. In keeping with this possibility, DHICA has been shown to be less cytotoxic than DHI (Pawelek and Lerner, 1978; Urabe et al., 1994). In any case, several mutant alleles of mouse Dct are associated with pigment dilution, but the phenotype is mild. Concerning human melanocytes, no mutations in the human gene have been described, and the levels of Dct activity appear to be lower than in mouse (Bernd et al., 1994).

The Tyrosinase Family and the Melanogenic Complex A number of studies from different laboratories have raised the possibility of the occurrence of a multimeric complex comprising several melanosomal proteins, and particularly tyrosinase and the Tyrps. Orlow et al. (1993a, b) first reported the identification by sucrose density gradient centrifugation of a high-molecular-weight complex including tyrosinase and likely other melanosomal proteins, which was not detected in extracts from platinum mice. Later on, it was shown that, in extracts from Cloudman mouse melanoma cells, antibodies directed to Tyrp1 and Tyrp2 could immunoprecipitate tyrosinase and that the three proteins comigrated in sucrose density gradients under appropriate conditions (Orlow et al., 1994). This suggested a stable interaction among these proteins, at least in detergent-solubilized extracts. In a study performed with purified tyrosinase and Tyrp1, a strong and close interaction between these proteins, with formation of heterodimers, could be demonstrated (Jiménez-Cervantes et al., 1998). This suggested a physical interaction between these proteins in a “melanogenic complex” that may involve other melanosomal components (Winder et al., 1994). The functional consequences of such a complex remain obscure. At least Tyrp1 (Kobayashi et al., 1998, Manga et al., 2000), and also likely Tyrp2 (Manga et al., 2000), stabilize tyrosinase in melanocytes and in transfected heterologous cells. This is best explained in terms of a physical interaction between the proteins within the melanosome. Concerning the enzymatic activities, interaction with Tyrp1 was reported to decrease tyrosinase activity (Manga et al., 2000), but other studies failed to demonstrate a similar effect (Jiménez-Cervantes et al., 1998). Further evidence for complex formation by the melanogenic enzymes comes from studies with enzymes from

fungi and insects (Sugumaran and Semensi, 1991; Sugumaran et al., 1995). It could be shown by a number of techniques that dopachrome “isomerase” and tyrosinase interacted with each other and mutually inhibited their enzymatic activity. It should be noted that complexes of sequential metabolic enzymes, such as the melanogenic complex, are referred to as “metabolons.” A number of evolutionary advantages for such structures have been proposed (Cascante et al., 1994; Srere, 1987). Whatever the case, the advantages of a melanogenic complex seem obvious in terms of optimization of the flow of substrates and products, and also probably as a means to minimize leakage of reactive and potentially cytotoxic intermediates from the melanosome.

Silver/Pmel-17 and Other Melanosomal Proteins Involved in the Regulation of Melanogenesis In addition to the tyrosinase family, there are other melanosomal-specific proteins with clear involvement in the regulation of melanogenesis, in spite of uncertainties as to their enzymatic role or mechanism of action. Donatien and Orlow (1995) found that certain melanosomal proteins interact more closely with melanin than tyrosinase, Tyrp1, and Tyrp2/Dct. Three of these proteins displaying a tighter interaction with the melanin polymer were identified by immunochemical techniques as the silver/gp100/gp87/Pmel-17, P (pink-eyed dilution), and MATP/AIM-1/underwhite proteins. Mutations in these three proteins lead to strong inhibition of melanin formation. Concerning Pmel-17, its mutation causes the silver phenotype in mice (Martinez-Esparza et al., 1999). It is a glycoprotein of molecular mass around 100 kDa in humans (Kobayashi et al., 1994; Kwon et al., 1991, 1994) and 87 kDa in mice (Martínez-Esparza et al., 2000) before its proteolytic cleavage within the melanosome. Chakraborty et al. (1996) proposed a potential enzymatic activity for the Pmel-17/silver protein. They reported that wheat germ agglutinin-purified extracts of both mouse and human melanoma cells contain an enzymatic activity that catalyzes the polymerization of DHICA to melanin in vitro. Similar results were described by Lee et al. (1996), who described a DHICA-converting activity in human Pmel-17. However, this enzymatic activity of Silver/Pmel-17 acting on DHICA polymerization overlaps the activity described for murine Tyrp1 and human tyrosinase (Olivares et al., 2001) and, as a matter of fact, it has never been unequivocally associated with Pmel-17. From the chemical point of view, DHICA is an o-diphenol so that its converting activity is more likely to reside in proteins such as Tyrp1 or tyrosinase, which are metalloenzymes with diphenol oxidase activity, than in Silver/Pmel-17. Furthermore, an opposite activity has also been proposed for this protein that, under certain conditions, could act as “stablin” by stabilizing DHICA and preventing its auto-oxidation and incorporation into melanin (Pawelek, 1991). This raised the possibility that the stablin and DHICA polymerase activities may reside in alternative transcripts of the Pmel-17/silver gene, but so far this has not been confirmed. What is clear is that cells overexpressing Pmel-17 display 269

CHAPTER 14

structures resembling premelanosomal striations. Therefore, Pmel-17 seems to be sufficient to drive the formation of striations within multivesicular bodies and is directly involved in the biogenesis of premelanosomes (Berson et al., 2001). Furthermore, it is also known that melanogenesis does not begin in melanosomes until this protein is processed from its membrane-bound original state to its soluble form and is integrated into the fibrillar matrix characteristic of stage II melanosomes (Kushimoto et al., 2001). Thus, the proteolytic product of Pmel-17 devoid of its C-terminal region seems to be essential for eumelanosome architecture and for initiation of melanosynthesis (Yasumoto et al., 2004). Intriguingly, Pmel-17 has a proline/serine/threonine-rich region in the central portion abundant in O-glycosylation signals similar to typical proteoglycans. One attractive hypothesis is that this saccharide moiety would contribute to the formation of the fibrillar matrix of Stage II premelanosomes and/or catalyze melanin deposition onto this matrix by binding dihydroxyindole-derived units (Solano et al., 2000), but experimental evidence in favor of this possibility is not yet available. In summary, it is clear that Pmel17 is an essential factor for the distal steps of melanogenesis under “in vivo” conditions (Fig. 14.1) but, up to now, there is no clear enzymatic activity associated with this protein. The other two melanosomal proteins mentioned above, p and underwhite, may have a key function in melanogenesis as their mutations also lead to oculocutaneous albinism, OCA2 and OCA4 respectively. However, these proteins do not show enzymatic activity. The P protein is probably an anionic transporter localized to the melanosomal membrane, and partially responsible for the control of the pH within the melanosome (Brilliant, 2001; Puri et al., 2000). Anionic transport should be coupled to vATPases and VDAC, two proton transporters also found in the melanosomal membrane (Basrur et al., 2003), in order to maintain charge neutrality. Early reports suggested that melanogenesis could be triggered by low pH (Bhatnagar et al., 1993). This was related to the observation that acidic pH favors an allosteric interconversion of tyrosinase (Devi et al., 1987; Tripathi et al., 1987). However, melanin synthesis in human pigment cell lysates is maximal at neutral pH, and is suppressed in Caucasian melanocytes when the melanosomal pH is low (Ancans et al., 2001). Moreover, inhibition of the v-type ATPases increases tyrosinase activity and melanin production in human and mouse melanoma cells (Ancans and Thody, 2000) and in melanocytes cultured from Caucasian, but not black, donors (Fuller et al., 2001). Therefore, the melanosomal pH could be critical for the control of tyrosinase activity and melanogenesis. In this scenario, the P protein could provide a key control point for skin pigmentation through its ability to mediate neutralization of the melanosomal pH (Ancans et al., 2001). On the other hand, other effects different from tyrosinase activation by an increase in pH would also be possible as mutations in the p gene cause mislocalization of melanosomal proteins (Boissy et al., 1998b; Manga et al., 2001). Concerning OCA4, related to the murine underwhite gene, 270

the encoded protein is MATP, a membrane-associated transporter protein also named AIM-1 (Newton et al., 2001). As in the case of the P protein, this protein is predicted to span the membrane 12 times, a usual topology in transport proteins. So far, no enzymatic activity has been assigned to MAPT/AIM-1/underwhite and, moreover, its actual substrate for transport is still unclear (Rundshagen et al., 2004). Similar to OCA2, in OCA4 melanocytes, tyrosinase processing and intracellular trafficking to the melanosome is disrupted, and tyrosinase is abnormally secreted from the cells in immature melanosomes (Costin et al., 2003). Therefore, Pmel-17, P, and underwhite proteins, present in mammalian melanocytes, are needed for normal melanogenesis but they lack well-defined enzymatic activity. An opposite situation holds for another protein proposed to play a role in melanogenesis, namely peroxidase, the enzymatic activity of which is well established but whose role in melanogenesis is doubtful. Okun et al. (1970) proposed that tyrosine might be oxidized to melanin by a peroxidase activity in mammalian melanocytes. Additional studies on the oxidation of tyrosine by horseradish peroxidase led these authors to propose that the initiating step in melanogenesis, tyrosine hydroxylation, might be catalyzed by a peroxidase. Today, it is clearly proven that mammalian tyrosinase catalyzes the conversion of ltyrosine to l-dopa, but the possibility still remains that a peroxidase activity might also be involved in the distal phases of melanogenesis. In this regard, the ability of horseradish peroxidase to oxidize DHI is well documented (d’Ischia et al., 1991), although this enzyme is much less efficient with DHICA. In fact, peroxidase seems to be important in melanogenesis in some nonmammalian systems, such as the melanin in the ink of cuttlefish, but its role in mammals should be confirmed after the identification of a protein with peroxidase activity within melanosomes. So far, proteomic analysis of this organelle (Basrur et al., 2003) has failed to achieve this identification. In addition, another critical point would be the origin of hydrogen peroxide needed for this enzyme. In this respect, we have observed that oxidative stress due to the addition of this peroxidase substrate to melanoma cell cultures led to an inhibition, rather than a stimulation, of mammalian melanogenesis (Jiménez-Cervantes et al., 2001). Although the mechanism is likely due to other factors different from enzymatic activities, this fact suggests that peroxidase is not an important factor in mammalian melanogenesis.

Methods for Determination of Melanogenic Enzymatic Activities The measurement of the melanogenic activities is not a trivial matter, as it is complicated by several factors, particularly when the biological sample under study contains more than one of the melanogenic enzymes. On the one hand, the product of one particular reaction can be the substrate of a subsequent and coupled enzymatic step. For instance, the tyrosinase-catalyzed oxidation of l-dopa to l-dopaquinone is

ENZYMOLOGY OF MELANIN FORMATION

rapidly followed by the formation of l-dopachrome, which is a substrate for Tyrp2/Dct, so that the presence of this enzyme in the reaction mixture can modify the rate of dopachrome accumulation. The situation is even more complex for the hydroxylation of l-tyrosine, where l-dopa acts as a necessary cofactor and, simultaneously, as a competing alternative substrate. On the other hand, the reaction specificities of the melanogenic enzymes are in some cases overlapping. The best example is provided by mouse Tyrp1, which is considered to be a pseudotyrosinase of low specific activity (JiménezCervantes et al., 1993a). Moreover, the substrate specificities of the melanogenic enzymes seem to be variable between species, and are sometimes still debated. For instance, mouse tyrosinase does not catalyze DHICA oxidation, whereas the human enzyme appears to be able to do so (Olivares et al., 2001). Finally, the substrates employed in many of the relevant assays are unstable in the presence of molecular oxygen, undergo metal ion-catalyzed transformations, or are not available commercially. Therefore, the interpretation of enzyme activity measurements is complex, and calls for an adequate knowledge of the chemistry of the reaction considered and the possible interferences in the sample under study. Not surprisingly, the first assays for melanogenic activity aimed at determining the rates of the tyrosinase-catalyzed reactions and took advantage of either the requirement for oxygen or the formation of reactive oxidized species, such as o-quinones. These methods consisted of the conventional Warburg technique (Dawson and Magee, 1955), the Clarktype electrode to measure oxygen consumption (Nelson and Mason, 1970), or the chronometric (Dawson and Magee, 1955) and spectrophotometric (El-Bayoumi and Frieden, 1957) assays employing ascorbate to reduce the quinones formed by tyrosinase back to catechols. These assays showed low sensitivity and low specificity. They were applied to samples with high tyrosinase activity from plants, insects, and amphibians, but were not sensitive and/or specific enough to measure tyrosinase activity accurately in mammalian samples where, in addition to a lower tyrosinase activity, the presence of the Tyrps must be taken into account. Thus, new methods of enzymatic analysis of the melanogenic pathway have been designed, aiming at: 1 increasing not only the sensitivity but also the specificity of the tyrosinase assays; 2 measuring the enzymatic activities of Tyrps; 3 minimizing the mutual interferences among these activities in crude extracts (Valverde et al., 1993), as they may catalyze consecutive reactions in the pathway; 4 distinguishing the relative contributions of different proteins to a single enzymatic activity. According to the type of reaction considered, the enzymatic activities of the melanogenic pathway can be classified into three groups: hydroxylation of monophenolic substrates; oxidation of diphenols or dihydroxyindoles; and tautomerizations. The more frequent and useful activity assays are presented below, according to this general classification. Experimental details are generally omitted, as they can be

found in the corresponding references. Table 14.1 summarizes these assay methods, with brief comments on their relative value. However, it should be taken into account that, owing to the presence of enzymes catalyzing coupled reactions in crude extracts, and to the overlapping catalytic potentials of tyrosinase and Tyrp1, there is not an easy and absolutely accurate method for the determination of these melanogenic activities.

Methods for the Measurement of Tyrosine Hydroxylase Activity Tyrosine hydroxylase activity measurements are often considered to be the methods of choice for the assay of tyrosinase. Compared with dopa oxidase activity measurements, they are more specific, but also more technically demanding because of the lower turnover number and the lag period of the tyrosine hydroxylase activity. A further complication derives from the impossibility of isolating tyrosine hydroxylation from dopa oxidation (see the catalytic cycle described in Fig. 14.2). By far, radiometric methods are the most frequently used, sensitive, and specific assays for the tyrosine hydroxylase activity of mammalian tyrosinase. The most versatile of these assays uses l-[3,5-3H]-Tyr as substrate to measure tritium release as water, according to the reaction depicted in Figure 14.4. It was first described by Pomerantz (1964, 1966) as a modification of other assays developed for different hydroxylases. l-Dopa is normally added to the reaction mixture as cofactor of the reaction (Lerner et al., 1949), and tritiated water is separated from the excess of l-tyrosine and other radiolabeled products by absorption on a charcoal–celite mixture. Modifications to standardize, speed up, and optimize the assay have been published (Hearing, 1987; Hearing and Ekel, 1976; Jara et al., 1988b; Townsend et al., 1984). The method can be extended to samples such as human hair bulbs (King and Witkop, 1976). The major limitation is the high background due to tritium exchange between the substrate and water, so that radiolabeled tyrosine should sometimes be repurified. On the other hand, only half the radioactivity present in the substrate is released upon hydroxylation. Accordingly, long incubation times should be avoided as the second tritium retained in the l-dopaquinone molecule could be released in subsequent polymerization reactions, without the direct involvement of tyrosinase. The presence of Dct in crude samples has no significant influence on this. The assay has been modified to allow for its in vivo application to melanocytes in culture. Essentially, this involves the addition of radiolabeled tyrosine to the culture media and the measurement of tritiated water after long incubation periods, usually at least 24 h (Auböck et al., 1983; Oikawa et al., 1972). This assay is used quite frequently, but some caution should be exercised because the results may be influenced by factors other than tyrosinase activity, such as l-tyrosine transport inside the cells. A spectrophotometric assay using the increase in A280 accompanying the oxidation of l-tyrosine has been used as an indicator of this activity in mushroom (Duckworth and 271

272

Dopa oxidase

Oxidation of l-dopa formed to yield fluorescent indoles

Separation and quantitation of l-dopa

Fluorometric

HPLC

Radiometric

HPLC

Spectrophotometric

Sensitive Use d-dopa as cofactor

Release of 14CO2 after oxidation of the reaction mixture

Radiometric

Sensitive

Sensitive

Sensitive and stereospecific

Rapid, simple, and continuous More sensitivity than above Dct interference avoided Sensitive and stereospecific

Detection of an adduct DQ-MBTH at 500 nm Separation and quantitation of l-dopachrome Formation of cysteinyldopa and electrometric detection Incorporation of 3-[14C]-dopa into acid-insoluble melanin Release of 3H from position 6 in 2,5,6-[3H]-Dopa

Rapid, simple, and continuous

Detection of dopachrome formation at 475 nm

Good resolution of substrate and product

Sensitive and nonradiometric

Sensitive and rather specific Can be used in situ for melanocytes in culture

Release of 3H2O from l-[3,5]-3H-Tyr

Radiometric

Tyrosine hydroxylase

Major advantages

Rationale

Technique

Activity

Table 14.1. Summary of methods for the determination of enzymes involved in melanogenesis.

Limited solubility of MBTH A proportion of DQ might evolve DC Technically more complex than spectrophotometric. Discontinuous Technically more complex than spectrophotometric. Discontinuous Dct and other factors influence the rate of polymerization of products High background due to the instability of the radioactive tracer

Thiols and Dct affect results

Interference at long incubation times due to polymerization High background due to 3H exchange Some carboxyl group is retained Evolved CO2 is difficult to trap completely High background but low specificity Affected by the DHI/DHICA ratio formed It needs l-dopa accumulation Less sensitive than radiometric methods

Major drawbacks

Pomerantz (1976)

Aroca et al. (1992)

Tsukamoto et al. (1992a, b) Agrup et al. (1983)

Mason (1948); Horowitz et al. (1970) Winder and Harris (1991)

Jergil et al. (1983)

Adachi and Halprin, (1967); Husain et al. (1982)

Winder and Harris (1991)

Pomerantz (1964)

References

273

Melanin formation

DHICA oxidase

DHI oxidase

Dopachrome tautomerase (Dct)

Radiometric

HPLC

Formation of acid-insoluble melanin from l-[14C]-Tyr

Detection of an IQCA-MBTH adduct at 490 nm Separation and quantitation of residual DHICA by UV

Separation and quantitation of residual DHI by UV

HPLC

Spectrophotometric

Detection of melanochrome at 540 nm

Separation of dopachrome and DHICA quantitation by UV absorption

HPLC

Spectrophotometric

Continuous interferences due to spontaneous DHI formation avoided

Increase in A308 due to DHICA formation

Spectrophotometric

Low background Good sensitivity It measures the global melanogenic activity

Specific for Tyrp1 in mouse samples

Rapid, simple and continuous

Interference of melanin is avoided

Rapid, simple, and continuous

More specific than spectrophotometric methods

Simple and continuous

Decoloration of l-dopachrome at 475 nm

Spectrophotometric

Results do not directly correlate with tyrosinase activity Tyrps and other factors (thiols, metal, detergents) affect results

Limited solubility of MBTH Limited sensitivity Long incubation times are required Substrate disappearance, rather than product formation, is determined

High background due to the DHI instability and rapid melanin formation High background Substrate disappearance, rather than product formation, is determined

Low specificity and sensitivity Time of recording limited by the formation of colored melanin-like products High initial absorbance Interference by UV-absorbing material Limited sensitivity Discontinuous

Chen and Chavin (1965); Jara et al. (1988b)

Jiménez-Cervantes et al. (1994) Kobayashi et al. (1994); Jiménez-Cervantes et al. (1994)

Tsukamoto et al. (1992a, b)

Miranda et al. (1985)

Palumbo et al. (1987)

Aroca et al. (1990b)

Pawelek et al. (1980)

CHAPTER 14 Dehydroascorbate

HO +

COO–

HN

HO

3

1 Ascorbate

O +

O

Fig. 14.4. Tyrosine hydroxylation: reaction measured in the Pomerantz assay by estimation of the tritium released as water when the hydroxylation at position ortho takes place.

COO–

HN 3

2

+Cys

4 3

HO

274

+

HN 3

N H

COO–

Cys-Dopa

lmax= 293 nm

+ MBTH +

HN 3

COO–

HO N+ H

HO

CH3

COO–

+

Reduction back to L-dopa Addition of Thiols

N MBTH-Dopa

N

C S

lmax= 500 nm

l

2

N

HO

L-Dopachrome = 305 & 475 nm max

1

COO–

S

–OOC HO

O

Coleman, 1970) as well as in human melanoma (Wood and Schallreuter, 1991). According to the UV absorption of the possible subsequent products, dopa, thiol-dopa adducts, and dopachrome, and the high interference due to the UV absorption of proteins, this assay is rarely used, and the choice of 280 nm as the wavelength employed to follow the reaction progress does not seem appropriate. If thiol adduct formation is expected, 295 nm (maximal absorption of cysteinyl-dopa and related chemicals) is better suited. If dopachrome formation is anticipated, 305 or 475 nm should be chosen. The fluorescence properties of indoles have been exploited as an alternative for measuring the tyrosine hydroxylase activity of tyrosinase. These methods are based on the accumulation of l-dopa when l-tyrosine is oxidized by tyrosinase in the presence of ascorbate (Fig. 14.5). Subsequent chemical oxidation of l-dopa with ferricyanide in the presence of traces of Zn(II), followed by the rapid stop of this reaction with ascorbate in concentrated NaOH yields a mixture of dihydroxyindoles that can be quantitated on the basis of their fluorescent emission at 490 nm upon excitation at 360 nm. Fluorescence intensity is proportional to the tyrosinase activity. The method was first described by Adachi and Halprin (1967) and later optimized by Husain et al. (1982). A complete description can be found in more recent literature (Hearing, 1987; Tripathi et al., 1992). The assay shows variability depending on the DHI/DHICA ratio formed, as the fluorescence of DHICA is higher than that of DHI. Other drawbacks are the high background due to the amount of l-dopa cofactor initially present in the assay and the putative chemical hydroxylation of ltyrosine catalyzed by ascorbate. Inclusion of metal chelators in the assay medium minimizes this reaction (Husain et al., 1982). HPLC assays have been developed to gain sensitivity and specificity. HPLC makes it possible to separate, identify, and quantitate the substrate remaining in the assay media as well as the products of the reaction. Most of the methods use reverse phase C18 columns for the separation. For tyrosine hydroxylase activity, HPLC was first used in 1983 (Jergil et al., 1983). The method is based on the determination of ldopa generation from l-tyrosine. Ascorbate should be added to allow for l-dopa accumulation (Fig. 14.5). Finally, another radiometric assay for tyrosine hydroxylase activity was reported using l-[HOO14C]-Tyr as substrate (Winder and Harris, 1991). It is based on the fact that, after tyrosinase action, l-[HOO14C]-dopa is formed. Oxidation

HO HO + NH3

3 4

Addition of Besthorn Hydrazone Internal cyclation

Fig. 14.5. Some reactions of l-dopaquinone involved in the different assays for the l-dopa oxidase activity of tyrosinase.

with ferricyanide promotes decarboxylation of dopa, but not tyrosine. Thus, one molecule of 14CO2 is released from the products of tyrosinase action for every molecule of tyrosine hydroxylated to dopa. The main drawback of this method could be the retention of some carboxyl groups in post-dopa products due to the presence of Dct or the incomplete oxidation of dopa.

Methods for the Measurement of Diphenol and Dihydroxyindole Oxidase Activities The dopa oxidase activity is easily measured by spectrophotometric recording of l-dopachrome formation at 475 nm. This rapid and easy to perform assay was introduced by Mason (1948), and it has been used for tyrosinases from all sources, from Neurospora crassa (Horowitz et al., 1970) to mammalian melanoma (Pomerantz and Li, 1970). A comparison with radiometric assays was published (Jara et al., 1988b). The sensitivity is limited by the absorption coefficient of l-dopachrome (e = 3700/M/cm), but it should be considered that the turnover number of tyrosinase as hydroxylase is around one order of magnitude lower than that as oxidase. The main limitations of the assay are: 1 The presence of thiols in the assay media prevents dopachrome formation (Jara et al., 1988a), as l-dopaquinone is trapped to yield thiol–dopa conjugates (Fig. 14.2). 2 Only half the l-dopa molecules oxidized by tyrosinase are transformed into l-dopachrome (Fig. 14.2). The other half is reverted back to l-dopa (García-Cánovas et al., 1982; Lerner and Fitzpatrick, 1950). 3 The assay should be performed in phosphate buffer at neutral pH and low ionic strength, because l-dopachrome stability is lower in other conditions. 4 Dct prevents the accumulation of l-dopachrome, leading to an underestimation of the dopa oxidase activity (Valverde et al., 1993).

ENZYMOLOGY OF MELANIN FORMATION

A modification of this assay (Winder and Harris, 1991) with improved sensitivity is based on the trapping of l-dopaquinone with the Besthorn’s hydrazone (MBTH, 3-methyl-2-benzothiazolinone hydrazone) (Pifferi and Baldassari, 1973) to form a dark pink pigment with maximal absorption at 500 nm (Fig. 14.5). The most important problem of this assay is the low solubility of MBTH and the relative efficiency of this hydrazone to capture l-dopaquinone, compared with other possible reactions such as the formation of thiol conjugates. Two HPLC procedures have also been used for dopa oxidase activity measurements. The first is based on the determination of l-dopa consumption and the appearance of dopachrome and other indoles at 280 nm (Tsukamoto et al., 1992a). The second is based on the trapping of dopaquinone by l-cysteine with the identification of formed cysteinyldopas by electrometric detection with an Ag/AgCl electrode (Agrup et al., 1983; Wittbjer et al., 1989). This last procedure was used to estimate the tyrosinase activity in human hair bulbs (Townsend et al., 1986) and in the serum of patients with malignant melanoma (Sonesson et al., 1995). Finally, the dopa oxidase activity of tyrosinase was assayed by a radiometric method using 2,5,6-[3H]-dopa as substrate (Pomerantz, 1976), and measuring tritium release from position 6 after dopaquinone cyclization (Fig. 14.5). This assay was rarely used, and we believe that the substrate is no longer available. Alternatively, the dopa oxidase activity can also be determined by the production of acid-insoluble products from l-[3-14C]-dopa (Aroca et al., 1992, 1993), although in this case, the likely effect of the Tyrps on the rate of product formation should be considered. Concerning DHICA oxidase activity, a spectrophotometric assay related to the MBTH method for dopa oxidase activity has been designed (Jiménez-Cervantes et al., 1994; Kobayashi et al., 1994). DHICA is used as substrate, and its oxidation product, IQCA, is trapped by MBTH, leading to a conjugate with maximal absorption around 490 nm. In mouse melanocyte extracts, the activity measured can be assigned to Tyrp1, as interference from tyrosinase is unlikely, because murine tyrosinase does not use DHICA as substrate (Olivares et al., 2001). The interpretation of the results may be more complex for human melanocytes because DHICA oxidase activity has been demonstrated for human tyrosinase, but not yet for TYRP1. A spectrophotometric method is also available for the DHI oxidase activity of tyrosinase (Körner and Pawelek, 1982), based on the measurement of melanochrome appearance from DHI at 540 nm (Miranda et al., 1985). Here, spontaneous DHI oxidation causes a high background. In any case, DHI/DHICA oxidase is better determined by HPLC methods. These methods have been designed to follow substrate disappearance after appropriate incubation times. For DHI, 1 h at 37∞C, pH 6.8 is appropriate (Kobayashi et al., 1994; Tsukamoto, 1992a). For DHICA, incubation times can be prolonged up to 24 h because of the greater stability of this indole. Control reaction mixtures should always be performed to subtract the rate of spontaneous auto-oxidation.

Methods for the Measurement of Dopachrome Tautomerase Activity The easiest method for monitoring the dopachrome tautomerase activity of Tyrp2/Dct is the spectrophotometric recording of l-dopachrome decoloration at 475 nm (Aroca et al., 1990a, b; Barber et al., 1984; Körner and Pawelek, 1980; Pawelek, 1990; Pawelek et al., 1980). Two important points should be considered concerning this assay: 1 Preparation of the substrate. l-dopachrome is not stable, and it should be prepared in situ. This is performed by rapid chemical oxidation of l-dopa. Three different agents have been used for this purpose: potassium ferricyanide (Wakamatsu and Ito, 1988), silver oxide (Barber et al., 1984; Körner and Pawelek, 1980; Leonard et al., 1988; Palumbo et al., 1987, 1991; Pawelek et al., 1980), and sodium periodate (Aroca et al., 1990a, b; Graham and Jeffs, 1977; Wilczek and Mishima, 1993). By far, sodium periodate is preferred because of the rapid and stoichiometric oxidation of l-dopa (molar ratio l-dopa:periodate 1:2). Thus, l-dopachrome solutions devoid of l-dopa can be easily obtained. In the other cases, l-dopa is not completely oxidized, and a significant proportion remains that can interfere with the Dct assay in crude extracts. 2 Spontaneous l-dopachrome decoloration. l-Dopachrome undergoes a slow but significant decarboxylation leading to DHI (Fig. 14.1). This reaction causes a background that should be subtracted from enzymatic measurements. The rate of this spontaneous reaction is highly dependent on the pH, ionic strength, and the presence of traces of metal ions (Palumbo et al., 1987). Recommended conditions include 10 mM phosphate buffer, pH 6.0 and 0.1 mM EDTA (Aroca et al., 1990b). As an alternative to the A475 decrease, another spectrophotometric assay for Dct was described (Aroca et al., 1990b). It takes advantage of the different absorption of dopachrome, DHI, and DHICA in the UVA region. Around 310 nm, the order of molar absorption coefficients is DHICA > dopachrome > DHI. Thus, at this wavelength, dopachrome decarboxylation leads to absorbance decreases, whereas Dct-catalyzed transformation of l-dopachrome into DHICA leads to absorbance increases. As for the oxidase activities, HPLC assays are more sensitive and specific for the determination of Dct activity. HPLC permits the simultaneous determination of l-dopachrome disappearance and DHI and DHICA appearance, although only the DHICA formed is proportional to the enzymatic activity. The original chromatographic conditions were reported by Palumbo et al. (1987, 1991), and involved an isocratic separation with 0.2 M sodium borate buffer, pH 2.5, containing 25% methanol. Slight modifications were introduced to improve the resolution of indole separation (Aroca et al., 1993; Leonard et al., 1988; Pawelek, 1990; Solano et al., 1994; Tsukamoto et al., 1992a; Wilczek and Mishima, 1993; Winder et al., 1993a, b). As described for the spectrophotometric Dct assay, periodate is preferred to silver oxide to prepare the l-dopachrome substrate by stoichiometric oxida275

CHAPTER 14

tion of l-dopa, but extra caution should be taken in this case as the iodate subproduct of the oxidation is still oxidant at very acidic pHs, and the indoles formed could be destroyed. Thus, periodate oxidation should be accompanied by mobile phases with pHs higher than 4 (Solano et al., 1994; Wilczek and Mishima, 1993).

Global Melanogenic Activity Measurements The overall melanogenic activity can be estimated by methods measuring melanin formation from l-tyrosine. Although the assay was first introduced (and is still often considered) as a tyrosinase assay, it should be borne in mind that it actually reflects the activity of the total pathway, including the contribution of the Tyrps, because the final product is determined. The assay was introduced by Lerner (1955) and then improved by Chen and Chavin (1965) and Hearing and Ekel (1976). Modifications to facilitate the assay in small samples were described by Hearing (1987) and Jara et al. (1988b). The assay is based on the incorporation of uniformly labeled tyrosine, l[U14C]-Tyr, into acid-insoluble melanin, which is absorbed onto a filter paper, washed, and counted. Owing to the low spontaneous oxidation of l-tyrosine and the insolubility of melanin, the background is low. In addition to the complexity in the interpretation of the results due to the contribution of more than one enzyme activity, another drawback of the method is related to the fate of the 14C-carboxyl group of radiolabeled tyrosine. The release of this group as 14CO2 is a potential hazard, and its rate and extent is dependent on the Dct activity present in the sample. This enzyme can accelerate or decrease the incorporation of indole units into melanin, depending on the incubation time (Aroca et al., 1990a, 1992). Attempts to improve the sensitivity of the method (Jara et al., 1988c) and to extend it to pheomelanin formation (Aroca et al., 1989) have been published.

Activity Stains in Electrophoresis Gels Taking advantage of the high resistance of tyrosinase to denaturing agents such as urea or SDS, and of the specificity of the enzyme, SDS-PAGE followed by activity stain can be used to visualize and compare tyrosinase activity in complex mixtures such as crude melanocyte extracts. The discontinuous system of Laemmli (1970) yields adequate separations. Bearing in mind that an active conformation of the melanogenic proteins must be preserved throughout the procedure, care should be taken to avoid denaturing conditions. SDS can be added to the sample to final concentrations as high as 3%, but b-mercaptoethanol even at low concentrations, or heating, should be avoided. Basically, those conditions were used in the characterization of tyrosinase isoforms (Burnett, 1971; Hearing et al., 1981; Miyazaki and Ohtaki, 1975; Quevedo et al., 1975), as well as in the establishment of the catalytic potentials of the enzyme and its distinction from peroxidase (White and Hu, 1977). This technique has been exploited to show that, in mouse melanoma cells, Tyrp1 behaves as a tyrosinase isoenzyme with lower specific activity for l-tyrosine, l-dopa, and DHI but higher activity for DHICA 276

(Jiménez-Cervantes et al., 1993a, 1994; Kobayashi et al., 1994). After the proteins in the sample under study are resolved by electrophoresis, the gels can be stained by several procedures exploiting the tyrosine hydroxylase or dopa oxidase activities of tyrosinase. Staining for tyrosine hydroxylase activity is difficult because of the low turnover of tyrosinase on l-tyrosine. However, the use of l-[U-14C]-tyrosine as substrate and the fluorographic detection of the radioactive melanin formed is sensitive enough to stain tyrosinase activity in extracts from mouse melanoma cells (Jiménez et al., 1991; Tsukamoto et al., 1992b). Nevertheless, the more frequent activity stain procedures involve incubation of the gels with l-dopa. As the pH of the resolving gels is basic, care should be taken to equilibrate the gels at pH around 6.5 before addition of l-dopa to avoid its rapid oxidation. The stain is reminiscent of the histochemical procedure developed by Bloch (1927) to detect tyrosinase in human skin specimens. Direct staining with ldopa usually requires long incubation times and displays limited sensitivity. Therefore, attempts were made to design specific methods with higher sensitivity and reduced incubation times. Fluorographic stain involving 14C-labeled l-dopa has been used successfully (Jiménez et al., 1991; JiménezCervantes et al., 1993b; Tsukamoto et al., 1992b). A colorimetric procedure based on the formation of colored adducts between dopaquinone and MBTH has also been reported (Jiménez-Cervantes et al., 1993b; Nellaiappan and Vinayagam, 1986). A reddish band is rapidly formed, with sensitivity comparable to the fluorographic procedures. As opposed to tyrosinase and Tyrp1, Dct is very sensitive to SDS (Aroca et al., 1990a). This problem has prevented the general use of SDS-PAGE for the study of mammalian Dct, although the technique has been used to identify isoenzymic forms of a related protein from insect hemolymph (Nellaiappan et al., 1994a, b; Sugumaran and Semensi, 1991). In our hands, the assay cannot be employed to stain authentic mammalian Dct on account of the lower activity and stability and the difference in the product formed.

Perspectives In spite of the cloning and characterization of tyrosinase and the Tyrps, a number of important questions concerning the enzymology of melanogenesis are still unanswered. The pheomelanogenic pathway probably involves still unknown enzymatic reactions. In its present form, it may not be complete in a situation reminiscent of the eumelanogenesis a decade ago. On the other hand, pheomelanin synthesis seems to be the default melanogenic pathway, occurring when tyrosinase activity is low and free thiol compounds are available within the melanosome. The regulatory factors responsible for the switch from pheo- to eumelanogenesis and controlling the size of the thiol pool are also poorly known. Concerning eumelanogenesis, the involvement of the Tyrps is now firmly established, but the actual basis for the relationship between

ENZYMOLOGY OF MELANIN FORMATION

Tyrps expression and eumelanin synthesis remains obscure. This is mainly due to uncertainties as to the actual role of Tyrp1, which may be more complex than the catalysis of DHICA oxidation. As a corollary, and as stated by J. Pawelek and A. K. Chakraborty in the previous edition of this book, we are still not able to explain properly, at the molecular level, the pigmentation phenotype associated with the slaty and brown mutations. Moreover, most of our knowledge of the enzymatic action of tyrosinase and the Tyrps comes from studies performed with purified preparations or under conditions in which noncovalent protein–protein interactions may be weakened. As there is good evidence that a melanogenic complex is formed in vivo, the question of subtle changes in the catalytic properties of individual proteins within the complex is still open. On the other hand, the renewed interest in the role of pH as a dynamic regulatory factor in melanogenesis should lead to a better understanding of its effects, not only on the rate of the enzymatic steps, but also as related to the polymerization of melanogenic intermediates. Another aspect that should yield new information is the role of structural melanosomal proteins such as silver in melanin deposition and polymerization of monomers. New developments in the role of the p and underwhite transporters are also anticipated. The proteomic analysis of the melanosome in its different maturation, and hence metabolic, stages is still just beginning (Basrur et al., 2003). We can expect that this powerful methodology will identify new melanogenic activities and/or effectors of already characterized enzymes. Concerning developments in the enzymatic analysis of melanogenesis, the progress since the first edition has been modest, which leaves room for further refinement of the currently employed methods to increase their sensitivity and specificity. This may need the design of specific substrates or inhibitors for Tyrp1 and tyrosinase that might allow for the specific determination of both enzymes in crude extracts. Progress in the development of sensitive and reliable assays for the last steps of the melanogenic pathway, allowing for an accurate estimation of the rate of polymerization of indolic intermediates, is needed in order to approach the identification of factors controlling the distal steps of melanogenesis. This is true for eumelanogenesis and, more importantly, for pheomelanogenesis. Here, the design of those assays seems to be a necessary prerequisite for the discovery of new pheomelanogenic enzymes.

Acknowledgments This chapter is an update of Chapters 28 and 33 in the first edition of this book, published some years ago. We are therefore indebted to the authors of the former Chapter 28, J. Pawelek and A. K. Chakraborty, especially to J. Pawelek, who was a pioneer in so many aspects of the enzymatic regulation of melanogenesis. The authors are also grateful to all graduate students who performed research on the role of tyrosinase and its related proteins in melanogenesis during approxi-

mately the last 20 years. We thank Dr Santiago Delgado for helpful suggestions concerning the mechanism of dopachrome tautomerization and for drawing Figures 14.2 and 14.3. We are also grateful for the continuous financial support of the Spanish government. Work in the authors’ laboratories is currently supported by grants BIO2001-140 and SAF2004-3411.

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15

Chemistry of Melanins Shosuke Ito and Kazumasa Wakamatsu

Summary 1 Melanin pigments can be classified into two major groups: the brown to black insoluble eumelanins and the alkali-soluble yellow to reddish-brown pheomelanins. Both pigments derive from the common precursor dopaquinone formed via the oxidation of l-tyrosine by tyrosinase. Trichochromes are a variety of pheomelanic pigments with structures that are fully elucidated. 2 Dopaquinone, a highly reactive ortho-quinone, plays pivotal roles in chemically controlling melanogenesis. When sulfhydryl compounds are absent, it undergoes intramolecular cyclization to form cyclodopa, which is rapidly oxidized by redox reaction with dopaquinone to give dopachrome (and dopa). Dopachrome then gradually rearranges to give mostly 5,6-dihydroxyindole (DHI) and a trace of 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Oxidation of these dihydroxyindoles leads to the production of eumelanins. However, intervention of cysteine with this process gives rise preferentially to the production of cysteinyldopa isomers. Cysteinyldopas are then oxidized through redox reaction with dopaquinone to cysteinyldopaquinones that eventually give rise to pheomelanins. 3 Kinetic data, provided by pulse radiolysis studies of the early stages of melanogenesis involving dopaquinone (and cysteine), indicate that the process of mixed melanogenesis proceeds in three distinctive steps: the production of cysteinyldopas, the oxidation of cysteinyldopas to give pheomelanins, followed finally by the production of eumelanins. The switching from pheomelanogenesis to eumelanogenesis is chemically controlled by the cysteine concentration. 4 Isolation and properties of natural and synthetic melanin pigments are discussed. Artificial modification of pigment structure should be cautioned when acid or base is employed in the isolation procedures. 5 Recent advances in the study of melanogenesis are summarized. In eumelanogenesis, dopachrome rearrangement to DHICA is catalyzed by dopachrome tautomerase (Dct) or by metal ions. DHI and DHICA can copolymerize on the way to eumelanic pigments. In pheomelanogenesis, cysteinyldopaquinone cyclizes to form the ortho-quinonimine intermediate, the rearrangement of which gives the benzothiazine derivative(s). Oxidative polymerization of the latter leads to the production of pheomelanins including the trichochrome pigments. 6 The biological significance of melanin-related metabolites, such as 5,6-dihydroxyindoles and cysteinyldopas, is addressed, with special emphasis given to their use as 282

melanoma markers, the mechanism of their cytotoxicity, and their possible role in photoprotection. 7 Melanins are difficult to characterize because of their intractable chemical properties, the heterogeneity in their structural features, and the lack of methods to split melanin polymers into monomer units. 8 Degradation studies carried out in the 1960s provided a number of useful degradation products, such as pyrrole-2,3,5tricarboxylic acid (PTCA) and 4-amino-3-hydroxyphenylalanine (4-AHP), arising from eumelanins by permanganate or peroxide oxidation and from pheomelanins by hydriodic acid hydrolysis. A rapid and sensitive method for quantitatively analyzing eumelanins and pheomelanins in tissue samples has been developed on the basis of the formation of PTCA and 4-AHP followed by their high-performance liquid chromatography (HPLC) determination. 9 The total amount of melanin (total melanin) in hair samples can be spectrophotometrically assayed by dissolving them in hot Soluene-350 plus water. The PTCA/total melanin and 4-AHP/total melanin ratios are useful in characterizing eumelanins and pheomelanins respectively. The former ratio reflects the DHICA/DHI ratio in various eumelanins. 10 In addition to PTCA and 4-AHP, the significance of which in pigment research has been established, several other degradation products are also useful in characterizing various types of melanin pigments. These products include thiazole-2,4,5tricarboxylic acid (TTCA) and pyrrole-2,3-dicarboxylic acid (PDCA) as markers of pheomelanic pigments and dopaminederived melanins respectively. This methodology has been used to analyze natural dopamine melanins, such as neuromelanin. Finally, 6-b-alanyl-2-carboxy-4-hydroxybenzothiazole (BTCA), a product of alkaline peroxide oxidation, may also be a good marker of pheomelanic pigments.

Historical Background Melanin and melanogenesis have been fascinating subjects for chemists not only because of the widespread presence of pigments in nature but also because of the complexity of their structures and functions. Landmark events in our understanding of the chemistry of melanogenesis are briefly summarized (revised from Prota, 1992; Prota et al., 1998a). 1885 Borquelot and Bertrand discovered the enzyme tyrosinase in fungi and, in 1886, Bertrand identified the amino acid tyrosine as the melanogenic substrate. 1926–27 Raper elucidated the early stages of the oxidation of tyrosine to melanin catalyzed by tyrosinase. He identified the red intermediate as dopachrome and isolated

CHEMISTRY OF MELANINS

dopa, 5,6-dihydroxyindole (DHI), and 5,6-dihydroxyindole2-carboxylic acid (DHICA). 1948 Beer and coworkers first synthesized DHI and DHICA and showed that DHI is more susceptible to oxidation than DHICA. 1948 Mason extended Raper’s studies to the later stages of melanogenesis and identified dopachrome by spectroscopy. 1952 Panizzi and Nicolaus identified pyrrole-2,3,5tricarboxylic acid (PTCA) as the most significant fragment from the degradation of Sepia melanin (PTCA is now used as a specific degradation product of DHICA-derived units in eumelanins). 1955 Leonhardi first recognized that a group of Thormählen-positive urinary melanogens are derivatives of DHI. 1962–66 Based on extensive analytical and degradative studies, Nicolaus, Piatelli, and their associates suggested that Sepia melanin is a heteropolymer derived from copolymerization of various intermediates in the Raper scheme. 1967 Duchón identified the Thormählen-negative melanogens in melanoma urine as metabolites of DHICA. 1967–68 Prota and Nicolaus isolated pheomelanins from red feathers and showed that they contain sulfur, arising by addition of cysteine to dopaquinone. They also isolated trichochromes and showed them to be related to pheomelanins. 1967–70 Fattorusso, Minale, and their associates carried out extensive degradative studies on pheomelanins and suggested that they are complex mixtures of polymers containing benzothiazine, benzothiazole, and isoquinoline units. 1968 Prota and associates synthesized 5-S-cysteinyldopa and showed that it is a precursor of pheomelanins and of trichochromes. 1968 Fattorusso, Minale, and their associates identified a number of characteristic degradation fragments of pheomelanins, including aminohydroxyphenylalanines (AHP) (which are now used as specific markers of pheomelanins). 1970–76 Swan and coworkers provided analytical and biosynthetic evidence in support of Nicolaus’ heteropolymer model of eumelanins. 1972 Rorsman and Rosengren discovered 5-S-cysteinyldopa in the urine of melanoma patients. 5-S-Cysteinyldopa is now widely used as a biochemical marker of melanoma progression. 1980 Pawelek and his associates discovered a new factor, now known as dopachrome tautomerase (Dct), which promotes tautomerization of dopachrome to give DHICA. 1985 Ito established microanalytical methods to quantitate eumelanins and pheomelanins, based on HPLC determination of the specific degradation products, PTCA and AHP. The original methods have been improved and are now commonly used as standard methods. 1985 Land, Chedekel, and colleagues introduced a pulse radiolysis method to study the fates of highly reactive orthoquinone intermediates (leading to the determination of kinetic constants for reactions in the early stages of melanogenesis in 2003 by Land and associates).

1986– Prota, d’Ischia, Palumbo, Napolitano, and their coworkers in Naples have carried out a long series of studies on the biogenesis and structure of eumelanins and pheomelanins. Metal ions were shown to modify the course of melanogenesis. The color of hair, skin, and eyes in animals mainly depends on the quantity, quality, and distribution of the pigment, melanin. Melanocytes are responsible for the synthesis of melanins within membrane-bound organelles, melanosomes, and the transport of melanosomes to surrounding epidermal cells, keratinocytes. Melanocytes in mammals and birds produce two chemically distinct types of melanin pigments, the black to brown eumelanins and the yellow to reddish pheomelanins (Ito, 1998; Ito et al., 2000; Prota, 1992; Prota et al., 1998a). Among the biopolymers, melanins are unique in many respects. The other essential biopolymers, i.e. proteins, nucleic acids, and carbohydrates, are chemically well characterized; their precursors (monomer units) and modes of connection between the monomer units are known, and sequences of their connection can be determined with well-established methodology (Table 15.1). On the other hand, we still do not have, for example, a method to determine accurately the ratio of various units present in melanins. This is due largely to the chemical properties of melanins, such as their insolubility over a broad range of pH, to heterogeneity in their structural features, and to the lack of methods to split melanin polymers into monomer units (all other biopolymers can be hydrolyzed to the corresponding monomer units). In this chapter, we review advances in the chemistry of melanins and melanogenesis and pay equal attention to the chemical analysis of melanins, with special emphasis on methodology to determine the quantity and quality of melanins present in pigmented tissues and cells (Wakamatsu and Ito, 2002). Characterization of synthetic and natural dopamine melanin is also described briefly. An excellent book (Prota, 1992) is available that deals extensively with the chemistry of melanins and melanogenesis, and more condensed information can also be found in several recent reviews (Ito, 1993a; Prota, 1988, 1993; Prota et al., 1998a).

Table 15.1. Comparison of melanins with other biopolymers. Biopolymer

Monomers

Covalent bond

Protein Polysaccharide Nucleic acid Melanin

Amino acids Glucose Nucleotide Dihydroxyindoles Benzothiazines

Peptide bond (C–N) Glucoside bond (C–O) Phosphate diester bond (P–O) Carbon–carbon bond (C–C)

283

CHAPTER 15 Tyrosinase

O

COOH

COOH NH2

O

NH2

HO

O2

Tyrosine

Dopaquinone (DQ)

Tyrosinase O2

COOH N H

HO

NH2

HO

Cyclodopa

Dopa O –O

N+ H

COOH

Dopachrome Dopachrome tautomerase (Tyrp2)

NH2

HO H 2N

HO

O

HO

HO

N H

O2

N H

O2

O

NH2

S N

+

S

O

NH2 COOH

(HOOC)

COOH

HO

COOH NH2

1,4-Benzothiazine Intermediates (O)

Pheomelanin

Current Concepts Melanogenesis and Melanins The Raper–Mason–Prota Pathway of Melanogenesis It is now well recognized that animal melanins can be classified into two major groups: the brown to black eumelanins that are insoluble in all solvents and the yellow to reddishbrown pheomelanins that are soluble in alkali. Nevertheless, most studies on melanins have so far been conducted on eumelanins. One of the reasons for the scantiness of research on pheomelanins is because it was formerly believed that pheomelanins are produced only in follicular and feather melanocytes. However, it has been shown recently that pheomelanins are also produced in melanomas (Prota et al., 1976; Rorsman et al., 1979) and in normal epidermis (Thody et al., 1991). Both eumelanins and pheomelanins derive from the common precursor dopaquinone, which is formed by tyrosinase oxidation of the common amino acid l-tyrosine (Fig. 15.1). Until recently, it was generally believed that dopa is first formed on the way to dopaquinone. However, using N,N-dimethyldopamine as a tyrosinase substrate, Cooksey et al. (1997) showed that ortho-quinones such as dopaquinone are formed directly during the initial stage of melanogenesis. Dopaquinone is a highly reactive intermediate and, in the absence of sulfhydryl compounds (thiols), it undergoes the intramolecular addition of the amino group giving cyclodopa (often called leukodopachrome). The redox exchange between cyclodopa and dopaquinone then gives rise to dopachrome, the red intermediate (Mason, 1948; Raper, 1927), and dopa. This latter is considered as a source of dopa formed during melanogenesis (Fig. 15.1). Dopachrome then gradually 284

+

NH2

N

Eumelanin

COOH S

CD-quinones

Tyrp1 (DHICA oxidase)

Tyrosinase

NH2

S

HO

(HOOC)

HO

COOH NH2

HOOC

5,6-Dihydroxyindole -2-carboxylic acid (DHICA)

HO

NH2 COOH

2-S-Cysteinyldopa (2-S-CD)

5-S-Cysteinyldopa (5-S-CD) DQ Dopa

COOH

5,6-Dihydroxyindole (DHI)

+

HOOC

O HO

S

S

H2N

CO2

COOH

COOH

HO

HO

COOH

HO

+Cysteine

-Cysteine

Fig. 15.1. The biosynthetic pathways to eumelanins and pheomelanins. Note that the activities of tyrosinase, Tyrp1, and Tyrp2 are involved in the production of eumelanins, whereas only tyrosinase (and the amino acid cysteine) is necessary for the production of pheomelanins.

rearranges to give mostly DHI and, to a lesser extent, DHICA (Palumbo et al., 1987a; Raper, 1927). Finally, these dihydroxyindoles are oxidized and polymerized to give eumelanins. On the other hand, intervention of sulfhydryl compounds (such as cysteine) with this process gives rise exclusively to thiol adducts of dopa, cysteinyldopas, among which 5-Scysteinyldopa (5-S-CD) is the major isomer (Ito and Prota, 1977). Further oxidation of the thiol adducts leads to pheomelanin formation via benzothiazine intermediates. In fact, most melanin pigments present in pigmented tissues appear to be mixtures or copolymers of eumelanins and pheomelanins (Prota, 1980; Ito, 1993b). In addition to tyrosinase, two tyrosinase-related proteins have recently been shown to regulate and promote eumelanogenesis (Hearing, 1993). Dopachrome tautomerase (Dct), the presence of which had been suggested for some years (Pawelek et al., 1980), catalyzes the tautomerization of dopachrome to DHICA (Tsukamoto et al., 1992a). Recently, this enzyme was shown to be identical to tyrosinase-related protein 2 (Tyrp2) (Jackson et al., 1992). Certain metal ions are also known to promote the tautomerization (i.e. the isomerization with a shift of hydrogen atom) of dopachrome to DHICA (Palumbo et al., 1987a, 1991). Oxidative polymerization of DHI is known to be catalyzed by mammalian tyrosinase (Tripathi et al., 1991). Another tyrosinase-related protein, Tyrp1, isolated from mouse melanoma (the brown locus protein) has recently been shown to oxidize DHICA (Jiménez-Cervantes et al., 1994; Kobayashi et al., 1994), although human Tyrp1 is unable to catalyze the same reaction (Boissy et al., 1998). Instead, human tyrosinase is able to oxidize DHICA, as well as tyrosine, dopa, and DHI. It now becomes clear that the activities of these tyrosinase-related proteins greatly affect the

CHEMISTRY OF MELANINS

quantity and quality (the ratio of DHI to DHICA and the degree of polymerization) of the eumelanins produced.

reactions are competitive. The reaction with amine compounds does not proceed so fast. However, only when the amino group is present within the same molecule may the amino group fairly rapidly undergo either an addition reaction to give aminochromes (such as dopachrome) or a condensation reaction to give ortho-quinonimines. An example of the latter type of reaction is found in the cyclization of cysteinyldopaquinones (see Fig. 15.1). It should be stressed that all these reactions are controlled by the intrinsic chemical reactivity of ortho-quinones.

The Intrinsic Reactivity of Ortho-quinones Dopaquinone belongs to the category of ortho-quinones, and it is essential to summarize the chemistry of ortho-quinones for a better understanding of melanogenesis. Figure 15.2 summarizes some of the most important reactions of orthoquinones, which are extremely reactive compounds. In 1976, Tse et al. showed that the addition of sulfhydryl compounds proceeds extremely fast to give thiol adducts. In a pulse radiolysis study on the reactivities of 4-substituted ortho-quinones with cysteine and glutathione, Cooksey et al. (1996) showed that the rate constants of thiol addition are over the range 4 ¥ 105 to 3 ¥ 107/M/s (in the case of cysteine at pH ~7) depending on the nature of the substituents. The reduction to parent catechols through redox exchange proceeds as fast as the thiol addition (Tse et al., 1976) and, therefore, these two

HO

R

HO

fast

R’-SH

R

O

Redox exchange

In 2003, Land et al. reported rate constants (r1–r4) for all of the four important steps in the early phase of melanogenesis, based on pulse radiolysis studies (Fig. 15.3; Land and Riley, 2000; Land et al., 2001, 2003). The pulse radiolysis method is a powerful tool to study the fates of highly reactive melanin precursors. The technique depends on the production of bromine radicals Br2•– by pulse radiolysis of an N2O-saturated aqueous buffer containing KBr. The bromine radical thus formed oxidizes dopa to dopasemiquinone, which then disproportionates to give dopaquinone (and dopa). This entire process proceeds within 2–3 ms so that the fate of dopaquinone can be followed by spectrophotometry in the presence (or absence) of a targeted molecule. The first step (r1 = 3.8/s) in eumelanogenesis is the intramolecular addition of the amino group giving cyclodopa, a fairly slow step. However, as soon as cyclodopa is formed, it is rapidly oxidized to dopachrome through a redox exchange (r2 = 5.3 ¥ 106/M/s). On the other hand, the first step in pheomelanogenesis (r3 = 3 ¥ 107/M/s), the addition of cysteine, proceeds very quickly. The second step, the redox exchange giving cysteinyldopaquinone, proceeds at a slower rate (r4 = 8.8 ¥ 105/M/s) (Land and Riley, 2000). From these kinetic data, several important conclusions can be drawn.

R

HO HO

fast

O

Catechols

Pivotal Roles of Dopaquinone in Controlling Melanogenesis

S-R’

ortho - Quinones

Thiol adducts R’-NH2

slow R

O HO

R

HO

NH-R’

or

Amine adducts (Aminochromes)

(+ H2O)

R’-N

ortho - Quinonimines

Fig. 15.2. Intrinsic chemical reactivities of ortho-quinones. Note that, unless the amino group is present in the same molecule, the addition of an amine does not proceed at biologically relevant rates.

COOH NH2

HO

Tyrosine O2

Tyrosinase HO

COOH N H

HO

Fig. 15.3. Schematic outline of the branching point of production of eumelanins and pheomelanins (Ito, 2003; Land et al., 2003). Note that the rate constants r1–r4 are all controlled by the intrinsic chemical reactivity of dopaquinone. No enzymes other than tyrosinase are necessary to promote these reactions. The intramolecular cyclization of dopaquinone to cyclodopa requires deprotonization of the amino group (actually present in the form of –NH3+).

COOH

Cysteine

HO O

r1 3.8

sec–1

Cyclodopa

O

O

3×

O2

r2

Tyrosinase

M–1sec–1

NH2

HO H2N

S

HOOC

r4

5-S -Cysteinyldopa (5-S -CD) 8.8 × 105 M–1sec–1 O

HO + N H

r3 107

Dopaquinone (DQ)

5.3 × 106 M–1sec–1

–O

COOH NH2

COOH

COOH

COOH

Dopachrome

Dopa

NH2

O

NH2

HO

H2N

S

HOOC

5-S -Cysteinyldopaquinone

Eumelanin

Pheomelanin 285

CHAPTER 15 COOH

HO

COOH

COOH

O

+

S

NH2

H 2N

NH2

O

6

HO

HS NH2

HOOC

Dopaquinone (DQ)

6-S-Cysteinyldopa (1%)

COOH S HO

COOH

COOH

H2N

5

NH2 COOH

S

NH2 HO

2

HO

+

HO

2

NH2

COOH

NH2

+

HO

S NH2

H2N

5 S

HO HOOC

5-S-Cysteinyldopa (74%)

HOOC

2-S-Cysteinyldopa (14%)

1 Comparison of the rate constant for the addition of cysteine to dopaquinone (r3) with the rate constant for the intramolecular cyclization (r1) shows that cysteinyldopa formation is preferred over cyclodopa formation as long as the cysteine concentration is higher than 0.13 mM. 2 The redox exchange giving cysteinyldopaquinone (r4) proceeds 30 times more slowly than the addition of cysteine (r3). Cysteinyldopas thus accumulate in the early phase of pheomelanogenesis. 3 Comparison of the rate constant for redox exchange giving dopachrome from dopaquinone (r2) with the rate constant for intramolecular cyclization (r1) shows that dopachrome production becomes faster than cyclodopa production when the cyclodopa concentration is higher than 0.7 mM. Cyclodopa thus does not accumulate in the early phase of eumelanogenesis. 4 Comparison of the rate constant for redox exchange giving cysteinyldopaquinone (r4) with the rate constant for dopachrome formation (2 ¥ r1; Land et al., 2003) shows that pheomelanogenesis is preferred over eumelanogenesis as long as the cysteinyldopa concentration is higher than 9 mM. 5 It is now possible to derive an “index of divergence” between eumelanogenesis and pheomelanogenesis. By taking dopachrome and cysteinyldopaquinone as representatives of the divergent pathways, Land et al. (2003) proposed an index of divergence (D): D = r3 ¥ r4 ¥ [cysteine]/r1 ¥ r2 This leads to a “crossover value” (i.e. for D = 1) for switching between eumelanogenesis and pheomelanogenesis when the cysteine concentration at the site of melanogenesis is 0.8 mM. The above kinetic data are useful in interpreting the early phase of pheomelanogenesis. Thus, tyrosinase oxidation of dopa in the presence of excess cysteine gives a high yield of 5S-cysteinyldopa (74%) and 2-S-cysteinyldopa (14%) in a ratio of about 5:1, together with a minor 6-S-isomer (1%) and a 286

2,5-S,S’-Dicysteinyldopa (5%)

Fig. 15.4. Formation of isomeric cysteinyldopas and 2,5-S,S¢-dicysteinyldopa by the addition of cysteine to enzymically produced dopaquinone (Ito and Prota, 1977).

diadduct, 2,5-S,S¢-dicysteinyldopa (5%) (Fig. 15.4; Ito and Prota, 1977). This result confirms the interpretation that cysteinyldopa formation is preferred as long as cysteine is present. It may also be pointed out that the ratio of these cysteinyldopa isomers is determined by the intrinsic chemical reactivity of dopaquinone. The ratio of r3 to r4 is ~30, indicating that only after the cysteine concentration decreases to 30 times lower than the 5-S-cysteinyldopa concentration does the formation of 5-Scysteinyldopaquinone become predominant. This interpretation is supported by the facts that cysteinyldopa monomers accumulate in the early phase of pheomelanogenesis and that the formation of the diadduct 2,5-S,S¢-dicysteinyldopa is only a minor pathway. This is why high levels of 5-S-cysteinyldopa are produced in melanoma tissues and secreted into the blood of melanoma patients, which makes 5-S-cysteinyldopa a useful biochemical marker of melanoma progression (Wakamatsu et al., 2002a). The proposed pathway of pheomelanogenesis was substantiated by the identification of 5-S-cysteinyldopa (Bjorklund et al., 1972) and other isomers, along with the diadduct, in melanoma urine (Prota et al., 1977) by Rorsman and his associates. The ratios between various cysteinyldopa isomers found in melanoma urine and tissues (Morishima et al., 1983) are close to that obtained in vitro by incubation of dopa with tyrosinase in the presence of cysteine (Ito and Prota, 1977). This indicates that cysteinyldopas originate in vivo by a similar route, involving the addition of cysteine to dopaquinone. As this process is the earliest event in the course of pigment metabolism, it is thus likely that a certain portion of cysteinyldopas formed are secreted into body fluid, regardless of the type of melanin eventually formed. An alternative route has been proposed for the biosynthesis of cysteinyldopas involving the addition of dopaquinone with glutathione (Ito et al., 1985), followed by the hydrolysis of the resultant glutathionyldopas. The latter step requires the action of two enzymes, g-glutamyltranspeptidase and peptidase, which

CHEMISTRY OF MELANINS

Fig. 15.5. Proposed pathway for mixed melanogenesis. Note that the course of melanogenesis proceeds in three distinct steps: cysteinyldopa genesis, pheomelanogenesis, followed by eumelanogenesis (Ito, 2003; Ito et al., 2000).

Melanogenesis

High tyrosine / low cysteine levels

Low tyrosine / high cysteine levels

EM genesis

EM genesis

PM genesis

PM genesis CD genesis (no melanin)

are present in melanoma cells (Agrup et al., 1975; Mojamdar et al., 1983). Which of the two pathways for cysteinyldopa formation is actually operative in vivo has been addressed. Inhibition of glutathione synthesis by the inhibitor l-buthionine sulfoximine led to a strong reduction in glutathione levels with some increase in cysteine levels in both melanoma cells (Benathan, 1996) and normal melanocytes (Benathan and Labidi, 1996). This modification of thiol levels results in a moderate increase in the cellular level of 5-S-cysteinyldopa. Thus, it is now generally believed that glutathione is not directly involved in the production of cysteinyldopas (Potterf et al., 1999).

The Proposed Pathway for Mixed Melanogenesis From the above interpretation of kinetic data, a pathway for mixed melanogenesis can be proposed as shown in Figure 15.5 (Ito, 2003; Ito et al., 2000). The amount of melanin produced is proportional to dopaquinone production, which is in turn proportional to tyrosinase activity. Melanogenesis proceeds in three distinctive steps. The initial step is the production of cysteinyldopas, which continues as long as cysteine is present (0.1 mM). The second step is the oxidation of cysteinyldopas to give pheomelanins, which continues as long as cysteinyldopas are present (9 mM). The last step is the production of eumelanins, which begins only after most of the cysteinyldopas (and cysteine) are depleted. Therefore, it appears that eumelanins deposit on the preformed pheomelanin (Agrup et al., 1982) and that the ratio of eumelanins to pheomelanins is determined by the tyrosinase activity and the cysteine concentration. This proposal has been supported by several studies. Ozeki et al. (1997a) examined the tyrosinase oxidation of tyrosine in the presence of cysteine and showed that the three steps proceed in sequence. By decreasing the extracellular concentration of cysteine in cultured human melanoma cells, del Marmol et al. (1996) showed a shift to more eumelanic cells as a result of a dramatic decrease in intracellular cysteine concentration. By changing concentrations of tyrosine and cysteine in cultured human melanocytes, Smit et al. (1997) found a twofold increase in melanin production with a decreased ratio of pheomelanin to total melanin when cultured at a higher concentration of tyrosine.

CD genesis (no melanin)

Tyrosinase activity (Dopaquinone production)

The proposal that tyrosinase activity plays a major role in controlling melanogenesis is also supported by several studies. It has been shown that tyrosinase activity is lower when pheomelanogenesis proceeds in viable yellow mice compared with eumelanogenesis (Burchill et al., 1986, 1993; Movaghar, 1989). Moreover, the switching to pheomelanogenesis is accompanied by a marked decrease in the melanin content of hair (Burchill et al., 1986; Granholm et al., 1990). However, the change in tyrosinase activity itself is not enough for the switching of melanogenesis as seen in chinchilla mice where tyrosinase activity is decreased to one-third from the wild type (Coleman, 1962; Lamoreux et al., 2001). The eumelanin content is reduced to one-half in black chinchilla without any increase in pheomelanin content compared with black mice, whereas the pheomelanin content is reduced nine-fold in lethal yellow chinchilla compared with lethal yellow mice (Lamoreux et al., 2001). Conversely, the proposal summarized in Figure 15.5 fits well with the above results that pheomelanogenesis is more strongly affected by the decrease in tyrosinase activity than eumelanogenesis. Barsh (1996) has put forth a similar proposal.

Classification and Structure of Eumelanins and Pheomelanins Table 15.2 summarizes criteria for classifying melanins (Prota et al., 1998a). Extensive studies carried out by Nicolaus’ group in Naples (Nicolaus, 1968) and Swan’s group in Newcastle (Swan, 1974; Swan and Waggott, 1970) led to the conclusion that eumelanins are highly heterogeneous polymers consisting of different oxidative states of DHI and DHICA units, and pyrrole units derived from their peroxidative cleavage (structure 1 in Fig. 15.6). Interestingly, a recent study indicates a high proportion of pyrrole units in Sepia melanin (Pezzella et al., 1997). However, how much those pyrrole units contribute to the structure of natural eumelanins in mammalian pigmentation remains to be studied. Most of the present knowledge on pheomelanin chemistry results from work in the late 1960s conducted by Prota, Nicolaus, and collaborators (Prota, 1972; Thomson, 1974). These, combined with some new findings, led us to formulate the representative structure 2 in Figure 15.6 for pheomelanins 287

CHAPTER 15

that consist mostly of benzothiazine units with minor contributions from benzothiazole and isoquinoline units. Some portions of the monomer units may be connected through ether bonds (Di Donato and Napolitano, 2003; Napolitano et al., 1996a). However, the contribution of ether bond formation should be minimal, because monomer units connected through ether bonds should be colorless, which is incompatible with the brownish color of natural pheomelanins. The isoquinoline units may be produced by post-polymerization modifications of freshly formed pheomelanin, because isoquinoline units are not found in the earlier stages of pheomelanogenesis (Fig. 15.3). The modification of alanyl side-chain to the aromatic character is consistent with biosynthetic studies using radioisotopes and 13C-NMR spectroscopy (Chedekel et al., 1987; Deibel and Chedekel, 1984). Trichochromes are the only melanin-like pigments with fully characterized structures (3–6; Fig. 15.6). The close similarity in structural features between trichochromes and pheomelanins and their coexistence in pigmented tissues suggest that they are formed oxidatively from the same monomer units,

Table 15.2. Main types of epidermal melanin pigments (from Prota et al., 1998a). Melanin

Criteria

Eumelanins

Black or brown nitrogenous pigments, insoluble in all solvents, which arise by oxidative polymerization of 5,6-dihydroxyindoles derived biogenetically from tyrosine via dopaquinone Alkali-soluble, yellow to reddish brown pigments, containing sulfur in addition to nitrogen and arising by oxidative polymerization of cysteinyldopas via 1,4-benzothiazine intermediates A variety of sulfur-containing pheomelanins, of low molecular weight, characterized by a pH-dependent bi(1,4-benzothiazine) chromophore

Pheomelanins

Trichochromes

OH H N

O

O OH

(COOH)

N

N H

O

(COOH)

S

HO S

(COOH)

HOOC

N H

HO

NH2 N

HOOC

OH

N

O S

(COOH)

HOOC N H

OH COOH

O

NH2

H2N

COOH

N

S

S

S

N H

HOOC

OH Trichochrome F (4)

NH2

OH N

COOH

N

COOH

S

O

NH2

OH

S

S N H

HOOC

COOH S N

NH2 OH

OH Trichochrome B (5)

288

COOH

N

NH2

Trichochrome C (3)

NH2

H2N

S

OH

HOOC

NH2

OH N

HOOC

HOOC Pheomelanins (2)

Eumelanins (1)

Trichochrome E (6)

Fig. 15.6. Structures of eumelanins (1), pheomelanins (2), and trichochromes (3–6). Structures of eumelanins and pheomelanins are the only representative ones formulated on the basis of biosynthetic and degradative studies. The positions with (–COOH) are connected either to –H or –COOH; these positions may also be available for attachment to other units. The arrows indicate sites for attachment to other units. The isoquinoline units in pheomelanin structure (2) may be produced by the postpolymerization modification of freshly formed pheomelanin, because those units are not produced in the early stages of pheomelanogenesis.

CHEMISTRY OF MELANINS

differing only in the mode of polymerization. Therefore, trichochromes may be regarded as a variety of pheomelanic pigment (Table 15.2). Prota’s group conducted a series of biosynthetic studies to clarify the stages beyond DHI and DHICA in eumelanogenesis and those of cysteinyldopas in pheomelanogenesis. Although considerable advances have been made in these respects (Di Donato and Napolitano, 2003; Napolitano et al., 1996a; Prota et al., 1998a), it appears at present unnecessary to change our basic views on the structures of eumelanins and pheomelanins, as depicted in Figure 15.6 (see Degradative Studies below for further discussion).

Isolation and Preparation of Melanins Natural Eumelanins One of the major problems in studying melanin is the lack of adequate methods to isolate pure melanin pigments. The most frequently used sources of natural eumelanins are ink sacs of cuttlefish Sepia officinalis (Sepia melanin) (Nicolaus, 1968), the uveal tract and the retinal pigment epithelium of bovine eyes (Dryja et al., 1979), and melanoma tumors. Eumelanins are considered to be firmly bound to proteinaceous components, through covalent or ionic bonds (Zeise, 1995). To remove proteins, treatment with hydrochloric acid is used, but complete removal requires a prolonged treatment in concentrated HCl at room temperature or 24 h of boiling in 6 M HCl (Nicolaus, 1968). As early as 1907, von Fürth and Jerusalem isolated “melanin” as an insoluble, black pigment by heating melanoma tissues in concentrated HCl. One should be aware that such harsh treatment causes irreversible structural alterations. For example, carbon dioxide and ammonia molecules are liberated under these conditions from various synthetic eumelanins (Ito, 1986). Also, the indole moiety is known to be labile in strong acid; tryptophan is decomposed during 6 M HCl hydrolysis of proteins. To circumvent these difficulties, treatment with proteinases such as pronase or collagenase may be preferable; but again, treatment with cold acid is often used to remove accompanying residual proteins (Novellino et al., 1981; Prota et al., 1976). Another approach is to solubilize melanins (melanoproteins) with alkali. Fortner (1910) was the first to report a systemic study to isolate melanin pigment by alkali solubilization. It involves extraction of melanins with NaOH followed by repeated precipitation of the melanins with HCl and redissolution in NaOH (Bolt, 1967). However, proteins cannot be removed by this method; approximately 50% of proteins were found to remain in the melanin preparations from human black hair and from red hair (Menon et al., 1983). In addition, treatment of melanins with alkaline solutions causes irreversible uptake of oxygen (Felix et al., 1978), suggesting that further modification in the melanin structure takes place. To overcome these difficulties, the use of intact Sepia melanin is preferable. However, some precautions should be taken when one wishes to use it as a representative of natural eumelanins. The intact Sepia melanin consumes little oxygen and displays poor redox properties, suggesting a high degree

of breakdown in the indolic moiety (Pezzella et al., 1997). Further, the intact, freshly prepared Sepia melanin differs in surface area depending on the isolation procedure employed (Liu and Simon, 2003). The different degree of aggregation may alter the photochemical properties of melanin granules. If one needs to isolate eumelanins of mammalian origin, preparation of melanosomes from bovine eyes seems to be the choice. Duchón et al. (1973) determined the contents of melanin and protein in melanosome preparations from 10 different biological sources and found the melanin contents to be fairly high, ranging between 20% and 70%. If one wishes to remove proteins from the eumelanin preparations, melanosome fractions should be used as starting materials, rather than whole tissues, to reduce the possibility of unnecessary reactions that might occur between melanins and tissue components. An example of such reactions is sulfur incorporation into eumelanins when heated in 6 M HCl in the presence of cysteine or cystine (Ito, 1986). Recently, Novellino et al. (2000) developed a new enzymatic procedure for isolation of eumelanin from black human hair involving digestion with protease, proteinase K, and papain in the presence of dithiothreitol. In an extension of this study, Liu et al. (2003) compared two different acid/base extractions and an enzymatic extraction to isolate eumelanin from black human hair. The data indicate that pigments obtained by the acid/base extractions contain significant protein (52% and 40%), destroy the melanosomes, and possess an altered molecular structure. The enzymatically extracted hair melanin (14% protein content), on the contrary, retains the morphology of intact melanosomes and is an excellent source of human eumelanin.

Natural Pheomelanins As a source of natural pheomelanins, red feathers of New Hampshire chickens have been commonly used (Minale et al., 1967; Prota, 1972; Thomson, 1974). The isolation procedure involves extraction in 0.1 M NaOH, acidification, and dialysis followed by gel chromatography on Sephadex G-50. Although the procedure is much less destructive than those employed for eumelanins, it is still harsh enough to affect the structural features. Trichochromes (3–6), dimeric pheomelanic pigments, have been isolated from red feathers (Prota and Nicolaus, 1967). Interestingly, trichochrome C (and its isomer trichochrome B) was also found in the urine of patients with melanoma metastases (Agrup et al., 1978; Rorsman et al., 1979). It should be noted that trichochrome F (and its isomer E) may be artificially formed from 5-S-cysteinyldopa (and a mixture of 5-S and 2-S isomers) during the extraction and work-up procedures (Prota, 1992; Rorsman et al., 1979).

Synthetic Melanins Synthetic eumelanins can be prepared by the oxidation of ltyrosine or l-dopa at neutral pH in the presence of tyrosinase, usually commercially available mushroom tyrosinase (Ito, 1986). Biosynthetic dopa melanin has long been considered as 289

CHAPTER 15

a good model of eumelanins. However, recent progress in eumelanin chemistry has proved that dopa melanin prepared by tyrosinase oxidation is quite different from natural eumelanins with respect to the carboxyl group content, reflecting a difference in the ratio of DHI to DHICA (Ito, 1986). A synthetic dopa melanin sample finely suspended in neutral buffer and exposed to oxygen showed a marked increase in the carboxyl content over time (Crescenzi et al., 1993). Similar changes may occur during the process of melanin preparation. Dopa melanin prepared by the auto-oxidation of dopa at alkaline pH appears to be degraded to a significant extent by oxidative cleavage of the ortho-quinone moiety, and the use of these preparations should be discouraged (Ito, 1986). Natural eumelanins are now believed to arise from copolymerization of DHI and DHICA in various ratios. Therefore, it is recommended that synthetic eumelanins prepared from DHI, DHICA, and various ratios of their mixture be used as eumelanin standards, at least for structural studies (Ozeki et al., 1997b). A one-step synthesis of DHI and DHICA for such studies is available (Wakamatsu and Ito, 1988). Synthetic pheomelanins can be prepared by tyrosinase oxidation either of a mixture of l-dopa and l-cysteine or of 5-S-cysteinyldopa in the presence of a catalytic amount of ldopa (Ito, 1989). Degradative experiments suggest that these biosynthetic pheomelanins resemble natural pheomelanins present in the yellow hair of rodents (Ito, 1989). Pheomelanins prepared from 5-S- or 2-S-cysteinyldopa are shown by isoelectric focusing to be more homogeneous than those prepared from dopa and cysteine (Deibel and Chedekel, 1984). In contrast to the availability of eumelanin standards, one faces difficulty in obtaining a valid pheomelanin standard, either natural or synthetic. We therefore refined the conditions for preparing pheomelanin by tyrosinase oxidation of l-dopa in the presence of an excess of l-cysteine (Ito, 1989). The method makes it unnecessary to synthesize pure 5-Scysteinyldopa as the starting material for pheomelanin preparation. When one needs to prepare pheomelanins from 5-S-cysteinyldopa or other isomers, both enzymic and chemical methods are available for the synthesis of cysteinyldopa isomers (Chioccara and Novellino, 1986; Ito, 1983; Ito and Prota, 1977). Copolymers of eumelanin and pheomelanin can be prepared by tyrosinase oxidation of l-dopa in the presence of varying ratios of l-cysteine (Ito and Fujita, 1985). When precise analytical data on melanins are required, differences in the water content of melanins prepared can no longer be neglected. It is thus recommended to use melanin standards that are kept in a desiccator of a constant moisture content (Napolitano et al., 1995).

Molecular Weight The unusual insolubility of eumelanins and their blackness suggest that eumelanins consist of several hundreds of monomeric units. However, there is no direct evidence to support this belief. Because of their insolubility, the direct measurement of molecular weight has been difficult. With soluble DHICA melanins, an estimate of molecular weight 290

using HPLC/molecular sieve analyses gave values in the range of 20 000 to 200 000 (Orlow et al., 1992), although the validity of that is questionable because of a possible interaction of melanins and the chromatographic matrix during the molecular weight determinations (Prota et al., 1998a). Recent application of matrix-assisted laser desorption ionization (MALDI) mass spectrometry shows that both DHI and DHICA melanins have a large proportion of oligomeric fractions of lower molecular weight, not exceeding 1.5 kDa (Napolitano et al., 1996b, c; Seraglia et al., 1993). However, whether polymeric melanins exceeding 1.5 kDa are not present in the samples or are not detectable by this method remains to be clarified. In contrast to eumelanins, the measurement of the molecular weights of pheomelanins is more feasible. A molecular weight of less than 2.0 kDa has been obtained for proteinfree gallopheomelanin 1, isolated from red chicken feathers (Fattorusso et al., 1968). However, that estimate is also subject to question because of the heterogeneity of the pigment and the irreversible binding of part of the material to the chromatographic gel (Deibel and Chedekel, 1982).

Recent Advances in the Study of Melanogenesis Late Stages of Eumelanogenesis Dopachrome accumulates in the early stages of eumelanogenesis, and stages beyond the formation of dopachrome are discussed here. The red pigment dopachrome is a fairly stable molecule with a half-life of about 30 min (first-order rate constant of 4.0 ¥ 10–4/s), and it spontaneously decomposes to give mostly DHI by decarboxylation at neutral pHs in the absence of Dct (or metal ions). The ratio of DHI to DHICA under these conditions is 70:1 (Palumbo et al., 1987a). On the other hand, in the presence of Dct (Tyrp2), dopachrome is catalyzed to undergo tautomerization preferentially to produce DHICA (Palumbo et al., 1991). The ratio of DHICA to DHI production is thus determined by the activity of Dct. The presence of metal ions, especially Cu2+, accelerates the rate of dopachrome rearrangement and also affects the DHICA/DHI ratio, but Dct seems to be more effective in catalyzing the tautomerization (Palumbo et al., 1987a, 1991). Until 1980, eumelanins had been believed to be DHI rich, because the decarboxylation of dopachrome is the major pathway taken in the absence of any extrinsic factor. Then, in 1980, Pawelek’s group discovered dopachrome conversion factor, now known as dopachrome tautomerase or Dct (Pawelek, 1991; Pawelek et al., 1980). Therefore, we analyzed the contents of DHICA-derived units in various types of eumelanins (Ito, 1986). Two analytical methods were used: acidcatalyzed decarboxylation and permanganate oxidation to give PTCA. The results showed that synthetic dopa melanin contained only trace amounts of DHICA-derived units, while melanins from Sepia, B16 melanoma, and mouse black hair consisted of about equal amounts of DHI- and DHICAderived units. This study thus confirmed the significance of DHICA-derived units in the structure of natural eumelanins. The same methodology was employed by Palumbo et al.

CHEMISTRY OF MELANINS H N

4 2

OH

HO

OH

HO

NH

NH

HO

HO

24 N H

HO

24 HO

HO

OH

N H

HO

7

8

COOH

HO

HO

COOH

4 4

NH COOH

HO

OH

H N

HO

HO

N H

7 2

HO

N H

7 4

HO

N H

COOH N H

HO

HO

OH

10

9

Fig. 15.7. Structures of representative oligomers formed in the early stages of enzymic conversion of DHI and DHICA to eumelanins. The homotrimers 7 and 8 are products of oxidation of DHI whereas the homotrimer 9 is from DHICA (Prota et al., 1998a). The heterodimer 10 is among products obtained by oxidation of a mixture of DHI and DHICA.

(1988) to show that dopa melanin prepared in the presence of metal ions, especially Cu2+, are more akin to natural eumelanins than those prepared in the absence of metal ions. The fact that DHICA is equally significant compared with DHI as a eumelanin precursor prompted Prota and his associates to carry out extensive, biomimetic studies on the mode of polymerization of these 5,6-dihydroxyindoles (for reviews, see d’Ischia et al., 1996; Prota et al., 1998a). Upon enzymic or chemical oxidation, DHI affords in the early stages a number of dimers and trimers (d’Ischia et al., 1990). Some representative structures, such as the DHI trimers 7 and 8, are shown in Figure 15.7. Judged from the structures of oligomers isolated, the most reactive position is the 2-position, followed by the 4- and 7-positions. Oxidation of DHICA, catalyzed by tyrosinase/O2 or by peroxidase/H2O2, produces mixtures of dimers and trimers in which the indole units are mostly linked through the 4- and 7-positions (Palumbo et al., 1987b; Pezzella et al., 1996). The 4-position appears to be more reactive than the 7-position, as exemplified by the isolation of the trimer 9. Interestingly, the mode of polymerization is influenced by the oxidant employed; Cu2+ ion-catalyzed oxidation of DHICA affords minor 3,4¢- and 3,7¢-coupled dimers arising from participation of the 3-position, in addition to the major 4,4¢-, 4,7¢-, and 7,7¢-dimers (Pezzella et al., 1996). Cooxidation of DHI and DHICA with peroxidase/H2O2 affords, in addition to homo-oligomers, some heterodimers including the dimer 10 (Napolitano et al., 1993a).

As shown above, the pulse radiolysis technique is very useful in following the fate of short-lived ortho-quinone intermediates. Using this technique, Lambert et al. (1989) were able to suggest the possibility that 5,6-indolequinone tautomerizes to its quinone-imine and quinone-methide tautomers, which then undergo nucleophilic addition of water, thus giving trihydroxyindole species. The role of Dct in melanogenesis has been examined using follicular melanocytes of congenic mice (Lamoreux et al., 2001; Ozeki et al., 1995). The slaty mutation in the mouse is known to decrease the activity of Dct. The effects of tyrosinase, Dct, and Tyrp1 on eumelanogenesis were compared (Ito, 2003). Chinchilla and slaty mouse hairs had total melanin values about 50% that of black hair, while brown hair had about 30% (Lamoreux et al., 2001). Black, chinchilla, and brown mouse hairs give similar PTCA to total melanin ratios. Comparison with synthetic eumelanins indicated that the DHI to DHICA ratios in these three mutants were close to 1:3. On the other hand, slaty hair gave a PTCA to total melanin ratio similar to that of synthetic eumelanin having a 3:1 ratio. Thus, Dct accelerates dopachrome tautomerization, increasing the ratio of DHICA in eumelanins and accelerating the production of eumelanins. Human hair (Ozeki et al., 1996a) and skin (Alaluf et al., 2001) appears to produce DHI-rich eumelanins, similar to mouse slaty hair. Then, what is the role of Tyrp1? There is some controversy regarding the role of Tyrp1 (Hearing, 2000), although it is known that mouse Tyrp1 is able to catalyze the oxidation of DHICA (Jiménez-Cervantes et al., 1994). DHICA has a higher oxidation potential than DHI. This means that DHICAquinone may be able to undergo a redox exchange with DHI yielding DHICA and DHI-quinone (Fig. 15.8). This proposal, however, does not exclude the possibility that DHI adds directly to DHICA-quinone to form heterodimers (Napolitano et al., 1993a). Copolymerization of these four intermediates should yield a series of heteropolymers of DHI and DHICA. Thus, the role of Tyrp1 appears to accelerate eumelanogenesis by oxidizing not only DHICA but also DHI indirectly. Our chemical analysis showed that follicular melanocytes of brown mice produce a eumelanin with a smaller molecular size compared with black mice (Ozeki et al., 1997b). As evidence to show that this type of copolymerization takes place, Prota’s group isolated a heterodimer of DHI and DHICA as the acetyl derivative from an oxidation mixture of DHI and DHICA (Napolitano et al., 1993a).

Late Stages of Pheomelanogenesis The early stages of pheomelanogenesis until the formation of cysteinyldopaquinones are well elucidated, as discussed above. In the later stages of pheomelanogenesis, 5-Scysteinyldopaquinone, once formed, then rapidly cyclizes via attack of the cysteinyl side-chain amino group on the carbonyl group to give a cyclic ortho-quinonimine intermediate (Fig. 15.9; Napolitano et al., 1994). The rate (r5) of quinonimine formation was determined by pulse radiolysis to be 5.5/s (Thompson et al., 1985). It should be stressed that the rate of 291

CHAPTER 15 O

HO

Polymerization of 4 intermediates

N H

O N H

HO

DHI-quinone

DHI

O

Eumelanin

HO COOH

O

N H

COOH Tyrp1

DHICA-quinone

DHICA

O

OH

O O H2N COOH

N

COOH

S NH2

N

COOH

r6

r5

HOOC

Fig. 15.8. Proposed role of Tyrp1. Copolymerization of DHI and DHICA is suggested (Ito, 2003).

N H

HO

S

5.5 sec–1 HOOC

NH2

HOOC

ortho-Quinonimine

5-S-Cysteinyldopaquinone (CDQ)

S

0.5 sec–1 NH2

1,4-Benzothiazines

CD

OH H N

COOH

S HOOC

NH2 Dihydrobenzothiazine 11

5-S-cysteinyldopaquinone formation is controlled by the rate of dopaquinone formation, because 5-S-cysteinyldopa itself is a much poorer substrate for tyrosinase than dopa (Agrup et al., 1982). An alternative pathway is possible for the metabolism of 5S-cysteinyldopaquinone; this ortho-quinone also undergoes the addition of cysteine with a rate constant of 1 ¥ 104/M/s to give the diadduct 2,5-S,S¢-dicysteinyldopa (Thompson et al., 1985). It is thus delineated that, unless the cysteine concentration is higher than 5 mM (an unlikely situation in vivo), the quinonimine formation predominates. The ortho-quinonimine then undergoes rearrangement to benzothiazine intermediate(s) with (85%) and without (15%) decarboxylation (Napolitano et al., 1994). The rate (r6) of decay (k = 0.5/s) of the cyclic ortho-quinonimine to the benzothiazines was recently determined by pulse radiolysis (Napolitano et al., 1999). As shown in Figure 15.9, an alternative pathway for ortho-quinonimine is also possible by redox exchange with 5-S-cysteinyldopa, leading to the production of a reduced form of the quinonimine (a 3,4dihydro-1,4-benzothiazine derivative 11) and 5-Scysteinyldopaquinone (Napolitano et al., 2000a). Whether the redox exchange or the rearrangement prevails is strongly influenced by many factors, including the nature of the oxidant and the concentration of the precursor 5-S-cysteinyldopa. It 292

Fig. 15.9. Kinetics in the late stages of the formation of pheomelanins from 5-Scysteinyldopaquinone. Note that the dihydrobenzothiazine 11 is produced via redox exchange whereas benzothiazine intermediates are by rearrangement (with/without decarboxylation).

therefore remains to be seen whether this redox change is a significant pathway in vivo. Reactions beyond the benzothiazines, which lead to pheomelanins, are rather complex, but nevertheless these have also been extensively studied by Prota and associates (reviewed by Di Donato and Napolitano, 2003). It is interesting to note that the presence of metal ions strongly affects the course of later stages; trichochrome-type dimers (reduced form of trichochrome pigments) are produced in the zinccatalyzed oxidation of 5-S-cysteinyldopa (Napolitano et al., 2001), while two monomeric amino acids, the benzothiazinone 12 and the benzothiazole 13 (R = H), are produced by chemical oxidants in the presence of Fe3+ or Cu2+ at neutral pH (Fig. 15.10; Di Donato et al., 2002). Notably, the formation of benzothiazole (13) suggests that the ring contraction of benzothiazines to benzothiazoles is a feasible process under physiological conditions. In this connection, high levels of zinc, iron, and copper ions are detected in melanosomes isolated from human hair (Liu et al., 2003) and in intact melanosomes from the eye (Samuelson et al., 1993). Interestingly, upon irradiation with UVA, the dihydroxybenzothiazine 11 undergoes ring contraction to give 2-methylbenzothiazole 13 (R = CH3) (Costantini et al., 1994a). The presence of benzothiazole and isoquinoline units in pheomelanins has been questioned by Prota (Prota et al.,

CHEMISTRY OF MELANINS OH

OH N

(COOH)

OH H N

O

N

Fe3+ or Cu2+

R

S

Fig. 15.10. Products of metal-catalyzed oxidation of 5-S-cysteinyldopa via the 1,4benzothiazine amino acid.

HOOC

NH2 1,4-Benzothiazines

1998a) based on the fact that the products derived from benzothiazole and isoquinoline units are obtained only when pheomelanins are subjected to drastic degradative reactions. However, as shown above, the ring contraction of benzothiazines giving benzothiazoles proceeds under very mild conditions that mimic in vivo situations. The same argument may hold true for the presence of the isoquinoline units (the unit located at the center of the structure 2). The isoquinoline structure would be readily formed through the Pictet–Spengler reaction (Manini et al., 2001) when an alanyl side-chain on a benzene ring and a carbonyl group are close to each other.

Chemical Properties of Melanins A major role of melanins in vivo is generally believed to be photoprotection of underlying tissues. However, this concept has been challenged from time to time (Hill, 1992; Wood et al., 1999), and this topic is dealt with extensively in Chapter 17 (Photobiology of melanins). However, it should be pointed out here that the measurement of eumelanin and pheomelanin contents is indispensable in these studies (De Leeuw et al., 2001; Tadokoro et al., 2003). Another important property of melanins is the ability to bind to various chemicals and metal ions. This property is discussed in Chapter 18 (Toxicological aspects of melanin and melanogenesis). Chapter 16 also addresses the physical properties of melanins, including free radical properties, redox state, and photo-oxidation.

Melanin-related Metabolites Melanin-related Metabolites as Markers of Melanoma (and Melanin Production) In addition to eumelanins and pheomelanins, normal and malignant melanocytes produce and excrete their precursors, 5,6-dihydroxyindoles and cysteinyldopas. These precursors and their metabolites are found in epidermal and melanoma tissues and in body fluids at variable levels. Therefore, those melanin-related metabolites have been extensively studied as markers of melanoma progression, to detect metastases, evaluate therapeutic effects, and to predict prognosis (for reviews, see Duchón, 1987; Hartleb and Arndt, 2002; Ito, 1992; Rorsman and Pavel, 1990; Rorsman et al., 1983). As for the 5,6-dihydroxyindoles, DHI, DHICA, and their metabolites in urine were once explored as possible markers for screening of metastasizing melanoma (Duchón et al., 1981). The indolic urinary melanogens were classified into two main groups based on the response to the Thormählen reaction (sodium nitroferricyanide). Thormählen-positive

S HOOC

NH2 Benzothiazinone 12

+

S

HOOC

NH2 Benzothiazole 13

melanogens were first recognized by Leonhardi (1955) as DHI derivatives. Through extensive studies by Duchón and his associates in Prague, this group of melanogens was shown to be glucuronides and sulfates of 5-hydroxy-6-methoxyindole and 6-hydroxy-5-methoxyindole (Matous et al., 1981; Pavel et al., 1981). The Thormählen-negative melanogens were shown to consist mainly of 5-hydroxy-6-methoxyindole-2carboxylic acid and 6-hydroxy-5-methoxy-indole-2-carboxylic acid (Duchón and Matous, 1967). These indolic melanogens are formed in melanocytes by O-methylation (Smit et al. 1990), followed by subsequent conjugation with glucuronic acid or sulfuric acid in the liver or kidney. The O-methylation appears to be a mechanism of detoxification of these cytotoxic 5,6-dihydroxyindoles. The potential usefulness of these indole melanogens as markers of melanoma progression (and of melanin production in normal subjects) has attracted the interest of clinicians. 5-Hydroxy-6-methoxyindole-2-carboxylic acid was found to be the best marker of melanin pigmentation in the urine (Westerhof et al., 1987). The level of 6-hydroxy-5-methoxyindole-2-carboxylic acid in plasma was proposed to be more sensitive and reliable than 5-S-cysteinyldopa (Hara et al., 1994), whereas other studies have reached the opposite conclusion (Horikoshi et al., 1994). In contrast to 5-S-cysteinyldopa, which is still drawing attention (Hartleb and Arndt 2001; Wakamatsu et al., 2002a), it appears likely that clinicians have lost interest in those indolic melanogens as melanoma markers. The growing interest in DHI and DHICA as equally important precursors of eumelanins and as markers of melanocyte activities has prompted us to establish more efficient methods to prepare these rather labile compounds, because previously reported methods of DHI and DHICA preparation require tedious, multistep procedures (Benigni and Minnis, 1965). Therefore, by taking advantage of the chemical reactivity of dopachrome, we developed biomimetic procedures to prepare DHI and DHICA in subgram quantities (Wakamatsu and Ito, 1988). Dopachrome generated in situ by ferricyanide oxidation of dopa at neutral pH undergoes spontaneous decarboxylation to give DHI, while treatment with alkali at pH 13 affords mostly DHICA. After recrystallization, DHI and DHICA are obtained in modest yields. As for the cysteinyldopas, the major isomer of cysteinyldopas, 5-S-cysteinyldopa, was first detected in melanoma tissues and in urine (Bjorklund et al., 1972). Subsequently, other minor isomers, i.e. 2-S- and 6-S-cysteinyldopas, along with the diadduct 2,5-S,S¢-dicysteinyldopa, have also been 293

CHAPTER 15

detected in the urine of melanoma patients (Morishima et al., 1983; Prota et al., 1977). Tyrosinase appears to be primarily responsible for the production of 5-S-cysteinyldopa. 5-S-Cysteinyldopa has thus been detected not only in melanoma tissues, in the serum, and in the urine of melanoma patients, but also in skin, hair, serum, and urine of normal subjects (Ito, 1992; Rorsman and Pavel, 1990; Rorsman et al., 1983). However, the detection of small amounts of 5-S-cysteinyldopa in plasma and urine from human and mouse albinos indicates that tyrosinaseindependent routes may also be present (Acquaron et al., 1981; Nimmo et al., 1985). In this connection, it should be noted that some biologically relevant oxidizing systems, in addition to tyrosinase, can mediate the formation of cysteinyldopas from dopa and cysteine, i.e. peroxidase/H2O2, ferrous ion, superoxide, and hydroxyl radicals (Ito, 1983; Ito and Fujita, 1981, 1982). In contrast to 5,6-dihydroxyindoles, 5-S-cysteinyldopa is not metabolized to any major extent; decarboxylation does not take place, and O-methylation (Agrup et al., 1977) and conjugation with glucuronic and sulfuric acids appear to be only minor pathways. These properties, coupled with the high renal clearance, make 5-S-cysteinyldopa a useful marker of pigmentation in normal and malignant melanocytes. Determination of plasma levels of 5-S-cysteinyldopa seems to be useful in predicting distant metastases in melanoma patients, and elevated levels are associated with a poor prognosis (Wakamatsu et al., 2002a). However, one of the drawbacks of 5-Scysteinyldopa as a melanoma marker is that the levels often rise two to several-fold in summer, occasionally to pathological levels, due to sun exposure (Rorsman et al., 1976; Wakamatsu and Ito, 1995), although this property may be used as a measure to assess the degree of sun exposure in normal subjects. The growing demand for 5-S-cysteinyldopa, not only for the study of pheomelanogenesis but also for clinical studies, has prompted us to develop convenient laboratory syntheses. For subgram scale preparation, the reaction of cysteine with dopaquinone produced by tyrosinase oxidation of dopa serves the purpose (Ito and Prota, 1977). For gram scale preparation, oxidation of dopa with ceric ammonium nitrate in sulfuric acid in the presence of cysteine seems a better choice (Chioccara and Novellino, 1986).

Cytotoxicity and Related Properties of Melanin Precursors The concept is generally accepted that melanin precursors are cytotoxic to the cells where melanin pigments are produced, i.e. melanocytes (Hochstein and Cohen, 1963). This concept leads to another concept: that the compartmentalization of melanogenesis in melanosomes represents a strategy by melanocytes to prevent the inherent cytotoxicity of melanin precursors (Prota et al., 1998a). Wick et al. (1977) were the first to explore the possibility of using the cytotoxicity of melanin precursors to develop chemotherapeutic agents against melanoma. They examined the cytotoxic effects of dopa and other related melanin pre294

cursors. These studies were followed by studies on the cytotoxicity of melanin precursors, DHI and 5-S-cysteinyldopa, to cultured melanoma cells (Fujita et al., 1980; Pawelek and Lerner, 1978). However, the mechanism of cytotoxicity of 5-S-cysteinyldopa to melanoma cells soon proved to involve the production of reactive oxygen species, such as H2O2, formed by auto-oxidation in culture media (Ito et al., 1983). The inherent cytotoxicity of DHI and DHICA was also reexamined (Urabe et al., 1994). The observed cytotoxic effects were found to be mainly due to the generation of reactive oxygen species outside the cells. It thus appears that DHI and DHICA may be less cytotoxic than one would imagine as long as they are produced and oxidized within melanosomes. Nevertheless, it is well known that stimulation of melanogenesis leads to the suppression of proliferation and eventually to cell death in cultured normal melanocytes (Hirobe et al., 2003). Further, ectopic expression of tyrosinase in the absence of Tyrp1 or Dct may cause severe cytotoxicity to nonmelanocytic cells in which no melanosomal compartmentalization is present (Singh and Jimbow, 1998). Then, what are the more toxic intermediates than DHI and DHICA that are produced in melanocytes? In the process of melanogenesis, a number of highly reactive ortho-quinones are produced including dopaquinone, dopachrome, DHI-quinone, and DHICA-quinone (Figs 15.2 and 15.8). Among these quinones, dopaquinone, DHI-quinone, and DHICA-quinone appear to be too reactive and would be polymerized within the melanosomes. Dopachrome is, however, quite stable in the absence of Dct or metal ions (Palumbo et al., 1987a). In fact, a recent study indicates that aminochromes such as dopachrome and dopaminechrome are much more toxic to cultured melanoma and neuroblastoma cells than l-dopa, DHI, and DHICA (Matsunaga et al., 2002). Dopachrome reacts with sulfhydryl compounds at the 4-position (d’Ischia et al., 1987). It now appears that dopachrome, although not extremely reactive, is able to react with sulfhydryl enzymes essential for melanocyte survival and to inactivate them, eventually leading to cell death. Supporting this view, Dct is a melanocyte-specific enzyme considered to be a “rescue” enzyme essential for melanocyte survival (Tsukamoto et al., 1992a). Mutations in Dct that decrease catalytic function affect DHICA production and are generally quite cytotoxic to melanocytes. Melanocytes typically express Dct before any of the other melanogenic enzymes, presumably to minimize such toxicity (Steel et al., 1992). Matsunaga et al. (1999) have recently shown that a related enzyme, called macrophage migration inhibitory factor (MIF), is able to catalyze the conversion of dopaminechrome to DHI. MIF is expressed in neuronal tissues and is believed to participate in a detoxification pathway for catecholamine oxidation products. The cytotoxicity of ortho-quinones could potentially lead to chemotherapeutic approaches to treat melanoma using phenolic melanin precursors (Prota et al., 1994; Riley et al., 1997). Phenolic compounds are expected to be less cytotoxic themselves than the corresponding catecholic compounds, yet they can be metabolized in melanocytes to highly reactive

CHEMISTRY OF MELANINS

those of ascorbic acid and glutathione. 5-S-Cysteinyldopa has also been shown to be a potent inhibitor of hydroxylation/ oxidation reactions mediated by H2O2 and the Fe2+/EDTA complex (Fenton reaction) (Napolitano et al., 1996d). Furthermore, DHI is highly efficient in inhibiting the generation of peroxidation products in in vitro models of UVinduced lipid peroxidation compared with 5-S-cysteinyldopa as well as eumelanin and pheomelanin samples (Schmitz et al., 1995). A recent kinetic study using laser flash photolysis also shows that the antioxidant properties of 5,6-dihydroxyindoles, in particular DHI, are as good as those of a-tocopherol (Zhang et al., 2000). The photoprotective role of melanin-related metabolites is also an interesting consideration. Photobiological and photochemical data indicate that DHI and DHICA have protective roles. Upon photoexcitation, these 5,6-dihydroxyindoles undergo photolysis with the generation of semiquinone radicals. DHI semiquinone can react with oxygen and related species, giving rise to hydroxylated oligomer species that can polymerize to eumelanic pigments (d’Ischia and Prota, 1987; Lambert et al., 1989). Thus, DHI and DHICA can contribute significantly to protect the skin from damaging UV radiation by quenching oxygen species and providing an additional amount of photoprotective pigment (Prota et al., 1998a). In contrast to 5,6-dihydroxyindoles, irradiation of 5-Scysteinyldopa with UVB results in the formation of dopa, arising by photolytic cleavage of the S–CH2 bond followed by desulfuration (Costantini et al., 1994b; Land et al., 1986). Thus, UV photolysis of 5-S-cysteinyldopa affords potentially toxic free radicals, capable of affecting important biological targets such as DNA (Chedekel and Zeize, 1988; Koch and Chedekel, 1986) or membrane lipids (Schmitz et al., 1995). However, how much these photoprotective and phototoxic events are functionally important in vivo remains to be studied. The significance of these events should depend on the concentrations of these melanin precursors in epidermal tissues, information that is scarce at present.

R

HO HO GSH R

Tyrosinase

S-G Catechol - GSH adducts

R

O O

HO Phenols OH radical

ortho-Quinones H2O2

(H)

Enz-SH

HO

O2

S-Enz

R

HO

R

HO

Catechol - Protein adducts HO Catechols

Fig. 15.11. Mechanism of melanocytotoxicity of phenolic and catecholic melanin precursors (Ito, 2003).

ortho-quinones by the action of tyrosinase (Fig. 15.11). The ortho-quinones thus formed may be detoxified by glutathione in the cytosol, but those that escape from this detoxification mechanism may enter the nucleus and inactivate sulfhydryl enzymes such as thymidylate synthase, thereby leading to cell death (Prezioso et al., 1992). Among the phenolic compounds so far examined, 4-S-cysteaminylphenol and its derivatives appear to be the most promising antimelanoma agents (Alena et al., 1990; Yukitake et al., 2003). The ultimate toxic metabolite of 4-S-cysteaminylphenol has been shown to be dihydro-1,4-benzothiazine-6,7-quinone, a sulfur homolog of dopaminechrome (Hasegawa et al., 1997; Mascagna et al., 1994). Catechols, such as dopa and DHI, are cytotoxic through two possible routes: one through the generation of ortho-quinones and the other through auto-oxidation producing H2O2 and hydroxyl radicals (Graham et al., 1978). To evaluate the binding of ortho-quinones to proteins through cysteine residues, we developed a method to measure the catechol–protein adducts. The method is based on the HPLC analysis of cysteinyl–catechols formed on HCl hydrolysis of the modified proteins (Ito et al., 1988a). Melanin precursors themselves may have protective roles in melanocytes (Prota et al., 1998a). DHI and, to a lesser extent, DHICA and 5-S-cysteinyldopa are capable of inhibiting lipid peroxidation in several in vitro model systems (Memoli et al., 1997; Napolitano et al., 1993b). These melanin precursors have been shown to have inhibitory effects much greater than

Degradative Studies on Melanins Eumelanins Biosynthetic and degradative studies indicate that natural eumelanins are highly heterogeneous polymers consisting of various monomer units, including DHI unit A, DHICA unit B, and the pyrrole units C and D derived from peroxidative cleavage of units A and B (Fig. 15.12; Prota, 1992).

HO

HO

COOH

N H

HO

A

N H

HO

B

O

O

O

HOOC

COOH

N H

N H

C

D

Fig. 15.12. Monomer units present in the eumelanin polymer. The carbon to nitrogen ratios are 8, 9, 6, and 7 for DHI unit (A), DHICA unit (B), pyrrole unit (C), and pyrrole-carboxylic acid unit (D) respectively. DHI and DHICA units may also be present in the oxidized, orthoquinone form.

295

CHAPTER 15 HOOC

HOOC COOH

HOOC N H

14 (PTCA)

COOH

HOOC N H

COOH

15

Extensive degradative studies provided a number of chemical degradative methods (Nicolaus, 1968; Swan and Waggott, 1970). Among them, oxidative degradation by permanganate or H2O2 appears to be most informative (Panizzi and Nicolaus, 1952; Piattelli and Nicolaus, 1961). Nicolaus (1968) repeated the oxidation of Sepia melanin with H2O2 at pH 7 followed by alkaline hydrolysis, which afforded a 6.5% yield of pyrrole2,3,5-tricarboxylic acid (PTCA; 14 in Fig. 15.13). In another preparative experiment in which 10 g of Sepia melanin was oxidized by H2O2 in acetic acid, 200 mg of PTCA and 61 mg of pyrrole-2,3,4,5-tetracarboxylic acid (15) were isolated (Nicolaus, 1968; Piattelli et al., 1962). In our studies, permanganate oxidation of natural eumelanins gave about a 2% yield of PTCA (Ito and Fujita, 1985). We also noticed that permanganate oxidation gave a considerable amount of the tetracarboxylic acid 15 only when the oxidation was conducted under alkaline conditions (Ito and Fujita, 1985). This suggests that, during oxidative degradation carried out in an alkaline medium, the 3-position of DHICA unit B is connected to another monomer unit, thus giving rise to the artificial formation of the tetracarboxylic acid 15. This represents an example of possible artifact formation during degradative reactions and calls for more attention to such possibilities. The origin of PTCA was interpreted in terms of oxidative breakdown of the DHICA-derived structures (units B and D) in the eumelanin polymer. Another pyrrolic acid, pyrrole-2,3dicarboxylic acid (PDCA, 16), can be expected to arise from DHI-derived structures (units A and C). In fact, small amounts of this pyrrolic acid were detected among oxidation products of Sepia melanin, dopa melanin, and DHI melanin (Piattelli et al., 1962). The significance of its formation has recently been re-examined (Napolitano et al., 1995). Other degradative reactions appeared to be much less informative. Alkaline fusion of Sepia melanin afforded both DHI and DHICA, but the yields were too low to make this reaction significant (Piattelli et al., 1963). Mild treatment of Sepia melanin with sodium borohydride in 0.1 M NaOH also afforded DHICA (d’Ischia et al., 1985). Again, the yield was very low. Some PTCA was also obtained by boiling Sepia melanin in 4% NaOH (Piattelli et al., 1962).

Carboxyl Content in Eumelanins The carboxyl group attached to the indole or pyrrole ring (in units B or D) is labile and may easily be split off as CO2 by heating melanin powder at a high temperature or by heating a melanin suspension in Vaseline (Piattelli et al., 1962) or in 6 M HCl (Ito, 1986). Our experience indicates that the acid decarboxylation is more reproducible and releases CO2 quan296

HOOC HOOC N H 16 (PDCA)

Fig. 15.13. Products of oxidation of eumelanins with acidic potassium permanganate or alkaline H2O2.

titatively (Ito, 1986). This methodology has been applied successfully to estimate the content of carboxyl groups in natural and synthetic eumelanins (Ito, 1986; Novellino et al., 2000; Palumbo et al., 1988; Pezzella et al., 1997). Using decarboxylation, substantial amounts of carboxyl groups were found in all the natural eumelanins examined, suggesting the common presence of DHICA-derived units (Prota, 1992; Prota et al., 1998a). Permanganate oxidation of eumelanins gives PTCA that arises from DHICA-derived structures (units B and D). Comparison of the yields of PTCA from a number of natural and synthetic eumelanins, coupled with results of the acid decarboxylation, indicates that units derived from DHICA comprise only 10% of synthetic dopa melanins, but as much as one-half of intact, natural eumelanins (Ito, 1986). The first unambiguous demonstration of the involvement of DHICA in eumelanogenesis came from a study in which 1-14C-dopa was injected into melanoma-bearing mice (Tsukamoto et al., 1992b). The pigment from the tumor was then isolated, purified, and chemically decarboxylated. Determination of the labeled CO2 evolved showed that at least 20% of the precursor incorporated into the melanin retains the labile isotope in the form of DHICA-linked carboxyl groups. In another approach to determine the carboxyl contents in melanins, Zeise and Chedekel (1992) used a titrimetric analysis to quantify the bioavailable carboxyl groups present on the surface of melanin particles. They found that the ratio of moderately acidic (–COOH) to weakly acidic (phenolic OH) groups was 0.86 in Sepia melanin. This method (Zeise and Chedekel, 1992) would be useful in comparing the functional groups present on the surface of various melanin pigments.

Pheomelanins Biosynthetic and degradative studies carried out on pheomelanic pigments including trichochromes indicate that pheomelanins are also highly heterogeneous polymers arising from the oxidative polymerization of 1,4-benzothiazines derived from cysteinyldopas (Prota, 1992; Prota et al., 1998a). Structural studies on pheomelanic pigments were greatly facilitated by reductive hydrolysis with hydriodic acid that yielded a number of informative degradation products. Although the conditions employed for the degradation were harsh, suggesting the possibility of artifact formation, the relatively high yield of degradation products makes the method indispensable in structural studies of pheomelanic pigments (Patil and Chedekel, 1984; Prota, 1992). When heated in hydriodic acid, the decarboxy derivative of trichochrome C (3) gives as major products 4-amino-3-

CHEMISTRY OF MELANINS

OH

R

NH2

N

NH2

Fig. 15.14. Products of hydriodic acid hydrolysis of trichochromes and pheomelanins. R = H or CH3.

HOOC

OH

HOOC

NH2

NH2

S

HOOC

18 (3-AHP)

17 (4-AHP)

OH OH

NH2

R

N

HOOC

NH2

20

19

OH COOH COOH

N HOOC

HOOC

N

N

COOH S

COOH

Fig. 15.15. Products of oxidation of pheomelanins with acidic potassium permanganate or alkaline H2O2.

HOOC

N

COOH

21

hydroxyphenylalanine (4-AHP; 17) and the benzothiazinone amino acid 12, along with the benzothiazole amino acid 13 (R = CH3) (Fig. 15.14; Nicolaus et al., 1969). Trichochrome F (4), which occurs concomitantly at a much lower concentration than trichochrome C, gives 4-AHP along with a much smaller amount of the benzothiazole 13 (R = H) on hydriodic acid treatment (Prota et al., 1969). On the other hand, degradation of trichochrome E (6) with hydriodic acid gives a mixture of two isomers, 4-AHP (17) and 3-AHP (18). The benzothiazole amino acids 12 (R = H and CH3) can arise readily from trichochromes by ring contraction under acidic or alkaline conditions; heating the benzothiazinone 12 in hydrochloric acid affords the benzothiazole 13 (R = H) (Fattorusso et al., 1968). Gallopheomelanin-1, the major, protein-free, pheomelanic pigment, was isolated from red chicken feathers by alkaline extraction and chromatography on Sephadex (Minale et al., 1967; Prota and Nicolaus, 1967). A most notable feature of gallopheomelanin-1 was its high sulfur content; it contained 9.0% nitrogen and 9.9% sulfur, the molar ratio of nitrogen to sulfur being 2.1:1 (Minale et al., 1967). On hydriodic acid hydrolysis, gallopheomelanin-1 affords the benzothiazole amino acids 13 and 19 (R = H and CH3) along with a comparable amount of AHP isomers, 4-AHP (17) and 3-AHP (18), with benzothiazole (19) and 3-AHP (18) being minor isomers (Fattorusso et al., 1968; Minale et al., 1967). These characteristic products were obtained in similar yields (total yields of about 20%) from synthetic pheomelanins prepared either from a mixture of dopa and cysteine or from 5-S-cysteinyldopa (Fattorusso et al., 1969a). Difficulties in interpreting these results arise from the fact that these natural and synthetic pheomelanin preparations were subjected to strongly alkaline pH during the isolation procedure or chromatographic separation. This would suggest that a major part of the benzothiazole units in pheomelanins arise artificially from the benzothiazine units. In support of this view, our recent study has shown that the benzothiazoles

HOOC

S 22 (TTCA)

HOOC

S

23 (TDCA)

HOOC

NH2 24 (BTCA)

13 and 19 (R = H) were obtained in yields approximately 1/10th those of AHP isomers on hydriodic hydrolysis of both natural and biosynthetic pheomelanins that were not subjected to alkaline conditions (Ito, 1989). The production of AHP isomers occurs specifically at the expense of benzothiazine units, but not benzothiazole units (Fattorusso et al., 1968; Ito, 1989). The reaction involves reductive fission of the aromatic C–S bond induced by the iodide anion. Other interesting products of reductive degradation with hydriodic acid are the isoquinolines 20 (R = H and CH3) (Fattorusso et al., 1970). Also, permanganate oxidation of pheomelanins gives a product arising from the isoquinoline unit, pyridine-2,3,4,6-tetracarboxylic acid (21), in addition to thiazole-2,4,5-tricarboxylic acid (TTCA; 22) and thiazole-4,5tricarboxylic acid (TDCA; 23), which arise from the benzothiazole units (Fig. 15.15; Fattorusso et al., 1969b). Red hair was recently found to be closely associated with loss-of-function mutations of the melanocortin-1 receptor (MC1R) (Valverde et al., 1995). After that discovery, there has been a growing interest in red hair as a risk factor for melanoma and for nonmelanoma skin cancers (Rees, 2000). Napolitano et al. (2000b) recently described another marker of pheomelanins, i.e. 6-b-alanyl-2-carboxy-4-hydroxybenzothiazole (BTCA; 24) in addition to TTCA (22), both of which are produced by alkaline H2O2 treatment of various hair samples. It is suggested that BTCA represents a new biogenetic marker for predicting individuals at high risk for skin cancer and melanoma. Based on these data, a representative structure (2) can be proposed for pheomelanins (Ito, 1993a, 1998). The benzothiazine unit should constitute at least 40% of the monomer units, as hydriodic acid hydrolysis of trichochrome F gives AHP in about a 50% yield, whereas the yields are about 20% from pheomelanins (Ito, 1989; Ito and Fujita, 1985). The other monomer units, such as the isoquinoline and benzothiazole units, may represent only minor constituents. Some of the alanyl and cysteinyl side-chains may be degraded during 297

CHAPTER 15 Table 15.3. Comparison of chemical and physical properties of eumelanins and pheomelanins. Property

Eumelanins

Pheomelanins

Specificity

Color of tissue Solubility Elemental composition IR spectrum UV-vis spectrum NMR spectrum EPR spectrum Chemical degradation

Dark brown to black Insoluble in all solvents C, H, O, N (6–9%), S (0–1%) No characteristic bands General absorption Potentially useful Single peak PTCA

Yellow to reddish brown Soluble in alkali C, H, O, N (8–11%), S (9–12%) No characteristic bands General absorption No data Two peaks AHP†

Low Low Low Low Low* ? High High

*Difference between eumelanins and pheomelanins can be used to differentiate them. †Recently, 4-AHP has been introduced as a more specific marker for pheomelanins (Wakamatsu et al., 2002b).

polymerization (Chedekel et al., 1987; Deibel and Chedekel, 1984).

Chemical Analysis of Melanins Spectrophotometric Analysis of Melanins: Historical Background Regulation of melanogenesis has been the subject of extensive studies. In most such studies, quantitation of melanins in pigmented tissues such as hair and skin and in cultured melanocytes is essential. Effects of the genetic background on hair pigmentation in mammals can also be assessed by analysis of the quantity and quality of the melanins produced. Despite these needs, no simple laboratory methods to quantify melanins have been developed. Most laboratory tests to quantify biomolecules such as proteins, carbohydrates, lipids, and nucleic acids utilize specific reagents that develop a characteristic color with a given biomolecule in solution. On the contrary, a major problem in quantifying melanins appears to be how to solubilize the melanin pigment. Once solubilized, melanin can easily be assayed by absorbance in the visible absorption spectra. Quantitation of melanins present in pigmented tissues is a challenge. By taking advantage of the insolubility of melanins in hydrochloric acid, Oikawa and Nakayasu (1973) reported a spectrophotometric assay of melanins based on solubilizing the deproteinized melanin in Soluene-100 (a 0.5 M solution of dimethyl-n-dodecyl-n-undecylammonium hydroxide in toluene). Eumelanins can be completely dispersed in Soluene100 giving a solution that shows no light scattering (Oikawa and Nakayasu, 1975). If the melanin content is sufficiently high, the dry weight of black pigment remaining after HCl hydrolysis also serves the purpose of quantitation (Borovansky, 1978). On the other hand, melanins present in cultured melanocytes can be solubilized in hot KOH or NaOH of about 1 M concentration, and the resulting solution is analyzed for absorbance between 400 to 500 nm (Whittaker, 1963). Treatments prior to the solubilization include precipitation of melanin (and protein) with perchloric acid or washing with a buffer followed by extraction of lipids with organic solvents. 298

Such pretreatments can be omitted when Soluene-350 is used as a solvent to dissolve all the constituents of cells, including the melanin pigment (Kable and Parsons, 1989). Soluene-350, a tissue solubilizer widely used in liquid scintillation counting, has a higher capacity to dissolve tissue constituents and to retain water than Soluene-100. Melanins can be oxidatively solubilized by heating in an alkaline, dilute H2O2 solution. The resulting solution has a characteristic fluorescence that can be used for quantitation of melanins (Rosenthal et al., 1973). Although the authors claimed that the assay could be applied directly to tissues or cell cultures, it still required a lengthy pretreatment. In another interesting method, sodium borohydride, a mild reducing agent, was used to solubilize melanins (Das et al., 1976), and this method was used to characterize neuromelanin isolated from the substantia nigra (Das et al., 1978).

Comparison of Chemical and Physical Properties of Eumelanins and Pheomelanins Because of the lack of adequate methods to isolate melanins from biochemical sources, their insolubility at neutral pH, and their structural heterogeneity, full characterization of melanins faces great obstacles. Table 15.3 compares various methods as to whether they can differentially characterize eumelanins and pheomelanins. As the physical analysis of melanins is dealt with in the following chapter, it is discussed here only briefly. The color of tissue containing eumelanins is considered to be dark brown to black, whereas that containing pheomelanins is yellow to reddish brown. However, one cannot differentiate between dark brown and reddish brown with certainty. In fact, HPLC analysis of hair samples indicates that the visual differentiation is not reliable (Jimbow et al., 1983). Differences in solubility are also not very specific (Prota et al., 1976), as eumelanins appear to be slightly soluble in alkaline solutions, the property serving as a basis for the spectrophotometric assay of melanins (Watt et al., 1981). Elemental analysis of melanins is often considered unreliable, as repeated analyses of a given preparation of melanin give variable results. However, we have found that this is due to the presence of loosely bound water in the melanin samples;

CHEMISTRY OF MELANINS

melanins are hygroscopic, containing from 10% to 20% water (Ito, 1986; Zeise et al., 1992). In addition, duplicate analyses of melanins from various sources showed satisfactory reproducibility (Dryja et al., 1979). Differences in the elemental composition, especially in sulfur content, serve to distinguish pheomelanins from eumelanins. Theoretically, eumelanins contain no sulfur while pheomelanins possess a sulfur to nitrogen molar ratio of 1:2. Novellino et al. (1981) attributed the high content of sulfur in some melanins isolated from hair, feathers, and melanomas to copolymerization of the two types of melanin pigments (Prota, 1988). However, the possibility that at least some of the sulfur may arise as an artifact was suggested by the finding that as much as 1–2% of the sulfur was incorporated into melanins during isolation procedures under commonly used acidic conditions (Ito et al., 1988b). Elemental analysis is also of considerable value in characterizing eumelanins; the carbon to nitrogen molar ratio may be indicative of the extent of retention of the carboxyl group originally present in dopa and of the extent of oxidative cleavage of 5,6-indolequinone units (Ito, 1986) (Fig. 15.12). Chedekel et al. (1992) prepared Sepia melanin and tyrosine melanin in the form of K+ salts under mild conditions. The elemental analyses of these preparations, corrected for amino acid contents, were C7.67H5.33NO3.68K0.18 for Sepia melanin and C7.88H4.53NO3.78K0.12 for tyrosine melanin. These analytical data indicate that at least 18% and 12%, respectively, of the monomer units contain carboxyl groups (in the form of –COOK), and considerable degrees of peroxidative cleavage of the ortho-quinone moiety have taken place. Infrared absorption spectroscopy appears to be of little value in characterizing melanins (Wilczok et al., 1984). Ultraviolet-visible absorption spectra of both types of melanins exhibit general absorption showing no distinctive absorption maxima. However, it should be pointed out that a solution of black hair melanin dissolved in Soluene-100 showed much higher absorbances at longer wavelengths than those of red hair melanin (Menon et al., 1983). This would suggest a possible use of the absorption spectra to differentiate eumelanins from pheomelanins (see below). Recently, solid-phase nuclear magnetic resonance (NMR) spectroscopy has been introduced to assess structural features surrounding carbon and nitrogen atoms using natural 13C and 15 N as probes (Duff et al., 1988; Hervé et al., 1994). The methodology has thus far been applied to the analysis of synthetic and natural eumelanins; no application has been reported so far for the characterization of pheomelanins. Interestingly, Sepia melanin, partially dissolved in D2O at pH 10, gives a surprisingly simple and well-resolved 1H-NMR spectrum (Katritzky et al., 2002). Thus, NMR would potentially be useful in characterizing both synthetic and natural eumelanins as well as pheomelanins. Among the spectroscopic methods, electron paramagnetic resonance (EPR) spectroscopy has proved most successful in distinguishing between eumelanins and pheomelanins. Both melanins contain radical centers in their polymer matrices; eumelanins are characterized by the O-centered ortho-

semiquinone radical (single peak in the EPR spectrum) and pheomelanins by the N-centered ortho-semiquinone-imine radical (two peaks). One can estimate the content of pheomelanin in mixtures or copolymers of eumelanins and pheomelanins (Sealy et al., 1982a, b; Vsevolodov et al., 1991). Extensive studies carried out in Naples resulted in the detection of many degradation products, some of which are specific to one type of melanin. Notably, permanganate oxidation of eumelanins gave pyrrole-2,3,5-tricarboxylic acid (PTCA, 13) (Nicolaus, 1968; Piattelli et al., 1963), while hydriodic acid hydrolysis of pheomelanins yielded aminohydroxyphenylalanine isomers, 4-AHP (16) and 3-AHP (17) (Minale et al., 1967; Prota, 1972; Thomson, 1974). We have developed a microanalytical method to quantitate eumelanins and pheomelanins in biological materials, based on HPLC analysis of these specific degradation products (Fig. 15.16; Ito and Fujita, 1985; Ito and Jimbow, 1983). The method is relatively simple and rapid and does not require the isolation of melanins from tissue samples. The details of this HPLC method are discussed below.

Quantitative Analysis of Eumelanins and Pheomelanins by Chemical Degradation Previous methods for the quantitation of melanins in pigmented tissues required the isolation of melanins. Moreover, none of those methods was suitable for distinguishing between eumelanins and pheomelanins. In 1983, we introduced a rapid method for quantitatively analyzing eumelanins and pheomelanins in tissue samples, which makes the isolation of melanin pigments unnecessary (Ito and Jimbow, 1983). The method is based on the formation of PTCA (14) by permanganate oxidation of eumelanins and of AHP isomers (17, 18) by hydriodic acid hydrolysis of pheomelanins respectively (Fig. 15.16). These specific degradation products are determined by HPLC; PTCA is quantified with UV detection while a mixture of AHP isomers is determined as a single peak with electrochemical detection. These degradation products were chosen because not only are they the major products from eumelanins and pheomelanins but they are also easily detected with high sensitivity. A similar approach using paper chromatography had been reported previously by Hackman and Goldberg (1971) and Novellino et al. (1981). The original method was later improved to increase the sensitivity and to reduce the time for pre-HPLC work-up (Ito and Fujita, 1985). It should be noted that the alkaline 1 M K2CO3 medium for permanganate oxidation was replaced with acidic 1 M H2SO4. With this modification, the artificial formation of pyrrole-2,3,4,5-tetracarboxylic acid (15) could be avoided. The improved method requires only 5 mg or less of tissue samples or 106 cultured cells for each analysis. Recently, the conditions for permanganate oxidation were refined so that the PTCA values become more linearly correlated to the melanin contents (Ito and Wakamatsu, 1994). The yields of PTCA and AHP (4-AHP and 3-AHP combined) are approximately 2% from natural eumelanins and 20% from synthetic pheomelanins (Fig. 15.16), and the tissue contents of 299

CHAPTER 15

HOOC

HO COOH

KMnO4/H+ HOOC

COOH

N H

2.8% yield

N H

HO

HO

KMnO4/H+

PTCA

DHICA-derived eumelanin

N H

HO

0.03% yield

DHI-derived eumelanin

(COOH) OH

N N

(COOH)

S

OH

+ S NH2

HOOC

HOOC

5-S-CD-derived pheomelanin

11% yield

NH2

2-S-CD-derived pheomelanin

HI

HI

OH

HOOC

NH2 4-AHP

NO2

NH2 NH2

OH

HOOC

NH2 3-AHP

eumelanins and pheomelanins can therefore be estimated by multiplying the PTCA and AHP contents by factors of 50 and 5 respectively (Ito and Fujita, 1985). Thus, a PTCA/AHP ratio of 0.1 indicates a mixed melanin consisting of equal amounts of eumelanins and pheomelanin. Our HPLC method is relatively simple, fairly rapid, and highly sensitive. It has been applied for quantitatively analyzing eumelanins and pheomelanins not only in synthetic melanins, isolated melanosomes, hair, feathers, skin, nevi, and melanomas, but also in human epidermis and in cultured melanocytes (Ito, 1993b, 1998; Ito and Wakamatsu, 2003; Wakamatsu and Ito, 2002). A typical application of the method was to demonstrate that a synthetic analog of a-MSH, Nle4DPhe7aMSH, induces significant increases in the eumelanin content of cultured human melanocytes (Hunt et al., 1995). Several other laboratories have employed the same method in studies on melanin and melanogenesis (reviewed by Ito and Wakamatsu, 2003). As an example of one study using a similar method, pigments present in iris pigment epithelium of human eyes were found to be eumelanic (Prota et al., 1998b). The EPR and HPLC methods have been compared in estimating the contents of eumelanins and pheomelanins in hair samples from newborn sheep. The results showed that the EPR method correlates well with the HPLC method, although the former lacks the sensitivity of the latter with respect to the analysis of pheomelanins (Vsevolodov et al., 1991). 300

OH

HI

HOOC

NH2 3-Nitrotyrosine

Fig. 15.16. Chemical degradation of eumelanins to form PTCA and of pheomelanins to form 4-AHP and 3-AHP. Note that the yield of PTCA from DHIderived eumelanins is extremely low, compared with that from DHICA-derived eumelanin. PTCA is thus a specific degradation product of DHICA-derived eumelanin. Reductive hydrolysis of pheomelanins with hydriodic acid gives two isomers, 4-AHP and 3-AHP, arising from 5S- and 2-S-cysteinyldopa-derived pheomelanins respectively. 3-AHP also arises from 3-nitrotyrosine-containing proteins (Wakamatsu and Ito, 2002).

The formation of PTCA has been interpreted in terms of the oxidative breakdown of indole units, either linked through the 2-position or bearing a carboxyl group at the same position (Napolitano et al., 1995). Permanganate oxidation of DHI melanin and DHICA melanin in acidic medium afforded PTCA in 0.03% and 2.8% yields respectively (Fig. 15.16; Ozeki et al., 1995; Ito, 1998). A similar result was also reported by Prota’s group (Napolitano et al., 1995; Novellino et al., 2000). These results have confirmed our proposal that PTCA is a specific product arising from DHICA-derived structures (units B and D).

4-Amino-3-hydroxyphenylalanine (4-AHP) as a Specific Marker for Pheomelanins In previous reports from our laboratory, the HPLC conditions used were such that 4-AHP (16) and 3-AHP (17) eluted in a single peak. This was based on the assumption that any natural pheomelanin pigment consists of a fixed ratio of 5-Scysteinyldopa and 2-S-cysteinyldopa because the ratio of these cysteinyldopa isomers in biological materials is chemically controlled to be approximately 5:1 (Ito and Prota, 1977; Morishima et al., 1983). In fact, the estimation of pheomelanin as the combined amount of 4-AHP and 3-AHP (“total AHP”) did not impose any serious problem in most cases. However, small amounts of background values of total AHP were found even in hairs from tyrosinase-negative, albino mice (Lamoreux et al., 2001; Ozeki et al., 1995).

CHEMISTRY OF MELANINS

However, one problem with using total AHP as a marker was that background levels of AHP seemed to originate from precursors other than pheomelanin. Considerable and variable amounts of background 3-AHP are produced from other sources, most likely nitrotyrosine residues in proteins. The nitration of tyrosine appears to be a common biological phenomenon originating from nitric oxide. Kolb et al. (1997) and Borges et al. (2001) described the separation of 4-AHP and 3-AHP in hydriodic hydrolysates of various tissue samples. However, their reported methods require ion-exchange chromatography prior to the HPLC separation, which is not only time-consuming but also very costly (because of using commercial, disposable ion-exchange columns). In order to overcome this problem, we developed HPLC conditions that enable the direct injection of the hydriodic acid hydrolysis products into the HPLC system allowing separation of 4-AHP and 3-AHP (Fig. 15.16). As the yield of 4-AHP from synthetic pheomelanin is 11%, the content of pheomelanin can be obtained by multiplying the content of 4AHP by a factor of 9 (Wakamatsu et al., 2002b). We are now using 4-AHP as a more specific marker of pheomelanins in subsequent studies (Naysmith et al., 2004; Tadokoro et al., 2003). For clinical studies, it is often preferable to use serum or urine specimens to monitor the degree of pigmentation. We recently applied the specific pheomelanin assay method to analyze pheomelanin in the serum and urine from melanoma patients. Wakamatsu et al. (2003a) reported that serum levels of 4-AHP in metastatic melanoma patients were sevenfold higher than in control subjects, and they correlated well with serum levels of 5-S-cysteinyldopa. Similarly, Takasaki et al. (2003) showed significant correlations of 4-AHP and 3-AHP in melanoma urine with the urinary levels of 5-S-cysteinyldopa. These results suggest that the levels of 4-AHP in serum and urine could be used to monitor the production of pheomelanin in human skin.

Dopamine Melanin, Cysteinyldopamine Melanin, and Nonmelanocytic Melanins Dark brown pigments, similar to eumelanins and pheomelanins, are also produced in cells other than melanocytes. For example, humans and primates produce neuromelanin in dopaminergic nigrostriatal neurons (Zecca et al., 2001). Recently, Napolitano et al. (1995) showed that peroxide oxidation of DHI melanin in 1 M K2CO3 produces PDCA (16) in a yield (~0.5%) that is much higher than that produced by acidic permanganate oxidation. To characterize the diverse types of melanins, especially to identify dopamine-derived melanins, we have improved the alkaline H2O2 oxidation method of Napolitano et al. (1995) in terms of speed and sample size required (Ito and Wakamatsu, 1998). The results with peroxide oxidation show that: (1) PDCA, a specific marker of DHI units in eumelanins, is produced in yields 10 times higher than by acidic permanganate oxidation; (2) PTCA is produced in higher yields as well, but is also artificially produced from pheomelanins; and (3) the PDCA/PTCA

NH2

OH

OH

NH2

NH2 25 (4-AHPEA)

NH2 26 (3-AHPEA)

Fig. 15.17. Products of hydriodic acid hydrolysis of cysteinyldopamine-derived melanins.

ratio may be useful in characterizing eumelanins with various ratios of the monomers DHI and DHICA. Analogous to pheomelanins, hydriodic acid hydrolysis of cysteinyldopamine melanin produces a high (12%) yield of a 5:1 mixture of 4-amino-3-hydroxyphenylethylamine (4AHPEA; 25) and 3-amino-4-hydroxyphenylethylamine (3AHPEA; 26) (Fig. 15.17). 4-AHPEA may thus serve as a specific indicator of cysteinyldopamine-derived melanin (Wakamatsu et al., 1991, 2003a). It is generally accepted that neuromelanin is produced from dopamine (Zecca et al., 2001). Cysteine may be incorporated into neuromelanin in a mechanism similar to pheomelanin production. However, our group and Rorsman’s group reached opposite conclusions as to whether cysteine (via cysteinyldopamine) is actually incorporated (Carstam et al., 1991; Odh et al., 1994a). To solve this discrepancy, a more accurate method was developed to characterize neuromelanin chemically (Wakamatsu et al., 2003b). We prepared synthetic models of neuromelanin by tyrosinase oxidation of dopamine and cysteine in various ratios (Wakamatsu et al., 1991). Alkaline peroxide oxidation of these model neuromelanins produces thiazole carboxylic acids, TTCA (21) and TDCA (22), in addition to PDCA (13) and PTCA (14). We found that the yield of PDCA is relatively constant in synthetic melanins with various dopamine and cysteine ratios, whereas the yield of TTCA is higher than that of TDCA and is proportional to the sulfur to nitrogen ratio. It is concluded that the TTCA/PDCA ratio is a useful indicator of cysteinyldopamine-derived units in neuromelanin, and that neuromelanin consists mainly of dopamine melanin with some contribution from cysteinyldopamine melanin (Wakamatsu et al., 2003b). Similarly, Odh et al. (1994a, b) used the TDCA/PTCA ratio as an indicator of the benzothiazine units in isolated neuromelanin and in pigment in cultured melanoma cells. These results also suggest that the same methodology should be useful for analyzing eumelanins and pheomelanins in various tissues. Melanin pigments may be characterized by contents of PTCA, PDCA, TTCA, TDCA, and the ratios among them. In particular, TTCA may serve as a specific marker of pheomelanins (Napolitano et al., 2000b). The dark pigment in butterfly wings is another example of a natural melanin whose chemical nature was mostly 301

CHAPTER 15

unknown. We applied peroxide oxidation and hydriodic acid hydrolysis to follow the developmental increase in melanin in wings from Precis coenia and found that cysteinyldopamine melanin was formed first, followed by more eumelanic, dopamine melanin (Wakamatsu et al., 1998). Certain bacteria and fungi also produce insoluble, dark brown, melanin-like pigments. Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening infections in brains of about 10% of AIDS patients. We have applied the alkaline peroxide oxidation to analyze melanin pigments produced in C. neoformans (Williamson et al., 1998). C. neoformans produces dark pigments on its cell wall when grown in media containing a diphenolic substrate, such as dopa or dopamine. We performed peroxide oxidation of C. neoformans cells grown on dopamine or dopa agar. Cells grown on dopamine agar gave a high ratio of PDCA/PTCA whereas cells grown on dopa agar gave a high ratio of PTCA/PDCA. These data provide direct chemical evidence for the formation of eumelanic pigments by oxidation of catecholic precursors by C. neoformans laccase.

indicator of DHI-derived units in eumelanin, and the PDCA/PTCA ratio is useful in characterizing various types of eumelanin, a ratio greater than 1 indicating dopamine melanin; (2) the calibration curves for PDCA and PTCA are linear; and (3) it is easier to perform than the permanganate oxidation. However, the alkaline peroxide oxidation method also has a certain disadvantage: yields of PTCA and PDCA from 5-S-cysteinyldopa melanin are abnormally high compared with those with permanganate oxidation. This indicates that indole units are formed artificially during the oxidation, because the postulated structure of 5-S-cysteinyldopa melanin does not contain an indole unit (Prota, 1992). Prota et al. (1995) and our group (Ito and Wakamatsu, 1998) also found abnormally high yields of PTCA and PDCA when lethal yellow and recessive yellow mouse hairs were subjected to alkaline peroxide oxidation. We therefore recommend that special care be taken when alkaline peroxide oxidation is used to analyze pigmented tissues containing pheomelanin.

Comparison of Permanganate Oxidation and Peroxide Oxidation

Although the melanin assay based on chemical degradation and HPLC determination is relatively simple, it still requires an HPLC system with UV and electrochemical detectors. In addition, PTCA arises from DHICA-derived units but not from DHI-derived units. Therefore, we have developed a spectrophotometric method that is specific to eumelanins but does not discriminate between the DHI- and DHICA-derived units (Ito et al., 1993). In this method, hair and melanoma samples are hydrolyzed in hot hydriodic acid to remove pheomelanic components, and the insoluble eumelanic pigments are subsequently solubilized in hot NaOH in the presence of H2O2 and analyzed for absorbance at 350 nm. Although much less sensitive, this spectrophotometric method can substitute for the PTCA method to measure eumelanin content when substantial amounts of samples are available. Total amounts of melanin pigment in tissue samples can be calculated by the use of conversion factors of 50 and 9 for PTCA and 4-AHP respectively. However, the accuracy of this estimation rests on the assumptions that the DHICA/DHI ratios in different eumelanins from various sources are constant and that the 11% yield of 4-AHP from synthetic pheomelanins holds for different natural pheomelanins. The DHICA/DHI ratio appears to vary from one species to another. These situations made it necessary to introduce a simple, reference method to estimate the total amounts of melanins, even if the method might not give accurate values. We have found that hot Soluene-350 (in the presence of 10–20% water) is able completely to dissolve mouse and human hairs and sheep wool. The resulting brown solutions are analyzed for absorbances at 500 nm (Ito et al., 1996; Ozeki et al., 1995, 1996a, b). Hair samples from different coat color phenotypes of mice and human hairs of various colors were analyzed with this spectrophotometric method. Excellent correlations were found between the absorbance at 500 nm (A500) and the melanin con-

Our HPLC methods for assaying eumelanin and pheomelanin are highly sensitive and specific and possess many advantages, but also have certain disadvantages. The acidic permanganate oxidation method that we have been using for quantitative analysis of eumelanin has a number of advantages (Table 15.4): (1) PTCA is formed primarily from DHICA-derived units in eumelanin, thus making PTCA a specific marker of DHICA content (Wakamatsu and Ito, 2002); and (2) PTCA is not artificially formed from pheomelanin. However, this method also has some disadvantages: (1) the yield of PDCA is too low to be used as a marker of DHI-derived units; and (2) the amount of PTCA formed gives a slightly concave calibration curve against the amount of melanin oxidized despite recent improvements (Ito and Wakamatsu, 1994). The alkaline peroxide oxidation method has several advantages (Ito and Wakamatsu, 1998): (1) PDCA is an excellent

Table 15.4. Advantages and disadvantages of the two oxidation methods. Comparison

Acidic KMnO4 oxidation

Alkaline H2O2 oxidation

PTCA

Specific for DHICA units

PDCA

Little produced

TTCA, TDCA

Produced, but hard to be extracted

Method

Requires some skills

Not specific for eumelanins Indicator of DHI units, but not specific for eumelanins Specific for CD-derived and Cys-DA-derived units Easier to perform

302

Combined Use of Spectrophotometric and Degradative Analyses

CHEMISTRY OF MELANINS

tents calculated from PTCA and AHP levels (Ozeki et al., 1996b). This indicates that the A500 value can serve as an indicator of the total amount of eumelanin and pheomelanin combined, regardless of the type of melanin. Using A500 values, we obtained a conversion factor of 160 for calculating eumelanin content from the PTCA value in human hair (Ozeki et al., 1996b; Wakamatsu and Ito, 2002). This high conversion factor suggests a low activity of Tyrp2 in humans compared with other species. Alaluf et al. (2001) recently reported that the HPLC method underestimates the melanin content in human epidermis by a factor of 3 compared with the spectrophotometric method. This discrepancy can be solved, however, using a conversion factor of 160 instead of 50. The PTCA/total melanin (A500) ratio appears to be a good indicator of the content of DHICA-derived units in eumelanins. The ratios were at similar levels in mouse black, brown, dilute black, and pink-eyed black hairs, whereas they were very low in pheomelanic hairs (Ozeki et al., 1995). In contrast, the opposite holds for the AHP/total melanin ratios. The PTCA/total melanin ratio in slaty hair was only one-fifth that of the black counterpart, the result paralleling the decreased activity of Tyrp2 in the slaty mutation (Krompouzos et al., 1994). It is interesting to note that black to brown hair eumelanins in humans contain low ratios of DHICA-derived units, comparable to the slaty mutation in mouse (Lamoreux et al., 2001; Ozeki et al., 1996a, b). Orlow et al. (1992) suggested that DHICA melanins are responsible for brown colors in the animal kingdom. However, our study using coat color mutants of mice indicates that the brown-type eumelanins differ from the black-type eumelanins in the degree of polymerization but not in the ratio of DHICA/DHI (Ozeki et al., 1995, 1997b). Pheomelanins are soluble in strong alkali solutions. By taking advantage of this property, we were able to solubilize pheomelanin in 8 M urea/1 M NaOH, although not completely, from the yellow hair of pheomelanic mice (Ozeki et al., 1995). Under the same alkaline conditions, brown eumelanins from brown, pink-eyed, black, and silver mutants were slightly soluble. On the basis of the ratios of absorbance at 400 nm of the alkaline solution to total melanin (A500), one can differentiate between pheomelanins, brown-type eumelanins, and black-type eumelanins, with the brown-type eumelanins being characterized by their partial solubility in strong alkali. Using a similar approach to characterizing melanins in human epidermis, Alaluf et al. (2002) showed that European skin contains as much as 40% alkali-soluble melanin compared with about 15% in African skin. When classification of melanin pigments into eumelanins and pheomelanins is not a major concern, the solubilization of melanins in Soluene-350 (or NaOH or KOH, if soluble) appears to be the choice for the quantitation. Absorbance at 500 nm of the Soluene-350 solution (total melanin) can be used to quantify melanin contents in hair samples (Ito et al., 1996; Ozeki et al., 1995, 1996a, b) and in cultured melanocytes (Kable and Parsons, 1989) without any pretreatment. However, before it is applied to tissue samples such as

melanomas, some refinements in pretreatment are required to remove hemoglobin and other interfering tissue components. Eumelanins and pheomelanins in hair show significantly different ratios of absorbances at 650 nm to 500 nm when solubilized in Soluene-350. This difference has been used to develop a simple and rapid spectrophotometric method to distinguish eumelanins from pheomelanins, at least for qualitative purposes (Ito et al., 1996; Ozeki et al., 1996b).

Perspectives The first part of this chapter described the chemistry of melanin pigments and related metabolites. Two types of melanin production, eumelanogenesis and pheomelanogenesis, have been extensively studied. Most of the pathway at the monomer level has been clarified, using biosynthetic and pulse radiolysis approaches. Both approaches have produced valuable information and have been complementary to each other. The former approach has the strength of isolating various monomeric and oligomeric melanin intermediates, while the latter approach is able to follow rapid reactions involving dopaquinone that could not be studied otherwise. Some unsolved problems in the chemistry of melanogenesis include: (1) the nature of post-polymerization modifications of eumelanins and pheomelanins; and (2) the nature of copolymerization of eumelanins and pheomelanins. Problem 1 has been addressed only sporadically (Crescenzi et al., 1993; Deibel and Chedekel, 1984), and is also relevant in clarifying the biodegradation of melanins and melanosomes (Borovansky and Elleder, 2003). No study has examined in depth the mode of copolymerization of the two types of melanin pigments (problem 2). The biological functions of melanin pigments are closely related to their structural features. Therefore, after the great progress in melanin chemistry over the past decade, more intimate cross-talk among specialists from chemistry, biochemistry, biophysics, cell biology, genetics, and dermatology is highly desirable in order to enjoy the fruits of growing chemical knowledge. One example of an unsolved problem in these multidisciplined areas is the mechanism of switching from eumelanogenesis to pheomelanogenesis (Fig. 15.3, 5), which appears to depend on the availability (rate and regulation of uptake) of cysteine in melanosomes. The latter part of this chapter dealt with the methodology to determine the quantity and quality of melanins present in pigmented tissues, an area that has also enjoyed fruitful collaborative studies (Ito et al., 2000; Ito and Wakamatsu, 2003). Extensive degradative studies have provided a number of useful (or potentially useful) markers. These include the contents of PTCA and AHP, the PTCA/AHP ratio, the PTCA/total melanin ratio, the AHP/total melanin ratio, the PTCA/TDCA ratio, the PTCA/PDCA ratio, and the TTCA/PDCA ratio. The significance of PTCA, AHP, and their ratio in the study of melanogenesis has been well established (Ito, 1993b; Ito et al., 303

CHAPTER 15

2000). Recently, AHP (“total AHP”) measurement has been replaced by a more specific 4-AHP measurement (Ito and Wakamatsu, 2003; Wakamatsu et al., 2002b). One problem in using PTCA and 4-AHP as markers of eumelanins and pheomelanins is that two different types of degradation are required for analyzing one sample. When alkaline peroxide oxidation is applied, TTCA and TDCA are formed specifically from pheomelanins, while PTCA and PDCA are derived from both types of melanin pigments (Ito and Wakamatsu, 1998; Wakamatsu and Ito, 2002). It is therefore expected that ratios such as the TTCA/PTCA ratio may be as useful as the 4-AHP/PTCA ratio in characterizing copolymers (or mixtures) of eumelanins and pheomelanins. For the characterization of neuromelanin, the PTCA/TDCA ratio has been used by Odh et al. (1994a, b) whereas the TTCA/PTCA ratio was used by Wakamatsu et al. (2003b). One problem in applying this approach is that TTCA and TDCA are difficult to extract in organic solvents and should thus be analyzed without purification and concentration (Wakamatsu et al., 2003b). This would lead to a lower sensitivity and specificity, unless melanin pigments are purified prior to the oxidation. Alkaline peroxide oxidation also produces BTCA (24) as a specific marker of pheomelanins (Napolitano et al., 2000). How much this marker is useful in pigment research remains to be explored. In the study of eumelanogenesis, the PTCA/PDCA ratio, analyzed by alkaline peroxide oxidation, may become a useful substitute for the PTCA/total melanin ratio as a marker to estimate the DHICA/DHI ratio in eumelanins. At present, only PDCA is available as an indicator specific for the DHI-derived units in eumelanins. A major problem with the alkaline peroxide oxidation is the artificially high yield of PTCA from pheomelanins present in the hair of genetically pheomelanic mice (Ito and Wakamatsu, 1998; Prota et al., 1995). The EPR method appears to be highly sensitive and specific in detecting melanin pigments (Enochs et al., 1993). It may also be able to differentiate between eumelanins and pheomelanins (Sealy et al., 1982a, b). Which of the two methods, the EPR method or the HPLC method, is more sensitive and specific has not been fully determined (Vsevolodov et al., 1991), although some groups claim that the HPLC method is not sufficiently sensitive or specific (Sarna et al., 2003). However, it should be stressed that the HPLC method to detect 4-AHP as the measure of pheomelanins is highly specific and sensitive.

Acknowledgments The authors wish to acknowledge the late Professor Giuseppe Prota and his associates for their inspiring chapter in the first edition of this book (Chapter 24, The chemistry of melanins and related metabolites) (Prota et al., 1998a).

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melanocytes can be substantially influenced by l-tyrosine and lcysteine. J. Invest. Dermatol. 109:796–800, 1997. Steel, K. P., D. R. Davidson, and I. J. Jackson. TRP-2/DT, a new early melanoblast marker, shows that steel growth factor (c-kit legand) is a survival factor. Development 115:1111–1119, 1992. Swan, G. A. Structure, chemistry, and biosynthesis of the melanins. Fortsch. Chem. Organ. Naturst. 31:521–582, 1974. Swan, G. A., and A. Waggott. Studies related to chemistry of melanins. Part X. Quantitative assessment of different types of units present in DOPA-melanin. Chem. Soc. J. Perkin I 10:1409–1418, 1970. Tadokoro, T., N. Kobayashi, B. Z. Zmudzka, S. Ito, K. Wakamatsu, Y. Yamaguchi, K. S. Korossy, S. A. Miller, Z. Z. Beer, and V. J. Hearing. UV-induced DNA damage and melanin content in human skin differing in racial/ethnic origin. FASEB J. 17:1177–1179, 2003. Takasaki, A., D. Nezirevic, K. Årstrand, K. Wakamatsu, S. Ito, and B. Kågedal. HPLC analysis of pheomelanin degradation products in human urine. Pigment Cell Res. 16:480–486, 2003. Thody, A. J., E. M. Higgins, K. Wakamatsu, S. Ito, S. A. Burchill, and J. M. Marks. Pheomelanin as well as eumelanin is present in human epidermis. J. Invest. Dermatol. 97:340–344, 1991. Thompson, A., E. J. Land, M. R. Chedekel, K. V. Subbarao, and T. G. Truscott. A pulse radiolysis investigation of the oxidation of the melanin precursors 3,4-dihydroxyphenylalanine (dopa) and the cysteinyldopas. Biochim. Biophys. Acta 843:49–57, 1985. Thomson, R. H. The pigments of reddish hair and feathers. Angew. Chem. Int. Ed. Engl. 13:305–312, 1974. Tripathi, R. K., V. J. Hearing, K. Urabe, P. Aroca, and R. A. Spritz. Mutational mapping of the catalytic activities of human tyrosinase. J. Biol. Chem. 267:23707–12, 1991. Tse, D. C. S., R. L. McCreery, and R. N. Adams. Potential oxidative pathways of brain catecholamines. J Med. Chem. 19:37–40, 1976. Tsukamoto, K., I. J. Jackson, K. Urabe, P. M. Montague, and V. J. Hearing. A second tyrosinase-related protein, TRP-2, is a melanogenic enzyme termed DOPAchrome tautomerase. EMBO J. 11:519–526, 1992a. Tsukamoto, K., A. Palumbo, M. d’Ischia, V. J. Hearing, and G. Prota. 5,6-Dihydroxyindole-2-carboxylic acid is incorporated in mammalian melanin. Biochem. J. 286:491–495, 1992b. Urabe, K., P. Aroca, K. Tsukamoto, D. Macagna, A. Palumbo, G. Prota, and V. J. Hearing. The inherent cytotoxicity of melanin precursors: a revision. Biochim. Biophys. Acta 1221:272–278, 1994. Valverde, P., E. Healy, I. Jackson, J. L. Rees, and A. J. Thody. Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin. Nature Genet. 11:328–330, 1995. von Fürth O., and E. Jerusalem. Zur Kenntnis der melanotischen Pigmente und der fermentativen Melaninbildung. Beitr. Chem. Physiol. Pathol. 10:131–171, 1907. Vsevolodov, E. B., S. Ito, K. Wakamatsu, I. I. Kuchina, and I. F. Latypov. Comparative analysis of hair melanins by chemical and electron spin resonance methods. Pigment Cell Res. 3:30–34, 1991. Wakamatsu, K., and S. Ito. Preparation of eumelanin-related metabolites, 5,6-dihydroxyindole, 5,6-dihydroxyindole-2-carboxylic acid, and their O-methyl derivatives. Anal. Biochem. 170:335–340, 1988. Wakamatsu, K., and S. Ito. Seasonal variation in serum concentration of 5-S-cysteinyldopa and 6-hydroxy-5-methoxyindole-2-carboxylic acid in healthy Japanese. Pigment Cell Res. 8:132–134, 1995. Wakamatsu, K., and S. Ito. Review: Innovative technology. Advanced chemical methods in melanin determination. Pigment Cell Res. 15:174–183, 2002. Wakamatsu, K., S. Ito, and T. Nagatsu. Cysteinyldopamine is not incorporated into neuromelanin. Neurosci. Lett. 131:57–60, 1991. Wakamatsu, K., S. Ito, and K. B. Koch. Chemical characterization of dopamine-melanin: application to identification of melanins in butterfly wing. Pigment Cell Res. 11:259, 1998.

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CHAPTER 15 Wakamatsu, K., T. Kageshita, M. Furue, N. Hatta, Y. Kiyohara, J. Nakayama, T. Ono, T. Saida, M. Takata, T. Tsuchida, H. Uhara, A. Yamamoto, N. Yamazaki, A. Naito, and S. Ito. Evaluation of 5-S-cysteinyldopa as a marker of melanoma progression: 10 years’ experience. Melanoma Res. 12:245–253, 2002a. Wakamatsu, K., S. Ito, and J. L. Rees. The usefulness of 4-amino-3hydroxyphenylalanine as a specific marker of pheomelanin. Pigment Cell Res. 15:225–232, 2002b. Wakamatsu, K., M. Yokochi, A. Naito, T. Kageshita, and S. Ito. Comparison of phaeomelanin and its precursor 5-S-cysteinyldopa in the serum of melanoma patients. Melanoma Res. 13:357–363, 2003a. Wakamatsu, K., K. Fujikawa, F. Zucca, L. Zecca, and S. Ito. The structure of neuromelanin as studied by chemical degradative methods. J. Neurochem. 86:1015–1023, 2003b. Watt, K. P., R. G. Fairchild, D. N. Slatkin, D. Greenberg, S. Packer, H. L. Atkins, and S. J. Hannon. Melanin content of hamster tissues, human tissues, and various melanomas. Cancer Res. 41:467–472, 1981. Westerhof, W., S. Pavel, A. Kammeyer, F. D. Beusenberg, and R. Cormane. Melanin-related metabolites as markers of the skin pigmentary system. J. Invest. Dermatol. 89:78–81, 1987. Whittaker, J. R. Changes in melanogenesis during dedifferentiation of chick retinal pigment cells in cell culture. Dev. Biol. 8:99–127, 1963. Wick, M. M., L. Byers, and E. Frei. L-DOPA: selective toxicity for melanoma cells in vitro. Science 197:468–469, 1977. Wilczok, T., B. Bilinska, E. Buszman, and M. Kopera. Spectroscopic studies of chemically modified synthetic melanins. Arch. Biochem. Biophys. 231:257–262, 1984.

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Williamson, P. R., K. Wakamatsu, and S. Ito. Melanin biosynthesis in Cryptococcus neoformans. J. Bacteriol. 180:1570–1572, 1998. Wood, J. M., K. Jimbow, R. E. Boissy, A. Slominski, P. M. Plonka, J. Slawinski, J. Wortsman, and J. Tosk. What’s the use of generating melanin? Exp. Dermatol. 8:153–164, 1999. Yukitake, J., H. Otake, S. Inoue, K. Wakamatsu, C. Olivares, F. Solano, K. Hasegawa, and S. Ito. Synthesis and selective in vitro anti-melanoma effect of enantiomeric a-methyl- and a-ethy-4-Scysteaminylphenol. Melanoma Res. 13:603–609, 2003. Zecca, L., F. A. Zucca, P. Costi, D. Tampellini, A. Gatti, M. Gerlach, P. Riederer, R. G. Fariello, S. Ito, M. Gallorini, and D. Sulzer. The neuromelanin of human substantia nigra: structure, synthesis and molecular behaviour. J. Neural Transm. (Suppl.) 65:145–155, 2003. Zeise, L. Analytical methods for characterization and identification of eumelanins. In: Melanin: Its Role in Human Photoprotection, L. Zeise, M. R. Chedekel, and T. B. Fitzpatrick (eds). Overland Park, KS: Valdenmar Publishing, 1995, pp. 65–79. Zeise, L., and M. R. Chedekel. Melanin standard method: Titrimetric analysis. Pigment Cell Res. 5:230–239, 1992. Zeise, L., R. B. Addison, and M. R. Chedekel. Bio-analytical studies of eumelanins. I. Characterization of melanin the particle. In: The Pigment Cell: From the Molecular to the Clinical Level. Proceedings from the XIVth International Pigment Cell Conference. Pigment Cell Research Supplement 2, Y. Mishima (ed.). Copenhagen: Munksgaard, 1992, pp. 48–53. Zhang, X., C. Erb, J. Flammer, and W. M. Nau. Absolute rate constants for the quenching of reactive excited states by melanin and related 5,6-dihydroxyindole metabolites: implications for their antioxidant activity. Photochem. Photobiol. 71:524–533, 2000.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

16

The Physical Properties of Melanins Tadeusz Sarna and Harold A. Swartz

Summary 1 The complexities of melanins make it difficult to reach very specific conclusions as to the properties and structure of these intractable pigments. 2 Different melanins can have different physicochemical properties, with the most important variables being the nature of the monomeric units. 3 Consequently, there are at least three different types of naturally occurring melanins: eumelanin, pheomelanin, and neuromelanin. 4 Although melanins have been viewed as large heterogeneous polymers and amorphous substances, there appears to be a short-range order within the melanin nanostructure, consisting of several layers of stacking 0.34 nm apart. A growing body of experimental evidence indicates that the assembly of melanin oligomers into nanoparticles, and their aggregation into larger particles, is a key process that determines basic physicochemical properties of melanin pigments. 5 The most important functional groups of the melanin oligomeric units are: aryl and a-amino acid carboxylic groups, fully oxidized, semi-reduced, and fully reduced o-quinone groups in eumelanins, and the corresponding o-quinonimine, o-semiquinonimine, and o-aminophenols in pheomelanins. 6 The observable optical properties of natural melanins are a complex function of different melanin monomers and oligomers to absorb light, and ability of melanin particles to scatter and reflect light at different wavelengths. 7 Melanins are poor fluorophores; however, the intensity of fluorescence increases upon their oxidative degradation. 8 Although several band models for melanin have been proposed to explain its optical absorption and electrical properties, there is at present no fully satisfactory theory for melanin to have amorphous semiconductor and related properties. 9 Melanins are the only known biopolymers that both in vivo and in vitro contain a significant amount of persistent free radical centers that are easily detectable by electron paramagnetic resonance (EPR) spectroscopy. 10 Many of the EPR characteristics and the associated free radical and redox reactions of melanin can be explained on the basis of a comproportionation equilibrium involving key redox groups of the melanin oligomers. 11 The ability of melanin to form complexes with multivalent metal ions is one of its basic physicochemical properties that affects the biological effects of this pigment. 12 Binding of transition metal ions by melanin may result in two opposite effects on its free radical EPR signal: while para-

magnetic ions induce magnetic quenching of the intensity of the observable EPR signal of the melanin, diamagnetic ions, such as zinc(II), enhance the EPR signal intensity by shifting the comproportionation equilibrium of the melanin subunits. 13 Melanin polymers are complex redox systems, the resultant properties of which are modified by pH, temperature, illumination with ultraviolet and visible light, and storage conditions. 14 Prolonged illumination of melanin with intense light, in the presence of oxygen, leads to irreversible bleaching of the pigment and its oxidative degradation. 15 Antioxidant properties of melanin, shown in model systems, can be explained by melanin’s ability to sequester redox-active metal ions, scavenge oxidizing free radicals, and quench electronically excited states of molecular oxygen and photosensitizing dye molecules. 16 Much is known about melanin as a result of the use of sophisticated physical and chemical approaches, and it is likely that continued progress will be made.

Historical Background Melanins are a complex group of pigments of different origins, the composition and structure of which depends very much on local conditions, including the type of synthesis (e.g. within specific organelles such as melanosomes vs. polymerization of naturally occurring high concentrations of monomers as in some regions of the brain), the monomeric units that are polymerized (e.g. whether or not cysteine derivatives are involved), the presence of other types of molecules at the time of formation (e.g. tyrosinase, other proteins, lipids, metal ions), and the subsequent history of the usually long-lived polymers (e.g. oxidation, complexing of metal ions). The resulting melanins are usually heterogeneous with very intractable physical properties, which resist attempts to characterize them by simple physical–chemical approaches. Consequently, attempts to characterize melanins have involved the use of a large number of different and often complex techniques. These have led to large amounts of data, but the nature of the data obtained from many different techniques, combined with the heterogeneity of the melanins that have been studied, has resulted in incomplete and sometimes apparently contradictory conclusions on the composition and structure of melanins. Since publication of the first edition of The Pigmentary System, significant advances in biophysical studies of melanin have been made. Particularly impressive are the results of recent studies, in which the ultrastructure of melanin 311

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was examined by powerful imaging techniques such as scanning tunneling and atomic force microscopies, and the photodynamics of melanin, after excitation with light, were analyzed by ultrafast emission and absorption spectroscopies. The aim of this chapter is to summarize representative data on the biophysics of melanin that have been published recently and to provide an overview of the existing data on melanins and, where possible, to indicate generally accepted conclusions on their significance. The reader will find, however, that the current state of knowledge, in spite of the recent advances, often does not lead to such conclusions and, therefore, only a summary of experimental findings is provided without fitting them into a comfortably unified picture of melanin. This chapter represents an updated progress report and a source of information that may facilitate understanding and experimental progress on this intriguing and important natural polymer.

Current Concepts Structure and Composition of Melanin The usual melanin pigments are amorphous substances with a distinct particulate character. In biological material, melanin is usually present in the form of discernible units such as pigment granules. The size and shape of the mature melanin granules are, to a significant degree, determined by the process that forms them. In most cells, melanin is synthesized in a specific organelle, the melanosome, the phenotype of which determines the geometry of the melanin particles (Seiji, 1981) (see Chapter 15). For example, melanosomes in human retinal pigment epithelium are typically elongated and relatively large (2–3 mm long and 1 mm wide; Feeney-Burns, 1980), while melanin granules in Harding–Passey melanoma cells are almost spherical in shape and much smaller in size (0.40 mm diameter) (Hach et al., 1977). In normal skin, the ultrastructure of melanosomes usually relates to the type of melanin they produce (Hearing et al., 1973; Jimbow et al., 1979). Typical eumelanosomes have an ellipsoidal–lamellar structure with melanin being deposited in a uniform pattern. Pheomelanosomes, on the other hand, are round and granular with uneven deposition of pigment within the melanosome. It should be noted, however, that another spherical melanosome with a distinct granular ultrastructure, which occurs in the Harding–Passey mouse melanoma, does not produce pheomelanin but mostly eumelanin, as determined by complex physicochemical analysis (Ito and Jimbow, 1983; Jimbow et al., 1984). In human epidermis, melanin granules are transferred to keratinocytes where they appear as single, nonaggregated pigment granules or as aggregates, often termed complex melanosomes (see Chapters 7 and 9). The size and distribution of melanosomes in epidermal keratinocytes depends predominantly on the type of human skin (Toda et al., 1973). Eventually, after fusion with lysosomes, the epidermal melanosomes are degraded and broken up into “melanin 312

dust” (Wolf, 1973). It remains to be determined to what extent the physicochemical properties of the melanin are modified by such a disassembly of the melanin granules. Very significant changes in melanin can occur with age; it has been shown by electron microscopy that, in human retinal pigment epithelium from donors over 90 years old, virtually all the melanin granules are enclosed in other material, thereby creating “melanolipofuscin” or “melanolysosomes” (FeeneyBurns et al., 1990). Neuromelanin has a distinctively different type of pigment granule. Neuromelanin is found in certain dopaminergic neurons of the substantia nigra and locus coeruleus of primate brains (Bazelon and Fenichel, 1967; Marsden, 1969; Van Woert and Ambani, 1974). These neuromelanin granules are irregular in shape and vary in size and, unlike melanin arising from melanosomes, they do not have distinct well-organized limiting membranes. Neuromelanin in situ is a material granule consisting of three different components — an electrondense melanin core, electron-translucent lipid vacuolae, and lipofuscin (Barden and Brizzee, 1987). It has been reported that neuromelanin accumulates with age in the substantia nigra of human brains until it reaches a maximum level at the age of 50–60 years, and then it gradually decreases (Mann and Yates, 1974). Based on scanning electron microscopy studies, it was proposed that the “primary particles” of natural melanin are spherical, nonporous particles with a diameter in the order of 30 nm, with an inherent specific surface area in the order of 160 m2/g (Kollias et al., 1991; Zeise et al., 1992). According to this view, a “melanin aggregate” is composed of strongly associated primary melanin particles placed together in such a fashion that the measured surface area is significantly less than the sum of the specific surface areas of the primary particles of which it is composed. Such aggregates may be similar to, if not identical with, melanin granules derived directly from Sepia officinalis melanosomes, which are spherical in shape, have an average diameter about 160 nm, and a specific surface area 29.31 m2/g (Kollias et al., 1991). Sepia melanin granules are uniformly electron dense and, under electron microscopy, exhibit no detectable ultrastructure. In this respect, it is an open question whether typical elongated eumelanosomes and round pheomelanosomes with distinct ultrastructure should be classified, according to this scheme, as “agglomerates,” i.e. loosely associated clusters of melanin aggregates. More recent scanning electron microscopy (SEM) study of Sepia melanin indicated the existence of much smaller particles with lateral dimensions of about 15 nm that adhered to larger melanin subunits (Nofsinger et al., 2000). This study also suggested significant structural differences between Sepia melanin and a synthetic melanin obtained by enzymatic oxidation of dopa. The presence of such small particles in purified melanin, isolated from the ink of cuttlefish, has been confirmed by another imaging technique. Taking advantage of the three-dimensional spatial resolution of atomic force microscopy (AFM) and the ability of the technique to cut the

THE PHYSICAL PROPERTIES OF MELANINS

imaged object or perform with it other mechanical manipulations, the Simon group demonstrated that, although the spherical particles 100–200 nm in diameter were stable structures, they were composed of tiny particles with lateral dimensions of 15–25 nm (Clancy et al., 2000). In a recent study, images of melanosomes from bovine retinal pigment epithelium and from human hair clearly indicate that these pigment granules are also aggregated assemblies of substructures about 20 nm in diameter (Liu and Simon, 2003a). In a related study, the authors demonstrated the importance of the isolation and purification procedure on the structure and chemical composition of a natural melanin (Liu and Simon, 2003b). Thus, while two different acid/base procedures, used for the isolation of melanin from human hair, yielded amorphous material without any apparent structure, only the enzymatic extraction preserved the normal morphology of the melanosomes. Even though synthetic melanins, from different substrates, often exist in aggregated forms, it has been concluded that neither chemically nor enzymatically prepared tyrosinemelanin is composed of discrete particles (Zeise et al., 1992). Indeed, in a comparative study, Nofsinger et al. (2000), using SEM, showed that, unlike Sepia melanin, a synthetic melanin, obtained by enzymatic oxidation of DOPA, was an amorphous material without any distinct substructure. On the other hand, light scattering experiments on colloidal aqueous suspensions of synthetic auto-oxidized dopa-melanin revealed a particulate character of this melanin preparation, with a particle size of less than 10 nm (Huang et al., 1989). Characterization of the structural units (nodules) of synthetic dopamelanin was also carried out by the X-ray small-angle scattering technique (XSAS) (Miyake and Izumi, 1984; Miyake et al., 1985, 1987). The radius of gyration of melanin in its aqueous solution, calculated from an initial slope of the Guinier’s plot, suggests that the elemental molecular unit of this synthetic melanin may be rod-like in shape, 4.8–8.5 nm long, and 0.6–0.8 nm in diameter. Qualitatively similar conclusions about the submolecular structure of melanin have been reached on the basis of ultrasonic measurements of synthetic diethylamine melanin and natural melanin from Sepia (Aconthosepian), and B16 and Harding–Passey melanomas (Kono, 1984; Kono and Jimbow, 1985; Kono and Yoshizaki, 1987). A pronounced particle wave resonance observed for all studied melanins at 220 MHz was related to a stiff-chain unit that could be approximated by a rod-like rigid molecule. Perhaps the most detailed structure of the synthetic tyrosine-melanin “protomolecule” has been proposed by Zajac et al. (1994). Based on wide-angle X-ray diffraction analysis of dried melanin and scanning tunneling microscopy measurements of monomolecular layers of the melanin deposited on highly oriented pyrolytic graphite, the authors constructed a model of the fundamental unit of synthetic melanin consisting of three stacked sheets with five to eight 5,6-indolequinone residues in each sheet. The spacing between stacks was assumed to be 0.34 nm in order to account for the

prominent features observed by X-ray diffraction and scanning tunneling microscopy. Therefore, the overall dimensions of the melanin protomolecule were calculated to be roughly 2.0 nm in lateral extent and 0.76 nm in height. The X-ray and STM results were verified by structure minimization and molecular orbital techniques. Systematic modeling of the data from X-ray diffraction studies of melanin performed by Cheng et al. (1994a, b) can be summarized as follows. The derived structure factor, S(q), typically shows six diffused peaks within the q-range 0.3/Å to 16/Å in reciprocal space (Fig. 16.1). Although some differences are apparent in the magnitude of S(q) oscillations for DOPA-melanin, tyrosine-melanin, and Sepia melanin, all these melanins are rather similar in the arrangements of their neighbors. The first peak in S(q) (at q = 1.74/Å) reflects mainly the number and spacing of the melanin monomer layers, suggesting that, in real space, the interlayer spacing is 3.4 Å. The plane-polymerized monomeric units produce the second peak at q = 3.0/Å, while the third peak at q = 5.6/Å refers to the number (four or five) of connected monomers in a layer. The last three peaks in the higher q-region are mainly produced by the single monomer structure, the average band length of which determines their locations. A 1.42-Å distance obtained in real space can be attributed to the average bond length of C–C, C–O, and C–N. The four-layer stacking of four to eight 5,6-dihydroxyindole (or 5,6-indolequinone) units gives a dimension of the fundamental melanin unit of 15 Å. Interestingly, a prepeak at q = 0.45/Å (which corresponds to the length 13–20 Å in real space) has also been observed in some melanin samples (Bridelli et al., 1990; Cheng et al., 1994b; Thathachari and Blois, 1969). The X-ray diffraction data revealed that melanin has a relatively low X-ray absorption coefficient (1 < m £ 2/cm), which is consistent with its elemental composition (Cheng et al., 1994b). The collective results of SEM and AFM studies of Sepia melanin, carried out by the Simon group, and of independent structural studies of synthetic melanins, in which the researchers used STM and AFM (Gallas et al., 2000), smallangle neutron scattering (Gallas et al., 1999), or synchrotron small-angle X-ray scattering (Littrell et al., 2003), as well as the results of matrix-assisted desorption ionization (MALDI) mass spectroscopy measurements, are consistent with the proposed model of ultrastructural organization of eumelanins, in which the major building block of eumelanin pigments is a small planar oligomer, probably highly cross-linked, with maximum dimensions 0.4 ¥ 1.0 nm, that is preferentially aggregated into fundamental aggregates of 3–4 p-stocked oligomers (Cheng et al., 1994a, b; Clancy and Simon, 2001; Gallas et al., 2000; Zajac et al., 1994). The macroscopic morphology of eumelanin pigment granules is a result of hierarchical self-assembly, in which the building blocks of eumelanin assemble into hundred-nanometer structures, which then aggregate to form the final pigment granules (Clancy et al., 2000). This model is a radical deviation from the existing model, in which melanin was pictured as a huge heteropolymer, consisting of a large number of different monomers 313

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1.6

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r (Å) Fig. 16.1. Structure factors S(q) (A) and the radial distribution functions (RDF) (B) of two synthetic melanins and Sepia melanin. Reproduced from Cheng et al. (1994b), with permission.

linked covalently by a variety of bonds (Nicolaus, 1968). According to the new model, melanin is built of very small building blocks — planar oligomers — consisting of as few as five to eight monomers, which assemble into nanoaggregates that form characteristic stocks. Such nanoaggregates assemble into larger structures, which then aggregate to macroscopic pigment granules. Although the exact nature of the forces that are involved in the assembly of nanoaggregates and of hundred-nanometer structures, as well as in their aggregation, remains unknown, it can be speculated that Van der Waals’, p–p, and hydrophobic interactions play a key role. Unfortunately, until now, no comparable structural studies of pheomelanin have been carried out. Much of our current knowledge about the chemical structure of melanins comes from chromatographic identification 314

and quantitation of the characteristic products arising from chemical degradation of the pigments (Ito, 1986, 1998; Ito and Fujita, 1985; Ito and Jimbow, 1983; Jimbow et al., 1984; Ozeki et al., 1995; Prota et al., 1998a, b). This approach has been applied to human neuromelanin to try to unravel its chemical structure (Carstam et al., 1991; Odh et al., 1994; Wakamatsu et al., 1991). Unfortunately, the conclusions reached by the authors have significant areas of apparent disagreement. The Swedish researchers found large quantities of 4-amino-3-hydroxyphenyl-ethylamine (AHPEA) in neuromelanin samples hydrolyzed with hydriodic acid, whereas the Japanese researchers were unable to detect any significant amount of AHPEA after they analyzed neuromelanins by degradation by hydriodic acid (HI). This discrepancy leaves open whether cysteinyldopamine is incorporated into neuromelanin. While detectable levels of pheomelanin are found in human skin, regardless of race, color, and skin type, eumelanin is always the major constituent of epidermal melanin (Ito and Wakamatsu, 2003). It appears that high levels of pheomelanin are found only in yellow and red hair of mammals and in red feathers of birds (Ito and Wakamatsu, 2003). It is believed that the key intermediates in the biosynthetic pathway for eumelanin are 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), as well as their fully oxidized forms that determine the nature of the fully formed melanin polymer (reviewed by Hearing, 1993; Hearing and Tsukamoto, 1991; Prota, 1992). In the biosynthetic pathway for pheomelanins, a similar role may be played by 1,4-benzothiazynylalanine, derived from cysteinyldopas (Deibel and Chedekel, 1984; Ito and Wakamatsu, 1989; Prota, 1988; Rorsman et al., 1979). The distributions of the functional groups (CHx, C–O, C=O, O–C=O, –NH2, and R2NH) of synthetic and natural eumelanins have been studied in solid-state samples by electron spectroscopy for chemical analysis (ESCA or X-ray photoelectron spectroscopy) (Clark et al., 1990). Qualitative and quantitative analysis, calibrated with model compounds (various precursors of eumelanin), revealed interesting differences between the synthetic polymers obtained from DOPA and DHI and Sepia melanin. Thus, although none of the synthetic melanins had elemental compositions identical to Sepia melanin, DOPA melanins (both auto-oxidized and enzymatically oxidized) were roughly comparable with the Sepia eumelanin in elemental composition and distribution of functional groups. The high content of nitrogen observed in Sepia melanin was attributed to proteinaceous material still associated with the natural eumelanin. Enzymatically produced DOPA melanin had a higher percentage of carbonyl-type functionalities than auto-oxidized DOPA melanin. As judged by their functional group distribution, the enzymatic DOPA melanin was a better model of natural eumelanin than autooxidized DOPA melanin. Infrared (IR) spectroscopy also has been used in the chemical analysis of melanin (Blois et al., 1964; Bridelli et al., 1980; Garcia-Borrón et al., 1985; Jimbow et al., 1984; Wilczok

THE PHYSICAL PROPERTIES OF MELANINS

et al., 1984; Zecca et al., 1992). Using solid pellets of melanin dispersed in KBr, broad absorption bands with varying intensities were observed at 3400/cm, 3200/cm, 3000– 2800/cm, 2700–2500/cm, 1700–1650/cm, 1600–1400/cm, and 1045/cm. The bands could arise from symmetric and asymmetric stretching and bending of bonds in a variety of functional groups such as amine, imine, carboxylic, carboxylate, phenolic, aliphatic CH3, CH2, C–H, aromatic C–H, etc.; however, precise assignment of these bands is still difficult. Quantum mechanical calculations of the vibrational structure of the key melanin monomers, carried out by Powell et al. (2004), showed that the three main redox forms of 5,6-dihydroxyindole had significantly different infrared and Raman signatures, suggesting that these spectra could be used in situ to identify nondestructively the monomeric content of various melanins. Melanin–protein complexes have been investigated by IR using absorption bands of amide groups. Based on detection of the distinct amide I and amide II bands in natural melanins and in synthetic melanin obtained by auto-oxidation of DOPA in the presence of bovine serum albumin, it was concluded that melanins in vivo exist as true melano-protein complexes with the protein moiety covalently bound to melanin (Bilinska et al., 1987; Garcia-Borrón et al., 1985). It should be noted, however, that no independent unequivocal evidence for covalent binding between melanin and protein has been obtained. IR also has been used to study the effect of potential degradative treatments on melanin. Characteristic changes in the IR spectra of DOPA melanin were observed upon treatment with HCl. The appearance of sharp methyl and methylene bands at 3000/cm and 2800/cm upon acid hydrolysis of this synthetic melanin was attributed to acid-induced decomposition of some indolic monomers, yielding noncyclic units with aliphatic side-chains (Garcia-Borrón et al., 1985). This is consistent with evidence from analysis by degradative chemistry. Ito (1986) found that acid-treated melanins gave much lower yields of pyrrol-2,3,5-tricarboxylic acid (after oxidation of melanin with permanganate) than the corresponding native melanins. The number of accessible aryl carboxylic acid and a-amino acid carboxylic groups in synthetic tyrosine melanins and natural Sepia melanin was estimated by titrimetric analysis using nonaqueous media — 2-propanol and acetic acid (Zeise and Chedekel, 1992). Titratable acidic groups were measured to be 180 mEq/q for Sepia melanin, 490 mEq/q for melanin prepared by oxidation of tyrosine with persulfate, and only 68 mEq/q for enzymatic tyrosine-melanin. It was concluded that, of the two synthetic melanins, only the enzymatic tyrosine-melanin was an adequate functional group model for the surface structure of eumelanin. Elemental analysis and quantitative amino acid analysis were employed for estimation of the elemental composition of the melanin backbone in Sepia melanin and two synthetic melanins (Chedekel et al., 1992a, b). Assuming only one nitro-

gen atom per monomeric unit of eumelanin, the authors were able to determine the average monomeric backbone chromophore of Sepia melanin. The elemental composition of the melanin chromophore backbone is: C, 45.91%; H, 2.66%; N, 6.98%; and O, 29.32%. Accordingly, the C/N and empirical formula for the Sepia melanin were 7.67 and C7.67H5.33NO3.68 respectively. Of course, the derived formula of any natural melanin will critically depend on accurate estimates of the contribution of any amino acids associated with the melanin, which can obscure the determination of the actual elemental composition of the melanin. Taking advantage of the enhanced spectral resolution offered by the NMR techniques of cross-polarization, magic angle-spinning (CP/MAS), and high-power proton decoupling, high-resolution 13C and 15N solid-state NMR have been used for the assignment of functional groups of various eumelanins (Aime and Crippa, 1988; Aime et al., 1991; Duff et al., 1988; Peter and Förster, 1989). Briefly, at a 13C Larmor frequency of 75.7 MHz, the main features of the 13C CPMAS spectrum of Sepia melanin consisted of an intense carboxylate resonance centered at 173 ppm as well as broad and strong absorptions in the aromatic and olephenic region (90–120 ppm). In natural melanins, but not in synthetic melanins, a number of variously substituted aliphatic regions (15–75 ppm) was observed, consistent with the presence of unreacted DOPA and a proteinaceous component (Fig. 16.2) (Herve et al., 1994). Although solid-state CP/MAS and 15N NMR were used recently for characterization of Sepia melanin and human melanin (Adhyaru et al., 2003), and high-resolution 1H NMR was employed for quantification of the aromatic protons of the polymer chain in Sepia melanin and human hair melanin (Katritzky et al., 2002), the application of these advanced analytical methods has not yet yielded any truly unique information about the chemical structure of melanin. Much of the information on the structure and composition of melanin derived by chemical and structural analysis needs to be considered critically and considered tentative because of the experimental complexities involved in such determinations. This is because of the heterogeneous nature of melanins and their difficult physical–chemical properties, and the possible presence of nonintrinsic proteinaceous material. The procedures required to make it feasible to analyze melanins can readily lead to artifacts. The analyses of its chemical composition often depend on the derivatization of relatively small parts of the macromolecule and so, even if the derivatization procedures are entirely valid from a chemical point of view, the portions of the molecule from which they are obtained may not be fully representative of the entire molecule; for example, they may be derived primarily from parts that are on the surface. Analogous considerations apply to many of the structural studies, especially when these require that the melanin be dried or otherwise altered. This can lead to very distinct changes in its physical properties [e.g. as monitored by electron paramagnetic resonance (EPR) or, equivalently, ESR], which may not reflect the fully hydrated state, etc. (Sealy et al., 1980). 315

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Fig. 16.2. 13C solid-state NMR spectra of Sepia melanin (A) and a synthetic melanin obtained by auto-oxidation of 5,6dihydroxyindole (C), and their Lorentzian peak fitting spectra (B) and (D) respectively; spectra obtained by conventional crosspolarization/magic-angle spinning (CP/MAS) technique are shown by solid lines, while those obtained by short-contact-time CP/MAS (protonated carbons) are shown by dotted lines and those obtained by dipolar dephasing (quaternary carbons) by broken lines. Reprinted from Hervé et al. (1994), with kind permission of Elsevier ScienceNL.

Optical Properties The absorption of light is one of the most obvious and important properties of melanins. This has proven to be an immensely complex topic, in terms of both understanding the mechanisms and consequences of optical absorption by melanin and exploiting these properties to enhance an understanding of melanins. In this section, we attempt to provide representative highlights of what appears to be a productive and growing area of study. The absorption spectrum of human skin melanin in vivo is a linear function of the wavelength in the range of 500–750 nm (Kollias and Baqer, 1985). Based on diffused reflectance spectra obtained from patients with vitiligo and normal volunteers, it has been proposed that human melanin absorbs visible radiation through two distinct mechanisms: one that is in effect over the entire visible range and is linear in wavelength, and a second one that is evident at wavelengths in the range 400–500 nm and is exponential in frequency (Kollias and Baqer, 1987; Kollias et al., 1991). As natural melanin in situ is in particulate form (Barden and Brizzee, 1987; Feeney-Burns, 1980; Kollias et al., 1991), its observable optical properties are a complex function of melanin’s ability to absorb, scatter, and reflect light at different wavelengths. 316

Therefore, it has been very useful to carry out optical studies on synthetic and isolated natural melanins as well as in situ. The optical density of soluble synthetic melanin, such as auto-oxidized DOPA melanin, increases almost monotonically with decreasing wavelength (Crippa et al., 1978; Sarna and Sealy, 1984a). The apparent absorption coefficient of DOPA melanin increases from about 4/mg/cm2 at 600 nm to over 30/mg/cm2 at 200 nm (Fig. 16.3). It is important to stress that these are only representative values because the detectable absorption spectra of melanin depend, among other factors, on the conditions used for its synthesis, the redox state of the polymer, the pH and the ionic strength of the aqueous media, temperature, and the handling of the sample (Sarna, 1992). Although the molecular origin of melanin determines many of its physicochemical properties, optical absorption of the polymers synthesized from dopa and cysteinyldopas is quite similar (Fig. 16.3). The smooth absorption curve of a solution of synthetic auto-oxidized DOPA melanin, with almost monotonic increase in the absorbance with decreasing wavelength, viewed as a characteristic feature of melanin, has been an enigma for years and is still not fully understood. Although different models have been considered to explain the unusual optical

THE PHYSICAL PROPERTIES OF MELANINS 50 A

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properties of melanin, including the solid-state model, in which melanin is treated as an amorphous semiconductor with distinct energy bands (Crippa et al., 1978; Galvao and Caldas, 1988; Jastrzebska et al., 1990; Longnet-Higgins, 1960; McGinness et al., 1974), none of the models proved to be satisfactory. One of the most interesting results of the theoretical studies by Galvao and Caldas (1990a, b) was the finding that key physicochemical properties of melanin start to emerge in systems consisting of a small number of monomeric units. Indeed, a remarkably smooth absorption spectrum in the region 300–800 nm for a random composition of basic

monomer units such as 5,6-dihydroxyindole, indole-5,6quinone, and their semiquinone form has been obtained by Bochenek and Gudowska-Nowak (2003a, b), using the intermediate neglect of differential overlap (INDO) and other semi-empirical methods. A similar trend, with significant absorption in the visible spectral region, has also been observed by Stark et al. (2003), who carried out much more advanced density functional theory (DFT) calculations for simple melanin oligomers. The relative stability of nine tautomers of 5,6-dihydroxyindole and 5,6-indolequinone and their excitation energies in both gas phase and solution were examined by Il’ichev and Simon (2003), who used DFT, timedependent DFT, and self-consistent reaction field calculations. The results of their calculations indicated that the indolequinone units could be responsible for a relatively strong absorption in near infrared. Finally, a first principle density functional theory calculation of the electronic and vibrational structure of key melanin monomers has been reported by Powell et al. (2004). The authors postulated that the difference in energies of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of the different monomers could lead to a large range of HOMO–LUMO gaps in eumelanin molecules and thus be related to the observed broadband optical absorption of melanin. In other words, it is the monomer diversity and chemical disorder that are responsible for the optical properties of melanin. Although the existing theoretical studies of melanin have not yet resulted in a satisfactory description of its optical properties, there is little doubt that advanced quantum mechanical calculations of various melanin oligomers should yield important new results and, ultimately, will lead to better understanding of its structure and physical properties. The degree of aggregation of the melanin is an especially important factor that will significantly modify the ability of melanin to transmit light at different wavelengths (Huang et al., 1989; Pilas and Sarna, 1985). This effect can also be exploited to study the aggregation of melanins. Using static and dynamic light scattering emitted by a NeHe laser, the dynamics of aggregation of a synthetic DOPA melanin in acidic aqueous solution was studied (Huang et al., 1989). It was found that, depending on the final pH of the solutions, slow and fast regimes of the kinetics of aggregation could be identified. The precipitates formed in these two regimes could be characterized by fractal structures. It was estimated that, in the fast, diffusion-limited aggregation regime, the aggregate was a fractal with dimension of 1.8, whereas the dimension of the fractal was 2.23 in the slow, reaction-limited aggregation regime. The fractal nature of melanin aggregates has been established over scales ranging from about 0.08 to 2.0 mm (Eisner, 1992). The slow regime of melanin aggregation, with the fractal dimension 2.2, was also observed in the presence of critical amounts of transition metal ions such as copper, nickel, and zinc. That aggregation of melanin affects its optical properties has also been found for very small melanin particles. Thus, Nofsinger et al. (1999) reported distinct differences 317

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in the optical absorption of Sepia melanin obtained by ultrafiltration of the bulk melanin using a series of different ultrafiltration disk membranes. The results of this study, perhaps predictably, showed a substantial reduction in melanin absorbance in the visible and near-UV spectral region when very low-molecular fractions of this eumelanin were examined. In a related study, the authors demonstrated that differences in the absorption bands were due to varying levels of melanin aggregation (Nofsinger and Simon, 2001). The optical absorption and scattering properties, and thermal diffusivity of melanin particles from Sepia officinalis were recently determined at 580 nm and 633 nm, using photometric and photothermal techniques (Vitkin et al., 1994). The absorption coefficient (ma) and the transport scattering coefficient (ms1) of Sepia melanin were determined from data of diffuse reflectance and transmittance. The scattering anisotropy was obtained from an additional measurement of the total attenuation coefficient and, independently, by goniometry. Pulsed photothermal radiometry was used to deduce the absorption and transport scattering coefficients by a model based on optical diffusion theory, and to provide the thermal diffusivity of solid melanin. At 633 nm, ma was found to be 127–157/cm/% and 162–176/cm/% at 580 nm. The corresponding transport scattering coefficient values were 24–28 and 28–30/cm/% at 633 nm and 580 nm respectively. The photometrically measured optical absorption and scattering properties of Sepia melanin were consistent with the Mie theory predictions, which indicated strong dependence on the size of melanin particles. The larger the size of melanin particles (melanosomes), the higher their albedo (and more forwardpeaked scattering phase functions). These effects are likely to be important for epidermal melanosomes and for retinal pigment epithelial granules, the size of which is significantly larger than that of Sepia melanin. Data from both optical and photothermal studies suggest that Sepia melanin is not a perfect black body absorber; the melanin significantly scatters light in the yellow–red region. The internal absorption coefficient of melanosomes in situ in human skin was determined, based on measurements of the threshold radiant exposure from a pulsed ruby laser that was necessary to achieve explosive vaporization of the melanosomes (Jacques and McAuliffe, 1991). This phenomenon is observed when the rate of radiant energy deposition within the melanin granule is substantially higher than the thermal relaxation rate of the pigment granule. As the thermal relaxation of melanosomes is estimated to be between 0.5 and 1 ms, threshold radiant exposures for melanosomal injury could easily be obtained using nanosecond Q-switched Nd:YAG or ruby lasers when the energy density exceeds 1 J/cm2 (Anderson et al., 1989; Watanabe et al., 1991). Based on their own data and the literature they provided, a summary of the absorption coefficient (ma) of the melanosome interior vs. wavelength was presented. The ma parameter was found to vary significantly with the wavelength; thus, the average ma values were around 80/cm at 1064 nm, 200/cm at 694 nm, 350/cm at 630 nm,

318

700/cm at 532 nm, and 1500–2000/cm at 355 nm. Dry melanosomes required lower threshold radiant exposure to achieve their explosive vaporization than wet melanosomes, indicating that the effective ma for dry melanosomes is higher (202/cm) than that for wet melanosomes (120/cm). The data have been interpreted in terms of swelling, with wet melanosomes having a 70% larger volume than dry melanosomes. The absorption spectrum for human epidermis in vivo, measured by an optical fiber spectrometer, matched quite well the relative changes with wavelength of the absorption coefficient of the interior of the melanosome. The solid-state appearance of typical melanin and its very efficient nonradiative de-excitation, following the absorption of ultraviolet or visible photons, would suggest photo-acoustic spectroscopy to be a method of choice for studying the optical properties of natural melanins. This spectroscopic technique has been applied, with great success, to many biological systems (Balasubramanian and Mohan Raa, 1986; Braslavsky, 1986; Fork and Herbert, 1993; Moore, 1983) but, surprisingly, has been used sparsely to study melanins. The only photo-acoustic spectrum of melanin reported to date that the authors are aware of is a spectrum of a synthetic DOPA melanin obtained in a limited spectral range (Wróbel et al., 1997). The PAS spectrum of DOPA melanin is similar to typical optical absorption spectra of solubilized eumelanins. Photo-acoustic techniques were used to determine the thermal properties of melanin and to study nonradiative relaxation of excited states in melanin (Crippa and Viappiani, 1990; Gallas et al., 1988). It was found that the acoustic signal induced in synthetic DOPA melanin by chopped light from a krypton laser was dependent on the chopping frequency (~w–1), as predicted by the Rosencwaig–Gersho theory for optically dense samples with a length of thermal diffusion that is greater than the optical extinction length (Rosencwaig and Gersho, 1976). The data suggested, rather unexpectedly, that the optical absorption coefficient in acid- or acetone-precipitated melanin was about 30 times smaller than that of “standard melanin.” Photo-acoustic measurements of dense aqueous melanin suspension made at various chopping frequencies with an argonion laser as the excitation source revealed strong dependence on pH and the redox state of melanin (Crippa and Viappiani, 1990). Control experiments, performed on natural and synthetic melanins in the form of powders or pastes, confirmed the results of Gallas et al. (1988) only to some extent; the photo-acoustic signal intensity varied with the chopping frequency as w–0.9–w–1.1. A very efficient electron–photon coupling, consistent with efficient energy transfer toward the internal degrees of freedom of the melanin macromolecule, was inferred from the effects of phonon amplification observed in a synthetic melanin upon the application of a pulsed electric field with increasing gradient (Crippa et al., 1991). This explains why melanin is a very poor fluorescence emitter. The first unambiguous report of melanin-specific fluorescence was reported only in 1984, using natural melanins, both intact and solubi-

THE PHYSICAL PROPERTIES OF MELANINS

Fig. 16.4. Fluorescence spectra of auto-oxidized dopa melanin (A) and of intact melanin granules from human RPEs of different age groups (B). (A) Fluorescence of melanin was observed as a function of its excitation wavelength: 340 nm (dotted line extending to the shortest wavelengths) and 400 nm (solid line in the longest wavelength region), with fluorescence induced by excitation at 360 nm and 380 nm being characterized by dotted lines in between. (B) Left curves (I) are excitation spectra with emission monitored at 570 nm, and right curves (II) are emission spectra with excitation at 364 nm. (a) Fetal, (b) 5–29 years, (c) 30–40 years, (d) > 50 years and (e) 1 year bovine melanin. Reprinted from Gallas and Eisner (1987), with kind permission; and from Boulton et al. (1990), with kind permission from Elsevier Science.

lized, after drying, in solid-state KBr (Kozikowski et al., 1984). The authors observed a weak broad luminescence, centered at about 540 nm, when natural melanins from human hair and Sepia ink were excited by an argon-ion laser emitting at 488 nm. A dramatic increase in the fluorescence emission intensity was brought about by the solubilization of the melanin, achieved by treatment with H2O2 at high pH. [Similar increases in the detectable fluorescence of synthetic DOPA melanin and melanoproteins, after early oxidative degradation of the melanin by treatment with H2O2, were also reported by Soviet workers (Korzhova et al., 1989).] An induction of a distinct fluorescence of melanin in situ in tissue sections was observed upon irradiation of the sections with UV

light (Elleder and Borovansky, 2001). The phenomenon was explained as being due to an oxidative breakdown of melanin induced by simultaneous action of UV and hydrogen peroxide (Elleder and Borovansky, 2001). Fluorescence of synthetic DOPA melanin was studied by Gallas and Eisner (1987) as a function of excitation wavelength and melanin concentration. Fluorescence of melanin, excited at 340 nm, corrected for attenuation of excitation and emission beams and removal of background Raman and impurity fluorescence signals, exhibited a broad signal. Upon deconvolution, the data indicated the presence of two emission bands, one with a maximum at about 430 nm and the other with a maximum at 510 nm (Fig. 16.4). When melanin

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was excited at longer wavelengths, such as 400 nm, one emission band (at 500 nm) was observed predominantly. Assuming two interacting species (quinone and hydroquinone moieties of melanin) that can form a complex, quinhydrone, the fluorescence data of melanin were interpreted by a simple model, in which excitation occurs mainly through the broad absorption band of the complex. The complex can then dissociate, leaving the quinone or hydroquinone groups in the excited state, or can decay by radiative processes after a series of vibronic transitions. The latter would lead to one band of fluorescence emission, while the radiative decay of the excited quinone (or hydroquinone) units would constitute another band of fluorescence emission. However, the validity of the conclusions discussed above has been questioned by Nofsinger and Simon (2001), who in a more recent study analyzed spectral and kinetic parameters of radiative relaxation of a natural eumelanin. Using different molecular-weight fractions of melanin, extracted from the ink sacs of Sepia officinalis, these authors measured excitation and emission spectra of the melanins. They found no structure for the corrected emission spectra in the wavelength range 400–550 nm for excitation wavelengths above 325 nm. On the other hand, the authors demonstrated that emission properties of Sepia melanin varied with the aggregation state of the melanin. Although different melanins were examined in these two studies, it seems unlikely that melanin origin was responsible for the observed differences in melanin fluorescence. Indeed, in a recent independent study, Meredith and Riesz (2004) reported on radiative relaxation quantum yields for a synthetic dopa-melanin. Using a strict renormalization procedure to correct for pump beam attenuation and heavy reabsorption of the emission, the authors found no evidence of a double-peaked feature in the emission spectrum of the melanin that was reported by Gallas and Eisner (1987). Meredith and Riesz (2004) observed that the position, width, and intensity of the emission maxima, as well as the quantum yield, varied as a function of the excitation wavelength. The emission quantum yield was calculated to be in the range 0.0005–0.0007, almost an order of magnitude lower than that determined by Nofsinger and Simon (2001) for Sepia melanin. The data may indicate that fluorescence emission in a synthetic eumelanin is derived from ensembles of small chemically distinct oligomeric units that can be selectively pumped. A detailed analysis of the photodynamics of melanin was carried out by the Simon group, using a number of complementary time-resolved techniques, such as femtosecond transient absorption spectroscopy, picosecond fluorescence spectroscopy, and nanosecond photo-acoustic calorimetry (Forrest et al., 2000; Nofsinger and Simon, 2001; Nofsinger et al., 1999, 2001; Ye and Simon, 2002, 2003). The main results could be summarized as follows. The emission dynamics of melanin are nonexponential and require a sum of exponentials to generate functional forms that provide a fit for experimental data. Thus, for Sepia melanin, four exponentials are required with the following lifetimes (and amplitudes): 56 ps (0.54), 0.51 ns (0.22), 2.9 ns (0.16), and 7.0 ns (0.08). 320

Emission decay of a synthetic pheomelanin is also nonexponential and can be fitted by three exponentials: 46 ps (0.66), 1.2 ns (0.166), and 6.2 ns (0.18). When different molecularweight fractions of Sepia melanin were examined, the authors found that, although large size fractions were the source of short emission decay (lifetimes less than 1 ns), small melanin fractions were responsible for long-lived emission dynamics (lifetimes greater than 1 ns). Nonexponential character of transient absorption decay dynamics has also been observed for eumelanin and pheomelanin following photoexcitation with an ultrashort laser pulse at 303 nm. Global fitting parameters, obtained from transient absorption data for Sepia melanin, give the following characteristic lifetimes: 0.56 ps, 3.2 ps, and 31 ps. Corresponding parameters for a synthetic pheomelanin are 0.46 ps, 2.9 ps, and 27 ps. The data clearly show that melanin is a system in which a very efficient thermal relaxation occurs. This is to say that energy absorbed by melanin photons is rapidly converted into heat via very fast internal conversion. Excitation and emission spectra of intact melanin granules of the retinal pigment epithelium (RPE) from human donors of different age groups were studied by continuous wave (CW) and time-resolved spectrofluorometry (Boulton et al., 1990; Docchio et al., 1991). Fluorescence spectra of RPE melanin exhibited distinct age-related changes; the excitation maximum shifted from 350 to 450 nm and became broader with increasing age of the RPE, and the emission spectrum developed a second peak (in addition to the main intensity at 440 nm) at about 560 nm, which grew in intensity with age. The overall fluorescence intensity of RPE melanin increased with increasing age of the donor. Using timeresolved fluorescence spectroscopy, four decay components in the fluorescence spectrum of RPE melanin have been identified (0.14–0.19 ns, 0.43–0.58 ns, 1.98–2.30 ns, and 5.49–7.90 ns). The resultant time-integrated and time-gated fluorescence spectra of RPE melanin also exhibited marked variations with age. Although the results of these impressive investigations are not completely understood, some of the observed age-related changes could be explained by partial oxidative degradation of the melanin and formation of complex pigment granules such as melanolipofuscin (Rózanowska et al., 1995).

Melanin as an Amorphous Semiconductor The remarkable ability of melanin to absorb near infrared, visible, and ultraviolet radiation almost indiscriminately has not been explained satisfactorily. Even though it cannot be ruled out that the observable optical properties of melanin result from the presence of many chromophores, the absorption bands of which overlap to the extent that an absorption continuum is formed in the entire UV-vis range, no evidence has been provided to prove this view. In fact, the existing relevant data are rather ambiguous in this respect. Thus, irreversible bleaching of melanin, induced by oxidative degradation, is at first accompanied by a gradual decrease in the absorbance by melanin in both the visible and the ultra-

THE PHYSICAL PROPERTIES OF MELANINS

violet regions (Korytowski and Sarna, 1990; Wolfram and Albrecht, 1987). This could be explained by assuming that all the chromophores in melanin are equally susceptible to oxidative degradation induced by light plus oxygen or by hydrogen peroxide. Synthetic melanin, which is extensively bleached, has a severely modified absorption spectrum compared with that of native melanin; the absorption in the visible region, particularly the red, is significantly reduced, whereas it increases in the UV. The absorption changes in bleached melanin seem to be correlated with changes in an important intrinsic molecular probe of melanin — its free radical centers (Sarna et al., 2003). In an attempt to explain the unusual optical behavior of melanin (as well as its electrical properties), a band model, viewing the melanin as an amorphous semiconductor, has been proposed (Crippa et al., 1978; Kurtz et al., 1987; McGinness and Proctor, 1973; Strzelecka, 1982a, b). An optical band gap value of 3.4 eV, reported for synthetic DOPA melanin, was based on optical absorption and photoconductivity measurements (Crippa et al., 1978). On the other hand, significantly lower values for the optical gap (1.4–1.73 eV) of several natural melanins and synthetic DOPA melanin were determined by Strzelecka (1982a, b). Band gaps in the range of 1.0–1.4 eV were also found by Kurtz et al. (1987) for various melanins. Finally, an optical gap value of 1.45 eV could be estimated from the photoconductivity edge of about 850 nm reported by Trukhan et al. (1973). The reason for significant discrepancies in the reported band gap values is not clear. It can be speculated that some of the differences may arise from variations in the types of melanin and/or the preparation of the samples that were used. A mechanism for band gaps in melanins, based on mobility gaps, typical for amorphous semiconductors, has been proposed by McGinness (1972), and unusual current–voltage (I–V) characteristics for a wet melanin were explained by amorphous semiconductor switching in the melanin (McGinness et al., 1974). This explanation was later questioned (Chio, 1977), but the hypothesis about melanin being an amorphous semiconductor has led to additional speculations and experiments. Measurements of electrical photoconductivity of melanin were reported in 1968 (Potts and Au, 1968), and photoconductivity of natural melanin from frog RPE was studied by the microwave dispersion technique (Trukhan et al., 1970, 1973). In the latter works, the current carrier was found to be of hole character, and its mobility was estimated to be 15 cm2/Vs. Measuring dark, DC, steady-state conductivity as a function of the applied voltage, and plotting the specific conductivity vs. inverse absolute temperature and optical absorption of melanin vs. the photon energy, basic semiconductor characteristics of synthetic DOPA melanin and several natural melanins were obtained (Strzelecka, 1982a, b, c). Specific conductivity of the natural melanins was in the range 10–11–10–10/W/cm, whereas it was almost 10–7/W/cm for the synthetic melanin. Thermal activation energies for natural melanins were found to be 0.93–1.04 eV at 298–333 K. Interestingly, the synthetic melanin appeared to have two values of

activation energies: below 311 K it was 0.1 eV and above 313 K it increased to 0.78 eV. The energy of 0.1 eV was taken as evidence for a band of states (0.2 eV wide) of the Fermi level. Unfortunately, it does not appear that these results, obtained with DOPA melanin, have been reproduced by an independent study. Dark and photoinduced, DC, steady-state conductivity measurements were also carried out on synthetic melanins prepared from dopamine, epinephrine (adrenaline), adrenochrome, and adrenolutin (Jastrzebska et al., 1990). Specific conductivities of the melanins were in the range 1.3 ¥ 10–12–1.5 ¥ 10–10/W/cm, which is significantly lower than that reported for DOPA melanin (Strzelecka, 1982a, b). Thermal activation energies determined for the catecholamine melanins, on the other hand, were rather similar to that of DOPA melanin: 0.62–0.73 eV. Except for adrenolutin melanin, no photocurrent was observed for other melanins that were tested. The importance of absorbed water on melanin conductivity has been demonstrated in a more recent study (Jastrzebska et al., 1995). The authors reported that thermal activation energy of dark conductivity varied in the range 47–73 kJ/mol, depending on the hydration state of melanin. Indeed, in a more recent study, Meredith et al. (2004) showed that the electrical conductivity of a synthetic eumelanin varied by five orders of magnitude if the melanin was exposed to relative humidity in the range 10–80%. The authors of the latter study concluded that the electronic contribution to the conductivity of melanin was very small and eumelanin should essentially be considered as an insulator. A polaron and hopping model for melanin conductivity, based on the results of dielectric spectroscopy and photoconductivity studies of synthetic DOPA melanin, has been proposed by Jastrzebska et al. (2002a, b). Charge transport in synthetic catechol melanin was studied by analyzing current–voltage characteristics and temperature dependence of DC steady-state conductivity (Osak et al., 1989a), and polarization and depolarization currents at different electric fields and temperatures (Osak et al., 1989b). The data indicated a deviation from Ohm’s law for voltages higher than several hundred volts. The temperature dependence of DC steady-state conductivity at an applied voltage of 85 V (for which Ohm’s law was obeyed in the entire temperature range studied) suggested two thermal activation energies: below 3∞C, the activation energy was 0.76 eV and, at higher temperatures, it was 1.58 eV. Long-lasting polarizing currents, detected in the melanin, were described in terms of the movement of charges trapped in deep states of the polymer. Polarization of the melanin samples had an activation character, with the thermal activation energy of 0.67 eV. According to the authors, the current carrier in the catechol melanin was predominantly of electron character. The authors further concluded that transport of charges involved both charges generated in the sample and charges injected from electrodes. The results of the studies on semiconductor properties of melanin, briefly reviewed in this section, are difficult to interpret. The observable DC dark and photoconduction of melanin are very low and depend critically on the water 321

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content in the tested samples. This is a serious consideration in strongly hygroscopic samples such as melanin. Thus, unless all experimental parameters are strictly controlled, including the preparation of melanin and its physicochemical state, DC conductivity measurements on melanin samples may lead to random results and erroneous conclusions. It therefore appears that, at this time, the semiconducting properties of melanin have not been rigorously determined. Even if melanin is an amorphous semiconductor, it remains to be established whether this description of melanin offers any new insight into our understanding of the structure and properties of melanin, and its biological functions. A band model for melanin that could explain some of its optical absorption, electron exchange, and paramagnetic properties has been suggested following theoretical calculations of the electronic structure of the melanin monomer and dimer units (Longnet-Higgins, 1960; Pullman and Pullman, 1961). Extrapolating the bonding character of the lowest unoccupied orbital (LUMO) of one particular dimer of 5,6-indolequinone to the lowest conduction band of the infinite polymer, the authors pointed out the tendency of such a melanin model to be an electron acceptor and that this would explain the trapping of free radicals. The semiconducting polymer model, however, was found to be inconsistent with EPR data that apparently ruled out the occurrence of any extensive p-electron delocalization in melanin (Blois et al., 1964). More recent theoretical investigations of model polymers for eumelanins have been carried out by Galvao and Caldas (1990a). Using the Hückel p-electron approximation, and the same parametrization used by Pullman and Pullman (1963), the authors studied the electronic structure of a family of ideal ordered polymers arising from 5,6-indolequinone in different redox states. The authors found that structural effects, such as direction of polymerization, began to emerge as the length of the polymer increased. The redox state of the melanin units seemingly played an important role in its band structure, e.g. a polymer built from 5,6-dihydroxyindole units consistently showed larger gaps and narrower bands, whereas finite chains of semiquinone units exhibited bonding character of their lowest unoccupied molecular orbital. An important conclusion, reached by the authors, was the inevitable occurrence of end-type defects in any finite melanin polymer. The existence of defects with deep gaps is consistent with the hypothetical electron acceptor properties of melanin. According to the authors, an electron injected at the surface of the pigment (by a donor molecule) could be trapped at an end-type defect state producing the observable electron paramagnetic resonance (EPR) signal. Furthermore, they argued that capture of a second electron at the same defect would not be favored because of electron–electron repulsion effects and, as a result, such an electron would be easily transferable to another empty defect center. In this model, unpaired electrons are likely to have rather uniform distribution among defects, and the spin concentration is expected to be roughly independent of temperature. The authors speculated that, for samples in solution,

322

the chains of the polymer would behave more like isolated molecules so that double occupation of defects was more probable and, therefore, an enhanced temperature dependence of the spin concentration was predicted for samples in solution. In an extension of their theoretical study of model polymers for eumelanins, Galvao and Caldas (1990b) investigated the effects of different kind of defects, such as the aggregation of carboxyl radicals into one skeleton monomer, aggregation of a host monomer in a lateral misplaced position, and faults in the polymerizing sequencing. The data indicated that, although the end-type defect is not deactivated by the introduction of other defects, new capture centers might be formed, which could enhance the electron-accepting properties of the melanin.

Free Radicals in Melanin Melanin is the only known biopolymer that both in vivo and in vitro contains relatively high concentrations of persistent free radical centers that can easily be detected by EPR spectroscopy (Blois et al., 1964; Enochs et al., 1993a; Mason et al., 1960; Sarna and Lukiewicz, 1971; Sarna and Swartz, 1978; Sealy et al., 1980). These free radicals have turned out to be very important experimental parameters both to explain the properties of melanin and to serve as analytical probes for investigating the structure and properties of melanins. The EPR signals of melanin are specific for the two main types of melanin pigments (Fig. 16.5). At X-band (~9.5 GHz), eumelanins have a single slightly asymmetric line 4–6 G wide with a g-factor close to 2.004. The EPR spectrum of pheomelanin typically consists of three spectral features with an overall width of about 30 G, and g = 2.005. It is important to stress that, even though the EPR signal of melanin is very persistent, and no physicochemical procedures are known to quench it irreversibly without decomposition of the material, the free radicals in melanin are by no means “stable.” In fact, it has been demonstrated that the concentration of free radicals can be changed reversibly by almost two orders of magnitude (Sarna et al., 1981). Several physicochemical agents have been shown to modify the amount and/or type of free radicals in melanin: ultraviolet and visible radiation (Cope et al., 1963; Felix et al., 1979; Ostrovsky and Kayushin, 1963; Sarna and Sealy, 1984b; Sarna et al., 1985a, b), pH (Chio et al., 1982; Grady and Borg, 1968), temperature (Arnaud et al., 1983; Chio et al., 1980), complexing of diamagnetic multivalent metal ions (Felix et al., 1978a), redox reactions of the melanin polymer (Dunford et al., 1995; Korytowski et al., 1986; Reszka and Chignell, 1993; Sarna and Swartz, 1993), and the degree of hydration of the melanin (Sealy et al., 1980). As discussed in a later section, it is believed that most of the changes in the free radicals induced by these agents are due to changes in the so-called comproportionation equilibrium, i.e. the equilibrium between fully reduced and oxidized subunits, and the intermediate semi-reduced (semi-oxidized) states that

THE PHYSICAL PROPERTIES OF MELANINS

Red chicken feather pheomelanin

Red hair pheomelanin

Eye melanoma eumelanin

Brown eye eumelanin

Black hair eumelanin 10 G

Fig. 16.5. EPR spectra from frozen suspensions of natural melanins (1 mg/ml) containing 3 mM Zn2+ (pH 4.5) at –196∞C. Spectra were recorded at X-band. Reproduced from Sealy et al. (1982b), with permission.

are free radicals (Felix et al., 1978a; Sealy, 1984). The equilibrium is significantly shifted toward the diamagnetic form of the melanin subunits, and the free radical content of synthetic DOPA melanin is around 2 ¥ 1018 spins/g (referred to mass of the dried melanin) under typical experimental conditions: pH 7, ambient temperature, no irradiation with ultraviolet or visible light, no metal ions, and fully hydrated samples. This corresponds to about one free radical per 1500 polymer units (assuming a molecular weight of 200 for the melanin subunit). The free radical content of purified melanin from the choroid

of bovine eyes was reported to be about half that of DOPA melanin (Chio et al., 1982). Although the number of melanin free radicals detected under typical experimental conditions is rather low because of the equilibria, the total number of participating units is likely to be substantially higher, similar to the maximum spin concentration obtained under any experimental conditions (Sarna et al., 1981; Sealy et al., 1980). It therefore appears that the comproportionation equilibrium, monitored by EPR in melanin, reflects a significant percentage of total monomer units (Chio et al., 1980). In addition to changes in the intensity of the EPR signal of melanin detected, there can be small, but distinct, changes in other spectral characteristics. For example, the g-factor of DOPA melanin is 2.0034 at pH 1, 2.0036 at pH 7, and 2.0042 at pH 12 (Chio et al., 1982). The corresponding changes in the spin concentration are 2 ¥ 1018–1.2 ¥ 1019 spins/g. The EPR signal of eumelanins (particularly synthetic DOPA melanin) at high pH becomes narrower and more asymmetric than at low pH. Dramatically different EPR spectra have been observed for pheomelanins (Sealy et al., 1982a). The spectra, showing distinct changes with varying pH, were interpreted as being due to the presence of a different type of melanin free radical. It has been proposed that, unlike eumelanin, pheomelanin contains ortho-semiquinonimine radicals, in which the unpaired electron is delocalized on both oxygen and nitrogen atoms. As a result, an immobilized, nitroxide-like EPR spectrum is observed with the parallel component of the hyperfine coupling (2 A||) being about 30 G. The ortho-semiquinonimine radical is in equilibrium with the pheomelanin subunits — fully reduced o-aminophenols and oxidized o-quinonimines. The free radical can predominantly be observed at low pH or, in the presence of complexing zinc(II) ions, at neutral and slightly acid pH. The effect of complexing of diamagnetic multivalent metal ions on the melanin EPR signal is an important diagnostic test that can be used to determine the molecular nature of the subunits. Changes in the EPR spectra are consistent with complex formation between the metal ion and chelating polymer radicals (Felix et al., 1978a). The structure of the chelating radicals can be inferred from line width changes that reflect hyperfine splittings associated with the metal ions. The magnitude of the hyperfine splitting from association with a particular metal ion is sensitive to the detailed structure of the free radicals in the melanin (Felix and Sealy, 1981), e.g. radicals complexed with the 113Cd(II) isotope in melanins derived from DOPA, catechol, and cysteinyldopa have splittings of about 3.5 G, 7 G, and 15 G respectively (Sealy, 1984). The established relationship between the amount of inhomogeneous broadening of the EPR signal of melanin induced by complexation of 113Cd(II) with melanin and the origin of the melanin also has an important practical implication because it can be used for unambiguous differentiation of natural melanins (Jimbow et al., 1984; Sealy et al., 1982a; Vsevolodov et al., 1991). Thus, EPR spectroscopy is a unique

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A

B

Fig. 16.6. The chemical structure of the monomer units of the melanin oligomers.

physical method that enables nondestructive analysis and characterization of melanins with high sensitivity and accuracy. The molecular nature of inducible melanin radicals (extrinsic free radical centers) appears to be well understood. It is most likely determined by the chemical structure of the monomer units of the melanin oligomers that can engage in redox equilibria (Fig. 16.6A): k1 Æ Q + QH2 ¨ 2SQ + 2H+ k–1 These monomers are o-quinones, o-hydroquinones, and osemiquinones in the case of eumelanin. Corresponding units for pheomelanin are o-quinonimines, o-aminophenols, and osemiquinonimines respectively. Any agent that can influence the equilibrium constant, kc/kd, may modify the detectable concentration of free radicals in melanin. A key aspect of the comproportionation equilibrium is stabilization of the radicals (Fig. 16.6B). Thus, diamagnetic metal ions that are able to form chelate complexes with the free radicals in the melanin shift the equilibrium toward the semiquinone free radicals. A similar phenomenon is observed 324

at high pH: the induced deprotonization of the radicals results in their enhanced stabilization. This, in turn, increases the observable concentration of free radicals. A careful analysis of the results of numerous EPR studies of melanin free radicals leads to the conclusion that the above may not be the only free radical centers present in the melanin polymer (Sarna, 1992; Sealy et al., 1980). In the authors’ experience, regardless of the experimental conditions under which melanin is examined, its free radical content does not decrease below a certain level (providing the melanin is not decomposed). This seems to indicate the existence of two independent pools of melanin free radicals: extrinsic and intrinsic radical centers. The extrinsic radicals can be viewed as a convenient molecular probe, reporting on the molecular nature of the melanin monomer units and the redox state of its functional groups. The intrinsic radicals, on the other hand, are somewhat less understood. They are probably paramagnetic centers that were generated during the formation of the melanin and trapped within the growing oligomers and aggregates of basic subunits. Because of severely restricted accessibility to any reactive extraneous agents and low chemical reactivity, these radicals are essentially “stable.” The intrinsic radicals being associated with the melanin core can be viewed as a unique endogenous spin label reporting on the molecular state of the melanin and its integrity. For example, it has been demonstrated that the magnitude of the low-pH EPR signal of DOPA melanin (which is a measure of the intrinsic free radicals in melanin), subjected to oxidative degradation, corresponds to the degree of bleaching of the melanin in a reproducible and consistent way (Sarna et al., 2003). Thus, the EPR spectrum of melanin, examined under nonextreme experimental conditions, is usually a superposition of two or more EPR signals arising from the corresponding free radical centers. Detailed analysis of EPR spectra of melanins of various origins, recorded at 35 GHz (Q-band) to increase spectral resolution, over a range of pH, led to a conclusion that the EPR spectrum of melanin at intermediate pH was a composite of two spectra arising from anionic and neutral radicals with different g-factors (Grady and Borg, 1968). Such an analysis has later been refined by recording second derivative Q-band EPR spectra of DOPA melanin at various pH and the use of more advanced computer simulations of the EPR spectra (Pasenkiewicz-Gierula and Sealy, 1986). The authors interpreted the EPR spectra of frozen aqueous dopa-melanin in terms of four different spectral species related to anion osemiquinone radicals with relatively localized unpaired electrons and cation radicals with extended delocalization of their unpaired spins. Recent advances in EPR spectroscopy made possible the examination and analysis of EPR spectra of various melanins at very high frequency (W-band, 95 GHz; Nilges, 1998). It is expected that such high-resolution EPR measurements and sophisticated computer simulations will make possible unambiguous identification of the molecular nature of all radicals in melanin, and determination of their role in the physicochemical activity of melanin.

THE PHYSICAL PROPERTIES OF MELANINS

As noted previously, dramatic changes in the intensity of the EPR spectrum of melanin free radicals can be induced by paramagnetic metal ions (Blois et al., 1964; Sarna et al., 1976). Even though the quenching effect of copper(II) ions on melanin EPR signal was originally interpreted as a chemical reaction between copper and free radicals (Blois et al., 1964), it has later been shown that the effect is purely magnetic in nature (Sarna et al., 1976). Using lanthanide ions, which have similar chemical properties but quite different magnetic properties, it was possible to observe consistent changes in the amplitude and microwave power saturation of the EPR signal of the radicals in melanin as a function of the type and concentration of the added metal ion. Lanthanides with a very short spin-lattice relaxation time had a strong effect on microwave power saturability of the melanin EPR signal, but only weakly quenched the signal amplitude. The effect can be understood by viewing the interacting melanin radical with neighboring metal ions as magnetic dipoles fixed in space. As a result of such an interaction, dipolar broadening of the narrow EPR signal of melanin occurs. As the magnitude of the broadening depends inversely on the cube of the distance between the melanin free radical and the metal ions, quenching of the melanin EPR signal is a distinct function of the concentration of paramagnetic metal ions in the melanin environment. This static dipolar interaction is modulated by spin-lattice relaxation of the metal ion, which efficiently decreases the amount of the dipolar broadening of the EPR signal of melanin. The faster the rate of relaxation of the metal ion, the weaker the dipolar broadening observed as the metal ion-induced quenching of the melanin EPR signal. The theory of such an unusual dipolar broadening has been described by Leigh (1970). For very rapidly relaxing paramagnetic metals, the fluctuating magnetic dipole field sensed by the melanin free radicals provides a powerful spin-lattice relaxation mechanism. Maximum relaxing efficiency of metal ions occurs when the rate of their spin-lattice relaxation approximates the microwave frequency. The quenching of the EPR signal of melanin is much more pronounced when relatively slowly relaxing metal ions are used, such as copper(II), iron(III), manganese(II) and, among lanthanides, gadolinium(III). It is important to emphasize that, in melanins, the dipolar broadening effect decreases the melanin EPR signal intensity without any apparent broadening of its line width. Thus, the effect can easily be misinterpreted by an inexperienced researcher as a real reduction in the free radical content of the examined melanin. This becomes a serious consideration when analyzing natural melanins, which can, both in vivo and in vitro, accumulate substantial amounts of transition metal ions, including paramagnetic metal ions (Enochs et al., 1993b; Zecca and Swartz, 1993; Zecca et al., 1994). One way to deal with such a problem is carefully to determine the microwave power saturability of the melanin samples and their signal amplitude before and after subjecting the samples to procedures that may stimulate the release of the metal ions that are bound to the melanin. In the authors’ experience, washing the melanin samples in an aqueous solution of

high concentrations of powerful metal ion chelators such as EDTA or DTPA and desferal for several hours is usually quite effective in this respect. Incubating melanin samples in solutions of hydrochloric or sulfuric acid (0.1–1.0 M) is also effective; however, the latter procedure may be unacceptable because of possible modifications of the melanin chemical structure induced by acids (Liu et al., 2003; Prota, 1988).

Ion Exchange Properties The ability of melanin to bind metal ions is one of its basic physicochemical properties that affects the biological effects of this pigment (reviewed by Enochs et al., 1994; Sarna, 1992; Sarna and Rozanowska, 1994; Swartz et al., 1992). As is the situation for other properties of melanins, the study of ion exchange properties of melanin has been valuable both to understand the biological effects of these interactions and as a tool to investigate the structure and properties of melanins. It is well known that melanin, both in vivo and in vitro, can accumulate substantial amounts of multivalent metal ions (Bruenger et al., 1967; Cotzias et al., 1964; Larsson and Tjälve, 1978; Lydén et al., 1984; Okazaki et al., 1985; Simonovic and Pirie, 1963; Valkovic et al., 1973; Zecca and Swartz, 1993). It has been estimated that the number of metal ion binding sites in eumelanins is about 20% of the number of monomeric units in the polymer (Potts and Au, 1976). Similar estimates were made after titrating the EPR signal of melanin with paramagnetic metal ions; plots of microwave power saturability vs. concentration of metal ions were sigmoidal and yielded a value of 6 ¥ 1020 total metal binding sites per gram of dried melanin (Sarna et al., 1976). Binding of multivalent metal ions by melanin is a pHdependent phenomenon; the amount of metal ions bound to melanin usually increases with pH in the pH range 1–7. This indicates that melanin behaves as a weak acid ion exchange resin. This is expected because the melanin polymer contains a number of functional groups (Larsson, 1998) that can serve as potential ligands for the interacting metal ion. Thorough analysis of complexing of metal ions with melanin requires precise control of the sample pH. This is because the pH of the sample (which is likely to change after adding substantial amounts of metal ions to an aqueous suspension of melanin) may determine not only the amount of metal ions that bind to melanin, but also the type of complexes that are formed. The radioactive isotope 54Mn(II) was used to determine binding parameters for interactions with bovine eye melanin (melanoprotein), human hair melanin, and synthetic dopamine melanin, and the binding was analyzed by the method of Scatchard (Lydén et al., 1984). Four classes of binding sites were found in bovine eye melanin, with the corresponding number of sites (in mmol/mg melanin) and the apparent association constants: n1 = 0.072, K1 = 5.2 ¥ 107; n2 = 0.195, K2 = 1.8 ¥ 106; n3 = 0.661, K3 = 2.0 ¥ 104; n4 = 0.398, K4 = 1.1 ¥ 103. The total binding capacity was 1.33 mmol/mg melanin, which is equivalent to about 20% of the number of all melanin units, assuming 200 as the molecular weight for an average melanin monomer unit. The 325

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Fig. 16.7. EPR spectra of copper (63Cu2+) bound to melanin from bovine eye choroid at: (A) pH 1.6, (B) pH 1.9, (C) pH 2.9, (D) pH 4.3, (E) pH 5.8, (F) pH 8.7, (G) pH 11, (H) pH 12.6. Spectra were recorded at –196∞C using an X-band EPR spectrometer equipped with second derivative capabilities. The abscissa indicates magnetic field strength in kilograms. The ratio of melanin isonomers to copper ions was 100:1 (assuming a monomeric molecular weight of 200). Reproduced from Sarna et al. (1980a), with permission.

authors indicated, however, that the binding capacity of the bovine eye melanin was rather high, as the total binding capacity was 0.23 mmol/mg melanin for human hair melanin and 0.15 mmol/mg melanin for dopamine melanin. Using X-band EPR spectroscopy and the isotope 63Cu(II) as a molecular probe, the molecular nature of the main binding sites in synthetic DOPA and catechol melanins, and natural melanin from bovine eye choroid have been studied (Froncisz et al., 1980; Sarna et al., 1980a). The results can be summarized as follows. Depending on the pH of the system, copper(II) can form a number of complexes with melanin that involve different functional groups of the polymer and exhibit different stabilities (Fig. 16.7). Nearly all complexes involve just one or two ligands from melanin; the others are presumably H2O or OH– groups. At pH < 7, binding is predominantly to monodentate carboxyl complexes and, in eumelanin, also to bidentate nitrogen–carboxyl complexes. The corresponding EPR spectral parameters are within the range: g|| = 2.26–2.34, A|| = 460–560 MHz, and g^ = 2.066–2.076. The complexing of copper ions by melanin can be observed at low pH (below pH 3) in the EPR spectra of frozen suspensions of melanin in solutions of copper(II) with a ratio of melanin monomers to copper ions of 100:1 (assuming a monomeric molecular 326

weight of 200). At pH 7 and above, binding is to phenolic hydroxyl groups, but the number of such sites is much less in natural melanin than in synthetic DOPA melanin. The corresponding magnetic parameters are: g|| = 2.24–2.26, A|| = 560–579 MHz, g^ = 2.054–2.064. At very high pH (above pH 11), the EPR signal of natural melanin with Cu(II) is unlike any found in synthetic melanins. Its spectroscopic parameters are: g|| = 2.18, A|| = 610–620 MHz, g^ = 2.050. The assignment of complexes of Cu(II) to functional groups of melanin has been further supported by EPR and elemental analysis of chemically modified synthetic DOPA melanin (Sarna et al., 1981). Blocking the melanin phenolic hydroxyl and carboxylic groups by methylation, ethylation, or acetylation was accompanied by consistent changes in the EPR spectra of melanin–Cu(II) complexes and reduction in the amount of Cu(II) that was bound to melanin. Using a potentiometric method, in combination with mathematical fitting and correlative spectroscopies, Szpoganicz et al. (2002) quantitated the binding sites of a colloidal suspension of synthetic melanin, and compared the metal-binding affinities of the melanin functionalities. One of the most interesting conclusions reached by the authors was the apparent role of a quinonimine functionality in metal binding by DHImelanins. On the other hand, recent quantum mechanical calculations of selected melanin monomer units indicate that the quinonimine tautomer of 5,6-indolequinone should not be present in melanin to any appreciable extent (Il’ichev and Simon, 2003). As virtually identical EPR spectra of melanin–copper(II) complexes have been observed for choroidal melanoprotein and for protein-free melanin (obtained from the melanoprotein by hydrolysis with cold hydrochloric acid), it has been concluded that the protein component does not play a significant role in metal ion binding. This conclusion was supported by results of the titration of the free radical signal of melanin with Cu(II) in melanoprotein and purified melanin; the corresponding titration curves were identical. Binding of copper ions to melanin is a time-dependent process. At moderately acidic pH, after addition to suspensions of melanin, cupric ions rapidly form an initial complex, which rearranges within hours to a more stable monodentate or bidentate complex. At pH 3.0, the complex is predominantly the monodentate complex with the carboxyl groups of melanin. Similar intramolecular rearrangements of iron(III) have been observed recently for synthetic neuromelanins (Shima et al., 1997). The binding of metal ions to melanin was also studied by the utilization of ferric ions as molecular probes (Sarna et al., 1981). The EPR spectra of Fe(III)–melanin complexes at neutral and weakly acidic pH (pH 3–7) were found to be almost indistinguishable (apart from signal intensity). The most prominent feature of the spectra was a single asymmetric line at g = 4.3. Similar EPR spectra of iron were detected in natural melanin from bovine eye choroid (Sarna et al., 1980a), in melanosomes from human and bovine retinal pigment epithelium (Zareba et al., 2005), and in neuro-

THE PHYSICAL PROPERTIES OF MELANINS

melanin (Enochs et al., 1993a; Zecca and Swartz, 1993). The EPR data of iron–melanin complexes can be compared with results obtained by another powerful spectroscopic technique — Mössbauer spectroscopy (Bardani et al., 1982; Gerlach et al., 1995; Kochanska-Dziurowicz et al., 1985; Sarna et al., 1981). For fully hydrated DOPA melanin samples incubated with the 57Fe(III) isotope at pH 3 and pH 7, the Mössbauer spectra at 77 K were essentially identical, with quadrupole splitting 0.33 ± 0.005 mm/s and isotopic shift 0.17 ± 0.04 mm/s (Sarna et al., 1981). The data from EPR and Mössbauer studies suggest that ferric ions bind to melanin predominantly via phenolic hydroxyl groups and form high-spin complexes with distorted octahedral or rhombic symmetry, with a coordination number of 4–6. It can be speculated that the functional groups of melanin provide the four planar ligands for Fe(III) complexes with melanin, while the two remaining ligation sites are occupied by OH– or H2O. A significant modification of melanin–iron complexes due to drying may be inferred from the fact that substantially different Mössbauer parameters were observed when examining fully hydrated (Sarna et al., 1981) and dried melanin samples (Bardani et al., 1982; Kochanska-Dziurowicz et al., 1985). Drying of neuromelanin, isolated from human substantia nigra seems to modify the state of iron ions bound to melanin, as their Mössbauer parameters become similar to those of human hemosiderin or ferritin (Galazka-Friedman et al., 1996; Gerlach et al., 1995). In a recent study of ion exchange properties of Sepia melanin, Liu et al. (2004) compared the ability of EDTA to remove Mg(II), Ca(II), Sr(II), and Cu(II) bound to the melanin granules. The authors found that the binding constants of Sepia melanin at pH 5.8 for all these ions were higher than that of EDTA. The binding of Fe(III) was concluded to involve coordination to o-dihydroxyl groups. The authors also found evidence that Ca(II) and Mg(II) were bound to melanin via coordination to carboxylic groups. It is worthwhile emphasizing that ionic binding to melanin is by no means restricted to multivalent metal ions. Strong complexes of melanin with organic cations, both in vivo and in vitro, have been observed (Bielec et al., 1986; D’Amato et al., 1986; Larsson and Tjalve, 1979; Larsson et al., 1977; Lindquist, 1973; Lindquist and Ullberg, 1974; Lindquist et al., 1988; Link et al., 1989; Lydén et al., 1983; Stepien and Wilczok, 1982). Among the molecules that exhibit very high binding affinity with melanin are cationic forms of porphyrin derivatives, phenothiazine derivatives, chloroquine and its derivatives, and quaternary bipyrridyllium salts (paraquat and diquat). Even though different types of interaction can be involved, electrostatic attraction is the dominant force that determines complexing of these organic cations with melanin. Accumulation of drug molecules by melanin was reviewed in depth by Larsson (1998).

Melanin as a Redox System The redox properties of melanin have been recognized for a long time (Figge, 1939). In fact, one of the principal histolog-

ical tests used to detect melanin in situ is based on its reducing power; the presence of melanin in biological samples is deduced from the ability of the specimen to reduce Ag+ to metallic silver (Lillie and Fullmer, 1976). The redox properties of melanin have been investigated extensively because of their potential biological roles, especially with regard to oxidative damage. The redox properties have also served as very valuable probes to elucidate the properties and structure of melanins. The redox properties of melanin are, to a large extent, determined by the redox properties of its monomer units (reviewed by Sarna and Swartz, 1993). The relevant functional groups in eumelanin are most likely 5,6-dihydroxyindole, 5,6-dihydroxyindole-2-carboxylic acid, and their fully oxidized (quinone) and semi-oxidized (semiquinone) forms. In pheomelanins, the relevant monomer units are probably o-aminophenols, such as 1,4-benzothiazine and the corresponding fully oxidized o-quinonimine and semi-oxidized o-semiquinonimine. Although the basic redox features of melanin can be described in terms of the chemical properties of its monomers, significant differences exist between the properties of the free monomers and when they are subunits in melanin. One of the most apparent differences is their chemical reactivity; while free o-quinones such as dopaquinone, cysteinyldopaquinone, 5,6-indolequinone, and dopaminequinone are very reactive and extremely unstable (Graham, 1978; Monks et al., 1992; Thompson et al., 1985), the related subunits are quite stable in melanin. The relative stability and moderate reactivity of melanin subunits are probably due to modifications of their redox potential, electron affinity, and restricted accessibility of these functional groups within the pigment granule as a result of intramolecular interactions and steric hindrance. Using bipyridinium quaternary salts as a redox probe, the one-electron reduction potential E01 (corresponding to the fully oxidized/semi-reduced couples of the melanin subunits) of synthetic DOPA and cysteinyldopa melanins was studied by the pulse radiolysis method (Rozanowska et al., 1999). It was found that a synthetic model of pheomelanin could be reduced by a milder reducing free radical than was required for the synthetic model of eumelanin. Even though quantitative determination of the one-electron reduction potential for the melanins studied was not possible (the melanins interacted with all redox probes without establishing an apparent equilibrium), a very approximate estimation of E01 suggested that the one-electron reduction potential of cysteinyldopa melanin was more positive than –350 mV, while the E01 for the major reactive sites of DOPA melanin was between –450 and –550 mV. Owing to the intrinsic heterogeneity of melanins, a moderate dispersion of the redox properties of the melanin functional group is to be expected and is probably more realistic than a single value. These preliminary data need to be verified by the use of other methods suitable for the determination of redox properties of other aggregated polymeric materials. It remains to be determined what is the second one-electron reduction 327

CHAPTER 16

potential (E01) of the semi-reduced/fully reduced couple of the melanin moieties, and it is necessary to show that data obtained with synthetic melanins can be extrapolated to natural melanins. An interesting attempt to study redox properties of several natural and synthetic melanins by the use of simultaneous electrochemical and EPR measurements has been described by Lukiewicz et al. (1980). Distinct changes in the EPR signal of melanin radicals, observed during electrochemical treatment of the sample induced by the applied voltage, were explained by direct interactions of the melanin polymer with the electrodes. The data were interpreted in terms of the melanin being electroreduced and electro-oxidized via discrete oneelectron steps. Unfortunately, no quantitative data were provided that could be used for estimation of the melanin redox potential or electron exchange capacity, and these very promising investigations have not been followed up by any systematic studies that could provide much needed reference data on the redox properties of various melanins. One-electron reduction (and oxidation) reactions, induced by synthetic DOPA melanins, have been unambiguously shown by EPR spectroscopy using several different nitroxide radicals as redox probes (Sarna et al., 1985a). The interaction between melanin and nitroxide probes was found to be strongly pH dependent, with the rate of reduction of nitroxides at pH 10 being about 20 times faster than that at pH 5 (Sarna et al., 1985a). The data indicate that hydroquinone groups of melanin may be involved in the reduction of the nitroxides. The reduction of the nitroxide radicals was reversible, indicating that a redox equilibrium was established. From equilibrium concentrations of nitroxides and the product of the one-electron reduction of nitroxides, hydroxylamines, the equilibrium constant can be estimated for the reaction between nitroxide and melanin, assuming reasonable values for the concentrations of oxidizing and reducing groups on the polymer. The values that were obtained were in good agreement for a range of nitroxide concentrations, suggesting that the assumptions inherent in the calculations were broadly correct. Thus, for DOPA melanin formed by auto-oxidation, the number of electron-donating groups was found to be 20–30 times higher than that of the electron-accepting groups (the total number of active redox sites on the polymer was assumed to be about 25% of all monomer units). The results are rather surprising; however, if verified, they would suggest that synthetic DOPA melanin occurs predominantly in the reduced state. On the other hand, an estimate, based on available data in the literature, suggested that the reducing and oxidizing capacities of dopa-melanin were about 5 and 3 mEq/g respectively (Sealy et al., 1980). Of course, the storage conditions of the sample, including age and exposure to light, high pH, and oxygen, could significantly modify the resultant redox state of melanins. Molecular oxygen is one of the most common, biologically important, electron acceptors. Although thermodynamically, this species is very reactive with many electron-donating molecules, as a result of spin restriction (its ground state is a 328

triplet), molecular oxygen is kinetically quite unreactive with typical diamagnetic molecules (Koppenol and Butler, 1985). Melanin is no exception in this respect, although its reactivity with O2 may vary significantly, depending on the experimental conditions. The effect of pH, temperature, and chemical modification of synthetic DOPA melanin on melanin-induced oxygen consumption has been studied using EPR oximetry (Sarna et al., 1980b). EPR oximetry is an indirect, physical method that has proved to be a very convenient tool for measuring the concentration of oxygen and its changes, in a variety of biological samples (reviewed by Hyde and Subczynski, 1989; Swartz et al., 1994). The investigation clearly demonstrated that pH is the most effective experimental parameter for enhancing the rate of melanin-induced oxygen consumption: at pH 5.5, the auto-oxidation rate was slow — 10–6 g of O2 per minute, per 1 g of DOPA melanin dissolved in 1 l; the rate increased several thousand times at pH 11. The rapid increase in the rate of consumption of oxygen, particularly evident between pH 9 and 10.5, was attributed to ionization of the phenolic hydroxyl groups of the polymer. The process was found to be thermally activated, with the thermal activation energy of the order 10 Kcal/mol. The inhibitory effect of catalase on the consumption of oxygen suggests the formation of hydrogen peroxide as a product of the interaction of melanin with molecular oxygen. This conclusion has been supported by the results of studies in which the formation of H2O2 during auto-oxidation of melanin pigment was shown, using an oxidase electrode (Hintz and Kalyanaraman, 1986; Korytowski et al., 1985). The amount of hydrogen peroxide produced upon autooxidation of melanin was found to be dependent on the type of melanin, with the melanin obtained by auto-oxidation of DOPA being five times more efficient than DOPA melanin synthesized in the presence of tyrosinase, and 10 times more efficient than purified melanin from bovine eye choroid. Superoxide dismutase accelerated the rate of production of H2O2, indicating the involvement of superoxide as an intermediate. The proposed overall scheme for auto-oxidation of melanin consists of one-electron reduction of molecular oxygen to superoxide anion, followed by reduction of superoxide to H2O2 and oxidation of superoxide to O2, and spontaneous dismutation of superoxide to equimolar H2O2 and O2. The data indicate that DOPA melanin can act as a pseudo-dismutase; it is able to oxidize and reduce superoxide anion, albeit the oxidation of superoxide to O2 seems to be the dominant process, accounting for approximately 80% of the reaction between superoxide anion and DOPA melanin. Auto-oxidation of melanin may be an important, ratelimiting process in coupled reactions in which melanin acts as an electron transfer agent. An example is the melanincatalyzed oxidation of NADH and p-phenyldiamine (Van Woert, 1968). Oxidation of NADH by melanin was later confirmed in an independent study (Gan et al., 1974), which also showed that proteins associated with the melanin polymer (either in natural melanoproteins or in synthetic model systems) efficiently inhibited electron transfer reactions of

THE PHYSICAL PROPERTIES OF MELANINS

melanin. An inhibition of the electron transfer ability of DOPA melanin has also been observed for several different drugs upon binding to melanin (Debing et al., 1988). As the degree of inhibition correlated with the extent of binding and did not show any consistent dependence on the chemical nature of the drugs, the inhibitory effect of the drugs was explained by simple shielding of the redox active sites in melanin. It should be emphasized that the oxidizing equivalents for regeneration of melanin, in its electron transfer reactions, can be provided not only by oxygen, but also by other electron-accepting molecules such as ferricyanide (Gan et al., 1976). Another oxidation reaction catalyzed by melanin has been reported by Baich and Schloz (1989): in the presence of synthetic and natural melanins, glycine was oxidized to glyoxylic acid and formic acid. It was not clear from this study, however, what exactly was the role of melanin and whether possible products of reduction of oxygen, such as superoxide and H2O2, were also involved in the oxidation of glycine.

Photoreactivity of Melanin Ultraviolet and visible light can significantly modify the physicochemical properties of melanin. The most noticeable examples of the effects of light in melanin are photoinduced free radicals and photomodification of the redox properties of melanin. Illumination of melanin samples in the EPR spectrometer resonant cavity at room temperature causes a reversible enhancement of the free radical signal of melanin (Cope et al., 1963; Ostrovsky and Kayushin, 1963; Stratton and Pathak, 1968). Steady-state spectra for light-induced radicals observed during continuous photolysis, obtained by subtracting the intrinsic (dark) spectrum from the composite spectrum, revealed that the intrinsic and light-induced species differed in several EPR parameters: line width, g-factor, and microwave saturation (Felix et al., 1979). After termination of the irradiation, the signal decayed with second-order kinetics, suggesting that recombination was the primary process responsible for the termination of the photoinduced radical (Fig. 16.8). As second-order kinetics are typically observed for free radicals

in solutions, the data seem to indicate that the free radicals in melanin at ambient temperature have a substantial degree of molecular mobility. Time-resolved EPR measurements also revealed a new transient species formed in photoirradiated melanin samples (Felix et al., 1979). This species, unlike the “usual” photoinduced radicals, decayed rapidly (in milliseconds) at both ambient and cryogenic temperature, and had an EPR spectrum with a time profile that indicated the occurrence of chemically induced dynamic electron polarization. The data were interpreted in terms of the involvement of a triplet state intermediate. Such interpretation, however, seems inconsistent with the results of recent studies of melanin photodynamics using femtosecond laser flash photolysis and nanosecond photo-acoustic calorimetry (Forrest and Simon, 1998; Nofsinger et al., 2001). The authors failed to detect any long-lived transients with triplet-like properties. On the other hand, the observed very fast repopulation of the melanin ground state was a clear indication of a very efficient mechanism of energy dissipation due to internal conversion. The increased efficiency of melanin for absorbing light at shorter wavelengths suggests that photoformation of melanin free radicals may be a wavelength-dependent phenomenon. Action spectra for photogeneration of free radicals from bovine eye melanin and synthetic DOPA melanin showed a strong wavelength dependence in the 230–600 nm wavelength range (Sarna and Sealy, 1984b). Quantum yields for the steady-state formation of radicals were generally low; for DOPA melanin, the quantum yield reached the value 0.01 only at the shortest wavelengths studied, and it was well below 0.001 in the visible range. The efficiency of production of radicals from natural eumelanin was about three times greater than for synthetic melanin. As action spectra for the production of radicals differed from the optical absorption spectra, it was concluded that the chromophore(s) most active in free radical production were not the major chromophore(s) that absorb light, particularly in the visible range. A new intriguing interpretation of the spectral dependence of the photoinduced formation of melanin radicals was proposed by

Fig. 16.8. Time-resolved changes in the intensity of EPR spectrum of photoinduced free radicals in melanin from bovine eye choroid (A), and second-order plot of the data (B); [R◊] denotes transient free radical concentration. Reproduced from Felix et al. (1979), with permission.

[R·]-1 (arbitrary units)

Light Off 8 7 6 5 4 3 2 1 B

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Nofsinger et al. (1999). Comparing optical properties of different particle-size fractions of Sepia melanin with the reported spectral dependence of photoformation of melanin radicals, Nofsinger et al. (1999) found striking similarities between the absorption spectrum of the lowest molecular size fraction examined (MW < 1000) and the action spectrum for the photogeneration of melanin radicals. The role of small melanin particles in photogeneration of radicals was further substantiated by photo-acoustic data for different size fractions using an excitation wavelength of 351 nm. Thus, the data revealed that, although the percentage of the absorbed energy that was released as heat by the MW > 10 000, 10 000 > MW > 3000, 3000 > MW > 1000, and MW < 1000 eumelanin fractions was above 90 for the first two fractions, it became about 80 for the third fraction and only about 40 for the smallest molecular weight fraction. The authors concluded that the photochemical properties of melanin are determined by the presence of small molecular size constituents that, unlike the larger melanin particles, retain high photoreactivity. Although the interpretation proposed by Nofsinger et al. (1999) is very attractive and seems probable considering the role of different size aggregates in the structure of melanin (Clancy and Simon, 2001), it remains to be determined how representative the smallest particles are among the building blocks of natural melanin. Photoionization and photohomolysis of melanins occur in the wavelength range 240–300 nm (Kalyanaraman et al., 1984). This was inferred from spin trapping experiments, in which melanin was irradiated in the EPR resonant cavity in the presence of the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). In the absence of oxygen, irradiation of the melanin samples resulted in the formation of characteristic spin adducts, DMPO–H◊, product of the interaction of DMPO with either H◊ or eaq–. Photoirradiation of melanin leads to the production of both reducing and oxidizing equivalents on the polymer, which below 400 nm seem to originate from a common precursor (or precursors) (Sarna et al., 1985a). The data indicate that irradiation of melanin with light enhances melanin’s reducing and oxidizing power. In aerated aqueous samples, photoexcitation of melanin leads to an enhanced consumption of molecular oxygen and the formation of superoxide anion and hydrogen peroxide (Felix et al., 1978b; Korytowski et al., 1987; Rózanowska et al., 1995; Sarna et al., 1980b; Tomita et al., 1984). Action spectra and quantum yields for photoinduced consumption of oxygen have been determined for eumelanins and pheomelanins using EPR oximetry (Sarna and Sealy, 1984a; Sarna et al., 1984). The results of these studies indicate that the reduction of oxygen induced in melanin by visible light has a low yield: the quantum yield in the visible range is below 0.001 and reaches 0.01 only at 230–220 nm. Action spectra for photoconsumption of oxygen by eumelanin and pheomelanin are comparable with each other and exhibit similarities to the action spectra for anaerobic photogeneration of free radicals in melanin (Fig. 16.9). Using EPR spin trapping, Rózanowska 330

Fig. 16.9. Action spectra for melanin free radical photoproduction (dotted line) and for oxygen photoconsumption (broken line) by eumelanins. For comparison, an apparent optical absorption spectrum of dopa melanin (solid line) is also shown. Reproduced from Sarna and Sealy (1984b), with permission.

et al. (2002) demonstrated a significant generation of superoxide anion when melanosomes, isolated from human retinal pigment epithelium, were irradiated with blue light. The authors also showed that such an aerobic photoreactivity of the human RPE melanosomes increased with the age of the donors. Although the data suggest an increased pro-oxidizing activity of RPE melanin in RPE from older individuals, at this point it is unclear whether this intriguing observation has any biological implications. A very interesting study regarding the ability of eumelanin to photogenerate reactive oxygen species was recently published by Nofsinger et al. (2002). Using a simple spectrophotometric technique, the authors observed that the efficiency of cytochrome c reduction, used to monitor the photogeneration of superoxide by different molecular size fractions of Sepia melanin, was an order of magnitude higher for the smallest unaggregated oligomers than that characteristic of the bulk pigment. The reduced efficiency of aggregated melanin to photogenerate superoxide anion and hydrogen peroxide was attributed to the decrease in surface concentration of melanin redox active sites upon aggregation. Using the nitroblue tetrazolium–superoxide dismutase assay, the action spectrum for photoproduction of superoxide from aerated aqueous solutions of pheomelanin was determined (Chedekel et al., 1980). In contrast to the results cited above, these results indicated higher quantum yields for the photogeneration of superoxide even in the visible range. The quantitative aspects of this work should be considered with significant caution, however; it is important to realize that nitroblue tetrazolium can be reduced by many electron donors (including melanin itself), and even the inhibitory effects of

THE PHYSICAL PROPERTIES OF MELANINS

superoxide dismutase may not be used as a specific indicator of primary formation of superoxide anion (Aulair and Voisin, 1985). A model for interfacial photoinduced electron transfer between melanin and oxygen molecules has been proposed by Crippa (2001). It involves adsorption of dioxygen on melanin solid surface and light-induced carriers. The process depends on the surface fractal characteristics and is described by the Marcus theory for electron transfer reactions. Prolonged aerobic photolysis of pheomelanin is accompanied by loss of a major melanin chromophore (Chedekel et al., 1977). Although a role for superoxide anion, hydrogen peroxide, and hydroxyl radicals in the photodestruction of pheomelanin was postulated (Chedekel et al., 1978), the mechanism of the observed phenomena has not been established. Although pheomelanin was viewed as being a particularly photolabile type of melanin (Chedekel et al., 1977), a comparative study of photobleaching of eumelanin and pheomelanin suggested that eumelanin was actually more susceptible to aerobic photodegradation (Wolfram and Albrecht, 1987). Bleaching of DOPA melanin induced by its aerobic illumination from near ultraviolet and visible radiation has been studied as a function of pH, concentration of exogenous hydrogen peroxide, oxidation state of melanin, and the presence of copper ions (Korytowski and Sarna, 1990). Based on detection of characteristic products of salicylate hydroxylation (2,3- and 2,5-dihydroxybenzoic acids), the formation of hydroxyl radicals in photolyzed melanin samples has been demonstrated. It was suggested that redox active metal ions that are bound to melanin might be involved in the generation of hydroxyl radicals via Fenton-type processes (Korytowski et al., 1987). Indeed, using direct EPR measurements of copper(II) complexes with melanin, it has been shown that copper(I) bound to melanin was rapidly oxidized by either H2O2 or oxygen (Korytowski and Sarna, 1990). It has been concluded that photobleaching of melanin involves two distinct stages: reversible oxidation of the hydroquinone moieties of melanin followed by irreversible reactions of the monomers that lead to degradation of the melanin polymer. An interesting melanin-mediated photo-oxidation of ascorbate has been reported (Glickman and Lam, 1992; Glickman et al., 1993). It was demonstrated that melanin granules that were isolated from retinal pigment epithelium of bovine eyes induced nonenzymatic oxidation of ascorbate when illuminated with a continuous-wave argon-ion laser. The photooxidation of ascorbate was explained in terms of two processes — one that might be mediated by melanin-induced oxygen radicals and another that was due to interaction of ascorbate with the free radicals of melanin. However, in a more recent study, it has been demonstrated that melanin acts predominantly as an electron transfer agent in the photooxidation of ascorbate, while the ultimate electron acceptor is molecular oxygen (Rózanowska et al., 1997). The results of the latter study also suggested that the primary electron transfer between ascorbate and melanin involved photoinduced melanin radicals.

Antioxidant Properties The discovery of free radical properties of melanin led to a hypothesis that melanin might act as a free radical trap, thereby protecting cells from the effects of free radicals formed in biological oxidation–reduction reactions (Mason et al., 1960). A growing body of experimental evidence suggests that the cellular melanin may be an important antioxidant system (Bustamante et al., 1993; Ostrovsky et al., 1987; PorebskaBudny et al., 1992; Reszka et al., 1998; Scalia et al., 1990; Slawinska et al., 1983; Stepien et al., 2000; also reviewed by Sarna, 1992). As the term “antioxidant” is not always used very rigorously in biomedical literature, it may be useful to define it. Following the definition given by Halliwell and Gutteridge (1989), we will consider an antioxidant as “any substance that, when present at low concentration compared to those of an oxidizable substrate, significantly delays or inhibits oxidation of that substrate.” Thus, melanin may act as an antioxidant by: 1 scavenging initiating radicals; 2 deactivating electronically excited oxidizing species such as singlet molecular oxygen (1O2); 3 sequestering redox active metal ions such as iron and copper (metal ions that are bound to melanin may be less efficient in generating diffusible damaging free radicals and/or decomposing lipid peroxides to form propagating radicals); 4 chain breaking, i.e. scavenging intermediate radicals such as peroxyl and alkoxyl. There is a fairly extensive but amorphous literature on potential antioxidant properties of melanin. The following is aimed at being an indicative, rather than a comprehensive, review of this subject. The antioxidant properties of melanin have been examined by studies of the abilities of melanin to quench electronically excited dye molecules, scavenge reactive free radicals, and sequester redox active metal ions. Using laser flash photolysis and EPR oximetry as well as conventional absorption and fluorescence spectroscopy, it has been shown that binding of two cationic dye molecules, tetra(N-methyl-4-pyrridyl)porphyrin and tetra(4-N,N,N,Ntrimethyl-anilinium)porphyrin, was accompanied by a broadening of their absorption band, fluorescence quenching, triplet state quenching, and reduction in the dye-photosensitized oxygen consumption (Bielec et al., 1986). For anionic dyes such as tetra(4-sulfonatophenyl)porphyrin, there was no binding and, consequently, melanin had little or no effect on the absorption, fluorescence, and triplet states of the dyes and on their photosensitizing abilities (Fig. 16.10). Thus, complexing of positively charged dye molecules via ionic interaction can lead to a very efficient deactivation of their electronically excited states and, as a result, to complete loss of the dyephotosensitizing activity. If not quenched, the dye molecules, via type I or type II photosensitized oxidation reactions, could oxidize many substrate molecules and generate potentially cytotoxic species such as singlet molecular oxygen, superoxide anion, hydrogen peroxide, and hydroxyl radicals (Bensasson et al., 1993; Nonell, 1994). An interaction of melanin with potentially photosensitizing molecules was later confirmed for 331

CHAPTER 16 A 1.0 emission intensity (relative units)

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Fig. 16.10. The effects of complexation of a positively charged porphyrin dye by synthetic melanin on the optical properties of the dye (top) and its photosensitizing ability (bottom). (A) Optical absorption (a and c) and fluorescence emission (b and d) spectra of an anionic (a and b) and a cationic (c and d) porphyrin in the presence of cysteinyldopa melanin (broken lines) and its absence (solid lines). (B) Oxygen consumption rates photosensitized by the anionic porphyrin (open symbols) and cationic porphyrin (solid symbols) are plotted as a function of concentration of dopa melanin (a) and cysteinyldopa melanin (b). Reprinted from Bielec et al. (1986), with kind permission from the Royal Society of Chemistry, UK.

ground-state complexes with cationic porphyrins (Ito et al., 1992) and demonstrated for the singlet excited state of 8methoxypsoralen (Losi et al., 1993). The mechanism of the quenching of excited states of melanin bound to tetra(4N,N,N,N-trimethylanilinium)porphyrin was studied recently by femtosecond absorption and picosecond emission spectroscopies (Ye et al., 2003). It has been concluded that such a binding facilitates an ultrafast energy transfer from the excited 332

porphyrin molecule to melanin. The excited energy is then rapidly converted into heat. Because of its speed, the process involves only singlet excited states with no triplet state formation. Melanin can also be an efficient quencher of singlet oxygen (Sarna et al., 1985b; Sealy et al., 1984). The rate constants of the interaction of 1O2 (generated by a Rose Bengal photosensitized reaction) with synthetic DOPA and cysteinyldopa melanins and natural melanins have been determined under steady-state conditions, using EPR oximetry. The data indicate that chemical quenching of 1O2 by melanin is a rapid reaction, with the corresponding rate constants of 1.3 ¥ 105 mg/ml/s and 6 ¥ 105 mg/ml/s for DOPA melanin and cysteinyldopa melanin respectively. Natural melanins interacted significantly slower with 1O2. This is not unexpected, considering the particulate form of such melanins; as singlet oxygen was generated uniformly in the sample volume, the aggregated melanin had little chance to interact with most molecules of 1O2 before they deactivated via competing processes. Similar values for the apparent rate constants for the interaction of synthetic melanins with 1O2 have been obtained most recently by direct time-resolved detection of the singlet oxygen after pulse laserinduced generation of 1O2 (Wielgus et al., unpublished). Superoxide anion is another “reactive oxygen species” that may be involved, directly or indirectly, in damage of key cellular constituents (Fridovich, 1983). Superoxide is a product of one-electron reduction of molecular oxygen and can be generated by the interaction of oxygen with suitable electron donors with one-electron reduction potential that is more negative than –0.16 V (Koppenol and Butler, 1985). One of the first reports indicating that melanin can scavenge superoxide anion appeared some 20 years ago (Goodchild et al., 1984). Unfortunately, the experimental details of this study were very sparse. The superoxide scavenging ability of eumelanins was subsequently demonstrated by direct EPR measurements of the characteristic EPR signal of frozen alkaline solutions of H2O2 and NaIO4 in the absence and presence of increasing amounts of melanin (Geremia et al., 1984). This method has been refined by the addition of acetone to the reacting mixture (Sichel et al., 1991). It must be stressed, however, that the free radical scavenging properties of melanin were tested by this method under extreme conditions (pH 13), which are not directly relevant in biological systems. Using stopped-flow EPR measurements of the induced melanin free radical signal and dimethyl sulfoxide (DMSO)/KO2 as the source of superoxide anion, its interaction with DOPA melanin and melanin from bovine eye choroid has been observed, and the rate constant of the interaction has been calculated (Korytowski et al., 1986). The superoxide-induced melanin radicals decayed with an effective bimolecular rate constant of 0.8–5.1 ¥ 104 mg/ml/s. The apparent rate of the interaction of melanin with superoxide has been determined from the observed inhibition of the DMPO–O2H radical adduct formed in the superoxidegenerating system in the presence of superoxide dismutase (SOD) and/or melanin. The rate constant was calculated to be

THE PHYSICAL PROPERTIES OF MELANINS Table 16.1. Apparent second-order rate constants of interaction of melanin with singlet oxygen and free radicals mg/ml/s. Species

Melanin DM

O2(1Dg) O2•¯ • OH

1.3 ¥ 105*

•

0.018 x 104‡ 7.3 ¥ 104‡

OOCH2OH CH2OH CH3 •CHOH

•

3

(2.0†–3.3‡) ¥ 10 107‡

– aq

6.7 ¥ 104‡ 1.7 ¥ 106‡

N3•

104–105‡ 1.2 ¥ 106‡

e CO2•¯

DMT

CDM

BEM

RHM

6.0 ¥ 105*

0.06 ¥ 105*

0.3 ¥ 105*

3

3

10 †

10 † 0.7 ¥ 107‡ 1.3 ¥ 104‡ 1.1 ¥ 106‡ 6

0.4 ¥ 10 ‡

0.8 ¥ 106‡ 2.4 ¥ 106‡ 1.5 ¥ 106‡

*Data obtained by EPR oximetry under steady-state illumination in the presence of Rose Bengal as the sensitizer. †Data obtained by direct EPR measurements and EPR spin trapping using DMSO/KO2 as the source of O2. ‡Data obtained by pulse radiolysis. DM, auto-oxidative DOPA melanin; DMT, DOPA melanin synthesized in the presence of tyrosinase; CDM, cysteinyldopa melanin; BEM, melanin from bovine eye choroid; RHM, red hair melanin.

4 ¥ 105/M/s, assuming a molecular weight of 200 for the DOPA melanin monomer unit. A similar value for the rate constant of DOPA melanin interaction with superoxide anion in fully aqueous media has been obtained by independent measurements using the pulse radiolysis method (6.5 ¥ 105/M/s) (Sarna et al., 1986). This powerful method has also been used for the determination of rate constants of the interaction of several different melanins with radicals from water radiolysis (Table 16.1). In addition, it has been shown that superoxide anion interacts with DOPA melanin predominantly by reducing it. Perhaps the most comprehensive study to date of the interaction of DOPA and cysteinyldopa melanins with a variety of oxidizing and reducing radicals has been published by Rózanowska et al. (1999). The authors, using pulse radiolysis to generate specifically selected free radicals, observed either directly or indirectly their interaction with both synthetic melanins. The results showed that the efficiency of the interaction depended, in a complex way, on the redox potential, the electric charge, and the lifetime of the radicals. Repetitive pulsing experiments, in which the free radicals, probing the melanin redox sites, were generated from four different viologens, indicated that the eumelanin model had more reduced than oxidized groups accessible to reaction with the radicals. Oxidation of DOPA melanin by oxidizing radicals appeared to be easier than that of cysteinyldopa melanin. Although with many radicals studied, melanin interacted via a simple one-electron process, the reaction of both melanins with the strongly oxidizing peroxyl radical from carbon tetrachloride involved radical addition. A very efficient scavenging by cysteinyldopa melanin of peroxyl radicals generated from hydroxyethyl and hydroxymethyl radicals with the corresponding rate constant 2.6 ¥ 106/M/s (3.5 ¥ 104/M/s for DOPA melanin) suggests that

melanin in vivo might be able to participate in chain breaking by scavenging intermediate radicals that are involved in the propagation of the peroxidation process (Dunford et al., 1995). Both synthetic melanins were good scavengers of carbon-centered radicals with corresponding rate constants in the range 107–108/M/s. The results of the reviewed investigations are consistent with melanin being an efficient scavenger of strongly oxidizing (and reducing) free radicals and a quencher of singlet molecular oxygen. It is rather unlikely, however, that these properties of melanin play a key role in the antioxidant action of the pigment. Unless “site-specific” formation of reactive free radicals or singlet oxygen is considered, natural melanin is a very inefficient scavenger and quencher of such short-lived species. This is because of the limited lifetime of randomly generated reactive species. These species would interact with many constituents of the pigmented cell before having a chance to diffuse to the proximity of the melanosomes (or pigment granules), where they would then need to penetrate the melanosome (pigment granule) surface and interact with the active groups in the polymer. A special case, in which the free radical scavenging and singlet oxygen quenching abilities of melanin might be of importance, is “site-specific” formation of such species, i.e. if the generation of reactive, short-lived species is predominantly within the melanin granule (melanosome) or in its proximity. This could happen if copper, iron, or manganese ions bound to melanin were redox activated and exposed to oxygen or H2O2, or when photosensitizing molecules, associated with the melanin granule (melanosome), were activated by light in the presence of oxygen. Under such circumstances, melanin would act as a very powerful antioxidant, and very little reactive species would escape the scavenging and quenching action of the melanin. 333

CHAPTER 16

Perspectives The complexities of melanins, due to their physical–chemical properties and inherent heterogeneity, make it difficult, at least at this time, to reach very specific conclusions as to the properties and structure of melanins. In spite of these limitations, some very useful generalizations can be drawn. It is necessary, however, to use a variety of different methods to try to characterize melanins and to recognize that, inevitably, the conclusions reached by the use of a single method need to be confirmed by other, independent methods. The physical state and experimental conditions of melanins can have profound effects on the apparent properties of the melanin. The most important variables of this type include the state of hydration of the molecule (it might be argued suc334

A 100

DMPO-OH (%)

80

60

40

20

0 0.0

0.2

0.4

0.6

0.8

1.0

DA-melanin (mg/ml) B

Rate of DMPO-OH formation (arb. units)

Melanin principally exerts its antioxidant activity by binding redox active metal ions. This has been demonstrated in model studies with DOPA melanin (Pilas et al., 1988) and dopamine melanin (Zareba et al., 1995). The data indicate that iron ions that are bound to melanin are inefficient catalysts for H2O2-dependent generation of free hydroxyl radicals (Fig. 16.11). Even though melanin complexes with ferrous ions are readily oxidized by H2O2 and oxygen, very few OH radicals leak out of the melanin polymer, as shown by EPR spin trapping and HPLC electrochemical detection of salicylate hydroxylation products. Ferric ions, on the other hand, after binding to melanin, become significantly more difficult to reduce by mild reductants. Sequestration of iron ions has been identified as a major mechanism for the inhibitory effects of melanin on lipid peroxidation (Korytowski et al., 1995), and an important role of the degree of iron binding to melanin on the antioxidant/pro-oxidant actions of melanin in protection against chemically and photochemically induced peroxidation of lipids has been considered in an independent study (Krol and Liebler, 1998). Melanin may lose part of its antioxidant activity, or even become pro-oxidant, when its metal ion-binding capacity is exceeded, or if redox active metal ions, present in melanotic systems, are bound to strong chelators (such as EDTA). The basal rate of lipid peroxidation induced by iron, which was enhanced by dopamine melanin (Ben-Shachar et al., 1991), may be explained by the inability of the melanin to complex all ferric ions. Under the conditions employed by the authors of the cited paper, it seems that the dopamine melanin was saturated with iron ions. Similar phenomena are probably involved in a recently reported pro-oxidant action of DOPA melanin in lipid peroxidation (Sotomatsu et al., 1994). An enhanced formation of hydroxyl radicals, observed in model systems containing ferric ions complexed with EDTA, induced by DOPA melanin (Pilas et al., 1988) and dopamine melanin (Zareba et al., 1995), can be explained by the reducing power of melanin, which is able to reduce and thereby activate ferric ions chelated by EDTA and, hence, drive a Fenton reaction (Fig. 16.11).

6

4

2

0 0.0

0.2

0.4

0.6

0.8

1.0

Melanin (mg/ml) Fig. 16.11. The effects of synthetic dopamine melanin on the generation of free hydroxyl radicals by a Fenton system. (A) shows the inhibitory effect of melanin when ferrous ions were complexed by a weak chelator such as citrate (open triangles); on the other hand, melanin exhibited a negligible effect if Fe(II) was chelated by a strong chelator such as DTPA (solid triangles). (B) illustrates the activating role of melanin, when ferric ions were present in the form of complexes with DTPA (solid circles) and with citrate (open circles). The inhibitory effect of melanin is expressed as normalized maximum levels of the DMPO–◊OH spin adduct formed (A) or its rate of formation (B). Reprinted from Zareba et al. (1995), with kind permission of Elsevier Science-NL.

cessfully that studies on dried melanins will usually have limited validity), the pH, and the present and past history of redox conditions for the sample. Another important consideration is the method of purification of the melanin. It is now recognized that treatment of melanin with strong acids or

THE PHYSICAL PROPERTIES OF MELANINS

alkali may irreversibly modify the physicochemical properties of the melanin polymer. Different melanins can have very different properties. The most important variables are the nature of the monomeric units, especially whether these include cysteine-containing subunits. As a consequence, there are at least three quite different types of naturally occurring melanins: eumelanin, pheomelanin, and neuromelanin. The biological effects and/or roles of melanin are significantly affected by interactions with metal ions as a result of the binding of metal ions by the melanin; the interactions affect the properties of both the metal ions and the melanin. Even though melanin can bind a variety of different types of molecules (with different forces being involved), the most common and important is binding of metal ions and organic cations via electrostatic interaction. In vivo it is likely that some melanins, perhaps most, have proteins associated with them and that these affect the properties of the melanin. The proteins probably include those associated with the synthesis of melanin (tyrosine and related enzymes, except for neuromelanin) and proteins derived from the immediate environment, especially from the melanosomes. The redox character of melanin is very complex and is greatly affected by the nature of the melanin and its environment. In order to understand the extent and even the direction of redox reactions that will occur, it is essential to specify the conditions, including the type of melanin, metal ions that are present, proteins associated with the melanin, the redox state of the melanin, pH, and the presence of other redox active species such as molecular oxygen. The ability of melanin to quench electronically excited states of certain photosensitizing dye molecules, scavenge reactive free radicals, and sequester redox active metal ions makes the pigment an efficient antioxidant. The occurrence of lipids with melanins is very incompletely understood. Melanins are unique biopolymers with persistent free radicals and a distinct EPR signal. Many of the EPR characteristics and the associated free radical and redox reactions occurring in the presence of melanins can be explained on the basis of a dynamic redox equilibrium involving quinones, hydroquinones, and semiquinones in the monomeric units of the melanin. The optical properties of melanin are very incompletely understood in spite of the extensive and often productive studies that have been carried out. In particular, we do not yet have a satisfactory and full explanation for the absorption spectrum of melanins and the biological significance of the photochemistry associated with melanins. Irradiation of melanin with ultraviolet or visible light generates transient free radicals and enhances redox reactivity of the electron exchange groups of melanin. Although intriguing, there is at present no fully satisfactory theory or evidence for melanin to have amorphous semiconducting and related properties. Much is known about melanin through the use of sophisticated physical and chemical approaches, and it is likely that continued progress will be made, eventually resulting in a thorough understanding of this important class of biopolymers.

Acknowledgments The work was supported in part by NIH (grant ROIEY 13722) and by the State Committee for Scientific Research.

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Fujimoto, and T. J. Flotte. Comparative studies of femtosecond to microsecond laser pulses on selective cell injury in skin. Photochem. Photobiol. 53:757–762, 1991. Wielgus, A., J. Bielec, and T. Sarna. Direct time-resolved measurements of quenching of singlet oxygen by synthetic melanins. Unpublished. Wilczok, T., B. Bilinska, E. Buszman, and M. Kopera. Spectroscopic studies of chemically modified synthetic melanins. Arch. Biochem. Biophys. 231:257–262, 1984. Wolf, K. Melanocyte-keratinocyte interactions in vivo. The fate of melanosomes. Yale J. Biol. Med. 46:384–396, 1973. Wolfram, L. J., and L. Albrecht. Chemical and photo-bleaching of brown and red hair. J. Soc. Cosmet. Chem. 82:179–191, 1987. Wróbel, D., A. Planner, I. Hanyz, A. Wielgus, and T. Sarna. Melanin–porphyrin interaction monitored by delayed luminescence and photoacoustics. J. Photochem. Photobiol. B Biol. 41:45–52, 1997. Ye, T., and J. D. Simon. Ultrafast spectroscopic study of pheomelanin: implications on the mechanism of superoxide anion formation. J. Phys. Chem. Biol. 106:6133–6135, 2002. Ye, T., and J. D. Simon. Comparison of the ultrafast absorption dynamics of eumelanin and pheomelanin. J. Phys. Chem. B 107:11240–11244, 2003. Ye, T., T. Sarna, and J. D. Simon. Ultrafast energy transfer from bound tetra(4-N,N,N,N-trimethylanilinium)porphyrin to synthetic dopa and cysteinyldopa melanins. Photochem. Photobiol. 77:1–4, 2003. Zajac, G. W., J. M. Gallas, J. Cheng, M. Eisner, S. C. Moss, and A. E. Alvarado-Swaisgood. The fundamental unit of synthetic melanin: a verification by tunneling microscopy of X-ray scattering results. Biochim. Biophys. Acta 1199:271–278, 1994. Zareba, M., A. Bober, W. Korytowski, L. Zecca, and T. Sarna. The effect of a synthetic neuromelanin on yield of free hydroxyl radicals generated in model systems. Biochim. Biophys. Acta 1271:343–348, 1995. Zareba, M., M. W. Raciti, M. M. Henry, T. Sarna, and J. M. Burke. Oxidative stress in ARPE-19 cultures: Do melanosomes offer cytoprotection? Free Rad. Biol. Med., 2005, in press. Zecca, L., and H. M. Swartz. Total and paramagnetic metals in human substantia nigra and its neuromelanin. J. Neural Transm. Park. Dis. Dement. Sect. 5:203–213, 1993. Zecca, L., C. Mecacci, R. Seraglia, and E. Parati. The chemical characterization of melanin contained in substantia nigra of human brain. Biochim. Biophys. Acta 1138:6–10, 1992. Zecca, L., R. Pietra, C. Goj, C. Mecacci, D. Radice, and E. Sabbioni. Iron and other metal in neuromelanin; substantia nigra and putamen of human brain. J. Neurochem. 62:1097–1101, 1994. Zeise, L., and M. R. Chedekel. Melanin standard method: titrimetric analysis. Pigment Cell Res. 5:230–239, 1992. Zeise, L., B. L. Murr, and M. R. Chedekel. Melanin standard method. Particle description. Pigment Cell Res. 5:132–142, 1992.

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17

Photobiology of Melanins Antony R. Young

Summary Exposure to ultraviolet radiation (UVR) results in long-term deleterious effects such as skin cancer. A well-recognized short-term consequence of UVR is increased skin pigmentation. Skin color is one of most conspicuous ways in which humans vary, yet many aspects regarding the function of melanin remain controversial. Pigmentation, whether constitutive or facultative, has been widely viewed as photoprotective, largely because darkly pigmented skin is at a lower risk of photocarcinogenesis than fair skin. Research is increasingly suggesting that the relationship between pigmentation and photoprotection may be far more complex than previously assumed. For example, photoprotection against erythema and DNA damage has been shown to be independent of the level of induced pigmentation in human white skin types. Growing evidence now suggests that UVR-induced DNA photodamage, and its repair, is one of the signals that stimulates melanogenesis. These findings suggest that tanning may be a measure of inducible DNA repair capacity, and it is this rather than pigment per se that results in the lower incidence of skin cancer observed in darker skinned individuals. This is supported by some studies that suggest that repeated UVR exposure in skin types IV, who tan well, results in faster DNA repair in comparison with skin types II, who tan poorly. It has been suggested that epidermal pigmentation may in fact be the mammalian equivalent of a bacterial SOS response.

Introduction The color of human skin is largely determined by its epidermal melanin content, whether this melanin is constitutively expressed or induced by UVR from the sun or a tanning device. Melanin production, or melanogenesis, occurs in highly specialized dendritic melanocytes that account for about 1% of epidermal cells. Each basal layer melanocyte is associated with about 36 keratinocytes and one Langerhans cell, and this is known as the epidermal melanin unit (EMU) (Fitzpatrick and Breathnach, 1963). Melanogenesis is described in detail in Chapter 14. The composition of melanin, the end-product of melanogenesis that is transferred to adjacent keratinocytes, is still incompletely understood but is a variable mixture of lighter/reddish/yellowish alkali-soluble, sulfur-containing pheomelanin and darker brownish/blackish insoluble eumelanin. In both cases, the rate-limiting step is the oxidation of tyrosine by tyrosinase in a series of reactions known as the Raper–Mason pathway. Eumelanogenesis leads 342

to the formation of indole derivatives that include 5,6dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), formed from the oxidation of the 1,4 addition product of tyrosinase-generated dopaquinone. Intermediates in pheomelanogenesis include cysteinyldopas. Indole and cysteinyldopa precursors and their related o-methyl derivatives are released into the epidermis during periods of melanogenic activity induced by UVR or psoralen plus UVA (PUVA). Their lipophilic nature suggests a long half-life in lipophilic dermal and epidermal tissue. Skin color is a consequence of the mix of melanin types and possibly also the way that melanin is packaged in melanosomes, highly specialized melanocyte-derived organelles that are used for the transfer of melanin to keratinocytes via the melanocyte dendrites. Caucasian melanosomes have a long axis of about 400 nm and tend to occur in groups of 3–8, whereas Negroid melanosomes are much longer (800 nm length) and exist singly (Kollias et al., 1991). Melanocyte density is independent of race (Halaban et al., 2003) but varies with body site, with densities ranging from 2000/mm2 in the head or forearm to 1000/mm2 elsewhere. Recent studies indicate that the distribution of melanosomes within keratinocytes (Thong et al., 2003) and melanosome size (Alaluf et al., 2003) play a role in skin color. Melanocyte numbers in non-sunexposed skin show an age-related decline with an approximately 8–10% reduction per decade (Halaban et al., 2003). There is considerable intercellular chemical communication between melanocytes and keratinocytes and, to lesser extent, fibroblasts, neurons, mast cells, and other skin cells. Keratinocytes produce a wide range of mitogenic [e.g. basic fibroblast growth factor (bFGF), transforming growth factor (TGF)a] and inhibitory [e.g. interleukin (IL)1, IL6, TGFb] factors for melanocytes. In addition, the proliferation of melanocytes, melanogenesis, and the transfer of pigment also rely on hormonal controls [a-melanocyte-stimulating hormone (MSH), sex hormones), agouti signal protein, and inflammatory mediators in skin (Chu et al., 2003; Gilchrest et al., 1996). The melanocyte plasma membrane is also thought to be a primary UVR target with the release of membrane-bound factors, such as arachidonic acid and diacylglycerol (DAG), leading to activation of tyrosinase via a protein kinase C (PKC)-mediated pathway (Gilchrest et al., 1996). The behavior of isolated melanocytes in vitro is different from their behavior as part of an EMU. For example, recent studies have shown that the pheomelanin/eumelanin ratio is regulated by keratinocytes (Duval et al., 2002). However, it has also been reported that melanosomes from different skin types maintain their melanin type preferences in

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melanocytes stimulated with tyrosine in vitro (van Nieuwpoort et al., 2004).

The Effects of Solar UVR on Human Skin The detrimental effects of solar UVR (~295–400 nm) on the skin are well established and are usually categorized as either acute or chronic. Acute effects include DNA (Bykov et al., 1999) and oxidative damage (Sander et al., 2004), mutation (Ziegler et al., 1994), immunosuppression (Ullrich, 2000), and erythema (sunburn) (Harrison and Young, 2002). Molecular biology is increasingly showing that UVR induces the expression of a large number of genes, the physiological consequence of which is poorly understood. Tanning is also an acute effect, and it is debatable whether this is a detrimental or beneficial effect. The chronic effects include skin cancers, which are thought to be a consequence of mutation and immunosuppression (Ullrich, 2002; Ziegler et al., 1994), and photoaging, which is thought to be a consequence of the induction of matrix metalloproteinases (MMPs) (Fisher et al., 2002). Skin darkening in response to solar UVR occurs via two distinct mechanisms: immediate pigment darkening (IPD) and delayed tanning (DT). Both processes are influenced by genetic factors and are more pronounced with darker constitutive pigmentation. The action spectrum for IPD shows a broad peak in the UVA region (Irwin et al., 1993) and is completely different from the action spectrum for DT (Parrish et al., 1982), which indicates that they are mechanistically different processes. There is a range of non-invasive optical techniques to measure skin pigmentation in vivo, but all have disadvantages of various types (Stamatas et al., 2004) and none is in routine use. IPD starts during UV irradiation as a grayish coloration that gradually fades to a brown color over a period of minutes to days depending on UVR dose and individual complexion. These changes are not due to new melanin synthesis but rather oxidation of pre-existing melanin and redistribution of melanosomes from a perinuclear to a peripheral dendritic location (Routaboul et al., 1999). The color change may be so subtle as to be almost undetectable in fair-skinned individuals but is easily observed in skin types IV (or darker). The transient nature of IPD has made understanding of this phenomenon difficult. In vivo reflectance spectroscopy showed a UVA (365 nm) dose-dependent induction of IPD with increased absorbance between 620 and 720 nm (Rosen et al., 1990), similar to that expected from increased native melanin. However, at shorter wavelengths (410–610 nm), the absorbance is less than expected from increased native melanin. Significantly, no photoprotective effect for IPD has been established; hence, its biological function remains unknown. DT, which results from melanogenesis, is associated with increased melanocyte activity and proliferation. It is evident

3–4 days after UVR exposure and is maximal from 10 days to 3–4 weeks depending on complexion and UVR dose. It may take several weeks for the skin to return to its base constitutive color. UVA-induced DT is two or three orders of magnitude less efficient per unit dose than UVB and has an earlier onset, often directly after IPD. Furthermore, it has a different pathophysiology (Eller and Gilchrest, 2000; Lavker and Kaidbey, 1982) that, unlike UVB, is oxygen dependent. The only widely recognized beneficial effect of solar UVR exposure is epidermal vitamin D photosynthesis. Sunlight is believed to be the body’s major source of vitamin D as few people consume enough food that is naturally rich in vitamin D to meet their dietary requirements. Solar UVB (295–310 nm) converts 7-dehydrocholesterol in the skin to vitamin D3 (cholecalciferol), a prohormone with no intrinsic biological activity. Vitamin D3 is metabolized in the liver to 25-hydroxyvitamin D (25(OH)D). Plasma levels of this metabolite are the hallmark for determining vitamin D status as it is used as the substrate for producing the biologically active steroid hormone 1,25-dihydroxyvitamin D. In temperate regions, there is insufficient UVB in the winter months to synthesize vitamin D, and therefore there is a seasonal variation in plasma 25-(OH)D concentrations, with winter levels dependent upon the amount of vitamin D stored in adipose tissue during the previous summer (Devgun et al., 1981; Vieth et al., 2001; Webb et al., 1988). Maintenance of optimal levels of 25(OH)D are essential for bone health, but some epidemiological and intervention trials suggest that vitamin D deficiency may increase the risk of some internal malignancies and autoimmune disorders (Zittermann, 2003).

Skin Chromophores and Their Relationship to Photobiology The UVR-absorbing properties of skin depend on its natural chromophores, which include DNA, amino acids, urocanic acid, and melanins and their precursors (Young, 1997). Absorption of UVR energy by chromophores may initiate photochemical events that are the basis of all skin photobiology. Chromophores, especially those in the upper epidermis, may also attenuate UVR and thus protect the structure beneath from photodamage. For example, nonsolar UVC radiation (100–280 nm) causes very little damage to DNA in cells of the basal layer because of its attenuation by DNA in the cells above and urocanic acid in the stratum corneum (Chadwick et al., 1995). In contrast, solar-range UVB and UVA radiation readily results in damage to DNA of keratinocytes and melanocytes in the basal layer of human skin in vivo (Young et al., 1998a). The emission spectrum of the sun is rich in UVA (315–400 nm) radiation with UVB (280–315 nm) radiation accounting for less than 5% of total UVR content under most conditions. However, because most skin chromophores are primarily UVB absorbers, it is that part of the solar spectrum 343

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that causes most of the biological effects described above. For example, action spectrum (wavelength dependence) studies have shown that UVB is three to four orders of magnitude more effective per unit physical dose (J/cm2) than UVA for DNA photodamage (Young et al., 1998b), erythema (Parrish et al., 1982; Young et al., 1998b), tanning (Parrish et al., 1982), and skin cancer in mice (de Gruijl, 1995). However, UVA may be more important for indirect damage to cells caused by oxidative stress. This is caused by reactive oxygen species (ROS) that are generated when UVA (and also UVB) (Sander et al., 2004) is absorbed by as yet poorly defined chromophores that may include flavins and porphyrins. The chromophore(s) for melanogenesis have not been established with certainty, but there is an increasing body of indirect and direct evidence that supports a major role for DNA (Eller and Gilchrest, 2000). Human studies have shown that the action spectra for DNA photodamage, as assessed by cyclobutane pyrimidine dimers (CPD), and tanning are very similar (Parrish et al., 1982; Young et al., 1998b) as shown in Figure 17.1. This suggests that the photochemical event that initiates tanning may be a consequence of DNA photodamage. Figure 17.2 summarizes some of the photobiological events that initiate melanogenesis.

1.0

Skin Type and Melanin The skin’s constitutive melanin content and its melanogenic response to solar UVR form part of the basis of the skin type clinical classification shown in Table 17.1. This scheme was originally devised to optimize UVR doses in phototherapy. Sensitivity to sunburn is routinely evaluated by minimal erythema dose (MED) determination, the MED being the lowest dose (J/cm2) of UVR that will cause erythema assessed at 24 h. In general, tanning capacity is inversely related to MED, but there is a considerable degree of MED overlap within white skin types I–IV (Harrison and Young, 2002). In other words, MED is not a reliable index for predicting skin type. Investigators have determined the quantitative and qualitative melanin content in human hair and skin in vivo and, in some cases, after skin exposure to UVR (for a review, see Ito and Wakamatsu, 2003). The first study of epidermis in vivo was by Thody et al. (1991) in skin types I, II, and III, in which there was a positive correlation between skin type and eumelanin but not pheomelanin. However, the ratio of eumelanin/pheomelanin shows considerable variation, especially in skin types II and III. The level of tanning by PUVA

Upper Epidermis TT Basal Layer TT

Log10 Relative Effect

0.0

MMD

–1.0

–2.0

–3.0 270

290

310

330

350

Membrane Damage

370

CPD

Wavelength (nm) Fig. 17.1. Human action spectra for epidermal DNA photodamage (TT = thymine dimers; data from Young et al., 1998b) and tanning expressed as minimal melanogenic dose (MMD) assessed at 7–14 days (data from Parrish et al., 1982). The TT action spectra show epidermal layer dependence at wavelengths less than 300 nm, probably because of marked UVR attenuation by epidermal chromophores at these wavelengths. These TT and MMD spectra are very similar, which suggests that DNA is an important chromophore for melanogenesis and in particular DNA from the upper epidermis. This suggests the possibility that damage to keratinocytes, with the release of melanogenic factors (see Fig. 17.2), may be more important than direct damage to melanocytes that reside in the basal layer region.

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Keratinocyte Factors

DNA Repair

Fig. 17.2. Multiple photobiological events initiate melanin synthesis. These include DNA photodamage (CPD) and its repair, membrane damage, and factors from other epidermal cells, especially keratinocytes.

PHOTOBIOLOGY OF MELANINS Table 17.1. Classification of human skin types with respect to relative response to acute and long-term solar exposure. Skin type

Susceptibility to sunburn

Constitutive skin color

Facultative tanning ability

Susceptibility to skin cancer

I II III IV V VI

High High Moderate Low Very low Very low

White White White Olive Brown Black

Very poor Poor Good Very good Very good Very good

High High Moderate Low Very low Very low

was correlated with eumelanin but not pheomelanin. Alaluf et al. (2001) evaluated melanin content in sun-protected and -exposed sites of skin types V and IV. In comparison with DHICA and DHI eumelanins, pheomelanin content was very low. Solar exposure enhanced eumelanin content, in particular predominant DHI eumelanin, but had no effect on pheomelanin levels. In further studies, these authors (Alaluf et al., 2002) assessed epidermal melanin content in five different ethnic groups using alkali solubility as a means of separating the lighter pheomelanin and DHICA eumelanin from the darker DHI eumelanin on sun-protected and -exposed skins. The difference in total melanin content between the lightest European skin and the darkest African skins was only about twofold in both skin sites, which is consistent with previously reported skin type-dependent differences in tyrosinase activity (Iwata et al., 1990; Pomerantz and Ances, 1975). The data also showed that darker skin was associated with greater quantities of alkali-insoluble melanins, and lighter skins were associated with greater quantities of alkali-soluble melanins. The level of melanin, however assessed, was always higher (P < 0.01) in all study populations on sun-exposed sites by factors ranging from 1.3 to 1.9, with the differences in alkalisoluble melanin (1.3–1.5) being smaller than those of alkaliinsoluble melanin (1.5–1.9). The 1.8-fold difference in total melanin in sun-exposed and -protected African skin is about the same as the differences in African and European skin on their respective sun-protected and -exposed sites. The darker skins have the lowest values when the data are expressed as % alkali-soluble melanin, and this is reduced slightly (1.2–1.3) by sun exposure in African and Indian skins only. Overall, these data support a role for eumelanin in sun tolerance as indicated by skin type. However, they also show that skin type differences in skin color and the degree of photoprotection are unlikely to be accounted for by differences in melanin alone, and Alaluf et al. (2002) have suggested that this may also be related to melanosome size.

Photoprotection by Melanin The primary function of skin melanin has not yet been established. A number of roles have been proposed that include photoprotection, thermoregulation, antibiotic, cation chelator,

free radical sink, and by-product of the scavenging of the superoxide radical in the skin by tyrosinase (Giacomoni, 1995; Hill and Hill, 2000; Morison, 1985). It is often stated that melanin is photoprotective because people with constitutively pigmented skins [e.g. types V (brown) or VI (black)] or skin that tans well (e.g. Mediterranean type IV) have much lower incidences of skin cancer that white-skinned people who tan poorly if at all (e.g. types I and II). Indeed, epidemiological studies confirm an inverse correlation between skin cancer incidence and pigmentation with age-adjusted male cancer incidence at 3.4 per 100 000 for blacks and 232.6 per 100 000 for Caucasian whites in the USA (Scotto and Fraummeni, 1982). At its simplest, this argument assumes that melanin or melanogenesis is the only factor that determines the level of a given photobiological response, e.g. skin cancer, to a given physical dose of UVR. However, there is clear evidence that this is not the case. For example, skin types I/II are more readily immunosuppressed than skin types III/IV when compared on the basis of physical or erythemal dose (Kelly et al., 2000). These studies showed that suberythemal doses of solar simulated radiation (SSR) suppressed the sun-sensitive skin types I/II but erythemal doses were necessary to suppress the sun-tolerant skin types III/IV. Furthermore, there is evidence for skin type-dependent differences in responses to oxidative stress (Kerb et al., 1997), which is thought to play a role in cancer (Sander et al., 2004). A photoprotective role for melanin, particularly against skin cancer, was first proposed by Home (1820). The Darwinian argument to support this hypothesis would require skin cancer to interfere with reproduction and child-rearing. Blum (1961) dismissed this argument and noted that nonmelanoma skin cancers generally occur after the reproductive age and are rarely fatal. Malignant melanomas are more fatal but account for only 4% of skin cancers and also usually occur after reproduction. Studies in Nigerian albinos show that, even in an extreme solar environment, skin cancer did not result in death below the age of 25 years (Okoro, 1975). In 2000, a landmark study examined the relationship between UVR levels, skin reflectance (a marker of melanin), vitamin D, and folate levels at different latitudes (Jablonski and Chaplin, 2000). Predicted skin reflectance based on UVR levels was noted to correlate closely, and a strong correlation between skin reflectance, absolute latitude, and UVR was

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demonstrated. It was concluded that the gradient between UVR and constitutive pigmentation was a compromise between the beneficial effect of vitamin D photosynthesis and the deleterious effect of folate photolysis that would interfere with reproduction. In reality, little is in fact known about the relationship between solar UVR and vitamin D status. Population surveys have shown that vitamin D deficiency is more common among institutionalized, elderly individuals with pigmented skin and those who habitually cover the skin with clothing (BischoffFerrari et al., 2004; Devgun et al., 1981; Vieth et al., 2001). Suberythemal UVB is a potent stimulus of vitamin D synthesis in white-skinned subjects in vivo, who show a ninefold increase in circulating vitamin D3 after a single whole-body exposure of about 0.75 MED (Matsuoka et al., 1989, 1990, 1991). Skin pigmentation is believed to greatly reduce the UVR-mediated synthesis of vitamin D3, with black subjects (n = 1) requiring at least a sixfold greater UVR dose to increase circulating levels of vitamin D3 than whites (n = 2) (Clemens et al., 1982). This finding, based on a very small study, was not confirmed in a later study. Matsuoka and colleagues (1991) found a significant association between skin color and vitamin D3 synthesis in different ethnic groups (n = 8) with white > Oriental > Asian > black, but reported only a twofold difference in vitamin D3 synthesis between white and black skin. The same skin type trend was seen for serum 25(OH)D levels, but it did not reach significance and no skin type effect was seen for serum 1,25 dihydroxyvitamin D. It should be noted that the single UVR challenge dose used in this study could readily be achieved after 10-min exposure to noonday UK summer sunlight. Therefore, it is unlikely that differences in cutaneous vitamin D3 synthesis would result in vitamin D deficiency in pigmented individuals. This conclusion is supported by Stamp et al. (1975), who showed that increases in serum 25(OH)D were similar in white (n = 4), Asian (n = 2), and black subjects (n = 1) after three daily exposures to the same physical UVR dose, and by Brazerol et al. (1988), who also found similar increases in 25(OH)D in white (n = 13) and black (n = 7) subjects exposed to suberythemal whole-body UVR twice a week for 6 weeks. It is possible that a higher incidence of vitamin D deficiency in subjects with pigmented skin results from other factors such as behavior or diet. For example, in the UK, low vitamin D status is relatively more common among Asians, especially children, adolescents, and women. A combination of factors, including the type of vegetarian diet, low calcium intake, and limited solar exposure, appears to underlie the risk (Hamson et al., 2003; Stamp, 1975). Some studies have attempted to determine the photoprotective properties of melanin against specific endpoints in human skin in vivo, and these are reviewed below. It must also be noted that melanogenesis and stratum corneum thickening occur concurrently during the normal tanning response, and this must be borne in mind when considering photoprotection by melanin alone. Although photoprotection may be considered to be a passive physical process, e.g. the attenuation of 346

UVR by melanin and/or stratum corneum thickening, as is the case with conventional sunscreens, it may also be considered as an active enzymatic process, e.g. as a means by which DNA repair is enhanced or ROS are inactivated. For example, chimeric epidermal reconstructs with melanocytes from one skin type added to keratinocyte cultures of a different skin type suggest keratinocyte/melanocyte interaction with both cell types regulating antioxidant defense in a skin typedependent way (Bessou-Touya et al., 1998).

Optical Properties of Melanin and Associated Molecules The absorption spectra of monocysteinyldopas (5-S-cysteinyldopa, 2-S-cysteinyldopa, 6-S-cysteinyldopa) show a maximum at 292 nm, whereas indoles, their methyl derivatives, and 2,5S-S¢-dicysteinyldopa show maxima at 302–330 nm (Kollias et al., 1991). Diffuse spectroscopy has been used to calculate melanin absorption in vivo by comparing normal with melanin-depleted skin in the same vitiligo patient. This approach reveals a maximum at 335 nm, with a steep decline at shorter wavelengths. Absorption is also noted within the visible range (400–800 nm). Differences in pigmentation induced by various bands of UVR (UVB, UVA, and PUVA) have also been analyzed using in vivo reflectance spectroscopy by comparing the results of tanned and untanned skin in the same individuals (Kollias et al., 1994). UVB- and PUVAinduced melanin showed an absorption peak at 305 nm whereas UVA resulted in a 360-nm-centered loss in UVR absorption with a concomitant relative increase in the absorption of visible light. These data suggest that different stimuli induce different pathways of melanogenesis. Interestingly, derivatives in the eumelanogenesis pathway such as DHI and DHICA also showed significant absorption in the solar UVR range (the latter in particular showing strong UVA absorption), and hence could possibly be classified as photoprotective. The particulate nature of melanin results in scattering as well as absorption of UVR. Studies on the cuttle fish melanin particles (size range 20–300 nm) found that the reported level of scatter in the wavelengths 580–633 nm did in fact correspond to the levels predicted (Vitkin et al., 1994). Melanosomes measuring ≥ 300-nm diameter mainly cause forward scatter of UVR in contrast to the smaller particles such as melanin dust found in keratinocytes (< 30 nm), which display symmetrical scattering profiles (Chedekel et al., 1995).

Erythema In the field, Cripps (1981) assessed the protection factor afforded by a Wisconsin (~45∞N) summer tan (obtained over 3.5 months) in skin types II, III, and IV by comparing the SSR MED on tanned and untanned buttock skin. Protection factors were 2.4 ± 0.65 (SD), 2.45 ± 0.5, and 2.1 ± 0.22, respectively, with a mean of 2.33 ± 0.05. Although not stated, it is presumed that tanning was according to skin type, and it seems that better tans did not afford any better protection against erythema. In laboratory studies, Sheehan et al. (1998)

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induced tanning in skin types II and III with repeated suberythemal exposure to SSR. Photoprotection was evaluated by assessing the MED on untanned and tanned sites, included tanned sites that had had the stratum corneum removed by tape stripping. As expected, the tanning response in skin type III was greater than that in skin type II, but there was no significant difference in the level of photoprotection with a protection factor of about 2 in both skin types. Thus, the degree of photoprotection could not be correlated with the level of the tan. Removal of the stratum corneum generally reduced the level of photoprotection by about 20%, which suggested that the stratum corneum was relatively unimportant in the tanning response. In a comparable later study but without the tape-stripping component, Sheehan et al. (2002) used a similar protocol in skin types II and IV. Despite the difference in the tanning response, the protection factor against erythema, assessed by MED determination, was again of the order of 2 with no difference between the skin types. Overall, the data of Sheehan et al. (1998, 2002) in skin types II, III, and IV show that the degree of photoprotection against erythema was very similar but could not be correlated with the level of tan, as was presumed to be the case in the field study by Cripps (1981). This suggests that other, as yet undefined, inducible forms of photoprotection may be in operation and that the level of melanin may be of less importance. Ha et al. (2003) used reflectance spectroscopy to assess the relationship between acute UVB-induced erythema and constitutive pigmentation. Regression analyses for a given UVB dose (119–300 mJ/cm2) were done on a panel of individuals and showed an inverse relationship, but that slope depends on the dose used. Although these data show that constitutive melanin is photoprotective, they also suggest that this protection is less effective at higher UVB doses. Gniadecka et al. (1996) specifically assessed the photoprotective role of the stratum corneum in vitiliginous and normal adjacent skin (skin type not specified) that had had no UVR exposure for 3 months. Sensitivity to erythema was compared with the thickness of the stratum corneum and the viable epidermis as well as pigmentation assessed by a reflectance device. Overall, the authors concluded that the stratum corneum accounted for almost two-thirds of the photoprotection of normal skin and was therefore more important than pigmentation, and that the thickness of the viable epidermis was not important in photoprotection.

DNA Photodamage UVR induces structural changes in DNA that include the formation of potentially mutagenic CPD and pyrimidine (6–4) pyrimidone photoproducts ((6–4) pp). Human studies have shown that epidermal CPD and ((6–4) pp) are readily induced with suberythemal exposure of SSR, UVB, and UVA (Chadwick et al., 1995; Young et al., 1998a, b). There is increasing evidence that epidermal DNA is a major chromophore for many of the acute and long-term effects of solar exposure (Young, 1996). Photodamage to DNA may result in highly characteristic gene mutations, e.g. p53 which is thought to be the initial

step in nonmelanoma skin cancer (Brash et al., 1996). Furthermore, there is also considerable evidence that DNA photodamage, especially CPD, initiates many of the immunological effects of UVR (Yarosh, 2004). Given the significance of DNA photodamage, it is not surprising that living organisms have developed highly effective DNA repair mechanisms. Studies in our laboratory and by others have shown that, after a single exposure of SSR, ((6–4) pp) repair in human skin in situ is relatively rapid and that CPD repair is much slower with many lesions persisting for at least 24 h (Bykov et al., 1999; Young et al., 1996). Such slow repair also means that lesion induction may be cumulative if skin is exposed to UVR the following day, as is the case in “real life.” There is also evidence that cytosinecontaining lesions (C = C, C = T) are repaired more rapidly than those with thymine only (T = T) (Bykov et al., 1999; Xu et al., 2000). Apart from the DNA repair response, it might also be expected that photoprotection by melanin would include protection against DNA photodamage in situ. One study compared the SSR induction of T = T in white and black skin ex vivo and came to the conclusion that constitutive pigmentation afforded DNA protection factors of 2–4 (Strickland et al., 1988). Bykov et al. (2000) reported an inverse relationship between constitutive pigmentation in white-skinned people and UVB-induced epidermal DNA photodamage. Tadokoro et al. (2003) reported fewer CPD in skins with high levels of constitutive melanin after a dose of about 1 MED. Some studies have assessed the ability of UVRinduced pigmentation in white skin to protect epidermal cells from CPD by a subsequent challenge dose of UVR. Gange et al. (1985) reported that UVB- and UVA-induced tans, in people who tanned well, resulted in protection factors of about 2–3 against the induction of epidermal CPD by UVB. Sheehan et al. (2002) treated skin types II and IV with 0.65 MED SSR for 2 weeks and exposed SSR-treated and untreated skin to 2 MED SSR 1 week after the last tanning treatment. An analysis of the data, taking into account CPD caused by the tanning treatment, showed that pigmentation was associated with a DNA protection factor of about 2–3. Surprisingly, despite the superior tan in skin type IV, the level of photoprotection was not significantly higher than in skin type II. Assessment of photoprotection against erythema was also made, and the protection factors for erythema and CPD were virtually identical, as might be expected in that DNA is the chromophore for erythema. These data are in contrast to those of Gange et al. (1985) who, as stated above, reported that comparable UVB- and UVA-induced tans gave comparable protection against UVB-induced CPD, but that only the UVB tan resulted in protection against UVB-induced erythema with a protection factor of 3. These data, as do those of Tadokoro et al. (2003) described above, suggest a lack of relationship between DNA photodamage and erythema. In a study of skin explants taken from habitually sunexposed skin from two people of skin type III, Kobayashi et al. (1998) showed an inverse relationship between the level of melanin in supranuclear caps and UVB-induced DNA photodamage in the corresponding keratinocytes. The authors 347

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showed DNA protection factors ranging from just over 1 to about 5. Barker et al. (1995) compared the UVB dose–responses for CPD in vitro in human melanocytes of different skin types. Melanocytes from skin types IV–VI showed much more tyrosinase activity and melanin than those from skin types I and II. However, the dose–response data show a relatively modest difference in CPD from a single UVB exposure that suggests a protection factor of less than 2. Melanocytes and adjacent basal layer keratinocytes in untanned human skin in situ show similar sensitivity to the induction of CPD by UVB and UVA, suggesting no inherent differences in sensitivity to DNA photodamage (Young et al., 1998a). In combination, the data from these two studies would suggest that melanocytes have no great advantage over keratinocytes in terms of photoprotection from DNA damage. Studies on reconstructed human skin with and without melanocytes from skin types II and III showed that the presence of melanocytes had no effect on the induction of CPD or ((6–4) pp) (Cario-Andre et al., 2000). This is perhaps not surprising, as melanin was not detected in the preparations. However, the presence of melanocytes inhibited the formation of apoptotic sunburn cells (SBC) that are considered to be a means of eliminating keratinocytes with mutagenic and therefore carcinogenic potential (Ziegler et al., 1994). In theory, a reduction in SBC is likely to enhance skin cancer risk. These data suggest that melanocytes might influence the effects of UVR in ways that are unrelated to melanin production. Rijken et al. (2004) compared the effects of a single 2-MED exposure of fluorescent SSR on white buttock skin (types I, II, and III) with comparable physical (i.e. suberythemal) doses on black buttock skin (type VI) in vivo. Subjective assessment of CPD by immunostaining showed comparable levels of CPD in all volunteers in the suprabasal epidermis when sampled immediately after irradiation. CPD was also seen in the basal epidermis and the dermis in the white skin types but not in skin type VI. Fisher et al. (2002) also reported similar differential of epidermal CPD in skin types I/III and V/VI after exposure to UVB plus UVA II (approximately fourfold higher doses in darker skin types) and UVA I (110 J/cm2 in both skin type groups) when samples were taken 24 h after irradiation, which allows time for repair. Both these studies also showed a reduction in other indicators of photodamage (e.g. induction of matrix metalloproteinases, infiltrating neutrophils) in black skin compared with white. Overall, these data suggest that melanin is photoprotective of basal layer DNA, but the experimental design does not provide any indication of the level of protection. However, the data of Fisher et al. (2002) could also be explained by possible skin type differential repair. Overall, several human studies suggest that the acute photoprotection afforded by constitutive and induced pigmentation against DNA photodamage in keratinocytes and melanocytes and erythema is equivalent to wearing a sunscreen with a sun protection factor (SPF) of 2–3. The generally comparable results with DNA photodamage and erythema provide additional evidence that DNA is an impor-

348

tant chromophore for erythema. Protection factors of 2–3 should result in a 50–60% reduction in biologically effective dose. Such a reduction, if maintained over long periods, would be significant in terms of long-term risk of skin cancer. However, the maintenance of a tan requires repeated exposure to UVR, from either the sun or an artificial source, and it is likely that this is associated with the accumulation of epidermal DNA photodamage (Sheehan et al., 2002) that may minimize the benefits of the protection. There are conflicting data about the role of a tan in affording protection against malignant melanoma in women, with Weinstock et al. (1991) suggesting that a tan may afford protection and Holly et al. (1995) reporting no benefit.

Is UVR-induced Melanogenesis an Indicator of DNA Repair? Extensive data suggest that DNA damage per se or DNA repair intermediates initiate melanogenesis, as summarized in Figure 17.2. Several studies have examined the effect of thymine dinucleotides (pTpT), as a model for thymine dimers, on pigmentation. Cloudman S91 mice melanoma cell lines show increased pigmentation in response to UVR. A sevenfold increase in melanin content was also observed after treatment with 50 mm of pTpT for 4 days compared with diluent-treated controls (Eller et al., 1994). Significantly, treatment of cells with pdApdA (a dinucleotide rarely seen as a photoproduct) showed only modest increases in melanin content of 20–30%. These results suggest that the response is specific to UVRinduced DNA damage products. However, agents that induce single-strand DNA breaks are also able to stimulate melanogenesis in vitro (Eller et al., 1996). Cells treated with pTpT displayed not only an increase in melanin content, but also a two- to threefold rise in mRNA for tyrosinase. Interestingly, a rise in tyrosinase levels was noted within 4 h of the addition of pTpT, which is well before mRNA changes were detectable. It would thus appear that pTpT influences gene expression at both the mRNA and the protein level (Eller et al., 1994). In addition to these in vitro studies, in vivo experiments on shaved guinea-pig skin have demonstrated that the topical application of pTpT twice daily for 5 days was able to induce a visible tanning after 1 week, reaching a maximum 1–2 weeks later. When skin sections were examined histologically, they demonstrated the presence of melanin, primarily in the basal epithelial layers, but also in suprabasal caps over nuclei (Eller et al., 1994). This is analogous to the picture seen in the human tanning response. The significance of the dipyrimidine form in inducing a tanning response was verified by Pedeux et al. (1998). Induction of pigmentation in human melanocyte and melanoma cells was demonstrable in response to the dinucleotide pTpT but not the monomer pT alone. Addition of pTpT in concentrations capable of inducing a pigmentary response was not cytotoxic to the cells, and no increase in apoptosis was observed. Treated melanoma cell lines did however show a

PHOTOBIOLOGY OF MELANINS

temporary arrest in the S phase of the cell cycle 24 h after the addition of pTpT, with resumption to normal cycling by 48 h. The mechanism and significance of this phenomenon are yet to be understood. The capacity of DNA photoproducts to induce pigmentation varies with oligonucleotide length and base composition. Although initial studies were largely carried out on pTpT, other DNA fragments are also able to stimulate pigmentation. For example, the p9-mer oligonucleotide pGpApGpTp ApTpGpApG and the p7-mer pApGpTpApTpGpA stimulated melanogenesis in Cloudman S91 murine melanoma cells by up to 800% compared with controls, whereas the p5-mer pCpApTpApC had no effect (Hadshiew et al., 2001). T4 endonuclease V (T4N5) is a bacterial phage enzyme which, apart from catalyzing the rate-limiting step in excision of CPDs, has no other recognized function (Grossman et al., 1988). T4N5 has been shown to accelerate repair of CPD in both cultured cells (Ceccoli et al., 1989; Yarosh et al., 1992) and intact skin (Yarosh, 1990). The effects of this enzyme on pigmentation following UVR have been studied in vitro. Both murine S91 melanoma cells and human melanocytes demonstrated greater melanogenesis (with an almost doubling of melanin content) when treated with T4N5 after irradiation compared with treatment with either diluent alone or heatinactivated enzyme (Gilchrest et al., 1993). These results suggest that accelerated and extensive excision of CPD enhances tanning. A photoprotective effect of DNA fragments has been shown in animal studies. Guinea pigs were treated with topical application of pTpT for 1 month to induce tanning (Gilchrest and Eller, 1999). Their shaved skin was then exposed to a previously determined 6 MED of UVB, and biopsies were taken from both UV-irradiated treated and untreated skin at the height of sunburn reaction (24 h after irradiation). Fontana Mason staining revealed higher melanin content in pTpT treated compared with untreated skin. Routine hematoxylin and eosin staining revealed extensive epidermal necrosis with intraepidermal blistering in UV-irradiated untreated skin. In contrast, pTpT-treated irradiated skin showed no histological damage and was almost indistinguishable from unirradiated skin apart from slight increases in basilar and suprabasilar melanin. Thus, it was concluded that pTpT was fully protective to 6 MED. This capacity of small DNA fragments, especially pTpT, to induce protective tanning responses in the absence of DNA damage has far-reaching therapeutic implications. Topical application of these products may potentially be used to provide a photoprotective tan without the harmful effects of UVR. Sheehan et al. (2002) reported that 10 repeated weekday doses of 0.65 MED SSR resulted in more cumulative CPD in skin type IV than in skin type II, almost certainly because skin types IV have higher MEDs than skin types II. When skin biopsies were examined 1 week after treatment, there was a nonsignificant reduction in lesions in skin type II but significant loss in skin type IV. These data suggest better DNA repair

in skin type IV, which is in accordance with the hypothesis that tanning is associated with DNA repair (Gilchrest and Eller, 1999). Such a hypothesis would suggest that the inverse relationship between skin type and skin cancer, as indicated in Table 17.1, is a consequence of DNA capacity rather than, or as well as, the ability of melanin to act as a photoprotective agent. Recent data suggest that aMSH, associated with the tanning process especially eumelanogenesis (Thody and Graham, 1998), may enhance repair of UVR-induced DNA damage (Böhm et al., 2004).

Mechanism of pTpT-induced Tanning Repair of DNA photolesions requires cell cycle arrest prior to replication and mitosis (Murray, 1992). The tumor suppressor gene p53, known as guardian of the genome, plays a vital role in the repair of DNA and apoptosis. pTpT directly activates p53, with treated cells demonstrating reduced proliferative rates and upregulation of p53-mediated p21, a protein known to mediate cell cycle arrest (Eller et al., 1997). Nuclear translocation of p53 was also observed, a known indicator of p53 activation. Thus, the data unequivocally suggest that pTpT acts at least partially via induction of p53, which regulates tyrosinase gene expression (Khlgation et al., 2002). It has been hypothesized that the DNA photodamage to the telomere 3¢ overhang (TTAGGG) may be a specific trigger for the cellular defense responses to UVR (Eller et al., 2003) and that this is the reason why oligonucleotides with homology (i.e. TT) to this sequence are able to induce such responses as p53 activation.

Melanin Synthesis may be a Damage Response System Although the major DNA photoproducts CPD and 6–4PP are excised by a well-recognized family of DNA repair proteins, the metabolic fate of the excised photoproduct containing a single-stranded DNA (ssDNA) fragment is relatively understudied. In bacterial studies, however, it has been demonstrated that ssDNA plays an important role in photoprotection. Single-strand DNA in prokaryote systems generated from DNA damage/repair interacts and activates a protease, the Rec A protein. Rec A is then responsible for the lifting of repression of over 40 genes important in DNA repair, replication, and cell survival (Courcelle and Hanawalt, 2003). This phenomenon, known as the SOS response, not only increases bacterial survival after irradiation, but also enhances resistance to subsequent UVR-induced DNA damage. Thus, repeat exposure to a sublethal dose of UVR in these bacteria would result in more efficient repair of DNA damage and enhanced survival (Crowley and Hanawalt, 1998). It has been postulated that tanning may form part of a mammalian SOS response (Eller and Gilchrest, 2000).

Immunosuppression by UVR There is considerable evidence that UVR-induced immunosuppression may play a role in skin cancer, and animal studies

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also suggest a role for susceptibility to infectious disease (Sleijffers et al., 2002). There have been two human studies on the photoprotective effects of melanin on UVR-induced immunosuppression (Selgrade et al., 2001; Vermeer et al., 1991), and both came to the conclusion that pigmentation, even in dark-skinned people, had no effect on the ability of UVR to suppress the induction phase of the contact hypersensitivity (CHS) response, which is regarded as a model for some of the photoimmunological events that are important in skin cancer. The reasons for this are not known, but one explanation would be that a superficial chromophore such as stratum corneum urocanic acid (UCA) is more important than epidermal DNA. Mouse studies have shown that photoimmunosuppression is initiated via either DNA or UCA depending on the viability of the antigen (Kim et al., 2003).

Photosensitization by Molecules Associated with Melanogenesis It has been reported that albinos, who have tyrosinasedeficient melanocytes (i.e. not producing melanin), are prone to nonmelanoma skin cancers but not malignant melanoma (Streutker et al., 2000). This suggests the possibility of melanogenesis-related photosensitization in malignant melanoma. Several in vitro studies have identified photosensitizing properties of melanins or their intermediates (Hill, 1992; Kvam and Dahle, 2004). 5-SCD photobinds to native DNA after exposure to 300-nm radiation and also induced single-strand breaks (SSB) in DNA (Koch and Chedekel, 1986). More recently, it has been reported that 5-SCD is photochemically unstable in the presence of UVA radiation and oxygen (Costantini et al., 1994) and that the eumelaninsoluble precursor DHICA sensitizes DNA SSB with 313-nm exposure, especially in the presence of oxygen (Routaboul et al., 1995). Kipp and Young (1999) showed that the addition of DHICA to human keratinocytes increased their sensitivity to UVA-induced SSB by the generation of ROS. Kvam and Tyrrell (1999) concluded that melanogenesis, but not melanin itself, was associated with oxidative base damage in human melanoma cells. Wenczl et al. (1998) compared the UVA sensitivity of melanocytes from skin type I and skin type IV that had been induced to synthesize melanins by a high concentration of l-tyrosine in the culture medium. The ratio of pheomelanin to total melanin remained the same in skin type IV, but relatively more pheomelanin was induced in skin type I. This was associated with an increase in UVA-induced SSB in DNA. In recent in vivo studies in different mouse strains, Takeuchi et al. (2004) have reported that melanins, in particular pheomelanin, are UVB and UVA photosensitizers in mammalian skin in vivo. It should be stressed that the clinical significance, if any, of these reactions is unknown, but they clearly demonstrate the photobiological potential of melanogenesis intermediates, and it is therefore possible that pheomelanin plays a role in the susceptibility of skin types to skin cancer and other types of photodamage. 350

Pheomelanin

Photosensitization

Eumelanin

Photoprotection

Fig. 17.3. Solar UVR activates melanocytes to initiate the synthesis of a mix of pheomelanin or eumelanin in specialized organelles called melanosomes (represented by the oval structures). The balance of melanin type depends on genetic factors such as skin type. Pheomelanin and eumelanin and their precursors interact in different ways with solar UVR. Pheomelanogenesis may favor photosensitization via ROS, whereas eumelanin is more likely to favor photoprotection.

Perspectives Exposure to solar UVR, especially UVB, results in epidermal DNA photodamage such as CPD that, if unrepaired, can result in mutation, immunosuppression, and consequent skin cancer, especially in people who do not tan readily. Despite concerted public health campaigns and increased awareness of the hazards of UVR, the incidence of skin cancer continues to rise in white-skinned populations. Indeed, a tan is still widely regarded as a desirable sign of health and well-being, especially by the young, and is often justified by its photoprotective properties, which are relatively modest even in those who tan well. The level of photoprotection may ultimately depend on the ratio of pheomelanin to eumelanin as indicated in Figure 17.3, as there is experimental evidence to suggest that pheomelanin may be associated with photosensitization.

PHOTOBIOLOGY OF MELANINS

There is increasing evidence that melanogenesis is initiated by CPD and its repair and may therefore be seen as a biomarker for DNA repair capacity which, in white-skinned people at least, may be more important in skin cancer prevention than in optical photoprotection. The relationship between DNA damage/repair potentially has major therapeutic implications given that topical application of small DNA fragments may induce melanogenesis and a measure of photoprotection, without the harmful effects of UVR. In effect, this may make a skin type II respond more like a skin type IV, and this is an area for further research from which there may be significant public health benefit.

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Toxicological Aspects of Melanin and Melanogenesis Edward J. Land, Christopher A. Ramsden, and Patrick A. Riley

Summary 1 The major contributor to surface pigmentation in vertebrates is melanin. The precise structure of melanin is not clear, but it comprises conjugated indolic moieties that endow the material with strong light-absorptive properties. Eumelanins are dark brown or black, whereas pheomelanins are lighter variant molecules incorporating sulfur atoms. 2 Mammalian melanin is derived from tyrosine by a series of reactions that initially involve oxidation to dopaquinone. The enzyme responsible for this step is tyrosinase, which is synthesized by specialized dendritic cells derived embryologically from the neural crest. Tyrosinase is active in specialized organelles within these melanocytes, and pigment is laid down on a protein stroma in these melanosomes. The fully melanized organelles (melanin granules) are distributed to adjacent cells by partial cytophagy. 3 From the toxicological standpoint, there are two main facets of the pigmentation process that demand attention. One is the process of melanogenesis, in which chemically reactive intermediates are generated that are potentially harmful to melanocytes and may also have toxic effects on adjacent tissues. The other relates to the properties of the product, melanin. Although some cells, such as the pigment epithelium of the retina, retain melanin, the pigment is generally transferred to adjacent recipient cells. Consequently, many of the toxicological sequelae associated with the properties of melanin are observed in epithelial cells. 4 Melanogenesis involves a metabolic pathway, the regulation of which is complex. The crucial enzyme is tyrosinase, a protein with an active site containing two copper atoms that bind molecular oxygen. Regulation of pigmentation incorporates signaling pathways that control the synthesis, posttranslational modification, metal incorporation, routing to organelles, and distribution to melanosomes of the enzyme. The mechanism of tyrosine oxidation requires the copper atoms at the active center to be in the reduced state, and the biochemical regulation of this step depends on the indirect formation of dopa from dopaquinone. The indirect reactions involve spontaneous cyclization of dopaquinone to form cyclodopa, which acts as a reductant in the eumelanogenic pathway, and this is in competition with nucleophilic addition of cysteine to dopaquinone to form cysteinyldopa in the 354

pheomelanic pathway. In this system, cysteinyldopa acts as a reductant of dopaquinone to yield dopa as the autoactivating substrate for tyrosinase. 5 The reactivity of dopaquinone and other intermediate orthoquinones formed during the biogenesis of melanin poses a threat to the survival of melanocytes, in particular by depleting melanogenic cells of antioxidant defenses such as glutathione. Several mechanisms have evolved that provide some protection against this cytotoxic hazard. These include the segregation of melanogenesis in membrane-bound organelles to minimize diffusion of reactive intermediates into the cytosol, and the presence of quinone reductases and catechol-O-methyl transferases in the cytoplasm. 6 Because of their relatively low population density, melanocytes are particularly susceptible to cytotoxic elimination. The visible result of this is depigmentation of epidermal structures, including hair. A wide array of stimuli associated with oxidative stress has been shown to cause local depigmentation by melanocyte depletion. 7 Melanogenesis has been exploited for both diagnostic and therapeutic purposes, mainly in connection with malignant melanoma. Detection of nascent melanin by radiolabeled probes that are incorporated by reaction with quinone intermediates has permitted localization of melanogenic tumors, and a similar principle has been applied to target radiotherapy to melanomas. 8 The introduction of analog substrates for tyrosinase has been pursued as a possible melanoma-targeting strategy. The first-generation analogs were based on 4-hydroxyanisole which, by virtue of its noncyclizing and lipophilic side-chain, results in the formation of a more stable and diffusible orthoquinone product when oxidized by tyrosinase. A more recent development has been to attach to cyclizable tyrosinase substrates known cytotoxic moieties that are released by hydrolysis on cyclization of the quinone oxidation product. 9 In relation to the toxicology of melanin, there has been continued interest in the selective uptake and binding of many materials including metals and a variety of xenobiotics, especially organic amines. The binding mechanisms in the case of cations are largely due to the carboxyl anion content of the pigment, and this also accounts for some of the binding capacity for other agents, including pharmaceuticals, although charge transfer and hydrophobic interactions are also involved.

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

10 The kinetics of uptake by and release of materials from melanin have been investigated by several techniques. The equilibrium conditions seem to favor the notion that, at relatively low concentrations of potentially noxious agents, melanin acts primarily as a cytoprotective pigment by sequestering harmful chemicals. Combined with the exfoliation of epithelial cells, this may even constitute an important excretory pathway for the organism, especially of heavy metals and, possibly, some carcinogens. 11 However, in tissues where melanin is retained, the accumulation of material during chronic exposure may be related to the induction of specific lesions in pigmented tissue. This appears to be the case in the eye where melanin-affinic drugs may cause retinopathies and cataract formation. Similarly, neurotoxicity in pigmented regions of the central nervous system may be related to the selective accumulation of certain harmful agents, as in parkinsonism. 12 Selective accumulation may constitute a therapeutic advantage. For example, the storage of local anesthetics in inner ear melanin has proved useful in the treatment of severe tinnitus. Similarly, it has been proposed that selective uptake by melanin of psoralens is advantageous in PUVA treatment of psoriasis. Melanin binding of methylene blue has been advocated as a targeting strategy for radiotherapy of melanoma using labels such as radioiodine or astatine-211, and some interesting results have been published of the use of neutron capture therapy of melanoma using boron-10 incorporated into melanin-binding agents. In addition, biological monitoring or forensic investigation of exposure to a variety of agents including industrial chemicals, drugs, or illicit narcotics has been facilitated by examination of pigmented hair samples in which these agents are accumulated.

Introduction Definition of Melanin and Description of Types The predominant surface pigmentation of vertebrates is melanin. Despite the widespread usage since it was first introduced in the eighteenth century, the term “melanin” covers a range of polymers with uncertain structure. It is now widely held that the variability of structure is the consequence of the incorporation of several precursors into the final polymer. In general, however, eumelanin, a black or yellow pigment, is predominantly an indolic polymer which is highly conjugated. The polymer possesses redox properties, an anionic character, and powerful metal-chelating properties. The physical properties of melanins include photon absorption, electron exchange, and cation binding as well as photon–phonon coupling, and are dealt with elsewhere. The chemical reactivity of the precursor intermediates permits the generation of polymers that incorporate other compounds, including especially thiol compounds, which form addition products with quinones and may modify the size and properties of the final polymer. Melanins with high sulfur content are generally reddish and are known as pheomelanins.

Location of Melanin Melanins are found in cutaneous structures. In humans, there is marked pigmentation of the epidermis and epidermal structures such as the hair. The important extracutaneous sites include the eye where there are two melanized structures: the iris and the retina. The choroidal pigment is synthesized by melanocytes, whereas the retinal melanin is synthesized and retained by the cells of the retinal pigment epithelium (RPE). Another site in which melanin is found is in the inner ear. Melanin is also found in the central nervous system (CNS) where it is present in the extrapyramidal nuclei, in particular the substantia nigra. The process involved in the formation of CNS melanin is apparently not dependent on tyrosinase-catalyzed oxidation and thus differs from the general group of these pigments. Melanocytes are found in the meninges, and melanin has been reported in many other locations.

Synthesis In vertebrates, most melanin is synthesized by melanocytes. These neural crest-derived dendritic cells uniquely express features that permit the biosynthesis of melanin. The only other cells that are normally able to do this in mammals are the cells of the RPE. In both cases, the process of melanogenesis involves the synthesis of tyrosinase and related enzymes, which are active in intracellular organelles (melanosomes) where the melanin polymer is formed and deposited on matrix proteins to form a melanin granule. It is likely that the process evolved from a more primitive system in which tyrosinase was exported by a secretory mechanism to act on external substrates present in the microenvironment. This mechanism seems to be an important defensive mechanism in plants and insects, both from an antibiotic point of view and also because of the structural reinforcement that is added by the polymer. A good example of this is the formation of the insect exoskeleton in which hardening is dependent on the action of tyrosinase. In higher animals, although the melanin-generating reactions are still topologically exterior to the cytosol, melanogenesis has been enabled to take place in cytoplasmic organelles by the fusion of enzyme-containing vesicles with matrix vesicles, which either contain the substrate or are able to concentrate it. The details of the segregation of the contents of the vesicles and the processes involved in their fusion have yet to be fully established. In some cells, these melanized granules are retained, for example in the RPE and dermal melanocytes and melanophores in fish and amphibia. But in the epidermis and associated structures such as hair, pigment is transferred to the adjacent keratocytes. The melanin granules that are transferred to recipient cells exhibit no tyrosinase activity so that, in the process of melanization, the enzyme is inactivated. Examination of enzyme activity in different granule fractions derived from melanocytes shows that there is virtually no tyrosinase activity in fully melanized melanosomes. This implies that the enzyme is inactivated by its product. It was shown by Wood and Ingraham (1965) that covalent binding of 14C-labeled 355

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phenol to tyrosinase occurs, and this is likely to be due to the interaction of the enzymatically derived orthoquinone with nucleophilic groups of the enzyme protein (Brooks and Dawson, 1966). Studies by Dietler and Lerch (1982) on Neurospora tyrosinase, however, indicated that the amount of binding was too small to account for the inactivation of the enzyme. They gave evidence that there was loss of copper from the enzyme, and it is possible that the metal-chelating property of melanin is responsible for the removal of copper from the active site. An oxidative modification of one of the tyrosinase histidine residues (His-306) was also noted, which may have been due to hydroxyl radical attack.

Distribution Transfer of melanin granules may take place singly, but the most frequent distribution process involves the wholesale transfer of subportions of the melanocyte cytoplasm to surrounding cells by phagocytosis of melanocyte dendrites — a process known as cytocrine transfer. Redistribution of melanin granules in the recipient cells then takes place by membrane fusion. Degradation of melanin is very slow or nonexistent, and turnover depends on the pigment being shed with the cells that have taken up the melanin, e.g. in keratinized epidermis such as the stratum corneum and hair. Secondary depigmentation may therefore accompany conditions in which there is a local increase in epidermal desquamation such as in eczema. Such a mechanism might also be responsible for the hypopigmentation of leprosy and the hypomelanotic macules that are found in association with tuberous sclerosis. In RPE, where the melanin is retained, what turnover occurs appears to involve incorporation of melanosomes into autophagosomes where melanin may contribute to the composition of lipofuscin granules.

Properties Despite many careful studies of melanins, the details of the composition of the pigment is still unclear. In principle, melanins are regarded as complex aggregates of oligomers composed principally of dihydroxyindoles and related oxidation intermediates of the melanogenic pathway. Melanins exhibit three fundamental properties: (1) strong photon absorption across a broad spectrum; (2) facile redox exchange properties; and (3) the capacity to bind chemicals. The binding may give rise to pronounced accumulation of drugs and other chemicals in melanin-containing tissues, especially after chronic exposure, and the melanin-affinic uptake in pigmented tissues is one of the strongest retention mechanisms in the body. Chloroquine, for example (Lindquist, 1973), and N,N-bis-acetanilidedimethylamine (Lyttkens et al., 1984) have been found in marked concentrations in the melanin of the eye 1 year after a single intravenous injection of a trace dose in pigmented mice (Fig. 18.1). The melanin affinity of certain drugs, e.g. phenothiazine derivatives and chloroquine, has turned out to be a main factor in the etiology of lesions affecting the pigmentary system of the body, but it also creates opportunities for tar356

Fig. 18.1. Whole-body autoradiogram of a pigmented mouse (C57BL) 1 year after a single intravenous injection of 3H-N,N-bisacetanilidedimethylamine (QX-572). Note the significant retention of label in the uveal tract of the eye, whereas no radioactivity can be detected in other organs.

geted therapy of malignant melanoma and other diseases, directly or indirectly related to the pigment cells.

Biological Function In man, normal levels of pigmentation differ between races and reflect quantitative differences in the constitutive rates of melanogenesis (Robins, 1991). The explanation given for the relative lack of epidermal pigment in less equatorial populations is based on the idea of the evolutionary value of a diminished light barrier in permitting the light-assisted synthesis of vitamin D in less strongly illuminated regions of the globe (Loomis, 1967). Whatever the genetically determined level of melanization, it is regulated by the control of a number of processes, including access of melanocytes to the skin, which may be important in modifying the degree of regional pigmentation, the nutritional and microenvironmental conditions of melanocytes, and the hormonal regulation of the rate of melanogenesis. In mammals, there does not appear to be direct neural control of pigment cells that permits the spectacular camouflage or communication displays of fish, amphibia, and some reptiles. These rapid reversible changes depend on alterations in the dispersal of melanin granules that are synthesized in and retained by melanophores. Melanophores in frogs are responsive to melanocyte-stimulating hormone (MSH), which binds to cell surface receptors that cause a rise in intracellular cyclic adenosine monophosphate (cAMP). The rise in cAMP causes granular dispersion in melanophores. In

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

mammals, a rise in cAMP is associated with increased tyrosinase synthesis. Camouflage and communication depends on pigment patterning and, in vertebrates, this is largely determined by the distribution of melanocytes. Relatively little is known about the microenvironmental conditions that regulate access of melanocyte precursors to the skin. The astounding migratory range of neural crest cells and the remarkable phenotypic repertoire of their differentiation products are evidence of the many possible mechanisms that could be implicated in modifying the extent of local pigmentation. Diminished migration into the skin of premelanocytes (melanoblasts) seems to account for the dorsoventral gradient of pigmentation in many vertebrates, and failure to colonize certain regions of the skin may give rise to certain patterns of depigmentation. Some forms of striping or mottling seem to be brought about in this way, for example the patterns resulting from X-chromosome inactivation. Other mechanisms, perhaps involving processes that influence the survival of melanocytes, seem to be involved in patterns of depigmentation or lack of pigmentation such as those in zebra stripes. These phenomena are genetically determined, and pathological variants of them are found in lethal white spotting in mice, which is associated with the megacolon pathology resulting from failure of migration of neural crest ganglion cell precursors in the large gut, and also in Waardenburg syndrome (Waardenburg, 1951), in which there is combined deafness and segmental cranial pigment deficiency due to the failure of neural crest cell migration. The segmental lack of pigment in piebaldism may represent a similar process, although the phenomenon may be viewed as a congenital variant of vitiligo. A good deal of emphasis has been given to the UVR protection afforded by epidermal melanin. Certainly, there is a strong inverse correlation between sunburn susceptibility and the degree of skin pigmentation, and the incidence of skin cancer is much lower in blacks than in whites. Pigmentation of the epidermis and associated structures is probably a manifestation of an evolutionarily significant property unrelated to the light absorption of melanin. It has been suggested that melanin could act as an excretory route for heavy metals. This may be of particular importance to marine mammals and other species with a significant dietary intake of shellfish, and may have proved to be an evolutionary advantage during the littoral evolution of Homo sapiens (Riley, 1992, 1994, 1995). However, eye pigmentation is probably a good example of the exploitation of the photon-absorbing property of melanin to increase visual acuity. Consequently, loss of melanin in the eye, for example in ocular albinism, constitutes a serious problem. The evolutionary benefit of melanin in other sites is unclear. In the inner ear, melanin may act as a phonon absorber, or its action may depend on its chelating properties. One possibility might be that the melanin serves as a device for the local regulation of endogenous cations, such as Ca2+ (Meyer zum Gottesberge-Orsulakova, 1985). Another conceivable function is that melanin is a reservoir for melanin-affinic biogenic

amines, such as dopamine, epinephrine (adrenaline), and norepinephrine (noradrenaline) (Lindquist, 1973), or endogenous polyamines, e.g. spermidine (Tjälve et al., 1981). As far as xenobiotics are concerned, it is tempting to consider melanin as a protective chemical filter. The presence of melanin in some very sensitive tissues evidently favors this hypothesis. The melanin would protect these or adjacent tissues by keeping potentially harmful substances bound and slowly releasing them in low, nontoxic concentrations (especially transient, high-concentration peaks of noxious chemicals are limited by the adsorption to melanin). In the eye, for example, the melanin is located in close proximity to the receptor cells, and the nutrients from the capillary bed of the choroid are filtered through the choroidal melanocytes and the pigment epithelium before reaching the retinal receptors for nourishment. A similar situation exists in the inner ear, because the receptors (the hair cells) are located relatively close to the melanin of the stria vascularis in the cochlea and in the vestibular ampullae. In the brain, neuromelanin is present in nerve cells in the extrapyramidal system, mainly in the substantia nigra and locus coeruleus, and the neuromelanin may be involved in chemical protection. Korytowski et al. (1995), for example, have provided strong experimental support for the idea that the neuromelanin from human substantia nigra can act as a natural antioxidant by sequestering redox-active metal ions. Under certain circumstances, however, the possible protection mechanism may be a threat to the pigment cell, as chronic exposure to certain toxic substances with melanin affinity may cause selective adverse effects in the melanin-containing tissues, as discussed in a subsequent section.

Modification of Melanin Cosmetically, the most important category of depigmentation is the “bleaching” of melanin in hair and in the epidermis. Bleaching of hair is generally achieved by the action of hydrogen peroxide. As has been mentioned previously, melanin is a powerful chelator of transition metals such as iron and copper. In the presence of hydrogen peroxide, these metals catalyze the Fenton reaction, which leads to the generation of hydroxyl radicals: H2O2 + Fe2+ Æ Fe3+ + HO– + HO◊ Hydroxyl radicals are highly reactive and attack any vicinal molecule at diffusion-controlled rates. Thus, melanin exposed to hydrogen peroxide is subjected to a high-density attack by hydroxyl radicals, which results in multiple scission of the polymer (Korytowski and Sarna, 1990). As the melanin is hydroxylated and broken down into shorter segments, the low-energy, photon absorption properties associated with the highly conjugated polymer are lost, and the peroxide-treated tissue (e.g. hair) becomes lighter in hue. Hydroxyl radical attack is also able to damage keratin, so there is some danger to the structural integrity of hair, but the relatively selective effect on melanin is dependent on transitional metal binding to the pigment. A similar approach cannot be used to 357

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depigment the epidermis because of the cytotoxic effect of peroxide-derived hydroxyl radicals which damage the skin. An alternative approach to epidermal depigmentation has been employed based on chemical reduction of epidermal melanin. It is known that the optical properties of melanin are affected by the redox status of the polymer because the photon absorption is a function of the number of carbonyl groups. Theoretically, therefore, increasing the reducing environment of the melanin will diminish its light-absorbing properties. The reverse of this process underlies the “immediate pigment darkening” (IPD) that occurs on exposure of melanin to blue or ultraviolet light. This results from an oxidation reaction in which electrons in the melanin are raised to an excited state by high-energy photon absorption and donated to oxygen. Oxidation of the melanin increases the number of carbonyls in the polymer and results in enhanced absorption of low-energy photons (the so-called “bathochromic” effect). The phenomenon is prevented by ischemia during exposure, and IPD is an oxygen-requiring phenomenon. Electron spin resonance spectroscopy of melanin during irradiation shows that there is an increase in the semiquinone content of the polymer. This may come about either by partial reduction of oxygen to form superoxide and the corresponding semiquinone or by delocalization of electrons in the melanin polymer which is facilitated by an increase in the oxidation state. To some extent, a reducing environment exists naturally in the cytoplasm of cells that are recipients of melanin, but it can be augmented, at least in the upper layers of the epidermis, by the external application of creams containing reducing agents such as ascorbic acid or hydroquinone. The main problems associated with this approach concern the uncertain levels of skin penetration of the agents and, in the case of hydroquinone, the complications introduced by the ability of this compound to form covalent addition products with melanin and melanogenic intermediates.

Toxicological Aspects In this chapter, we attempt to summarize the toxicological aspects of melanins and melanogenesis. We distinguish between the process of pigment formation and the properties of the product and, in particular, between cells that elaborate melanin, and are thus prone to the toxic effects of melanogenic intermediates, and cells that are recipients of melanin, which are susceptible to toxicological hazards arising from the properties of the pigment. Melanogenesis has long been recognized as a potential source of cytotoxicity because of the reactivity of the intermediates formed, and several mechanisms are involved in protecting melanogenic cells from damage. We outline potentially hazardous reactions of melanogenic intermediates such as orthoquinones and discuss the mechanisms that have evolved to protect melanocytes. In this context, we discuss the idea of utilizing melanogenesis as a targeted cytotoxic strategy in the treatment of disseminated melanoma. 358

As noted above, melanin has structural properties that endow it with a range of chemical reactivity with toxicological implications. Notably, the high degree of conjugation of the pigment makes it a strong absorber of photons, and part of its protective function results from its photochemical reactivity. Also, the balance between quinone and hydroquinone functions, with the equilibrium permitting intermediate semiquinone steady-state conditions, makes melanin a facile redox exchanger able to act either in a protective mode or as a source of damage. In addition, the presence of anionic functions, such as carboxyl groups, makes melanin a cationic exchanger. The tendency of van der Waals’ stacked ring structures and the presence of nitrogen-containing heterocyclic rings (in the form of indoles or indolines) give rise to the possibility of hydrophobic interactions and charge transfer complexes, thus enabling melanins to act as a sink or depot of a wide range of xenobiotics. We discuss these reactivities in relation to the toxicology of melanin-containing tissues including the eye, ear, and brain in addition to the skin. Finally, we mention attempts to employ melanin-directed toxicology in various therapeutic modalities including melanoma treatment.

Melanogenesis Surface pigmentation by melanin is the result of a chain of events beginning with the migration of neural crest precursors to populate the epidermis and its associated structures, such as hair bulbs, with melanoblasts which undergo differentiation to give rise to the melanocyte population of the skin. Melanocytes possess the specialized cytoplasmic organization and the biosynthetic apparatus to generate melanin. In vertebrates, the stages involved in surface pigmentation include: (1) migration of pigment cell precursors from the neural crest; (2) clonal population of the skin by melanocytes; (3) induction of melanogenic genes; (4) synthesis of melanogenic enzymes and matrix components; (5) post-translational processing of melanogenic proteins; (6) fusion of vesicles to form melanosomes; (7) control of tyrosinase and related melanogenic enzymes: (8) control of post-tyrosinase modification of the synthetic pathway; (9) transfer of pigment granules to recipient cells; and (10) modification of melanin.

Melanocyte Origin, Migration, and Differentiation Relatively little is known about the control of migration of pigment cell precursors. Clearly, the process involves the expression of genes in both the migrating cells and those tissues that are invaded by melanoblasts that control motility and direction. Failure of the appropriate inductive effects results in abnormalities. One such is the premature cessation of migration, which is probably the origin of dermal accumulations of pigment cells as in Mongolian spots. Another example of significance in human pathology is Waardenburg syndrome in which there is a combination of defects including cranial depigmentation ascribable to failure of immigration of neural crest cells.

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

Dispersion of cells from the neural crest has been clarified by the studies of Mintz (1967) using chimeric mice composed of cells carrying different coat color genes mixed at the blastocyst stage. She concluded that the various striping patterns observed in the mice conformed to a basic distribution of pigment cell precursors into 34 independent regions, i.e. 17 bands on either side of the midline. This implies that all the melanocytes responsible for hair color in mice are derived from 34 cells whose descendants migrate to specific areas of the skin. Further diversification can take place within these clonal areas, accounting for the finer grain patterning exhibited by Xchromosome inactivation (Lyon, 1963) such as in the case of the X-linked genes “dappled”, “brindled,” and “mottled” (Modp, Mob–, Mo), which inhibit pigmentation possibly by reducing the viability of melanocytes expressing the allele. Spotting, piebaldness, or variegation of pigmentation also occur in animals with normal coat color genes and in distributions that do not correspond with the clonal pattern of melanocyte migration. In these cases, there is evidence that the melanoblasts are influenced by the local tissue environment and either fail to enter a region or fail to survive or are not induced to express their differentiated function (Mayer, 1967). The skin can be regarded as a mosaic of zones in which different titers of a factor influencing melanocyte development exist. Spotting genes may therefore determine the distribution of such factors. Some of these effects may, however, reflect abnormal levels of sensitivity of melanocytes to environmental factors. Phenomena related to this are considered elsewhere. During embryogenesis, melanoblasts migrate to sites where they will later synthesize pigment. During this migratory and proliferative phase, no melanogenesis occurs, and the onset of melanin synthesis is the result of a differentiation process in which the biosynthetic apparatus for melanin production is induced. It is important to note that melanogenesis is in no way a “terminal” differentiation either in RPE or in epidermal melanocytes as fully melanized cells are able to proliferate. It seems likely therefore that the genetic controls of melanogenesis are separate from those that determine the degree of expression of melanogenesis. However, experimental systems involving isolated melanocytes have demonstrated that rapidly proliferating cells often become progressively less pigmented. This phenomenon is principally the consequence of pigment dilution. Manifestly, if the cytoplasmic expansion necessary for cell doubling is more rapid than the rate of melanin synthesis, the amount of pigment per cell will be progressively diminished at each cell division. Conversely, any agent that reduces or arrests proliferation will increase the degree of pigmentation even if no direct effect is exerted on the rate of melanogenesis (Riley, 1987). Care is therefore necessary in ascribing changes in the level of pigmentation to “differentiation” or expression of a differentiated phenotype (Abdel-Malek et al., 1992; Abe et al., 1987; Durkacz et al., 1992; Fisher et al., 1985; Kiguchi et al., 1990; Lauharanta et al., 1985; Orlow et al., 1990; Osman et al., 1985; Sunkara et al., 1985).

Having entered the region to be pigmented, the melanoblasts are converted to active melanocytes by the induction of melanin synthesis. A good deal is known not only about tyrosinase and related genes, but also about the regulatory regions of the genome that control their expression. However, at present, the details of the operation of the transcriptional controls remain to be elucidated. Nevertheless, several studies have demonstrated the action of a wide range of agents that appear to stimulate or inhibit transcription of melanogenic genes, in particular tyrosinase. The main stimulatory pathway involves activation of protein kinase A (PKA) through a cAMP-dependent mechanism. Another hormonal effect on melanogenesis (possibly acting at the transcriptional level) is through the catecholamine receptor pathway. Burchill and Thody (1986) showed that the specific D2 receptor agonist LY 171555 inhibited tyrosinase activity in explanted hair bulbs, an effect blocked by sulpiridine, a D2 receptor antagonist. In the case of MSH, the pathway involves MSH receptor-linked G protein activation. The direct action of UVB on melanocytes appears to be cAMP independent (Friedmann and Gilchrest, 1987), and it appears that the stimulating action of prostaglandin E1 may be cAMP independent, although it does raise cAMP levels in Cloudman S91 cells (Abdel-Malek et al., 1987). Inhibitory effects on melanogenesis at the transcriptional level have been observed in the case of a variety of agents including lactic acid (Ando et al., 1993), linoleic acid (Ando et al., 1990), retinoic acid, interleukin (IL)-6, IL1a, tumor necrosis factor (TNF)a (Swope et al., 1991), and epidermal cell-derived thymocyteactivating factor (ETAF) (Swope et al., 1989). It is possible that several of these agents exert their effects through the intermediacy of aPKC (Oka et al., 1993), which may in turn interfere with the stimulatory action of PKA. There is evidence that the stimulatory action of 12-O-tetradecanoylphorbol-13acetate (TPA) is by down-regulation of aPKC (Carsberg et al., 1994) and, conversely, 1-oleoyl-2-acetyl glycerol and linoleate (Ando et al., 1990) exert their inhibitory action by stimulation of aPKC. Undoubtedly, when the transcriptional controls are better comprehended, the complex interactions by which normal melanogenesis is regulated will become clearer.

Melanogenesis — Pathway and Regulation Physiological Control of Melanogenesis Although the entire process of pigment formation involves many complex interactions, including vesicle trafficking and fusion, the intercession of tyrosinase-related proteins, and matrix generating processes, it is clear from the depigmentation of albinism that the crucial element of the melanogenic pathway is the action of the principal oxidizing enzyme, tyrosinase. At present, relatively little is known about the control of expression or the requirement of the tyrosinase-related proteins TRP-1 and TRP-2. The function of TRP-1 is unknown, 359

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but TRP-2 has an enzymatic action in catalyzing the conversion of dopachrome to its tautomer 5,6-dihydroxyindole-2carboxylic acid and thus plays a part in the melanogenic pathway. The suggestion has been made that the biosynthesis of melanin in melanosomes is dependent on the cooperative action of tyrosinase and the tyrosinase-related proteins in the form of a physical aggregate (Orlow et al., 1994). Thus, the efficiency of the process could be modified by changes in the composition of the aggregate structure. If this model of melanogenesis is correct, then control of pigmentation may be exerted by components without catalytic activity. Whether this proves to be the case remains to be seen. An interesting observation is the apparent inverse correlation between tyrosinase and TRP-2 activity in MSH-stimulated melanocytes (Martínez-Liarte et al., 1992), which suggests that our present comprehension of the regulatory mechanisms of melanogenesis is extremely tenuous. It is a moot point whether, under normal circumstances, there exists a physiological regulator of tyrosinase activity that acts by preventing access of the substrate to the enzyme (see Chen and Chavin, 1975). As several products of the process of melanogenesis bear a close resemblance to the substrates for both tyrosinase and TRP-2, it would not be surprising if there is some element of feedback inhibition by competition between products and initial or intermediate substrates. Such a phenomenon has been shown in an in vitro system (Wilczek and Mishima, 1995), and a compound having the property of inhibiting tyrosinase has been extracted from melanoma cells (Chian and Wilgram, 1967). The lack of melanogenesis in extramelanosomal sites has been ascribed to binding to tyrosinase of an inhibitor that is diluted or inactivated when the enzyme is taken up by premelanosomes (Hearing and Jiménez, 1987; Martinez et al., 1987). Kameyama et al. (1989) have obtained a highly stable inhibitor of melanogenesis from nonpigmented cells that acts directly on tyrosinase. At present, the identity of this inhibitor is not known (Kameyama et al., 1995), but it may correspond with that isolated from human skin xenografts by Farooqui et al. (1995).

Tyrosinase Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme that catalyzes the oxidation of monohydric phenols and dihydric phenols (catechols) to their corresponding orthoquinones. In vertebrates (and also some invertebrate species), the major natural substrate is tyrosine, which is oxidized with the utilization of molecular oxygen to dopaquinone. Mammalian tyrosinase has an amino acid composition consistent with the DNA sequence established by Kwon et al. (1988), Terao et al. (1989), and Yamamoto et al. (1989), which predicts a protein of 515 amino acids with a molecular mass of 58.5 kDa after cleavage of the leading peptide. This structure includes a hydrophobic transmembrane domain of 23 amino acids and a further 37 amino acids that may act as a signal peptide. Proteolytic cleavage of the transmembrane portion of the enzyme gives an expected molecular weight of about 53 kDa for the unmodified “soluble” form of tyrosinase. 360

This corresponds closely with the lower molecular weight range of the unglycosylated form of the enzyme as ascertained by several laboratories (Burnett, 1971; Hearing et al., 1981).

Substrate Specificity Although the details of the three-dimensional arrangement of the substrate binding site of tyrosinase remain to be confirmed by X-ray crystallographic analysis, it is clear from amino acid sequence data and, by inference, from the structure of the closely related copper protein hemocyanin, that the active site is situated in a hydrophobic pocket into which the phenyl ring of the substrate can be inserted. As tyrosinase is stereospecific, it must be assumed that there are secondary binding domains for the carboxyl and amino groups of tyrosine that exclude binding by the d-isomer. The carboxyl binding site may recognize the carbonyl group (hydrogen bond acceptor) or, if the carboxyl group is ionized at the relevant pH, forms an ionic bond with the negatively charged oxygen. As the amino group of the tyrosine side-chain is unlikely to be protonated under normal conditions, this group may form one (or two) hydrogen bonds with a vicinal acceptor group on the enzyme. The third restriction is presumably spatial because otherwise free rotation of the asymmetrical b carbon would permit the accommodation of either optical isomer of tyrosine. There is also a spatial restriction imposed by the distance between the phenyl ring and the groups on the side-chain conferring specificity of binding and by the maximum size of the substituents at the a carbon of the side-chains. Compounds resembling ltyrosine may act as competitive inhibitors of tyrosinase (e.g. l-phenylalanine) or may themselves be melanogenic substrates (e.g. l-dopa or l-dopamine). A large number of compounds have been investigated as potential substrates for tyrosinase-catalyzed oxidation. Analysis of the data (mostly obtained with tyrosinase from Agaricus bisporus) suggests that a carboxyl or carbonyl groups prevent substrate binding, but the enzyme is apparently tolerant of many side-chain variations. Shortened side-chains (e.g. –CH3 or –C2H5) permit binding to and oxidation of phenols by the enzyme. Many of the shortened side-chain phenols are oxidized more rapidly than tyrosine (Naish-Byfield et al., 1991). Although they may be effective as competitors of tyrosine binding to the enzyme, the majority of these agents are not competitive inhibitors of melanogenesis but exert their depigmenting effect by a cytotoxic action on melanocytes. Generally speaking, the rate of phenol (or catechol) oxidation is determined by the relative ease of electron loss from the phenolic ring so that side-chain substituents that are electron donating in nature increase the tyrosinase-catalyzed oxidation rate (Passi and Nazzaro-Porro, 1981; Passi et al., 1987; Riley, 1975). For example, a methoxy side-chain results in a rapid tyrosinase-catalyzed oxidation, the rate of which exceeds tyrosine oxidation by several fold. Conversely, electronwithdrawing substituents can greatly reduce the oxidation rate. Trifluoromethylphenol is not oxidized by tyrosinase (Riley et al., 1997), whereas the hydrogen-containing analog

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

p-cresol is a rapidly oxidized substrate. In vitro, trifluoromethylphenol is a competitive inhibitor of tyrosine oxidation, i.e. it competes for occupancy of the substrate binding site but is not itself oxidized. An interesting possibility concerns agents that may bind to the secondary (side-chain) binding sites of tyrosinase and prevent access of the substrate to the active site. Of these, the simplest is probably glycine because its structure (CH2(NH2)COOH) would enable it to occupy the secondary binding site of tyrosinase without impinging on the active site pocket. The same considerations apply to l-alanine (CH3CH(NH2)COOH). Other amino acids could also be considered in this category, but structural considerations suggest that there could be several impediments to their binding to the enzyme. Recently, there has been interest in novel cyclic peptide inhibitors of tyrosinase (Morita et al., 1994a, b). It is interesting to note that glycine is a component of the tripeptide glutathione (GSH) and that some of the influence of GSH on tyrosinase activity (Jara et al., 1988) might be mediated by competitive substrate binding. If binding to either the amino or the carboxyl “receptor” sites were sufficient significantly to block the access of tyrosine to the active site, a host of potential competitive inhibitors is presented including agents such as polyamines and carboxylic acids. Among these could be counted dicarboxylic acids such as azelaic acid (Nazzaro-Porro and Passi, 1978), although the structural requirements with respect to the number of carbons separating the carboxyl groups imply that additional criteria need to be satisfied.

H2NCSCSNH2

dithio-oxamide

HSCH2CHOHCHOHCH2SH dithiothreitol (DTT) (CH3CH2)2NCSSH

diethyldithiocarbamate (DDC)

CH3CHSHCHSHOH

2,3-dithiopropanol

(CH3)2NCH2CH2SH

2-mercaptoethyldimethylamine (MEDA)

HSCH2CH2NH2

cysteamine

HSCH2CHNH2COOH

cysteine

Fig. 18.2. Structural formulae of some potential copper-chelating agents.

Copper Incorporation The essential requirement for copper at the active site of tyrosinase was shown by Keilin and Mann (1938) and Kubowitz (1938). Their studies demonstrated that tyrosinase activity was inhibited by agents such as carbon monoxide, cyanide, diethyldithiocarbonate, and salicylaldoxime that react with copper and render it incapable of binding oxygen (see above). This inhibition was reversible by the addition of excess copper salts to the preparation. Later work by Lerner et al. (1950) and Nelson and Dawson (1955) demonstrated that the copper requirement was general and applied not only to tyrosinases of plant origin but also to the mammalian enzyme. It is now widely agreed that, although there are some differences in the detailed structure of the enzyme on an evolutionary scale (Morrison et al., 1994), the family of tyrosinases all possess copper at the active site probably with a configuration closely similar to that found in the enzyme from Neurospora crassa studied by Lerch and colleagues (Lerch, 1981, 1988; Winkler et al., 1981). In this model, the active site contains two copper atoms bound to histidine residues of the protein and positioned about 3.6 Å apart. The evidence reviewed by Mason (1956) indicated that monohydric phenol oxidation depended on these copper atoms being in the Cu(I) state in order to bind molecular oxygen (Mason, 1955). If oxygen binding is prevented, the enzyme is inactive against phenolic substrates such as tyrosine. It seems likely that oxygen binding is prevented by copper chelation by cyanide

Fig. 18.3. Structural formulae of 8-hydroxyquinoline and analogs.

and carbon monoxide, and it is possible that, if there is no impediment to access, dithiol reagents such as diethyldithiocarbonate and dithiothreitol can form inhibitory complexes with the active site copper atoms (Fig. 18.2). It has been reported that thioredoxin and 2,3-dithiopropanol inhibit tyrosinase by forming bis-cysteinate complexes with one of the copper atoms at the active site by a mechanism involving reduction of a disulfide bridge between cysteine residues located vicinal to the active site (Wood and Schallreuter, 1991). Searle (1970, 1972) demonstrated that local applications of the copper-binding agent 8-hydroxyquinoline produced striking patterns of hair depigmentation in C57Bl mice. A similar effect was exhibited by 8-hydroxyquinaldine (Fig. 18.3). Depigmentation was confined to hair in the growth phase, and 361

CHAPTER 18

Fig. 18.4. Structural formulae of some substrate analog tyrosinase inhibitors.

hair clippings showed that the affected hair was completely devoid of melanin except for occasional dark tips. Cessation of application was followed by the regrowth of normally pigmented hair, demonstrating that the inhibition of melanogenesis was not due to cytotoxic obliteration of the hair bulb melanocytes. 8-Hydroxyquinoline is widely used as an analytical reagent for copper and has antiseptic properties ascribed to its copper-chelating action (Albert, 1985). It seems likely that its depigmenting action is related to rendering tyrosinase copper deficient or binding to the active site. This latter alternative may be favored by the failure of other copper chelators to produce a similar effect; perhaps surprisingly, dithio-oxamide, 2,2¢-biquinoline, and 2,9-dimethyl-1,10phenanthroline (Fig. 18.3) produced no depigmentation. However, these agents, although efficient copper-binding compounds, do not possess a phenolic group and are less likely to gain access to the active site of tyrosinase. Another group of tyrosinase inhibitors includes agents that are able to bind to the active site copper atoms but are able to release only one electron. This group includes mimosine, a naturally occurring pyridone analog of dopa (Hashiguchi and Takahashi, 1977), kojic acid, a Streptomyces-derived antibiotic with pronounced depigmenting activity (Mishima et al., 1988), and tropolone (Kahn and Andrawis, 1985) (see Fig. 18.4). Early observations on the inhibitory effects of carboxylic acids on tyrosinase were interpreted, on the basis of their pHdependent effects, to act by interaction with the histidine ligands of the active site copper atoms (Krüger, 1955). It is difficult to envisage the circumstances in which such a reaction could occur, although compounds having such an action may be included in the group of weak inhibitors in the classification of Wilcox et al. (1985). Relatively powerful carboxylic acid inhibitors include substrate analogs such as benzoic acid, which probably act by occupying the substrate binding site. Benzyl hydroxamic acid (Rich et al., 1978) probably acts as an inhibitor by a mechanism similar to that of phenylalanine 362

and tryptophan (Chakraborty and Chakraborty, 1993). Another melanogenic inhibitor, phenylthiourea, acts in a noncompetitive manner on tyrosinase (Laskin and Piccinini, 1986), suggesting that it belongs to the class of “post-tyrosinase” inhibitors. This also probably applies to the histamine H2 agonists related to nordimaprit (S-(2-(N,N-dimethylamine)ethyl)isothiourea). A series of analogs was examined by Fechner et al. (1993), some of which had depigmenting activity despite increasing the level of tyrosinase activity. Some of the depigmenting action of these compounds was ascribable to a cytotoxic effect (see above). Conversely, the histamine H2 antagonists, cimetidine and ranitidine, were shown to stimulate tyrosinase activity and melanogenesis in B16 cells (Ucar, 1991). Melanogenesis inhibitors of unknown function include two compounds isolated from Streptomyces. These agents, known as OH-3984 K1 and K2, inhibit melanogenesis in B16 melanoma cells at low concentrations by a mechanism that is not thought to involve a direct action on tyrosinase (Komiyama et al., 1993). Currently, little is known about the details of copper insertion into the active site of the enzyme. It is possible that it occurs during protein synthesis in association with the three-dimensional folding of the polypeptide chain. However, the relatively large distances between the putative copperchelating histidine residues make it more likely that the apoenzyme is synthesized first, followed by metal insertion. It is unclear how the copper is transported to the requisite site, or where and how it is added to the apotyrosinase (Martinez et al., 1987). Free copper ions are normally kept at very low concentrations in the cell by scavenging by metallothionein. There may be specific pathways for the delivery of copper to enzymecontaining vesicles. Inhibition of such a mechanism could comprise a hitherto unexplored regulatory mechanism for melanogenesis. As the copper binding is by coordination bonds, it is conceivable that other metals could be incorporated into the enzyme. For example, it has been proposed that the active site of TRP-2 normally contains coordinated zinc atoms (Solano et al., 1994) and, in view of the structural similarity between tyrosinase and TRP-2, there seems to be no obvious structural impediment to the occupancy of the metal binding site by metal ions of appropriate size. Whether competition for the copper binding site of tyrosinase by exposure to high concentrations of metal ions such as iron, nickel, or cobalt influences the activity of the enzyme is not clear. However, Jara et al. (1990) have evidence that high concentrations of zinc are inhibitory to tyrosinase.

Glycosylation The major post-translational modification of the enzyme consists of asparagine-linked glycosylation producing an almost continuous spectrum of isoenzymes on electrophoresis (Ferrini et al., 1987). Carbohydrate addition increases the molecular weight to about 65 kDa (Hearing and Jiménez, 1989), which is consistent with the trypsin-treated material extracted from human melanoma cells (IGR1) by Wittbjer et al. (1990). The shorthand nomenclature of the main molecular forms of mam-

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

malian tyrosinase permits the stages in its synthesis and distribution to be summarized in the following terms: T3 = unglycosylated, newly translated tyrosinase complete with transmembrane domain [molecular weight (MW) ~58 kDa]; T1 = fully glycosylated enzyme with transmembrane domain (MW ~73 kDa); T1¢ and T2 = partially glycosylated forms of T3; T4 = soluble glycosylated tyrosinase (probably the active form in melanosomes, MW ~65 kDa). At present, little is known about the possible carbohydrate signals that may be involved in the protein sorting in the Golgi region of the cell. What is clear is that inhibition of glycosylation by a variety of agents results in inhibition of melanogenesis. Some of these effects may be exerted by modification of the carbohydrate moieties of the matrix proteins of premelanosomes, but the major feature of melanocytes treated with glycosylation inhibitors is loss of T3. This suggests that an important function of glycosylation is to prevent degradation of newly synthesized enzyme, although Imokawa and Mishima (1984) reported that the effect of glucosamine (a glycosylation inhibitor) was unmodified by the protease inhibitors phenylmethylsulfonyl fluoride (PMSF) or leupeptin. It is not clear to what extent the glycosylation of tyrosinase affects proteolytic attack at specific sites or the general degradation of the protein by masking vulnerable peptide bonds. It seems likely that the release of the soluble enzyme from the membrane-bound form is required as an activation step after fusion with matrix granules of the coated vesicles carrying the bound form of the enzyme. This activation process envisages proteolytic cleavage, the specificity of which may be ensured by glycosylation of other vulnerable sites. An important series of studies by Imokawa and Mishima have demonstrated the depigmenting action of a wide range of glycosylation inhibitors of varying degrees of specificity (Imokawa, 1990; Imokawa and Mishima, 1984, 1985, 1986). In brief, these show that tunicamycin and glucosamine produce reversible loss of melanogenesis associated not only with loss of sialic acid-rich T1 but also with a reduction in T3. These changes are associated with alteration in mannose-specific binding of tyrosinase to the lectin concanavalin A. Recovery of melanogenesis can be interrupted by deoxynojirimycin, castanospermine, swainsonine, and monensin, which inhibit carbohydrate chain elongation at different points (Imokawa, 1990). More recently, Terao et al. (1992) have investigated the effect of some novel glycosylation inhibitors on pigmentation in cultured melanocytes. The compound BMY-28565 inhibited melanogenesis without diminishing the levels of tyrosinase messenger RNA. Although degradation of the unglycosylated protein is the most probable explanation for the reported data, some misrouting of partially glycosylated enzyme cannot be excluded.

Effect of Thiols The regulatory action of thiols with regard to melanogenesis has been the subject of comment for many years. De Léobardy and Labesse (1934) reported some reduction in skin pigmentation in a patient with Addison’s disease treated by daily

injections with 100 mg of cysteine. Although this effect might have resulted from interference with the course of the disease, some other publications report the direct depigmenting action on skin or hair of relatively simple thiols. Cysteamine and 2mercaptoethyldimethylamine (MEDA) were shown to cause depigmentation in black goldfish (Chavin and Schlesinger, 1966) and in black guinea-pig skin (Pathak et al., 1966). Frenk et al. (1968) and Bleehen et al. (1968) found that MEDA was more effective than cysteamine. There are at least four mechanisms that might be invoked to explain these results: (1) thiols will bind to the copper atoms at the active site of tyrosinase and inhibit the enzyme; (2) there are 14 cysteine residues in the mammalian T4 tyrosinase according to the sequence published by Kwon et al. (1988). Some of these may be involved in intramolecular disulfide bridge, modification of which could have significant mechanistic sequelae; (3) thiols react with quinone intermediates of melanogenesis (see below) to modify the product; (4) some thiols may interfere with glutathione generation of the cell and render melanocytes vulnerable to the action of melanogenic intermediates. In addition, an effect of glutathione on recovery of melanogenesis in glucosamine-treated cells was reported by Imokawa (1989), suggesting that quite low concentrations (0.2 mM) of glutathione (GSH) are able to block the translocation of tyrosine to premelanosomes. In this context, it is of interest (and, perhaps, of significance) to note the existence of a cysteine residue in the cytosolic portion of the enzyme. If this intracytoplasmic “tail” acts as a signal peptide that determines the specificity of organelle fusion, it may be that access of tyrosinase to premelanosomes can be indirectly controlled by the redox status of the cell. Halprin and Ohkawara (1966) claimed that differences in the skin color of negroes and Caucasians were inversely proportional to their glutathione level. Benedetto et al. (1981, 1982) reported that the skin levels of reduced glutathione and glutathione reductase were inversely related to the darkness of the melanin in tortoiseshell (tricolor) guinea pigs. It seems likely that these latter effects are largely due to the posttyrosinase modification of melanogenesis, i.e. the production of pheomelanin by combination of thiol compounds with dopaquinone (see below). However, direct effects on enzyme activity may play a part in the regulation of melanogenesis. Thioredoxin reductase has been implicated in such a function (Wood et al., 1995), possibly by the action of reduced thioredoxin in altering the tyrosinase conformation by reducing intramolecular disulfide bonds and, in this connection, it has been shown that thioredoxin reductase is susceptible to inactivation by UVA and UVB (Schallreuter and Wood, 1989), thus suggesting a mechanism for light-stimulated tyrosinase activity. Richter and Clisby (1941) investigated the action on black rats of phenylthiourea. Although 1–2 mg was usually lethal, tolerance could be built up to a dose, administered in the drinking water, of 18 mg per day. After 27 days of administration, graying of the hair was seen. This occurred first behind the neck, then spread along the back and around the head, 363

CHAPTER 18

but the top of the head remained black. Normal pigmentation of the hair returned when the dosage was discontinued. Dieke (1947) studied the effects of three thioureas on black rats that had their hair clipped on one side only. Phenylthiourea in the drinking water again produced almost complete depigmentation of the hair at doses of 25 mg/kg, without affecting hair growth. Thiourea at 200 mg/kg had no effect on hair growth or pigmentation, whereas a-naphthylthiourea, given in the diet, suppressed hair growth, leaving the skin unpigmented. The effects of a-naphthylthiourea, but not those of phenylthiourea, were reversed by cysteine. Although thyroid hormone is of importance in pigmentation, both phenylthiourea and the ineffective thiourea produced similar degrees of hyperplasia and loss of colloid in the thyroid glands of treated animals, supporting the view that phenylthiourea causes depigmentation by inhibiting tyrosinase, which it does in vitro more effectively than thiourea. Phenylthiourea has also been tested as an inhibitor of repigmentation in regenerating fins of the platyfish Xiphophorus (Kull et al., 1954).

Tyrosinase Activity Phase I Melanogenesis, Divergence and the Lag Period The initial step in the biogenesis of melanin consists of the oxidation of tyrosine to dopaquinone (DQ), and this oxidation, together with a set of related reactions, is known as phase I melanogenesis (see Fig. 18.5). Since the first edition of this volume was published, new data on the rates of the chemical reactions involved in phase I melanogenesis have been obtained by pulse radiolysis. The orthoquinone, dopaquinone, formed by the action of tyrosinase on tyrosine, is very susceptible to spontaneous intramolecular and intermolecular nucleophilic attack (Land et al., 2004). Intramolecular Michael addition of the amino group in the side-chain of dopaquinone (reaction A, Fig. 18.5) leads to cyclization, resulting in the formation of cyclodopa. This indoline is rapidly oxidized by residual dopaquinone in a redox exchange (reaction B, Fig. 18.5), giving rise to the corre-

COOH NH2

HO tyrosine

tyrosinase

HO

O

A

COOH

COOH N H cyclodopa

HO

NH2

O

HO N

NH2

HO

COOH NH2

O H 2N

dopa

dopachrome

S

O

COOH

COOH

NH2

COOH 5-S-cysteinyldopa

D

O

COOH

HO HO H2N

dopaquinone

B

HO

C

S

COOH 5-S-cysteinyldopaquinone E

EUMELANIN HO

COOH

PHEOMELANIN

NH2

N HOOC

S

benzothiazine

364

O

F

COOH NH2

N HOOC

S

quinonimine

Fig. 18.5. Reactions of phase I melanogenesis and corresponding rate constants. The rate constants for the spontaneous reactions are as follows: A. 3.8/s (Land et al., 2003c); B. 5.3 ¥ 106/M/s (Land et al., 2003c); C. 3 ¥ 107/M/s (Thompson et al., 1985); D. 8.8 ¥ 105/M/s (Land and Riley, 2000); E. 10/s (Napolitano et al., 1999); F. 5/s (Napolitano et al., 1999).

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

sponding aminochrome, dopachrome. This redox exchange yields concurrently the catechol, dopa, which is essential to reduce the active site of tyrosinase, allowing it to become active as a phenol oxidase. Tertiary amine analogs of dopaquinone also cyclize but, in this case, yield indolium olate betaines that do not undergo redox exchange to form the corresponding aminochrome and enzyme-activating catechol (Clews et al., 2000; Cooksey et al., 1997). The fact that these tertiary amine derivatives of tyrosine are not oxidized by inactivated tyrosinase provides strong evidence that direct enzymatic catechol formation does not occur under normal conditions. The indirect formation of dopa accounts for the features of the “lag period” observed in vitro for tyrosinasecatalyzed oxidation of tyrosine, during which the rate of oxidation slowly accelerates to reach the maximum velocity. During this lag time, the concentration of catechol formed by the disproportionation of dopaquinone and cyclodopa gradually becomes sufficient to reduce all the inactive mettyrosinase to active deoxy-tyrosinase (Land et al., 2003a). Other factors influencing tyrosinase activation have been elucidated by pulse radiolysis studies of a series of secondary quinone amines (Land et al., 2003b). Such studies showed that the failure of tertiary amine-derived betaines to undergo redox exchange results from the absence of a second exchangeable proton. Intermolecular nucleophilic addition of cysteine to dopaquinone results in the formation of mainly 5-Scysteinyldopa (reaction C, Fig. 18.5) which, like cyclodopa, can undergo redox exchange with residual dopaquinone (reaction D, Fig. 18.5) yielding, initially, 5-S-cysteinyldopaquinone. Over a few tenths of a second, the latter isomerizes spontaneously to a benzothiazine via a metastable quinonimine (reactions E and F, see Fig. 18.5). Both eumelanogenesis and pheomelanogenesis stem from dopaquinone, and the divergent pathways of biogenesis may be described by the balance between the rate constants of the nonenzymatic reactions involved in the initial steps. Taking dopachrome and cysteinyldopaquinone as representative of the divergent pathways, it is possible to derive an “index of divergence” between eumelanogenesis and pheomelanogenesis using the experimentally determined rate constants of reactions A, B, C, and D. From the ratio of the products of the first two rate constants of eumelanogenesis (A and B) and pheomelanogenesis (C and D), it can be deduced that a crossover value for switching between predominance of

eumelanogenesis to predominance of pheomelanogenesis occurs when the cysteine concentration is 7.6 ¥ 10–7 M (Land et al., 2003c). As emphasized above, the essential oxidative step in melanogenesis consists of the tyrosinase-catalyzed generation of orthoquinones, the most reactive of which is dopaquinone. It has been proposed that some subsequent oxidations in the melanogenic pathway may be brought about by redox exchange reactions with dopaquinone and that therefore the regulation of the levels of dopaquinone is crucial to the control of melanogenesis (Riley, 1993). As the reduction product of dopaquinone is dopa, which is rapidly reoxidized by tyrosinase, factors affecting the abundance of dopaquinone exert an important influence on melanin biosynthesis. The formation of dopa is of particular significance in relation to the activation of tyrosinase. Tyrosinase with the active site copper atoms in the oxidized [Cu(II)] state is unable to bind molecular oxygen and therefore cannot catalyze the oxidation of tyrosine. According to the “recruitment” hypothesis, the activity of tyrosinase is enhanced by the reduction of met-tyrosinase, which is accomplished by electron donation from dopa (Fig. 18.6). As dopa is formed as an intermediate product of tyrosine oxidation, this process is considered to account for the “lag” or induction period observed when monophenolic substrates, such as tyrosine, are oxidized by tyrosinase. This view is supported by the action of increasing tyrosine concentrations in lengthening the lag period, which argues that there is competition with the reducing substrate (dopa) for the substrate binding site. Also, the pH dependency of the effect is consistent with met-tyrosinase reduction being inhibited by increasing proton concentrations (Naish-Byfield and Riley, 1992). The generation of dopa in a tyrosine–tyrosinase reaction system is the result of the spontaneous reductive cyclization of dopaquinone to form cyclodopa, which is rapidly oxidized by redox exchange with dopaquinone to yield dopachrome and dopa (reaction B, Fig. 18.5). This process was first described by Evans and Raper in 1937, and the kinetics of the redox exchange were established by pulse radiolysis by Chedekel et al. (1984). Thus, dopa is formed effectively by the disproportionation of the tyrosine oxidation product dopaquinone (Fig. 18.5). Dopaquinone may undergo a redox exchange resulting in the oxidation of a reducing agent, which does not readily form a melanoid polymer. Compounds in this category include

Fig. 18.6. Schematic outline of dopa oxidation by electron donation to the copper atoms in met-tyrosinase.

365

CHAPTER 18

ascorbate and hydroquinone, although the details of the chemistry are not straightforward. It has even been suggested that metal ions could be oxidized by such a mechanism (Palumbo et al., 1987). If the activation of tyrosinase is regulated by the prevalence of dopa, then removal of dopa would constitute an important regulator of melanogenesis. It has been suggested that dopa methylation by the enzyme catechol-O-methyl transferase (COMT) may play such a role (Smit et al., 1994). Catechol methylation (Westerhof et al., 1987) plays a dual role in promoting removal and excretion of catechols and in preventing their oxidation (either enzymatic or auto-oxidation) to the potentially toxic quinone derivatives.

Biochemical Control of Tyrosinase Activity Oxidation of tyrosine to the corresponding orthoquinone involves an ortho hydroxylation and a dehydrogenation and takes place in a single step. For this reaction to occur, the active site must be oxygenated. Oxygen is bound in a peroxy conformation to the two copper atoms at the active site. This oxygen binding can only occur when the copper atoms are in the reduced form [Cu(I)]. This is because each copper atom at the active site of the enzyme donates an electron to molecular oxygen, resulting in the peroxy conformation. The peroxy group forms coordination bonds with the resultant Cu(II) copper atoms. This arrangement is usually indicated as a symmetrical binding with partial coordination bonds to the two copper atoms. Tyrosinase with oxidized copper atoms [Cu(II)] is unable to bind oxygen and is inactive toward tyrosine because its oxidation requires oxygen insertion into the ring (Mason et al., 1955). In this form, it is known as met-tyrosinase, by analogy with met-hemoglobin. The investigations of Lerch (1981) and Solomon and Lowery (1993) have demonstrated the reaction mechanism of monohydric phenol oxidation. This consists of a cycle in which deoxy-tyrosinase first binds molecular oxygen and subsequently the substrate is coordinated to the copper atoms at the active site. A series of proton exchanges results in oxygen insertion in the phenolic ring (Mason et al., 1955), and electron donation from the copper atoms yields the orthoquinone and one molecule of water. In this process, the deoxyenzyme is regenerated. The cycle involved in catechol oxidation is different. Coordination of the catechol to the copper atoms to which oxygen is already coordinated results in the breakdown of the complex with transfer of electrons from catechol to the oxygen to give the corresponding orthoquinone and two hydroxyl ions, leaving the copper of the enzyme in the oxidized (met) state. The met-enzyme is able to coordinate a further molecule of a catecholic substrate and oxidize it to the orthoquinone by electron donation to the cupric copper atoms, regenerating the Cu(I) form of deoxy-tyrosinase that is able to bind molecular oxygen. There are, therefore, two potentially competing oxidation cycles catalyzed by tyrosinase. In both cases, the substrate binding site is the same, involving the two copper atoms, and the products in each case are orthoquinone 366

and water. As, in the first stage of catechol oxidation, two hydroxyl ions are formed and in the second-stage reaction two protons are generated, there is the potential for a pH dependency of the reaction rate, which has complications regarding the lag period, and hence the maximum velocity of monophenol oxidation. There is evidence that newly synthesized tyrosinase is routed to lysosome-like vesicles, and it has been argued that the natural microenvironment of the enzyme contains a high proton concentration (Bhatnagar et al., 1993; Devi et al., 1987, 1989). It may be that pH shifts contribute to the physiological regulation of melanogenesis and the matter has not finally been resolved. An interesting phenomenon is the stimulation of tyrosinase activity in vivo by polyamine antimetabolites (Kapyaho et al., 1985). This may indicate that polyamines inhibit melanogenesis by a lysosomotropic action, although arguments have been advanced in refutation of this proposal. Kapyaho and Janne (1983) showed that stimulation of melanogenesis by 2-difluoromethylornithine (DFMO) was suppressed by putrescine, whereas MSH-stimulated tyrosinase activity was unaffected, arguing for an effect on transcriptional control.

Reactions of Intermediates and the Hazard to Melanocyte Survival An important aspect of the melanogenic pathway involves addition reactions. There are several reactive orthoquinone intermediates that can take part in such reactions, but dopaquinone is particularly susceptible to attack by nucleophiles such as thiol compounds. When these reactions are confined to the melanosomes, they merely constitute aspects of the formation of different variants of melanin. But, should the intermediates leak into the melanocyte cytosol, reductive addition reactions may constitute a cytotoxic hazard to melanocytes, as pointed out by Hochstein and Cohen (1963). There is evidence that some catecholic compounds formed by reductive addition are capable of being reoxidized by tyrosinase but whether they act directly as reducing substrates for the enzyme or are oxidized secondarily by a dopa-DQ redox cycle is not clear, although some in vitro evidence favors direct substrate action. Under conditions in which only small amounts of dopa are available, the addition of a suitable thiol reagent (e.g. cysteine, GSH) is a mechanism for accelerating tyrosine oxidation by the provision of a reducing substrate able to recruit mettyrosinase (Naish and Riley, 1989; Naish-Byfield et al., 1994). Such a process may underlie some aspects of the physiological control of melanogenesis by agencies modifying the access of thiols, such as glutathione, to the melanosomes. This mechanism appears to be implicated in the control of melanogenesis, e.g. in agouti hair, and may have other, more general significance. Conditions in which there is a relative thiol deficiency, such as in cystinosis (Lignac–Fanconi syndrome), may therefore exhibit hypomelanosis by virtue of interference with a thiol-dependent activating mechanism for tyrosinase. The reoxidation of catecholic thiol adducts by tyrosinase appears to be the basis of the synthesis of pheomelanins

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

(Prota, 1980, 1992). This pathway has been well characterized in the case of the benzothiazine-based pigments found in red chicken feathers, and a similar, though partial, process is regarded as the route to the sulfur-containing red–brown melanins, although the details remain unclear in most cases. It is likely that the diminished absorption of red light by pheomelanins is a reflection of shorter polymer length and the relative paucity of carbonyl groups in the fully oxidized form of the pigment. This implies that immediate pigment darkening (IPD) is a less prominent feature of pheomelanins with the concomitant generation of free radical products. An inhibitory action on melanogenesis by several 2-aryl-1,3thiazolidines has been shown by Napolitano et al. (1991). The main material studied was 2(2-hydroxyphenyl)-1,3thiazolidine-4-carboxylic acid, which prevented equimolar concentrations of tyrosine from tyrosinase-catalyzed conversion to melanin by the formation of a colorless product identified as b-(7-(3-carboxy-5-hydroxy-3,4-dihydro-2H-1,4benzothiazinyl) alanine. This was formed through an intermediate adduct. The authors conclude that 1,3-thiazolidines inhibit melanogenesis by trapping the tyrosinase-generated dopaquinone by the thiol-containing Schiff’s base arising from cleavage of the thiazolidine ring, followed by hydrolysis and ring closure to form a colorless product. An interesting aspect of the reaction of dopaquinone with thiols is the observation that certain thiourea derivatives selectively bind to nascent melanin. By analogy with the reaction of cysteine, glutathione, and dithiothreitol (DTT) with DQ, the process consists predominantly of thiolate ion addition to the 2- or 5-position of the ring. This has been confirmed in the case of thiourea (Palumbo et al., 1990). Few studies have examined the kinetic or regulatory effects of the binding of these compounds. Their major interest has been in connection with their use as carriers of radioisotopes (Broxterman et al., 1983), which can be used to label sites of active melanogenesis.

Protective Mechanisms It is likely that the evolutionary development of pigmentation, which includes the retention of the oxidative enzymes in the cell (as opposed to their secretion as occurs in lower eukaryotes), necessitated the fusion of the tyrosinase-containing vesicles with another membrane-bound structure in which the process of melanogenesis could be contained. In normal circumstances, the membrane surrounding these organelles (melanosomes) serves to isolate the diffusible water-soluble reactive products such as orthoquinones from the cytosol. The formation of melanosomes involves the fusion of at least two vesicular components, one that contains stromal material that forms the framework for subsequent melanin deposition and the other containing tyrosinase and associated melanogenic enzymes. Clearly, this fusion is a potential target for both physiological and pathological inhibition of melanogenesis. At present, little is known about the controlling processes. It is possible that modification of vesicular membrane structure and fluidity or cytoskeletal elements could

interfere with normal fusion. This might account for some of the actions of retinoic acid (Orlow et al., 1990; Ortonne, 1992), although the melanogenic inhibition exhibited in UVBstimulated melanocytes exposed to both cis- and trans-retinoic acid has been shown to be associated with diminished tyrosinase (and TRP-1) synthesis (Romero et al., 1994). Also, some effects of flavonoid extracts of St John’s wort on melanogenesis have been reported (Sattler and Schutt, 1994), which might act through an effect on vesicular membranes. Another potential control point is the intramelanosomal availability of the melanogenic substrate. A study of tyrosine transport in a human melanoma cell line (SK-mel 23) by Pankovich and Jimbow (1991) showed that tyrosine uptake is largely through system L transport, which supplies the tyrosine for both protein synthesis and melanogenesis. Tyrosine uptake by cells was inhibited by the analog substrate 4-Scysteinylphenol and by tryptophan as well as the specific system L inhibitor 2-amino-bicyclo-2,2,1-heptane-2-carboxylic acid. Similar data were published by Jara et al. (1991). Although this mechanism is responsible for the cellular uptake of tyrosine, there may be separate permeases to regulate the access of tyrosine to the melanosome because the process is more analogous to amino acid export. Outward diffusion of potentially cytotoxic products of melanogenesis is thought to be minimal in normal melanocytes, although the existence of melanogens in the blood and urine suggests that some leakage does occur, or that there is extramelanosomal catechol oxidation. There are several cytosolic processes that are invoked as cytoprotective mechanisms and have been reviewed by Smit et al. (2000). The intermediates that may reach the cytosol consist essentially of quinones liberated directly or are generated by oxidation from hydroquinones that leak through the melanosomal membrane. The main pathway of quinone detoxification is by quenching by glutathione with the formation of S-glutathionyl adducts, which are subsequently degraded. In some cells, quinone reductase (DT-diaphorase) may be invoked as a defense mechanism (see Smit et al., 2000). However, one of the major defense mechanisms appears to involve the inactivation of dihydroxyindoles by O-methylation. This pathway, first demonstrated by Axelrod and Lerner in 1963, is catalyzed by the enzyme catechol-O-methyl transferase. It was suggested that this methylation might act as a regulatory mechanism in melanogenesis, but the location of the enzyme outside the melanosome makes this unlikely. However, as a protective mechanism, O-methylation of catechols is likely to be important not only in preventing the formation of reactive orthoquinones but also in interrupting the possibility of free radical reactions generated by one-electron redox cycling involving semiquinones. There is direct evidence that O-methylation is related to the degree of melanogenesis (Smit et al., 1990, 1994). Elimination pathways probably involve glucuronidation and sulfonation as these derivatives of indoles are found in urine (Pavel et al., 1986), but it is likely that this is largely due to hepatic metabolism of the methylated derivatives formed in melanogenic cells. 367

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Toxicological Aspects of Melanogenesis In the context of acquired loss of pigmentation, there are good reasons to believe that the initiation of vitiligo may result from damage to melanocytes produced by a local factor. A model of vitiliginous depigmentation is provided by the cytotoxic action of a range of phenolic compounds. The first reported incidence of local depigmentation in man shown to be the result of phenols was in Negroes working in a tanning factory (Oliver et al., 1940). They developed patchy depigmentation of the hands and forearms due to contact with the monobenzyl ether of hydroquinone, which was used as an antioxidant in the production of the rubber gloves with which they were issued. Although the original authors demonstrated the absence of dopa-positive cells in the depigmented zones, it was initially assumed that the effect on melanogenesis was the result of competitive inhibition of tyrosinase (Denton et al., 1952; Lea, 1951; Lorincz, 1950; Peck and Sobotka, 1941). In an extensive survey of locally applied antioxidants, Brun (1961) showed that 4-hydroxyanisole (monomethyl ether of hydroquinone) had a powerful depigmenting action, and studies of this compound, its structural isomers, and a range of related phenols (Riley, 1969a, b) demonstrated that the depigmentation was the consequence of a cytotoxic action on melanocytes. Moreover, 4-hydroxyanisole (4HA) was shown to be a rapidly oxidized substrate of tyrosinase and the cytotoxic action to be due to one or more of the oxidation products (Riley, 1970, 1975). Subsequent investigations have established that the principal cytotoxic species is the corresponding orthoquinone (Naish et al., 1988a, b), which probably initiates melanocyte damage by thiol depletion (Cooksey et al., 1995). An account of these studies is to be found in Riley (1985) and Searle and Riley (1991). Because of the possibility that this tyrosinase-mediated cytotoxicity could provide a targeting mechanism for systemic antimelanoma treatment (Riley, 1970, 1984, 1991), there have been many studies of tyrosine analogs, and several alternative candidate compounds have been investigated (Jimbow et al., 1989; Mascagna et al., 1992). In the early work that demonstrated the cytotoxicity of tyrosine analogs (Riley, 1969a, b, 1970), attention was drawn to the potential hazard posed to the cell by the normal process of melanogenesis, as pointed out by Hochstein and Cohen (1963), and the consequent importance of the containment of the process in melanosomes (Borovansky et al., 1991; Riley, 1975). This led to the mistaken belief in some circles that a cytotoxic action on melanocytes could be achieved by augmentation of melanogenesis by the supply of normal tyrosinase substrates such as dopa and, in vitro, melanocytes exposed to relatively high concentrations of dopa are damaged (Wick, 1977). This effect is due to the oxidation products of dopa (i.e. dopaquinone or DHI) being formed in the medium, partly by auto-oxidation (Picardo et al., 1987) and partly by the action of small amounts of tyrosinase secreted by the cells. Some of the toxic effects elicited by this process may result from the generation of reactive oxygen species such as hydrogen peroxide (Kable and Parsons, 1988; Karg et al., 1991). 368

It has also to be borne in mind that dopa-like catechols may have significant pharmacological effects and that alternative metabolism may result in a plethora of toxic effects unrelated to melanogenesis (Inoue et al., 1990; Passi et al., 1987; Riley, 1984). There is no evidence of cytotoxic action or depigmentation by normal melanogenic substrates in vivo; no case of depigmentation has been recorded among patients receiving long-term, high-dosage l-dopa for the treatment of Parkinson disease. Of the wide range of phenols and catechols that are oxidized by tyrosinase (briefly reviewed by Prota, 1992), only a few exhibit significant melanocytotoxicity. In general, these are agents with side-chains that do not permit facile cyclization, that do not impede diffusion through phospholipid membranes, and that endow the quinone resulting from tyrosinase-catalyzed oxidation with an appropriate level of reactivity with regard to nucleophiles (Cooksey et al., 1992, 1995). Such materials are potential depigmenting agents, producing their effects by depleting the population of melanocytes in the epidermis or hair bulbs.

Diagnostic and Therapeutic Exploitation of Melanogenesis Detection of Nascent Melanin Some melanin-affinic substances, mainly thioureylenes, have turned out to be selectively accumulated solely in nascent melanin (reviewed by Larsson, 1991). The most studied substance in this regard is 2-thiouracil, and Whittaker (1971) was the first to demonstrate the incorporation into the melanin of the retinal pigment cell epithelium of chick embryo in vitro; he also found that the incorporation was tyrosinasedependent. By autoradiography, it was subsequently demonstrated that thiouracil accumulates strongly in the pigmented structures of murine fetal eyes in vivo, where the rate of melanin synthesis is high, whereas in the adult eye, where melanin synthesis is relatively low, only minute amounts of thiouracil were found (Dencker et al., 1979). The uptake in melanin was thus related to the melanin synthesis rather than to the occurrence of preformed melanin — thiouracil is lacking ordinary affinity to melanin (Dencker et al., 1981). Extended studies in melanoma-bearing mice demonstrated a corresponding uptake of thiouracil in the growing melanin of melanotic, but not of amelanotic, melanoma (Dencker et al., 1982). The specific localization of thiouracil and its analogous thioureylenes in nascent melanin has mainly focused interest on the possibility of targeted melanoma therapy, but the uptake may also implicate certain toxicological risks in connection with high-dose administration (see below). The sulfur ligand of thiouracil is apparently of crucial importance for the uptake in melanin, as uracil, which lacks sulfur, and 2-benzylthiouracil, where the sulfur is blocked, do not attach to the melanin (Olander et al., 1983). The thiouracil seems to be incorporated into the melanin structure as a false precursor, presumably through a nucleophilic attack on inter-

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

Fig. 18.7. Conjugation of 2-thiouracil with dopaquinone giving 6-S-thiouracildopa (3,4dihydroxy-6-(4¢-hydroxypyrimidinyl-2¢thio)-phenylalanine) — cf. Palumbo et al. (1990).

mediates of the melanin synthetic pathway, but the detailed biochemical mechanism behind the incorporation is still unclear. Palumbo et al. (1990) have shown previously that thiouracil is bound covalently to dopaquinone with the formation on 6-S-thiouracildopa (Fig. 18.7), and they concluded that the incorporation into melanin might be due to inglobation of the 6-S-thiouracildopa adduct within the structure of the melanin. More recently, they found that 6-S-thiouracildopa is rapidly oxidized by the melanogenic enzyme tyrosinase to a yellow chromophore, which ultimately forms an insoluble brown pigment (Palumbo et al., 1994). They also provided evidence for the ability of thiouracil to affect melanogenesis by the interaction with melanin intermediates beyond the dopaquinone stage, suggesting other possible modes for the incorporation into melanin. Recent studies in vitro have shown that, besides the thioureylenes, thioamides, 2-mercaptothiazoles, and 2mercaptoxazoles also interact with intermediates of the melanin synthetic pathway, which strongly indicates possible incorporation into growing melanin (Mårs and Larsson, 1995). A complicating finding, however, was that some of these substances acted as tyrosinase inhibitors which, in principle, might counteract the incorporation in vivo.

Toxicology of Nascent Melanin Toxicological risks associated with the incorporation of thioureylenes into nascent melanin, for example in growing fetal pigmented tissues, or in tanning skin and hair follicles, have been poorly evaluated so far. A change in the physicochemical properties of melanins, resulting from a significant incorporation of thioureylenes, might in principle impair the protective role of melanin (see above). In preliminary experiments, it has been found that the uptake of thiouracil and thiourea in synthetic dopa-melanin substantially increases the solubility (B. Larsson, unpublished results) and, with regard to the ability of melanin to oxidize nicotinamide adenine dinucleotide (NADH) (Gan et al., 1976; Van Woert, 1967), it has been demonstrated that the incorporation of thiouracil markedly decreases the oxidative capacity of the melanin in a dose-related manner (Larsson and Mårs, 1994). There are some indications on adverse effects in the living organism connected with the incorporation of thiouracil into growing melanin. Whittaker (1966) reported that thiouracil may cause a specific anomalous gigantism of the melanin granules in the otolith cells of ascidian larvae, reared throughout

embryogenesis in the presence of the drug, and the morphological changes were also accompanied by lighter color of the pigment granules. It is also known that propylthiouracil and methimazol, which are incorporated into melanin (Larsson et al., 1982; Olander et al., 1983), may cause loss or depigmentation of hair in humans (Goodman et al., 1985), indicating a toxic effect in the hair follicles where the rate of melanin synthesis is normally high.

Toxicity of Analog Substrates As discussed above, the potential hazard to melanogenic cells from extramelanosomal leakage of reactive intermediates is, in normal circumstances, contained by quenching mechanisms in the cytosol. However, the steady state may be disturbed by alterations in the propensity of intermediates to diffuse out of the melanosomes as a result of changes in concentration or diffusibility. This appears to be the case for a range of analog substrates for tyrosinase, of which 4-hydroxyanisole (4-HA) is a well-established example. The lack of a cyclizable sidechain increases the stability of the lipophilic quinone product of tyrosinase-catalyzed oxidation aiding its outward diffusion. Several studies have concluded that the major cytotoxic pathway involves the depletion of cellular GSH and attack on crucial nucleophiles, such as the cysteine residues in proteins with thiol-dependent functions (Cooksey et al., 1987, 1995, 1996; Land et al., 1990; Riley et al., 1997). The shortcomings of this approach as a possible anti-melanoma treatment seem to reside in the need for very elevated drug doses to overcome the cellular defenses and, recently, an alternative possibility has been investigated using analog substrates for tyrosinase that release cytotoxic drugs on cyclization of the derived quinone (Riley, 2003). This chemotherapeutic modality, known as melanocyte-directed, enzyme-activated prodrug therapy (MDEPT), is currently under development (Jordan et al., 1999, 2001).

Depigmentation Depigmentation may be either partial or complete, but the difference between hypopigmentation and total depigmentation may not distinguish between the mechanisms involved. Depigmentation may also be generalized or segmental. Of the pathologically significant depigmentations, the congenital category includes generalized depigmentation such as is found in those cases of albinism in which tyrosinase is totally inactive, the lesions being characterized by the presence of melanocytes that 369

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contain melanosomes but are not pigmented. In the segmental depigmentation characteristic of piebaldism, melanocytes are absent in the depigmented zones, but tyrosinase activity is normal in the cells in the pigmented areas. Generalized hypopigmentation is associated with phenylketonuria and, to a lesser extent, with cystinosis and homocysteinuria. These lesions are characterized by normal melanocytes with normal tyrosinase activity but in which the total amount of melanin synthesized is much reduced. Phenylketonuria was described by Følling in 1934 and was one of the earliest recognized disorders of amino acid metabolism. The basic lesion is a reduction in phenylalanine hydroxylase activity, and the conversion of phenylalanine to tyrosine proceeds at only about 1/10th of the normal rate in affected individuals (Coleman, 1962). The condition is inherited as a recessive trait and is characterized by decreased pigmentation of the hair and eyes (Fitzpatrick et al., 1961). If untreated, the condition is associated with mental deficiency (Jervis, 1937). The evidence that tyrosine acts as an inducer for tyrosinase (Wilde, 1955) is not strong and, therefore, the suggestion that the high phenylalanine concentrations in these patients may be responsible for depigmentation by repression of tyrosinase synthesis is probably not correct. Coleman (1962) has shown that a molar excess of phenylalanine over tyrosine as high as 70-fold is necessary to produce detectable inhibition of pigment synthesis by tyrosinase. As it is likely that it is the raised concentrations of phenylalanine found in untreated phenylketonuria that are responsible for the dilution of pigmentation in these patients, it may be proposed that the inhibition is brought about by preventing access of tyrosine to the melanosome (Farishian and Whittaker, 1980). Recent evidence has suggested the existence of melanosomal permeases responsible for the uptake and concentration of tyrosine in these organelles, and the similarity in structure of tyrosine and phenylalanine might suggest that inhibition of the permease is the means by which pigment dilution in these cases is brought about. In cystinosis, another inherited metabolic disorder, there is accumulation of cysteine in tissues, and this is associated with hypopigmentation of the skin and eye (Stenson et al., 1983). It is not clear what the mechanism of inhibition of pigment synthesis is, but it is possible that it involves the direct inhibition of tyrosinase by chelation of the copper at the active site of the enzyme. Homocystinuria, an abnormality of methionine metabolism, usually caused by a deficiency in cystathione synthetase, is also associated with hypopigmentation (Ortonne et al., 1983). Affected patients have blond hair, blue eyes, and fair skin. It is not clear whether the inhibition of melanin synthesis that is exhibited by these patients results from an effect on tyrosinase activity or an interaction with dopaquinone (see below). Among the acquired pathological depigmentations are the segmental depigmentation characterized by the partial or complete absence of melanocytes in the affected areas. This condition is known as vitiligo. The predisposition to this type

370

of depigmentation is inherited as an autosomal dominant trait and may be regarded as the acquired version of piebaldism. Although the degree of hypomelanosis is not strictly correlated with the total number of surviving melanocytes in the skin, complete depigmentation is associated with the total loss of functional melanocytes in the affected area. These have been designated absolute and relative vitiligo, respectively, depending on the complete or relative absence of dopa-positive melanocytes in the otherwise normal epidermis (Jarrett and Szabo, 1956). Vitiligo, like albinism, has been recognized for over 2000 years, and descriptions of it occur in the Indian sacred book Atharva Veda. There are many references to white skin in the writings of Buddhism, Islam, and Shintoism. It is likely that vitiligo was the cause of the depigmentation incorrectly referred to as leprosy in translations of the Hebrew Scriptures and, possibly as a result of this, patients with vitiligo were stigmatized as outcasts in many societies. Many vitiligo patients are acutely self-conscious of their cosmetic disfigurement, and Pandit Nehru regarded vitiligo as one of the conditions requiring urgent attention in India. Although vitiligo is more evident in the more darkly pigmented races, it is a disease affecting all ethnic groups with a prevalence of approximately 1%. Once the lesions appear, they tend to spread, and there is usually little chance of effective repigmentation, although strenuous efforts using photosensitizers have been made. Vitiligo generally occurs at sites of friction and trauma and in hyperpigmented areas. The segmental distribution is sometimes very dramatic. Although the pathogenesis of the condition is not well understood, there are two main etiological proposals: one based on the demonstration in some cases of autoantibodies to melanocytes, suggesting that the loss of melanocytes in certain areas is the result of an autoimmune reaction; the alternative is that the loss of melanocytes is due to an autotoxic mechanism resulting from the inability of the melanocytes to detoxify the intermediates of melanogenesis. A third proposal combining these two hypotheses would be that the initiation of the abnormality is caused by an autotoxic action resulting in the release of neoantigens from the melanocytes and the elicitation of a secondary autoimmune response by the host. Such a proposal would enable an immunological distinction to be made between melanocytes in different locations and would therefore account for the nonhomogeneous distribution of the lesions. Acquired pathological hypopigmentations should include the generalized hypopigmentation associated with hypopituitarism, in which there seems to be a generalized loss of MSH and adrenocorticotropic hormone (ACTH) stimulation of the melanocyte population with consequent reduction in overall tyrosinase expression. A number of agents have been reported to have depigmenting action on skin and hair. In some cases, the action may be indirect through the stimulation of inflammatory or immune reactions but, in the majority of instances, the mech-

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

anism is unknown. Spillane (1963) reported that the muscle relaxant mephensin caused reversible bleaching of hair in some patients, and local depigmentation has been described after intravenous administration of aldhesine (Coote and Abeysiri, 1979). Frenk (1980) described depigmentation from unidentified components of adhesive plaster, and reversible perioral depigmentation attributed to an ingredient of toothpaste (Mathias et al., 1980) has been reported. Poulsen (1981) reported reversible loss of hair pigment in patients taking the tranquilizer dexirosine. Acquired segmental hypopigmentations include a large group of conditions in which localized nutrition is compromised or where there is significant local ischemia. This condition occurs, for example, in chronic hypovascularization of the epidermis as in scleroderma and other collagen diseases. Acute ischemia is also sufficient to cause depigmentation. During experiments on cattle, Findlay and Jenkinson (1964) observed that a single injection of epinephrine (adrenaline) caused the local growth of white hair, and a similar observation was made in rats by Shelley and Ohman (1969). The effect in each instance was ascribed to the intense vasoconstriction caused by the epinephrine injection. Selye (1967) showed that ischemia produced by clamping skinfolds of rats for 8 h was sufficient to cause the regrowth of white hair at the site. A similar interpretation was put forward by Arnold et al. (1975) for the depigmenting effect of 33% triamcinolone in view of the powerful vasoconstricting effect of fluorinated steroids. The relatively low population density of melanocytes and their normally limited rate of proliferation in the adult may render them sensitive to damage. There is evidence that the melanocyte population is especially sensitive to low temperatures, and depigmentation by clamping or freezing is often used for branding animals. It is possible that the selective loss of pigmented hair is responsible for the many anecdotal reports of hair graying or whitening overnight. Ephraim (1959) reviewed a collection of cases of sudden hair whitening that were preceded by stressful experiences such as shipwrecks, railway catastrophes, and terrifying battle experiences and others by neurological disorders such as seizures, strokes, and brain injury. The beard of St Thomas More became white on the night before his execution, and the hair of Ludovicio Sforza is alleged to have turned white after his capture by Louis XII. It is reputed that Marie Antoinette’s hair turned white after the insults and abuse suffered by her family on their enforced return to Paris during the French Revolution. The most reasonable explanation for this phenomenon is that whitening is due to the selective loss of pigmented hair. Jelinek (1972) has shown that hair loss in alopecia areata is frequently confined to the pigmented hairs with white hairs remaining intact. One may postulate that the various traumas involved result in a prolonged peripheral vasoconstriction affecting the arterioles of the upper dermis followed by severe reperfusion injury damaging the pigmented hair bulbs.

Melanin Binding Mechanisms The mechanism of the binding, which has turned out to be complex, is dominated by electrostatic forces. Melanins are polyanions with a relatively high content of negatively charged carboxyl groups and ortho-semiquinones at physiological pH (Felix et al., 1978; Ito, 1986; Prota, 1992), and substances with cationic properties such as amines and metal ions are readily bound to the melanin by ionic interaction (Larsson and Tjälve, 1978, 1979). Not only aromatic amines (especially the polycyclic ones) but also aliphatic amines are bound (Tjälve et al., 1981). The ionic binding is apparently strengthened by additional forces, such as van der Waals’ attraction at the close apposition between the aromatic rings of the compounds and the melanin structure, as well as hydrophobic interaction, which may be rather pronounced (Larsson et al., 1988a; Stepien and Wilczok, 1982). In addition, involvement of charge transfer interaction has been indicated for electrondonating substances, mainly phenothiazine derivatives such as chlorpromazine (Larsson and Tjälve, 1979; Potts, 1964a). Using electron paramagnetic resonance spectroscopy and 63 Cu(II) as a probe in vitro, it was shown that synthetic dopamelanin and isolated beef-eye melanin have several different metal ion binding sites (Froncisz et al., 1980; Sarna et al., 1980). At pH 3, the interaction was dominated by a monodentate complex with the carboxylic groups of the melanin but, at higher pH, the binding was rearranged to a more stable mono- or bidentate complex with the carboxylic amine or carboxylic imine groups of the melanin. Other studies have shown that ferric ions bind predominantly to ortho-phenolic hydroxyls of the melanin (Sarna et al., 1980, 1981). The complexity of the binding has been demonstrated by Scatchard analysis, which normally shows that more than one binding class is involved for individual substances, including metal ions, indicating the presence of cooperating binding mechanisms as well as the influence of steric factors (Larsson and Tjälve, 1979). The binding energies are inversely proportional to the percentage binding, according to conformational analyses and molecular graphics used for the modeling of a representative melanin structure (Raghavan et al., 1990). Chlorpromazine, chloroquine, and methylene blue, which are strongly bound to melanin in vitro (Larsson and Tjälve, 1979; Potts, 1964a), accordingly showed the lowest binding energies, whereas the reverse was found for phenol and pyridine, which lack experimentally provable melanin affinity. Ionic interaction, however, was not taken into account in this study. The binding to melanin is normally reversible, but there are some interesting exceptions. Chlorpromazine and chloroquine, for example, are partly irreversibly bound in vitro (Larsson and Tjälve, 1979) and, as mentioned above, significant fractions of chloroquine and N,N-bis-acetanilidedimethylamine are retained in rodent uveal melanin 1 year after

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injection (see Fig. 18.1). At present, the mechanisms responsible for the irreversible interaction are poorly understood, but one possibility might be covalent binding. Another example concerns certain metal ions (copper, iron, and manganese) accumulated in natural melanin in situ, which cannot be removed by either prolonged acid treatment or exposure to strong chelators (Sarna et al., 1980), possibly because of strong multidentate complex formation. The exploration of the binding mechanisms and binding parameters has preferably been performed in vitro on natural and synthetic eumelanins. A few studies, however, have shown that even synthetic dopamine melanin (Ben-Shachar and Youdim, 1993; D’Amato et al., 1987; Larsson, 1979; Lydén et al., 1983, 1984; Wu et al., 1986), as well as natural human neuromelanin (D’Amato et al., 1987; Lindquist, 1973) and synthetic cysteinyldopa melanin (unpublished results), which is a model of pheomelanin, interact similarly with chemicals. The binding properties thus seem to be a general characteristic of melanins. The variety of chemicals with melanin affinity is large. Various drugs of quite different categories are represented, e.g. psychotropic drugs such as phenothiazines and other neuroleptics and tricyclic antidepressants, drugs for rheumatoid arthritis and malaria, local anesthetics, aminoglycoside antibiotics and so forth, and also other kinds of chemicals, for example herbicides, dyes, alkaloids, and metal ions (Larsson, 1979). The techniques used for in vitro studies of melanin affinity basically emanate from Potts (1964a), who used a suspension of isolated beef-eye melanin, which was incubated with test substance dissolved in buffer. Typically, 2.5 mmol of test substance is mixed with 10 mg of melanin suspended in 7 ml of buffer, pH 7.0. The incubation is performed at room temperature for 45 min, when equilibrium has occurred (cf. Larsson et al., 1977), and the pigment granules are then sedimented by centrifugation at 35 000 ¥ g for 10 min. The concentration of the unbound fraction in the supernatant is measured by spectrophotometry, or impulse counting when radiolabeled substances are used. The difference between the initial concentration of test substance and the remaining concentration in the supernatant gives the fraction bound to the melanin. In principle, any purified natural or synthetic melanin may be used for these studies and, if the test substance is poorly water soluble, buffered 33% ethanol may be used as solvent (Larsson et al., 1988b). Scatchard analysis (Scatchard et al., 1957) has proved to be useful for the estimation of detailed binding parameters, i.e. association constants and the number of binding sites on the melanin (Larsson and Tjälve, 1979). The in vitro model for studying melanin affinity has in most cases proved to be suitable for the prediction of melanin affinity in vivo (cf. below). Potts (1962a, b) was the first to demonstrate that phenothiazines are accumulated in the uveal tract of pigmented experimental animals. Whole-body autoradiography (Dencker et al., 1991; Ullberg et al., 1982) has been the most extensively used technique for mapping melanin affinity in vivo. Autoradiography differs from other biological 372

radioisotope techniques in providing more detailed information on the localization in tissues of a labeled substance, without laborious preselection of samples including risks of contamination. Using whole-body autoradiography, Lindquist and Ullberg (1972) found that chloroquine and chlorpromazine are strongly and selectively accumulated and retained in the melanin-containing tissues of pigmented mice, not only in the uveal tract of the eye, but also in the melanincontaining structures of the inner ear and the skin. A corresponding accumulation in fetal tissues was also demonstrated. In these studies, albino mice were used as controls, and they lacked corresponding uptake of label (Lindquist and Ullberg, 1972). During the following years, the melanin affinity in vivo of a great number of substances has been documented, mainly by autoradiography but also by impulse counting of excised tissue samples (for a review, see Larsson, 1979, 1995; Lindquist, 1973; Lindquist et al., 1987; Lydén-Sokolowski, 1990; Tammela, 1985; Wästerström, 1984). In most studies, pigmented mice have been used, but also rats, guinea pigs, hamsters, monkeys, frogs, and fish have been employed.

Kinetics of Uptake As mentioned above, the accumulation of certain melaninaffinic substances in pigmented tissues may be very pronounced. The highest concentrations in the body are often seen in the pigment cells soon after the injection and, about 2 days after the administration, the concentration of most xenobiotics has normally decreased to low levels due to excretion, except from the pigmented tissues where significant retention persists for weeks or longer. The importance of melanin in hair as a repository of substances to which a subject was exposed is important in both clinical and forensic toxicology. An interesting study by Testorf et al. (2001) employed the displacement from Sepia melanin of tritiated flunitrazepam in vitro as a means of measuring the binding and retention of a group of benzodiazepine tranquilizers. They observed rapid Langmuir binding followed by slower diffusion-limited uptake. They interpreted this as evidence of rapid surface binding with subsequent “bulk” binding. The competition by unlabeled drugs enabled the measurement of amounts of drug bound to melanin at low concentrations. Studies on the melanin affinity in vitro usually give accurate data of the intrinsic interaction, but the corresponding outcome of studies in vivo may still be different. This anomaly is mainly due to the interference of additional kinetic factors such as biotransformation and transfer through membranes. Aminoglycoside antibiotics, for example, have a pronounced melanin affinity in vitro but, because of their relatively high molecular weight, combined with positive charges at physiological pH, they show a marked extracellular biodistribution (with a few exceptions, such as endocytosis in the kidney) and do not reach the melanin in vivo (Larsson et al., 1981). Metal ions also show high melanin affinity in vitro but, in the living organism, rather few metals are accumulated in the pigmented tissues after a single injection (Lydén et al., 1984; Tjälve et al.,

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

1982), apparently because of membrane barriers or possibly metal binding competition between the melanin and proteins. On the other hand, significant amounts of various metals, e.g. Cu, Mn, Pb, Ti, and Zn, are normally found in pigmented tissues, indicating cumulative accumulation during chronic exposure (Zecca and Swartz, 1993). In this connection, it is of interest that the biodistribution of metals may undergo a considerable change after exposure to chelating agents, due to the formation of lipophilic complexes. Lead, for example, is not accumulated in the pigmented tissues of mice after a single injection but, after coexposure to dithiocarbamates, a pronounced melanin binding, in both fetal and adult eyes, has been found (Danielsson et al., 1984). Other examples are nickel combined with pyridinethione (Jasim and Tjälve, 1986) and thiram (Borg and Tjälve, 1988). The coexposure to metals and chelating agents is a potential toxicological problem, as organic compounds with chelating properties are relatively frequent, e.g. in the chemical industry, as pesticides, and even as drugs. The lipophilic metal complexes also readily pass across the blood–brain barrier, which increases the risk of metal binding to neuromelanin and the induction of central adverse effects. In this regard, recent studies by Tjälve et al. (1995) demonstrated that manganese cations are transported at a constant rate along the primary olfactory neurons of pike into the brain, thus circumventing the blood–brain barrier and gaining direct access to the central nervous system. Manganese is a neurotoxic metal that can induce extrapyramidal motor system dysfunctions in man, apparently associated with occupational inhalation of manganese-containing dusts or fumes.

Toxicology of Melanin-affinic Compounds There are many indications that the long-term accumulation of melanin-affinic drugs in pigmented tissues is a major cause behind the development of chronic lesions in the eye and the inner ear, in the skin, and possibly in the neuromelanincontaining nerve cells of the brain stem. The selective chemical stress on the pigment cells, due to a substantial increase in the local concentration of noxious substances, may also add to, or even be the main cause behind, the early aging processes that often are seen in melanin-containing cells. The most striking example is perhaps the early graying of hair, due to the deterioration of the melanocytes in the hair bulbs. Other examples are senile or presenile impairment of melanized structures in the eye and in the inner ear, with the development of certain types of cataracts or retinopathies, secondary to degeneration of the retinal pigment epithelium, and hearing loss due to strial atrophy. In the substantia nigra of the brain stem, the number of pigmented nerve cells normally decreases at the age of 55–65 years, without impairment of the extrapyramidal system, but an additional loss of neurons, induced by chemicals stored in neuromelanin, may ultimately cause extrapyramidal disturbances such as Parkinson disease or tardive dyskinesia (Lindquist, 1973). Although, in general, the uptake of chemicals by melanin affinity affects recipient cells in which the topological distrib-

ution of melanin in the cytosol renders them more susceptible to their effects, there are reports of melanocyte toxicity. A number of studies have shown that melanocytes are susceptible to damage by mutagens and carcinogens. During early experiments on the antitumor action of alkylating agents, Boyland et al. (1948) noted long-lasting bleaching of hair at the site of injection of nitrogen mustard in mice. A similarly localized depigmentation was found by Schoental (1971) in mice injected subcutaneously with N-methyl-Nnitrosourethane, a potent nitroso-carcinogen, and the injection of the same material intravenously into rabbit ears caused the appearance of depigmented hair over the ear veins, still visible after 18 months. Schoental (1971) suggested that the depigmentation was the result of degeneration of melanocytes. Schoental et al. (1978) found that T2 toxin, a carcinogenic metabolite of some Fusarium species, had a necrotizing action on topical application to the skin of mice and caused lasting depigmentation. Aw and Boyland (1978) found permanent depigmentation of hair in black mice at the site of injection of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which they ascribed to selective melanocyte toxicity and, in later experiments (Aw and Boyland, 1981; Aw et al., 1982), they showed that depigmentation of hair in mice injected subcutaneously with a range of alkylating agents, including chlorambucil, dimethylsulfonate, and methyl nitrosourea, was similar to that produced by phenols and some other substances including chloroform and carbon dioxide snow. Aubert and Bohuon (1970) reported depigmentation of hair in hamsters treated with dibenzanthracene. Animals treated with this agent orally became totally depigmented after about 1 month. These actions are consistent with the view that, as the melanocyte population density is low, pigment cells are particularly sensitive to the toxic action of a wide range of agents. The depigmenting effects of these agents should be distinguished from the mutagenic action of the agents, many of which have been shown to produce hair color alterations in the offspring of treated animals (see, for example, Carr, 1947; Lang, 1978; Russell, 1983; Strong, 1945, 1948). Most studies so far on chemically induced lesions in pigmented tissues have been focused on the eye for two reasons: the eye contains a rather high concentration of melanin, restricted to a few well-visible structures, making the eye a suitable model for the demonstration of melanin affinity in vivo. The other reason is that much of the original knowledge on melanin-related adverse effects of drugs mainly originated from the eye and, to some extent, from the skin and the inner ear. During the last decade, however, more attention has been directed toward central effects, mainly Parkinson disease, related to the neuromelanin.

Toxicological Mechanisms The development of specific lesions in pigment cells is obviously the consequence of a combination of selective retention, resulting from melanin binding, and the toxicity of a particular compound. For example, substances with low toxicity 373

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(based on the local molar dose) in practice only very rarely induce lesions, in spite of high melanin affinity, whereas substances with a more expressed or specific toxicity may induce the adverse effects in the pigment cells. The melanin thus serves as a chemical depot, from which the stored substances are released and slowly enter the cytoplasm (the binding is normally reversible), and the degenerative course in the cell is ultimately determined by the intrinsic toxicity of the individual substance. Chloroquine, for example, which induces chorioretinopathy and hearing impairment (cf. below), is known to be an intercalating agent inhibiting various biochemical reactions such as protein synthesis (Roskoski and Jaskunas, 1972) and the digestive activity of lysosomes (Homewood et al., 1972). The toxicity of a particular compound may also depend on biotransformation and possible bioactivation reactions, which may take place within the pigment cell or in its close vicinity. Below, some examples of melanin binding combined with bioactivation are given in connection with the discussion of separate target organs. The mechanism so far discussed is based on the premise that the melanin serves passively as a storing device for the toxic chemicals. There are many indications, however, that the binding per se between a substance and melanin may also change the physicochemical properties of the melanin with modification or impairment of its physiological function, ultimately leading to adverse effects. It has been proposed that an important role of melanins might be the protection of pigment cells from the attack of chemically reactive molecules such as free radicals, electrophilic metabolites, and excited, potentially harmful species induced by photochemical processes (for a review, see Larsson, 1979; Swartz et al., 1992). It is well known, for example, that melanins may participate in redox reactions as an electron transfer agent (Gan et al., 1976), which apparently underlies the protective capacity, but it may also involve the oxidation of drugs with the formation of noxious radical cations (Bolt and Forrest, 1967). The binding of certain chemicals, especially electron-donating molecules such as phenothiazines, may block the redox properties of the melanin, because of charge transfer interaction, with a possible decrease in the protective capacity. On the other hand, it has been proposed that the melanin in the retinal pigment epithelium can exert its scavenging and antioxidant properties by the binding of redox-active metals, e.g. iron, copper, and manganese, and the sequestration of potential photosensitizing agents (Sarna, 1992). Another possibility that has been proposed in various connections seems to be the participation of melanin in redox-cycling reactions with the production of reactive oxygen species such as superoxide anions, hydroxyl radicals, peroxides, or singlet oxygen (Swartz et al., 1992). The redox properties of melanin and its interaction with molecular oxygen have been reviewed (Sarna, 1992). The adverse effects normally have a chronic character, and they are related in most cases to high-dose/long-term exposure. A prominent feature of the lesions is that the histological changes are found initially in the melanin-containing cells, and successively in adjacent tissues such as receptor cells. The 374

onset of the adverse effects is often delayed, and the entire manifestation of the lesions may occur even years after cessation of the offending substance (Burns, 1966). The exposure to various toxic compounds with melanin affinity may also cause additive effects. The histopathological changes (reviewed by Lindquist, 1973) are usually characterized by enlargement of the cells, a marked increase in the number of melanosomes, and pigment deposits. Degeneration of the pigment cells occurs gradually with release of melanosomes and other cellular debris, which migrate into surrounding tissues where secondary side-effects can be induced.

The Skin Pigment deposits in the eye, induced by phenothiazines, may appear together with hyperpigmentation of the skin, the socalled eye–skin syndrome (Greiner and Berry, 1964). Chloroquine has also been reported to induce pigment disturbances in the form of deeply pigmented, usually blue–black macules and bleaching of the hair (Stewart et al., 1968). Excessive pigmentation of illuminated areas of the skin appear routinely in most patients treated with chloroquine during the summer, and the pigmentation slowly and incompletely disappears during the winter (Drew, 1962). These findings indicate the presence of photochemical reactions. The high accumulation in the skin and the hair follicles in pigmented (but not in albino) animals strongly indicates that the pigment disturbances are associated with the accumulation of these drugs in the epidermal melanocytes and the hair follicles (Lindquist, 1973). The main interest in the pathology of the pigment cells of the skin, however, has been focused on malignant melanoma. Melanoma induction is generally considered to be causally connected with UV exposure, combined with certain individual characteristics such as number of nevi, ability to tan, skin color, age, etc. (Evans et al., 1988; MacKie and Aitchison, 1982). According to critical examination of the literature, however, the traditional UV-oriented hypothesis seems not to be fully consistent (Koh et al., 1990), and chemical carcinogenesis has been suggested to be an additional cause of the disease (Larsson et al., 1993; Rampen and Fleuren, 1987). This idea is supported by a number of epidemiological studies on melanoma incidence among workers employed in chemical industries and the like (Albert et al., 1980; Austin and Reynolds, 1986; Barthel, 1985; Heldaas et al., 1987; Magnani et al., 1987; Pell et al., 1978; Thomas and Decoufle, 1979). In autoradiographic studies on the biodistribution of various carcinogenic compounds in experimental animals, a pronounced and specific accumulation of potent carcinogens in the melanin-containing cells has been demonstrated, for example aflatoxin B1 (Larsson et al., 1988a), tobacco-specific N-nitrosamines, benzidine, polycyclic hydrocarbons such as dimethylbenzo-[a]anthracene and benzo[a]pyrene (Larsson et al., 1989a; Roberto et al., 1996), and some mutagenic food pyrolysis products (Bergman, 1985; Brandt et al., 1983, 1989). Most carcinogenic substances, however, are procarcinogens, which need metabolic activation to become car-

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

cinogenic. It is well known that the epidermis and upper dermis, including hair follicles and sebaceous glands, contain inducible enzymes, e.g. aryl hydrocarbon hydroxylase, capable of bioactivating xenobiotics to electrophilic reactive intermediates (Pannatier et al., 1978). Of particular interest in this respect, it has been found that human melanocytes can metabolize benzo[a]pyrene to a number of metabolites, including the proximate carcinogen benzo[a]pyrene-7,8-diol, which demonstrates the presence of cytochrome p450 IA1,2 in the melanocytes (Agarwal et al., 1991). The combination of the specific retention, due to melanin affinity, and the presence of bioactivating enzymes in pigmented tissues strongly supports the idea of chemical carcinogenesis as an etiological factor in malignant melanoma. There are some reports on chemical induction of melanoma in experimental animals (e.g. Anders et al., 1991; Berkelhammer et al., 1982; Goerttler et al., 1980), but a problem in this regard is that most studies on chemical carcinogenesis have routinely been performed in albino animals, which are refractory to melanin-related risks; this is also a general problem concerning drug-induced adverse effects in pigmented tissues.

Ocular Tissues Chorioretinotoxic effects of phenothiazines were first reported from clinical tests of piperidylchlorophenothiazine (NP-207) (Goar and Fletcher, 1957; Kinross-Wright, 1956). Later on, other phenothiazines such as thioridazine and especially chlorpromazine were found to induce similar side-effects (for a review, see Lindquist, 1973). The effects were dependent on relatively high total doses; thioridazine, for example, was considered to be safe in this respect when used in daily doses of less than 800 mg (Siddall, 1966). Many of the patients who receive high-dose therapy over long periods with phenothiazines, especially chlorpromazine, develop pigment disturbances in the eye characterized by pigment deposits in the lens,

which can grow into irreversible cataracts in the most severe cases (Barsa et al., 1965; Siddall, 1966). Pigment deposits on the cornea and the anterior surface of the iris have also been reported (Edler, 1966). The pigment disturbances in the anterior part of the eye are secondary to damage of the melanincontaining cells of the iris, which gives rise to a release into the aqueous humor of pigment granules that are deposited on the corneal endothelium and the frontal surface of the lens. It is unclear why the pigment deposits on the lens surface may occasionally induce opacities and even cataracts, but one possibility that has hitherto been overlooked is that it might result from a transfer of noxious chemicals, stored on the melanin of the pigment deposits, into the lens parenchyma. Chloroquine is considered to be a more potent chorioretinotoxic substance than the clinically used phenothiazines (Meier-Ruge, 1965; Rubin, 1968). The primary lesions are normally seen in the retinal pigment epithelium, where the cells become significantly enlarged with substantial increase in the number of melanosomes, and pigment-laden cells are successively found in the retina (Bernstein and Ginsberg, 1964; Wetterholm and Winter, 1964). Large pigment deposits occurring in the choroid, ciliary body, and the optic nerve have also been reported (Dale et al., 1965). The basic research on the drug-induced, ocular side-effects were performed during the 1960s, and several studies have since confirmed the results. The chemically induced ocular injuries were better understood when the pronounced melanin affinity of the offending drugs, e.g. chlorpromazine and chloroquine (Fig. 18.8), was revealed (Lindquist, 1973; Lindquist and Ullberg, 1972; Potts, 1962a, b, 1964a, b). The retention of the drugs was always restricted to the pigment layers of the uveal tract (the choroid and the iris stroma) and the pigment epithelium, and was absent from the nonpigmented ocular tissues. No corresponding accumulation was seen in albino animals, which are also considerably less sensitive to the adverse effects. Long-

Fig. 18.8. Whole-body autoradiogram of a pregnant pigmented mouse (C57BL) 24 h after intravenous injection of 14C-chloroquine. The highest concentration of radioactivity is found in the maternal and fetal eyes. Some accumulation can also be seen in the skin, kidney, liver, gastric mucosa, and the pancreas, especially in the pancreatic islets.

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term oral administration of high doses of chlorpromazine to albino rats, for example, has been found to have no observable effect on the retina (Dewar et al., 1978). Subsequent studies have revealed a great number of drugs with melanin affinity (Larsson, 1979; Menon et al., 1982; Persad et al., 1986; Salazar et al., 1976; Zane et al., 1990), and the significance of the affinity for melanin in the etiology of toxic chorioretinopathy and ocular pigment disturbances has become well established. However, not all melanin-affinic compounds may be related to ocular toxicity. Leblanc et al. (1998) claimed that many drug-related toxic effects on the retina described in humans and animal models are unrelated to melanin binding and that melanin binding and retinal toxicity are separate entities, the toxicity being largely a factor determined by intrinsic tissue toxicity rather than melanin affinity. However, as discussed below, it is clear that slow release of melanin-binding drugs from depots in sites such as the eye is important in the regulation of ocular pharmacology (Salazar-Bookaman et al., 1994). As emphasized by Hu et al. (2002), it is clear that, in some cases, the melanin binding acts as a protective shield, but accumulation of potentially toxic agents by binding to melanin may expose ocular pigmented tissue to increased hazard.

Inner Ear The first demonstration of selective retention of drugs in the inner ear due to melanin affinity was reported by Lindquist and Ullberg (1972), and they concluded that there might be a causal connection between the melanin affinity and chemically induced ototoxic lesions. The melanin of the inner ear is present in melanocytes in the connective tissues of the labyrinth and in the modiolus, in epithelial cells of the stria vascularis in the cochlea, and in the vestibular part, mainly in the planum semilunatum adjacent to the crista ampullaris, and in the walls of the utricle and saccule (for a review, see Lyttkens et al., 1979). For the most part, the melanocytes are localized to well-vascularized areas of apparent metabolic and secretory importance, in close contact with capillaries (Savin, 1965). Stria vascularis and planum semilunatum, for example, are locations for the secretion and absorption of endolymph. It is well documented that the antimalarial agent quinine exerts toxic effects on hearing, which becomes permanent when given in large doses, and developing embryos appear to be exceedingly sensitive (Schuknecht, 1974). Prolonged use of chloroquine, which, like quinine, is a quinoline derivative, in the treatment of arthritis and other chronic diseases may also lead to ototoxicity (Dewar and Mann, 1954). Hart and Naunton (1964) reported ototoxic lesions in children whose mother had been treated with chloroquine throughout the pregnancies. Both chloroquine and quinine are strongly bound to the melanin of the inner ear (Dencker and Lindquist, 1975; Dencker et al., 1973; Lindquist, 1973). It is particularly interesting to note that the otic and the ocular lesions caused by these drugs have typical features in common, such as an association with high-dose/long-term therapy, late onset of the pathological course, and primary lesions induced in the 376

pigment cells. These points of agreement strongly favor the idea that the ototoxic effects are related to the specific uptake and retention of the drugs in melanin. The accumulation of chloroquine and quinine in the stria vascularis and planum semilunatum causes a local disturbance or even degeneration of these areas, with migration and deposits of pigment granules (even into the endolymph and to the organ of Corti), followed by secondary lesions in adjacent tissues, including the hair cells (reviewed by Lindquist, 1973). The degeneration of the stria vascularis and the planum semilunatum may impair the secretion and electrolytic composition of the endolymph (a high K+/Na+ concentration ratio), which is injurious to the hair cells (Johnsson and Hawkins, 1972; Müsebeck, 1963). Both dilation and narrowing of the strial capillaries have been reported in experimental animals treated with quinine (Rüedi, 1951), which is obviously connected with the morphologically close contact between the pigment cells and the blood vessels of the stria vascularis. Aminoglycoside antibiotics constitute another group of ototoxic drugs known to produce lesions in the receptor cells of the cochlea and the vestibular tract (Rüedi, 1951), possibly due to secondary alterations of the endolymph (Johnsson and Hawkins, 1972). According to clinical experience, kanamycin, neomycin, and dihydrostreptomycin exert their deleterious effect mainly in the cochlea, whereas streptomycin and gentamicin preferentially cause vestibular damage (reviewed by Wästerström, 1984). Several of these antibiotics are strongly bound to melanin in vitro (Larsson et al., 1981; Lindquist, 1973), but not in vivo after a single administration (Larsson et al., 1981; Nilsson Tammela and Tjälve, 1986). In these studies, it was found that gentamicin, injected intravenously in mice, was mainly localized in the perilymph and the membranous linings of the cochlea and in the floor of the cochlear duct, and dihydrostreptomycin showed a similar distribution in rats and guinea pigs. The toxicokinetic outcome of chronic exposure is unknown. On the other hand, it has been demonstrated that kanamycin is significantly more ototoxic in pigmented than in albino guinea pigs (Wästerström et al., 1986). Degenerative changes (Johnsson and Hawkins, 1972) and increased pigmentation (Nakamura, 1957) in the stria vascularis in animals given ototoxic antibiotics have also been reported. The results are thus in conflict as regards a possible role of the melanin for the ototoxic effects of aminoglycosides, and the problem needs to be investigated further.

Central Nervous System The observation by Mann and Yates (1983) that the most heavily pigmented nerve cells of the substantia nigra are the first to degenerate in parkinsonian patients strongly indicates that neuromelanin is somehow directly involved in the etiology of the disease. Several factors of importance for the development of parkinsonism have been proposed, e.g. heredity, aging, virus infections, drugs, and other environmental chemicals, but the latter, i.e. chemical factors, are considered to be most likely (for a review, see Lydén-Sokolowski, 1990). Phenothiazines and other neuroleptics, for example, are known

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

to be associated with extrapyramidal disorders such as parkinsonism, occasionally with irreversible symptoms, and tardive dyskinesia (Richardson and Craig, 1982; Schmidt and Jarcho, 1966). The syndrome has been claimed to be a hyperdopaminergic condition of the postsynaptic dopamine receptors (Carlsson, 1970), but this hypothesis has been questioned, for example by Christensen et al. (1970), who found structural degeneration in both the striatum and the substantia nigra of patients suffering from tardive dyskinesia. Although most phenothiazines are bound to melanin (Larsson, 1979), including chlorpromazine, which has been demonstrated to interact with human neuromelanin (Lindquist, 1973), it is unclear to what extent the melanin affinity is involved in the extrapyramidal disorders. A complicating factor so far has been the difficulty in obtaining suitable animal models for studies of melanin-related central effects. Most experimental animals are poorly pigmented in the substantia nigra and locus coeruleus, and laboratory animals such as mice, rats, guinea pigs, and rabbits have been reported to lack, or have only minute amounts of, neuromelanin (Barden and Levin, 1983; Marsden, 1969). In primates, the concentration of neuromelanin increases with age and with the relation to man, but the general impression in conjunction with histological examination of the brain of relatively young or adolescent monkeys, which are the most commonly used primates for experiments, is the presence of a rather low pigmentation in the substantia nigra. In addition, the use of monkeys for experiments is very expensive and ethically controversial. An animal model of potential interest has turned out to be the frog (Barbeau et al., 1985). In the ventral motor regions of the brain, frogs have pigmented neurons that correspond functionally to the pigmented nerve cells in mammals (Kemali and Gioffré, 1985). The nature of the amphibian neuronal pigment is not clearly understood, but histochemical staining has indicated that it is a melanin (Lydén-Sokolowski et al., 1989). A decisive event in the idea of chemical factors behind the etiology of Parkinson disease was the observation some years ago that drug abusers were affected with severe irreversible motor disturbances, similar to those seen in Parkinson disease, after administration of a synthetic heroin (Langston et al., 1983). Chemical analysis showed that the illicit narcotic preparation was contaminated with 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), which was found to induce selective destruction of the melanin-containing nerve cells of the substantia nigra (reviewed by Lindquist et al., 1987), without involvement of the dopamine receptors of the striatum, as revealed by positron emission tomography (Hartvig et al., 1986). MPTP is strongly bound to melanin in vitro, including synthetic dopamine melanin (Lydén et al., 1983) and human neuromelanin (D’Amato et al., 1987), and to neuromelanin of frogs in vivo (Lindquist et al., 1986). The degree of MPTP-induced neurotoxicity is apparently related to the amount of neuromelanin present in the substantia nigra. Man and other primates are considerably more sensitive to the neurotoxic effects than laboratory animals such as guinea pigs and rats (Chiueh et al., 1983) and mice (Hallman et al., 1984;

Heikkila et al., 1984), which are almost lacking neuromelanin (cf. above). The preferential destruction of pigmented nerve cells by the MPTP is dependent on local bioactivation in the brain. MPTP is oxidized to 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+) and ultimately to the pyridinium cation 1-methyl-4-phenylpyridine (MPP+) by monoamine oxidase (Chiba et al., 1984). Owing to its water solubility, MPP+ is retained in the dopaminergic zone of the brain where it is formed (Lydén-Sokolowski et al., 1988). MPP+ is bound to neuromelanin (D’Amato et al., 1986), and it has been shown that MPP+ is the species responsible for the neurotoxic effects (Langston et al., 1984; Markey et al., 1984). It has been found that MPP+ is concentrated in mitochondria by energydependent mechanisms, and that it inhibits NADH oxidation, leading to ATP depletion and possible cell death (Ramsay et al., 1986). At high acute dose levels, this mechanism should apply to all experimental animals, irrespective of the neuronal content of melanin but, at subchronic or chronic exposure to low concentrations, the local storage of MPP+ on the neuromelanin is apparently a prerequisite for the toxic effects (cf. Lindquist et al., 1987). In this connection, it is interesting to note that synthetic dopamine melanin can oxidize MPDP+ to MPP+, with the generation of hydrogen peroxide and hydroxyl radicals (Korytowski et al., 1987, 1988; Wu et al., 1986). The reaction is promoted by iron chelates, and it has been proposed that iron plays an important role in the development of parkinsonism (Ben-Shachar and Youdim, 1993; Zecca and Swartz, 1993). In the search for possible parkinsonism-inducing candidates, interest has been focused on the herbicide paraquat, which is structurally related to MPP+ and shows high melanin affinity (Larsson et al., 1977). Paraquat has been found to induce parkinsonian symptoms in frogs (Barbeau et al., 1985) and, by autoradiography, a selective accumulation of paraquat in the pigmented nerve cells of frogs (Fig. 18.9) has been demonstrated (Lindquist et al., 1988). It has been proposed that paraquat may participate in redox cycling (Johannessen

Fig. 18.9. Whole-body autoradiogram of 14C-paraquat in a frog (Rana temporaria) 1 day after intraperitoneal injection. Accumulation is evident in neuromelanin-containing nerve cells and in melanin-bearing cells in the meninges.

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et al., 1985), continuously generating noxious oxygen species, which is actually the mechanism for the lung toxicity of paraquat (Bus et al., 1976). During recent years, the main interest has been concentrated on neurotoxic metals. Manganese, for example, is known to induce an extrapyramidal disorder resembling parkinsonism (Mena et al., 1967; Rodier, 1955), with selective injury of the pigmented nerve cells of the substantia nigra (Gupta et al., 1980). Like MPTP, manganese is bound to melanin in experimental animals (Lydén et al., 1984), and it seems likely that the redox properties of manganese (similar to those of iron; see above) are of importance for the neurotoxicity. As mentioned above, neuromelanin may act as an antioxidant as a result of the ability to sequester redox-active metal ions such as iron (Korytowski et al., 1995) but it was also demonstrated that dopamine melanin saturated with ferric ions could enhance the formation of free hydroxyl radicals by redox activation of the ions (Zareba et al., 1995). It was concluded that neuromelanin may become an efficient cytotoxic pro-oxidant under conditions that stimulate the release of accumulated iron.

Fig. 18.10. Detail of an autoradiogram showing the cochlea of a young pigmented hooded rat 4 days after intraperitoneal injection of 3H-N,N-bis-acetanilidedimethylamine (QX-572). There is high uptake of radioactivity in the stria vascularis, basal membrane, and mediolus, corresponding to the presence of melanin.

Exploitation of Melanin Binding Pharmacology Specific accumulation of drugs in melanin-containing tissues has mainly been related to adverse effects, but it also implies certain potential pharmacological benefits. The most obvious example is the possibility of using melanin-affinic compounds for melanoma targeting, which is discussed separately below, but there are other potential applications as well. The basic difference between toxicology and pharmacology is a matter of dose, which means that the combined outcome of the therapeutic index of a drug and the stored concentration on melanin is decisive for a local toxicological vs. pharmacological effect (or even no effect whatever). As mentioned previously, melanin may temporarily protect pigment cells from the noxious effects of chemicals by acting as an adsorbing filter. A corresponding effect, from a pharmacological approach, is illustrated by the melanin-dependent mydriatic effect of atropine after topical application, described by Salazar et al. (1976). They found that atropine, which is bound to melanin, exerts a significantly stronger mydriatic effect in a light human iris than in a dark iris, which was explained on the basis of the accumulation of the atropine on the melanin, giving reduced concentrations of the drug in the vicinity of the muscarinic receptors. They also demonstrated a significantly increased duration of the atropine mydriasis in the pigmented iris compared with an albino iris, which neatly illustrates the significance of a drug depot retained on melanin.

Treatment of Tinnitus The storage of drugs on melanin for local therapeutic effects has not been put into ordinary practice so far, mainly because of the delicate and potentially hazardous balance between 378

toxic and pharmacological dose levels that need to be adjusted, but a few studies have been undertaken according to this principle. Lidocaine, which is a local anesthetic drug, is known to have a mitigating effect on tinnitus (Israel et al., 1982), and it is also bound to melanin, e.g. in the modiolus of the cochlea in rats (Englesson et al., 1976; Lyttkens et al., 1979). A few additional drugs with the combined properties of melanin affinity and alleviating effect on tinnitus have also been identified, viz. bupivacain and chlorpromazine (Lyttkens et al., 1979), tocainide (Larsson et al., 1984), and N,Nbis-acetanilidedimethylamine (QX-572), which is the quaternary analog of lidocaine (Lyttkens et al., 1984), and the results from autoradiographic experiments on rats and clinical trials on patients have indicated a connection between the storage of the drugs on the melanin of the inner ear and the relieving effect on severe tinnitus. A fundamental question discussed in this regard is whether lidocaine reduces tinnitus by a central or a peripheral mechanism of action. To elucidate this problem, the effect of lidocaine on tinnitus was compared with that of QX-572 (Lyttkens et al., 1984). As QX-572 is a quaternary ammonium compound, it does not pass the blood–brain barrier, which eliminates the possibility of a central effect. The results of the investigation, including both autoradiographic experiments as well as clinical studies, showed that the effects of lidocaine and QX-572 on tinnitus are mediated by a peripheral mechanism, apparently related to their accumulation on the inner ear melanin (Fig. 18.10) — both drugs exerted similar mitigating effects on tinnitus and uptake on the melanin of the inner ear, but only lidocaine passed the blood–brain barrier. As indicated above, it is of crucial importance that the therapeutic effects of the local

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

anesthetics strongly outweighs the toxicity of the drugs. Clinical tests have shown that no acute or chronic functional disturbances on hearing, related to lidocaine, are induced at dose levels of 1000–1500 mg, given at a therapeutic dose rate of 6 mg/kg/h (Englesson et al., 1976).

Photochemotherapy Skin conditions, such as vitiligo and psoriasis, may be treated by photochemotherapy (PUVA), which involves the combined effect of a photosensitizer and UV radiation, especially UVA (Honigsmann et al., 1987). Psoralens are commonly used as photosensitizers, and the acronym PUVA originally referred to the potentiating interaction between orally administered 8methoxypsoralen (methoxsalen) and UVA radiation. Methoxsalen is the most commonly used photosensitizer, but related furocoumarins such as 5-methoxypsoralen and 4,5¢,8trimethylpsoralen are occasionally preferred. PUVA therapy is generally regarded as the most effective and safe method for achieving repigmentation in vitiligo, even though interest in grafting is increasing. The effects of PUVA therapy are characterized by an increased number of viable melanocytes and melanosomes, activation of tyrosinase, and gradual migration of melanocytes at the border of the vitiliginous areas inward to form homogeneous pigmentation of the entire area (reviewed by Honig et al., 1994). The mode of action of the psoralens is not fully understood. According to the classical concept, the psoralens intercalate in the DNA, and the absorption of UVA photons leads in two steps to firm covalent bonding to pyrimidine bases, creating a cross-linked bifunctional photoadduct that inhibits DNA synthesis and delays epidermal cell division (Walter et al., 1973). After cell repair, the melanocytes divide into functionally active cells with enhanced pigmentation, but the full explanation of the beneficial effects in vitiligo is probably complex, with the potential additions of selective destruction of lymphocytes or suppression of an autoimmune response playing some role (Honig et al., 1994). It has also been proposed that PUVA therapy induces the formation of reactive oxygen species, which can damage membranes and cause deleterious metabolic changes (Pathak and Joshi, 1984). More recent studies have shown that PUVA treatment of hairless mice induces cutaneous depletion of GSH (Connor and Wheeler, 1987), and this finding was subsequently supported by experiments in vitro, demonstrating PUVA-induced photooxidation of GSH (d’Ischia et al., 1989), which indicates a direct connection between PUVA therapy and the critical role of GSH in melanogenesis (Prota, 1992). Turning now to the melanin and its possible role in PUVA therapy, it has been demonstrated by autoradiography that methoxsalen is bound to melanin, resulting in pronounced accumulation in the uvea of pigmented rats (Wulf and Hart, 1978). In similar studies on mice, a selective but transient uptake of methoxsalen was found in the pigment cells of the eye and skin, with a decrease in the concentration starting about 4 h after injection (Larsson and Mårs, 1993). The melanin affinity of methoxsalen, which is quite surprising

because the drug lacks functional cationic groups, was also demonstrated in vitro (Larsson and Mårs, 1993). The interaction is probably of hydrophobic character, which should explain the limited retention period after a single injection. By the means outlined above, the storing of methoxsalen on the melanin may in principle improve the outcome of PUVA therapy in pigment cells due to increased local concentrations of the drug but, as the mechanism of action obviously involves DNA cross-linking and other possible biochemical insults (cf. above), the melanin affinity may underlie side-effects as well. It has been reported, for example, that geese and ducks, chronically fed with Ammi majus seeds, which naturally contain 5and 8-methoxypsoralen, may develop uveal atrophy (Barishak et al., 1975) and pigmentary retinopathies (Egyed et al., 1975). These effects may in fact be the result of the interaction between the psoralens and UV radiation, as thorough measurements have shown that 30% UVA (365 nm) is transmitted by the lens (Bachem, 1956). Long-term exposure to PUVA is known to be associated with risks of skin cancer (reviewed by Stern, 1992), and there are a number of case reports that suggest a possible connection between PUVA therapy and melanoma. More extensive epidemiological studies have indicated a slightly higher incidence of melanoma in PUVA-treated patients than expected in the general population, but the increased risk found was not statistically significant (Gupta et al., 1988). It seems to be too early to state whether exposure to PUVA is associated with an increased risk of melanoma or not. Additional exposure to other skin carcinogens, e.g. arsenic or methotrexate, may complicate the assessment of epidemiological studies, and there is also some question as to whether sufficient latency time has passed from first exposure to PUVA for its full carcinogenic effect to be realized.

Monitoring Biological monitoring of the exposure to xenobiotics is in principle subordinated to both toxicology and pharmacology. The main areas so far have been the monitoring of exposure to occupational hazardous chemicals and environmental pollution, individual drug screening, the control of illicit abuse of narcotics and doping, and the analyses of tissue samples in forensic medicine. The body levels of foreign substances in urine and blood samples, which are easily accessible, may normally be determined within only a couple of days after exposure, solely giving information on acute exposure. Screening and quantitative assessment of chronic exposure or intermittent administration, on the other hand, is much more complicated and implies specific retention of a compound in a tissue that has easy access for biopsy and analysis. Hair has proved to meet these requirements for certain organic compounds, mainly because of interaction with melanin and keratin. Metals have been investigated most closely in this connection, and the binding to melanin has been established explicitly in a few studies (Cotzias et al., 1964). Subsequently, however, interest has been more concentrated on the accumulation of organic 379

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compounds in the hair, e.g. morphine for the determination of opiate abuse histories (Baumgartner et al., 1979; Marigo et al., 1986). Baumgartner et al. (1979) reported that differences in morphine concentration in sections of hair near the scalp and near the distal end correlated with the length of time the drug had been used, but the mechanism behind the accumulation is still obscure. There are a growing number of reports, however, on melanin affinity as the basis for the uptake, for example of industrial chemicals, monitored by mass spectrometry of hydrolyzed hair samples of workers (Larsson et al., 1988b), detrimental airborne impurities such as nicotine (Ishiyama et al., 1983), and various drugs (Ishiyama et al., 1983; Uematsu et al., 1990; Viala et al., 1983). Recently, a promising technique for the detection of illegal use of the beta-agonist clenbuterol as a repartitioning agent in meat production was developed (Appelgren et al., 1994a; Dürsch et al., 1995). Doses given for this purpose are 5–10 times higher than the therapeutic ones, and the illegal use of the drug has caused several cases of food poisoning (cf. Appelgren et al., 1994a). Clenbuterol is bound to melanin (Appelgren et al., 1994b), and the results from experiments on calves, fed with relevant doses of clenbuterol, have shown that it is possible to detect the drug in hair samples up to 14 days after cessation of the administration (Appelgren et al., 1994a). Most likely, it would be possible to detect the drug until the hair is shed, as the clenbuterol will probably stay in the hair once it has been incorporated. Similar experiments by others have shown that the concentration ratio of clenbuterol between black and white hair of calves is approximately 50:1, illustrating the importance of melanin-related retention (Dürsch et al., 1995).

Applied Toxicity of Melanin-affinic Agents A unique characteristic of melanotic melanoma is the presence of melanin, which may serve as a target for the diagnosis and treatment of the disease. Drugs with melanin affinity, e.g. quinoline derivatives labeled with radioiodine, have been used for melanoma scanning (Beierwaltes et al., 1968; Blois, 1968). A potential problem, however, is the corresponding uptake of label in normal pigmented tissues, for example in the eye and the skin. Some reagents may, however, prove valuable as diagnostic probes. For example, 123I-labeled iodoamphetamine and benzamides have been used successfully (Cohen et al., 1988; Labarre et al., 1999; Moins et al., 2002). In recent years, possible therapeutic application of melaninbinding substances has been attempted using methylene blue (MTB) as carrier of radionuclides (Link, 1992; Link and Carpenter, 1990, 1992; Link et al., 1989). Radioiodinated MTB was demonstrated to be suitable for scintimetric detection of melanoma and, when labeled with the a-emitter astatine-211 (with a half-life of 7.2 h), promising therapeutic effects on human melanotic melanoma xenografts in mice were found. In further studies of the histopathology in melanomabearing mice after the administration of therapeutic doses of 211 At-MTB, it was found that the radiation effects were restricted to the tumor cells, whereas no macro- or microscopic 380

lesions could be observed in normal tissues, including the melanin-containing portion of the eye (Michalowski et al., 1993). This may be explained by the dual contributions of the exposed position of tumor cells in general, due to rapid cell turnover (cf. above), and the finding that the accumulation of 211 At-MTB in the eye is delayed for several hours, i.e. one to two times the half-life of astatine-211 (Link, 1994). Another approach, tried mainly in experimental animals, is the use of radiolabeled melanin precursors, at least as far as melanotic melanoma is concerned, where the melanin formation is usually rather pronounced. Physiological precursors, such as tyrosine, dopa, and dopamine, have been examined with reference to their possible specificity for developing melanin (Blois and Kallman, 1964; Hutton and Pentland, 1976; Meier et al., 1967), but a limitation of this approach has been the uptake of the precursors in the synthesis of proteins and monoamines in normal tissues, which gives poor selectivity for the growing tumoral melanin. Artificial melanin precursors such as the thioureylenes, on the other hand, are more promising melanoma seekers because of their high specificity for nascent melanin (Larsson, 1991). Autoradiographic distribution studies on melanoma-bearing mice have demonstrated a selective and pronounced accumulation of label in the tumors, not only of 2-thiouracil (Fig. 18.11) (Dencker et al., 1979, 1982; Fairchild et al., 1982; Levin et al., 1983), but also of the radioiodinated analogs 5iodo-2-thiouracil and 5-iodo-6-n-propyl-2-thiouracil (Larsson et al., 1982; Van Langevelde et al., 1983), methimazole (Olander et al., 1983), thiourea (Mårs and Larsson, 1996), and 2-mercaptobenzothiazole (Fig. 18.12), but the latter was also found to be a strong tyrosinase inhibitor, which decreases the uptake in melanoma (Mårs and Larsson, 1995). Thiouracil and iodothiouracil are the most explored thioureylenes so far as regards possible clinical application. Their selectivity for melanotic melanoma, including metastases (Fairchild et al., 1982; Yamada et al., 1988), is striking, and the only normal tissue in which a significant accumulation and retention occurs is the thyroid gland as a result of the thyrostatic properties of thioureylenes in general. Uptake in the thyroid is essentially decreased by pretreatment with thyroxine, which acts through a feedback depression of the glandular activity and, if radioiodine is used as label, the uptake of radioactivity in the thyroid is prevented by pretreatment with potassium iodide (Larsson et al., 1982). The accumulation of thiouracil in murine melanoma increases almost linearly with the injected dose up to subtoxic levels, indicating nonsaturable incorporation into the melanin in vivo (Dencker et al., 1982). The clinical use is thus favored, especially when the specific activity of the radiolabeling is high, which is easily obtained with the use of short-lived radioisotopes. Palumbo et al. (1994) reported that the incorporation of thiouracil into melanoma tumors in mice follows very rapid kinetics, with the maximum being reached within 30 min. Previous studies have shown that the highest relative concentration in murine melanoma, compared with normal tissues, is attained at 24–48 h after injection (Dencker et al., 1982). If properly labeled with a clinically useful

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

Fig. 18.11. Whole-body autoradiogram of a mouse, subcutaneously transplanted with Harding–Passey melanoma, 24 h after two daily intramuscular injections of 14C-thiouracil. Highly selective retention of the radiolabel can be seen in the melanoma (the lower part of the tumor is necrotic). The concentration in normal tissues, such as the liver, kidney, lung, and eye, is relatively low.

2-Thiouracil

Fig. 18.12. Structural formulae of substances that are selectively incorporated into melanoma melanin in vivo.

Methimazole

radionuclide, diagnosis by radioscanning may thus be started soon after the injection, and the optimal time for differential diagnosis should be 1–2 days after the administration. Preparatory clinical studies on iodothiouracil have shown that the uptake in cultured human melanoma cells is time dependent and increases with the melanin content (Broxterman et al., 1983; Larsson et al., 1985). In both mice (Larsson et al., 1985) and Syrian golden hamsters (Franken et al., 1985), carrying transplantable melanoma, the accumulation of radioactivity was demonstrated by gamma scintigraphy after the injection of radioiodinated thiouracil. A couple of clinical pilot trials have also been performed. Franken et al. (1986a) compared the clinical outcome of ocular melanoma detection with 123I-thiouracil and 67Ga-citrate respectively.

5-Iodo-2-thiouracil

Thiourea

5-Dihydroxyboryl-2-thiouracil

2-Mercaptobezothiazole

Using a single eye probe collimator for the eye examinations, they demonstrated a significantly increased uptake of label in 7 out of 10 patients, with no false-positive results, and they concluded that 123I-thiouracil was at least as useful as 67Gacitrate for ocular melanoma diagnosis. A preliminary clinical experiment on gamma scintigraphy of cutaneous melanoma in patients injected with 131I-thiouracil has also been performed (Olander et al., 1991). The scanning was complemented by impulse counting of tumor and skin samples dissected out at surgery. Imaging was unsuccessful because of the use of too low radiodoses, but the results indicated that total doses of the order 150–200 MBq would be enough for the imaging. The extrapolation of data from experimental animal models to malignant melanoma of humans may in principle be 381

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unreliable with regard to factors such as the heterogeneity of pigment formation, the rate of proliferation, tumoral blood supply, and necrosis. These factors are usually rather reproducible in experimental melanomas, as distinguished from the tumors of patients, and the main problem of using thiouracils for melanoma targeting in patients is in fact the tendency of the metastases to lose pigmentation and become amelanotic. It has been estimated that about 7% of the primary cutaneous melanoma tumors are amelanotic, whereas 13% of the regional lymph node metastases and 31% of the distant metastases lack melanin (Shaw et al., 1985), but variations may occur between metastatic sites, as well as within single tumors (Watts et al., 1981). Amelanotic melanomas, however, may contain minor amounts of melanin (Sarna and Swartz, 1978) and, in view of this observation, the diagnostic potential of the thiouracils becomes more promising. In addition to melanoma detection, the thiouracils may be used for the assessment of the viability of treated melanoma, for example after X-ray irradiation therapy. In experiments with hamsters carrying ocular melanoma, Franken and coworkers (1986b) demonstrated that the uptake of 125Ithiouracil, after irradiation with 40 Gy X-ray, was reduced by 90% 12 days after the treatment. The results may contribute to a more adequate prognosis, as the regression of choroidal melanoma following radiation therapy is usually slow and may take months during which careful observations are necessary to indicate any possible reaction process (Lommatzsch, 1983). The usefulness of the thioureylenes for treatment of malignant melanoma is a matter of uncertainty, mainly because of the occurrence of amelanotic or poorly pigmented cells or metastases, but a few animal studies have been performed. To evaluate the therapeutic potential of thiouracil, mice transplanted with Harding–Passey melanoma were injected with high radiodoses of 35S-2-thiouracil in two independent studies (Fairchild et al., 1989; Olander, 1988). At total doses ranging from about 2 to 8 MBq/g body weight (Olander, 1988), there was on average a halving of the volume and weight of the tumors compared with untreated controls. At doses of about 18 MBq/g body weight (Fairchild et al., 1989), complete tumor regression with no regrowth was observed in some of the animals. It is clear that the radiodoses in these experiments were extremely high — the corresponding dose for a human patient, weighing 70 kg, would be of the order 130–1300 GBq, but comparable doses are more related to the body surface than to the body weight, which may in practice reduce the radiodoses required for the treatment of patients. The idea of using thiouracil as a vehicle for cytostatic agents has been tested in vitro by Wätjen et al. (1982). They investigated the effects of arotenoids and retinoids as 5-substituents of 2-thiouracil on Cloudman S91 melanoma cells. Despite selective uptake of the adducts in the tumor cells, no toxic effects could be noted. In a pilot study the chemotherapeutic effect of 5-(nitrogen mustard)-2-thiouracil on mice transplanted with Harding–Passey melanoma was tested, but 382

the results of this study were negative too. It might appear at first sight that this approach is doomed to failure, but it deserves to be explored in more detail with other cytotoxic ligands, because the discouraging results so far obtained are very preliminary. Thioureylenes may also be used as carriers of boron-10 for boron neutron capture therapy (BNCT). Boron-10, which is a stable nuclide, readily captures thermal neutrons and undergoes instantaneous nuclear fission into an alpha particle and a lithium ion with pronounced ionizing capacities (for a review, see Carlsson et al., 1992). The technique is based on the irradiation of tumors with a thermal neutron beam from an external source, after the accumulation of a boronated compound in tumor tissue and clearance from surrounding normal tissues. The range of the emitted particles is less than a cell diameter and, because of high linear energy transfer, the particles are very efficient in cell killing. The potential of this approach for melanoma treatment was first recognized by Mishima (1973) using a boronated chlorpromazine derivative for BNCT in melanoma-bearing pigs. Thermal neutrons may reach a distance into the body of about 4 cm, but the target depth may be doubled by the use of epithermal neutrons, which are moderated to thermal energies during the penetration of the top layer of tissue. At present, several reactors with epithermal neutron beams that meet with the clinical requirements as to purity and high flux density of neutrons are available, but the bottleneck of the technique is the poor availability of specific tumor seekers for the delivery of boron-10. Clinical experience of BNCT mainly originates from the treatment of brain tumors (reviewed by Carlsson et al., 1992), but trials on melanoma patients have also been performed. Mishima et al. (1989) used 10B1-p-boronophenylalanine (BPA) as a melanoma seeker, and they reported on successful treatment of a patient suffering from an inoperable malignant melanoma lesion on the left occiput. Additional patients have been treated more recently (Mishima et al., 1992). It was proposed that BPA accumulates selectively similarly to the melanin precursor tyrosine but, according to Coderre et al. (1987), BPA uptake in melanoma is dependent on active amino acid transport rather than melanin synthesis — they reported on accumulation of BPA in both melanotic and amelanotic rodent melanomas. Various thioureylenes have also been boronated and tested for melanoma specificity in experimental animals. 5-Dihydroxyboryl-2-thiouracil (Fig. 18.7) and its 6-propyl derivative, for example, were found to be selectively accumulated in B16 and Harding–Passey melanomas transplanted to mice, and the tumoral boron-10 levels as well as the melanoma/blood concentration ratios were in the range necessary for possible treatment by BNCT (Gabel, 1989; Tjarks, 1989). In these experiments, 10B-enriched preparations were used — naturally occurring boron consists of about 20% boron-10, but precursors for clinical use with an isotopic enrichment to 98–99% boron-10 are commercially available. In other experiments, thioureylenes have been boronated by adduct formation with

TOXICOLOGICAL ASPECTS OF MELANIN AND MELANOGENESIS

decaborane. Studies in vitro have shown that complexes between decaborane and, for example, 5-(diethylamino) methyl-2-thiouracil and 1H-1,2,4-triazole-3-thiol, respectively, are readily incorporated into dopa-melanin during its synthesis (Roberto and Larsson, 1989) and selectively accumulated in melanotic melanoma transplanted in mice (Larsson et al., 1989b). No boronated thioureylenes have come into clinical use so far, but the results from the animal experiments are quite promising and qualify for possible clinical application. As mentioned above, however, the use of boronated thioureylenes is limited to melanotic tumors, but it might be possible to optimize the treatment, i.e. to target all tumor cells, by combining the boronated thioureylenes with other melanoma seekers, e.g. BPA and/or boronated monoclonal antibodies. It should also be stressed that the use of BNCT is generally restricted to the treatment of metastases with limited spread, solitary metastases, and inoperable primary tumors, or to adjuvant therapy. The reason is the practical impossibility of giving whole-body exposure to neutrons (cf. Carlsson et al., 1992) and, as regards generalized melanoma, BNCT has to be combined with other suitable modalities, e.g. surgery, external radiation, or chemotherapy.

Perspectives The highly visible nature of surface pigmentation has made it a topic of interest to biologists for centuries, and the variations in human pigmentation are both remarkable and have many ramifications beyond the biological sphere. It is astounding that, despite the degree of interest in melanin pigmentation and the fact that the major pathway of melanogenesis was elucidated 80 years ago, our comprehension of the process and its control is so tentative. The fact that, in vertebrates, melanogenesis is the prerogative of specialized dendritic cells of neural crest origin is in itself an interesting feature and, in unraveling the mechanisms involved in the biogenesis of melanin, many insights have been gained into the complexities of subcellular organization, particularly with regard to vesicular traffic. Apart from leading to a greater understanding of phenomena associated with underlying systemic disease, the pragmatic importance of understanding the control of pigmentation is connected with the physiological functions of melanin, of which the best known is photoprotection. Modification of the degree of cutaneous pigmentation may be useful in the future, on the one hand providing adequate photoprotection to compulsive sun-bathers and possibly, on the other hand, in reducing the need for vitamin D supplementation of the diet necessary for highly pigmented individuals domiciled in regions of high latitude. Another area that could benefit from advances in the knowledge of melanogenesis is that concerned with cosmetic implications of surface pigmentation, particularly as there tends to be much anxiety created by regional pigmentary changes.

Modification of the degree of pigmentation could have important psychological benefits to patients. The research on melanin-related adverse effects of chemicals is presently most focused on neuromelanin and its possible role in the development of Parkinson disease and other extrapyramidal disorders, especially with regard to the interaction between redox-active metals and neuromelanin. The uptake of chemicals related to MPTP and, for example, neuroleptic drugs on neuromelanin is also of great interest in this respect. As mentioned previously, however, the lack of suitable animal models complicates the experiments, and another serious problem in this connection concerns the incomplete insight into the chemical structure of neuromelanin. Considerable efforts are presently being directed to the latter problem to obtain a reliable neuromelanin model for in vitro experiments. Not only the detailed nature and binding properties of neuromelanin, but also the properties of other extracutaneous melanins such as the melanins of the eye and the inner ear, which are found in both epithelial cells and melanocytes, are presently subjected to more extensive investigations. The binding of carcinogenic substances to melanin, as an etiological factor behind the induction of malignant melanoma, is another area of increasing interest. The role of enzymatic activation of procarcinogens stored on melanin has turned out to be of crucial importance in this connection, and it may be further elucidated by molecular biological or histochemical techniques for the mapping of bioactivating enzymes in pigment cells in situ. A related, and possibly underestimated, problem concerns the significance of photochemical reactions between substances stored on melanin and ultraviolet radiation. The development of melanoma seekers is presently found to be in a dynamic phase, mainly as the result of a better understanding of the mechanisms behind the incorporation of thioureylenes into nascent melanin, and the identification of new derivatives by screening techniques. The pharmacokinetic and dynamic properties of these substances need to be investigated in more detail before clinical application is advisable. Pharmacological applications of melanin affinity, in addition to the development of melanoma seekers, are more speculative. The use of melanin affinity for storage of drugs in pigmented tissues for local treatment of diseases, e.g. tinnitus, is complicated because of the delicate balance between therapeutic and toxic doses that need to be clarified and managed. The possibility of using melanin affinity for biological monitoring, on the other hand, is more realistic and presently subjected to further examinations. An important aspect of the study of pigmentation is the possibility of applying this process to chemotherapy for metastatic melanoma using the unique metabolic pathway of melanogenesis as the targeting modality. It is possible that the apparent sensitivity of melanocytes to freezing and reperfusion damage could be used in a similar way. The commercial advantage of being able to determine the hair color of, for example, wool-bearing animals may prove to be valuable, and 383

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advances in knowledge of mechanisms of branding by depigmentation may appeal to those governments wishing to barcode their citizens for easy identification.

Acknowledgments This chapter is based on two precursors: a general chapter on depigmentation by one of us (PAR) and a chapter by the late Bengt Larsson on the toxicology of melanin-affinic agents. We wish to acknowledge the great debt that we owe to Bengt Larsson in providing the firm foundation of his profound understanding of the binding of substances to melanins and his careful, balanced, and comprehensive review of the field.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Regulation of Pigment Type Switching by Agouti, Melanocortin Signaling, Attractin, and Mahoganoid Gregory S. Barsh

Summary 1 There are two major types of melanin pigment produced by mammalian pigment cells: black/brown eumelanin or red/yellow pheomelanin. Both require the enzymatic oxidation of tyrosine to form dopaquinone. During pheomelanin synthesis, dopaquinone is produced at relatively low levels and becomes incorporated into sulfhydryl derivatives. In contrast, eumelanin synthesis is associated with a high rate of dopaquinone production and subsequent enzymatic oxidation into indole derivatives. Studies based on coat color mutations in laboratory mice have identified several genes that regulate whether melanocytes produce pheomelanin or eumelanin; these genes and their homologs in other species are the subject of this chapter. A focal point for pigment type switching is the Melanocortin 1 receptor (Mc1r) gene (formerly known as Extension), which encodes a seven-transmembrane-domain receptor expressed by hair follicle melanocytes; gain-offunction Mc1r mutations cause exclusive production of eumelanin, whereas loss-of-function mutations cause exclusive production of pheomelanin. The Agouti gene (A) encodes the ligand for the Mc1r and is a paracrine signaling molecule secreted from mesenchymal cells in dermal papillae. Agouti protein inhibits Mc1r function such that gain-of-function Agouti mutations cause exclusive production of pheomelanin, whereas loss-of-function mutations cause exclusive production of eumelanin. Two additional molecules required for Agouti inhibition of Mc1r function are Attractin (Atrn), which acts as an accessory receptor for Agouti protein, and Mahoganoid (Mgrn1), an intracellular protein with E3 ubiquitin ligase activity. 2 The Mc1r is coupled to adenylate cyclase and is named after a family of receptors, Mc1r–Mc5r, that bind peptide ligands such as alpha-melanocyte-stimulating hormone (a-MSH) or adrenocorticotropic hormone (ACTH), which are derived from the same precursor, preproopiomelanocortin (Pomc). Activation of Mc1r by gain-of-function mutations or by addition of a-MSH causes increased accumulation of cyclic adenosine monophosphate (cAMP). However, Pomc is expressed primarily in the brain and pituitary gland, and most evidence suggests that basal levels of Mc1r activity in the absence of stimulatory melanocortin ligands are sufficient to induce

constitutive eumelanin synthesis. In most furry mammals, pigmentation patterns that depend on specific deposition of eumelanin vs. pheomelanin are controlled by dynamic changes in local levels of Agouti protein. In humans, however, a physiologic role for Agouti has not yet been established; the human Mc1r has a relatively high level of constitutive basal activity, and individual differences in the ratio of pheomelanin to eumelanin are controlled mostly by allelic variation of Mc1r. Most humans with red hair and fair skin carry a Mc1r lossof-function mutation; population genetic studies suggest that Mc1r function has been under positive selection in African populations, and that Mc1r-induced red hair and fair skin results mostly from genetic drift. 3 Homologs for Agouti and Mc1r are found in genomes from a variety of distantly related vertebrates, but not in invertebrate or Ascidian genomes. 4 Allelic variants of Agouti or Mc1r associated with altered pigment type switching phenotypes within populations of natural or domestic animals have been identified in sheep, cattle, horses, dogs, cats, pigs, foxes, bears, and several avian species. In addition, domestic cats and Syrian hamsters carry an X-linked coat color mutation referred to as Tortoiseshell or Orange, the effects of which are similar to that of the Mc1r but which is likely to represent a different gene. Finally, domestic dogs carry an autosomal coat color mutation referred to as Dominant Black (K) that affects the same pathway but is distinct from Agouti and Mc1r.

Historical Background Almost 100 years ago, studies by Sewall Wright on color inheritance in guinea pigs laid the groundwork for recognizing that mammalian melanocytes synthesize pigment of two different types, black/brown eumelanin or yellow/red pheomelanin (Wright, 1917a, b). Wright suggested that certain genes such as Agouti (A) or Extension (E) determined which of these two pigment types would be synthesized, while others such as Pink-eyed dilution (P) affected the quality of eumelanin but not that of pheomelanin. As described in Chapter 10, an important landmark in melanin research was the recognition in the 1920s that eumelanin synthesis involved oxidation of tyrosine to form an 395

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indole-based polymer, by a process now known as the Raper–Mason pathway. Because pheomelanin is soluble in dilute alkali and has a relatively high cysteine content, it was recognized to have a different chemical structure from eumelanin. Studies in the 1950s suggested that alternative amino acids might underlie differences between eumelanin and pheomelanin, but it is now clear that both pigment types are produced solely from tyrosine, and both depend on tyrosinasecatalyzed formation of dopaquinone. Thus, diversion of dopaquinone into the pheomelanic pathway serves as the fulcrum upon which genes that control eumelanin/pheomelanin switching are balanced (Fig. 19.1A). Interestingly, Sewall Wright suggested a biochemical pathway for pigment synthesis in 1917 that was based solely on genetic data (Wright, 1917a). Although details of his hypothesis later proved incorrect, an essential component — that eumelanin and pheomelanin shared both a common precursor and initial steps of oxidative metabolism — was confirmed nearly 50 years later. While the earliest studies of coat color genetics were carried out with guinea pigs as described above, mice are much better suited to breeding studies and so have provided most of the depth in mammalian pigmentation genetics (Wright, 1917c). This resource has arisen from three different sources. Many coat color variants that previously existed among communities of mouse fanciers or wild mice were incorporated into inbred strains during the early part of this century, such as the nonagouti (a) and tobacco (Etob or Mc1rtob) alleles at the Agouti and Mc1r loci respectively (reviewed by Morse, 1978; Silver, 1995). In addition, a large number of spontaneous mutations have arisen during the propagation of inbred strains, such as the mahogany (Atrnmg) or mahoganoid (Mgrn1md) alleles. Finally, systematic large-scale mutagenesis studies carried out at national laboratories such as Harwell (Lyon, 2002), Oak Ridge (Davis and Justice, 1998; Russell and Russell, 1992), and Neuherberg (Ehling et al., 1985; Favor and Neuhauser-Klaus, 2000) have focused on a small number of specific genes — the so-called specific locus test — and have therefore given rise to a large number of alleles for Agouti, Tyrp1 (formerly known as brown), Tyr (formerly known as albino), Myo5a (formerly known as dilute), Pinkeyed dilute, and Ednrb (formerly known as s or piebald spotting). The large number of mouse coat color mutations developed during the last half of the twentieth century provided the raw materials for classical studies of gene action and interaction, as described carefully and thoughtfully in the similarly titled book by Willys Silvers (Silvers, 1979). As molecular genetic tools became increasingly available in the 1980s and 1990s, most of the “classical” mouse coat color genes were cloned, leading to sophisticated explanations and models for how different genetic pathways give rise to specific cellular, tissue, and organismal phenotypes (Bennett and Lamoreux, 2003). A recurring theme from this work is that many of the genes and pathways used by the pigmentary system in laboratory mice are representative of physiologic processes used by other organ systems in all mammals; thus, studying the biology of mouse 396

Fig. 19.1. Biochemistry and genetics of pigment type switching. (A) In most biologic situations, synthesis of the two different pigment types behaves as a binary switch; either pheomelanin or eumelanin is synthesized, but not both. Genetic requirements and factors thought to “tip” the balance are described in the text. (B) Gene products and symbols are indicated together with their likely positions in melanogenesis. Dct (dopachrome tautomerase) and Tyrp1 (tyrosinase-related protein 1) catalyze oxidation of additional compounds after the formation of dopachrome (see Fig. 19.2A) that contribute to eumelanin. Several lines of evidence indicate that Dct and Tyrp1 are required for eumelanin but not pheomelanin synthesis (Kobayashi et al., 1995; Lamoreux et al., 2001; Prota et al., 1995). The P (Pink-eyed dilution) gene and MATP (membraneassociated transporter) are thought to facilitate the transport of small molecules across the melanosomal membrane as described in Chapter 12. Not shown in the diagram is the observation that P and MATP are required for normal levels of pheomelanin as well as eumelanin; however, studies of different coat color mutants indicate that loss-of-function for the P gene (Lamoreux et al., 2001; Prota et al., 1995) impairs synthesis of eumelanin more so than that of pheomelanin. As described in the text, the mechanism by which Mgrn1 is required for pheomelanin synthesis is not yet clear. One possibility (1) is that Mgrn1 normally inhibits the stimulatory activity of Mc1r, such that, in the absence of Mgrn1, Agouti is unable to overcome increased Mc1r signaling. In addition (2), Mgrn1 may be required to communicate signals brought about by Mc1r inhibition to the biochemical apparatus used for pheomelanin synthesis. (C) Summary of coat color phenotypes for various combinations of loss-of-function (lof) or gain-of-function (gof) mutants.

REGULATION OF PIGMENT TYPE SWITCHING BY AGOUTI

coat color helps to understand many aspects of human biology and disease. In the case of pigment type switching, molecular genetic studies of Agouti and Mc1r revealed that a homologous system in the brain, represented by Agouti-related protein and Mc4r, plays a key role in homeostatic control of energy balance and body weight (reviewed by Barsh and Schwartz, 2002). In addition, recent studies of Attractin and Mahoganoid suggest that studies of pigment type switching will prove helpful in understanding the pathogenesis of certain types of neurodegeneration (reviewed by He et al., 2003a). With increasing amounts of genome sequence and annotation available over the last several years, attention has once again turned to pigmentary genetics in animals outside the laboratory, including those of agricultural significance, such as cattle, horses, sheep, and pigs, and those in which unique aspects of their population history provide insight into the role of pigmentary phenotypes in ecology and evolution, including bananaquits, geese, pocket mice, domestic dogs, and humans (Andersson, 2003; Hoekstra et al., 2004; Kerns et al., 2003; Klungland and Vage, 2003; Mundy et al., 2003; Rees, 2003).

Current Concepts Eumelanin and Pheomelanin One of the most interesting features of this system is that, in many biologic settings regardless of scale, one finds eumelanin or pheomelanin, but not both. Pigment granules are generally classified as eumelanosomes or pheomelanosomes, hair follicle melanocytes switch abruptly and precisely between synthesis of the two pigment types, and color patterns in many different mammals are formed by juxtaposition of hairs that contain exclusively eumelanin or pheomelanin. Spots on a leopard, stripes on a tiger, dorsal–ventral differences in rodent coloration, and facial markings characteristic of many different dog breeds are each caused by regulation of an intercellular signaling mechanism and biochemical pathway that switches between synthesis of eumelanin and pheomelanin. Fundamental differences in chemical composition, ultrastructure, and biogenesis distinguish these two types of pigment and, although the differences are also frequently described in terms of visible reflectance — brownish-black (eumelanin) vs. reddish-yellow (pheomelanin) — this is an oversimplification. In particular, low levels of eumelanin (but an absence of pheomelanin) are characteristic of blond hair in many humans, whereas low levels of pheomelanin (but an absence of eumelanin) are characteristic of cream-colored hair in many animals (Ito, 1993, 2003; Ito and Fujita, 1985; Ito and Wakamatsu, 2003; Lamoreux et al., 2001; Ozeki et al., 1995; Prota et al., 1995). Thus, both the pigment type itself as well as the density and distribution of pigment granules in surrounding cells help to determine overall reflectance qualities; in situations where there are low levels of total melanin, visible characteristics can be deceiving with regard to the underlying type of pigment (Fig. 19.1B).

As described in Chapter 10, both pigment types require the enzymatic oxidation of tyrosine to form dopaquinone (by tyrosinase); however, during pheomelanin synthesis, dopaquinone is produced at relatively low levels and serves as a substrate for nonenzymatic addition of cysteine and redox exchange to produce cysteinyldopaquinone (Fig. 19.2A). In contrast, eumelanin synthesis is associated with a higher rate of dopaquinone production followed by nonenzymatic cyclization and redox exchange to yield dopachrome; subsequent enzymatic oxidation yields additional indole derivatives that serve as the building blocks for eumelanin (Ito, 2003; Land et al., 2003, 2004; Land and Riley, 2000; Ozeki et al., 1997). Thus, from a biochemical perspective, two cardinal characteristics of pheomelanin synthesis are low rates of dopaquinone production and a readily available supply of free cysteine. Indeed, a widely proposed theory to account for the ability of melanocytes to switch between eumelanin and pheomelanin synthesis posits that these features — low levels of tyrosinase activity and high levels of free cysteine — are necessary and sufficient for pheomelanin synthesis (Ito, 1993, 2003; Land et al., 2003, 2004). As initially proposed by Ito and colleagues (Ito, 1993), tyrosinase activity (and, thus, the rate of dopaquinone production) was suggested to be the primary determinant of whether or not dopaquinone gave rise to pheomelanin or eumelanin. In part, this proposal was based on the idea that reduced glutathione could serve as a thiol donor for dopaquinone, and that high levels of dopaquinone would inhibit glutathione reductase. More recently, studies of melanosomal transport indicated that cysteine rather than glutathione was likely to be the source of the thiol in cysteinyldopa (Potterf et al., 1999). Together with sophisticated techniques for measuring the kinetics of reactions with unstable intermediates, a more refined view of melanogenesis was reached (Land et al., 2003; Land and Riley, 2000), in which the ratio of pheomelanin to eumelanin production, or an “index of divergence,” could be estimated from rate constants of individual reactions in solution (Fig. 19.2A). At first glance, this approach predicts that the index of divergence depends only on cysteine availability, with approximately equivalent amounts of pheomelanin and eumelanin being produced at cysteine levels of ~ 1 micromolar, independent of dopaquinone concentration (Land and Riley, 2000). However, the underlying chemistry — in which a common precursor is utilized in a first-order reaction for one pathway but a second-order reaction for the other pathway — means that influx of dopaquinone indirectly controls the ratio of pheomelanin to eumelanin synthesis through its consumption of cysteine. Thus, only if cysteine levels were “clamped,” with influx rates rising and falling to maintain a constant steady-state level, would the ratio of pheomelanin to eumelanin be truly independent of dopaquinone production and, in practice, “the sensitivity of the system to the availability of cysteine is influenced by tyrosine uptake and tyrosinase activity” (Land et al., 2003). Advances in melanin chemistry notwithstanding, the preceding discussion does not adequately explain the abrupt 397

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Fig. 19.2. Contribution of cysteine and tyrosinase activity to pigment type switching. (A) As described by Land and Riley (Land et al., 2003; Land and Riley, 2000), formation of cysteinyldopa or cyclodopa from dopaquinone occurs spontaneously, and represents the initial step in pheomelanin or eumelanin synthesis respectively. In subsequent steps, cysteinyldopa or cyclodopa is oxidized to yield cysteinyldopaquinone or dopachrome respectively; both reactions proceed spontaneously and utilize dopaquinone as the oxidizing agent. Rate constants for these reactions in solution (r1–r4) have been measured by Riley and colleagues, and used to generate a model of how alterations in the availability of cysteine and the influx of dopaquinone influence the balance between synthesis of the two pigment types (Land et al., 2003; Land and Riley, 2000). As described in the text, this model predicts that cysteine concentration should exhibit a linear relationship with the relative rates of pheomelanin vs. eumelanin production, but does not easily explain how the “switch” between the two types of pigment occurs abruptly in many biologic situations. (B) The left-hand panel illustrates how changes in levels of Agouti expression are related to pigment synthesis. As Agouti expression increases, Mc1r signaling decreases and, at a midpoint, there is an abrupt switch from eumelanin to pheomelanin synthesis. In general, tyrosinase activity correlates with levels of Mc1r signaling. However, the exact shape of the curve is not known, and is shown here purely for purposes of illustration. In animals that carry a hypomorphic tyrosinase allele such as chinchilla (Tyrch), tyrosinase activity is reduced during the entirety of pigment type switching and, at high levels of Agouti expression, becomes insufficient to support any pigment synthesis. The right-hand panel illustrates this principle. Animals that express high levels of Agouti protein and carry a normal tyrosinase allele are reddish-yellow; animals that express no Agouti protein and carry a hypomorphic tyrosinase allele are black, but animals that express high levels of Agouti protein and carry a hypomorphic tyrosinase allele are cream colored. Quantitative measurements of melanin synthesis by Ito and colleagues support this conclusion (Lamoreux et al., 2001). In the absence of Agouti, the chinchilla mutation reduces total melanin from a level of 0.775 to 0.452 (absorbance 500 units/mg hair), almost all of which is pheomelanin; however, in the presence of Agouti (Ay/a), the chinchilla mutation reduces total melanin from a level of 0.093 to 0.023 (absorbance 500 units/mg hair); the latter value is so low that animals appear almost white. See also Plate 19.1, pp. 494–495.

nature of the “switch” itself, as the relationship between tyrosinase activity and the ratio of eumelanin to pheomelanin production is more logistic than linear (Fig. 19.2B). It also does not speak to the biogenesis of pheomelanosomes vs. eumelanosomes, which differ in their structure as well as in their melanin content. The former tends to be spherical with a granular and/or microvesicular lumen that shows little evidence of internal structure, whereas the latter tends to be 398

ovoid with a highly organized internal structure thought to be based on a proteinaceous matrix (Liu et al., 2004). Other than melanin itself, surprisingly little is known about the molecular anatomy that distinguishes pheomelanosomes from eumelanosomes. As described below, genetic observations point to the existence of components specific to eumelanogenesis vs. pheomelanogenesis that are clearly distinct from the pathways that modulate the switch itself; however, it remains to be deter-

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mined whether these components act by modulating the formation of dopachrome vs. cysteinyldopaquinone, by modulating specialized aspects of melanosome biogenesis, or both.

Melanocortin Receptors and Agouti Protein Ligands Molecular Genetics and Biology of Melanocortin Signaling Much of our current understanding of melanocortin receptor signaling is based on studies carried out on hormonal control of pigmentation in amphibians and reptiles (Lerner, 1993). Melanin-containing cells in these animals, melanophores, exhibit rapid changes in their absorption of visible light mediated by the intracellular dispersion or aggregation of microscopic pigment granules. This phenomenon underlies the ability of many reptiles and amphibians to adapt to their environmental background, and is mediated by a variety of different receptors for catecholamine, serotonergic, and peptide hormones. Recognition that extracts of mammalian pituitary glands could reproduce this process led to the isolation of aMSH, a 13-amino-acid peptide produced by proteolytic cleavage from a multifunctional larger precursor, Pomc (reviewed by Bertagna, 1994; De Wied and Jolles, 1982; Eberle, 1988). Besides a-MSH, Pomc also gives rise to gamma-MSH (gMSH), ACTH, and beta-endorphin. a-MSH, g-MSH, and ACTH (the melanocortins) each contain a common “core” His–Phe–Arg–Trp sequence thought to be responsible for the binding and activation of one or more of the five melanocortin receptors, Mc1r–Mc5r (reviewed by Cone et al., 1996; Schioth, 2001). These receptors were first isolated by screening human melanoma cDNAs for seven transmembrane receptor genes using a polymerase chain reaction (PCR)-based strategy, and identifying two receptors, Mc1r and Mc2r, that would cause intracellular accumulation of cAMP in response to melanocortin peptides (Chhajlani et al., 1993; Chhajlani and Wikberg, 1992; Mountjoy et al., 1992). Mc3r, Mc4r, and Mc5r were then isolated by sequence similarity; all five receptors couple to adenylate cyclase but exhibit unique patterns of agonist selectivity and tissue-specific expression that account for different biologic roles. Melanocytes express only the Mc1r, which is sometimes referred to as the “a-MSH receptor” even though a-MSH is probably more relevant physiologically to Mc3r and Mc4r activity. Some insects have been reported to produce Pomc-related peptides (Schoofs et al., 1993), but there is no obvious melanocortin receptor apparent among invertebrate genomes. Recent studies indicate that homologs for each of the five mammalian receptors exist in fugu and zebrafish (Klovins et al., 2004; Logan et al., 2003a; Logan et al., 2003b; Ringholm et al., 2002), but there is no obvious melanocortin receptor homolog in Ciona; thus, divergence of a single melanocortin receptor into ancestors of the five currently recognized sub-

types is likely to have occurred in primitive vertebrates between 400 and 500 million years ago.

Biology of Agouti Protein and Agouti-Related Protein Agouti protein is an ~ 18-kDa paracrine signaling molecule that acts as the primary physiologic ligand for the Mc1r, but via inhibition rather than stimulation of adenylate cyclase. The activity of Agouti protein in vitro is most easily measured by pharmacologic antagonism of a-MSH binding or receptor activation; however, as described below, several lines of evidence suggest that Agouti protein normally acts in vivo not as an a-MSH antagonist but instead as an inverse agonist, inhibiting high basal levels of Mc1r signaling that occur in the absence of any ligand. Agouti protein was first isolated by positional cloning of a mouse coat color gene that goes by the same name, in which allelic variation had long been recognized to control pigment type switching (Bultman et al., 1992; Miller et al., 1993). The name Agouti comes from a native South American language, where it refers to the rodent Dasyprocta leporina, also known as Dasyprocta aguti. These rodents, as well as many other furred mammals, display a characteristic pigmentation pattern in which individual hairs contain a subapical band of pheomelanic pigment while the tip and the base contain eumelanic pigment. In many cases, both within and among different species, these so-called “Agouti banded hairs” are present on the dorsal surface, whereas ventral hairs contain mostly pheomelanin. Both patterns — the presence of pheomelanic banding on individual hairs and the presence of ventral hairs that are almost entirely pheomelanic — relate directly to specific spatiotemporal patterns of Agouti gene expression directed by different promoters and untranslated first exons (Vrieling et al., 1994). The banding pattern correlates with transient expression of a “hair cycle-specific” Agouti mRNA isoform during early anagen, causing hair follicle melanocytes to switch from eumelanin synthesis to pheomelanin synthesis, and then back again to eumelanin synthesis. In contrast, the presence of ventral hairs that are entirely pheomelanic correlates with expression of a “ventral-specific” Agouti mRNA isoform throughout almost all of anagen, but in ventral rather in dorsal skin. In laboratory mice, ventral-specific isoforms contain an untranslated first exon, 1A, located approximately 118 kb 5¢ of the translational initiation codon, whereas hair cycle-specific isoforms contain one of two alternative untranslated first exons, 1B or 1C, located approximately 18 kb 5¢ of the translational initiation codon (Fig. 19.3). The situation described above is based on studies in laboratory mice, where the different Agouti mRNA isoforms are subject to independent genetic control and exhibit an additive phenotype (Chen et al., 1996; Vrieling et al., 1994). Thus, animals with banded hair on their dorsal surface and pheomelanic hair on their ventral surface are said to carry the whitebellied Agouti (AW) allele, in which both isoforms are active; animals with banded hair on both dorsal and ventral body 399

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Fig. 19.3. Region-specific expression of Agouti. As described in the text, expression of Agouti is controlled by two separate promoters that each have their own 5¢ untranslated first exon or exons. Exons 1A and 1A¢ are expressed throughout the entire hair growth cycle, but only in ventral skin, and therefore account for the pale ventrum observed in mice that carry the AW or at alleles. Exons 1B and 1C are expressed in both dorsum and ventrum, but only in the midphase of the hair growth cycle, and therefore account for the subapical band of pheomelanin present in individual hairs of animals that carry the AW or A alleles. See also Plate 19.2, pp. 494–495.

surfaces are said to carry the Agouti (A) allele, in which hair cycle-specific isoforms are present but the ventral-specific isoform is not; finally, animals with eumelanic hair on their dorsal surface and pheomelanic hair on their ventral surface are said to carry the black-and-tan (at) allele, in which the ventral-specific isoform is present but hair cycle-specific isoforms are not (Fig. 19.3). Both types of isoform are expressed in the dermal papillae of hair follicles (Candille et al., 2004; Millar et al., 1995), and help to confirm the notion, originally suggested from transplantation studies (Silvers and Russell, 1955), that Agouti protein has a small sphere of action limited to the hair follicle within which it is synthesized. Although molecular biologic and genetic studies of pigment type switching in animals other than laboratory mice are not extensive, available evidence suggests that the system described above is conserved across most vertebrate phyla. Loss-of-function Agouti alleles that cause a uniform black coat have been described in the rat (Kuramoto et al., 2001a), the domestic dog (Kerns et al., 2004), the domestic cat (Eizirik et al., 2003), horses (Rieder et al., 2001), and Arctic foxes (Vage et al., 1997); furthermore, canids probably use different Agouti isoforms for specific regions of the body (Kerns et al., 2004). The situation in nonmammalian vertebrates is somewhat more complicated, mainly because studies to date have been based on teleosts, in which a whole-genome duplication took place after divergence from the lineage leading to mammals. However, certain lizards carry Mc1r polymorphisms that correlate with color changes (Rosenblum et al., 2004). Finally, recent studies in goldfish have identified a likely ortholog of Agouti, which acts as an antagonist at the fugu

400

Mc1r, and is expressed in ventral but not dorsal skin (CerdaReverter et al., 2005). It should be noted that fish, amphibians, and reptiles have, in general, a pigmentary system that is more complicated than that of mammals, with multiple types of chromatophores and pigment biosynthetic pathways based on xanthines and pteridines as well as on tyrosine (Bagnara, 2003; Kelsh, 2004; Mellgren and Johnson, 2002). In avians, reddish-yellow feather coloration is caused by pheomelanin (Agrup et al., 1978; Haase et al., 1992, 1995; Prota, 1992; Thomson, 1974) but, surprisingly, pheomelanin has not been identified in fish (Ito and Wakamatsu, 2003), suggesting that the “pigment type switching pathway” in poikilotherms may function more to regulate pigment cell behavior via adenylate cyclase activity than to regulate different types of pigment synthesis. In either case, these observations point to a common theme of melanocortin signaling and pigmentation over 400 million years of evolution, whereby localized expression of Agouti protein gives rise to complex pigmentation patterns that distinguish between ventral and dorsal body surfaces in all vertebrates. Ironically, a role for Agouti signaling in humans and higher primates remains uncertain. The human Agouti gene (also referred to as ASIP for Agouti signaling protein) is capable of stimulating pheomelanin synthesis in transgenic mice, and is expressed at low levels in multiple tissues (Kwon et al., 1994; Wilson et al., 1995). Careful studies of Agouti expression in human skin have not been carried out, but putative regulatory sequences associated with the ventral-specific dog and mouse Agouti mRNA isoforms are also conserved in the human

REGULATION OF PIGMENT TYPE SWITCHING BY AGOUTI

genome (Kerns et al., 2004). Unlike the situation with MC1R (see below), population-based studies have failed to identify polymorphisms of human ASIP that support a definitive role in human pigmentation, although there is some evidence of a weak association between a single nucleotide polymorphism (SNP) in the 3¢ untranslated region (UTR) and different pigmentary phenotypes (Bonilla et al., 2005; Kanetsky et al., 2002; Voisey et al., 2001; Zeigler-Johnson et al., 2004); by the same token, the absence of ASIP sequence variation suggests that the gene has been under positive selection during recent primate evolution. Like many aspects of mouse coat color genetics, studies of pigment type switching have also played an important role in understanding basic aspects of mammalian biology that extend outside the pigmentary system. In particular, a set of unusual Agouti alleles in laboratory mice, including lethal yellow (Ay), viable yellow (Avy), and hypervariable yellow (Ahvy), are caused by genomic rearrangements that cause ubiquitous expression of Agouti protein coding sequence, leading to pleiotropic effects that include obesity, diabetes, and increased body size (Duhl et al., 1994). These observations led to an appreciation of a critical role for Mc3r and Mc4r signaling in energy balance, and the discovery of Agouti-related protein (Agrp), a neuropeptide that marks a key subset of hypothalamic neurons which sense and respond to changes in peripheral energy stores (reviewed by Barsh et al., 2000).

Mc1r Allelic Variation and Pigmentary Phenotypes A role for the a-MSH receptor in pigment type switching was suggested almost 30 years before Mc1r was cloned, based on experiments by Takeuchi and colleagues with organ culture of hair bulb melanocytes taken from mice with pheomelanic hairs (Tamate and Takeuchi, 1964). In cultures from animals carrying the Ay mutation (in which, as now known, Agouti protein is expressed at high levels), production of eumelanin could be stimulated by treatment with dibutyryl cAMP or aMSH. In contrast, in cultures from animals homozygous for the recessive yellow mutation of the Extension locus (e/e), production of eumelanin could be stimulated by dibutyryl cAMP but not by a-MSH. Over the last decade, a large number of molecular genetic studies have been carried out to investigate the role of Mc1r variation in different pigmentary phenotypes. An underlying theme of this work is that loss-of-function Mc1r mutations cause a predominantly pheomelanic color — in mice (Robbins et al., 1993), bears (Ritland et al., 2001), cattle (Joerg et al., 1996; Klungland et al., 1995), pigs (Kijas et al., 1998), horses (Marklund et al., 1996), humans (Valverde et al., 1995), dogs (Everts et al., 2000; Newton et al., 2000), and chickens (Takeuchi et al., 1996) — whereas gain-of-function Mc1r mutations cause a predominantly eumelanic color — in sheep (Vage et al., 1999), cattle (Klungland et al., 1995), pigs (Kijas et al., 1998), foxes (Vage et al., 1997), jaguars and jaguarundis (Eizirik et al., 2003), and several avian species (Ling et al., 2003; Mundy et al., 2003, 2004; Theron et al., 2001).

The Mc1r seems to be a relatively common source of normal pigmentary variation in many different species (Hoekstra et al., 2004; Mundy et al., 2004; Rees, 2003; Rosenblum et al., 2004), and has been a frequent subject of population and evolutionary genetic studies. In several instances, Mc1r polymorphisms have been strongly associated with melanism, in which dark individuals appear in a natural population that is mostly light colored. However, the exact nature of the lightcolored population varies considerably. Jaguars that normally display a striking rosette pattern (Eizirik et al., 2003), pocket mice that normally show Agouti banding (Nachman et al., 2003), and Arctic skuas that normally have pale-colored breast plumage (Mundy et al., 2004) are all changed into melanistic forms in association with Mc1r substitutions. These observations underscore the general theme that pigment type switching can give rise to considerable diversity in both color and pattern. In some of these cases, there is strong evidence that the melanistic forms are under selection, suggesting that mutations that constitutively activate the Mc1r have been used as an adaptive mechanism several times during vertebrate evolution. In contrast to rodents, cats, and birds, humans with dark hair and dark skin appear to have a “normal” MC1R that responds to both agonists and antagonists in vitro, and exhibits little variation in African populations. In fact, African individuals show a surprising paucity of noncoding variation in the MC1R (compared with most other genes), which suggests that “purifying selection” has helped to maintain strong constraint for a functional protein during recent human evolution (Harding et al., 2000; John et al., 2003; Rana et al., 1999). That is not to say that MC1R variation does not help to determine differences in human pigmentation phenotypes. Indeed, MC1R was the first gene identified that clearly influences normal variation in human skin and hair color, but via loss-of-function rather than gain-of-function alterations (Valverde et al., 1995). Chemical analyses show that, in general, black, brown, and blond human hair is composed mostly of eumelanin, with the various shades representing different amounts of melanin and/or different structures and arrangements of melanin granules (Ito and Wakamatsu, 2003; Liu et al., 2004). However, red human hair contains high levels of pheomelanin, and many studies have now shown that most individuals with red hair and fair skin carry one or more loss-of-function MC1R alleles (Box et al., 1997; Flanagan et al., 2000; Naysmith et al., 2004; reviewed by Rees, 2003; Sturm et al., 2003). Loss-of-function MC1R alleles are most common in individuals of European continental ancestry, but population genetic studies show an abundance of different alleles and haplotypes, suggesting that these variants are maintained by genetic drift rather than by selection (Harding et al., 2000; Rana et al., 1999).

Accessory Proteins for Melanocortin Signaling: Attractin and Mahoganoid In addition to Agouti and Extension, studies of the coat color

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mutations mahogany and mahoganoid implicate additional mechanisms in pigment type switching. Both mahogany and mahoganoid were recognized by their ability to suppress pheomelanin synthesis; animals that would otherwise have hairs banded with eumelanin and pheomelanin — the Agouti phenotype — instead show a dark coat color that is almost all eumelanin if they are homozygous for mahogany or mahoganoid. This phenotype is similar to that caused by lossof-function Agouti mutations or gain-of-function constitutively activating Mc1r mutations (Fig. 19.2) and, a priori, could be explained by decreased production or cell surface binding of Agouti protein, increased production of a-MSH, or defects in the melanocyte enzymes or structural proteins required to produce yellow pigment granules. Double mutant studies helped to distinguish among these alternatives, and to establish a genetic pathway for pigment type switching. The results are summarized in Figure 19.1, and indicate that the genes mutated in mahogany and mahoganoid are genetically downstream of Agouti but genetically upstream of Mc1r (Miller et al., 1997). Positional cloning of the gene mutated in mahogany revealed a cysteine-rich 1428-amino-acid type I transmembrane protein that was widely expressed and whose ectodomain contained several domains characteristic of axon guidance and cell adhesion molecules (Gunn et al., 1999; Nagle et al., 1999). The gene was named Attractin (Atrn) to correspond with its human homolog, discovered by DukeCohan and colleagues as a glycoprotein that would “attract” T lymphocytes to monocytes in vitro (Duke-Cohan et al., 1998, 2000); thus, mutant alleles are now referred to as Atrnmg, Atrnmg-L, and Atrnmg-3J (Gunn et al., 2001). Detailed studies of immune function in Attractin mutant mice have not been described; however, mutant animals develop a progressive neurodegenerative disease with dysmyelination, spongiform degeneration, and motor abnormalities (Gunn et al., 2001; Kuramoto et al., 2001b). Similar phenotypes are apparent in two spontaneous rat mutations of Attractin, zitter (Kuramoto et al., 2001b) and myelin vacuolation (Kuwamura et al., 2002), as well as a hamster mutation, black tremor (Kuramoto et al., 2002). Ironically, both the rat zitter mutation and the mouse mahogany mutation were extensively studied over the last decade for completely different reasons; the fact that the same gene underlies both mutations was recognized only in hindsight. In the case of zitter, mutant rats were discovered because of a pronounced tremor (Rehm et al., 1982), and were examined further because of their potential relevance to the pathogenesis of neurodegeneration (Gomi et al., 1990; Kondo et al., 1991; Kuramoto et al., 1994). However, the zitter mutation was maintained on an albino background; thus, the effects on pigment type switching (and the relationship to Attractin) remained obscure until both genes were cloned. In the case of mahogany, mutant mice were examined further because of a potential involvement in central melanocortin signaling: in Ay animals that express Agouti protein ubiquitously, mahogany suppresses the effects of Agouti on both pigmentation and 402

obesity (Miller et al., 1997). However, the neurodegenerative phenotype of mahogany is not readily apparent, in part because the original Atrnmg allele is hypomorphic, and in part because of modifier genes that ameliorate the tremor in some genetic backgrounds (Gunn et al., 2001). The gene mutated in mahoganoid (the official name of which is Mgrn1) encodes an intracellular C3HC4 RING domain protein that has E3 ubiquitin ligase activity in vitro, but whose normal substrate(s) has(ve) yet to be identified (He et al., 2003b; Phan et al., 2002). Like Attractin mutant mice, Mgrn1 mutant mice develop progressive spongiform degeneration, albeit with milder symptoms. Atrn and Mgrn1 have nearly identical patterns of mRNA expression, and are found in many, although not all, tissues and cell types examined; the two genes also have a similar pattern of evolutionary conservation, with homologs in worms and flies, but not in yeast. Although the molecular pathogenesis of neurodegeneration in Atrn and Mgrn1 mutant mice is still unclear, biochemical and genetic studies indicate that Attractin functions in pigment type switching as an obligate accessory receptor for Agouti protein on melanocytes. In transgenic animals, expression of an Atrn cDNA in melanocytes rescues the mahogany coat color phenotype, but expression in keratinocytes does not. Thus, Atrn function is melanocyte autonomous, as is that of the Mc1r. Furthermore, while the effect of Atrn is to facilitate Agouti-induced pheomelanogenesis, overexpression of Atrn has no effect on coat color by itself, suggesting a permissive rather than an active role in pigment type switching. Finally, structure–function studies have shown that different domains of Agouti protein interact with Atrn and the Mc1r. The cysteine-rich carboxy-terminal domain of Agouti protein binds with high (nanomolar) affinity to the Mc1r. In cultured cells, this interaction is sufficient to inhibit adenylate cyclase activity but, in vivo, the carboxy-terminal domain of Agouti protein has no effect on pigmentation. The positively charged amino-terminal domain of Agouti protein binds to Atrn with low (micromolar) affinity, but no consistent effect on cell behavior or signaling has been reported. However, the interaction is biologically significant, because Agrp, which is similar to Agouti in the carboxy-terminal but not in the amino-terminal domain, does not interact with Atrn in vitro or in vivo: although the effects of Agouti protein on obesity (and pigmentation) are suppressed by mahogany, the effects of Agrp are not. Taken together, these observations suggest that Attractin and Mgrn1 are linked components of a genetic pathway that is conserved among all metazoans, and whose original function may have been to ubiquitinate an as yet unidentified substrate or substrates, likely in response to signals from the extracellular environment. During vertebrate evolution, this pathway was “borrowed” by the Agouti-melanocortin system because ubiquitination of the Mgrn target could facilitate pheomelanin synthesis. According to this hypothesis, the ligand for Atrn is different in melanocytes and neurons, but the target for Mgrn1 ubiquitination is the same; thus, identi-

REGULATION OF PIGMENT TYPE SWITCHING BY AGOUTI

fication of this ligand could provide insight into both pigment type switching and the pathogenesis of neurodegeneration.

Perspectives and Future Directions How Does a Pigment Cell Make Pheomelanin? The foregoing discussion underscores the gap that remains in our understanding of pheomelanin synthesis. Outside the melanocyte, Agouti-induced inhibition of Mc1r signaling is the physiologic signal that normally triggers pheomelanin synthesis, but the way in which Atrn and Mgrn1 fit into this pathway inside the melanocyte is not yet clear. The ~100residue intracellular domain of Atrn is required for its effects on both pigmentation and neuronal function (Kuramoto et al., 2001b), which, together with the similar patterns of expression and evolutionary conservation between Atrn and Mgrn1, suggests that Atrn plays a signaling role by activating or facilitating Mgrn1 activity. However, Mgrn1 (and Atrn) are also genetically upstream of Mc1r inhibition, as animals doubly mutant for Mc1re and either Atrnmg or Mgrn1md are phenotypically identical to single Mc1re mutants. This suggests that Mgrn1 plays a role in the levels, subcellular distribution, and/or signaling activity of Mc1r, perhaps by ubiquitinating a substrate that modulates Mc1r turnover or recycling. It is also unclear how Mc1r inhibition and reduced levels of intracellular cAMP are connected to reduction in tyrosinase activity and availability of premelanosomal cysteine that are thought to be required for cysteinyldopa formation. For tyrosinase, one potential mechanism is based on the observation that Mitf serves as a transcriptional activator of tyrosinase, and that the cAMP–protein kinase A–CREB pathway promotes Mitf expression (Busca and Ballotti, 2000; Gaggioli et al., 2003; Price et al., 1998). However, reduced activity of tyrosinase during pheomelanin synthesis is regulated at the post-translational as well as the transcriptional level (Burchill et al., 1989; Kobayashi et al., 1995), and it is not at all clear that Mitf is downregulated during pheomelanin synthesis in vivo. In addition, hypomorphic alleles of tyrosinase have a much greater effect on dilution of pheomelanin than they do on eumelanin (Fig. 19.2B), but hypomorphic alleles of Mitf do not; thus, regulation of Mitf by cAMP signaling cannot easily explain changes in tyrosinase activity that occur during pigment type switching. Finally, it should be noted that regulation of pheomelanin synthesis in humans differs in several respects from that in mice. In addition to questions about the function, if any, of the human Agouti gene, the inverse relationship between tyrosinase activity and pheomelanin synthesis discovered from studies of mouse coat color mutants is not so obvious in studies of human skin and hair (Burchill et al., 1991). In part, this apparent discrepancy could be explained by differences in the distribution and biology of melanocytes in different skin compartments. In mice, the majority of melanocytes are located within hair follicles; there are a few regions where

melanocytes accumulate outside hair follicles (in both the extrafollicular epidermis and the dermis), and these tend to be in areas where hair follicles are either absent or at low density, such as the footpads, the ears, and the tail. In all these cases, however, pheomelanin is synthesized only by intrafollicular melanocytes; even in Ay/a mice, melanocytes in the dermis or extrafollicular epidermis produce only eumelanin (Fitch et al., 2003; Lamoreux and Russell, 1979; Van Raamsdonk et al., 2004). In human skin, however, hair follicle density over most regions of the body is much less than that of mice; extrafollicular epidermal melanocytes are the major determinant of skin color, and may contain substantial amounts of pheomelanin (Thody et al., 1991). In addition to the questions surrounding what happens between Mc1r inhibition and cysteinyldopa formation, a potential gap exists between cysteinyldopa formation and the production of pheomelanosomes, as additional, as yet unidentified, cellular components may be necessary for biogenesis and/or granule maturation. In particular, there is a series of coat color mutants that, at a whole-animal level, interfere with pheomelanin but not eumelanin synthesis — grizzled, subtle gray, gray lethal, and gray intense — and could lie upstream or downstream of cysteinyldopa formation. Answers to these questions may come, in part, from the molecular identity and characterization of the aforementioned mutations. The gene mutated in gray lethal, Ostm1, encodes a single transmembrane spanning protein that appears to be located in an intracellular compartment (Chalhoub et al., 2003). Its function is not yet clear, although a different group who identified the same gene (and named it Gipn) noted limited sequence similarity with a RING domain and suggested that the protein modulated signaling through Gicoupled receptors by ubiquitination of RGS proteins (Fischer et al., 2003). A related approach would be to perturb tyrosinase activity and/or intracellular cysteine levels artificially (using genetics or small molecules) and ask how such manipulations might affect pigment type switching. An experiment of this sort was reported by Lieberman and colleagues (Lieberman et al., 1996) by knocking out gamma-glutamyl transpeptidase (Ggt), a cell surface enzyme that initiates catabolism of glutathione and, when mutated, leads to cysteine deficiency. Mutant animals have a pleiotropic phenotype that includes alterations of the Agouti coat color pattern, possibly due to defective pheomelanin synthesis. However, previous literature linking Ggt activity to pigment synthesis suggests that changes in Ggt activity regulate overall levels of melanogenesis by modulating tyrosinase (Chaubal et al., 2002; Hu, 1982) and, as described above, the two “cardinal characteristics” of pheomelanin synthesis (Fig. 19.1) are not independent. Identification of additional mutations, such as gray intense and grizzled, and application of additional approaches to perturb (and uncouple) tyrosinase activity from changes in intracellular cysteine availability should lead to a more complete understanding of pheomelanin synthesis and pigment type switching. 403

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Mc1r Basal Activity and the Potential Role of a-MSH in Normal Pigmentation As described above, interactions between Agouti protein and Mc1r play a key role in pigment type switching across many different vertebrate phyla. Perhaps surprisingly, the same is not true of the Mc1r ligand, a-MSH. In pharmacologic studies of cultured cells, Agouti (or Agrp) acts as a so-called inverse agonist, inhibiting a relatively high level of basal Mc1r (or Mc4r) signaling in the absence of exogenous a-MSH (Adan and Kas, 2003; Chai et al., 2003; Nijenhuis et al., 2001; Siegrist et al., 1997). Initial physiologic studies of a-MSH biosynthesis and possible effects on pigmentation focused mainly on a-MSH as a possible endocrine hormone produced by the pituitary gland. Exogenous administration of a-MSH causes a switch from pheomelanin to eumelanin synthesis in Ay/a mice (Geschwind, 1966), and increased pituitary-derived a-MSH and/or ACTH are responsible for the darkening of skin that occurs in adrenal insufficiency (Beamer et al., 1994). However, plasma levels of a-MSH do not correlate with alterations in coat color that occur when certain strains of mice carrying Avy darken with age, and hypophysectomy does not cause pheomelanogenesis in nonagouti mice, even though small increases in plasma aMSH levels have dramatic effects on coat color (Geschwind et al., 1972; Thody et al., 1983). Thus, circulating a-MSH probably does not act as a major endocrine hormone in physiologic control of pigment synthesis in the mouse. More recently, a potential role for a-MSH in pigment synthesis has focused on a paracrine or autocrine role (reviewed by Pawelek, 1993; Slominski et al., 1993; Wintzen and Gilchrest, 1996), based on the finding that expression of Pomc RNA can be detected in keratinocytes and melanocytes (Farooqui et al., 1993; Slominski et al., 1992, 1995). In most of these studies, it has been difficult to determine whether the RNA detected actually gives rise to physiologically significant levels of aMSH. Targeted deletion of Pomc causes a lighter coat in A/A mice (Barsh, 1999; Yaswen et al., 1999), presumably due to a slight reduction in a-MSH released from plasma and/or skin, but the same targeted allele in a genetic background with very little Agouti expression has no effect on pigmentation; C57BL/6 animals are almost completely black as a result of exclusive production of eumelanin, and that phenotype is not altered by deleting the Pomc gene (Slominski et al., 2005). The situation in humans is less clear. Congenital deficiency of Pomc in humans is associated with red hair (Krude et al., 1998, 2003a, b), but the small number of affected individuals, the absence of quantitative studies, and the heterogeneity of human populations makes it difficult to compare the pigmentary effects caused by loss-of-function for Pomc with those caused by loss-of-function for Mc1r. In culture, human melanocytes exhibit a robust agonist or antagonist response to a-MSH or Agouti protein, respectively, but transgenic mice carrying the human Mc1r placed under the control of normal mouse regulatory elements exhibit a dose-dependent suppression of Agouti-induced pheomelanin synthesis (Healy et al., 2001). Taken together, these data suggest that the effect of 404

Mc1r signaling on human pigmentation includes contributions from high levels of basal receptor activity as well as Pomcderived agonists, but that the former component accounts for most of the pigmentary variation present in human populations.

Genetics of Pigment Type Switching in Cats and Dogs Given the very high level of conservation among mammalian genomes, and the remarkable utility of laboratory mice as a tool for experimental genetics, it may come as something of a surprise that color variation in other mammals harbors unsolved pigmentary mysteries. Among the most interesting group of coat color phenotypes are those associated with regular patterns of stripes or spots, as in zebras, tigers, leopards, or giraffes. Although chemical or biochemical studies have not been carried out, the components of such patterns are likely to be eumelanin alternating with pheomelanin (as in tigers or leopards), or eumelanin alternating with no pigment (as in zebras) (Searle, 1968). In domestic cats, Tabby is an important determinant of pheomelanin/eumelanin-based coat color patterns, but a homologous allelic series has not been identified in rodents (Robinson, 1991; Searle, 1968). Tabby is thought to take its name from the Attabiah region of Baghdad, which is known for weaving a type of silk with elaborate stripe patterns. The stereotypic Tabby contains stripes of Agouti-banded hair alternating with stripes of black (eumelanic) hair. Genetic segregation analysis carried out by cat breeders suggests that variations on this pattern such as large blotches and/or spots represent an allelic series in which the most dominant allele, Abyssinian (Ta), is associated with no stripes, an intermediate allele, mackerel (T), accounts for tiger stripes, and the most recessive allele, blotched (tb), is associated with large coalescent dark areas (Lomax and Robinson, 1988). Based on the pathway of pigment type switching as described earlier, these observations suggest that the Tabby gene product is normally required for Agouti banding, and that different alleles cause the gene to become inactivated in a pattern in which the largest area of inactivation corresponds to the most recessive allele. From this perspective, the gene mutated in Tabby might encode a protein such as Attractin or Mahoganoid, required for normal Agouti signaling, and the molecular basis of the mutant alleles would account for the intriguing nature of the underlying pattern. Comparative zoologic studies suggest that the same mechanism underlies Tabby patterns in domestic and wild cats (Weigel, 1961). Furthermore, a common variant in domestic cats is the Silver tabby, in which stripes of dark hairs alternate with stripes of cream-colored instead of Agouti-banded or yellow hairs. This phenomenon is reminiscent of chinchilla, in which Agouti-induced inhibition of Mc1r signaling gives rise to an absence of pigment rather than pheomelanin per se (Fig. 19.2B). Whether a similar phenomenon explains alternating patterns of black and white in ungulates, i.e. zebras, is

REGULATION OF PIGMENT TYPE SWITCHING BY AGOUTI

less clear, however, as the Tabby gene is clearly recognized only in the Carnivora. Also well known in domestic cats, Sex-linked orange (O) or tortoiseshell is an oft-cited example for teaching principles of mammalian genetics, as patches of yellow and black hair in Tortoiseshell or Calico cats are a dramatic manifestation of clonal patterns of X-inactivation, found only in females and rare XXY males (Centerwall and Benirschke, 1975; Leaman et al., 1999). In cats, Sex-linked orange is epistatic to nonagouti and therefore somewhat analogous to recessive yellow (Mc1re), but it would be extremely unusual to find that the Mc1r gene is responsible for Sex-linked orange as there are almost no exceptions to the conservation of X-linkage among eutherian mammals. An additional source of pigmentary mysteries is apparent from studies of color variation in domestic dogs. Many breeds with uniform pheomelanic coats such as Golden Retrievers, Irish Setters, or yellow Labrador Retrievers carry a loss-offunction Mc1r allele as in laboratory mice and other mammals (Newton et al., 2000). However, a similar phenotype in other breeds such as Rhodesian Ridgebacks, Dachshunds, Great Danes, Chows, and French Bulldogs is probably caused by an Agouti allele known as fawn (ay) (Berryere et al., 2005). As in laboratory mice, the dog ay allele is dominant to other Agouti alleles associated with eumelanin synthesis such as nonagouti (a) or black-and-tan (at). However, unlike mice carrying a “yellow” allele, the dog ay allele does not cause effects outside the pigmentary system, suggesting an underlying molecular basis in which variation in the hair cycle-specific promoter expands the timing of expression to include all of anagen. One of the most intriguing aspects of pigment type switching in domestic dogs is “dominant black,” in which a uniform eumelanic coat color is inherited in a simple dominant fashion, but is caused by variation in neither Mc1r nor Agouti (Kerns et al., 2003, 2004), and has been referred to as “the K locus” based on kurokami, the Japanese word for black hair. Mc1r is epistatic to K, as Labrador Retrievers homozygous for both dominant black (K) and an Mc1r loss-of-function allele are yellow colored; however, the epistasis relationship between K and Agouti is less certain. Together with Sex-linked orange and Tabby, identification of the K locus may not only further our understanding of pigmentary biology but also provide insight into basic aspects of melanocortin receptor signaling used for a variety of physiologic processes.

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Human Pigmentation: Its Regulation by Ultraviolet Light and by Endocrine, Paracrine, and Autocrine Factors Zalfa Abdel-Malek and Ana Luisa Kadekaro

Summary

Historical Background

1 Cutaneous pigmentation has cosmetic and cultural implications. Melanin in the skin plays a photoprotective role. 2 The tanning response to sun exposure correlates directly with constitutive melanin content, which in turn is determined by the rate of synthesis in melanocytes, and the rate and manner of melanosome delivery to keratinocytes. Cultured human melanocytes respond to ultraviolet radiation (UVR) with growth arrest and increased melanogenesis; however, melanocytes derived from skin types I or II have a slower recovery from the growth arrest, more cyclobutane pyrimidine dimers, and less melanogenic response than melanocytes from skin types V or VI. 3 Exposure to UVR induces the synthesis of a variety of epidermal cytokines and growth factors, many of which affect the proliferation of melanocytes and/or melanogenesis. 4 Human melanocytes respond to certain inflammatory mediators, which include the immune inflammatory cytokines interleukin-1, -6 and tumor necrosis factor-a, and a variety of eicosanoids. 5 Basic fibroblast growth factor is a paracrine factor that is mitogenic for melanocytes. 6 Endothelin-1 is a keratinocyte-derived factor that stimulates melanocyte proliferation and melanogenesis, and promotes survival after exposure to UVR. 7 The proopiomelanocortin-derived melanotropic peptides, particularly a-melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH), are mitogenic and melanogenic for human melanocytes and function as transducers for the melanogenic effect of UVR and as survival factors for melanocytes. 8 The proopiomelanocortin derivative b-endorphin stimulates the proliferation and melanogenesis of human epidermal and follicular melanocytes. 9 Agouti signaling protein antagonizes the effects of a-MSH on human melanocytes. 10 Human melanocytes are targets for vitamin D3 and are affected by estrogens.

The Role of Melanin

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Cutaneous pigmentation is a genetic trait that distinguishes humans from different ethnic backgrounds, yet is unique to every individual. The growing interest in understanding how human pigmentation is regulated stems from increasing concern about the photocarcinogenic and photoaging effects of sun exposure and the role of melanin in photoprotection. It also stems from cultural preferences for dark-tanned skin (as in Western societies) or light complexion (as in the Orient). Melanin in the skin is known to reduce the penetration of ultraviolet radiation (UVR) through the epidermal layers and to quench reactive oxygen radicals that contribute to the suninduced DNA damage (Kaidbey et al., 1979; Menon and Haberman, 1977; Morison, 1985). Many epidemiologic studies have shown that the frequency of skin cancers is higher in individuals with lightly pigmented skin than in individuals with dark skin (Epstein, 1983; Sober et al., 1991). The correlation between cutaneous melanin content and the susceptibility to sun-induced carcinogenesis has given skin pigmentation a special significance. With the increase in the frequency of sun-induced skin cancers due to the depletion of the ozone layer, understanding the role of melanin in preventing the photodamaging and carcinogenic effects of sun exposure has become an important public health issue.

Current Concepts Constitutive Pigmentation and the Response to UV Radiation One of the most obvious effects of sun exposure is increased skin darkening (i.e. tanning). This response, however, varies among individuals with different pigmentary phenotypes (i.e. skin types). Those with very light skin (skin types I or II) burn and do not tan, whereas those with dark skin (skin types IV–VI) tan well when exposed to the sun. The pigmentary response, particularly the delayed tanning response that results from the stimulation of melanin synthesis, is determined to a large extent by constitutive melanin content in the skin (Pathak et al., 1980). In addition, this response is affected by a wide variety of hormones and cytokines, the production of

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UV

Growth arrest Endocrine factors

Melanosomes

e.g.

KC Pituitary gland

Adrenal gland

Ovary

Testis

MC Other tissues

Melanogenesis

Paracrine factors that include: 1) α-MSH and ACTH 2) ET-1 3) ASP 1) + 2), 1) + 3)

cAMP, PKA activity PKC, tyrosine phosphorylation antagonist for α-MSH, other (?) signaling pathway regulation of melanocyte proliferation and function

Fig. 20.1. Schematic representation of the regulation of human melanocytes by sun exposure and hormonal factors.

which is augmented by UVR (Fig. 20.1). It was suggested that the increase in pigmentation following exposure to UVR is a consequence of DNA repair (Eller et al., 1994). If this is the case, then dark skin that tans readily would be expected to be more proficient than lightly pigmented skin in repairing DNA damage. However, unequivocal evidence linking skin pigmentation and DNA repair is not available. So far, only one study has demonstrated that multiple irradiations with UVR enhanced the rate of removal of DNA photoproducts in skin type IV but not in skin type I or II individuals (Sheehan et al., 2002). For decades, it has been recognized that differences in constitutive skin pigmentation are not due to differences in the number of melanocytes among different skin types, but to differences in the melanogenic activity of the melanocyte, the number and size of melanosomes, the type of melanin deposited onto melanosomes, and the donation of mature melanosomes to surrounding keratinocytes (Pathak et al., 1980). In Caucasian skin, several melanosomes are packaged and transferred as a single unit or entity to the keratinocytes. However, in dark skin, melanosomes are larger in size than those in Caucasian skin, and each melanosome is transferred individually from a melanocyte to surrounding keratinocytes. Furthermore, it has been demonstrated that the activity of tyrosinase, the rate-limiting enzyme in the melanogenic pathway, correlated directly with skin pigmentation and melanin content and is higher in melanocytes derived from deeply pigmented skin than in melanocytes from lightly pigmented skin (Abdel-Malek et al., 1994; Halaban et al., 1983;

Iwata et al., 1990). With the discovery of the two melanogenic enzymes, TRP-1 and TRP-2, it became evident that they too contribute to constitutive melanization (Hearing and Tsukamoto, 1991; Jackson, 1988; Vijayasaradhi and Houghton, 1991). Using specific antibodies that recognize tyrosinase, TRP-1, and TRP-2, respectively, it was demonstrated by Western blot analysis that the amount of each of these proteins in cutaneous human melanocytes correlates directly with the pigmentary phenotype from which these melanocytes were derived (Abdel-Malek et al., 1993). Additionally, the levels of tyrosinase, TRP-1, and TRP-2 could be increased or decreased by agents that stimulate or inhibit melanogenesis respectively (Abdel-Malek et al., 1993, 1994, 1995). One of the most obvious effects of sun exposure is skin darkening (i.e. tanning). It was demonstrated years ago that the number of DOPA-positive (i.e. active) melanocytes is significantly higher in chronically sun-exposed skin than in unexposed skin from the same individual (Gilchrest et al., 1979). The first evidence for direct responsiveness of human melanocytes in vitro to UV irradiation was provided by Friedmann and Gilchrest (1987), who demonstrated that these cells responded to UVR from a solar simulator with a dose-dependent decrease in proliferation and increase in pigmentation. Libow et al. (1988) then showed that melanocytes responded to UVR with increased proliferation and melanogenesis. Neither study referred to any differences in the responses of melanocytes with different melanin contents to UVR. We have reported that irradiation of melanocytes with a fractionated 411

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dose of UVR (mainly UVB with a minor UVC component) that did not cause cell death resulted in inhibition of melanocyte proliferation as a result of arrest in G2 phase of the cell cycle, and in increased tyrosinase activity and melanin content (Abdel-Malek et al., 1994). These effects were the same in melanocytes derived from different skin types. Yohn et al. (1992) found that melanocytes from white skin were more sensitive to the DNA-damaging effects of UVA light. Barker et al. (1995) compared the responses of human melanocytes derived from different pigmentary phenotypes to a single irradiation with increasing doses of UVR. Melanocytes, regardless of their melanin content, exhibited increased levels of the tumor suppressor gene product p53 and became arrested in G1 phase of the cell cycle following UVR irradiation. However, there were distinct differences between the responses of very lightly (skin type I or II derived) and very heavily (skin type V or VI derived) pigmented melanocytes. Only the latter but not the former demonstrated a melanogenic response following exposure to UVR. Furthermore, lightly pigmented melanocytes had a slower recovery from the UVR-induced G1 arrest, and a more prolonged increase in the p53 protein, and encountered more cyclobutane pyrimidine dimers than heavily pigmented melanocytes. The differences in the induction of DNA photoproducts were corroborated by a study in which the responses to UVR of individuals from different ethnic backgrounds and pigmentary phenotypes were compared in vivo (Tadokoro et al., 2003). In further support of these results, Musk and Parsons (1987) noted that human melanoma cells with a low melanin content were more sensitive to killing by solar irradiation or UVC light than melanoma cells that were more melanotic. Additionally, Kobayashi et al. (1993) demonstrated increased formation of cyclobutane pyrimidine dimers and pyrimidine (6,4) pyrimidone photoproducts following irradiation with UVC radiation in lightly pigmented, compared with heavily pigmented, human melanoma cells. Also, irradiated melanoma cells with a high melanin content were found to be more resistant to cell killing than their less melanotic counterparts. Interestingly, however, no difference in the DNA repair of pyrimidine dimers or (6,4) photoproducts was detected among melanoma cells with different melanin contents. Collectively, these studies clearly illustrate distinct differences in the responses of melanocytes from different skin types to UVR and show that the induction of DNA photoproducts correlates with melanin content. As stimulation of melanogenesis is accompanied by increased transfer of melanosomes to adjacent keratinocytes, and thus increased skin pigmentation and photoprotection, the above results offer an explanation for the differences in the cutaneous responses of individuals with different pigmentary phenotypes to sun exposure and in their susceptibility to the development of skin cancers.

Eumelanin, Pheomelanin, and Photoprotection Human epidermal melanocytes synthesize the main two forms of melanin, the brown–black eumelanin and the red–yellow pheomelanin. The relative amounts of eumelanin and 412

pheomelanin are an important determinant of constitutive cutaneous pigmentation. The ratio of eumelanin to pheomelanin is substantially higher in melanocytes in dark skin than in melanocytes in lightly pigmented skin, and seems to correlate directly with total melanin content (Kadekaro et al., 2003). It has become evident that regulation of synthesis of these two forms of melanin in human melanocytes is mainly carried out by activation of the melanocortin 1 receptor by its ligands a-MSH or ACTH, or inactivation of the receptor by mutations that disrupt its binding or signaling, or possibly by binding of the physiological antagonist agouti signaling protein (Abdel-Malek et al., 1995; Box et al., 1997; Hunt et al., 1995; Scott et al., 2002a; Smith et al., 1998; Suzuki et al., 1997). Eumelanin is thought to be superior to pheomelanin in its photoprotective properties. Eumelanin is more resistant than pheomelanin to degradation and is more efficient in scavenging reactive oxygen species that are produced by UVR exposure (Bustamante et al., 1993; Menon et al., 1985). Moreover, pheomelanin is thought to contribute to the damaging effects of UVR because it can generate hydroxyl radicals and superoxide anions (Chedekel et al., 1978; Felix et al., 1978). Eumelanin is synthesized and deposited onto elliptical melanosomes, whereas pheomelanin is synthesized in smaller round melanosomes. In situ, elliptical melanosomes in dark skin remain intact throughout the epidermal layers, and form supranuclear caps in keratinocytes, reducing exposure of nuclear DNA to impinging UVR (Pathak et al., 1971). In contrast, in lightly pigmented skin, melanosomes are degraded, and only “melanin dust” is evident in the suprabasal layers. The absence or reduction in melanosomes in the epidermis is expected to compromise the photoprotection of the skin, rendering it more vulnerable to UVR-induced damage, and hence carcinogenesis. Indeed, studies on cultured human melanocytes revealed an inverse relationship between eumelanin content and the ratio of eumelanin to pheomelanin, on one hand, and the induction of DNA photoproducts on the other (Smit et al., 2001; Tadokoro et al., 2003).

Induction of Epidermally Synthesized Factors by UVR Several groups of investigators have demonstrated that exposure to UVR stimulates the synthesis of a variety of hormones, cytokines, and growth factors by epidermal cells. Irradiation of keratinocytes with UVR was shown to increase the synthesis of the cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)-a, and the latter cytokine was found to play an important role in the formation of sunburn cells (i.e. apoptotic keratinocytes) (Köck et al., 1990; Kupper et al., 1987; Oxholm et al., 1988; Urbanski et al., 1992). Human melanocytes in culture also synthesize IL-1a and IL-1b (Swope et al., 1994). Basic fibroblast growth factor (bFGF) is synthesized by keratinocytes, and its synthesis is enhanced by UVR (Halaban, 1988). UV irradiation of keratinocytes in vitro, or human skin in vivo, increased the synthesis of endothelin (ET)1 (Imokawa et al., 1992, 1995; Yohn et al., 1993), as well as

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proopiomelanocortin and its derivatives, a-melanocyte stimulating hormone (a-melanotropin, a-MSH) and adrenocorticotropic hormone (ACTH), b-lipotropic hormone, and its derivative b-endorphin (Kippenberger et al., 1995; Schauer et al., 1994; Suzuki et al., 2002; Wakamatsu et al., 1997; Wintzen et al., 1996). The synthesis of a-MSH and ACTH by cultured human keratinocytes or melanocytes and the synthesis of ET-1 by cultured human keratinocytes were enhanced by IL-1a. Another factor whose synthesis is increased upon irradiation of the epidermis with UVR is stem cell factor (Hachiya et al., 2001). Prostaglandins (PGs) and leukotrienes are two classes of inflammatory mediators that are derived from the metabolism of arachidonic acid by the cyclooxygenase and lipooxygenase pathways respectively. The levels of prostaglandins D2, E2, and F2a have been found to increase in the skin following irradiation with UVR (Black et al., 1980; Kobza Black et al., 1978). We have found that human melanocytes synthesize PGF2a and the lipooxygenase derivatives LTB4 and 12-HETE (Leikauf et al., 1994). Recently, it was demonstrated that activation of the proteinase-activated receptor 2 (PAR-2) stimulates the synthesis of PGE2 and PGF2a by cultured human keratinocytes (Scott et al., 2004). PAR-2 is an important participant in melanosome transfer from melanocytes and keratinocytes, and is activated by UVR. Thus, UVR seems to increase the production of specific arachidonic acid derivatives indirectly by activating PAR-2. The significance of keratinocyte-derived factors in the regulation of melanocyte function was first demonstrated by the observation that media conditioned by cultured human keratinocytes stimulated the proliferation, melanogenesis, and dendricity of cultured human melanocytes (Gordon et al., 1989). Evidence for the participation of keratinocytes in the response of melanocytes to UVR was provided by the observation that melanogenesis was more markedly stimulated by UVR when melanocytes were cocultured with keratinocytes than when melanocytes were grown in monoculture (Duval et al., 2001). There is also evidence for autocrine regulation of human melanocytes. For example, we have reported that human melanocytes synthesize IL-1a and IL-b, and also PGF2a, LTB4, and 12-HETE (Leikauf et al., 1994). Most of the keratinocyte- and melanocyte-derived factors listed above affect the proliferation and/or melanogenesis of human melanocytes in vitro. Based on these findings, we hypothesize that the response of human melanocytes to sun exposure is direct, as well as indirect through the effects of UVR-induced paracrine and autocrine factors.

IL-1a, IL-1b, IL-6, and TNF-a on cultured human melanocytes (Swope et al., 1991). All four cytokines dosedependently decreased the proliferation and tyrosinase activity, and reduced the levels of tyrosinase, TRP-1, and TRP-2 in human melanocytes, as determined by Western blot analysis (Abdel-Malek et al., 1993). The biological effects of these cytokines on human melanocytes strongly suggest that they potentially function as paracrine factors. Additionally, the demonstration that human melanocytes respond to, and synthesize, IL-1a and IL-1b suggests that these two cytokines function as autocrine, as well as paracrine, regulators of human melanocytes (Swope et al., 1994). As UVR has been shown to stimulate the synthesis of IL-1 and TNF-a by keratinocytes, we speculate that these cytokines might be part of a negative feedback loop that downregulates the pigmentary effect of UVR. Moreover, these primary cytokines induce the synthesis of other cutaneous factors that in turn mediate the response of melanocytes to sun exposure or inflammation. For example, IL-1 has been shown to stimulate the synthesis of ET-1 and b-endorphin by keratinocytes (Imokawa et al., 1992; Wintzen et al., 1996), as well as a-MSH and ACTH by human keratinocytes and melanocytes (Chakraborty et al., 1996; Schauer et al., 1994). Another group of inflammatory mediators is the eicosanoids, metabolites of arachidonic acid. We have shown that PGs affect melanogenesis in murine Cloudman melanoma cells, with the E series (PGE1 and E2) being potent stimulators, whereas PGA1 and PGD2 act as potent inhibitors (AbdelMalek et al., 1987). As for normal human melanocytes, Tomita et al. (1987) reported that they respond to PGE1 with increased melanogenesis and dendrite formation. Recently, it was demonstrated that PGE2 and PGF2a increase the dendricity of human melanocytes, an effect that is expected to increase skin pigmentation by facilitating the transfer of melanosomes to keratinocytes (Scott et al., 2004). An important finding by Morelli et al. (1989) was that leukotrienes (LT) C4 and D4, two metabolites of the lipooxygenase pathway, were mitogenic for human neonatal melanocytes. Medrano et al. (1993) went further to demonstrate that normal adult melanocytes were induced to proliferate and form tight spheroids in the presence of LTC4 in the culture medium. Evidently, LTC4 resulted in loss of contact inhibition in the melanocyte cultures, which led to the speculation that LTC4 plays a role in the induction of early events of malignant transformation.

Role of Basic Fibroblast Growth Factor

Regulation of Human Melanocytes by Paracrine and Autocrine Factors Effects of Inflammatory Mediators The clinical observation of post-inflammatory hyperpigmentation has implicated immune inflammatory mediators in this phenomenon (Nordlund and Abdel-Malek, 1988). We have investigated the effects of the immune inflammatory cytokines

One growth factor that has been amply described as a mitogen for normal human melanocytes and as an autocrine stimulator of human melanoma cell growth is bFGF (Halaban et al., 1988a, b). Basic FGF was the first paracrine factor for human melanocytes to be identified. The mitogenic effect of bFGF was observed when it was present concomitantly with an agent that elevates intracellular cyclic adenosine monophosphate (cAMP) levels (e.g. cholera toxin) (Abdel-Malek et al., 1992; Halaban et al., 1988b). Basic FGF elicited its biological 413

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effects by binding to a tyrosine kinase receptor that is expressed on human melanocytes (Pittelkow and Shipley, 1989). Unlike most other keratinocyte-derived growth factors, bFGF is not secreted by keratinocytes, and direct contact of melanocytes with keratinocytes is required for its mitogenic effect.

Endothelins and Human Melanocytes Endothelins represent a family of three related peptides, ET-1, -2, and -3, each of which consists of 21 amino acids and is the product of a distinct gene (Fig. 20.1) (Inoue et al., 1989). These peptides have been shown to be synthesized by, and to influence, many cell types in a paracrine manner (Rubanyi and Polokoff, 1994). Yohn et al. (1993) and Imokawa et al. (1992) showed that ET-1 is produced by human keratinocytes. The latter group also reported that ET-1 is mitogenic and melanogenic for human melanocytes that express specific receptors for ET-1 (Yada et al., 1991). They also showed that ET-1 increases the expression of the two melanogenic enzymes tyrosinase and TRP-1 (Imokawa et al., 1995). The physiological relevance of endothelins to pigmentation is clearly illustrated by the findings that mutations in the genes that encode for ET-3, or the ET-B receptor that binds all three endothelins with a similar affinity, result in piebald spotting and megacolon in mice (Greenstein Baynash et al., 1994; Hosoda et al., 1994). These defects are also evident in humans with Hirschsprung’s disease (Puffenberger et al., 1994). These correlations emphasize the importance of normal ET-3 and ET-B receptor expression in the migration of neural crest derivatives, including the melanocytes, during embryonic development. We have reported that ET-1 interacts synergistically with aMSH and bFGF to stimulate human melanocyte proliferation (Swope et al., 1995). We have also found that ET-3 has identical effects to ET-1 and that both peptides bind the ET receptor on melanocytes with the same affinity (Tada et al., 1998). The latter results suggested that human melanocytes express the ET-B receptor (Tada et al., 1998). Northern blot analysis confirmed that human melanocytes predominantly express ET-B and not ET-A receptors. The production of ET-1 by keratinocytes, particularly in response to UVR and IL-1 treatment, suggests a role for this hormone in mediating the response of melanocytes to sun exposure and inflammation. We have found that ET-1 is an important mediator of the response of human melanocytes to UVR. Treatment of UVirradiated cultured human melanocytes with ET-1 enabled them to overcome the UVR-induced G1 arrest, proliferate, and increase melanin synthesis (Tada et al., 1998). Further evidence for the importance of ET-1 in the response of human melanocytes to UVR was provided by the finding that irradiation of human skin in vivo with UVR resulted in increased expression of ET-1 and tyrosinase genes (Imokawa et al., 1995). Additionally, the stimulatory effect of conditioned medium from UV-irradiated keratinocytes on human melanocytes in vitro was greatly abrogated by a neutralizing

414

antibody to ET-1. Recently, we described a survival effect of ET-1 on UV-irradiated human melanocytes. Treatment of human melanocytes with ET-1 markedly reduced the UVRinduced apoptosis and promoted melanocyte survival (Kadekaro et al., 2003, 2005). Melanocytes have a limited self-renewal capacity, and maintaining their survival is crucial, given their importance in conferring photoprotection to the skin.

Role of Proopiomelanocortin Derivatives: Melanocortins and b-Endorphins The melanocortins are a family of structurally related peptides that include a-, b-, and g-MSH and ACTH, all of which are derived from one precursor peptide, proopiomelanocortin (POMC). In particular, a- and b-MSH have been best known for their melanogenic effects on the integument of many vertebrate species, including mammals (Sawyer et al., 1983). Several decades ago, Lerner and McGuire (1964) reported that injection of humans with high concentrations of a-MSH and b-MSH or ACTH resulted in skin darkening. More recently, Levine et al. (1991) demonstrated that injection of humans with a potent synthetic analog of a-MSH, namely [Nle4,DPhe7]-a-MSH, caused increased pigmentation, mostly in habitually sun-exposed skin. A physiological role for melanocortins in regulating human pigmentation was controversial (Fuller et al., 1993; Halaban et al., 1983). However, with the cloning and characterization of the melanocortin receptors, it became evident that human melanocytes express the melanocortin 1 receptor (MC1R) that binds a-MSH and ACTH with the same affinity (Chhajlani and Wikberg, 1992; Mountjoy, 1994; Suzuki et al., 1996). Using receptor binding assays, Donatien et al. (1992) found that human melanocytes express a low number of MSH receptors, and that receptor expression is upregulated by cholera toxin, but downregulated by 12-Otetradecanoylphorbol-13-acetate (TPA). De Luca et al. (1993) confirmed the presence of MSH receptors on human melanocytes, and showed that these cells respond to a-MSH with increased proliferation, without any change in their pigmentation. Hunt et al. (1994a, b) then reported that human melanocytes respond to a-MSH or ACTH with increased melanogenesis. We have demonstrated that human melanocytes maintained in culture in the absence of any cAMP inducer respond equally to a-MSH and ACTH with an increase in both proliferation and melanogenesis (AbdelMalek et al., 1995; Suzuki et al., 1996). The similarity in the responses of human melanocytes to a-MSH and ACTH confirmed previous pharmacological data obtained using the cloned MC1R, which showed that both hormones have similar binding affinities (Chhajlani and Wikberg, 1992). By comparing the relative affinities of a-, b-, g-MSH, and ACTH for the MC1R and their abilities to stimulate the proliferation and melanogenesis of human melanocytes, we found that aMSH and ACTH were equally potent, b-MSH was less effective than either, and g-MSH was the least effective (Suzuki et al., 1996). These results suggest that a-MSH, ACTH, and

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possibly b-MSH, but not g-MSH, might have a physiological role in human pigmentation. Using Northern blot analysis, we found that human melanocytes express the mRNA transcript for the MC1, but not for the MC2, MC3, MC4, or MC5R. Additionally, we found that the level of the MC1R transcript was increased by brief treatment with a-MSH or ACTH, but decreased by treatment with the MC1R antagonist agouti signaling protein or exposure to UVR (Scott et al., 2002b; Suzuki et al., 1996). Interestingly, treatment of human melanocytes with ET-1 tremendously increased MC1R mRNA levels (Scott et al., 2002b; Tada et al., 1998). This effect of ET-1 suggests that its regulation of human melanocyte proliferation and melanogenesis is not exclusively mediated by binding to the ET receptors, but also by indirectly enhancing the response to melanocortins via upregulating MC1R expression. The observed increase in MC1R mRNA levels following treatment with its agonists a-MSH or ACTH or with ET-1 is expected to sustain, or even enhance, the response of melanocytes to melanocortins. Treatment of human melanocytes with bestradiol increased the expression of MC1R mRNA and augmented the upregulation of the MC1R mRNA level. This suggests a mechanism by which b-estradiol might increase human pigmentation. Studies on the murine Cloudman S91 melanoma cells concluded that UVR upregulates the expression of the MSH receptor, suggesting that the MSH receptor functions as a transducer of the UVR effects on melanoma cells (Chakraborty and Pawelek, 1993). We have found that treatment of normal human melanocytes with a-MSH immediately after irradiation with UVR results in partial recovery from UVR-induced G1 arrest and stimulation of melanogenesis (Im et al., 1998). Recently, we discovered a novel role for melanocortins as survival factors for human melanocytes (Kadekaro et al., 2003, 2005). We have observed that treatment of human melanocytes with either a-MSH or ACTH promotes survival following irradiation with UVR by inhibiting UVR-induced apoptosis and activating survival pathways that are mediated by Akt and the transcription factor Mitf (Kadekaro et al., 2005). Therefore, melanocortins contribute to photoprotection of the skin by maintaining the survival of the melanocyte in the epidermis and stimulating eumelanin synthesis. In addition to a-MSH and ACTH, another POMC-derived peptide, the opioid peptide b-endorphin, was recently shown to stimulate the proliferation, melanogenesis, and dendricity of epidermal human melanocytes (Kauser et al., 2003). bEndorphin may function as a paracrine and autocrine factor for epidermal melanocytes, because it is expressed in the skin as well as in cultured keratinocytes and melanocytes. Moreover, expression of the m-opiate receptor by melanocytes and keratinocytes was also demonstrated, suggesting that the effects of b-endorphin are directly mediated by activating its own specific receptor. Similar findings have just been reported in human follicular melanocytes, which express m-opiate receptor and respond to b-endorphin with increased melano-

genesis, dendricity, and proliferation (Kauser et al., 2004). These recent findings underscore the significance of POMC in the regulation of human skin and hair pigmentation.

Regulation of Eumelanin and Pheomelanin Synthesis by a-MSH and Agouti Protein Genetic studies on the regulation of mouse coat color revealed that the switch from eumelanin to pheomelanin synthesis by follicular melanocytes is mainly regulated by the extension locus and the agouti locus (Quevedo et al., 1981; Robbins et al., 1993; Silvers, 1979). The extension locus codes for the MC1R which, upon activation by a-MSH, stimulates the synthesis of eumelanin (Robbins et al., 1993). The agouti locus codes for a soluble factor, agouti signaling protein, which is temporally expressed in dermal papilla cells within the hair follicles. In mice, expression of different alleles of the agouti and extension loci determines the amount and distribution of pheomelanin and eumelanin in the hair follicles (Quevedo et al., 1981; Silvers, 1979). Five alleles for the murine MC1R have been characterized and found to alter the binding affinity to a-MSH or the coupling to, and activation of, adenylate cyclase and, ultimately, the extent of eumelanin synthesis in follicular melanocytes (Robbins et al., 1993). Five alleles of the agouti locus have been identified and shown to influence the pigmentary phenotype of mice (Silvers, 1979). The wildtype agouti allele results in a narrow subapical band of hair containing pheomelanin. The agouti protein is known to regulate dorsal and ventral pigmentation in the mouse. The difference between dorsal and ventral pigmentation was found to result from the expression of different isoforms of the agouti gene with different sets of 5¢ untranslated exons (Vrieling et al., 1994). Although the role of the agouti locus in the mouse is well defined, its physiological role in the regulation of human pigmentation is to be explored further. The human MC1R gene is homologous to the extension locus in mice. Activation of the human MC1R by its ligand a-MSH increases eumelanin synthesis (Hunt et al., 1995). The MC1R gene is highly polymorphic, with about 65 different allelic variants identified so far (Box et al., 1997; Smith et al., 1998). Many of these alleles were identified in northern European countries and Australia, with some having strong and others having weak association with red hair phenotype. The alleles that are strongly associated with red hair phenotype, namely Arg151Cys, Arg160Trp, and Asp294His substitutions, result in loss of function of the MC1R, and are highly associated with poor tanning ability and increased risk of melanoma (Palmer et al., 2000; Scott et al., 2002a). Expression of the above three alleles in the homozygous or compound heterozygous state increases the sensitivity of human melanocytes to UVR-induced apoptosis, possibly as a result of the inability of these melanocytes to withstand UVR-induced damage (Kadekaro et al., 2005; Scott et al., 2002a). The extensive polymorphism of the MC1R has suggested its significance in the diversity of constitutive human pigmentation and the response of human melanocytes to UVR.

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The mouse agouti gene has been cloned, and its product, a 131-amino-acid peptide, has been purified (Bultman et al., 1992). The human homolog for the mouse agouti gene has also been cloned and found to share 85% homology in its nucleotide sequence with the mouse gene (Kwon et al., 1994). The product of the human agouti gene is a 132-amino-acid peptide termed agouti signaling protein (ASP), which has 80% homology with the mouse protein (Kwon et al., 1994). Expression of ASP in transgenic mice resulted in a yellow coat color, thus providing evidence for the function of the human agouti gene in the regulation of pheomelanin synthesis (Wilson et al., 1995). The availability of purified mouse agouti protein and ASP provided the opportunity to investigate their effects and the mechanisms of action on melanocytes. Agouti protein acts as an antagonist for a-MSH on the mouse follicular melanocytes (Takeuchi et al., 1989; Yen et al., 1994). Agouti protein functions as a competitive inhibitor for a-MSH binding to the mouse MC1R that is expressed in heterologous cells or B16 melanoma cells (Blanchard et al., 1995; Lu et al., 1994). It has also been proposed that the agouti protein reduces the availability of a-MSH to its receptor. A third possibility is that ASP binds a specific, yet unidentified, receptor that counteracts the signal transduction pathway induced by a-MSH. Both recombinant human and murine agouti proteins were found to inhibit the a-MSH-induced stimulation of cAMP formation by murine B16 melanoma cells (Blanchard et al., 1995; Wilson et al., 1995). We have investigated the possible effects of the mouse and human agouti proteins on cultured human melanocytes. Both proteins completely blocked the mitogenic and melanogenic effects of a-MSH (Suzuki et al., 1997). ASP competed with aMSH for binding to the MC1R, and blocked the stimulation of cAMP formation by a-MSH in human melanocytes. These results clearly demonstrated that ASP functions as an antagonist of the human MC1R. However, the exact role of ASP in human pigmentation remains to be explored. Unlike the human MC1R gene which is highly polymorphic, only one variant of the human agouti gene has been identified (Kanetsky et al., 2002; Voisey et al., 2001). This argues against the significance of the agouti gene in the diversity of human pigmentation. The antagonistic effects of ASP are predominantly mediated by binding to the MC1R. Studies on primary cultures of follicular melanocytes established from three different isogenic mouse strains, namely C57 BL6 E+/E+ (which expresses the wild-type MC1R), e/e (recessive yellow mice that express lossof-function MC1R), and ESO/ESO (somber mice that express constitutively active MC1R), that cannot be activated by aMSH clearly showed that the effects of agouti are only evident in melanocytes that express functional MC1R (Abdel-Malek et al., 2001). These results argue against the existence of a receptor other than MC1R that mediates the effects of ASP.

Role of Steroid Hormones Many studies have described the responses of melanoma cells 416

to a wide variety of hormones, including the steroid hormones estrogen and glucocorticoids (Abdel-Malek et al., 1988; Bhakoo et al., 1981; Sadoff et al., 1973). Some studies have shown that normal human melanocytes are a target for different steroid hormones (McLeod et al., 1994; Ranson et al., 1988). We and others have investigated the effects of vitamin D3 (cholecalciferol) and its hydroxylated metabolites on human melanocytes (Abdel-Malek et al., 1988; Mansur et al., 1988; Ranson et al., 1988). Interest in this hormone stemmed from the fact that it is synthesized in the skin upon sun exposure, and from the possibility that active hydroxylated vitamin D3 metabolites might mediate the melanogenic effects of UVR (Bikle et al., 1986; Holick, 1981). We found, using Western blot analysis, that human melanocytes express specific receptors for vitamin D3 (Abdel-Malek et al., 1988). We also showed that melanocytes respond to 1,25(OH)2 vitamin D3 with a decrease in tyrosinase activity. A similar, yet less remarkable, effect was observed using 25(OH)vitamin D3, and no effect was produced by cholecalciferol. In contrast, in vivo studies in which 1,25(OH)2 vitamin D3 was applied topically onto mice demonstrated that the hormone increases the number of DOPA-positive melanocytes, indicating an increase in their melanogenic activity. Additionally, 1,25(OH)2 vitamin D3 augmented the melanogenic effect of UVR on murine skin. Contrary to these findings, Mansur et al. (1988) found no effect of 1,25(OH)2 vitamin D3, cholecalciferol, previtamin D3, lumisterol, or provitamin D3 on human melanocytes in vitro. Also, they could not detect any specific binding of vitamin D3 using sucrose density gradient analysis, or receptor binding assays utilizing radiolabeled 1,25(OH)2 vitamin D3. In yet another study, Ranson et al. (1988) reported that human melanocytes bound 1,25(OH)2 vitamin D3 with a high affinity and responded to this hormone with increased tyrosinase activity and stimulation of the activity of 25(OH)D3-24hydroxylase. The differences among these findings could be partially attributed to the differences in the melanocyte culture conditions used by the different investigators. The cutaneous effects of vitamin D3, particularly on human pigmentation, deserve to be reinvestigated, as this hormone is now used clinically, e.g. for the treatment of psoriasis. The effects of sex steroid hormones (androgens and estrogens) on cutaneous pigmentation have been recognized for a long time (Hamilton, 1941; Hamilton and Hubert, 1938). The increased pigmentation of the areola and genitalia has mostly been attributed to these hormones (Snell, 1964). Changes in the levels of female sex hormones during pregnancy have been implicated in the skin darkening seen in melasma (Mosher et al., 1987). Estrogens have also been investigated for their effects on cultured human melanocytes. Ranson et al. (1988) first reported that b-estradiol resulted in a dose-dependent increase in tyrosinase activity that was accompanied by a reduction in cellular proliferation. More recently, the same investigative group again reported similar responses of human melanocytes to 17-b-estradiol, effects that were evident at a concentration as low as 10–11 M (McLeod et al., 1994). Both a- and b-estradiol had similar effects, whereas estriol was less

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potent than either, and estrone was the least effective. Surprisingly, no specific receptors for estrogens could be detected in melanocytes. Among the different melanocyte lines tested, 65% responded as described above to estrogens, and the response did not correlate with constitutive pigmentation. Whether or not melanocytes from male or female donors differ in their responses to sex hormones is worthy of investigation.

Perspectives Cutaneous pigmentation is a genetically determined trait that is regulated by a complexity of intracellular enzymes and factors. In addition, extracellular endocrine, paracrine, and autocrine factors contribute to the regulation of the melanocyte (Fig. 20.1). A major environmental regulator of cutaneous pigmentation is UVR from sun exposure. It is becoming recognized that the response of melanocytes to UVR is not only direct, but also indirect, as UVR induces the synthesis of a wide array of paracrine and autocrine factors. Molecular and cellular studies, exemplified by the demonstrations that human melanocytes express the MC1R and respond to melanocortins, and that these cells respond to endothelins, provided unequivocal evidence that such factors play a crucial role in the regulation of melanocyte function and response to UVR. The network of cutaneous factors activated by UVR may prove to be critical for the maintenance of genomic stability of human melanocytes in the face of UVR-induced DNA damage, and thus prevention of photocarcinogenesis. The lessons learned from mouse genetic studies linking abnormalities of coat color to specific genetic loci are enabling us to unravel the role of genes coding for particular hormones or their receptors in an array of human pigmentary disorders.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

21

Paracrine Interactions of Melanocytes in Pigmentary Disorders Genji Imokawa

Introduction Over the past two decades since Eisinger and Marko (1982) first established human melanocytes in culture by supplementing them with a tumor promoter, phorbol ester, many cytokines, chemokines, chemicals, and eicosanoids have been discovered to be mitogens and/or melanogens for human melanocytes. In parallel, various kinds of cells in the skin have been shown to produce and secrete or release those growth factors in response to several stimuli, including ultraviolet irradiation. In relation to the tissue localization of cells secreting or releasing those factors and epidermal melanocytes as responders, melanogenic paracrine cytokine networks have been discovered to exist between melanocytes and other types of skin cells, including keratinocytes and fibroblasts, which regulate melanocyte function via the corresponding receptors (Imokawa et al., 1992a, 1995, 1996a, b, c, 1998a, b; Yada et al., 1991). Although these studies have usually used cultured skin cells, they have facilitated research directed toward elucidating regulatory mechanisms involved in melanocyte activation, as seen in several types of experimentally induced hyperpigmentation or in hyperpigmentary disorders. Thus, in vivo studies directed toward identifying intrinsic paracrine cytokines involved in hyperpigmentary disorders have been difficult in the absence of information about whether the up- or downregulation of cytokines and/or chemokines in the lesional skin is responsible for the constitutive activation or inactivation of melanocytes in those lesions. In part, this is due to the wide variety of cytokines or chemokines that are not directly related to melanocyte stimulation but are highly expressed in hyperpigmentary disorders because of the concomitant presence of other types of abnormal epidermal cells in addition to melanocytes. Thus, during our research to identify paracrine cytokines intrinsically involved in experimentally induced hyperpigmentation or those responsible for the stimulatory effects of conditioned medium from cultured keratinocytes or fibroblasts, we used six criteria to determine whether a cytokine is an intrinsic factor involved in epidermal hyperpigmentation, as follows: 1 The cytokine(s) should be highly expressed in the surrounding cells in response to several stimuli. 2 The cytokine(s) should exist in supernatants of cultures or in the epidermis at concentrations sufficient to stimulate melanocytes.

3 The stimulatory effect of culture supernatants on melanocytes should be neutralized by an antibody to the cytokine if it is secretable. 4 The cytokine(s) should have the potential to activate melanocytes at physiological concentrations in vitro. 5 The hyperpigmentation induced should be suppressed by antibodies that inhibit the corresponding receptor or by receptor antagonists in vivo. 6 Mutations in the gene encoding the cytokine or its receptor should produce an aberrant phenotype. According to these six criteria, we have characterized the melanogenic paracrine network in the skin associated with the stimulation of melanocyte function in the epidermis, the activation or inactivation of which leads to the hyper- or hypopigmentation associated with epidermal pigmentary disorders. Figure 21.1 depicts such a melanogenic paracrine network between skin cells, which serves to upregulate melanocyte functions. With respect to melanocyte/keratinocyte interactions, these include endothelin-1 (ET-1) (which is secreted by keratinocytes in response to UVB irradiation) (Imokawa et al., 1992a, 1995; Yada et al., 1991), membrane-bound stem cell factor (mSCF) (which is stimulated in production by keratinocytes in response to UVB irradiation) (Hachiya et al., 2001), granulocyte–macrophage colony-stimulating factor (GM-CSF) (which is secreted by keratinocytes in response to UVA irradiation) (Imokawa et al., 1996c), and growth-related oncogene a (GROa) (which has increased secretion by keratinocytes in response to allergic inflammation) (Imokawa et al., 1998a). With respect to melanocyte/fibroblast interactions, they include soluble-type stem cell factor (sSCF) and hepatocyte growth factor (HGF) (which are secreted by proliferating dermal fibroblasts) (Imokawa et al., 1998b). Consistent with these in vitro findings on melanogenic paracrine cytokine networks, we have found that the upregulation of such networks is intrinsically involved in vivo in the stimulation of melanocyte functions in several hyperpigmentary disorders, such as UVB melanosis (Imokawa et al., 1995), lentigo senilis (LS) (Kadono et al., 2001), seborrheic keratosis (SK) (Manaka et al., 2001; Teraki et al., 1996), Riehl’s melanosis (Imokawa and Kawai, 1987; Imokawa et al., 1992a, 1998a), café-au-lait macules (Okazaki et al., 2003), and dermatofibroma (DF) (Shishido et al., 2001). This chapter focuses on epidermal hyper- or hypopigmentary disorders, including UVB melanosis. The activated paracrine interactions between keratinocytes or fibroblasts and melanocytes responsible for increased pigmentation in the 421

CHAPTER 21

Keratinocyte UVA UVB

Melanocyte GM–CSF

TK–R

Cell response DNA synthesis Melanin synthesis

c–met IL–1α

IL–1 PKA

ET–1

MAPkinase

R Gs cAMP

GROa

Gi

R G

AC

c–met ATP PKC

PLC

Ca

HGF

c–kit bFGF

DG

Inflammation

SCF

IP3 PIP2

mSCF

Aging

c–kit

Fibroblast

Fig. 21.1. Melanogenic paracrine cytokine networks between cultured skin cells.

epidermis are described with special reference to the expression of paracrine cytokines and their corresponding receptors.

Endothelin-1/ETB Receptor Interactions between Keratinocytes and Melanocytes ET-1 was first identified in the culture media of porcine endothelial cells and is a 21-residue peptide with a potent vasoconstrictive activity (Yanagisawa et al., 1988). To date, three distinct genes encoding three closely related peptides, ET1, ET-2, and ET-3, have been identified (Inoue et al., 1989). ETs have been reported to bind to two types of receptors, endothelin A receptor (ETAR) and endothelin B receptor (ETBR), which bind all three ETs with different or similar affinities respectively (Arai et al., 1993; Sakamoto et al., 1993; Sakurai et al., 1990). Our study using ETAR and ETBR antagonists to study the proliferation of ET-stimulated cultured human melanocytes revealed that ETBR is predominantly expressed in human melanocytes, in contrast to the predominant expression of ETAR in human endothelial cells. It has been reported that ETs have hormonal regulatory activities in 422

various types of cells, including melanocytes, and in target organs via a receptor-mediated biochemical mechanism (Brenner et al., 1989; Resink et al., 1989; Reynolds et al., 1989; Watanabe et al., 1989). ET isopeptides are first expressed as corresponding ~200residue inactive prepropolypeptides (prepro-endothelins) that are encoded by distinct genes (Inoue et al., 1989). After removal of their signal peptides during their early processing, the propeptides are cleaved at pairs of basic amino acids to yield the intermediate Big ETs. This early processing step is presumably involved with furin, a prohormone convertase of the constitutive secretory pathway (Laporte et al., 1993). Big ETs are then further cleaved by an endopeptidase termed endothelin-converting enzyme (ECE) at Trp-21-Val-/Ile-22 to produce biologically active ETs, which are mature 21-residue peptides (Yanagisawa et al., 1988). We have recently characterized the properties of ECE-1a in human keratinocytes, which are identical to those in endothelial cells (Hachiya et al., 2002).

UVB Melanosis In 1991, we found for the first time that ET-1 is produced and

Tyrosinase TRP-1 GAPDH β-actin ET-1 bFGF IL-1α TNFα IFNγ

Tyrosinase TRP-1 GAPDH β-actin ET-1 bFGF IL-1α TNFα IFNγ

Fig. 21.2. RT-PCR analysis of UVB (2 MED)exposed human epidermis for tyrosinase, ET-1, IL-1a, and TNFa.

Tyrosinase TRP-1 GAPDH β-actin ET-1 bFGF IL-1α TNFα IFNγ

PARACRINE INTERACTIONS OF MELANOCYTES IN PIGMENTARY DISORDERS

Day 0

Day 2

Day 5

secreted by cultured human keratinocytes and can act as a mitogen and a melanogen for human melanocytes (Imokawa et al., 1992a; Yada et al., 1991). The production and subsequent secretion of ET-1 by human keratinocytes was markedly stimulated by UVB irradiation. This evidence prompted us to investigate the possibility that an accentuated ET-1/ETBR cascade is responsible for the increased pigmentation in the epidermis of UVB melanosis. As expected, in real-time quantitative or semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of UVB-exposed human epidermis, as late as 5–7 days after irradiation, there is a significant increase in the expression of ET-1 mRNA transcripts, which is accompanied by an increased expression in mRNA transcripts encoding the key melanogenic enzyme, tyrosinase (Fig. 21.2) (Hachiya et al., 2004; Imokawa et al., 1995, 1997). A little earlier than that, the gene expression of interleukin (IL)-1a (known to be primarily responsible for stimulating ET-1 secretion in UVB-exposed human keratinocytes) is also accentuated in UVB-exposed human epidermis. Consistent with this, ET-1 secretion is also increased in UVB (2 MED)exposed human epidermis in organ culture in concert with the increased secretion of IL-1a, compared with nonUVB-exposed human epidermis. Immunohistochemistry with an antibody to ET-1 demonstrates a marked increase in immunostaining throughout the UVB-exposed epidermis (at 7 days after irradiation) compared with nonUVB-exposed human epidermis (Hachiya et al., 2004) (Fig. 21.3). Real-time quantitative RTPCR analysis demonstrates that levels of ET-1 mRNA transcripts are significantly upregulated with a peak at 7 days after UVB irradiation, and remain at a higher level than before irradiation until 25 days after irradiation. In concert with the increased expression of ET-1, mRNA transcript levels for its receptor, ETBR, are also significantly upregulated with a peak at 7 days after irradiation in the UVB-exposed human epidermis compared with the nonexposed human epidermis. Western blotting analysis also reveals an increased expression of ETBR protein in the UVB-exposed human epidermis at

UVB

NonUVB

Fig. 21.3. Immunohistochemistry of ET-1 in UVB (2 MED)-exposed human forearm skin at 7 days after irradiation (see also Plate 21.1, pp. 494–495).

7 days after irradiation (Hachiya et al., 2004). Interestingly, the expression pattern of the ET-1/ETBR cascade following UVB irradiation is considerably delayed compared with the time course of the expression of SCF, as described below. The time course of the expression of ET-1/ETBR mRNA transcripts is shown in Figure 21.4 in comparison with those encoding SCF/c-KIT (as described below). The preferential role of the late expression of ET-1 following the expression of SCF in stimulating melanogenesis in melanocytes may be substantiated by the fact that the exogenous addition of sSCF stimulates the expression of ETBR in human melanocytes (Hachiya et al., 2004), resulting in acceleration of the binding of ET-1 to its receptor on melanocytes. 423

CHAPTER 21

Analysis UVB irradiation (288 mJ/cm2)

(day)

0

1

ACK2 injection (5 mg/50 ml)

2

3

4

Skin color Numbers of DOPA-positive cells

5

6

7

8

SCF

9

10

25

ET-1

ETBR

C-Kit

Fig. 21.4. The time course of expression of SCF/c-KIT and ET-1/ETBR mRNA transcripts following 2 MED UVB-exposed human epidermis in comparison with tyrosinase expression and pigmentation.

Tyrosinase

Pigmentation

Lentigo Senilis Lentigo senilis (LS) is the skin condition of common aging spots with accentuated epidermal pigmentation. In LS lesional skin, the number of tyrosinase-immunopositive melanocytes is increased twofold over the perilesional epidermis (Kadono et al., 2001). Consistent with the increased amount of tyrosinase within the lesional melanocytes, RT-PCR analysis for tyrosinase mRNA transcripts demonstrates that there is an increased expression of those transcripts (average 2.3-fold, P < 0.005, n = 7) in the lesional epidermis compared with the perilesional controls. As there are some similarities in the histopathology and localized eruptions, which predominantly affect sun-exposed areas in UVB-induced melanosis and in LS, and as among the known keratinocyte-derived cytokines pro424

10 nMET-1 300 ET-1(–) ET-1(+)

Thymidine incorporation (% control)

The specific contribution of the ET-1/ETBR linkage to melanocyte activation in UVB-exposed human epidermis is corroborated by the fact that an extract of M. chamomilla, which is a potent antagonist of the ETBR (Imokawa et al., 1997) and which abrogates ET-1-stimulated DNA synthesis in human melanocytes (Fig. 21.5) but does not inhibit tyrosinase in human melanocytes (Imokawa et al., 1997), is significantly effective in preventing the UVB-induced pigmentation of human forearm skin when applied topically immediately after UVB irradiation (Fig. 21.6) (Imokawa et al., 1997). Taken together, these results strongly suggest that UVB irradiation causes epidermal keratinocytes to produce and secrete ET-1, which triggers melanocytes in their vicinity to transduce intracellular signaling via accentuated ETBR function and leads to the stimulation of melanogenesis in UVB-induced melanosis.

200

100

0 0

3

15

30

M. chamomilla concentration (µg/ml) Fig. 21.5. Inhibitory effect of an M. chamomilla extract on ET-1stimulated DNA synthesis in cultured human melanocytes.

PARACRINE INTERACTIONS OF MELANOCYTES IN PIGMENTARY DISORDERS

duced in response to their proliferation, ET-1 is the only cytokine that has dual stimulatory effects on DNA synthesis and melanogenesis of human melanocytes (Imokawa et al., 1996a; Yada et al., 1991), we examined whether ET production and secretion is accentuated in the epidermis of LS. RTPCR analysis demonstrates accentuated expression of transcripts for ET-1 (average 3.2-fold, P < 0.05, n = 6) n=8 P < 0.01

Pigmentation (dl)

0

0.5% M. chamomilla

5 0%

10 21

28

35

42

Days after UVB irradiation with 2 MED dose Fig. 21.6. The preventive effect of a topically applied M. chamomilla extract on UVB-induced human pigmentation. Pigmentation was measured by a color difference meter and is expressed as delta L.

(Fig. 21.7) and for ETBR (average 6.8-fold, P < 0.05, n = 7) in LS lesional epidermis (Kadono et al., 2001). Consistent with this, immunohistochemistry using antibodies to ET-1 or ETBR demonstrates relatively stronger staining in the lesional epidermis (Fig. 21.8) or melanocytes than in the perilesional epidermis or melanocytes respectively. Our previous study demonstrated that, among more than approximately 50 cytokines or chemokines, only three (IL-1a, IL-1b, and TNFa) are ET-1-inducible cytokines (Imokawa et al., 1992a). In focusing on the mechanism(s) involved in the increased expression of ET-1, this prompted us to determine the expression level of these cytokines in the epidermis of LS (except for IL1b, which is not secreted by keratinocytes). The ET-1inducible cytokine, TNFa, is consistently upregulated in the LS lesional epidermis, as determined at the transcriptional level (average 5.5-fold, P < 0.05, n = 3) and by immunostaining, whereas IL-1a (known as a stimulator of ET-1 secretion in UVB-exposed human keratinocytes) is downregulated (average 0.26-fold, n = 2) at the transcriptional level and by immunohistochemistry (Kadono et al., 2001). In contrast, levels of ECE-1a mRNA transcripts (another factor responsible for stimulating the secretion of ET-1) were not substantially increased in the lesional epidermis. Thus, it seems likely that, in contrast to the increased expression of IL-1a as a factor stimulating ET-1 secretion in UVB melanosis, the increased expression of TNFa triggers the stimulation of ET-1 secretion in the LS lesional epidermis. These findings suggest that an accentuation of the epidermal ET cascade, especially with respect to the expression of ET and the ETBR, which is probably triggered by the stimulated secretion of TNFa, plays an important role in the mechanism involved in the hyperpigmentation of LS.

Seborrheic Keratosis Seborrheic keratosis (SK) is a common benign tumor with

Fluorogram A

Densitometric analysis

Fig. 21.7. RT-PCR analysis of ET-1 mRNA in the epidermis of LS and in perilesional skin. (A) Fluorogram. (B) Densitometric analysis. NL, non-lesional epidermis; L, lesional epidermis of LS. Fluorograms are shown at 35 or 37 cycles of PCR for ET-1 and at 22 or 23 cycles for G3PDH. *P < 0.05.

Relative intensity

B

1.4 1.2 1

* 3.2 fold

0.8 0.6 0.4 0.2 0

Patient

NL L 1

NL L NL L NL L NL L NL L 2 3 4 5 6

NL L NL L NL L intensity 7 8 averaged n=8

425

CHAPTER 21 A

Lesion B

Fig. 21.9. Immunostaining with anti-ET-1 in the epidermis of SK (¥180) (see also Plate 21.3, pp. 494–495). Non-lesion Fig. 21.8. Immunostaining with anti-ET-1 in the epidermis of LS. (A) Lesion (¥180). (B) Nonlesion (¥180). See also Plate 21.2, pp. 494–495.

accentuated epidermal pigmentation, which is thought to develop clinically from LS. In the tumor region, there are increased numbers of dopa-positive melanocytes in concert with an increased expression of tyrosinase mRNA as measured by RT-PCR analysis (Teraki et al., 1996). Based upon the observation that increased levels of melanin-producing melanocytes are located in the vicinity of highly proliferating keratinocytes (as seen in hair follicles), we hypothesized that the proliferating keratinocytes in SK trigger the activation of neighboring melanocytes by secreting melanocyte-stimulating cytokines. Thus, the accentuated melanization observed in SK may be associated with the increased production of ETs by the highly proliferating keratinocytes. Therefore, using RT-PCR and immunohistochemistry, we determined whether the production of ET-1 is accentuated in SK of acanthotic and deeply pigmented types. RT-PCR of ET-1 mRNA demonstrates increased levels of ET-1 mRNA transcripts (average 1.7-fold, P < 0.01, n = 5) in SK compared with levels in the perilesional normal controls, which is accompanied by a similarly accentuated expression of tyrosinase mRNA (average 1.7-fold, P < 0.01, n = 5) (Manaka et al., 2001; Teraki et al., 1996). In parallel, immunohistochemical analysis in SK reveals marked immunostaining for ET-1 in almost all basaloid cells (Fig. 21.9) compared with the perilesional normal epidermis. These findings indicate that, in nonUV-associated hyperpigmentary disorders such as SK, there is an overstimulation of ET production and subsequent secretion by keratinocytes, which results in the activation of melanocytes and leads to hyperpigmentation. Although ET secretion by keratinocytes is stimulated following exposure to UVB (Imokawa et al., 1992a), it is con426

ceivable that the increased pigmentation in SK is not directly related to UV exposure because it also appears in nonUVexposed areas. Therefore, it is of considerable interest to know how the secretion of ET-1 is accentuated in SK epidermis via nonUV-associated mechanisms. The production and secretion of ETs by keratinocytes is known to be generally augmented by several inflammatory cytokines, such as IL-1a or TNFa. In UVB-exposed human keratinocytes (Imokawa et al., 1992a; Tsuboi et al., 1994), IL-1a plays an essential role as an autocrine factor enhancing the production and secretion of ET-1. In UVB-exposed epidermis, IL-1a gene expression is consistently augmented prior to the increase in ET-1 gene expression (Imokawa et al., 1995). In contrast, the role of TNFa in UVB-stimulated ET secretion remains unclear because of the absence of increased gene expression in UVBexposed epidermis (Imokawa et al., 1995), although the exogenous addition of TNFa (10 ng/ml) to human keratinocytes in culture stimulates the transcription and eventual secretion of ET-1 (Tsuboi et al., 1994). Thus, it is of importance to determine whether some endogenous factors (known or unknown) are involved in the increase in ET secretion in SK. For this reason, we focused on ET-inducible factors, including IL-1a and TNFa to assess their expression in SK epidermis at the gene and protein levels. RT-PCR of TNFa mRNA demonstrates an increased expression of TNFa transcripts in SK (average 2.6-fold, n = 5) in comparison with the perilesional normal skin (Manaka et al., 2001), whereas there is a marked increase in ET-1 (average 1.7-fold, n = 5) and tyrosinase mRNA transcripts (average 1.7-fold, n = 5) in the lesional skin of SK. In contrast, there is a marked decrease in IL-1a mRNA transcripts (average 0.43-fold, n = 6) in SK in comparison with perilesional normal skin (Manaka et al., 2001). To confirm changes in the production of TNFa and IL1a in SK, we examined whether the production of these cytokines is reflected by changes in their transcripts. Staining with antibodies against TNFa reveals a distinct positive immunoreactivity throughout the basal layers and along the

PARACRINE INTERACTIONS OF MELANOCYTES IN PIGMENTARY DISORDERS

lower epidermis in SK, whereas there is only a weakly positive staining in the perilesional normal control (Manaka et al., 2001). In contrast, there is decreased staining for IL-1a in SK relative to the perilesional skin (Manaka et al., 2001). As TNFa is a potent stimulator of ET secretion in keratinocytes (Tsuboi et al., 1994), it is conceivable that the high production of TNFa in SK is a major factor responsible for the upregulation of ET secretion, although the precise mechanism leading to the increased secretion of TNFa in SK epidermis remains to be elucidated. In contrast to the ET-stimulatory mechanism involved in UVB-exposed keratinocytes or epidermis, it is unlikely that IL-1a is associated with the increased secretion of ET-1 in SK epidermis. Thus, it should be noted that there is a different secretion pattern for two primary inflammatory cytokines, IL-1a and TNFa, between UVBexposed and SK epidermis, with both cytokines and TNFa only, respectively, being stimulated. The increased secretion of TNFa in SK epidermis may be related to the tumorigenicity of SK epidermal cells. The secretion of ET-1 is regulated biologically by the proteolytic converting enzyme (ECE-1a), which degrades Big ET-1 to yield its active form, ET-1. Thus, the production of the active form of ET depends on the activity of ECE-1a, which is probably based on its expression levels in the epidermis. Although there have been no reports describing the presence of ECE-1a in human epidermis, we attempted to determine whether the same ECE-1a as in endothelial cells is expressed in human keratinocytes, and if there is increased expression of ECE-1a in SK epidermis that might account for the mechanism involved in the increased secretion of ET-1. RTPCR analysis of ECE-1a demonstrates that human keratinocytes in culture express the same ECE-1a transcript as endothelial cells but that the expression level is not altered by UVB exposure at 48 h after irradiation, which is in contrast to the increased expression of ET-1 transcripts (Manaka et al., 2001). Our biochemical characterization of ECE-1a in cultured human keratinocytes demonstrated that it has the same enzymatic properties as human endothelial cells (Hachiya et al., 2002). This suggests that, as in endothelial cells, ECE-1a plays an important role in the processing of ET in human keratinocytes. RT-PCR analysis of ECE-1a mRNA reveals a remarkably accentuated expression of those transcripts in the lesional skin of SK (average 15.2-fold, n = 3) (Manaka et al., 2001). Reactivity with antibodies to ECE-1a demonstrates a diffuse positive staining in basaloid cells and in basal layers of SK, whereas no staining with ECE-1a antibodies could be seen in the perilesional normal controls (Manaka et al., 2001). This strongly suggests that the increased expression of ECE-1a also plays a pivotal role in accelerating the secretion of ET-1 in SK, although the mechanism(s) involved in the accentuated expression of ECE-1a in SK epidermis remain(s) unclear. Thus, these findings suggest that there is a general upregulation of the ET-1 inductive linkage in SK, which triggers the high level of secretion of ET-1. This also indicates that the expression of cytokines and/or enzymes is very similar in LS and SK, except for the expression of ECE1a, which is specifically upregulated

in SK but not in LS. Taken together, our results suggest that at least the accentuated expression of the ET cascade (including ECE-1a) is associated with the stimulation of melanocytes leading to the hyperpigmentation in SK.

Membrane-bound SCF/c-KIT Interactions between Keratinocytes and Melanocytes SCF is known as steel factor, kit ligand, and mast cell growth factor, and is encoded by the steel (Sl) locus; its receptor (ckit) is encoded by the dominant white spotting (W) locus. Mutations in either of these loci elicit very similar phenotypes, which are characterized by the loss of neural crest-derived pigment cells, hematopoietic stem cells, and primordial germ cells (Bernstein et al., 1991; Besmer et al., 1993; Galli et al., 1993; Halaban and Moellmann, 1993; Matsui et al., 1990; Orr-Urtreger et al., 1990; Williams et al., 1992). The involvement of SCF/c-kit signaling in melanocyte development at embryonal stages has been delineated by phenotype analysis of Sl and W mice. Experiments using a monoclonal antibody to c-kit (ACK2), an antagonistic blocker of c-kit function, also demonstrated the importance of SCF/c-kit signaling in the development of murine melanocytes (Nishikawa et al., 1991; Okura et al., 1995; Yoshida et al., 1996). Piebaldism, a disorder presenting at birth that is characterized by amelanotic patches on acral and/or ventral skin surfaces, but apparently lacking detectable defects in germ cells or in the hematologic system, is caused by mutations in the genes encoding c-kit (Ezoe et al., 1995; Giebel and Spritz, 1991; Spritz et al., 1992) or ETBR (Amiel et al., 1996; Puffenberger et al., 1994). Thus, it seems likely that the SCF/c-kit linkage plays a pivotal role in developing and regulating melanocyte survival and function, defects in which lead to hypopigmentation.

UVB Melanosis The interaction of SCF with its receptor, c-kit, is well known to be critical to the survival of melanocytes. However, little is known about the role(s) of SCF/c-kit interactions in regulating epidermal pigmentation. Because no data were available concerning the expression of SCF in human keratinocytes in response to UVB irradiation, we determined whether UVB stimulates the expression of SCF in human keratinocytes at the gene and/or protein levels. Our results showed that the exposure of cultured human keratinocytes and melanocytes to UVB upregulates transcription and protein expression of SCF and c-kit respectively (Hachiya et al., 2001). This prompted us to determine whether the exposure of human epidermis to UVB in vivo stimulates the expression of SCF or c-kit at the gene and/or protein levels. In that study (Hachiya et al., 2001, 2004), human volar forearms of normal volunteers were exposed to UVB radiation at 2 MED. At various times after irradiation, suction blisters were induced at the irradiated sites, and epidermal sheets were removed for subsequent studies. Real-time quantitative RT-PCR analysis of UVB-exposed human epidermis at several 427

CHAPTER 21

A

UVB

Control

B UVB

3 days after 2MED UVB irradiation

Control

Membranebound SCF

31 kD

Soluble SCF

18 kD

UVB

Control

30000 25000 20000 15000 10000 5000 0 Control

B

UVB

A

Recombinant SCF

well to several stimuli (including UVB radiation) to induce hyperpigmentation (Imokawa et al., 1986), we used those guinea pigs as a useful model to determine whether interruption of SCF/c-kit signaling abolishes the UVB-induced pigmentation. Prior to that, we evaluated the cross-reactivity of the c-kit antibody ACK2 (Nishikawa et al., 1991) to brownish guinea pig c-kit by examining the stimulatory effect of murine SCF on the proliferation of bone marrow cells from brownish guinea pigs and the prevention of that effect fol-

Intensity

days after irradiation demonstrates that the expression of SCF mRNA transcripts increases significantly 3 days after the irradiation with a peak on day 5 compared with the nonexposed epidermis, after which SCF levels return to the nonirradiated control level by day 25. In a parallel study, pigmentation levels increased significantly by day 7 and reached a plateau at day 10. Expression of tyrosinase mRNA transcripts did not change by day 3, but had increased at day 5 with a plateau at day 10, which paralleled the time course of pigmentation levels. Tyrosinase protein expression assessed by Western blotting analysis was remarkably augmented 7 days after the UV exposure. This time course of expression of SCF/c-KIT mRNA transcripts is summarized in Figure 21.4 and, when compared with the expression of ET-1/ETBR mRNA transcripts, indicates an earlier expression of SCF mRNA transcripts compared with the ET-1/ETBR linkage. Western blotting analysis of mSCF shows a marked increase in UVB-exposed human epidermis compared with nonexposed epidermis, whereas sSCF is not detectable in either type of epidermis (Fig. 21.10). Immunohistochemistry of UVB-exposed human skin with SCF antibodies shows a strong immunostaining located in the stratum spinosum, accompanied by epidermal hyperplasia, compared with nonexposed epidermis (Fig. 21.11). On the other hand, there was no immunostaining with control nonspecific immunoglobulin G (IgG) in the UVB-exposed epidermis. To verify the contribution of the SCF/c-KIT linkage to melanocyte activation during UVB-induced melanosis, it is important to determine whether blockage of that ligand/receptor interaction would result in inhibition or prevention of the UVB-induced pigmentation. Because brownish guinea pigs have functional melanocytes in their epidermis that respond

A B Fig. 21.10. Membrane-bound SCF is markedly increased by UVB irradiation of human epidermis in vivo. (A) Western blotting. (B) Densitometric analysis.

3 days after 2MED UVB irradiation Anti-SCF

Non-specific IgG

Non UVB

100µm Anti-SCF

Non-specific IgG

UVB

100µm

428

Fig. 21.11. Immunostaining with anti-SCF in the epidermis of UVB (2 MED)-exposed human forearm skin (see also Plate 21.4, pp. 494–495).

PARACRINE INTERACTIONS OF MELANOCYTES IN PIGMENTARY DISORDERS

UVB irradiation (288 mJ/cm2)

0

1 Day 6

Fig. 21.12. The c-kit inhibitory antibody, ACK2, abolishes the UVB-induced pigmentation on the dorsal skin of brownish guinea pigs. Guinea pigs were exposed twice to UVB and then injected subepidermally with ACK2 or purified rat IgG 24 h and 48 h after the final UVB irradiation. See also Plate 21.5, pp. 494–495.

lowing treatment with ACK2. The addition of murine SCF stimulated bone marrow cells to synthesize DNA in a dosedependent manner, and that increase could be completely abrogated by ACK2. Control IgG had no such effect, indicating that ACK2 can inhibit the c-kit receptor function of brownish guinea pigs (Hachiya et al., 2001). Further, immunohistochemistry of brownish guinea-pig skin with an antibody to human c-kit revealed that melanocytes in the epidermis of brownish guinea pigs express that receptor. In order to interrupt SCF binding to c-kit, guinea pigs were exposed twice to UVB at a dose of 288 mJ/cm2 and were then injected subepidermally with 5 mg/50 ml ACK2 (or purified rat IgG as a control) 24 h and 48 h after the second UVB irradiation. When observed 6 days after the first UVB irradiation, treatment with ACK2 remarkably abolished the UVB induction of pigmentation, and the color of the UVB-irradiated skin remained similar to the nonexposed area (measured by a color difference meter), whereas a similar injection with nonspecific IgG did not elicit such inhibition (Fig. 21.12) (Hachiya et al., 2001). When observed 10 days after the first irradiation, the inhibitory effect was only partial and a slight increase in skin pigmentation was measured with the color difference meter (Hachiya et al., 2001). In parallel, the numbers of dopapositive melanocytes are comparable on day 6 in the ACK2-injected site and in the nonexposed area in contrast to significant increases in the numbers of dopa-positive melanocytes in the nonspecific IgG-injected site (Fig. 21.13) (Hachiya et al., 2001). On day 10, the number of melanocytes in the ACK2-treated site had increased slightly, but was still significantly less than in the nonspecific IgG-treated site (Fig. 21.13). Continuous observations of skin color changes revealed that a significant inhibition of pigmentation remained at a level similar to that at day 10 until several weeks after

ACK2 injection (5 mg/50 ml)

3

2

Measurement

4

5

6

7

8

A

B

C

Non UVB

UVB + ACK2

UVB + IgG

9

10 (day)

irradiation. These findings strongly suggest that upregulation of SCF/c-kit signaling between keratinocytes and melanocytes is associated with the increased pigmentation in the UVBexposed skin. One of the most important issues addressed concerning the mode of action of SCF refers to how SCF acts on melanocytes during the upregulation of melanogenesis to stimulate their proliferation and melanization. Thus, the observed inhibition of UVB-induced pigmentation by antibodies to c-kit was complete at day 6 but not at day 10, indicating that SCF functions in an early phase of melanogenic stimulation (Hachiya et al., 2001). This was also corroborated by the increased number of dopa-positive melanocytes at day 10, even in the antibodyinjected site (Fig. 21.13), which was paralleled by the skin color changes, although there was still a significant inhibition in melanocyte number and in skin color at the antibodyinjected site compared with the control. In our experiments using hairless mouse epidermis, the expression of SCF, as measured by immunohistochemistry and by enzyme-linked immunosorbent assay (ELISA) reached a peak as early as 24 h after the second daily irradiation with UVB. This contrasts with the later phase of production and secretion for the UVB pigmentation-associated intrinsic cytokine, ET-1, under similar UVB exposure conditions, which takes as long as 5 days or more (Imokawa et al., 1995). Therefore, it is likely that the incomplete inhibition of pigmentation observed at day 10 following the blockage of SCF/c-kit binding is due to an essential role of the SCF/c-kit signaling in an early phase of melanocyte activation as depicted in Figure 21.4. In the later phases of melanogenesis, ET-1 may play an essential role, as described previously (Imokawa et al., 1995). In conclusion, these findings suggest that, in addition to the ET-1/ETBR signaling, SCF/c-kit signaling is also involved in the 429

Day 6

Mean+SD (n=6)

1400 1200 1000

P<0.01

800 600 400 200 0 Non UVB

UVB + ACK2

UVB + IgG

Numbers of DOPA–positive melanocytes (per mm2)

Numbers of DOPA–positive melanocytes (per mm2)

CHAPTER 21

Day 10

Mean+SD (n=8) P<0.01

1400 1200 1000 800 600 400 200 0 Non UVB

biological mechanism(s) of the early phase of UVB pigmentation, both as a mitogen and as a melanogen for human melanocytes.

UVB + ACK2

UVB + IgG

Fig. 21.13. The c-kit inhibitory antibody, ACK2, suppresses the increase in the number of dopa-positive melanocytes in the epidermis of brownish guinea-pig skin. The number of dopa-positive melanocytes was measured in epidermal sheets on day 6 and on day 10, and are expressed as numbers per mm2. The values represent means ± SD from six or eight independent experiments. **P < 0.01.

A

Lentigo Senilis There are some similarities in the histopathology and localized eruptions that predominantly affect sun-exposed areas in UVB-induced hyperpigmentation and in LS. Subsequently, we demonstrated that, in addition to the ET cascade, SCF and its receptor KIT are also constitutively associated with UVBinduced hyperpigmentation of the epidermis (Hachiya et al., 2001). This prompted us to determine whether SCF and c-KIT expression is also upregulated in the epidermis of LS, and might play another important role in stimulating melanocytes to produce large numbers of melanin granules. In the epidermis of LS lesions, SCF expression is markedly accentuated. Thus, LS lesional epidermis expresses increased levels of SCF mRNA transcripts (average 3.9-fold, P < 0.01, n = 7) concomitantly with the increased levels of SCF protein (average 1.6-fold, P < 0.05, n = 6) by Western blotting compared with nonlesional controls (Hattori et al., 2004). mRNA transcripts for the SCF receptor, c-KIT, are upregulated in the LS lesional epidermis compared with the nonlesional control (average 2.14-fold, n = 3), although further data are required for a conclusion on the expression of SCF receptor mRNA. Consistent with this, by immunohistochemistry, the SCF receptor is markedly increased in staining throughout the LS lesional epidermis (Fig. 21.14). In contrast, two other melanogenic paracrine cytokines (GROa and bFGF) occur at similar levels of mRNA expression or immunostaining in the LS lesional and in the nonlesional skin. By immunohistochemistry, SCF expression is markedly increased in staining throughout the LS lesional epidermis (Fig. 21.15), which is corroborated by Western blotting using an antibody to SCF 430

Lesion

B

Nonlesion Fig. 21.14. Immunostaining with anti-c-KIT in the epidermis of LS. (A) Lesion (¥180). (B) Nonlesion (¥180). See also Plate 21.6, pp. 494–495.

(Hattori et al., 2004). Based on previous evidence on the association of the SCF/c-kit cascade with the activation of melanocytes in vitro (Imokawa et al., 2000), these findings strongly suggest that the stimulated melanogenesis in LS lesional epidermis is involved in the accentuated expression of SCF in LS lesional keratinocytes. In epidermal keratinocytes, SCF is expressed as a membrane-bound form, not in a secretory or soluble form as is ET1 (Imokawa et al., 1992a). Even in stimulated conditions such

PARACRINE INTERACTIONS OF MELANOCYTES IN PIGMENTARY DISORDERS

A

B

Fig. 21.15. Immunostaining with anti-SCF in the epidermis of LS. (A) Lesion (¥180). (B) Nonlesion (¥180). See also Plate 21.7, pp. 494–495.

as following UVB exposure, only the membrane-bound form of SCF is increased, and the soluble form is not detected (Hachiya et al., 2001). In contrast, dermal fibroblasts can secrete sSCF, probably as a result of the action of proteolytic enzymes capable of cleaving mSCF to release sSCF (Imokawa et al., 1996a, 1998b). In epidermal hyperpigmentation of DF (Shishido et al., 2001) or café-au-lait macules (Okazaki et al., 2003), as described below, sSCF secreted by dermal fibroblasts plays an important role in activating epidermal melanocytes via a dispersion pathway through the basement membrane from the dermis toward the epidermis. In cases where sSCF is involved in the activation of epidermal melanocytes, dermal mast cells also accumulate or increase in the upper dermis because sSCF is basement membrane permeable (and is also known as mast cell growth factor). Consistent with this, Longley et al. (1993) demonstrated that abnormalities in the proteolytic processing of SCF tend to produce sSCF in the epidermis, which leads to the phenotype of mastocytosis. Further, Kunisada et al. (1998) reported that the overexpression of sSCF in murine epidermis induces the accumulation of mast cells in the dermis in addition to increasing the number of melanocytes in the epidermis. In our study, as the number of mast cells is not increased in the lesional dermis of LS and as sSCF is not detected in LS lesional epidermis by Western blotting (Hattori et al., 2004) (indicating no mutation in the gene for the SCF in LS), it is likely that the accentuated production of mSCF plays an essential role in the increased proliferation and pigmentation of melanocytes, and that this leads to the epidermal hyperpigmentation in LS. This is consistent with a

report (Kunisada et al., 1998) using mouse skin in which the expression of mSCF alone resulted in epidermal melanocytosis and melanin production, but did not by itself cause mastocytosis. Thus, we suppose that a stimulation of two epidermal cascades, consisting of ET-1/ETBR and SCF/c-KIT, plays an important role in the hyperpigmentation mechanism of LS. The concomitant accentuated expression of both linkages is also confirmed for ET-1, ETBR, and tyrosinase by RT-PCR analysis and for ET-1 and tyrosinase by immunohistochemistry in the same donor as tested for SCF. The preference for enhanced expression of ET-1 and SCF in the LS lesional epidermis and the melanogenic stimulation of melanocytes is corroborated by the existence of the synergistic effects of SCF and ET-1 in prompting melanogenesis and mitogenesis by cultured melanocytes, which occurs via transactivation of SCF-stimulated KIT protein as a result of protein kinase C activation by ET-1 (Imokawa et al., 2000). These marked synergistic effects are restricted to the cross-talk type of interactions only between ET-1 and SCF among the cytokines tested. Thus, it is likely that, although there are multiple and complex mechanisms involved in the accentuated pigmentation of LS lesions, the coordinated and synergistic roles of SCF and ET1 in intracellular signaling leading to melanogenesis (Imokawa et al., 2000) may support the rationale for such ligand specificity in the melanogenic stimulation of LS lesions. Our findings collectively suggest that, in addition to the stimulated ET cascade, the accentuated SCF/c-kit cascade also contributes to the epidermal hyperpigmentation seen in LS lesions.

Vitiligo Vulgaris Vitiligo is an acquired idiopathic disorder of the skin (Nordlund et al., 1998). Although several hypotheses have been proposed for the loss of functional melanocytes in vitiligo skin, its etiology is still unknown. In the hypopigmented skin of vitiligo patients, it has been argued whether melanocytes remain but have lost their ability to synthesize melanin in melanosomes (Hann and Nordlund, 2000; Nordlund et al., 1998; Tobin et al., 2000). However, whatever mechanisms underlie the loss of pigmentation in vitiligo lesions, it is quite conceivable that the deterioration of melanocyte functions, including melanogenesis, and their survival within the epidermis are early events leading to the eventual and complete loss of epidermal pigmentation. Several hypotheses have been proposed for the pathophysiological mechanism(s) involved in the dysfunction or degeneration of melanocytes in vitiligo skin. These include: (1) an autoimmune mechanism in which antibodies or cytotoxic T cells reactive with melanocytes or their specific organelles (melanosomes) are produced in vitiligo patients and cause the death or apoptosis of melanocytes (van den Wijngaard et al., 2001); (2) an autocytotoxic mechanism in which superoxides, including hydroxyperoxide, are generated in abundance in the skin of vitiligo patients, which are toxic to melanocytes (Maresca et al., 1997; Passi et al., 1998; Schallreuter et al., 1991); and (3) an abnormality in melanocytes or in surrounding keratinocytes in producing 431

ETBR

S-100 α Tyrosinase

c-kit

S-100 α Tyrosinase ETBR

c-kit

S-100 α Tyrosinase ETBR

c-kit

S-100 α Tyrosinase ETBR

c-kit

S-100 α Tyrosinase ETBR

140

c-kit

CHAPTER 21

Remaining melanocytes (%)

120 100 80 60 40 20 0

Peri A Nonlesion

Peri B

Edge C-1

Edge C-2 Lesion

factors or their receptors that are necessary for the survival or function of melanocytes within the epidermis (Morretti et al., 2002). Based on the presence of paracrine cytokine networks in regulating melanogenesis in the epidermis, we hypothesize that some paracrine cytokines and their receptors may be involved in the melanocyte dysfunction or loss that occurs in vitiligo epidermis. In RT-PCR analysis of vitiligo epidermis separated by the suction blister technique, we found that there is an increased expression of ET-1 (average 2.44-fold, P < 0.05, n = 6) and SCF mRNA transcripts (average 2.52-fold, P < 0.05, n = 6) in lesional vitiligo epidermis compared with nonlesional epidermis, and that SCF protein expression is increased in the lesional epidermis (Kitamura et al., 2004). Unexpectedly, this indicates upregulated cytokine production in the lesional epidermis, which suggests that there is no diminished ability of vitiligo keratinocytes to produce these melanogenic cytokines. That finding led us to determine whether the SCF receptor (KIT protein) or the ETBR is altered in expression or in their ability to respond to their ligands, which would result in a deficiency for inducing melanogenesis or maintaining melanocyte function in vitiligo skin. Following assessment of the distinct border between nonlesional and lesional skin by Fontana–Masson staining, immunohistochemistry with antibodies to several melanocyte factors demonstrated that, although there is a complete loss of immunoreactive melanocytes in the center of the vitiligo lesional epidermis, there is an almost normal number of melanocytes expressing tyrosinase, ETBR, and S100a protein at the edge of the lesional nonpigmented epidermis, compared with the nonlesional pigmented epidermis. In contrast, there are very few melanocytes expressing KIT protein at the same edge (Kitamura et al., 432

Center D

Fig. 21.16. Quantitative analysis of melanocytes immunopositive for ETBR, KIT protein, tyrosinase, S100a over basal cells. n = 6. *P < 0.05, ***P < 0.005 compared with Peri A. Melanin deposits were detected by Fontana–Masson staining to determine the border of the lesion, and the numbers of melanocytes positively stained with antiETBR, c-KIT, tyrosinase, or S100a antibodies were counted and are expressed per 200 basal cells at the border of the vitiligo lesion and at the center of the vitiligo lesion.

2004). Quantitation of the number of melanocytes expressing such melanocytic factors reveals that the number of cells expressing KIT protein is significantly decreased at the edge of the lesional epidermis compared with the nonlesional epidermis, while there is only a slight decrease in the number of S100a, ETBR, and tyrosinase immunopositive cells at the same edge (Fig. 21.16). Consistently, by Western blotting, KIT protein expression is significantly decreased at the edge of vitiligo lesions compared with the nonlesional epidermis (Fig. 21.17). This suggests that a selective deficiency in KIT protein expression, among the melanocyte-associated molecules tested, occurs in melanocytes localized at the edge of the vitiligo lesional epidermis. Collectively, our results indicate that, in vitiligo epidermis, SCF/c-kit interactions are particularly disrupted because of the loss or deficiency of SCF receptor function on melanocytes, despite the increased production of its ligand, SCF, the interruption of which may lead to complete abolition of the melanogenic potential of melanocytes. In summary, the expression patterns observed for KIT protein or tyrosinase in melanocytes located at the border between the lesional and the nonlesional vitiligo epidermis indicate that, during the sequential process leading to the dysfunction or the loss of melanocytes, the initial event may result from a deficiency in KIT protein function within melanocytes in the nonlesional epidermis. Such a deficiency would lead to a decrease in the expression of its target molecule, tyrosinase, via activation of microphthalmia-associated transcription factor (MITF) by mitogen-activated protein (MAP) kinase. The diminished signaling via the SCF/c-KIT linkage and the distinct decrease in tyrosinase expression may underlie the complete loss of melanocyte function at the border between the lesional and the nonlesional vitiligo skin. In conclusion,

PARACRINE INTERACTIONS OF MELANOCYTES IN PIGMENTARY DISORDERS

Lesion

Nonlesion

0.04

Nonlesion

Patient 1

0.09

Relative intensity

Center D

0.83

Edge C

1.0

Edge B

0.15

Peri A

0.03

Center D

0.82 Edge B

1.0 Peri A

Normal human melanocyte

1.64

Edge C

c-kit 135 kD

Lesion Patient 2

Fig. 21.17. Western blotting of KIT protein in the epidermis of vitiligo lesions and in nonlesional skin. KIT protein expression is markedly decreased at the edges (Edge C) as well as at the center (Center D) of the vitiligo lesion compared with the nonlesional epidermis (Peri A and Edge B).

we hypothesize that the decreased expression of KIT protein in melanocytes may be responsible for the dysfunction and/or the loss of melanocytes in vitiligo epidermis.

Soluble-type SCF/c-KIT and HGF/c-MET Interactions between Fibroblasts and Melanocytes In epidermal keratinocytes, SCF is expressed in the membranebound form, not in a secretory or soluble cytokine form as is ET-1 (Hachiya et al., 2001). Even in stimulated conditions such as following UVB exposure and as seen in LS, only the membrane-bound form of SCF is increased without the appearance of sSCF (Hachiya et al., 2001; Hattori et al., 2004). In contrast, dermal fibroblasts can secrete sSCF, probably due to the action of proteolytic enzymes capable of cleaving mSCF to release sSCF (Imokawa et al., 1996a). Previously, we found that the conditioned medium from proliferating fibroblasts in culture has a potent ability to stimulate DNA synthesis in cultured human melanocytes. A study using neutralizing antibodies to SCF revealed that the DNA synthesis stimulated in human melanocytes is markedly abolished by antibodies to SCF and/or to HGF and, to a lesser extent, to bFGF (Imokawa et al., 1996a, 1998b). This suggests that SCF and/or HGF secreted by cultured human fibroblasts is responsible for the stimulation of DNA synthesis in cultured human melanocytes. Consistent with this, human fibroblasts secrete larger amounts of SCF and of HGF than of bFGF. This evidence leads to the notion that the major melanogenic cytokines secreted by proliferating human fibroblasts are SCF and HGF.

Dermatofibroma Dermatofibroma (DF) is a benign dermal tumor of fibroblastlike cells (Heenan, 1997). DF is also known as histiocytoma, benign fibrous histiocytoma, and sclerosing hemangioma. It has been reported that some cases of multiple DF are associated with autoimmune diseases such as systemic lupus erythematosus (SLE) (Lin, 1986; Newman and Walter, 1973). In lesional DF skin, there is an abnormal accumulation of proliferating fibroblast-like cells and histiocytes surrounded by mature collagen and increased numbers of capillaries. Of particular interest is the fact that the epidermis overlying DF tumors is markedly hyperpigmented with a slight acanthosis (Heenan, 1997), although the mechanism behind the pathogenesis of that epidermal hyperpigmentation remains to be clarified. Consistently, the number of tyrosinase-immunopositive melanocytes in the pigmented DF epidermis is significantly increased by twofold compared with the nonlesional normal epidermis (Shishido et al., 2001). The characteristic features of DF, including fibroblastic tumors in the dermis and hyperpigmentation in the overlying epidermis, allow us to speculate that paracrine cytokines produced by highly proliferative fibroblast-like cells in the dermis might be primarily responsible for the melanogenic stimulation of melanocytes in the epidermis. Although there is little evidence for their paracrine roles among skin cells, SCF and HGF have also been implicated as mitogens for human melanocytes in vitro and in vivo (Grichnik et al., 1995; Imokawa et al., 1996a, 2000; Matsumoto et al., 1991). Treatment with HGF at a concentration of 10 nM has been reported to stimulate the proliferation of cultured human melanocytes (Matsumoto et al., 1991). Using an in vivo injection protocol, Grichnik et al. (1995) and Costa 433

CHAPTER 21

A

B

Nonlesion

et al. (1996) demonstrated a potent capacity for SCF to induce hyperpigmentation in the epidermis, which was accompanied by an increased proliferation of epidermal melanocytes. Consistent with its stimulatory potential, increases in the numbers of mast cells and their degranulation are also observed concomitant with melanocyte activation (note that SCF is also known as mast cell growth factor). Thus, based upon the available evidence on the stimulatory effects of several skin cell-derived cytokines or chemokines on human melanocytes, it is most likely that SCF and/or HGF derived from the fibroblastic tumors is responsible for the stimulation of epidermal melanocytes, which leads to the hyperpigmentation restricted to the epidermis overlying these DF tumors. Our results demonstrate that, despite the lack of a significant difference in the expression of SCF and HGF mRNA transcripts in lesional and nonlesional DF epidermis, there is an increased expression of these transcripts (SCF: average 1.68fold, P < 0.001, n = 6; HGF: average 2.27-fold, P < 0.001, n = 6) in the lesional DF dermis relative to the nonlesional dermis (Shishido et al., 2001). In contrast, mRNA transcripts of other melanogenic cytokines, such as GROa, bFGF, and ET-1, are expressed similarly in lesional and in nonlesional dermis and epidermis of DF. Because the gene expression is expressed per cell (standardized by G3PDH), the increased expression of SCF and HGF transcripts implies that cells of the fibroblastic DF tumors produce more of these proteins than do fibroblasts in the nonlesional dermis. In support of this, immunostaining with antibodies to SCF or HGF of fibroblasts in the nonlesional DF dermis was substantially negative, indicating that these nonproliferating normal fibroblasts produce no or undetectable amounts of SCF and HGF. Based upon this assumption, it is conceivable that there are copious amounts of SCF and HGF secreted throughout the lesional DF dermis, being sufficient to reach and activate 434

Lesion

Fig. 21.18. Immunohistochemistry with an antibody to SCF in DF lesional skin. (A) Perilesional area (¥150). (B) Lesional area, (¥150). See also Plate 21.8, pp. 494–495.

melanocytes in the overlying epidermis. In contrast, the other melanogenic cytokines tested (including GROa, bFGF, and ET-1) might also be secreted by fibroblastic tumors, but at concentrations insufficient to contact and activate melanocytes. The expression of SCF and HGF, as evidenced by immunohistochemistry, is markedly elevated in the lesional DF dermis compared with the nonlesional dermis (Fig. 21.18), whereas for the other melanogenic cytokines tested, the levels of GROa, bFGF, and ET-1 are expressed similarly in the lesional and in the nonlesional DF dermis and epidermis (Shishido et al., 2001). Taken together, our evidence suggests that, in the lesional DF dermis, SCF and HGF are selectively produced and secreted by fibroblastic tumors at concentrations sufficient to activate melanocytes located in the adjacent epidermis, which results in the hyperpigmentation of the overlying skin. Although there may be other possible explanations as to how melanocytes in the overlying epidermis are activated in DF lesions, the ligand specificity against different types of cells would help to clarify the results of this study. Thus, the fact that, in addition to the increased numbers of activated melanocytes in the overlying epidermis, the number of mast cells located beneath the epidermis is increased fivefold over that in the nonlesional skin (Shishido et al., 2001) supports the suggestion that at least SCF is secreted in a soluble and diffusible form by fibroblastic tumors at concentrations capable of directly influencing mast cells to increase their number. This strongly suggests that at least sSCF plays an important role in the epidermal hyperpigmentation of DF. A similar type of epidermal hyperpigmentation associated with SCF has been reported in mastocytosis in which the abnormal and excessive conversion of the mSCF to sSCF in the epidermis plays an essential role in stimulating epidermal melanocytes and proliferating dermal mast cells (Longley et

PARACRINE INTERACTIONS OF MELANOCYTES IN PIGMENTARY DISORDERS

al., 1993). Thus, it is conceivable that the threshold concentrations of SCF needed to stimulate melanocytes and mast cells are similar. If mast cells are activated beneath the epidermis by SCF, it is reasonable that melanocytes can also be activated by the same SCF. Our finding that the numbers of mast cells are increased fivefold in DF skin compared with nonlesional skin may indicate that the stimulation of epidermal melanocytes in DF can be ascribed to the action of SCF, which is largely secreted as a soluble isoform by fibroblastic tumors, but not by lesional epidermal cells. This is further supported by the significant increase in SCF gene and protein expression in the lesional dermis but not in the lesional epidermis compared with nonlesional controls. This can be concluded because of the existence of soluble SCF that is released into the medium by cultured human fibroblasts but not by cultured human keratinocytes at a level detectable by ELISA (Imokawa et al., 1996b), and which diffuses to the nearby epidermis. Thus, it is unlikely that the expression of sSCF in the lesional epidermis is stimulated by unknown factors derived from fibroblastic tumors, which results in the stimulation of melanocytes and mast cells (as seen for mastocytosis). Unfortunately, as HGF is known just as a stimulator for hepatocytes and melanocytes (Matsumoto et al., 1991) and as there are no other types of skin cells that are known to respond to HGF, it is difficult to define the role of HGF in the hyperpigmentation seen in DF. However, the high expression of HGF in the dermis supports a distinct role for HGF in stimulating epidermal melanocytes in DF. In conclusion, taken together, the above results support a pathogenic mechanism for DF in which the proliferating fibroblast-like cells have a potent ability to secrete SCF and HGF, which then diffuse toward the epidermis. These factors then stimulate melanocytes located at the basal layer of the epidermis, which leads in turn to hyperpigmentation limited to the overlying epidermis, presumably accompanied by a concomitant accumulation of mast cells.

Café-au-lait Macules Café-au-lait macules (CALMs) are light to dark brown, wellcircumscribed cutaneous macular areas with no hair. CALMs are the best-known cutaneous sign of neurofibromatosis type 1 (NF1; von Recklinghausen’s disease), and they are present in almost 100% of NF1 patients (Kang and Sorber, 1992). Histological studies of CALMs in NF1 skin show increased epidermal melanization and increased numbers of melanocytes with normal amounts of tyrosinase activity (Benedict et al., 1986; Frenk and Marazzi, 1984; Moscher et al., 1993; Okazaki et al., 2003). The NF1 gene is found on chromosome 17 (Barker et al., 1987), and encodes neurofibromin, a tumor suppressor protein (Cawthorn et al., 1990; Viskochil et al., 1990; Wallance et al., 1990). It has been suggested that reduced neurofibromin levels in the epidermis of NF1 patients is responsible for the elevated melanogenesis and the increased density of melanocytes (Griesser et al., 1995). However, this cannot be the complete mechanism by which melanization is locally stimulated in CALMs of NF1 skin, because the reduc-

tion in neurofibromin levels is systemic, not localized. Based upon the paracrine cytokine network now known to function within the skin to regulate epidermal pigmentation, it is intriguing to examine the patterns of cytokine secretion in cells located in CALMs of NF1 skin. Histological studies of CALMs in the skin of NF1 patients showed increased numbers of melanocytes with normal morphology (Cawthorn et al., 1990; Viskochil et al., 1990; Wallance et al., 1990). As NF1 is a congenital skin disease and as CALMs of NF1 skin have an accentuated melanization in their epidermis due to increased numbers of epidermal melanocytes undergoing stimulated melanogenesis, we first examined the possibility that keratinocytes in the lesional epidermis secrete larger amounts of melanogenic cytokines than do keratinocytes in the nonlesional or in healthy control epidermis. As for known cases where epidermal keratinocytes produce and secrete larger amounts of melanogenic cytokines that lead to epidermal hyperpigmentation, we reported recently that such a mechanism occurs in vivo in UVB melanosis (Imokawa et al., 1992a, 1995) and in lentigo senilis (Kadono et al., 2001), where ET-1 functions as a paracrine melanogenic cytokine. Further, we have hypothesized that UVA melanosis primarily results from the stimulated secretion of GM-CSF by irradiated epidermal keratinocytes based upon an in vitro study, which showed the stimulatory effect of conditioned medium from UVA-exposed keratinocytes on human melanocytes (Imokawa et al., 1996c). Those studies have shown that melanogenic cytokines secreted by cultured human keratinocytes have so far been confined to ET-1 and GM-CSF. The present study consistently demonstrated that SCF and HGF proteins were not detectable in conditioned medium from lesional keratinocytes. Further, comparison of amounts of ET-1 and GM-CSF secreted revealed that there was no significant difference in the levels of ET-1 or GM-CSF secreted by keratinocytes isolated from CALMs of NF1 skin, from nonlesional NF1 skin, and from healthy control subjects (Okazaki et al., 2003). This suggests that lesional keratinocytes have no significant alteration in their potential to secrete melanogenic cytokines and may not be involved in the hyperpigmentary mechanism of CALMs in NF1, although the alternative possibility that lesional melanocytes have increased sensitivity to cytokines remains to be clarified. As epidermal melanogenic factors derived from dermal fibroblasts are known to be mainly SCF, HGF, and/or bFGF (Imokawa et al., 1996a, b), we next compared the secretion of SCF, HGF, and bFGF as intrinsic cytokines leading to epidermal pigmentation by fibroblasts derived from CALM, from nonCALM, and from healthy control skin. As for known cases where dermal fibroblasts produce and secrete larger amounts of melanogenic cytokines that lead to accentuated pigmentation in epidermis overlying fibroblasts in the dermis, we have found recently that such a mechanism occurs in vivo in DF, as discussed above. Determination of cytokine levels demonstrated that, in patients with NF1, the potential of dermal fibroblasts localized in CALMs to secrete HGF and/or SCF was significantly higher than that of fibroblasts derived from 435

CHAPTER 21 HGF

Group

RC * RN

*

NN 0

500

1000

1500

2000

2500

*

P < 0.05

*

P < 0.05

pg ml–1

SCF

Group

RC *

RN

*

NN 0

200

400 pg

600

800

ml–1

bFGF

Group

RC RN NN 0

2

4

6

8

10

pg ml–1

nonCALM skin or from normal control skin (Fig. 21.19) (Okazaki et al., 2003). These increases are accompanied by increased expression of mRNAs encoding HGF (average 14.4-fold, n = 2) and SCF (average 4.6-fold, n = 2) by fibroblasts cultured from CALMs of NF1 skin compared with healthy control skin (Fig. 21.20). In contrast, the secretion of bFGF was not increased in fibroblasts derived from CALMs of NF1 skin compared with those derived from nonCALM skin and from healthy control skin. These findings suggest the hypothesis that high levels of HGF and SCF secreted by dermal fibroblasts located in CALMs correlates with the stimulated melanogenesis in epidermal melanocytes. The NF1 gene was cloned in 1990 (Cawthorn et al., 1990; Viskochil et al., 1990; Wallance et al., 1990), and belongs to the family of tumor suppressor genes. Neurofibromin is a major negative regulator of the Ras pathway, a key signal transduction pathway. Loss of neurofibromin leads to 436

Fig. 21.19. Cytokine secretion by fibroblasts after 4 days in culture. After human fibroblasts were cultured for 4 days at 37∞C in a 5% CO2 atmosphere, the fibroblast-conditioned medium was collected and used to quantify secreted cytokines by ELISA. Values reported are means ± S.D. *P < 0.05 to NN (group RC, n = 5; group RN, n = 5; group NN, n = 19).

increased levels of activated Ras, and thus increased downstream mitogenic signaling. It has been suggested that the reduction in neurofibromin in the epidermis of NF1 patients is responsible for the abnormal physiology such as the elevated melanogenesis and the increased density of melanocytes (Griesser et al., 1995). However, this does not necessarily account for the localized hyperpigmented areas (CALMs) seen in NF1 because the reduction in neurofibromin is systemic, not localized. Our observation that fibroblasts localized beneath the CALMs, but not those in other areas, are stimulated to secrete SCF and HGF suggests that the reduction in neurofibromin is not directly associated with the hyperpigmentation of CALM skin, and that additional unknown factors associated with neurofibromin cause fibroblasts to stimulate their secretion of such cytokines. Thus, the relation between reduced neurofibromin in patients with NF1 and the mechanism(s) of increased secretion of HGF and SCF by dermal fibroblasts remains unclear.

PARACRINE INTERACTIONS OF MELANOCYTES IN PIGMENTARY DISORDERS

GROa/IL-8 Receptor Interactions between Keratinocytes and Melanocytes

Group

HGF

RC

NN

0

10

20

30

Group

SCF

RC

NN

0

5

10

15

20

25

Fig. 21.20. Real-time RT-PCR of HGF and SCF transcripts.

In our study (Okazaki et al., 2003), there was a significant increase in mast cells in CALM (mean 28 cells per 0.2 mm2, n = 20) and in nonCALM (mean 10 cells per 0.2 mm2, n = 20) skin in neurofibromas of NF1 and NF5 patients compared with the dermis of normal individuals (mean 3 cells per 0.2 mm2, n = 20). The density of mast cells in neurofibromas is independent of the NF type and the age of patients (Nurnberger and Moll, 1994). Increased secretion of SCF by dermal fibroblasts derived from CALM skin of patients with NF1 may lead to the increased numbers of mast cells in neurofibromas of NF1. This is also corroborated by the fact that, in DF, there are increased numbers of mast cells in the dermis overlying the fibroblastic tumors, and these cells secrete larger amounts of SCF than in the nonlesional dermis (Shishido et al., 2001). Thus, the fact that, in addition to the increased numbers of activated melanocytes in the CALM epidermis, the number of mast cells located beneath the epidermis is increased over that in the nonlesional skin supports the suggestion that at least SCF is secreted in a soluble and diffusible form by fibroblasts at concentrations capable of directly influencing mast cells to proliferate. This strongly suggests that at least sSCF plays an important role in the epidermal hyperpigmentation of CALM skin. In conclusion, our findings suggest that the increased secretion of HGF and SCF by dermal fibroblasts in CALM skin of NF1 patients is associated with the accentuated cutaneous pigmentation seen in those lesions. However, the association of reduced neurofibromin levels in NF1 patients and the mechanism(s) of increased HGF and SCF secretion by dermal fibroblasts remain to be further clarified.

GRO was identified originally as melanoma growthstimulating activity for its ability to induce the proliferation of human melanoma Hs294T cells (Castor et al., 1983). GRO is a member of a gene family encoding secretory proteins associated with the inflammatory response (Luster et al., 1985; Walz et al., 1987), and GRO is structurally related to a group of inflammatory proteins that includes IL-8, platelet factor 4, and b-thromboglobulin (Wen et al., 1989). In addition to its ability to stimulate the proliferation of melanoma cells, GRO has a variety of other biological effects including the ability to act as a neutrophil chemotactic factor (Moser et al., 1991) and to induce calcium mobilization in a variety of different target cells (Richmond et al., 1983). These biological relationships suggest that GRO may also have a function in the inflammatory response. The receptor for GROa is known to be the same as that for IL-8. We demonstrated for the first time the ability of GROa to stimulate the proliferation and melanogenesis of cultured melanocytes (Imokawa et al., 1998a). This provides an in vivo validation that keratinocytes, through production and secretion of GROa, may play an important role in regulating epidermal pigmentation. It has been reported that GROa mRNA is expressed in cultured human keratinocytes and that its expression is stimulated in response to UVB exposure (Venner et al., 1995) or to IL-1a treatment (Kojima et al., 1993). In inflammatory and hyperproliferative skin diseases such as psoriasis, GROa mRNA is overexpressed selectively in the epidermis as a keratinocyte response to activated T cells (Kojima et al., 1993). On the other hand, fibroblasts in human foreskins express a 10-fold elevation in their steady-state levels of GRO mRNA in response to serum or phorbol 12-myristate 13-acetate (PMA) stimulation (Anisowicz et al., 1988). It is interesting to note that IL-1 induces at least a 100-fold elevation of GROa mRNA expression without changing c-myc or c-fos gene expression (Griesser et al., 1995).

Riehl’s Melanosis Phenyazonaphthol (PAN) allergy-induced, late-appearing hyperpigmentation is known as pigmented cosmetic dermatitis or Riehl’s. Thus, PAN allergy-induced hyperpigmentation begins to appear on post-challenge day 19 and becomes well defined on day 28 (Fig. 21.21), whereas dinitrochlorobenzene (DNCB) allergy does not elicit hyperpigmentation during the same period of time (Imokawa and Kawai, 1987; Imokawa et al., 1992b). To examine the possibility that soluble factors released from PAN-sensitized skin are responsible for inducing the hyperpigmentation, skin extracts obtained from PAN-challenged areas on several post-challenge days were added at the indicated concentrations to melanocytes to evaluate the effect on melanogenesis (Imokawa et al., 1992b). Melanogenesis, as measured by 3H2O release and by 14C437

CHAPTER 21 PAN 19 days

16 days

Cont

28 days

DNCB

19 days

28 days

13 days

16 days

13 days

Cont

thiouracil incorporation, was slightly enhanced by the addition of skin extracts obtained on post-challenge days 7, 16, and 19, and markedly by an extract obtained on post-challenge day 28 (Fig. 21.22). In contrast to the stimulating effect of PAN-challenged skin, the addition of DNCB-challenged skin extracts to melanocytes demonstrated that there was no significant stimulation of melanogenesis on any of the postchallenge days tested. Experiments at different concentrations of the PAN allergy skin extract at post-challenge day 28 indicated a dose-dependent stimulation of melanogenesis (Imokawa et al., 1992b). The active skin extract was further fractionated by gel chromatography to clarify its biological properties when added to guinea pig melanocytes. Analysis of each fraction for effects on melanogenic activity demonstrated that the skin extract obtained from PAN allergy at 29 days after challenge contains three fractions of different molecular weights (approximately 56, 31, and 9 kDa) that stimulate melanogenesis (Fig. 21.23). Furthermore, these three fractions were also found to stimulate DNA synthesis of guinea pig melanocytes. In contrast, similar gel chromatographic analysis using a skin extract obtained at day 0 after challenge (without the challenge procedure) indicated that the skin extract also contained melanogenic fractions with 56 and 31 kDa but lacked the 9-kDa fraction compared with that seen at day 28. This suggests that the 9-kDa fraction is responsible for stimulating epidermal 438

7 days

1 day

7 days

1 day

Fig. 21.21. The appearance of hyperpigmentation induced by PAN allergy at several post-challenge days in contrast to DNCB allergy, which shows no hyperpigmentation. Each number of days represents days following the last challenge procedure. Cont, no challenge treatment. See also Plate 21.9, pp. 494–495.

pigmentation in PAN allergy. Thus, PAN allergy-induced hyperpigmentation clearly differs in its time course from ETor GM-CSF-associated pigmentation such as UVB or UVA melanosis respectively (Imokawa et al., 1992b). Further, there is no similarity in chromatographic properties between the relevant cytokines in that the allergy-evoked soluble factor exhibits an apparent molecular mass of about 8–9 kDa estimated by gel filtration, which is different from that of ET-1 (2.3–2.7 kDa) or GM-CSF (14.5–15.5 kDa) (Imokawa et al., 1996c). Subsequently, PAN-induced melanogenic stimulating factor (PIMSF) was purified from a hyperpigmented skin extract to apparent homogeneity by a combination of sequential column chromatography and electrophoresis, and showed a single band with an apparent molecular mass of 7.9 kDa (Imokawa et al., 1998). The PIMSF was similar in molecular profile to rat GROa (molecular mass of 7.9 kDa) upon sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 21.24). Immunoblotting analysis consistently revealed that the purified PIMSF cross-reacted with an anti-GROa antibody and that the bioactivities (ex. DNA synthesis) of PIMSF could be abrogated by the addition of the anti-GROa antibody (Fig. 21.25). These findings suggested that purified PIMSF is identical to GRO family proteins or is very closely related to them. The similarity of the purified PIMSF to the GRO protein is also corroborated by their comparable

Melanogenic activity of skin extract from PAN allergy

0.2

Release of tritiated H2O (dpm/105 cells)

1600

4500 3H O 2

3500

14

1100

C-TU

2500 600 1500 500 Control

Nontreated

100 1

7

13

16

19

28

Days after PAN challenge

F—I

10 8 6 4 2 0

F—II

F—III

0 day (nontreated)

10 8 6 4 2 0

5 4 3 2 1 0

(

0.1

5500

( ) 14C-thiouracil incorporation (dpm×10–3)

(—) Absorbance at 280nm

A

) 3H-thymidine (······) 3H2O release incorporation (dpm×10–3) –4 (dpm×10 )

Fig. 21.22. Melanogenic activity of a skin extract from PAN allergy at several days post-challenge, as shown by release of 3H2O and incorporation of 14C-thiouracil (TU) into cells when incubated for 24 h with skin extract at 100 ml/ml concentration in guinea pig melanocyte culture. Bar: SD from three experiments. Control, medium control; nontreated: nonchallenge procedure (equal to 0 day).

2100 6500

Incorporation of 14C-thiouracil (TU) (dpm/105 cells)

7500

0

20 F—I

10 8 6 4 2 0

30

40

F—II

50

60

70

80 28 days

F—III

10 8 6 4 2 0

5 4 3 2 1 0

(

0.1

) 3H-thymidine (······) 3H2O release incorporation (dpm×10–3) –4 (dpm×10 )

0.2

10

( ) 14C-thiouracil incorporation (dpm×10–3)

(—) Absorbance at 280nm

B

1

0

1

10

20

30 40 Fraction number

50

60

70

80

Fig. 21.23. Gel chromatographic analysis (TSK-2000SW) of skin extract from PAN allergy for melanogenic and mitogenic activities of each fraction as measured by 3H2O release into the medium, by incorporation of 14C-thiouracil into cells, and by incorporation of 14C-thymidine into cells when incubated at 100 ml/ml concentration for 24 h and the last 4 h with fractions from guinea pig melanocyte cultures respectively. (A) Skin extract at day 0 post-challenge (without challenge procedure). (B) Skin extract at day 28 post-challenge.

439

CHAPTER 21 DNA synthesis (% of control) 0

100

200

300

Control PIMSF

13.7KD

PIMSF +anti–GROα GROα

9.7KD 7.9KD 6.6KD 4.5KD

3.1KD 2.1KD PIMSF

GRO

Fig. 21.24. SDS-PAGE of the purified active fraction from TSK 2000SW gel chromatography. The applied concentrations were 10 ng per lane for the purified PIMSF and rat GROa. Migration of molecular standards is shown in kDa. Protein bands were visualized by means of Coomassie blue staining. Molecular mass markers in kDa: 9.2, collagen peptide A; 6.6, aprotinin; 4.5, sauvagine; 3.1, secretin; 2.1, atriopeptin; 1.4, bacitracin.

cellular effects, including a marked stimulation of DNA synthesis in cultured guinea pig melanocytes at similar concentrations (which can be abolished by the neutralizing antibody to GRO) accompanied by the release of inositol triphosphate and intracellular calcium mobilization, which was in turn followed by protein kinase C (PKC) translocation. As for mechanisms involved in the stimulated secretion of GROa, as IL-1a is known as a trigger for stimulating the secretion of GROa, we determined the time course of the secretion of IL-1a in the epidermal extract following the sensitization and challenge with PAN. IL-1a secretion was increased with a lag time and a peak at 28 days postchallenge, which almost paralleled the time course of GROa secretion as evaluated by its melanogenic activity. This suggests that PAN allergy causes keratinocytes to stimulate the secretion of IL-1a in a late phase, which subsequently triggers the secretion of GROa in an autocrine fashion. Therefore, it seems likely that the melanogenic stimulation induced by PAN allergy is mediated by GROa secreted during biological processes, including the increased secretion of IL-1a subsequent to cutaneous allergic reaction.

Summary and Perspectives The expression patterns of cytokines/chemokines and their 440

GROα +anti–GROα PIMSF +anti–bFGF PIMSF +anti–ET–1 Fig. 21.25. Mitogenic activities of purified PIMSF were abolished markedly by the addition of anti-GROa antibody but not by antibFGF or anti-ET antibodies. Purified PIMSF or GROa and antibodies were mixed and added to the guinea pig melanocyte cultures. The applied concentrations were as follows (in ml/ml): PIMSF, 1; GROa, 1; anti-GROa, 100; anti-bFGF, 120; anti-ET-1, 90. Mitogenic activity (DNA synthesis) was measured by incorporation of 3H-thymidine into cells. Melanocytes were incubated for 24 h with purified PIMSF and assayed. Bar, SD from three experiments.

corresponding receptors in several epidermal pigmentary disorders as described in this chapter are summarized in Table 21.1 and are depicted in Figure 21.26. This comparison led us to suggest that there is a similarity in cytokine secretion between UVB melanosis and LS/SK, and between DF and CALMs. In general, the increased secretion or production of cytokines leads to hyperpigmentation, but the lack or deficiency of corresponding receptors is critically associated with the loss of pigmentation, as seen in vitiligo vulgaris. These upor downregulated expressions of melanogenic cytokines or their receptors are mainly manipulated by primary inflammatory cytokines (such as IL-1a or TNFa) or by melanogenic cytokines themselves. However, why a single primary cytokine can trigger different melanogenic cytokines leading to different types of pigmentary disorders remains to be clarified. As far as hyperpigmentary disorders are concerned, their abnormalities are derived not from melanocytes but from nonmelanocytic cells such as keratinocytes, fibroblasts, or tumor cells in terms of the production of abnormal cytokines or excessive amounts of normal cytokines. Based upon the newly discovered paracrine or autocrine mechanisms involved in hyper- or hypopigmentary disorders, we anticipate the development of more efficient regulation of pigmentary disorders. Of special interest are the associated ligand receptors, which can be manipulated by cutaneous permeable chemicals with small molecules as antagonists against specific receptors. An ET receptor antagonist has already been used to treat UVB-induced pigmentation. Furthermore, it would be worthwhile to determine whether

PARACRINE INTERACTIONS OF MELANOCYTES IN PIGMENTARY DISORDERS Table 21.1. Summary of paracrine interactions on intrinsic cytokines/chemokines and corresponding receptors in several epidermal pigmentary disorders. Æ Epidermis

Cytokine/chemokine

Receptor

Æ Dermis

ET-1

SCF

GROa

HGF

bFGF

IL-1a

TNFa

ETBR

c-Kit

UVB melanosis

≠

≠

Æ

Æ

≠

≠

Æ

≠

Æ

Lentigo senilis

≠

≠

Æ

?

Æ

Ø

≠

≠

Æ

Seborrheic keratosis

≠

?

?

?

?

Ø

≠

?

?

Dermatofibroma

Æ

≠

Æ

≠

Æ

?

?

?

≠

Café-au-lait macules

Æ Æ

≠

?

≠

Æ

?

?

?

?

Riehl’s melanosis

?

?

≠

?

?

≠

?

?

?

Vitiligo vulgaris

≠

≠

?

?

?

?

?

Æ

Ø

UVB-melanosis Dermatofibroma Vitiligo

Seborrheic keratosis

Café-au-lait macules

Riehl’s melanosis (PAN allergy)

Lentigo senilis TNFα

IL-1α mSCF

IL-1α

Keratinocyte ET-1 mSCF

xxxx mSCF ET-1

Melanocyte

cKIT

GROα

Mast cell

Mast cell sSCF HGF

sSCF HGF Mast cell

Fibroblast

Fibroblast Fig. 21.26. Simplified paracrine cytokine model for epidermal hyperpigmentation in UVB melanosis, UVA melanosis, lentigo senilis, seborrheic keratosis, Riehl’s melanosis (PAN allergy), dermatofibroma, and café-au-lait macules. mSCF, membrane-type SCF; sSCF, soluble-type SCF.

441

CHAPTER 21

paracrine or autocrine regulation of melanocyte function contributes to cancer genesis and the development of malignant melanoma.

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22

Growth Factor Receptors and Signal Transduction Regulating the Proliferation and Differentiation of Melanocytes Ruth Halaban and Gisela Moellmann

Summary 1 Until two decades ago, the identity of growth factors that stimulate proliferation and differentiation of melanocytes was largely unknown. 2 Advances in melanocyte culture techniques, and the molecular elucidation of mutations that cause pigmentation disorders in humans and mice, led to the conclusion that melanocytes respond to a number of known growth factors that stimulate classical receptor tyrosine kinases and G protein-coupled seven-transmembrane-spanning receptors. 3 The biological responses to activation of some cell surface receptors include not only proliferation and motility but, concomitantly, enhancement of differentiated functions, such as pigmentation and dendricity. 4 Data derived both in vitro and in vivo indicate that sustained responses require the simultaneous activity of at least two complementary growth factors. 5 The signal pathways for both proliferation and differentiation share as intermediates activated protein kinases, such as mitogen-activated protein kinase (MAPK) and ribosomal kinase (RSK), which in turn leads to activation of transcription factors, stimulating growth and differentiation. 6 Several mouse models have been constructed in which the fate of neural crest melanocyte progenitors can be studied in the context of the whole organism. 7 Advances have also been made in elucidating growth factors, receptors, and activated intermediates that lead to the transformation of melanocytes to melanoma. 8 The application of high-throughput genomic and proteomic approaches is likely to address in an unbiased fashion the biochemical networks that characterize normal melanocytes and set in motion the uncontrolled growth of melanoma cells.

transduction have been obtained over the past 20 years from normal melanocyte cultures, lines of metastatic melanoma cells and from inherited pigmentary disorders. In previous studies, rodent melanoma cells had been the primary investigative tools, and knowledge of intracellular responses was largely restricted to the effects of melanocyte-stimulating hormone (MSH) on adenylate cyclase activation, cyclic adenosine monophosphate (cAMP) production, and cAMP phosphodiesterase activity on pigmentation. Untransformed immortalized murine and avian melanocytes, as well as neural crest-derived early melanocyte progenitors, or melanoblasts, were subsequently added to the pigment cell research arsenal (Bennett et al., 1987; Boissy et al., 1987; Dupin and Le Douarin, 2003; Opdecamp et al., 1998; Stocker et al., 1991; Tamura et al., 1987), paving the way for the analysis of melanocytes with defined genetic backgrounds. In addition, neural crest from the zebrafish (Dorsky et al., 1998, 2000) and the macromelanophore tumors of the platyfish Xiphophorus (see, for example, Wellbrock et al., 1998) have yielded valuable information on pigment cell lineage and signal transduction in a nonmammalian setting. A Xenopus frog melanophore line has been used extensively as an indicator tool for cloning cell surface receptors and testing drugs and a variety of agents for their ability to activate the Gsprotein/cAMP pathway and inhibitory pathways (Carrithers et al., 1999; Quillan et al., 1995; Tanaka et al., 2003). The classical frog skin or lizard skin color assays are still in use to test, for example, for potential agonists and antagonists of MSH and identification of ligands for orphan receptors (Castrucci et al., 1997; Ghanem et al., 1992; Haskell-Luevano et al., 1995; Hruby et al., 1995; Tammler et al., 2003).

Current Concepts

Historical Background

Biological Growth Factors for Normal Mammalian Melanocytes: The Need for Synergistic Stimulation

Most basic findings regarding the regulation of proliferation and differentiation of pigment cells via receptors and signal

To study growth control and normal signal transduction, pure cultures of nonmalignant melanocytes were prerequisite, but melanocytes did not grow or even survive in media used for 445

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fibroblasts, melanoma cells, or keratinocytes. Unlike many other cell types, melanocytes do not produce any of the synergistic growth factors known to stimulate them (Halaban, 2000 and references therein). A break came serendipitously in 1982 and involved two nonphysiologic stimulants, i.e., a phorbol ester (e.g. TPA, 12O-tetradecanoyl phorbol-13-acetate) acting in synergy with cholera toxin (CT) (Eisinger and Marko, 1982). Cholera toxin stimulates adenylate cyclase to produce cAMP. The phorbol ester, in addition to stimulating protein kinase C (PKC) and supporting melanocyte growth, caused differential lifting of keratinocytes from primary mixed cultures of epidermal cells, leaving melanocytes attached to the culture dish (Eisinger and Marko, 1982). Finally, preferential elimination of contaminating fibroblasts by short exposures of melanocyte cultures to geneticin enabled the establishment of pure pigment cell populations (Halaban and Alfano, 1984). Over the ensuing decade, intracellular levels of cAMP were also raised by means other than CT, mainly by dibutyryl cAMP and/or inhibitors of cAMP phosphodiesterases such as IBMX (isobutyl methylxanthine) (Halaban et al., 1986), but a phorbol ester remained a necessary staple of melanocyte growth media. None of these ingredients, however, gave a clue to the physiologic substances that stimulated normal human melanocytes or to their receptors. After an intensive search and testing of a plethora of known growth factors, the first highly effective natural melanocyte mitogen was identified as basic fibroblast growth factor (bFGF, currently termed FGF2) (Halaban et al., 1987; reviewed by Halaban et al., 2003; Hirobe, 1992), which acts through the receptor tyrosine kinase FGFR1 (Baird and Klagsbrun, 1991). Other FGF family members could substitute for FGF2 (Halaban, 1991; Halaban and Moellmann, 1993; Halaban et al., 1991). Basic FGF2 is the main active ingredient in extracts of brain (Wilkins et al., 1985), skin (Horikawa et al., 1991), as well as other tissues with melanocyte mitogenic activity (Halaban et al., 1987). As more mitogens for melanocytes were identified, it became clear that a combination of synergistic growth factors was required to release quiescent and, in culture, moribund melanocytes into the cycling mode (Böhm et al., 1995; Halaban, 1991, 2000; reviewed by Halaban et al., 2003). The additional peptides, besides FGF family members, that act as melanocyte mitogens include mast cell growth factor/stem cell factor (M/SCF, also known as Kit ligand and Steel factor) and hepatocyte growth factor/scatter factor (HGF/SF), endothelins (ET-1 to ET-3) and, to a limited degree, MSH (melanocyte-stimulating hormone, melanotropin). These peptides proved to act synergistically with FGF2 in the presence of cAMP, TPA, or with each other (Böhm et al., 1995; Halaban, 2000; reviewed by Halaban et al., 2003; Hirobe, 2001; Hirobe et al., 2004; Imokawa et al., 1997, 1998; Opdecamp et al., 1998; Swope et al., 1995; Tada et al., 1998). More recently, leukemia inhibitory factor (LIF) was added to the list of synergistic growth factors for mouse melanocytes (Hirobe,

446

2002). Most of these factors, with the exception of FGF2, also promote differentiation, as demonstrated for M/SCF, HGF/SF, ET-1, ET-3, and MSH (Halaban et al., 1993; Hirobe, 2001; Kos et al., 1999; Longley et al., 1993; Opdecamp et al., 1998). ET-1 has the additional capability of sustaining the viability of human melanocytes, if not their proliferation, in the absence of other growth factors and promoting, alone or in combination with others, the formation and elongation of melanocytic dendrites (Böhm et al., 1995; Hara et al., 1995). Like FGFs, both M/SCF and HGF/SF act through receptor tyrosine kinases, Kit and Met, respectively, and the endothelins, like MSH, through a typical G protein-coupled seven transmembrane-spanning receptor (GPCR) (Sakurai et al., 1992) (Table 22.1). The stringent requirement for synergistic growth factors may explain the relatively quiescent nature of melanocytes in the skin. Although keratinocytes produce FGF2 continuously (Halaban et al., 1988a), and FGF2 is present in the basal layer of keratinocytes in the skin (Fig. 22.1A), other growth factors are in limited supply as melanocytes do not proliferate on a keratinocyte monolayer (Fig. 22.1B). It is possible that additional mitogens are induced in vivo only in response to external stimuli such as UVB irradiation (Halaban et al., 1993; Imokawa et al., 1995; Jamal and Schneider, 2002; Tada et al., 1998), injury, and inflammation (reviewed by Hill and Treisman, 1999). Pigmentation mutants, genetically modified mice, and receptor/ligand manipulations revealed the importance of growth factors and their receptors in early melanoblast/melanocyte development, and later on in the adult skin. The classical example is piebaldism, a condition characterized by white patches devoid of melanocytes. Inactivation mutations in the Kit receptor kinase were the first to be identified as the cause for piebaldism in humans and mice (reviewed by Fleischman, 1993; Spritz, 1994; Yoshida et al., 2001), and new mutations are still being discovered (see for example Richards et al., 2001; Syrris et al., 2002). A more recent addition is the ETRB and its cognate ligand ET-3 (Lahav et al., 1996, 1998 and references therein). Each of these receptor/ligand systems is required for melanoblast/melanocyte viability, division, and migration during embryogenesis, and melanocytes fail to reach their destination when either receptor or ligand activity is impaired (Dupin and Le Douarin, 2003; reviewed by Kurihara

Table 22.1. Growth factors/receptors for normal human melanocytes. Growth factor

Receptor

Fibroblast growth factors (FGF) Mast/stem cell growth factor (M/SCF) Hepatocyte growth factor/scatter factor (HGF/SF) Endothelins (ET-1, ET-2, ET-3) Melanocyte-stimulating hormone/melanocortin (MSH)

FGFR1 Kit Met ETAR/ETBR MC1R

GROWTH FACTOR RECEPTORS AND PROLIFERATION AND DIFFERENTIATION OF MELANOCYTES

A

B

Fig. 22.1. Support of melanocytes by keratinocytes. (A) Human epidermis immunostained for FGF2. Immunostaining of basal layer of keratinocytes with FGF2 antibodies. The figure shows basal keratinocytes with peroxidase reaction indicating the presence of immunoreactive FGF2. Arrows point to melanocytes. Courtesy of Dr Glynis Scott, Department of Dermatology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA. Taken from Halaban et al., 2003. (B) Melanocytes cultured over keratinocytes. Light micrograph showing dendritic melanocytes from a black donor loaded with melanin and adjacent pigmented keratinocytes due to transfer of melanosomes. Taken from Halaban et al., 1988a. See also Plate 22.1, pp. 494–495.

et al., 1999; Lee et al., 2003; McCallion and Chakravarti, 2001; Pla and Larue, 2003). Proper homeostasis is also critical because over- or misproduction of the Kit ligand by neighboring cells during early mouse development causes redistribution of melanocytes to ectopic sites (Kunisada et al., 1998a, b; Wehrle-Haller and Weston, 1999; Wehrle-Haller et al., 2001; reviewed by Yoshida et al., 2001) (see Chapters 5 and 6, Regulation of Melanoblast Migration and Differentiation, and Melanoblast Development and Associated Disorders, respectively). Both human fibroblasts and keratinocytes, the natural cellular neighbors of melanocytes in vivo, produce melanocyte growth factors. This was first observed by coculture techniques, demonstrating that melanocytes survive and even multiply in the presence of these cells, or a layer of growthinhibited feeder cells, in medium supplemented with only a single growth factor (reviewed by Halaban, 2000; Halaban et al., 2003). The role of M/SCF in adult human skin is underscored by the manipulation of the receptor activity during cancer therapy. Intramuscular injections of the cognate ligand M/SCF administrated to patients with cancer or hematologic disorders to enhance immune responses by activating mast cells induced pigmentation (Bellet et al., 2003; Costa et al., 1996; Grichnik et al., 1995). The injection of M/SCF increased the number of melanocytes, melanocytic dendrite extension, and melanin compared with noninjected tissue. The opposite effect was observed in response to inhibition of Kit kinase activity by imatinib mesylate. This agent is a tyrosine kinase inhibitor that is undergoing phase II trials for the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors, because it suppresses the activity of BCR-ABL protein kinase, the PDGFR, and Kit. Patients treated with imatinib mesylate developed skin depigmentation (Tsao et al., 2003). Signaling by HGF/SF and its receptor Met is also critical for

melanocyte development. Targeted disruption of the Met gene in mice caused loss of melanoblasts from the forelimbs to the back (Kos et al., 1997). On the other hand, abnormal expression of HGF/SF in transgenic mice caused ectopically localized melanoblasts, such as within the neural tube and among dorsal root ganglia cells, and patterned hyperpigmentation of the skin (Takayama et al., 1996). The HGF/SF transgenic mice developed a broad array of tumors including melanomas. Most neoplasms, especially melanomas, overexpressed the HGF/SF transgene and endogenous c-Met, and had enhanced Met kinase activity, strongly suggesting that autocrine signaling promoted tumorigenesis (reviewed by Noonan et al., 2003). Neonatal UV irradiation of the HGF/SF transgenic mice increased the frequency of melanomas in the adults, thus recapitulating the etiology of melanoma (reviewed by Noonan et al., 2001, 2003). Interestingly, in spite of the critical function of HGF/SF and FGFs in melanocyte proliferation, Met abnormalities have not yet been identified in any human inherited depigmentation syndrome, and interference with FGF or its receptor in humans and mice does not cause changes in pigmentation (see, for example, Celli et al., 1998; reviewed in Coumoul and Deng, 2003). It is still possible that disabled (or superactive) Met receptor may occur in certain conditions for which no mutations in established target genes have been discovered. On the other hand, the ineffectiveness of mutations in the FGFR system can be explained by its redundancy. Fibroblast growth factor receptors constitute a family of four membranespanning tyrosine kinases (FGFR1–4), which serve as highaffinity receptors for over 22 FGF ligands (reviewed by Coumoul and Deng, 2003; Wilkie et al., 2002). The FGFR/FGF system is critical for limb development, and mutations in three of the FGFR members are associated with human congenital disorders of limb patterning and skeletal development leading to dwarfism and other defects (reviewed

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by Coumoul and Deng, 2003). The FGFR-specific tyrosine kinase inhibitors SU5402 and SU10991 are currently under evaluation as therapeutic agents for multiple myeloma, in which expression of FGFR3 mutants is an oncogenic event (Paterson et al., 2004). The introduction of these agents to the clinical setting would provide the opportunity to assess their effects on human pigmentation. The transgenic mice experience shows that overexpression of a strong cell surface receptor kinase that is not even relevant to melanocyte development or to the induction of human melanomas produces a phenotype that is similar to overstimulation of endogenous receptors, such as Kit and Met. In fact, the first receptor kinase reported to induce melanoma when overexpressed in mouse melanocytes is the Ret oncogene (Iwamoto et al., 1991; Kato et al., 1998; Taniguchi et al., 1992). Since this discovery, Ret mutations have been identified in multiple endocrine neoplasia type 2 and in familial medullary thyroid carcinoma, but not in melanomas (reviewed by Bugalho et al., 2003; Eng and Mulligan, 1997; Hunt, 2002; Takami, 2003). RET is a membrane tyrosine kinase receptor expressed in cells derived from the neural crest (but not melanoblasts or melanocytes) whose ligands are glial cellderived neurotropic factor and neurturin. The expression of activated Ret in mouse melanocytes is sufficient to enhance MAPK activity and thus to free the cells from the stringent control of normal cues in the mouse skin (Kato et al., 1998).

Alpha-MSH and the Melanocortin Receptor 1 Currently known predominantly as a neuropeptide influencing learning, memory, and attention (Butler and Cone, 2002; Ellacott and Cone, 2004; reviewed by Voisey et al., 2003), aMSH was originally isolated and named on the basis of its darkening effect on the skin color of lower vertebrates (Harris and Lerner, 1957; Lerner et al., 1964; Lerner and Lee, 1955). MSH is also a potent mitogen for normal mouse melanocytes in vitro (Tamura et al., 1987). Because of the extensive and productive use of MSH in pigmentation studies in rodent melanoma cells and the skin of nonmammalian lower vertebrates in the 1960s and 1970s, and because of the ability of MSH to darken the coat color of the agouti mouse (reviewed by Geschwind et al., 1972), a tan dog (Johnson et al., 1994), and human skin following systemic or local administration (reviewed by Hadley et al., 1993; Lerner and McGuire, 1961), the role of this anterior-lobe, or, in lower vertebrates, intermediate-lobe pituitary hormone in the proliferation of normal melanocytes was investigated as soon as pure cultures became available. MSH stimulates DNA synthesis transiently, and, as expected, only when supplied with synergistic melanocyte growth factors to pooled populations of human melanocytes (Halaban, 1993). However, FGF2 and the other peptides discussed above are far more effective than MSH. It turns out that melanocytes from individual donors have differing capac448

ities to respond to the hormone (Abdel-Malek et al., 1995; Hunt et al., 1996), a finding made earlier on human subjects (Lerner and McGuire, 1961). In one study, as many as 24% of melanocyte cultures from Caucasians were totally unresponsive to MSH (Hunt et al., 1996). The lack of response was particularly prevalent in individuals with red hair, probably due to a variant, less active MSH receptor (reviewed by Rees, 2003; Valverde et al., 1995). It is, however, important to distinguish in the large number of studies on the effectiveness of MSH whether the peptide was used in its natural form, a-MSH or b-MSH, racemized MSH, or a superpotent substituted analog, such as Ac-Nle4-cyclo[Asp5, d-Phe7, Lys10]aMSH(4–10)-NH2. The last is considerably longer lasting and, therefore, more effective than a-MSH (see, for example, Bool et al., 1981; Hruby et al., 1995; Lande and Lerner, 1971). For more information about MSH and its receptor, see Chapter 19, Regulation of Pigment Type Switching by Agouti, Melanocortin Signaling, Attractin, and Mahoganoid.

Nonmitogenic Growth Factors There are factors that do not directly trigger melanocyte proliferation but are critical for establishing the melanocyte lineage (see Chapters 5 and 6, Regulation of Melanoblast Migration and Differentiation, and Melanoblast Development, respectively). Neural crest cells destined to become melanocytes are guided by discrete diffusible signals that act as negative or positive activators of melanocyte-specific gene expression. Signaling by Wnt has been implicated genetically in melanocyte development by the severe impact on pigmentation of genetically engineered mice in which Wnt1 and Wnt3a were eliminated (reviewed by Christiansen et al., 2000; Larue et al., 2003). In addition, manipulation of neural crest in vivo and in vitro showed that overexpression of Wnt enhances the expansion of melanoblasts from neural crest in avian species (Jin et al., 2001), mouse (Dunn et al., 2000), and zebrafish (Dorsky et al., 1998). Wnts are secreted glycoproteins that activate the Frizzled receptor family. One of the main intermediates of this pathway is b-catenin, which mediates Wnt activation in the neural crest system (Dunn et al., 2000; Hari et al., 2002). The subsequent stabilization of bcatenin allows its accumulation in the nucleus, where it binds to the Tcf/Lef complex and mediates the induction of Mitf, the transcription factor critical for melanocyte-specific gene expression (Fig. 22.2). The production of Mitf is an early event during embryogenesis and is required for the differentiation of neural crest progenitors into the melanoblast/melanocyte lineage. The Wnt pathway and its downstream intracellular intermediates have been implicated in several cancers (Fearnhead et al., 2001; Polakis, 2000, 2001), and in melanoma metastasis (Weeraratna et al., 2002). There are several known growth factors investigated in the melanocyte system and shown not to stimulate their proliferation. Among these are EGF (epidermal growth factor), TGFa

GROWTH FACTOR RECEPTORS AND PROLIFERATION AND DIFFERENTIATION OF MELANOCYTES Growth factor

Ligand GPCR

RTK Shc

Ras

P1

3K

Src

PTEN

AKT P13K

Grb2

Gq/PLCγ

SOST

IKK PDK1 DAG/Ca+ PKC BAD GSK3 Ras/RAF NFκB PLCγ p70S6K Rap1/BRAF β-catenin Protein MAPK PKC+ Ca+ RSK synthesis RSK GAB1

P13K

STAT RAF MEK MAPK

MAPK RSK β-catenin

Tcf/Lef CREB MITF

RSK STAT NFκB

MAPK

SRF c-Fos, CBP Elk-1 TCF

Differentiation Anti-apoptosis

Proliferation

Fig. 22.2. A simplified diagram showing intracellular signal transduction initiated by cell surface receptor activation with particular emphasis on information related to the melanocytic cell system. Ligand activation of a prototypical receptor kinase (RTK) at the cell membrane (double line) induces activation of multiple pathways through the autophosphorylation of intracellular domains that serve as binding sites for scaffolding proteins (Shc), adaptors (Grb2), and kinases (PI3K, Src). Tyrosine phosphorylation of these intermediates mediates membrane recruitment of the guanine nucleotide releasing factor SOS, initiating the activation of a cascade of phosphorylation events known as the Ras/MAP kinase signaling pathway. Activation of MAPK leads to phosphorylation and activation of RSK, and both kinases migrate to the nucleus (empty speared arrows) and activate several transcription factors critical for proliferation (TCF, Elk-1, SRF, c-Fos) and differentiation (MITF and CREB), leading to induction of early response genes (such as Erk-1) and melanocyte-specific genes (tyrosinase, Tyrp1, and others). The endothelin receptor also activates the MAPK cascade via the second-messenger calcium and DAC (diacylglycerol), the Ras/Raf effector, or another small G protein, Rap1, and its effector B-Raf (Schinelli et al., 2001). On the other hand, PI3K, a heterodimer composed of the p85 regulatory and p110 catalytic subunits (dark spheres; see Plate 22.2) is activated by direct and indirect (Grb2/Gab1-mediated) recruitment to the receptor via the p85 SH2 domain, or by Ras and Src binding to the 110 kinase subunit. At the membrane, PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to PIP3 which, in turn, recruits other downstream molecules, particularly the serine-threonine kinases Akt and PDK1. PDK1 activates p70S6K and subsequent upregulation of protein synthesis which ensures cell growth. Akt controls several pathways. It inhibits BAD and anti-apoptotic signaling. It suppresses the otherwise active GSK3 (glycogen synthase kinase 3), stabilizing the unphosphorylated form of b-catenin allowing its translocation to the nucleus, which facilitates the activation of Tcf/Lef complexes in the MITF promoter, upregulating its transcriptional activity of melanocyte-specific genes. The NFkB pro-survival transcription factor is activated through the phosphorylation of IKK (IkB kinases) allowing its migration to the nucleus. It also promotes the translocation of MDM2 to the nucleus and downregulation of p53 tumor suppressor. The seven membrane-spanning domain G protein-coupled receptor (GPCR) of the chemokine CXCL1 (MGSA) also activates NFkB in response to upregulation of chemokines (Dhawan and Richmond, 2002). The lipid phosphatase PTEN terminates Akt activity by converting PIP3 back to PIP2. Src and PLCg are also activated by RTK, and PLCg is activated by GPCRs such as the ETR. Various stimuli signal through STAT (signal transducer and activator of transcription). STATs are direct or indirect substrates for RTK and interleukin receptors. Activated STATs migrate to the nucleus and activate growth regulatory genes such as cyclindependent kinases (CDKs), inhibitors of CDKs, and G1 cyclins, and genes whose products suppress apoptosis. In the color version of this diagram (Plate 22.2), activated enzymes are in green, inactivated intermediates are in purple, signal-activated transcription factors that translocate to the nucleus are in red, and nuclear transcription factors modulated by incoming kinases are in blue. SRF, serum-responsive factor; Tcf, T-cell transcription factor; TCF, ternary complex factors; CBP, co-activator, CREB binding protein. For more details, see the following reviews (Bertotti and Comoglio, 2003; Cantley, 2002; Pearson et al., 2001; Raman and Cobb, 2003; Schlessinger, 2000; Schlessinger and Lemmon, 2003). See also Plate 22.2, pp. 494–495.

(transforming growth factor a), VEGF (vascular endothelial growth factor; Gitay-Goren et al., 1993), and NGF (nerve growth factor; Halaban, 1989; reviewed by Halaban et al., 2003). In some of these cases, the lack of response was due to the absence of the cognate receptor (e.g. the receptors for EGF or TGFa). A role for NGF in normal melanocytes has been researched since NGF receptors were found on human melanoma cells over 25 years ago (Fabricant et al., 1977). The rationale behind this search is the neural crest origin of melanocytes that is shared with NGF-responsive neurons

(reviewed by Dupin and Le Douarin, 2003). According to such studies, NGF is chemotactic for melanocytes and melanoma cells, promotes melanocyte viability, and enhances their dendricity (Shonukan et al., 2003; Yaar et al., 1991, 1994). Keratinocytes are the immediate source because they express NGF and exert a protective role for melanocytes by upregulating the anti-apoptotic factor Bcl-2 (Pincelli and Yaar, 1997). The chemotactic effect on melanoma cells is mediated by the actinbundling protein fascin (Shonukan et al., 2003). Fascin specifically interacts with the low-affinity NGF receptor p75(NTR) 449

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in an NGF-dependent manner, and expression of mutant fascin lacking the serine phosphorylation site that regulates actin binding abrogated neurotropin-induced migration (Shonukan et al., 2003). However, NGF is probably not involved in melanocyte lineage determination and viability during early development because mice lacking TrkA, TrkB, or TrkC (NGF receptor tyrosine kinases A, B, and C) have severe neuropathies resulting from malfunctions of their sensory and sympathetic nervous systems, but no abnormalities in coat color (Klein et al., 1993, 1994; Smeyne et al., 1994).

From Cell Surface Receptors to Nuclear Transcription Factors Signal Transduction Cascade(s) in Human Melanocytes On the basis of the above findings, it became possible to identify some molecular controls of melanocyte proliferation and differentiation in terms of signal transduction intermediates and transcription factors, because considerable knowledge had already been derived from other cellular systems, and a plethora of molecular probes had become available. Two salient principles governing melanocyte growth and differentiation have been evolving. First, the requisite synergism among melanocyte growth factors is manifested by an increase in and prolongation of the tyrosine-phosphorylated, activated state of some universal signal transduction intermediates (Böhm et al., 1995; Imokawa et al., 1996, 2000; Tada et al., 2002), indicating that, in order to sustain melanocyte proliferation, a critical level of such intermediates for a necessary effector time is required and is unattainable via one type of receptor alone. The second principle involves the activation of transcription factors (TFs), such as cyclic-AMP-responsive element binding protein (CREB) (Böhm et al., 1995; Tada et al., 2002), microphthalmia (Mitf) (reviewed by Widlund and Fisher, 2003), and b-catenin (Dunn et al., 2000; Larue et al., 2003; reviewed by Saito et al., 2003). In Figure 22.2, we give a simplified composite of the pathways that may lead to modulation of transcriptional activation via growth factor-stimulated cell surface receptors in normal melanocytes. The binding of FGF, M/SCF, and HGF/SF to cognate receptors activates multiple intracellular signal transduction pathways, mainly the Ras/MAPK (MAPK, mitogen-activated protein kinase, also known as extracellular signal-regulated kinase or ERK) (reviewed by Pearson et al., 2001; Raman and Cobb, 2003), the phosphatidylinositol 3-kinase (PI3K) and Akt (PI3K/Akt), Src, and phospholipase Cg (PLC-g) pathways. The receptor kinases perform this function via self-phosphorylation of several tyrosyl residues in the intracellular domain that provide docking sites for an assembly of proteins that mediate signal transduction (reviewed by Bertotti and Comoglio, 2003; Pawson, 2004; Schlessinger, 2000). The GPCR endothelin receptor also activates MAPK,

450

stimulates protein kinase C (PKC), PI-3 kinase, increases intracellular Ca2+, and also inhibits adenylate cyclase via Gs (ETA) or Gi (ETB). The phosphorylation of some of the intermediates, such as MAPK and p90RSK, serves to transport them to the nucleus, where they phosphorylate nuclear factors such as ternary complex transcription factors (Elk-1), Mitf, and CREB, and change their transcriptional activity. These events cause rapid transcriptional induction of selected genes, known as early response genes (an example is c-Fos) required for cell proliferation (reviewed by Blume-Jensen and Hunter, 2001; Hill and Treisman, 1999; Pawson, 2004; Pawson and Saxton, 1999; Pearson et al., 2001). In the case of melanocytes, activation of the MAPK cascade by signaling emanating from the Kit receptor also upregulates genes controlled by microphthalmia (Mitf), such as tyrosinase, Tyrp1, DCT, silver, AIM-1, and melan A (Du et al., 2003; reviewed by Widlund and Fisher, 2003). Activated MAPK phosphorylates Mitf specifically on Ser-73, leading to upregulation of transcriptional activity as measured by transient cotransfection with luciferase gene under the control of the tyrosinase promoter (Hemesath et al., 1998). In addition, MAPK activates a second kinase, p90RSK, to phosphorylate Mitf at another serine residue (S409). These phosphorylations have the dual effect of enhancing binding by co-activators of the p300 family (thus enhancing gene expression) and targeting Mitf for rapid degradation by the ubiquitin–proteasome pathway (Wu et al., 2000). Rsk activation is also important because one of its known targets is CREB (Böhm et al., 1995), the transcription factor that upregulates the Mitf promoter in melanocytes (Widlund and Fisher, 2003). Other ligand/receptor systems also activate MAPK, such as HGF/Met, endothelin 3/ETRB, and a-MSH/melanocortin, suggesting that Mitf is a common target for extracellular signals (reviewed by Goding, 2000; Widlund and Fisher, 2003). For more details, see the legend to Figure 22.2. In human melanocytes, a single growth factor is sufficient to trigger the MAPK cascade, but is not able to sustain melanocyte proliferation and, with the exception of ET-1, not even viability. This failure is due, at least in part, to a shortlived rise in activated intermediates (Böhm et al., 1995). Only costimulation with synergistic growth factors prolongs and sustains the phosphorylated state and activity of MAPK and RSK, and leads to the accumulation of these kinases in the nucleus. Senescing melanocytes that have stopped proliferating in culture likewise fail to phosphorylate the tyrosine residues of MAPK in response to external growth factors, and the kinase is not translocated to the nucleus, reinforcing the critical role of MAPK in proliferation (reviewed by Bennett and Medrano, 2002; Medrano et al., 1994) (see Chapter 23, Aging and Senescence of Melanocytes). The curious role of PKC was highlighted by the observation that both activators and inhibitors of PKC were able to stimulate cultured mouse melanoblasts to proliferate, but that proliferation failed when activators and inhibitors were supplied

GROWTH FACTOR RECEPTORS AND PROLIFERATION AND DIFFERENTIATION OF MELANOCYTES

simultaneously (Hirobe, 1994). Moreover, TPA, an activator of several isoforms of PKC and a potent growth stimulator of normal melanocytes (see above), inhibits the proliferation of human melanoma cells (Arita et al., 1994; Halaban et al., 1986; Powell et al., 1993; Ziegler-Heitbrock et al., 1985). Such disparate behaviors undoubtedly reflect multiple levels of response, due not only to selective relative representation of the numerous isozymes of PKC in different cell types, but also to the presence or absence of PKC substrate proteins, e.g. oncogenes, that differ in their sensitivities to activation by PKC (Borner et al., 1995).

Signals Arising from within Pigment Cells In contrast to normal melanocytes and primary melanoma cells from thin early lesions, most metastatic melanoma cells proliferate in routine, serum-supplemented (in some cases even serum-free) media without the addition of external growth factor(s). To this day, constitutive activation of a receptor tyrosine kinase due to activating mutations has not yet been identified in melanomas. Instead, one of the hallmarks of melanomas is ectopic expression of growth factors and/or receptors that are not produced by normal human melanocytes (Table 22.2) (Easty and Bennett, 2000; Halaban, 1996) or upregulation of a receptor kinase, such as the insulin-like growth factor (IGF)-1 receptor (IGF-1R) (KanterLewensohn et al., 2000). The growth factors FGF2 and CXCL1/MGSA work via autocrine loops because both ligand and cognate receptor are expressed (Halaban et al., 1991; Richmond et al., 2004). Others, such as PDGF (plateletderived growth factor) or EGF, may promote tumor growth indirectly by stimulating cells in the immediate environment (keratinocytes or stromal cells) to elaborate melanocyte growth factors (Ellis et al., 1987; Forsberg et al., 1993).

FGF2 in Human Melanomas Just as FGF2 plays an effective role in the proliferation of normal human melanocytes, this peptide is instrumental in the

Table 22.2. Growth factors/receptor in melanoma. Ligand/receptor

Impact

FGF/FGFR1 IGF1-R Insulin receptor Met PDGF, VEGF CXCL1/MGSA

Autocrine growth, angiogenesis Autocrine growth, tumorigenesis Proliferation/metabolic changes Cell migration, metastasis Paracrine, metastatic growth, effect on stroma Paracrine, inflammation, angiogenesis, tumorigenesis Anchorage-independent growth, receptor signaling, anti-apoptotic pathway Cell interactions and communications Metastasis Apoptosis

Integrins ETAR/ETBR Wnt Kit

growth of human melanomas. Melanoma cells elaborate a variety of growth factors, cytokines, and chemokines not produced by normal melanocytes; among them are FGF2 (Halaban et al., 1988b; Scott et al., 1991), the chemokine CXCL1 (originally known as MGSA, melanoma growth stimulatory activity) (reviewed by Dhawan and Richmond, 2002; Haghnegahdar et al., 2000), and transforming growth factor (TGFb). The expression of FGF2 is an early event in the development of melanomas because it is present in situ in precursor cells such as the melanocytes of atypical nevi (Albino et al., 1991; Reed et al., 1994; Ribatti et al., 2003; Scott et al., 1991). The expression of FGF2 in nevi with melanocytic atypia enhances their survival in threedimensional type 1 collagen culture (Alanko et al., 1999), and may also enhance angiogenesis and microvascularization of the melanocytic lesions in vivo (Ribatti et al., 2003), but does not confer tumorigenicity. Mimicking melanomas to some extent are the FGF2transduced normal melanocytes. High expression of FGF2 in immortalized mouse melanocytes conferred independence from TPA and other growth factors, and loss of all melanocytic differentiated characteristics, including their ultrastructural melanogenic apparatus (Dotto et al., 1989; Halaban et al., 1996). However, these cells did not form tumors when injected into histocompatible mice, where they resumed their melanogenic activity (Dotto et al., 1989). Similarly, high ectopic expression of FGF2 via an adenoviral vector in human melanocytes conferred growth independence from exogenous FGF2, insulin/insulin-like growth factor 1, and cAMP enhancers, but not from TPA (Nesbit et al., 1999). These melanocytes, however, unlike their mouse counterparts, remained pigmented (Nesbit et al., 1999). Furthermore, the overexpression of FGF2 promoted the progression of melanocytes to melanoma in dermal reconstructs (Meier et al., 2000, 2003). In contrast, forced expression of low levels of FGF2 in human melanocytes by a retroviral vector did not alter their appearance and growth properties (Coleman and Lugo, 1998). Because human melanoma cells in most cases produce low levels of FGF2 (10¥ less than human fibroblasts), it is likely that release from external melanocyte mitogens requires additional activated signaling events, i.e. the production of a synergistic growth factor and/or the expression of mutated activated intermediates, such as B-Raf or N-Ras (Alsina et al., 2003; Satyamoorthy et al., 2003). Downregulation of pigmentation often observed in melanoma cells is in most cases due to enhanced degradation of tyrosinase with some suppression of melanocytic gene expression, and is likely not mediated through FGF2 production (Halaban, 2002; Halaban et al., 2002; Watabe et al., 2004). The critical role of intact FGF2/FGFR1 signaling in melanoma cell uncontrolled growth has been demonstrated by several investigators. In spite of the fact that multiple mitogens and cytokines are produced, neutralization of FGF2/FGFR1 alone arrests melanoma cell growth. Melanoma cell proliferation is inhibited by synthetic FGF2 peptides that

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span the receptor’s ligand-binding domain and act as FGF2 antagonists (Halaban et al., 1988b), by anti-FGF or antiphosphotyrosine antibodies (Halaban et al., 1988b), by antisense FGF2 or FGFR1 mRNA (Becker et al., 1989, 1992; Graeven et al., 2001; Wang and Becker, 1997), and by dominant-interfering FGFR1 (Yayon et al., 1997). As FGF2 was secreted by only 2 out of 10 melanoma cell lines so tested (Halaban et al., 1991), simply adding the FGF-neutralizing agents to the culture medium did not block FGF2 activity in the majority of melanoma cell lines. These agents were, therefore, “injected” into the cells by osmotic shock manipulations (Halaban et al., 1988b). It is possible that FGF2 acts through a nonconventional autocrine route of stimulation, utilizing heparan sulfate, which is known to be required for FGF2 and FGFR1 receptor activity (Aviezer et al., 1995; Schlessinger et al., 2000; Spivak-Kroizman et al., 1994). The ability to inhibit melanoma cell growth by interfering with FGF2/ FGFR1 activity alone is even more striking on account of the observations that up to 70% of melanomas harbor a mutation in B-Raf, a serine-threonine kinase activated by cell surface receptor signaling (reviewed by Tuveson et al., 2003). Depletion of B-Raf in melanomas or inhibition of its kinase activity also suppresses melanoma cell growth (reviewed by Tuveson et al., 2003). In fact, evidence exists that synergistic pathways are required for melanoma cell growth as well (Satyamoorthy et al., 2003), and thus neutralizing one may be sufficient to suppress growth.

Possible Mechanisms for the Aberrant Expression of FGF2 in Melanomas Because FGF2 is a potent mitogen, its regulated expression in normal melanocytes must be well guarded to prevent uncontrolled proliferation and to allow melanocytes to respond to secreted growth factors elaborated by a variety of cutaneous cells depending on the physiologic state of the skin (De Luca et al., 1988; Grichnik et al., 1998; Halaban et al., 1988a, c; reviewed by Imokawa, 2004). The aberrant expression of FGF2 in melanomas is due to gene activation rather than amplification or rearrangement (Birnbaum and Gaudray, 1989; Halaban et al., 1991; Yamanishi et al., 1992). The mechanism(s) by which FGF2 is aberrantly expressed in melanomas is/are not yet resolved. Growth factors and direct stimulation of adenylate cyclase or PKC induced FGF2 gene expression in adrenal chromaffin cells and primary astrocytes (Biesiada et al., 1996; Stachowiak et al., 1994). This activation was suggested to be mediated by Egr-1 (immediate early gene 1), a transcription factor induced by stress or injury, mitogens, and differentiation factors. Indeed, the core promoter region of FGF2 contains two binding sites for Egr-1, and FGF2 autoregulates its own transcription via Egr-1 binding to these sites (Ueba et al., 1999 and references therein; Wang et al., 1997). Interestingly, Egr-1 is expressed in human melanomas, and stimulation with HGF/SF increased its levels further in human and mouse melanoma cells but not in normal human melanocytes (Gaggioli et al., 2003; Recio and Merlino, 2003). In both reports, activation of Egr-1 expression was 452

mediated by the MAPK pathway. Because binding of ternary complex factors (TCFs) on six serum response elements (SREs) of the Egr-1 promoter is important for gene expression (Xi and Kersh, 2003 and references therein), it is possible that normal melanocytes lack critical components required for Egr-1 activation, if indeed this transcription factor is required for FGF2 expression in melanoma cells. The human FGF2 promoter also contains several other potential transcription factor binding sites, including those for SP1, AP1, and MYB. Recently, the HOXB7 homeodomain transcription factor was reported to transactivate the FGF2 promoter in cotransfected HeLa cells (Carè et al., 1996). However, similar levels of HOXB7 mRNA were detected in melanoma cells and proliferating melanocytes, indicating that HOXB7 expression correlates with neither the transformed phenotype nor the expression of FGF2. The presence of a consensus Myb binding site (MBS) in the human bFGF promoter prompted us to investigate whether this transcription factor might regulate FGF2 expression in melanomas. We found that c-MYB and B-MYB are overexpressed in melanoma cell lines compared with normal melanocytes and that murine c-Myb and B-Myb stimulated FGF2 promoter activity in transfected SK MEL-2 human melanoma cells (Miglarese et al., 1997). Rearrangements near or within the c-MYB locus have been detected in several melanomas, leading to speculation that c-MYB may play a role in the deregulated growth of transformed melanocytes (Dasgupta et al., 1989; Linnenbach et al., 1988; Trent et al., 1989). Therefore, c-MYB and/or B-MYB may contribute to FGF2-mediated autocrine growth of melanomas by stimulating FGF2 promoter activity. An alternative scenario is release of repression of FGF2 gene transcription in normal human melanocytes as they progress to malignancy. Deletion analysis suggests the presence of two negative regulatory elements (Shibata et al., 1991). In glioblastoma and hepatocellular carcinoma cells, the FGF2 promoter is negatively regulated by wild-type p53 tumor suppressor and positively by mutant p53 (Ueba et al., 1994). However, these findings may not be relevant for melanomas, because p53 mutations are very rare (Albino et al., 1994; Florenes et al., 1994; reviewed by McNutt et al., 1994; Volkenandt et al., 1991). Another possibility is transcriptional repression of chromatin modification. Gage and collaborators have cloned a gene termed regulator of FGF2 transcription (RFT) by virtue of binding to the basal core region GCCGAAC site in the FGF2 promoter (Ueba et al., 1999). Sequence analysis revealed that the protein is methyl-CpG-binding domain protein 1 (MBD1) isoform 3. Normally, MBD1 (and other family members) selectively recognizes and bind methylated CpG dinucleotides in promoter regions and represses genes by recruitment of chromatin-modifying enzymes, such as histone deacetylases (HDAC) (reviewed by Jones and Baylin, 2002). However, it is not clear whether MBD1 suppresses FGF2 promoter activity via binding to methylated regions, because the protein could bind unmethylated DNA as well, and a mutant deleted for the MeCpG binding motif was active in FGF2 promoter repression. Therefore, it is possible that FGF2 promoter

GROWTH FACTOR RECEPTORS AND PROLIFERATION AND DIFFERENTIATION OF MELANOCYTES

regulation by MBD1 does not require prior methylation (Ueba et al., 1999). It is not yet clear whether promoter-bound MBD1 directly blocks the binding of RNA polymerase II to the transcriptional initiation site, or whether it acts through its known function of recruitment of chromatin modification enzymes.

Other Cell Surface Receptors in Human Melanomas The expression and activity of the other melanocyte growth factors and their receptors in melanomas were also examined intensively. Both Kit (M/SCF receptor) and Met (HGF/SF receptor) are potent receptor kinases in normal human melanocytes, and oncogenic counterparts have been identified in nonmelanocytic tumors. Examples are mutations in Kit in gastrointestinal stromal tumors (Kitamura et al., 2003) and in Met in several types of human carcinoma (see, for example, Chiara et al., 2003 and references therein). These receptors were, therefore, of particular interest. However, Kit and Met are not constitutively active in melanomas (Funasaka et al., 1992; Zakut et al., 1993), and Kit protein and its kinase activity are progressively lost as melanocytic lesions advance to melanoma (Montone et al., 1997; Natali et al., 1992; Zakut et al., 1993), whereas KIT mRNA is expressed, at most, in 40% of metastatic melanomas (Lassam and Bickford, 1992; Zakut et al., 1993). M/SCF mRNA is detected in some melanoma strains but not necessarily those expressing Kit (Funasaka et al., 1992). Moreover, in melanoma cells expressing KIT, external M/SCF inhibited proliferation in a KIT kinase-dependent manner (Zakut et al., 1993). Enforced c-KIT expression into c-KIT-negative, highly metastatic human melanoma cells significantly inhibited tumor growth and metastasis (Bar-Eli, 1997). Furthermore, exposure of c-KITpositive melanoma cells in vitro and in vivo to M/SCF triggered apoptosis of these cells but not of normal melanocytes (BarEli, 1997). These results suggest that in vivo tumor growth is not necessarily promoted by Kit kinase either. On the contrary, transformed melanocytes might be held in check by M/SCF in the environment. This conclusion was confirmed recently using the inhibitor imatinib mesylate (also known as Gleevac), an inhibitor of PDGFR and c-Kit. Gleevac did not suppress the tumorigenic growth of PDGFR-positive, c-Kit-negative or -positive melanoma cells grown as xenografts in athymic nude mice in spite of the fact that PDGFR-alpha and PDGFR-beta activity was blocked by the drug (McGary et al., 2004). Met in human melanoma cells and tumors is expressed in a normal fashion (Halaban et al., 1992; Prat et al., 1991), but HGF/SF may play disparate regulatory roles in different melanomas. HGF/SF was slightly mitogenic toward two of four melanoma cell lines tested and inhibitory toward one (Halaban et al., 1992). The type 1 insulin-like growth factor receptor (IGF-1R) is overexpressed in melanoma cells as they progress from benign nevi to metastatic melanoma (Demunter et al., 2001; Kanter-Lewensohn et al., 2000; Quong et al., 1994; von Willebrand et al., 2003), increasing the responsiveness to the

cognate ligand, insulin and insulin-like growth factors I (IGF-I) respectively (Rodeck and Herlyn, 1988; Rodeck et al., 1987). The importance of the IGF-R for melanoma cell proliferation is underscored by the ability of IGF-1R antisense to inhibit melanoma tumor growth in nude mice (Resnicoff et al., 1994). In addition, the tyrphostin AG1024 that acts by inhibiting receptor autophosphorylation (Parrizas et al., 1997) is a highly effective inhibitor of melanoma cell growth in vitro (von Willebrand et al., 2003). Functional IGF-IR is required for cell cycle progression and malignant transformation, as cells lacking this receptor cannot be transformed by a number of oncogenes, including SV40 large T antigen and Ras (Baserga, 1995). In addition, IGF-IR is important in anchorage-independent proliferation (Baserga, 1999), a characteristic of metastatic melanoma cells (Herlyn et al., 1985; Rodeck et al., 1987). A role for the endothelin receptor in melanoma was suggested by two independent groups demonstrating that specific ETBR (but not ETAR) antagonists suppressed melanoma cell growth in vitro and in nude mice (Bagnato et al., 2004; Lahav et al., 1999). This effect might be mediated by decreasing E-cadherin and compromising the interactions between tumor cells and the surrounding normal microenvironment (Bagnato et al., 2004). However, expression studies provide conflicting results. In one study, ETAR was not expressed and ETBR was downregulated in melanoma cells compared with normal melanocytes in a pattern similar to the downregulation of melanocyte-specific genes (Eberle et al., 1999). In another, both receptors could be detected in melanoma cell lines by reverse transcriptase polymerase chain reaction (RT-PCR), and a functional ETBR by stimulation with ET1 (Bagnato et al., 2004). Immunohistochemistry of melanocytic lesions demonstrated increased expression of ETBR as the lesions progress to melanoma (Demunter et al., 2001). Altogether, it is possible that the jury is still out regarding the role of ETBR as a tumor promoter for melanomas.

Intermediates and Cell Cycle Regulators Sustained MAPK activity in melanoma cells was reported by several investigators (Eisenmann et al., 2003; Funasaka et al., 1992; Govindarajan et al., 2003; Halaban et al., 1992; Lefevre et al., 2003). The likely culprit is constitutive activity of the cell surface receptors described above. In addition, activating mutations in downstream receptor kinase intermediates (Fig. 22.2) are also implicated. These include Ras and BRaf (Alsina et al., 2003; Calipel et al., 2003; reviewed by Mercer and Pritchard, 2003; Tuveson et al., 2003). The transforming ability of persistent MAPK activation was validated by its experimental manipulation. The introduction of constitutively active MEK (MAPK kinase) into immortalized melanocytes facilitated their growth as tumors in nude mice (Govindarajan et al., 2003). Similarly, inhibiting MAPK signaling in melanoma cells directly or via downregulation of activated BRaf caused growth arrest and promoted apoptosis (Eisenmann et al., 2003; Hingorani et al., 2003). The activated MAPK cascade regulates cell proliferation by influencing the transcription of early response genes required 453

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for the activation of genes whose products control the transition through the cell cycle. These regulators, in turn, impact the retinoblastoma (Rb) and p53 tumor suppressor proteins. Rb and p53 are involved in regulating a variety of cellular functions, including cell division, differentiation, senescence, and apoptosis. A cooperating factor in malignant transformation of melanocytes is intermediates that suppress the activity of Rb (and p53) and free E2F, the transcription factor responsible for progression from G1 to the S phase of the cell cycle (reviewed by Kaelin, 2003; Ma et al., 2003; Sherr, 2004). Sustained inactivation of Rb and two additional family members, p107 and p130 (collectively known as “pocket proteins”), is common in human cancers, including melanomas (Halaban, 1999; Halaban et al., 2000). Inactivation by mutation is observed in retinoblastoma but is rare in sporadic tumors and melanomas (Bartkova et al., 1996; Horowitz et al., 1990), although surviving retinoblastoma patients and their families frequently suffer from melanomas (Bataille et al., 1995; Kefford et al., 1999; Moll et al., 1997). Sequestration of Rb by tumor viruses such as SV40 and E1a, described above, has not been shown so far as the mechanism operative in human melanomas. The major form of Rb inactivation in most cancers including melanomas is through site-specific phosphorylation catalyzed by CDKs (cyclin-dependent kinases). The CDKs are controlled by the cofactors cyclins and the inhibitors from the INK/CIP and KIP families. Under normal conditions, the fluctuating cyclins activate CDKs at different points in the cell cycle. Cyclin D/CDK4–CDK6 become active in mid- to late G1 phase, cyclin E/CDK2 in late G1 phase, and cyclin A/CDK2 in S phase. CDKs phosphorylate Rb, p107, and p130 causing dissociation from E2F transcription factors, release of E2F transcriptional activity, and progression through the cell cycle (Brown et al., 1999). Rb is persistently inactivated in melanoma cells by CDK4/6 and CDK2 (Bartkova et al., 1996; Halaban et al., 1998, 2000). In melanoma cells, cyclins D1, E, and A are maintained at high levels. This is likely to be the result of continuous cell surface receptor activity that maintains uninterrupted cyclin gene transcription (and CDK activity), because cyclin gene amplifications are rare. Only about 10% of melanoma tumors harbor cyclin D1 amplification (Adelaide et al., 1988; Bastian et al., 1998). Loss of the negative regulators is also a major contributor to unrestricted CDK activity. Inhibitors from the INK, KIP, and CIP families suppress the function of CDK4/6 and CDK2. Most melanomas do not express p16INK4a because of deletion of chromosome band 9p21, a region containing the CDKN2A locus encoding p16INK4a (Dracopoli and Fountain, 1996; Kamb et al., 1994a, b), or promoter silencing through hypermethylation (van der Velden et al., 2001). Furthermore, families carrying inactivation mutations in CDKN2A are predisposed to develop melanoma (Kefford et al., 1999). The mutations in p16INK4a release restraints on CDK4/6 activity because they reduce the ability of the protein to bind and inhibit CDK4/6. Likewise, a mutation in CDK4, R24C, which renders the protein insensitive to inhibition by p16INK4a, was also found in three melanoma families 454

(Kefford et al., 1999; Wolfel et al., 1995; Zuo et al., 1996). So far, no such mutations have been detected in CDK2 (Flores et al., 1997). The knockout mouse with deleted p16INK4a, and knockin mice expressing the CDK4 R24C allele, validate the importance of restraining CDK4 activity in melanoma genesis. These mice are susceptible to melanoma development, in particular after carcinogenic treatments (Krimpenfort et al., 2001; Sharpless et al., 2001; Sotillo et al., 2001). The Rb pathway was demonstrated to be involved in UV-induced melanomas because exposure of p19ARF-deleted mice expressing H-RAS in melanocytes accelerated melanoma genesis, with nearly half these tumors harboring amplification of CDK6 (Chin, 2003; Kannan et al., 2003; reviewed by Yang et al., 2001).

Nonmammalian Pigment Cell Systems Melanomas in Platyfish/Swordtail Hybrids The hereditary macromelanophore tumors and melanomas of the swordtail hybridized platyfish Xiphophorus have yielded pioneering genetic and molecular information on carcinogenesis via the first identified oncogene at the Tu melanoma tumor locus (Anders et al., 1984; Schartl et al., 1995), and have given a clue to the existence — and led to the conception — of tumor regulatory genes that possess tumor suppressor functions. Ironically, to date, no tumor suppressor gene has been unequivocally identified in the platyfish/swordtail melanoma system (Wellbrock et al., 1995). Tu encodes an activated growth factor receptor tyrosine kinase termed ONC-Xmrk (oncogenic Xiphophorus melanoma receptor kinase) that is the fish epidermal growth factor receptor (EGFR) ortholog (Volff and Schartl, 2003; reviewed by Wellbrock et al., 2002a). So far, three EGFR orthologs have been identified in Xiphophorus, egfra, egfrb [originally termed INVXmrk (invariably) present in all teleost fish species], and Tu, renamed ONC-Xmrk (Gómez et al., 2004). ONC-Xmrk activation is the result of overexpression and mutational alteration (C578S and G359R) in the extracellular region of the receptor. Either mutation leads to constitutive dimer formation via intermolecular disulfide bonding (Gómez et al., 2001). In the melanoma-prone hybrids, Tu is overexpressed via a promoter that is inactive in the nonhybridized platyfish because of loss of regulatory (R)-containing chromosome (reviewed by Volff and Schartl, 2003). R may encode a DNAmodifying enzyme such as DNA methylase. The analysis of the ONC-Xmrk promoter revealed CpG-rich island that is highly methylated in nontransformed tissues but completely unmethylated in the melanoma cell line (Altschmied et al., 1997). This effect was oncogene specific, as the Xmrk protooncogene showed CpG methylation in these cells. The presence of methylated cytosines may block the binding of some transcription factors, thereby influencing gene expression (Bird, 2002). Aberrant methylation patterns of DNA are observed in imprinted genes and in a variety of cancers (Costello et al., 2000; Esteller, 2002; Esteller et al., 2001; Esteller and Herman, 2002; reviewed by Jones and Baylin,

GROWTH FACTOR RECEPTORS AND PROLIFERATION AND DIFFERENTIATION OF MELANOCYTES

2002; Paulsen and Ferguson-Smith, 2001; Smiraglia et al., 2001). Therefore, future research on the mechanism by which selective promoters of Xmrk are methylated or demethylated is likely to shed light on the process that leads to overexpression of the Xmrk oncogene. The ligand for Xmrk has not yet been identified, and thus Xmrk-induced signal transduction has been investigated with a chimeric Xmrk receptor composed of the extracellular domain of human HER (the EGF receptor) and the intracellular domain of Xmrk (HER-mrk). This chimeric protein was ectopically expressed in fish melanocytes, in the nonmelanocytic pro B-cell line Ba/F3, or in immortalized melan-a melanocytes (see, for example, Morcinek et al., 2002 and references therein). In these cellular systems, stimulation of HERmrk with EGF activated the MAPK and PI3 kinase signaling pathways described above for mammalian cell surface receptors (Fig. 22.2). In addition, HER-mrk activated the Src kinase p59Fyn (Wellbrock et al., 1999, 2002b) and STAT5 transcription factor. Interestingly, ONC-Xmrk-transformed fish melanophores are highly pigmented as a result of upregulation of melanocyte-specific genes by Mitf. However, stimulation of ectopically expressed HER-mrk in mouse melanocytes led to Mitf degradation and loss of pigmentation due to persistent MAPK activity (Wellbrock et al., 2002b), suggesting that the EGF receptor-mediated signaling is not compatible with melanogenesis in the mammalian system. This conclusion was validated by the loss of melanocytic functions in mouse melanocytes transformed with the Her2/Neu oncogene (Dotto et al., 1989; Halaban et al., 1996).

A Frog Melanophore Line Of particular interest to the topic of signal transduction is a frog (Xenopus laevis) melanophore line, by virtue of its hormone-sensitive titrable pigment granule dispersing-&aggregating apparatus (see, for example, Gross et al., 2002; reviewed by Tuma and Gelfand, 1999). An immortalized melanophore has been used as an indicator tool for testing drugs and other agents, including light and odorants, for their ability to activate the Gs protein/cAMP pathway (Daniolos et al., 1990; reviewed by Lerner et al., 1993; McClintock et al., 1993; Potenza and Lerner, 1992). The baseline for the tests are cells whose pigment granules have been aggregated by melatonin, the melanin-dispersing hormone par excellence that activates this pathway (Abe et al., 1969a, b). In order to identify ligands and antagonists for potential use in clinical settings, transfected Xenopus melanophores have been used to study medically relevant human receptors, such as the D2 and D3 dopamine receptors (Potenza et al., 1994) and the a-2 adrenergic receptor (Lerner, 1994; Potenza et al., 1992). The cell line has also been used to find antagonists to MSH (Jayawickreme et al., 1994; Quillan et al., 1995), serotonin receptor (Potenza and Lerner, 1994), endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8 (Tanaka et al., 2003), cytokine receptors (Carrithers et al., 1999), the tyrosine kinase-coupled receptor PDGF (Graminski and Lerner, 1994), and to clone several endoge-

nous receptors: the receptor for melatonin (Ebisawa et al., 1994), a receptor specific for endothelin-3 (Karne et al., 1993), and the serotonin receptor (Potenza and Lerner, 1994).

Perspectives Until recently, discoveries have been made piecemeal governed mainly by individual investigators’ insights. The feasibility of applying high-throughput approaches to answer biological questions in an unbiased fashion on a genome-wide basis has changed this situation. The full impact of a mitogen can now be investigated by assessing changes in transcript levels of thousands of genes (reviewed by Hill and Treisman, 1999). Comparative genomic hybridization (CGH) has been applied to identify chromosomal aberrations in melanomas (Bastian, 2003), and oligonucleotide chips to assess global differences in gene expression in normal melanocytes vs. melanoma (Hoek et al., 2004 and references therein). These studies have already yielded important information not anticipated before. They revealed novel possible pathways activated in melanomas, new growth factors that contribute to proliferation and metastasis, as well as novel growth suppressors whose elimination in melanoma cells may enhance tumor growth. Applications of high-throughput approaches to identify mutations are also being developed and will be important tools in identifying novel genes that control pigmentation and cell proliferation. The challenges will be to catalog in a meaningful manner the intricate biochemical networks that characterize the normal and malignant states and to validate each aspect experimentally.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Aging and Senescence of Melanocytes Debdutta Bandyopadhyay and Estela E. Medrano

Summary 1 Basic concepts on aging and cellular senescence. 2 Aging of melanocytes in vivo. 3 Critical differences between replicative senescence of human melanocytes and senescence in mouse melanocytes. 4 Signal transduction pathways involved in human melanocyte senescence: role of the extracellular signalregulated kinases. 5 Melanin synthesis and melanocyte senescence. 6 New concepts on cellular aging: epigenetics and chromatin remodeling.

Introduction Replicative senescence, originally described by Leonard Hayflick almost four decades ago (Hayflick, 1965), is defined as the irreversible loss of the proliferative capacity of cells with maintenance of metabolic functions necessary for cell survival. Senescent cells accumulate with aging and have been detected at sites of age-related pathologies (Castro et al., 2003; Dimri et al., 1995). The molecular pathways that regulate replicative senescence are complex, cell type dependent (Bandyopadhyay et al., 2001) and, in many cases, redundant to insure the irreversibility of the senescent phenotype. Examples of critical changes in gene expression include inability to upregulate the early response gene c-fos, downregulation of the basic helix–loop–helix transcription factors Id1 and Id2, repression of G1/S proteins including thymidine kinase, thymidilate synthetase, and dihydrofolate reductase, and repression of selected cyclins (reviewed by Campisi, 1997a; Smith and Pereira-Smith, 1996). In turn, expression of the cyclindependent kinase inhibitors, including p21Waf-1, p27Kip-1, and p16INK4a, increases in a cell cycle-dependent manner (Bennett and Medrano, 2002). Other changes in gene expression associated with cellular motility and function are altered in a cell-specific manner. For example, stromal matrix molecules such as collagen and elastin are decreased in dermal fibroblasts, whereas matrix metalloproteinases and inflammatory molecules such as interleukin-1 are increased (reviewed by Campisi, 2003). Telomeres, the termini of linear chromosomes, shorten progressively with every round of DNA replication because of the classical end replication problem (Levy et al., 1992). Telomere attrition provides a possible explanation for the cell “counting” mechanism that is known to exist after each cell division (reviewed by Blasco, 2003; Blasco et al., 1997; 464

Hornsby, 2002; von Zglinicki, 2003; Wright and Shay, 2001, 2002). Telomerase, a DNA polymerase that adds telomeric repeats to chromosome ends de novo, prevents telomere attrition. However, normal somatic cells express extremely low levels of telomerase. In contrast, germ and tumor cells express substantial amounts of this enzyme (Greider, 1998). Severe telomere dysfunction triggers chromosomal breakage–fusion– bridge events and gross genomic imbalances. Telomere dysfunction may also cause a failure of cytokinesis, resulting in tetraploidization and the accumulation of supernumerary centrosomes. In turn, this could cause multipolar cell division and gross aberrations in chromosome number (reviewed by DePinho, 2000; Gisselsson, 2003). Such DNA changes in proliferating tissues might help to explain why cancer incidence rises exponentially with age in humans and many other mammalian species (Campisi, 2003; DePinho, 2000). A high frequency of chromosomal aberrations in melanomas and their relative absence in nevi suggest that melanocytes of melanomas went through telomeric dysfunction, whereas melanocytes in nevi did not. Thus, replicative senescence may be a tumor suppressor mechanism in melanocytic neoplasia (Bastian, 2003). New evidence indicates that senescent cells display molecular markers characteristic of cells bearing DNA double-strand breaks, suggesting that the telomere-initiated senescence reflects a DNA damage checkpoint response that is activated with a direct contribution from dysfunctional telomeres (d’Adda et al., 2003; von Zglinicki, 2002; von Zglinicki et al., 2003). In addition to the telomere attrition-mediated replicative senescence, several other stimuli including DNA damage, expression of a subset of oncogenes, and disruption of chromatin structure also trigger irreversible growth arrest and senescence but without extensive cell division (“premature senescence”). Among complex organisms, mounting evidence indicates that the senescence response is important for preventing the development of cancer (reviewed by Campisi, 1997b; Greider, 2000). The altered functions of senescent cells are cell type specific, but often include secretion of molecules that can disrupt tissue microenvironment. Thus, the accumulation of dysfunctional senescent cells may contribute to the decline in tissue function and integrity that is a hallmark of aging (Campisi, 2003).

Old Data and Current Concepts Aging of Melanocytes in vivo Melanocytes were the subject of aging studies well before any

AGING AND SENESCENCE OF MELANOCYTES

Fig. 23.1. Melanocyte mosaicism is apparent in the skin from an old Caucasian individual. Note that areas of hypopigmentation coexist with hyperpigmentation and lentigines.

damage. Alternatively, pigment dilution in hair may result from a reduction in tyrosinase activity of hair bulb melanocytes. This hypothesis is supported by data indicating that melanocytes are still present in the outer root sheath of senile hair, but they have lost their differentiated phenotype and become amelanotic (Takada et al., 1992). Alternative mechanisms for hair graying are suboptimal melanocyte– cortical keratinocyte interactions, and defective migration of melanocytes from a reservoir in the upper outer root sheath to the melanocyte microenvironment localized close to the dermal papilla of the hair bulb (reviewed by Tobin and Paus, 2001).

Biochemistry, Molecular Biology, and Genetics of Melanocyte Senescence in vitro culture conditions were available. It was found that there is a gradual decrease in the number of epidermal melanocytes and a decline in the number of melanosomes synthesized in old individuals (Montagna and Carlisle, 1990; Nordlund, 1986). In Caucasian individuals, the number of DOPA-positive melanocytes decreases from 8% to 20% per decade after 30 years of age. A similar decrease was observed in black skin (Herzberg and Dinehart, 1989). Despite the decrease in active melanocytes, older skin has irregular pigmentation frequently associated with hyperpigmentation (Fig. 23.1). The skin becomes mottled, and the ability to tan is also reduced (Herzberg and Dinehart, 1989). Solar lentigines, which appears in the sun-exposed areas, is considered to be a hallmark of older skin (Fig. 23.1). However, nothing is known regarding the genesis, proliferative potential, and evolution of these lesions. Normal pigmentation during wound healing is also affected by age. Older individuals have poorly pigmented, healed wounds, which are probably more susceptible to ultraviolet damage. Age-related changes in skin color have also been observed in species other than humans. In particular, the number of DOPA-positive melanocytes in the hairless Mexican dog also decreases with advancing age (Kimura and Doi, 1994). Intriguingly, when human skin derived from old individuals is grafted into nude mice, there is an increase in the number of DOPA-positive melanocytes compared with ungrafted skin (Gilhar et al., 1991), suggesting that factors derived from the mice stimulate either melanocyte proliferation or melanin synthesis in pre-existing, undifferentiated melanocytes. The hair growth cycle involves periods of melanocyte proliferation during early anagen, maturation in mid- to late anagen, and cell death by apoptosis in early catagen (reviewed by Tobin and Paus, 2001). Thus, follicular melanogenesis differs from the continuous melanogenesis in the epidermis. Melanin synthesis in hair bulbs is reduced or completely lost in senile old hair. Loss of pigmented hair with aging suggests an age-related, genetically regulated exhaustion of the pigmentary potential of each individual hair follicle. Melanocyte aging may result from increased reactive oxygen species and a consequent increase in nuclear and mitochondrial DNA

Lifespan of Human Melanocytes Tissue culture conditions suitable for the proliferation of neonatal melanocytes were established two decades ago (Eisinger and Marko, 1982; Gilchrest et al., 1984; Halaban, 1988). However, long-term proliferation of melanocytes isolated from adult individuals were unsuccessful as these cells were unable to grow beyond the third passage in a growth medium supplemented with the cyclic adenosine monophosphate (cAMP) inducer cholera toxin and hypothalamic extracts (Gilchrest et al., 1984). Omission of cholera toxin from the culture medium and low levels of tyrosine, Ca2+, and phorbol esters resulted in substantial proliferation of adult melanocytes (Medrano and Nordlund, 1990). Leukotriene C4 is also a potent mitogen for adult melanocytes, but prolonged culture with this inflammatory mediator results in loss of contact inhibition, expression of constitutively high levels of the c-fos protein, and formation of melanocyte aggregates resembling tumor spheroids (Medrano et al., 1993). Progression through the cell cycle is regulated by the synthesis, activation, and degradation of a family of proteins named the cyclin-dependent kinases (CDKs) (reviewed by Sherr, 1996, 2000). In turn, the proteins named cyclin-dependent kinase inhibitors (CDK-I), which include p21Waf-1, p27Kip-1, p16INK4a, and p15INK4b, regulate cell cycle exit (Sherr and Roberts, 1999). p16INK4a is a major tumor suppressor disrupted in many human cancers including familial melanoma. p16INK4a is upregulated in senescent cells including melanocytes (reviewed by Bennett and Medrano, 2002). Senescent melanocytes also show downregulation of cyclin E protein and mRNA levels, dephosphorylation of the protein RB, downregulation of CDK2 and CDK4 kinase activities, increased association of the histone deacetylase 1 (HDAC1) with E2F4/RB complexes, and downregulation of the histone acetyltransferases p300 and CBP (Fig. 23.2) (Bandyopadhyay et al., 2001). Paradoxically, the CDK inhibitor p21Waf-1, which is considered a senescence effector protein in fibroblasts (reviewed by Pereira-Smith and Ning, 1992), is downregulated in normal human senescent melanocytes. As expected from these results, p53 displays negligible DNA-binding activity, 465

CHAPTER 23

Lifespan of Mouse Melanocytes

40–60 PD +TPA (no cAMP inducers)

A Human neonatal melanocytes

↑↑ ± ↓ ↓↓ ±

ERK2 activity MITF TRP1 TRP2 Melanin

Telomere Erosion

SENESCENCE Effectors ???

(Partial differentiation)

13–15 PD (+ cAMP inducers)

B Human neonatal melanocytes

Telomere Erosion ↑↑ MITF ↑↑ TRP1 ↑↑ TRP2 ↑↑ Melanin (Terminal differentiation)

SENESCENCE ↓↓ MITF ↓↓ TRP2 ↑↑↑ Melanin ↓↓ cyclin E ↑↑ p16INK4a ↑↑ RB/HDAC1 complexes ↓↓ p300/CBP ↓↓ p53 ↓↓ p21Waf-1 ↓↓ ERK2 activity ↑↑ Osteonectin/SPARC

Fig. 23.2. Altered gene expression and signal transduction pathways in human melanocytes (modified from Bennett and Medrano, 2002).

but it can still be induced by ultraviolet B irradiation (E. E. Medrano, unpublished). Thus, normal melanocyte senescence seems to have aspects in common with the M0 model, with activation of the RB/p16 pathway but not the p53 pathway (reviewed by Bennett and Medrano, 2002). Intriguingly, p53 and p21Waf-1 are elevated in senescent human melanocytes derived from individuals displaying homozygous deletions for p16INK4a (Sviderskaya et al., 2003). It has been suggested that activation of the p53 pathway in these mutant melanocytes results from telomere shortening. However, substantial telomere shortening also occurs in normal human melanocytes in the absence of p53 activation (Bandyopadhyay et al., 2001). Thus, additional experiments are needed to clarify these issues. Senescence by the telomere dysfunction/p53 pathways is reversible in fibroblasts (Beausejour et al., 2003). Thus, it will be important to determine whether melanocyte senescence in the absence of p16INK4a can be reversed by p53 inactivation. If so, this can provide a mechanistic view of familial melanoma, as a “leaky senescence” may result in unrestricted proliferation and transformation of the p16INK4a null melanocytes. Finally, it is important to mention that the changes described in senescent melanocytes (Fig. 23.2) were obtained using nonpooled cultures displaying intermediate melanin levels. We have observed dramatic differences in the levels of cell cycle regulators and, consequently, in the lifespan of light vs. heavily pigmented melanocytes (Bandyopadhyay and Medrano, 2000). 466

As mentioned earlier, replicative senescence in human cells is associated with telomere attrition. This is not the case for murine cells, as these cells senesce rapidly in culture despite having long telomeres (Campisi, 2001; Sviderskaya et al., 2002). Hypotheses regarding the short lifespan of murine cells include correlations between short lifespan in culture and a short lifespan in vivo and tissue culture stresses among others (Ben Porath and Weinberg, 2004). However, it was demonstrated recently that mouse cells are highly sensitive to oxygen levels, which dramatically decrease their lifespan. Thus, these results explain the proliferative differences in culture between human and mouse cells (Parrinello et al., 2003). The lifespan of normal mouse melanocytes in culture appears to be quite variable. Whereas some reports demonstrated that mouse melanocytes senescence after 4–5 weeks in culture (Sviderskaya et al., 2002), others have found that these cells immortalize in a rather spontaneous manner (E. E. Medrano, unpublished results; R. Halaban, personal communication). It is likely that culture conditions, including oxygen, antioxidant, and tyrosine levels, and origin of serum are responsible for the extreme discrepancies in mouse melanocyte lifespan observed in different laboratories. Tyrosine is particularly interesting, as high levels of this amino acid in the culture medium also shortened the lifespan of human melanocytes (Schwahn et al., 2001). Absence of an intact INK4a/ARF pathway results in immortalization of mouse melanocytes (Sviderskaya et al., 2002). Thus, levels of p16INK4a induced by the different culture media may dictate whether the melanocytes enter rapid senescence or become immortal.

Signal Transduction Pathways: the MAP Kinases The mitogen-activated protein kinases (MAPKs), also called ERKs (extracellular signal-regulated kinases), are considered a major signal transduction pathway because they are rapidly activated in response to ligand binding with receptors that have tyrosine kinase activity, protein-coupled receptors, or activation of protein kinase C. The melanocyte mitogens, fibroblast growth factor, tetradecanoyl-phorbol-myristate acetate (TPA), leukotriene C4, endothelin-1, hepatocyte growth factor, and stem cell factor (Halaban et al., 1992; Medrano et al., 1994; Swope et al., 1995), either directly or cooperatively activated the kinase ERK2 in early passage cultures derived from neonatal or adult skin. This suggests that activation of the MAP kinase pathway is obligatory for the proliferation of human melanocytes. Supporting this hypothesis, a functional kinase assay demonstrated that ERK2 activity decreases dramatically as cells increase their population doublings (Medrano et al., 1994), which correlates with its inability to translocate to the nuclear compartment. Similar results were subsequently obtained using other cell types including fibroblasts (Lim et al., 2000) and hepatocytes (Liu et al., 1996). Apparently, the lack of ERK2 activity in senescent cells results from increased levels and activity of the nuclear MAP kinase phosphatase MKP-2 (Torres et al., 2003).

AGING AND SENESCENCE OF MELANOCYTES

Interestingly, constitutive activation of the MAP kinase pathway in mouse immortal melanocytes implanted into nude mice results in activation of the angiogenic switch and tumorigenesis (Govindarajan et al., 2003). Melanoma growth, invasion, and metastasis have been attributed to constitutively activated ERKs, which are apparently mediated by excessive growth factors and BRAF activation (Satyamoorthy et al., 2003), or by constitutive BRAF activity resulting from mutations (reviewed by Tuveson et al., 2003). Thus, in melanocytes, the ERK kinases are a paradigm of proteins targeted by both senescence and malignant transformation pathways.

Aging, Replicative Senescence, and Melanin Synthesis The altered differentiated function of a senescent cell may have a profound impact on tissue function. Senescent human fibroblasts switch from matrix-producing to matrix-degrading cells, they overexpress collagenase and underexpress collagenase inhibitors, which may contribute to collagen breakdown and thinning of the dermis as observed in vivo (Campisi, 1996). Mosaicism defines genetically different cell populations that arose from the same cell lineage. With aging, melanocytes in the skin become a mosaic population manifested by the appearance of large and small melanocytes (Ortonne, 1990). Heterogeneity of pigmentation in the skin of old individuals could result from uneven distribution of pigment cells, greater a-dihydroxyphenylalanine (DOPA) positivity of the individual melanocytes (Gilchrest et al., 1979), a local loss of melanocytes, and/or alterations in the interaction between

A

melanocytes and keratinocytes (Ortonne, 1990). Interestingly, mosaicism is also observed in late-passage, cultured melanocytes. At the end of their proliferative lifespan, melanocytes produce very small, aborted colonies that display dramatically distinct melanin levels (Fig. 23.3A). It is presently unknown whether such phenotypes arise from mosaic transcripts of tyrosinase and/or other melanogenic enzymes, or from actual genetic mosaicism in putative melanocyte precursors. A characteristic feature of melanocytes from older humans and monkeys is the formation of melanosome complexes in the cytoplasm (Hu, 1979). Melanosomes from terminally differentiated, choroidal monkey melanocytes have extensive alterations with age. These melanosomes have vacuoles filled either completely or partially with homogeneous material of a low electron density. Some melanosomes are fused together as a melanosome complex enclosed by a unit membrane. About 10–15% of the melanosomes had changes at age 7, 50% at age 13, and more than 60% at age 15. It remains to be established whether nonterminally differentiated, uveal melanocytes are affected by age in humans and higher mammals. In vitro, senescent melanocytes display a high degree of melanosome heterogeneity. Some cells have dispersed melanosomes whereas others present large melanosome aggregates filled with hundreds of melanosomes, which occupy onethird to one-quarter of the cytoplasm. Identical results were obtained in primate uveal melanocytes treated with theophylline for 2–3 weeks (Hu et al., 1987). Accumulation of dysfunctional melanocytes in the skin with aging may result in uneven pigmentation and lentigines.

B

Sen.

2w

1w

4d

TPA

CT

MITF

Tyros

DCT Fig. 23.3. Loss of MITF in senescent melanocytes does not prevent expression of tyrosinase, TRP-2, or Pmel17. (A) Melanocyte mosaicism is clearly observed in senescent human melanocytes in culture. (B) Changes in the expression of MITF and the melanogenic enzymes tyrosinase, DCT, and Pmel17 with increased population doublings.

Pmel-17

β-actin

467

CHAPTER 23

The retinal pigmented epithelium (RPE) also shows dramatic changes with aging in humans. Comparison of maculas from middle-aged and elderly groups showed a significant loss of both pigment epithelial and photoreceptor cells (Gilchrest et al., 1979). A quantification of melanin by electron spin resonance (ESR) spectroscopy in human RPE cells from donors of different age indicated an age-related melanin loss, as the melanin content diminished 2.5-fold between the first and the ninth decades of life. Irradiation of RPE with intense visible light was found to produce a time-dependent photobleaching of melanosomes that was accompanied by the formation of hydrogen peroxide, suggesting that photo-oxidation may play a role in the age-related changes in the human RPE (Sarna et al., 2003). Blue light-induced photoreactivity of melanosomes increases with age, perhaps providing a source of reactive oxygen species and leading to depletion of vital cellular reductants, which, together with lipofuscin, may contribute to cellular dysfunction (Rozanowska et al., 2002). In contrast, there seems to be active melanin synthesis in the adult eye of the mouse, most pronounced in iridial melanocytes and in the iridial pigment epithelium (Lindquist et al., 1998). Melanin has been found in the stria vascularis in humans and other mammals. The intermediate cells in the stria vascularis are believed to be neural crest-derived melanocytes (Hilding and Ginzberg, 1977), which make contact with the capillary endothelium and other perivascular structures. Old pigmented guinea pigs have a greater incidence of deafness than aged-matched albino animals (Conlee et al., 1988), suggesting that melanin may cause ototoxicity. Interestingly, studies performed in wild-type and albino mice showed that melanin is dispensable for hearing acuity. However, absence of melanocytes or intermediate cells causes severe hearing loss in mutant microphthalmia (mi) mice, presumably due to a cochlear dysfunction. Based on the hearing loss observed in these animals (Steingrimsson et al., 1994) and in Waardenburg type 2 patients with a mutant MITF (the human mi homolog) (Tassabehji et al., 1994), hearing impairment in the elderly could result from a decrease in melanocyte number and/or function.

Loss of MITF Does not Affect Expression of the Major Melanogenic Enzymes in Senescent Melanocytes: a Paradox or New Paradigm? Melanocyte mitogens have different effects on the expression of MITF and the melanogenic proteins dopachrome tautomerase (DCT) and Pmel17. For example, the DCT protein is absent in melanocytes cultured with TPA, whether it is expressed at high levels in melanocytes cultured with amelanocyte-stimulating hormone (MSH) or cholera toxin (CT) (Fig. 23.3B) (Schwahn et al., 2001). Intriguingly, the MITF protein is dramatically downregulated in senescent melanocytes (Fig. 23.3B). However, the expression levels of the major melanogenic enzymes including tyrosinase, DCT, and pMel-17 are not altered. These results suggest that either MITF is irrelevant for transcription of these genes in senescent melanocytes, or minimal levels of MITF (beyond detection by 468

the current Western blotting system) are sufficient for melanogenic gene transcription. Thus, which transcription factor does that job? Presently, we can discard other candidate proteins including USF-1 (also downregulated in senescent melanocytes) or TFE-3 (not expressed under any growth condition).

Epigenetics and the Replicative Lifespan of Human Melanocytes In addition to telomere dysfunction, other genome defects resulting from epigenetic modifications of chromatin can lead to cellular aging and cancer (reviewed by Bandyopadhyay and Medrano, 2003; Jaenisch and Bird, 2003). Epigenetic modifications consist of DNA methylation, histone modifications including acetylation, methylation, and phosphorylations, and adenosine triphosphate (ATP)-dependent structural modifications of chromatin. A distinct heterochromatic structure named senescenceassociated heterochromatic foci accumulates in senescent but not in quiescent fibroblasts (Narita et al., 2003) and melanocytes (D. Bandyopadhyay and E. E. Medrano, unpublished results), which coincides with the recruitment of RB into histone deacetylase 1 (HDAC1) and heterochromatin 1 (HP1) complexes. Senescent melanocytes display increased association of RB with HDAC1 complexes and decreased levels of the histone acetyltransferases p300 and CBP with increasing population doublings (Bandyopadhyay et al., 2002). p300 appears to be essential for the proliferation of human melanocytes and melanoma cells, as overexpression of a dominant-negative p300 HAT protein (DN p300 HAT) or treatment with Lys-CoA, a specific chemical inhibitor of p300 HAT, triggers early senescence (Fig. 23.4), as measured by irreversible loss of cell division and SA-b-gal staining. This suggests that sustained levels of histone H3 and H4 acetylation may be required for lifespan extension in cultured cells. This hypothesis is supported by recent studies demonstrating that feeding Drosophila with 4-phenylbutyrate (PBA), a histone deacetylase inhibitor, results in substantial extension of its lifespan (Kang et al., 2002).

Perspectives Defining the molecular events responsible for senescence and terminal differentiation should help to define in the future how melanocytes respond to the environment in vivo, with aging. Is a solar lentigine a proliferative/terminally differentiated response to ultraviolet radiation? What are the cellular consequences of altered checkpoint controls in senescent melanocytes? How important is cell cycle control during differentiation and apoptosis? Do epidermal keratinocytes contribute to melanocyte senescence and/or terminal differentiation in vivo? Do alterations in keratinocyte differentiation contribute to the formation of senile lentigines?

AGING AND SENESCENCE OF MELANOCYTES

Fig. 23.4. Proposed mechanism for senescence in human melanocytes mediated by downregulation of p300 and recruitment of HDAC1 in RB-containing complexes. Histone deacetylation favors methylation of Lys-9 of histone H3 by the histone methylase SUV39H1, followed by recruitment of the heterochromatin protein H1-b (modified from Bandyopadhyay et al., 2001).

Intriguingly, melanocytes from dark skin senesce more rapidly than their lighter pigmented counterparts (Bandyopadhyay and Medrano, 2000). Murine melanocyte senescence is also associated with increased pigmentation and requires an intact INK4a-ARF locus (Sviderskaya et al., 2002). Thus, is the ability to accumulate high levels of melanin part of a tumor suppressor pathway that includes RB/p16? If so, the melanocyte-stimulating hormone receptor (MC1R) may function as a tumor suppressor, whereas MC1R variants and/or mutants associated with pheomelanin may be prooncogenic. Determining whether melanin is or is not relevant for melanocyte senescence in vivo may provide clues for understanding how malignant lesions arise in melanocytes. In addition, the molecular analysis of altered differentiated functions in the melanocyte may help us to determine the reasons for the increased susceptibility to nonmelanoma skin cancers observed with aging in humans.

Acknowledgments Space limitations have prevented us from referencing a large

number of key original papers and reviews. E. E. Medrano wishes to thank past and present members of her laboratory for their contributions to this work. Work in the authors’ laboratory is supported by grants P01 AG19254 and R01CA84282 (E.E.M.) from the National Institutes of Health.

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Sviderskaya, E. V., V. C. Gray-Schopfer, S. P. Hill, N. P. Smit, T. J. Evans-Whipp, J. Bond, L. Hill, V. Bataille, G. Peters, D. Kipling, D. Wynford-Thomas, and D.C. Bennett. p16/cyclin-dependent kinase inhibitor 2A deficiency in human melanocyte senescence, apoptosis, and immortalization: possible implications for melanoma progression. J. Natl. Cancer Inst. 95:723–732, 2003. Swope, V. B., E. E. Medrano, D. Smalara, and Z. A. Abdel-Malek. Long-term proliferation of human melanocytes is supported by the physiologic mitogens alpha-melanotropin, endothelin-1, and basic fibroblast growth factor. Exp. Cell Res. 217:453–459, 1995. Takada, K., K. Sugiyama, I. Yamamoto, K. Oba, and T. Takeuchi. Presence of amelanotic melanocytes within the outer root sheath in senile white hair. J. Invest Dermatol. 99:629–633, 1992. Tassabehji, M., V. E. Newton, and A. P. Read. Waardenburg syndrome type 2 caused by mutations in the human microphthalmia (MITF) gene. Nature Genet. 8:251–255, 1994. Tobin, D. J., and R. Paus. Graying: gerontobiology of the hair follicle pigmentary unit. Exp. Gerontol. 36:29–54, 2001. Torres, C., M. K. Francis, A. Lorenzini, M. Tresini, and V. J. Cristofalo. Metabolic stabilization of MAP kinase phosphatase-2 in senescence of human fibroblasts. Exp. Cell Res. 290:195–206, 2003. Tuveson, D. A., B. L. Weber, and M. Herlyn. BRAF as a potential therapeutic target in melanoma and other malignancies. Cancer Cell 4:95–98, 2003. von Zglinicki, T. Oxidative stress shortens telomeres. Trends Biochem. Sci. 27:339–344, 2002. von Zglinicki, T. Replicative senescence and the art of counting. Exp. Gerontol. 38:1259–1264, 2003. von Zglinicki, T., J. Petrie, and T. B. Kirkwood. Telomere-driven replicative senescence is a stress response. Nature Biotechnol. 21:229–230, 2003. Wright, W. E., and J. W. Shay. Cellular senescence as a tumorprotection mechanism: the essential role of counting. Curr. Opin. Genet. Dev. 11:98–103, 2001. Wright, W. E., and J. W. Shay. Historical claims and current interpretations of replicative aging. Nature Biotechnol. 20:682–688, 2002.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

24

The Genetics of Melanoma Vanessa C. Gray-Schopfer and Dorothy C. Bennett

Summary 1 Melanoma denotes any malignant tumor of pigment cells. This chapter reviews the genetics of cutaneous melanoma, including both germline susceptibility and the somatic cell genetics of sporadic melanoma. 2 Familial melanoma is sometimes, but not always, associated with an inherited tendency to develop multiple nevi and atypical nevi. 3 The commonest locus for familial melanoma is CDKN2A (chromosome 9p), unusual in encoding two unrelated proteins with partly overlapping coding sequences. These are p16, an inhibitor of cyclin-dependent kinase 4 (CDK4) and activator of retinoblastoma (RB), and ARF, an activator of p53. Melanoma susceptibility is most often associated with the p16 coding sequence, but apparently also occasionally with that for ARF. 4 Two other specific high-penetrance melanoma loci are known: the CDK4 gene (chromosome 12q; rare) and an uncloned locus CMM4 (chromosome 1p). 5 A number of other loci may act as low-penetrance melanoma loci and/or as modifiers of penetrance for CDKN2A mutations. These include MC1R (chromosome 16q), encoding the melanocortin 1 receptor, mutations in which are associated with red hair and pale skin. Environmental factors, especially exposure to sunlight, also affect the penetrance of CDKN2A mutations. 6 Studies of somatic genetic alterations in sporadic melanomas show that around 100% of cultured melanoma cell lines have a genetic alteration in a component of the p16 pathway, namely p16, CDK4, cyclin D, or RB. Fewer uncultured melanomas have such mutations, but these frequently show reduced expression of p16 compared with benign nevi. 7 Melanomas differ from most cancers in that somatic mutations of p53 are quite rare (around 10%). Alterations in p53’s apoptotic effector APAF1 are quite common however. 8 Around 70–80% of melanomas show activating mutations of the signaling kinase BRAF, which activates the mitogenactivated protein kinase (MAPK) pathway. NRAS mutations (functionally similar) are less common but are found in many melanomas that lack a BRAF mutation. These mutations are equally common in benign nevi, suggesting that they occur early in progression. 9 No common mutations of protein tyrosine kinases are known in melanoma (unlike the case in many solid cancers). However, expression levels of several of these kinases are frequently abnormal in melanoma cell lines. 472

10 Approximately 10–15% of uncultured melanomas, especially advanced lesions, have genetic aberrations of PTEN (phosphatase and tensin homolog), a known tumor suppressor and promoter of apoptosis. 11 Mutations suppressing apoptosis seem to be a common feature of advanced melanomas. Telomerase activity is detectable in most melanomas. We have proposed a genetic model for primary melanoma progression, which would explain these features and which includes roles for cell senescence and the p16 pathway in arresting the growth of nevi and suppressing melanoma development.

Historical Background Cutaneous malignant melanoma, the fearsome black spot (Greek melas, black, -oma, outcome, later tumor), has been identified in Inca mummies, and was probably documented by Hippocrates in the fifth century bc (see Davis et al., 2004 for a historical review). The first published case description of melanoma is thought to be that by John Hunter (of St George’s Hospital Medical School, London) in 1787, while the first clear identification of the disease entity is attributed to R. Laennec in 1806. Both an association with noticeable numbers of moles and the tendency for melanoma to cluster in some families were already noted by W. Norris in the nineteenth century (Davis et al., 2004; Kefford et al., 2004). By definition, melanoma is any malignant tumor of pigment cells, and it can thus develop from the pigmented retina, choroid or iris of the eye, as well as in the skin. However, this chapter will refer almost exclusively to cutaneous melanomas, with which most genetic analysis has been concerned. Cancer geneticists are familiar with the idea that the same genes may be mutated both in the germline in cancer-susceptible families and somatically in actual cancers. This is a theme that is well supported by the data on melanoma, and this chapter will cover both the germline genetics of familial melanoma and the somatic cell genetics of sporadic melanoma development and progression. While our knowledge is still far from complete, there is now substantial genetic information on melanoma, which is beginning to shed some light on the etiology of the disease and the differences between types of pigmented lesions.

Current Concepts Cutaneous melanoma may be completely cured by excision if recognized early. It is notorious among cancers, however, as

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one of the most rapidly progressing and the least treatable once it has metastasized. This rapid progression is part of the circumstantial evidence that suggests that few genetic events are required to produce an invasive melanoma. Another is the tendency for this tumor to develop at relatively early ages; it is the third commonest cancer diagnosed in young people in the UK, according to online statistics (http://www. cancerresearchuk.org/aboutcancer/statistics/incidence). More substantial evidence came from mathematical analysis of age incidence curves, which indicated that, of all solid cancers analyzed, melanoma requires the fewest events for its development (Cook et al., 1969). Molecular genetic analysis of melanoma is thus scientifically attractive, as it may be one of the simplest cancers to “solve” genetically, and may provide a model for the understanding of more complex malignancies.

Classifications of Pigmented Lesions The following terminology is introduced to help in discussing genetic factors and their effects. Currently, four main clinical subtypes of melanoma are widely recognized (Crotty et al., 2004; Langley et al., 2003). These are: (1) superficial spreading melanoma (SSM), the commonest type, with a flat intraepidermal portion at least at the edges, although a thicker element may also be present; (2) nodular melanoma, a raised nodular lesion with no significant flat portion; and two rarer types not considered here (3) lentigo maligna and (4) acral lentiginous melanoma. An alternative classification also widely recognized for prognostic reporting is that of Clark and colleagues (Clark et al., 1984, 1989; Crotty et al., 2004) (Fig. 24.1). Including precursor or benign pigmented lesions, one can distinguish the common mole or benign nevus; the dysplastic (atypical) nevus, definitions of which vary somewhat, but which is generally agreed to be larger than normal (over 5 mm in diameter) and often to have indistinct or irregular edges; the radial growth phase (RGP) melanoma, which is purely intraepidermal or with only microinvasion close to the epidermis; and the vertical growth phase (VGP) melanoma with nodule(s) or large nests of cells in the dermis, considered competent for metastasis. VGP melanomas develop at least sometimes from RGP lesions, and both can develop from nevi (Clark et al., 1984, 1989; Crotty et al., 2004). SSM melanomas may either be

Fig. 24.1. The Clark model or classification of primary cutaneous melanocytic lesions (see also text). A variety of transitions from a less to a more advanced lesion are reported, without all these intermediates necessarily being seen in a given case. In addition, each lesion may arise apparently directly from normal skin.

Benign nevus

RGP or contain both RGP and VGP areas, while nodular melanomas are VGP only.

Melanoma Etiology and Risk Factors A brief summary of known etiological factors will help in understanding the role of genes in melanoma. The incidence of cutaneous malignant melanoma has risen steadily for over 60 years in Caucasian populations throughout the world, although a plateau has been reported more recently in Australia, possibly due to increasing avoidance of sun exposure (Langley et al., 2003; Tucker and Goldstein, 2003). This rise is attributed very largely to increased sunbathing, there being much evidence for intermittent exposure to sunlight as a causal factor in melanoma, such as increased incidence at low latitude and high altitude, and following a history of sunburn (Elwood, 1996; Langley et al., 2003; Tucker and Goldstein, 2003). Ultraviolet radiation (UVR), known to be mutagenic, is assumed to be the responsible waveband (Tucker and Goldstein, 2003). UVR has been shown to induce melanocytic lesions in skin grafts and in mouse models (Jhappan et al., 2003). Direct UVR molecular targets in human melanoma have so far not been identified, however. Of wavelengths that penetrate the earth’s atmosphere, it is the UVB waveband (290–320 nm) that interacts most efficiently with DNA, typically producing substitutions at dipyrimidine sites. However, the longer UVA waveband (320–400 nm) can also cause mutational damage in cells, by indirect mechanisms such as production of reactive oxygen species from cellular components (Joshi et al., 1987; Scott et al., 2002). This seems to be important because less than 5% of UVR in sunlight at ground level is UVB, while Caucasian human epidermis on average absorbs about 56% of UVB and 27% of UVA radiation. Thus, in Caucasians, around 98% of UVR reaching the basal Caucasian human epidermis and melanocytes is UVA, varying with skin thickness (Garland et al., 2003). The known risk factors for melanoma other than sunlight are host factors. These include red or fair hair, pale skin, blue eyes, the presence of freckles (solar lentigines), the presence of numerous benign nevi or any number of dysplastic nevi, and a family history of melanoma (Bauer and Garbe, 2003; Langley et al., 2003; Tucker and Goldstein, 2003). There is strong evidence that sun exposure leads to increased nevus

Dysplastic nevus

RGP melanoma

VGP melanoma

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counts, so these two risk factors are connected (Bauer and Garbe, 2003; Dulon et al., 2002). All the host factors can now be understood at least partially in terms of molecular genetics. We now review current knowledge of melanoma genetics, considering first familial predisposition to melanoma and then somatic mutations involved in sporadic melanoma.

Genetics of Familial Melanoma There are already some excellent and detailed recent reviews of this topic, including those by Hayward (2003) and Kefford et al. (2004). Around 10–11% of melanoma patients have one or more relatives with melanoma (Hayward, 2003; Kefford et al., 2004). Inherited susceptibility to melanoma is associated in a proportion of families with the “atypical mole syndrome” (AMS) or “dysplastic nevus syndrome,” in which both dysplastic (atypical) nevi and numerous benign nevi are present (Carey et al., 1994; Kefford et al., 2004; Newton et al., 1993). However, this is far from invariable; only about a third of melanoma-susceptible families worldwide seem to have AMS, and even then the AMS phenotype is incompletely correlated with the melanoma-associated mutation where known, suggesting a degree of separate inheritance (Kefford et al., 2004). AMS can also be found in some apparently sporadic melanoma patients (Newton et al., 1993). A high-penetrance gene or mutation is one that produces a phenotype in a high proportion of individuals who carry it. Inheritance of a high-penetrance gene conferring susceptibility to cancer is typically associated with early age of onset, the development of multiple primary tumors, and multiplex families (with more than one affected individual). These criteria have been applied in the search for high-penetrance melanoma susceptibility genes, except that melanoma is common enough in some populations that two cases may readily occur in one family by chance, and so families with three or more cases are preferable for linkage analysis (Tucker and Goldstein, 2003). To date, three susceptibility genes at two loci have been identified, together with one additional uncloned locus for a high-penetrance gene. A good many lower-penetrance genes or modifier genes are also known. We now consider the known susceptibility genes in turn.

CDKN2A CDKN2A and Human Melanoma One locus, CDKN2A (cyclin-dependent kinase inhibitor 2A), dominates the list of known familial melanoma genes. This locus on chromosome 9p21 (OMIM #600160) has also been called MTS1 (multiple tumor suppressor 1), INK4A (inhibitor of kinase 4A), and INK4A-ARF (Hussussian et al., 1994; Kamb et al., 1994; Sharpless and DePinho, 1999). The gene products and their functions are discussed below. The CDKN2A locus is reported to account for around 10–60% of melanoma-prone families, the percentage varying between regions with different climates, and rising with the number of affected family members (with melanoma) taken to define a 474

Fig. 24.2. Patient heterozygous for a CDKN2A mutation that affects p16 and not ARF function, showing atypical mole syndrome. (Illustration kindly provided by Dr Nelleke Gruis, Leiden University Medical School.) See also Plate 24.1, pp. 494–495.

melanoma-prone family (Bishop et al., 2002; Hayward, 2000, 2003); also depending whether the more recently reported deep intronic mutation (Harland et al., 2001) is included in the analysis. CDKN2A was the first identified melanoma susceptibility gene (Hussussian et al., 1994; Kamb et al., 1994). Since then, numerous different CDKN2A mutations and deletions in hundreds of families have been reported and analyzed in detail by Ruas and Peters (1998) and Sharpless and DePinho (1999). Some, but not all, of these families also have the AMS (Kefford et al., 2004) (Fig. 24.2). Mutations in CDKN2A tend to be highly penetrant (although penetrance is modulated by environmental factors) and transmitted in an autosomal dominant fashion. CDKN2A somatic mutations are also common in many types of sporadic cancers including melanomas (more in a later section). In spite of this, germline CDKN2A mutations are associated almost solely with melanoma susceptibility, the only other cancer demonstrated to have significantly increased incidence being pancreatic cancer (Hayward, 2000; Tucker and Goldstein, 2003).

CDKN2A: a Gene with Two Products CDKN2A appears to be unique in vertebrates in encoding two

THE GENETICS OF MELANOMA

RB G1

S

E2F

RB

E2F

cyclin D

Fig. 24.3. The p16/RB pathway. “RB” jointly represents the three RB family proteins RB1, RBL1 (p107), and RBL2 (p130). “CDK4” indicates CDK4 and CDK6, and “cyclin D” and “E2F” also represent protein families.

CDK4

CDK4 p16

unrelated protein products, p16 (also called INK4A or inhibitor A of kinase 4) and ARF (alternative reading frame). p16 and ARF are transcribed partly from different first exons and then in different reading frames from exons 2 and 3 (Ruas and Peters, 1998). ARF was initially thought to be an unimportant by-product of the locus, but we now know that, remarkably, both proteins have separate and significant roles in the cell. Both p16 and ARF sequences have now been implicated in familial melanoma, although nearly all reported CDKN2A mutations affect either p16 only or both products, with just a few recently identified as affecting ARF only (Hayward, 2003). The cellular functions of p16 and ARF have been reviewed by various authors (Bennett, 2003; Sharpless and DePinho, 1999, 2004; Sherr, 2001). In short, both are potent inhibitors of cell proliferation. p16: Normal Functions and Role in Melanoma The first product, p16, is responsible for the name CDKN2A, being an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4, CDK6) (Serrano et al., 1993) (Fig. 24.3), which in turn inactivate the growth suppressor function of the key tumor suppressor RB1 (retinoblastoma) and the other two related proteins of the RB family, RBL1/p107 and RBL2/p130 (Sage et al., 2000), collectively designated RB in Figure 24.3. Cell cycle arrest by p16 is RB dependent (Liggett and Sidransky, 1998). It is clearly inhibition of CDK4 and CDK6 that is important in suppression of melanoma and other cancers by p16, as melanoma-associated and other cancer-associated mutations in p16 disrupt the binding of p16 to CDK4 (Ruas and Peters, 1998), and CDK4 is another melanoma susceptibility gene (see below). However, loss of p16 function does not promote cancer simply by deregulation of normal cell proliferation (as is often implied). p16 is not expressed in most normal tissues (Zindy et al., 1997), and both humans and mice with germline deletion of both copies of p16 are born appar-

Transcription of critical cell cycle genes, proliferation

P

CDK4

p16

Cell cycle arrest

ently normal, with no defect in normal cell proliferation (Gruis et al., 1995; Huot et al., 2002; Krimpenfort et al., 2001; Sharpless et al., 2001). Instead, it seems that p16 functions primarily as an effector in the proliferative barrier and tumor suppressor mechanism of cell senescence. Cell senescence is the acquired inability of normal somatic cells to proliferate after a finite number of divisions (Bennett, 2003; Smith and Pereira-Smith, 1996; Wynford-Thomas, 1999; see also Chapter 23). It is recognized as a central mechanism of tumor suppression. Cancer cells have typically overridden cell senescence and are thus immortal (grow indefinitely if cultured), and key tumor suppressors RB1 and p53 are required for normal senescence (Bennett, 2003; Sharpless and DePinho, 2004; Wynford-Thomas, 1999). Cell senescence is disrupted in mouse fibroblasts that are null for Cdkn2a (Serrano et al., 1996) or for RB family members (Sage et al., 2000), and in mouse melanocytes null or hemizygous for Cdkn2a (Sviderskaya et al., 2002), as well as human melanocytes null only for the p16 sequence of CDKN2A (Sviderskaya et al., 2003). A normal p16 sequence can restore senescence to immortal cells, including melanocytes (Sviderskaya et al., 2002). While there is extensive evidence for this function (Bennett, 2003), it is not certain that cell senescence is the only function of p16. It is possible that p16 deficiency may lead to deficient cell cycle arrest in response to DNA damage for example, as UV irradiation was reported to induce cell cycle arrest associated with p16 upregulation in skin (Pavey et al., 2001). Nonetheless, recent evidence for activation of p16 expression and other markers of senescence in uncultured benign nevi, and the known association of germline p16 deficiency with AMS, support cell senescence in nevi as the likely mechanism for melanoma suppression by p16 (Gray-Schopfer et al., submitted). It is interesting that melanocytes normally seem to undergo the “M0” form of senescence, which depends solely on p16/RB, whereas most 475

CHAPTER 24

other cells studied (at least under favorable growth conditions) undergo “M1” senescence involving activation of p53 as well as p16/RB (Sviderskaya et al., 2003). This may provide a clue to the specificity of p16 mutations for melanoma. ARF: Normal Functions and Role in Melanoma ARF is the alternative transcript from CDKN2A, and is also known as p14ARF in the human and p19Arf in the mouse. Just three examples are currently known of apparently specific ARF mutations or deletions in familial melanoma (Hewitt et al., 2002; Randerson-Moor et al., 2001; Rizos et al., 2001a), suggesting a lesser importance for ARF than for p16 in melanoma susceptibility. Around 36% of the CDKN2A mutations analyzed by Ruas and Peters (1998) affected the ARF sequence as well as p16, but it is notable that none of those tested altered the growth inhibitory function of ARF. A trend toward higher penetrance of CDKN2A mutations was reported for mutations that affect both p16 and ARF coding sequences, compared with those that affect p16 only (Bishop et al., 2002), but a further analysis did not yield any significant difference between these two sets (Tucker and Goldstein, 2003). ARF-specific germline mutations seem to be associated with a modified spectrum of tumors in addition to melanoma, including neural tumors (Hayward, 2003; Tucker and Goldstein, 2003). There seems to be an emerging role for ARF in melanoma, although further examples of mutations would help us to be certain that the apparently specific mutations reported to date do not affect p16 in some cryptic way, for instance through modified transcriptional control. In discussing functional studies of ARF, one should note that differences are reported between the mouse and the human in its biological functions, although not its functions at the molecular level. ARF is in effect an activator of p53, and ARFinduced cell cycle arrest is largely p53 dependent (Vaziri and Benchimol, 1999). The mechanism is via the ubiquitin ligase MDM2 (mouse double minute 2 homolog, sometimes called HDM2 in human), which can bind p53 and target it for proteolysis. ARF interacts with MDM2 to inhibit this destruction of p53 (Momand et al., 2000; Pomerantz et al., 1998). As MDM2 can also interact with RB1 and inhibit its growtharresting function (Hsieh et al., 1999; Xiao et al., 1995), ARF may also act through the RB pathway. At the biological level, Arf null mice with intact p16 are susceptible to a number of tumor types, and Arf is essential for senescence of mouse fibroblasts (Kamijo et al., 1997). However, human ARF seems to have a different role. It appears to play no part in the normal senescence of human fibroblasts (Wei et al., 2001). Speculatively, this may be connected with the fact that p53 is activated by telomere shortening in human cell senescence, whereas mouse cells have much longer telomeres, such that telomere shortening normally plays no part in their senescence. Arf upregulation may be a substitute for telomere shortening in mouse cells. Possible functions of human ARF are discussed later.

476

CDK4 The importance of the RB pathway in melanoma suppression was further confirmed when a second high-penetrance locus for melanoma susceptibility was identified as CDK4 (chromosome 12q14), the kinase that phosphorylates RB and is inhibited by p16 (Fig. 24.3). Families with CDK4 mutations are rare, however: only three have been reported to date (Soufir et al., 1998; Zuo et al., 1996). These families show two mutations that disrupt p16 binding to CDK4, by substitutions in the same codon (R24C or R24H). The functional significance of these mutations is the same as for p16, except that the p16-insensitive form of CDK4 provides one of the very few known examples of human germline transmission of a dominant oncogene (most cancer susceptibility genes are tumor suppressor genes).

CMM4 A new locus representing a fourth high-penetrance melanoma susceptibility gene has now been mapped but not cloned, namely CMM4 (cutaneous malignant melanoma 4; chromosome 1p22; OMIM #608035) (Gillanders et al., 2003). This was identified by genome scanning of over 80 families with at least three cases of melanoma, from several countries. Increased significance (probability of linkage) was obtained by restricting the analysis to families with early age at diagnosis. Although investigation of candidate genes within this interval is in progress, the actual gene is not yet known (Gillanders et al., 2003). Because at least 40% of melanoma families remain apparently unaccounted for by CDKN2A or CDK4 mutations, it will be of great interest to determine the proportion that have mutations in CMM4. It seems likely that yet other high-penetrance melanoma susceptibility genes remain to be identified. The Melanoma Genetics Consortium, a worldwide collaborative group (Gillanders et al., 2003), continues to search actively for additional loci and has stated its wish to recruit more families from further collaborators.

Xeroderma Pigmentosum Genes There are some inherited disorders that increase melanoma susceptibility, although not specifically. In one set of such disorders, the xeroderma pigmentosum (XP) family of defects in DNA excision repair (XPA, XPB, etc.), the frequency of melanoma as well as nonmelanoma skin cancer is markedly increased (Wei et al., 2003), so that these loci would have high penetrance with respect to melanoma. XP patients also have many benign, freckle-like pigmented lesions (hence “pigmentosum”). This is interesting as an indication that defective excision repair can promote freckle-like lesions, consistent with the observation that freckles (ephelides) in normal humans appear in sun-exposed areas, and supporting a role for UVR in the etiology of freckles.

THE GENETICS OF MELANOMA

Low-Penetrance Genes and Modifier Genes in Familial Melanoma Several low-penetrance genes increasing melanoma susceptibility have now been identified, and other candidate genes are under investigation, as reviewed by Hayward (2003) and discussed in this section. There are also modifier genes, which may not affect melanoma susceptibility on their own, but modify the risk associated with another gene.

nucleotide polymorphism) was reported by one group to be associated with increased risk of melanoma in German males (Meyer et al., 2003a). However, no mechanism was identified as to how this noncoding polymorphism would produce a phenotype, and the report should be taken with caution, as other studies have failed to find evidence of any association of BRAF with familial melanoma (Lang et al., 2003; Laud et al., 2003; Meyer et al., 2003b).

Other Low-penetrance Genes MC1R The best-known low-penetrance melanoma susceptibility gene is MC1R (melanocortin 1 receptor, chromosome 16q24), encoding a G protein-coupled receptor for melanocortins, principally a-melanocyte-stimulating hormone (a-MSH). Binding of a-MSH to MC1R results in activation of adenylate cyclase and upregulation of cyclic adenosine monophosphate (cAMP), which increases melanin production and the ratio of eumelanin to pheomelanin in mammals (see Chapter 19). MC1R is quite polymorphic, and some variant alleles are associated with red hair, pale skin, and freckles in humans (Bastiaens et al., 2001; Valverde et al., 1995). Some MC1R alleles are also low-penetrance susceptibility genes for cutaneous melanoma (Kennedy et al., 2001; Palmer et al., 2000; Valverde et al., 1995). One might suppose that the pigmentary effects of MC1R mutations, which include impaired ability to tan, would explain the connection with melanoma, but it has been reported that the effect of different alleles on melanoma risk is not directly correlated with that on pigmentation (Kennedy et al., 2001). MC1R alleles have also been found to modify the melanoma risk associated with CDKN2A mutations (Box et al., 2001).

Glutathione-S-Transferases Glutathione S-transferases (GSTs) are involved in the detoxification of reactive oxygen species (which can be produced by the action of UVR on skin) and other carcinogens. GSTM1 (chromosome 1p13.3) and GSTT1 (chromosome 22q11.2) encode two GSTs. A large proportion of people in Caucasian populations lack these enzymes, owing to germline homozygous deletions (Kanetsky et al., 2001). The lack of GSTM1 has been linked with cancer risk (Lafuente et al., 1995). Data suggest that, among fair-skinned and fair-haired individuals, already at increased risk of melanoma, deletion of both GSTM1 and GSTT1 may elevate melanoma risk further (Kanetsky et al., 2001).

BRAF BRAF (v-raf murine sarcoma viral oncogene homolog B1, chromosome 7q34) emerged recently as important in sporadic melanoma and is discussed at more length below. It was thus intriguing that a germline BRAF variant (an intronic single-

One might expect that other genes controlling normal pigmentation would be likely, like MC1R, to modify melanoma risk, but there seems to be little evidence on this point. On the other hand, there are some known tumor suppressor genes for which melanoma is one of the cancers for which the risk is increased. One, not surprisingly, is RB1: melanoma is one of the second primary cancers reported to occur at above expected frequency in survivors of familial retinoblastoma (a childhood cancer) (Draper et al., 1986). Another such gene is TP53 (encoding p53). Germline mutations in TP53 lead to the multiple cancer syndrome Li–Fraumeni syndrome, and one of the cancers said to be associated with this syndrome is melanoma, although a large study listed only one melanoma among 200 tumors in these patients (Hisada et al., 1998). Interestingly, a rarer locus for Li–Fraumeni syndrome has recently proved to be CHK2, encoding checkpoint kinase 2, with melanoma again listed among the associated tumors (Bell et al., 1999). CHK2 is involved in activating p53 during DNA repair and in cell senescence (d’Adda di Fagagna et al., 2003). An epidermal growth factor (EGF) polymorphism that was described as a possible low-penetrance gene for melanoma has been suggested to act by forcing constitutive EGF signaling (Shahbazi et al., 2002), presumably in melanocytes, although in this case one wonders why a general effect on pigmentation is not reported. One group reported that vitamin D receptor polymorphisms were associated with altered prognosis in patients with malignant melanoma (Hutchinson et al., 2000). CYP2D (chromosome 22q13.1) encodes cytochrome P450, a drug-metabolizing enzyme that is inactive in up to 10% of Caucasians, owing to deletions, frameshift, and splice site mutations (Cascorbi, 2003). Homozygosity for mutant CYP2D alleles is reportedly associated with susceptibility and outcome in malignant melanoma, and is significantly more common in melanoma patients with red or blonde hair than in those with brown or black hair (Cascorbi, 2003). Lastly there is BRCA2 (breast cancer 2: chromosome 13q12.3). Mutations in this gene are best known in association with susceptibility to breast and ovarian cancer. However, BRCA2 mutation carriers were found in a large study to be susceptible also to several other tumor types, including an increased relative risk of melanoma of around 2.6 (Breast Cancer Linkage Consortium, 1999). BRCA2 protein functions in DNA repair, so that dysfunction would be likely to increase DNA mutability (Venkitaraman, 2003). 477

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The variety of loci affecting melanoma susceptibility suggests that we should be careful before assuming that a melanoma is completely sporadic. We will take the term simply to indicate melanomas that occur in the absence of an observed familial context. As mentioned earlier, the genes found to be somatically altered in sporadic melanomas overlap with those responsible for familial melanoma, and research on each of these two sets has provided material for the other — for example, a somatic deletion was invaluable in the identification of the CDKN2A gene on chromosome 9p21 (Fountain et al., 1992). The study of cancer cytogenetics was perhaps the earliest contributor to this set of knowledge. Before the identification of any familial melanoma gene, certain frequent chromosomal aberrations were recognized in melanomas. A combination of cytogenetic, molecular, and functional studies has suggested that genes involved in melanoma development are located to chromosomal regions 1p, 6q, 7p, 10q, 11q, and possibly also an additional 9p locus (Pollock and Trent, 2000). Molecular genetic studies have now provided strong potential candidate genes to explain all of these except perhaps the chromosome 6 locus. Evidence is accumulating for additional common alterations that do not lead to visible cytogenetic abnormalities; there are now numerous genes reported to be altered somatically in melanoma. We will first consider these aberrations in functionally related groups, and then discuss how the various types of functional alterations may fit with the clinical progression of melanoma.

some dysplastic nevi (Papp et al., 2003; Park et al., 1998), but not in benign nevi where tested (Papp et al., 1999), suggesting that RB pathway defects in pigmented lesions are associated at least with dysplasia. Another way that p16/RB defects can arise is indirectly: for example, p16 expression can be suppressed as a result of alteration of a different gene or as an epigenetic change. An epigenetic change is one that cells inherit without alteration in DNA base sequence, and the best known mechanism is DNA methylation, which occurs on cytosine within the sequence CG (“CpG”). Methylation is copied to daughter cells and tends to silence gene expression. The p16 sequence (Serrano et al., 1993) is very rich in CpG doublets, and is frequently methylated in cancers in general (Rocco and Sidransky, 2001) but, unexpectedly, not very frequently in melanoma (Gonzalgo et al., 1997). Nonetheless, several groups have reported frequent reduction in or loss of p16 protein expression in primary melanomas compared with nevi (Gray-Schopfer et al., submitted; Keller-Melchior et al., 1998; Pavey et al., 2002; Polsky et al., 2001; Straume and Akslen, 1997; Straume et al., 2000). Expression of the transcription factor ID1 (inhibitor of differentiation 1), which downregulates p16 expression, has been reported to correlate with p16 loss in RGP (in situ) melanoma, although not in VGP (Polsky et al., 2001). The mechanism for activation of ID1 expression is not known, however. Other factors that repress p16 transcription, such as ETS and BMI1, are potentially capable of a similar action (Huot et al., 2002; Itahana et al., 2003; Jacobs et al., 1999; Rothhammer et al., 2004). In summary, it is possible, although not proven, that all uncultured melanomas have some defect in the p16/RB pathway, as do cultured melanoma lines. The possible role of this in melanoma progression is considered later.

p16/RB Pathway

p53 Pathways

The pathway affected by the two best-known familial melanoma genes (p16 and CDK4) is frequently also aberrant in sporadic melanoma. In carcinogenesis in general, p16 is an important somatic target, rivaled in frequency of alteration only by p53, and with a tendency to be altered early in tumor progression (Brookes et al., 2002; Liggett and Sidransky, 1998; Ruas and Peters, 1998). In uncultured sporadic melanomas, however, the rate of detection of p16 mutations and deletions is surprisingly low, at around 10–25% (Ruas and Peters, 1998). However, a number of variant CDKN2A sequences in coding or noncoding regions, which may impinge on tumor development and are thus candidate mutations, have been reported (Kumar et al., 1998; Liu et al., 1999; Sauroja et al., 2000). Moreover, other components of the RB pathway can also be altered, with equivalent effect. Bartkova et al. (1996) reported that 100% of cultured melanoma lines examined had a p16 defect, overexpression of CDK4, or a defect in RB1. Others have found a similar spectrum of changes, also including overexpression of cyclin D1 (Halaban, 1999; Sauter et al., 2002) and somatic mutation of CDK4 (Zuo et al., 1996). p16 alterations and deletions have been reported in

It is well known that the p53 sequence is aberrant in over half of all human cancers. However, again melanoma is different, with only around 10% of even advanced and metastatic melanomas showing p53 sequence aberrations (Albino et al., 1994; Castellano and Parmiani, 1999; Straume and Akslen, 1997; Straume et al., 2000). The reason for this is still unknown. The melanomas that do have p53 mutations indicate that some growth advantage was conferred in those cases. As with the RB pathway, it is possible that mutations upstream or downstream in p53 signaling can substitute functionally. Some melanoma cell lines have been reported with specific deletions in the ARF sequence (exon 1b) of CDKN2A and intact p16 (Kumar et al., 1998; Rizos et al., 2001b). The role of ARF in human melanoma may not include mediation of cell senescence, as mentioned, but ARF can be induced in human cells by the E2F transcription factor family, which is negatively regulated by RB (Bates et al., 1998), thus potentially leading to p53 activation when RB function is reduced. ARF is also upregulated by proliferative signaling through MYC, human cells included (Drayton et al., 2003), which could mediate another negative feedback loop.

Somatic Cell Genetics of Sporadic Melanoma

478

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A key downstream mediator of p53 is APAF1 (apoptotic protease activating factor 1; chromosome 12q23), a component of the apoptosome that initiates the caspase cascade in apoptosis and is transcriptionally activated by p53 (Robles et al., 2001). APAF1 is reported to be frequently somatically altered in melanoma: its promoter is commonly methylated, and loss of heterozygosity of the locus is also frequent at around 40% (Baldi et al., 2004; Soengas et al., 2001). Moreover, APAF1 is reported to be transcriptionally induced by E2F1 (from the E2F family), and to be required for an apoptotic response to E2F1 (Furukawa et al., 2002; Moroni et al., 2001). Melanocytes do show a high rate of apoptosis when they lack normal p16 (impairing RB function and so activating E2F) (Sviderskaya et al., 2003). Thus, an APAF1 defect in melanoma may provide a double selective advantage: it may block the apoptosis associated with RB dysfunction as well as a potential apoptotic response to normal p53 activation. Other mutations suppressing apoptosis are considered later.

Protein Tyrosine Kinases Protein tyrosine kinases (PTKs) form a large superfamily of over 100 cellular signaling molecules, of which some are plasma membrane receptors for growth factors and other bioactive factors, whereas others are intracellular, although generally involved in signaling from other receptors (Blume-Jensen and Hunter, 2001). PTKs are frequently mutated in cancer cells, a correlate of their importance in the control of key cellular processes including proliferation, apoptosis, differentiation, attachment, and migration (BlumeJensen and Hunter, 2001). The roles of PTKs in melanoma were reviewed in detail by Easty and Bennett (2000). However, most available data are on expression levels rather than genetic alterations, so they will not be considered in detail here. Moreover, these data tend to refer to cultured cell lines only, so that conclusions still need to be confirmed by studies of uncultured lesions. The most frequent alterations, with overexpression seen in over 90% in melanoma cell lines, were in EPHA2 and EPHB3. These are members of the EPH family of receptors, which double as ligands (while the ligands, ephrins, double as receptors). EPHs are mitogens and angiogenic factors, with identified normal roles in, for example, neural crest migration (see Easty and Bennett, 2000). Most PTK receptors activate the RAS–RAF–MAPK mitogenic signaling pathway (Fig. 24.4 and see below), and also the phosphoinositide-3-kinase (PI3K) anti-apoptotic pathway, both advantageous to tumor growth (Blume-Jensen and Hunter, 2001). It is thus quite surprising that no particular PTK gene is yet numbered among the genes known to be commonly somatically mutated in melanoma (unlike the case with other tumor types). It is still a mystery that the key PTK required for normal melanocyte growth and development, KIT (the SCF receptor), is actually silenced rather than further activated in the great majority of advanced human melanomas (Natali et al., 1992). On the other hand, autocrine signaling through other PTKs has been shown to be capable of pro-

moting experimental melanoma development, for example in the transgenic mice that express hepatocyte growth factor in melanocytes, from the metallothionein promoter (Noonan et al., 2001), or in xenografted human skin carrying melanocytes that express FGF2, SCF, and endothelin 3, where melanoma development is observed upon UVR treatment (Berking et al., 2004). FGF2 also signals through a PTK receptor, while endothelin receptors activate the cytoplasmic PTK, FAK (focal adhesion kinase).

RAS–RAF–MAPK Pathway The MAPK (mitogen-activated protein kinase) pathway (Fig. 24.4) is a key signaling cascade that transduces extracellular stimuli through surface receptors (often PTKs) into cellular processes including proliferation and suppression of apoptosis (also cell differentiation and migration). The signals are amplified through a cascade of protein phosphorylations (Fig. 24.4; Adjei, 2001). The RAS family of small G proteins, at the top of this cascade, can thus produce large signals from a few molecules and are potentially powerful oncoproteins. Single amino acid substitutions can result in constitutively activated, oncogenic RAS mutations, which can stimulate proliferation and inhibit apoptosis. These mutations are some of the most commonly reported in human cancers (Adjei, 2001). An activated Hras sequence was sufficient to produce melanomas in combination with Ink4a-Arf (Cdkn2a) mutations in transgenic mice (Chin et al., 1997). RAS mutations are found in sporadic human melanomas, although less commonly than in some cancers. It is generally the NRAS form that becomes activated here, in 15–30% of uncultured melanomas according to larger studies (estimates vary) (Borner et al., 1999; Demunter et al., 2001). NRAS mutations are more commonly detected in melanomas from sun-exposed areas of the body, suggesting sunlight as a cause (Jafari et al., 1995; Jiveskog et al., 1998; van’t Veer et al., 1989). Experiments in mice also indicated a link between UV exposure and NRAS activation (Pierceall et al., 1992). NRAS mutations are also reportedly frequent in congenital nevi, however (56%), where sunlight cannot be the cause as these lesions originate before birth (Demunter et al., 2001; Papp et al., 1999). The same studies described NRAS mutations in 33% of primary melanomas and 26% of metastatic melanoma (Demunter et al., 2001; Papp et al., 1999). The frequency of NRAS mutations in dysplastic nevi appears to be possibly lower, at 0–5% (Albino et al., 1989; Jafari et al., 1995; Papp et al., 2003), than in benign nevi (around 15%) (Pollock et al., 2003), tentatively suggesting that dysplastic nevi do not always arise from a pre-existing benign nevus. Intense interest was generated by the report by Davies et al. (2002) that BRAF is subject to activating mutations in nearly 70% of melanomas, both cultured and uncultured. BRAF is a RAF family protein downstream of RAS signaling (Fig. 24.4). Over 80% of the BRAF mutations in melanoma were the same phosphomimetic (acidic) substitution, V600E. This mutation has now been shown directly to be both mitogenic and 479

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Growth factor/ PTK receptor

P13K

P

P

GRB

SOS

PIP3

MSH

RAS

MC1R

RAF

PTEN

AK cAMP

AKT

BAD

Gs

APC

MEK PKA

GSK3 MAPKs

Eumelanin

β-catenin Apoptosis

Proliferation

Fig. 24.4. The MAPK pathway and selected interacting pathways regulating cell proliferation and survival, believed to be important in melanoma. Arrows indicate stimulatory actions, and rectangles indicate pro-apoptotic proteins. AK, adenylate kinase; GSK3, glycogen synthase kinase 3; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; PKA, protein kinase A. AKT/PKB (protein kinase B) phosphorylates and inhibits the activity of a number of targets, including those shown and also growth inhibitor p21. Information from references cited in the text, some based on fibroblasts. Many other interacting pathways are omitted for clarity, for example GSK3 is also inhibited by WNT signaling, and the MAPK and PI3K pathways can also be activated by endothelin receptors through the cytosolic protein tyrosine kinase FAK (focal adhesion kinase). See text for other details.

oncogenic in mouse melanocytes (Wellbrock et al., 2004). There was a tendency for melanomas to show either BRAF or NRAS mutations rather than both, consistent with these proteins acting in a common pathway (Davies et al., 2002; Omholt et al., 2003). It will be of interest to know whether normal BRAF is indeed downstream of NRAS in melanocytes and, if so, which principal receptor(s) signal through NRAS and BRAF in these cells, as such receptors would be further candidates for mutations in melanoma. Moreover, BRAF mutations have also been identified in over 80% of nevi, including benign acquired, dysplastic, and congenital nevi (Pollock et al., 2003). These findings establish BRAF and NRAS mutations (and MAPK pathway activation more generally) as candidates for the principal initiating mutations in nevi, with the potential to contribute to melanoma development, although evidently not sufficient alone to cause melanoma (discussed further below). Where melanomas and associated underlying nevi were analyzed separately, either both or neither had a BRAF mutation, consistent with this idea (Yazdi et al., 2003). Likewise, the NRAS and BRAF mutation status of metastases corresponded with that of their primary tumor and, in 8/8 melanomas with both an RGP and a VGP component, the same BRAF mutation was present in both components, supporting the idea that RGP tumors can be precursors of VGP melanomas (Omholt et al., 2003). On the other hand, there are reports that BRAF mutations are less frequent overall in RGP (or “in situ”) melanomas, at around 0–10%, than in advanced melanomas and nevi (Dong et al., 2003; Yazdi et al., 2003), suggesting that RGP melanomas may often be a separate clinical entity from VGP tumors or nevi rather than an intermediate between both. 480

A point of interest about the common V600E mutation is that the DNA base change involved is not of the type induced directly in DNA by UVB. Nonetheless, there is strong evidence linking nevi and sporadic melanoma with intermittent sun exposure, including their distributions on the skin, as discussed above. Moreover, BRAF mutations themselves have been reported to be more frequent in melanomas that arise in areas subject to intermittent sun exposure (Maldonado et al., 2003). It thus seems likely that the V600E mutation can be caused by sunlight, even if not by direct action on DNA. This recalls the idea discussed above that mutagenesis in melanocytes by UV light may occur in part indirectly, through the generation of reactive oxygen species. In this case, UVA radiation would be one candidate cause for BRAF-V600E mutations. BRAF is located on chromosome 7q34. There is evidence that this location explains the common trisomy for chromosome 7: in melanomas with this trisomy, it appears in general to be a chromosome carrying a mutant BRAF that has been duplicated (Maldonado et al., 2003). This also indicates that two copies of the mutant gene are even more favorable for melanoma growth than one copy.

PTEN Loss of heterozygosity for chromosome 10q has been observed in up to 50% of sporadic melanomas (Wu et al., 2003). PTEN (phosphatase and tensin homolog; chromosome 10q23.31) accounts for at least some of these aberrations, and has been suggested to be an important tumor suppressor gene for

THE GENETICS OF MELANOMA

human melanoma, as deletion or mutation was detected in 23% of melanoma cell lines (Pollock et al., 2002; Walker and Hayward, 2002). The frequency was somewhat lower, however, in uncultured melanomas, at around 10–15% (Guldberg et al., 1997; Poetsch et al., 2001; Pollock et al., 2002; Teng et al., 1997). The region of deletions spans a large section of chromosome 10q, suggesting the likelihood of other melanoma suppressors in this area, but PTEN is the only one yet identified. PTEN is a known tumor suppressor and an enhancer of cellular apoptosis (with possible implication also in cell migration) (Yamada and Araki, 2001). The apparent principal function of PTEN (Fig. 24.4) is dephosphorylation (effectively degradation) of the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) and other lipids in the phosphoinositide cascade. This cascade promotes cell proliferation and reduces apoptosis (Maehama and Dixon, 1998). Downregulation of PIP3 by PTEN reduces the activity of AKT/protein kinase B, a central inhibitory regulator of apoptosis (Fig. 24.4; Stambolic et al., 1998). Conversely, activated RAS can increase PIP3 and AKT activities through stimulation of phosphoinositide-3-kinase (Fig. 24.4; Adjei, 2001). As expected from this pathway, there is some evidence that PTEN abrogation and NRAS activation are functionally overlapping with respect to melanoma genesis (Tsao et al., 2000). Deletions of PTEN are, however, reported predominantly in metastatic melanoma (Poetsch et al., 2001), whereas NRAS and BRAF mutations are common in nevi, suggesting that this overlap is not complete. Ectopic expression of PTEN in PTEN-deficient melanoma cells reversed their tumorigenic potential (Hwang et al., 2001; Robertson et al., 1998).

Other Alterations Suppressing Apoptosis Several genetic alterations common in melanoma and already mentioned have the effect of reducing apoptosis, including disruption of genes for p53, ARF, APAF1, and PTEN, also PTK overexpression and activating mutations of NRAS and BRAF. Most of these changes also promote cell proliferation, not surprisingly as the main signaling pathways controlling proliferation also impinge on apoptosis. However, it is interesting to note how many of the alterations commonly found in advanced melanoma suppress apoptosis. Others not yet mentioned include upregulation of the b-catenin pathway (see Fig. 24.4) by loss or, more usually, silencing of APC (Worm et al., 2004), by rare activating mutations of b-catenin itself (CTNNB1) (Omholt et al., 2001), or by overexpression of proto-oncoprotein SKI (Chen et al., 2003). Normal melanocytes and melanoblasts already have high expression levels of the mitochondrial anti-apoptotic protein BCL2 (Arita et al., 2000; Plettenberg et al., 1995; Yamamura et al., 1996). BCL2 appears to be important in melanocyte maintenance because, in Bcl2 knockout mice, levels of pigmentation and numbers of melanocytes are reported to fall postnatally (Yamamura et al., 1996). (It is possible that BCL2 provides protection against toxic effects of melanin interme-

diates or reactive oxygen species generated during melanogenesis and/or ultraviolet irradiation.) There are disparate reports of BCL2 expression during melanoma progression but, taken together, they suggest that there is no consistent directional change in expression (Bush and Li, 2003). Likewise, reports differ on changes in expression of the pro-apoptotic family member BAX; but interestingly, nearly all studies of another important anti-apoptotic family member, BCL2L1 (BCL-XL), indicate increased expression in primary and metastatic melanoma (Bush and Li, 2003). It may be thought surprising that anti-apoptotic mutations occur in melanoma, given the abundant expression of BCL2 in normal melanocytes. However, a possible reason for such changes is discussed later. There is various general evidence for high resistance to apoptosis in late melanoma cells. Melanoma cells demonstrate low levels of spontaneous apoptosis in vivo compared with other tumor cell types (Mooney et al., 1995; Staunton and Gaffney, 1995), and are relatively resistant to drug-induced apoptosis in vitro (Li et al., 1998). The dismal prognosis for patients with advanced melanoma is associated with lack of therapeutic response to conventional chemotherapy and radiotherapy (Koh, 1991). Most chemotherapeutic drugs function by inducing apoptosis in malignant cells (Fisher, 1994), so that resistance to apoptosis is a likely explanation for drug resistance in melanoma (Satyamoorthy et al., 2001).

AP2 and Downstream Effects AP2 is a transcription factor expressed in various normal cells including melanocytes and other neural crest-derived cells, but silenced in the great majority of advanced melanomas (reviewed by Bar-Eli, 1999, 2001). While not itself a genetic change, this is presumably a result of some unknown genetic (or possibly epigenetic) change, common in melanoma. AP2 repression is included here because the expected downstream effects include a number of important changes in gene expression already reported in melanomas (Bar-Eli, 2001). Reduction in AP2 expression in melanoma is expected to produce the following modulations of AP2 targets. (1) KIT expression should be reduced (as indeed it usually is in melanoma). As KIT is the receptor for the essential melanocyte growth factor, SCF, this loss might be expected to inhibit melanoma growth, yet restoration of KIT expression by genetic manipulation is reported to have a tumor-suppressive effect (Huang et al., 1996). An additional role has been ascribed to KIT, in melanocyte homing and attachment to the epidermis (Wehrle-Haller, 2003), so it is possible that KIT loss is required for melanoma cells to leave the epidermis and invade other tissues. (2) Expression of cell adhesion molecule MCAM (MUC18) should be increased. MCAM expression is elevated in metastatic melanoma cells, and engineered MCAM expression was followed by increased tumor growth and metastasis (Bar-Eli, 2001). (3) Another adhesion molecule, Ecadherin (cadherin 1, CDH1), should be downregulated. 481

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E-cadherin loss is indeed common in advanced melanoma, together with gain of N-cadherin (CDH2) (Hsu et al., 2000; Li et al., 2001). This incidentally reverses the developmental acquisition of E-cadherin expression, seen just before embryonic melanoblasts enter the epidermis (Nishimura et al., 1999). E-cadherin mediates adhesion to keratinocytes, so that its loss could facilitate invasion, whereas expression of Ncadherin promotes melanoma cell adhesion to endothelial cells, potentially aiding migration across endothelium during metastasis (Jahroudi and Greenberger, 1995). (4) Several other proteins potentially important in progression have promoters with AP2 sites and thus may be regulated by AP2, including CDK inhibitor p21, growth factor receptor HER2, apoptotic mediators BCL2 and FAS, protease MMP2, and angiogenic factor VEGF among others (Bar-Eli, 2001). It now seems important to find out the mechanism for the frequent loss of AP2 in melanoma.

Telomerase/Telomere Lengthening This section again refers to a gene product that is subject to frequent changes in expression in melanoma which seem to be functionally very important, and so is included although common genetic alterations have not been identified. Telomeres are the ends of chromosomes, consisting of thousands of copies of a hexamer DNA repeat. In normal somatic cells, telomeres shorten with each cell division because their ends are not fully replicated. However, in germ cells and some embryonic cells, the telomeres are maintained by the enzyme telomerase (Sharpless and DePinho, 2004). Somatic cells cease to express hTERT, the catalytic subunit of telomerase, and therefore lack active telomerase. In human cells, telomere shortening appears to be the initiating stimulus for cell senescence in most cell types (Sharpless and DePinho, 2004). Telomere shortening and the consequent cell senescence are believed to act as a barrier against cancer development. Some 90% of all human cancers appear to have telomerase activity, and the rest can also extend their telomeres through an activity called ALT (alternative lengthening of telomeres) (Londono-Vallejo et al., 2004). Senescence can be prevented by the expression of exogenous hTERT in some cell types, resulting in “immortal” cells that can divide indefinitely. In human melanocytes, however, this can be done only by interference with (or deficiency of) the p16/RB pathway in addition to hTERT expression (Gray-Schopfer et al., submitted; Sviderskaya et al., 2003). Evidence on melanomas appears to fit the generality that cancer cells can extend their telomeres. Up to 90% of primary and metastatic melanomas are strongly positive for telomerase activity, whereas some dysplastic nevi and under a third of benign melanocytic nevi express weak telomerase activity, and none is detected in normal skin (Glaessl et al., 1999; Rudolph et al., 2000; Villa et al., 2001). This point seems to be significant for the following model.

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Model for Relation of Genetic Changes to Melanoma Progression We have recently described a genetic model for melanoma progression (Bennett, 2003; Bennett and Medrano, 2002), in which a number of the known common somatic (and also germline) genetic changes can be related to the clinical series of pigmented lesions of increasing aggressiveness described by Clark et al. (1989) and introduced above (Fig. 24.1). The proposed steps or elements in the model, each based on evidence mostly already discussed above, are as follows: 1 A benign nevus typically results from a single mutation in a melanocyte that leads to cell proliferation to form a clonal colony, the mutation usually being an activation of BRAF, sometimes NRAS and, more rarely, other mutations with the same net effect. 2 Cells of a benign nevus stop growing after a certain number of divisions, through p16-dependent (M0) cell senescence. The idea that nevi are senescent has also been suggested by others (Bastian, 2003; Mooi and Peeper, 2002). We have shown that benign nevi express high levels of p16 and another marker of senescence, acidic b-galactosidase, together with morphological features of senescent cells (Gray-Schopfer et al., submitted). None of these markers was seen in normal epidermal melanocytes. Other evidence that nevus cells are senescent comes from cell cultures (Bennett and Medrano, 2002). 3 Dysplastic nevi, whether they develop from benign nevi or via some other route, have a lesion in at least one copy of p16 and are therefore able to grow larger. The original model proposed that growth of dysplastic nevi might be limited by p53-dependent (M1) senescence, as observed in p16-deficient cultured melanocytes (Sviderskaya et al., 2003). However, the expected expression of p53 and p21 was not usually detected in dysplastic nevi (Gray-Schopfer et al., submitted), suggesting that these lesions (at least those that get excised) are generally not in M1 senescence but may still be growing, although perhaps able to senesce eventually. 4 RGP melanomas have both a lesion in the p16/RB pathway and activated telomerase (or ALT), so that their cells are immortal and able to grow progressively. It is important to note here that melanocytes lacking p16 but with a normal p53 pathway show a high rate of apoptosis, which is suppressed by keratinocytes or by two soluble factors produced by keratinocytes (stem cell factor and endothelin 1) (Sviderskaya et al., 2003). We therefore hypothesize that the radial growth pattern of RGP melanomas — only in or near the epidermis — occurs because their cells are deficient in the p16/RB pathway, and therefore undergo apoptosis if they move away from keratinocytes. The ability of benign nevus cells to populate the dermis is consistent with the model, because these are not p16 deficient. 5 VGP melanoma cells have the same changes as in RGP, but also have a mutation(s) (or epigenetic change) that suppresses apoptosis, such as PTEN, APAF1, or APC mutation, an activating mutation of p53 or b-catenin, additional activation of

THE GENETICS OF MELANOMA

the PTK–RAS–RAF–PI3K–AKT pathway, BCL2L1 overexpression, AP2 downregulation, and so on. As noted above, such mutations are common in advanced melanoma. They would enable survival of melanoma cells that move away from the epidermis. If such suppression of apoptosis occurred prior to immortalization, then a melanoma with no RGP — the nodular form — would be expected.

Perspectives Three familial melanoma genes and one additional mapped locus are now known but, in many families, the susceptibility does not map to at least the three cloned genes. It seems likely that other melanoma susceptibility gene(s) remain to be identified, although these seem unlikely to be as commonly mutated as CDKN2A. Moreover, not all individuals carrying melanoma-associated mutations get melanoma, indicating the importance of modifier genes. One of these (MC1R) is known, together with some other candidates, and it may well be that a number of such lower penetrance genes will be found to confer lower risk or act as modifiers for high-penetrance genes. It is an exciting period in the study of somatic cell genetics of melanoma. We appear to be close to an initial understanding of the full set of steps required to produce an invasive (potentially metastatic) melanoma from a normal cell, which has not yet been achieved for a solid tumor type, to our knowledge. We still do not fully understand the genetic basis of a dysplastic nevus, and it remains to be tested whether suppression of apoptosis is indeed the definitive difference between RGP and VGP melanomas. Other important points yet to be firmly demonstrated are how p16 expression and AP2 expression are both silenced in a large proportion of melanomas, the role of ARF, and which of Clark’s series of lesions are liable to progress to which others. However, we have already gained much understanding, which may lead to applications in diagnosis and therapy. For example, markers of senescence and immortalization are under investigation in the Bennett laboratory and others for differential diagnosis of dysplastic nevi and early melanomas. The mutations frequently seen in melanomas are potential targets for therapy. Melanoma is one of the most aggressive forms of cancer, and is resistant to current therapies, perhaps because of its intrinsic resistance to apoptosis. Specific inhibitors of mutant BRAF may be useful therapeutic agents, and various RAF inhibitors are currently in phase I to phase III clinical trials for melanoma and other cancers. A better understanding of potential common factors in the mechanisms limiting apoptosis in melanoma may help us to devise strategies to compensate or bypass the cell death defects. Lastly, the positive elements in cell immortalization, CDK4, cyclin D1, and hTERT, also provide potential therapeutic targets, the pursuit of which may also impinge on numerous other cancer types.

Acknowledgments Research by the authors cited here was supported by European Commission contract QLK4-1999-01084.

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Straume, O., and L. A. Akslen. Alterations and prognostic significance of p16 and p53 protein expression in subgroups of cutaneous melanoma. Int. J. Cancer 74:535–539, 1997. Straume, O., L. Sviland, and L. A. Akslen. Loss of nuclear p16 protein expression correlates with increased tumor cell proliferation (Ki-67) and poor prognosis in patients with vertical growth phase melanoma. Clin. Cancer Res. 6:1845–1853, 2000. Sviderskaya, E. V., S. P. Hill, T. J. Evans-Whipp, L. Chin, S. J. Orlow, D. J. Easty, S. C. Cheong, D. Beach, R. A. DePinho, and D. C. Bennett. p16Ink4a in melanocyte senescence and differentiation. J. Natl. Cancer Inst. 94:446–454, 2002. Sviderskaya, E. V., V. C. Gray-Schopfer, S. P. Hill, N. P. Smit, T. J. Evans-Whipp, J. Bond, L. Hill, V. Bataille, G. Peters, D. Kipling, D. Wynford-Thomas, and D. C. Bennett. p16/cyclin-dependent kinase inhibitor 2A deficiency in human melanocyte senescence, apoptosis, and immortalization: possible implications for melanoma progression. J. Natl. Cancer Inst. 95:723–732, 2003. Teng, D. H., R. Hu, H. Lin, T. Davis, D. Iliev, C. Frye, B. Swedlund, K. L. Hansen, V. L. Vinson, K. L. Gumpper, L. Ellis, A. El Naggar, M. Frazier, S. Jasser, L. A. Langford, J. Lee, G. B. Mills, M. A. Pershouse, R. E. Pollack, C. Tornos, P. Troncoso, W. K. Yung, G. Fujii, A. Berson, and P. A. Steck. MMAC1/PTEN mutations in primary tumor specimens and tumor cell lines. Cancer Res. 57:5221–5225, 1997. Tsao, H., X. Zhang, K. Fowlkes, and F. G. Haluska. Relative reciprocity of NRAS and PTEN/MMAC1 alterations in cutaneous melanoma cell lines. Cancer Res. 60:1800–1804, 2000. Tucker, M. A., and A. M. Goldstein. Melanoma etiology: where are we? Oncogene 22:3042–3052, 2003. Valverde, P., E. Healy, I. Jackson, J. L. Rees, and A. J. Thody. Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans. Nature Genet. 11:328–330, 1995. van’t Veer, L. J., B. M. T. Burgering, R. Versteeg, A. J. M. Boot, D. J. Ruiter, S. Osanto, P. I. Schrier, and J. L. Bos. N-ras mutations in human cutaneous melanoma from sun-exposed body sites. Mol. Cell. Biol. 9:3114–3116, 1989. Vaziri, H., and S. Benchimol. Alternative pathways for the extension of cellular life span: inactivation of p53/pRb and expression of telomerase. Oncogene 18:7676–7680, 1999. Venkitaraman, A. R. A growing network of cancer-susceptibility genes. N. Engl. J. Med. 348:1917–1919, 2003. Villa, R., C. D. Porta, M. Folini, M. G. Daidone, and N. Zaffaroni. Possible regulation of telomerase activity by transcription and alternative splicing of telomerase reverse transcriptase in human melanoma. J. Invest. Dermatol. 116:867–873, 2001. Walker, G. J., and N. K. Hayward. Pathways to melanoma development: lessons from the mouse. J. Invest. Dermatol. 119:783–792, 2002. Wehrle-Haller, B. The role of Kit-ligand in melanocyte development and epidermal homeostasis. Pigment Cell Res. 16:287–296, 2003. Wei, Q., J. E. Lee, J. E. Gershenwald, M. I. Ross, P. F. Mansfield, S. S. Strom, L. E. Wang, Z. Guo, Y. Qiao, C. I. Amos, M. R. Spitz, and M. Duvic. Repair of UV light-induced DNA damage and risk of cutaneous malignant melanoma. J. Natl. Cancer Inst. 95:308–315, 2003. Wei, W., R. M. Hemmer, and J. M. Sedivy. Role of p14ARF in replicative and induced senescence of human fibroblasts. Mol. Cell. Biol. 21:6748–6757, 2001. Wellbrock, C., L. Ogilvie, D. Hedley, M. Karasarides, J. Martin, D. Niculescu-Duvaz, C. J. Springer, and R. Marais. V599EB-RAF is an oncogene in melanocytes. Cancer Res. 64:2338–2342, 2004. Worm, J., C. Christensen, K. Gronbaek, E. Tulchinsky, and P. Guldberg. Genetic and epigenetic alterations of the APC gene in malignant melanoma. Oncogene 23:5215–5226, 2004. Wu, H., V. Goel, and F. G. Haluska. PTEN signaling pathways in melanoma. Oncogene 22:3113–3122, 2003.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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The Transformed Phenotype of Melanocytes Dong Fang and Meenhard Herlyn

Summary 1 Cutaneous melanoma is a tumor derived from epidermal melanocytes, and is the result of complex interactions between environmental, constitutive, and genetic factors (Liu and Herlyn, 2004). 2 Melanoma has become one of the most rapidly increasing malignancies in the Caucasian population with 2.5–3% more cases each year in the US. The research field is developing rapidly, and our concepts on etiology and pathogenesis of melanoma evolve continuously. 3 This chapter covers recent advances in the study of growth properties of melanoma cells, their signal transduction pathways, and genetic abnormalities that may open new strategies for therapy.

The Clinical and Pathological Stages of Tumor Progression Melanoma development involves a multistep process. Based on clinical and histopathological studies, five steps of melanoma progression have been delineated (Fig. 25.1): Step 0, normal melanocytes; Step 1, common acquired and congenital nevi with structurally normal melanocytes; Step 2, dysplastic nevi with structural and architectural atypia; Step 3, RGP (radial growth phase) primary melanomas that are nontumorigenic and without metastatic competence; Step 4, VGP (vertical growth phase) tumorigenic primary melanomas with competence for metastasis; and Step 5, metastatic melanomas. These progression steps can be clearly delineated in approximately 40% of all melanoma cases, but it is not clear whether and how transformation occurs for the majority of melanomas. Summary: Melanoma progression includes several welldefined steps, which are clinically and pathologically defined. When melanomas are recognized early, surgical excision is curative. Invasion of tumor cells into the dermis and expansion into a nodule is associated with metastastic competence.

Stem Cells and Melanoma Epidermal melanocytes are derived from the neural crest during embryonic development. Experimentally, we are begin-

ning to distinguish several populations of cells that can give rise to melanocytes: (1) pluripotent stem cells (totipotent embryonic stem cells that have differentiation potential, including for melanocytes); (2) melanocyte stem cells that have the capacity to differentiate into melanocytes and potentially other cell types; (3) melanocyte precursor cells or melanoblasts that are nonpigmented; and (4) differentiated melanocytes that are pigmented. It is not clear whether melanocytes at each differentiation step can transform to melanoma. Initial work on transformation of precursor cells has been done in the hematopoietic cell system because monocytes or neutrophils are terminally differentiated and rarely transform (Bonnet and Dick, 1997). Very recently, it has become clear that gliomas and breast carcinomas arise from their respective stem cells instead of fully differentiated cells (Al-Hajj et al., 2003; Singh et al., 2003), suggesting that melanomas are also likely derived from melanocyte stem cells (Fig. 25.1; Herlyn, 2002). Similar to epidermal keratinocytes-derived tumors, it is likely that melanocytes from all differentiation stages give rise to melanoma, but that melanocyte stem cell-derived tumors are more prevalent. In leukemias, breast carcinomas, and gliomas, it is suggested that lesions contain a subpopulation of malignant cells with differing properties from the main population (Al-Hajj et al., 2003; Singh et al., 2003). These “tumor stem cells” have selfrenewal capacity and can differentiate into cell type(s) expressing differentiation markers, and having limited and invasive capacity. It is likely that melanoma lesions contain “tumor stem cell” populations, but the majority of tumor cells represent cells that have differentiated. The degree of differentiation from the stem cell phenotype or the “dedifferentiation” from a mature phenotype may vary depending on the clinical stage of progression, genotypic instability, and microenvironmental factors that cannot yet be defined. It is likely that tumor stem cells show differences in clinical responses to conventional therapies. The nonresponsiveness of tumor stem cells may come from relative quiescence, which could be analogous to the “label-retaining populations” of keratinocytes in the epidermis (Liu et al., 2003; Morris et al., 2004; Tumbar et al., 2004; Xu et al., 2003). The concept of divergent chemotherapy responses for cells within one lesion will have great importance for eliminating all malignant cells from a tumor. In the following, we will discuss the properties of cultured melanoma cells isolated from patients. Whether these cells represent the stem-like or the more differentiated melanoma cell type is not yet clear. Thus, in the future, we 489

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close communicative networks with fibroblasts and endothelial cells (Bogenrieder and Herlyn, 2003; Hsu et al., 2000; Li et al., 2002). VGP primary melanoma cells grow anchorage independently in soft agar, and are tumorigenic in immunodeficient mice. They have metastatic competence in both patients and experimental animals (Juhasz et al., 1993; Kath et al., 1991). Summary: The transition from RGP to VGP is critical for melanoma progression. Understanding the molecular shifts during the transition will help us to reveal how transformed melanocytes gain the capacity to survive and proliferate in the dermal microenvironment.

Stimulation of Melanoma Survival and Growth by Autocrine Growth Factors Fig. 25.1. Schematic of melanoma progression sequence.

may need to reclassify and regroup melanoma cells that are workhorses for experimental studies. Summary: It is possible that a subpopulation representing tumor stem cells exists in melanoma lesions. Characterizing this subpopulation will provide a better understanding of the biology of melanoma and may help to develop better treatment strategies.

Biological Properties of Melanoma Cells During the transition of primary melanoma cells from the superficial location in the epidermis with only a few nontumorigenic cells invading into the papillary dermis (RGP primary melanoma) to an invasive, expanding tumor nodule in the dermis (VGP primary melanoma), cells change their stromal partners from keratinocytes to fibroblasts (reviewed by Li and Herlyn, 2000). The transition is critical for expansion of the tumor mass and for further dissemination. Melanoma cells from the biological early RGP stage are still dependent on support from exogenous growth factors supplied to them by normal skin cells because they have limited capacity for production of autocrine growth factors. When RGP melanoma cells are placed in organotypic cultures of human skin, they cannot invade from the epidermis into the dermis. Those cells that are invading into the dermis undergo apoptosis (Meier et al., 2000). RGP melanoma cells do not grow in soft agar, or grow only poorly, and are generally not tumorigenic or grow very slowly in vivo. VGP primary melanoma cells represent the biologically advanced stage of primary melanoma. They have completely escaped the control by keratinocytes and have invaded the dermis to establish

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All VGP primary and metastatic melanomas produce growth factors constitutively, i.e. without stimulation by exogenous growth factors. A growth factor is defined as autocrine if (a) it is produced by the malignant cells; (b) tumor cells express the respective receptor; (c) exogenous growth factor stimulates the tumor cells; and (d) growth of tumor cells is inhibited by an antagonist such as antibodies against the factor, blocking agents of the receptor, or downmodulators of the expression of either factor or receptor through antisense oligodeoxynucleotides or RNA interference. In melanoma cells, basic fibroblast growth factor (bFGF) has been identified as a major autocrine growth factor (reviewed by Bogenrieder and Herlyn, 2003). Several other growth factors may also have autocrine functions including hepatocyte growth factor (HGF) (Li et al., 2001), interleukin (IL)-8 (Schadendorf et al., 1993), melanoma growth stimulatory activity or MGSA/GROa (Middleman et al., 2002; Richmond and Thomas, 1986), or PDGF-A (Barnhill et al., 1996; Stiles, 1983), but bFGF appears to be the dominant factor. bFGF is found already in nonmalignant nevus cells and RGP primary melanoma cells (al-Alousi et al., 1996a, b; Scott et al., 1991) but not in normal melanocytes. It appears to act as a “stimulation factor” of melanocytic cells of all stages because its overexpression in normal melanocytes allows growth in serum-free media in the absence of not only bFGF but also insulin or insulin-like growth factor (IGF)-1. It allows melanocytes to survive and proliferate in the human dermis in vivo (Berking et al., 2001). Whether melanoma-derived bFGF has paracrine functions to stimulate stromal fibroblasts and endothelial cells is unclear because the lack of a signal sequence does not allow protein secretion. However, it may be released from cells through alternative mechanisms, such as transport by the heparin binding protein, FGF-BP (Czubayko et al., 1994), or after cell death. Summary: The major autocrine growth factors linked with melanoma development are bFGF, HGF, MGSA/GROa, and PDGF-A. They facilitate survival of transforming melanocytes in the dermal environment.

THE TRANSFORMED PHENOTYPE OF MELANOCYTES

Transformation of Melanocytes with Growth Factors Overexpression of bFGF by adenoviral gene transfer in human skin xenografts led to black pigmented macules within 3 weeks of treatment, which persisted for 8 months if bFGF injections were done once per week (Berking et al., 2001). When bFGF was combined with UVB treatment, pigmented lesions with hyperplastic melanocytes were detected, including a lesion with high-grade atypia resembling the lentiginous form of malignant melanoma. When a combination of bFGF, stem cell factor (SCF), and endothelin-3 (ET-3) was overexpressed in human skin, melanocyte clusters developed in the epidermal–dermal junction zone, and single melanocytes began to migrate into the upper layers of the epidermis within 2–4 weeks (Berking et al., 2004). When the weekly induced expression of all three growth factors was combined with UVB irradiation, severe pigmented lesions developed after 3–4 weeks. Histologically, these lesions were composed of cytological atypical melanocytic cells and represented in situ or invasive melanomas resembling invasive and tumorigenic melanomas in patients’ skin. In adult human skin grafts, the induction of in situ melanomas was achieved with the combination of growth factors only and in the absence of UVB. These studies suggest that growth factors that are physiologically expressed in human skin, for example during inflammation, can disrupt the homeostatic balance and initiate the transformation process. We do not yet know why we need the combination of three growth factors at the same time, nor has it been elucidated which signaling pathways have to be activated for transformation. A significant role for UVB could be demonstrated in these studies only in skin from newborns, not in adult skin, suggesting that, in adult skin, molecular changes have occurred that allow transformation by growth factors only. We also do not know the molecular switches that have to occur from a dependence on exogenous growth factors by normal melanocytes to constitutive endogenous production in melanoma. In our experimental melanoma model (Berking et al., 2004), isolated melanocytic cells grew vigorously in vitro, including in soft agar. However, they remained dependent on exogenous growth factors, and they senesced over time. These results suggest that genetic alterations are required before cells fulfill all requirements for the “hallmarks of cancer,” i.e. autonomous growth and immortality (Hanahan and Weinberg, 2000). Thus, we can now delineate what the most critical environmental and genetic factors are that lead to malignant transformation. Summary: In combination with UVB, ectopic expression of a group of growth factors can transform melanocytes. However, genetic mutations are essential for malignancy. Searching for environmental and genetic transformation factors remains a major task for melanoma biologists.

Paracrine Growth Factors Produced by Stromal Cells and Stimulating Melanoma Cells The autocrine growth factors produced by the malignant cells are also released by the tumor cells to attract stromal fibroblasts, endothelial cells, and inflammatory cells. In turn, one of the major functions of tumor-infiltrating fibroblasts is the production of growth factors for stimulation of the malignant cells. Fibroblasts need positive feedback for tumor cells because they cannot produce IGF-1, which is essential for melanocytic cells at all stages of progression (Rodeck et al., 1987). Melanocytes, nevus cells, and RGP melanoma cells require IGF-1 (or insulin, which also binds to IGFR-1) for proliferation in vitro (Rodeck et al., 1987). VGP melanoma cells require IGF-1 as the only exogenous growth factor unless adapted to grow without it (Bogenrieder and Herlyn 2003; Graeven and Herlyn, 1992). In metastatic melanoma cells, Baserga and coworkers did not observe a difference in in vitro growth between cells transduced with an antisense IGFR-1 vector and controls (Resnicoff et al., 1994). On the other hand, tumor cells with suppressed IGFR-1 could no longer proliferate in immunodeficient mice, suggesting that IGFR-1 signaling is critical for survival in vivo. We have demonstrated that IGF-1 in biologically early primary melanoma cells activates both the mitogen-activated protein (MAP) kinase and the phosphoinositide (PI)-3 kinase pathways (Satyamoorthy et al., 2001). Activation of the PI-3 kinase pathway leads to decreased apoptosis and stabilization of b-catenin. IGF-1 also induces IL-8, which acts as a migratory factor for the tumor cells (Satyamoorthy et al., 2002). Whether additional growth factors are similarly as important as paracrine growth factors in stimulating the melanoma cells is not clear. Potential candidates are HGF, bFGF, and PDGF-A. The tumor stroma invariably contains inflammatory and immune cells, including monocytes, neutrophils, mast cells, and lymphocytes, because tumors secrete chemoattractants specific for each cell type. Through transfection experiments, it became apparent that inflammatory and immune cells have differing roles that are greatly influenced by the ratios of host to tumor cells. Melanomas secreting monocytes–chemoattractive protein-1 (MCP-1) attracted monocytes/macrophages (Nesbit et al., 2001). When small numbers of these inflammatory cells invade tumors, they stimulate the malignant cells through secretion of tumor-stimulating growth factors such as colonystimulating factors or PDGF, or through secretion of angiogenic growth factors such as TNFa. Similar dose-dependent effects of inflammatory cells were observed with melanoma-derived IL-8, which is a strong chemoattractant for neutrophils (Choi et al., 2003). These experimental studies are related to the infiltration of lymphocytes into melanomas. Strong (brisk) lymphocytic infiltration into the tumor environment correlates with increased survival and decreased progression (Clark et al., 1989; Sondergaard and Schou, 1985), suggesting that

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lymphocytes also require a certain threshold to have a biological impact on clinical outcome. Summary: The tumor microenvironment plays an important role in melanoma development and progression. The balance between tumor and stromal cell interactions determines the aggressiveness of the tumor.

Stimulation of Tumor Cells Through Aberrations in Signal Transduction Pathways Activating mutations in the BRAF gene have recently been described in the majority of melanomas and benign nevi, suggesting a role for this oncogene in melanocyte transformation (Brose et al., 2002; Davies et al., 2002; Pollock et al., 2003). BRAF is a member of the RAF kinase family that includes CRAF, BRAF, and ARAF. A diverse number of stimuli, which include mitogens, hormones, and neurotransmitters, promote the activation of RAF kinases by first triggering increases in the levels of RAS-GTP in cells. The guanosine 5¢-triphosphate (GTP)-bound forms of RAF interact directly with, and thereby facilitate, the translocation of the cytoplasmic RAF kinase to the plasma membrane. The activation state of RAF is further modulated by phosphorylation from other kinases and, potentially, autophosphorylation (reviewed by Chong et al., 2003). Fully activated RAF then assembles the MAP kinase signaling complex which consists of two classes of kinases, MEK and ERK, and scaffolding proteins such as KSR (Roy et al., 2002). Active RAF phosphorylates and activates MEK, which subsequently phosphorylates and activates ERK. This phosphorylated and activated ERK dissociates from the RAF–MEK–ERK complex and translocates into the nucleus where it phosphorylates and activates several transcription factors, such as ELK-1, C-JUN, ATF-2, and GABP (reviewed by Tuveson et al., 2003). Gene expression changes by these activated transcription factors increase survival and transformation depending on the cellular context. The demonstration that most melanomas harbor the BRAF-V600E allele provides a novel mechanism for oncogenesis through the MAP-kinase cascade in melanoma. V600E mutation destabilizes the inactive form of BRAF, shifting the equilibrium towards the active form of the kinase. BRAF mutations may induce different molecular and biological responses in nevi compared with melanomas, as suggested by the inability to detect MAP kinase activation in nevi despite the observation that over 80% of nevi harbor this oncogene. In melanomas, between 40% and 70% of lesions harbor the V600E mutation, as has now been demonstrated independently in many different laboratories (Wan et al., 2004). This finding helps to explain the constitutive activation of the MAP kinase pathway. However, there are still major gaps in our knowledge. For example, N-Ras, which is upstream of BRAF, is also activated by currently unknown mechanisms (Satyamoorthy et al., 2003). We also do not know how other signaling pathways are activated in melanoma, potentially through mutational events, but we also have to 492

consider biological triggers for activation. Activated pathways include AKT, STAT-3, NFkB, PKC, and PKA. Whether these pathways are activated in all melanomas or only in a subset of melanomas is not clear. However, many studies point to constitutive activation of few and defined pathways in this malignancy, which could provide us with a chance to target these with selected inhibitors. Indeed, the first inhibitor for a RAF kinase, BAY-43-9006, is already being used successfully in clinical trials, promising that targeting of signaling pathways in melanoma is a new strategy for therapy. Summary: As the search for additional mutations is ongoing in melanoma, we hope to understand in the next few years which genetic alterations are most critical for growth, survival, and invasion. We are also beginning to learn how we can target these abnormalities by either increasing the functions of tumor suppressor genes or inhibiting those of oncogenes. It is most likely that targeting of signaling pathways will need to be done through a combination of drugs.

Perspectives The melanoma research field is exploding with new concepts on melanocyte transformation and melanoma etiology. We expect new diagnostic techniques and therapeutic strategies. Studying tumor stem cells in melanoma may identify a subpopulation that is resistant to conventional therapeutic approaches. Understanding growth factor requirements for melanocytes and melanoma will elucidate the microenvironmental factors important for transformation. Aberrant signal transduction pathways will provide new targets for therapy. It is fortunate that several major pharmaceutical companies have developed an interest in melanoma, promising a new era in the control of this disease. However, coordinating the findings of diverse groups with differing interests will be a major challenge.

References al-Alousi, S., R. Barnhill, K. Blessing, and S. Barksdale. The prognostic significance of basic fibroblast growth factor in cutaneous malignant melanoma. J. Cutan. Pathol. 23:506–510, 1996a. al-Alousi, S., J. A. Carlson, K. Blessing, M. Cook, T. Karaoli, and R. L. Barnhill. Expression of basic fibroblast growth factor in desmoplastic melanoma. J. Cutan. Pathol. 23:118–125, 1996b. Al-Hajj, M., M. S. Wicha, A. Benito-Hernandez, S. J. Morrison, and M. F. Clarke. Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. USA 100:3983–3988, 2003. Barnhill, R. L., M. Xiao, D. Graves, and H. N. Antoniades. Expression of platelet-derived growth factor (PDGF)-A, PDGF-B and the PDGF-alpha receptor, but not the PDGF-beta receptor, in human malignant melanoma in vivo. Br. J. Dermatol. 135:898–904, 1996. Berking, C., R. Takemoto, K. Satyamoorthy, R. Elenitsas, and M. Herlyn. Basic fibroblast growth factor and ultraviolet B transform melanocytes in human skin. Am. J. Pathol. 158:943–953, 2001. Berking, C., R. Takemoto, K. Satyamoorthy, T. Shirakawa, M. Eskandarpour, J. Hansson, P. A. VanBelle, D. E. Elder, and M. Herlyn. Induction of melanoma phenotypes in human skin by growth factors and ultraviolet B. Cancer Res. 64:807–811, 2004.

THE TRANSFORMED PHENOTYPE OF MELANOCYTES Bogenrieder, T., and M. Herlyn. Axis of evil: molecular mechanisms of cancer metastasis. Oncogene 22:6524–6536, 2003. Bonnett, D., and J. E. Dick. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 3:730–737, 1997. Brose, M. S., P. Volpe, M. Feldman, M. Kumar, I. Rishi, R. Gerrero, E. Einhorn, M. Herlyn, J. Minna, A. Nicholson, J. A. Roth, S. M. Albelda, H. Davies, C. Cox, G. Brignell, P. Stephens, P. A. Futreal, R. Wooster, M. R. Stratton, and B. L. Weber. BRAF and RAS mutations in human lung cancer and melanoma. Cancer Res. 62:6997–7000, 2002. Choi, M., S. Rolle, M. Wellner, M. C. Cardoso, C. Scheidereit, F. C. Luft, and R. Kettritz. Inhibition of NF-kappaB by a TAT-NEMObinding domain peptide accelerates constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosis. Blood 102:2259–2267, 2003. Chong, H., H. G. Vikis, and K. L. Guan. Mechanisms of regulating the Raf kinase family. Cell Signal 15:463–469, 2003. Clark, W. H., Jr., D. E. Elder, D. T. Guerry, L. E. Braitman, B. J. Trock, D. Schultz, M. Synnestvedt, and A. C. Halpern. Model predicting survival in stage I melanoma based on tumor progression. J. Natl. Cancer Inst. 81:1893–904, 1989. Czubayko, F., R. V. Smith, H. C. Chung, and A. Wellstein. Tumor growth and angiogenesis induced by a secreted binding protein for fibroblast growth factors. J. Biol. Chem. 269:28243–28248, 1994. Davies, H., G. R. Bignell, C. Cox, P. Stephens, S. Edkins, S. Clegg, J. Teague, H. Woffendin, M. J. Garnett, W. Bottomley, N. Davis, E. Dicks, R. Ewing, Y. Floyd, K. Gray, S. Hall, R. Hawes, J. Hughes, V. Kosmidou, A. Menzies, C. Mould, A. Parker, C. Stevens, S. Watt, S. Hooper, R. Wilson, H. Jayatilake, B. A. Gusterson, C. Cooper, J. Shipley, D. Hargrave, K. Pritchard-Jones, N. Maitland, G. Chenevix-Trench, G. J. Riggins, D. D. Bigner, G. Palmieri, A. Cossu, A. Flanagan, A. Nicholson, J. W. Ho, S. Y. Leung, S. T. Yuen, B. L. Weber, H. F. Seigler, T. L. Darrow, H. Paterson, R. Marais, C. J. Marshall, R. Wooster, M. R. Stratton, and P. A. Futreal. Mutations of the BRAF gene in human cancer. Nature 417:949–954, 2002. Graeven, U., and M. Herlyn. In vitro growth patterns of normal human melanocytes and melanocytes from different stages of melanoma progression. J. Immunother. 12:199–202, 1992. Hanahan, D., and R. A. Weinberg. The hallmarks of cancer. Cell 100:57–70, 2000. Herlyn, M. Emerging concepts and technologies in melanoma research. Melanoma Res. 12:3–8, 2002. Hsu, M. Y., F. E. Meier, M. Nesbit, J. Y. Hsu, P. Van Belle, D. E. Elder, and M. Herlyn. E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors. Am. J. Pathol. 156:1515–1525, 2000. Juhasz, I., S. M. Albelda, D. E. Elder, G. F. Murphy, K. Adachi, D. Herlyn, I. T. Valyi-Nagy, and M. Herlyn. Growth and invasion of human melanomas in human skin grafted to immunodeficient mice. Am. J. Pathol. 143:528–537, 1993. Kath, R., J. A. Jambrosic, L. Holland, U. Rodeck, and M. Herlyn. Development of invasive and growth factor-independent cell variants from primary human melanomas. Cancer Res. 51:2205–2211, 1991. Li, G., and M. Herlyn. Dynamics of intercellular communication during melanoma development. Mol. Med. Today 6:163–169, 2000. Li, G., H. Schaider, K. Satyamoorthy, Y. Hanakawa, K. Hashimoto, and M. Herlyn. Downregulation of E-cadherin and Desmoglein 1 by autocrine hepatocyte growth factor during melanoma development. Oncogene 20:8125–8135, 2001. Li, G., K. Satyamoorthy, and M. Herlyn. Dynamics of cell interactions and communications during melanoma development. Crit. Rev. Oral Biol. Med. 13:62–70, 2002.

Liu, Y., S. Lyle, Z. Yang, and G. Cotsarelis. Keratin 15 promoter targets putative epithelial stem cells in the hair follicle bulge. J. Invest. Dermatol. 121:963–968, 2003. Liu, Z. J., and M. Herlyn. Molecular biology of cutaneous melanoma. In: Cancer: Principles and Practice of Oncology, 7th edn, V. T. DeVita, Jr., S. Hellman, and S. A. Rosenberg (eds). Philadelphia, PA, Lippincott Williams & Wilkins, 2005. Meier, F., M. Nesbit, M. Y. Hsu, B. Martin, P. Van Belle, D. E. Elder, G. Schaumburg-Lever, C. Garbe, T. M. Walz, P. Donatien, T. M. Crombleholme, and M. Herlyn. Human melanoma progression in skin reconstructs: biological significance of bFGF. Am. J. Pathol. 156:193–200, 2000. Middleman, B. R., M. Friedman, D. H. Lawson, P. B. DeRose, and C. Cohen. Melanoma growth stimulatory activity in primary malignant melanoma: prognostic significance. Mod. Pathol. 15:532–537, 2002. Morris, R. J., Y. Liu, L. Marles, Z. Yang, C. Trempus, S. Li, J. S. Lin, J. A. Sawicki, and G. Cotsarelis. Capturing and profiling adult hair follicle stem cells. Nature Biotechnol. 22:411–417, 2004. Nesbit, M., H. Schaider, T. H. Miller, and M. Herlyn. Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells. J. Immunol. 166: 6483–690, 2001. Pollock, P. M., U. L. Harper, K. S. Hansen, L. M. Yudt, M. Stark, C. M. Robbins, T. Y. Moses, G. Hostetter, U. Wagner, J. Kakareka, G. Salem, T. Pohida, P. Heenan, P. Duray, O. Kallioniemi, N. K. Hayward, J. M. Trent, and P. S. Meltzer. High frequency of BRAF mutations in nevi. Nature Genet. 33:19–20, 2003. Resnicoff, M., D. Coppola, C. Sell, R. Rubin, S. Ferrone, and R. Baserga. Growth inhibition of human melanoma cells in nude mice by antisense strategies to the type 1 insulin-like growth factor receptor. Cancer Res. 54:4848–4850, 1994. Richmond, A., and H. G. Thomas. Purification of melanoma growth stimulatory activity. J. Cell Physiol. 129:375–384, 1986. Rodeck, U., M. Herlyn, H. D. Menssen, R. W. Furlanetto, and H. Koprowsk. Metastatic but not primary melanoma cell lines grow in vitro independently of exogenous growth factors. Int. J. Cancer 40:687–690, 1987. Roy, F., G. Laberge, M. Douziech, D. Ferland-McCollough, and M. Therrien. KSR is a scaffold required for activation of the ERK/MAPK module. Genes Dev. 16:427–438, 2002. Satyamoorthy, K., G. Li, B. Vaidya, J. Kalabis, and M. Herlyn. Insulinlike growth factor-I-induced migration of melanoma cells is mediated by interleukin-8 induction. Cell Growth Differ. 13:87–93, 2002. Satyamoorthy, K., G. Li, B. Vaidya, D. Patel, and M. Herlyn. Insulinlike growth factor-1 induces survival and growth of biologically early melanoma cells through both the mitogen-activated protein kinase and beta-catenin pathways. Cancer Res. 61:7318–7324, 2001. Schadendorf, D., A. Moller, B. Algermissen, M. Worm, M. Sticherling, and B. M. Czarnetzki. IL-8 produced by human malignant melanoma cells in vitro is an essential autocrine growth factor. J. Immunol. 151:2667–2675, 1993. Scott, G., M. Stoler, S. Sarkar, and R. Halaban. Localization of basic fibroblast growth factor mRNA in melanocytic lesions by in situ hybridization. J. Invest. Dermatol. 96:318–322, 1991. Singh, S. K., I. D. Clarke, M. Terasaki, V. E. Bonn, C. Hawkins, J. Squire, and P. B. Dirks. Identification of a cancer stem cell in human brain tumors. Cancer Res. 63:5821–5828, 2003. Sondergaard, K., and G. Schou. Survival with primary cutaneous malignant melanoma, evaluated from 2012 cases. A multivariate regression analysis. Virchows Arch. A Pathol. Anat. Histopathol. 406:179–195, 1985. Stiles, C. D. The molecular biology of platelet-derived growth factor. Cell 33:653–655, 1983.

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CHAPTER 25 Tumbar, T., G. Guasch, V. Greco, C. Blanpain, W. E. Lowry, M. Rendl, and E. Fuchs. Defining the epithelial stem cell niche in skin. Science 303:359–363, 2004. Tuveson, D. A., B. L. Weber, and M. Herlyn. BRAF as a potential therapeutic target in melanoma and other malignancies. Cancer Cell 4:95–98, 2003. Wan, P. T., M. J. Garnett, S. M. Roe, S. Lee, D. Niculescu-Duvaz, V.

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M. Good, C. M. Jones, C. J. Marshall, C. J. Springer, D. Barford, and R. Marais. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116:855–867, 2004. Xu, X., S. Lyle, Y. Liu, B. Solky, and G. Cotsarelis. Differential expression of cyclin D1 in the human hair follicle. Am. J. Pathol. 163:969–978, 2003.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

II

The Pathophysiology of Pigmentary Disorders

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

3

An Overview of Human Skin Color and its Disorders

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

26

A More Precise Lexicon for Pigmentation, Pigmentary Disorders, and “Chromatic” Abnormalities James J. Nordlund, Tania Cestari, Pearl Grimes, Henry Chan, and Jean-Paul Ortonne

Skin color is an extraordinarily complex subject. There is no standard definition of usual or normal skin color. Abnormal skin color is a result of a vast array of alterations in the skin, from blood flow to deposition of chemicals, to alterations in the morphology of the skin and more. To make the subject even more difficult, there is no standard language, jargon, or lexicon used throughout the dermatological community, much less the medical community at large, to describe these abnormalities. Yet precise, accurate language is the basis for education, for communication, for investigative and therapeutic strategies. The critical need for a precise lexicon is strongly emphasized by the National Institutes of Health (NIH) lexicon project that seeks to develop a consistent lexicon for dermatology as a discipline (Papier et al., 2004).

Skin Color A number of factors contribute to skin color (Table 26.1; see also Chapters 3, 27, and 28). A major determinant of skin color is melanin, its quantity, type, distribution, and location of melanin within the epidermis and dermis (Chapter 3). Other significant determinants are the capillary blood flow (Hajizadeh-Saffar et al., 1990; Rise et al., 1989), chromophores such as carotene or lycopene (Alaluf et al. 2002a; Lascari 1981), and collagen in the dermis. Physical factors are also involved in the observed skin color (Alaluf et al., 2002b; Dawson et al., 1980; Rise et al., 1989). These include the spectrum of light striking the skin, the reflection, refraction, and absorption of light, and the transparency of the stratum corneum and epidermis. There is no color that defines normal skin. Ethnic populations with various skin colors have evolved over time so that skin color varies from very light with red (Celtic) or blond (Scandinavian) hair to very dark (African) or almost black (Australian aborigine) (Nordlund et al., 1998; Nordlund and Ortonne, 1998) (Frontispiece A–I). In between are the peoples of India who have a brown–black skin color. Asians and indigenous American peoples have a yellow tan to reddish tan coloration. The evolutionary value of skin color has been studied and discussed in depth. Recent reviews suggest that, for peoples living between the tropics of Cancer and

Capricorn, a dark skin provides a natural advantage against the deleterious effects of the sun such as sunburn (Aoki, 2002; Jablonski and Chaplin, 2000). Sunburn is an impediment to gathering or hunting for food. Melanin also protected against photolysis of essential nutrients such as folic acid that were likely available in limited quantities especially during prehistoric times (Jablonski and Chaplin, 2000). For those living north or south of these tropical latitudes, it has been postulated that sexual selection chose lighter skin as preferable (Jablonski and Chaplin, 2000).

The Chemistry of Skin Color and Ethnic Pigmentation For the most part, the color of skin is determined by the pigmentary system, i.e. the quantity, type, and distribution of melanin in the skin. There are two main types of melanin: eumelanin, which is brown or black in color (Chapters 10 and 15); and pheomelanin, which has a reddish hue. The synthesis of either type of melanin is a complex multistep process catalyzed by at least one or several enzymes, the most well known being tyrosinase. The biochemistry of melanin and its synthesis are the subject of chapters in the first part of this book. The type of melanin synthesized is not a random event. It is proposed that the first humans had deeply pigmented skin and hair color. Results of recent studies suggest that red hair and freckles are a manifestation of one of several mutations in the receptor for melanocyte-stimulating hormone (a-MSH), which preferentially stimulates the production of eumelanin (Valverde et al., 1995) (see Chapters 20 and 21). The mutated receptor cannot respond to a-MSH and produces pheomelanin as a default. Other than the type and quantity of melanin, epidermis obtained from individuals of all ethnic groups morphologically appears to be the same. The population density of melanocytes in dark skin is similar to that in light skin (Szabo, 1957, 1959, 1967) (see Chapter 3). There is considerable discussion as to whether dark and light skin function in identical manners (Agner, 1991; Berardesca and Maibach, 1988, 1996; Singh et al., 1987; Srinivas et al., 1987; van de Kerkhof and Fokkink, 1989). Skin disorders such as dermatitis or 499

CHAPTER 26 Table 26.1. Chromophores that contribute to skin color. A) Melanotic

B) Nonmelanotic (chromatics)

Melanin Eumelanin and/or pheomelanin Melanocytes Melanin in melanophages

Collagen Carotene or lycopene Other chemicals or drugs Oxyhemoglobin Reduced hemoglobin Other

pityriasis rosea have a different appearance in light and dark skin, an observation that suggests that there are subtle functional differences.

The Physics of Skin Color The color of any object as perceived by an individual is determined by the spectrum of light that is reflected or emitted from the object and impinges on the retina of the observer. Chemicals or chromophores in the skin absorb or reflect incident light to produce the colors we observe. The importance of reflected and absorbed light in humans is underscored by the phenomenon termed iridescence (Simon, 1971). Blue light is reflected preferentially from the stratum corneum, whereas red light penetrates more deeply into the dermis. Human skin can take on a blue hue due to iridescence. Some patients taking minocycline acquire a blue discoloration on the tibial surfaces (Plate 54.6, pp. 494–495) due to deposition of minocycline chelated with divalent cations such as iron (chemically black in color) in the dermis (see Chapter 54). Blue light not absorbed by epidermal melanin is reflected from the skin; the red light penetrates into the dermis where it is absorbed by the black metal chelates. There are many examples of iridescence such as the blue of irides, the sacral spot in babies (Plates 52.1, 52.2, and 52.7, pp. 494–495), the nevus of Ota (Plate 52.3, pp. 494–495) and Ito, and tattoos (Plate 54.10–54.12, pp. 494–495).

Abnormalities of Skin Color There are many mechanisms by which skin color can become abnormal (see Chapter 28). Any of the components that contribute to skin color (Table 26.1) can be deficient or excessive within the skin and produce abnormal skin color (Ortonne and Nordlund, 1998). Excessive capillary blood flow produces rosacea or a nevus flammeus, manifested as a red discoloration of the skin. b-Carotene or lycopene imparts a yellowish cast to the skin. Collagen in the dermis makes the skin look whitish. Excessive or deficient amounts of melanin caused by any mechanism cause hyperpigmentation or hypopigmentation respectively. Language must be able to distinguish the various forms of skin color changes in a clear, precise, and simple manner. 500

Fig. 26.1. Patient with poikiloderma of Civatte referred for evaluation of “depigmentation,” possibly vitiligo vulgaris. The white macules are caused by scarring and fibrosis in the dermis. The erythema is caused by dermal capillary engorgement. The quantity of melanin in the epidermis in a biopsy taken from the whitecolored skin was equal to that in the red-colored skin determined by Fontana–Masson stains. This type of leukoderma is not a pigmentary problem but a form of nonmelanotic achromia. See also Plate 26.1, pp. 494–495.

Confusing Terms How clinicians describe the appearance of a skin disorder is a major determinant of how that disorder is classified (Papier et al., 2004). The classification of a disorder is the basis for therapeutic strategies. Normal and abnormal discoloration of the skin is often described inaccurately. A nevus depigmentosus is rarely depigmented (total absence of melanin) but is typically hypopigmented (partial loss of melanin; see also Chapter 32). Various forms of leukoderma attributable to deposition of collagen in the dermis such as scars are described as hypopigmented (Fig. 26.1). Histology often confirms that the quantity of melanin overlying the scar is equivalent to that in surrounding skin (Velangi and Rees, 2001). Thus, the whitish color is not attributable to abnormalities of the pigmentary system. Yet treatments such as PUVA to repigment the skin are prescribed. It is very common to hear a dermatologist label hydroquinone as a bleach. Hydroquinone is an inhibitor for production of new melanin and has no bleaching effects on existing melanin whether it be in the epidermis or in the dermis. Thus, it can be effective only for eliminating excessive melanin in the epidermis by blocking the formation of new melanin. The improper use of the word “bleach” causes the confusion. Segmental vitiligo affects a part of the body, typically on one side. The words “segmental vitiligo” are considered equivalent to “dermatomal vitiligo.” The terms have become so interchangeable that theories for the pathogenesis of segmental vitiligo are based on the innervation of the skin. Treatments have been proposed based on proposed abnormalities

A MORE PRECISE LEXICON FOR PIGMENTATION, PIGMENTARY DISORDERS, AND “CHROMATIC” ABNORMALITIES Table 26.2. Hyperchromias. A) Melanotic (hyperpigmentation) Hypermelanosis

Hypermelanocytosis

1) Congenital

2) Acquired

a) Localized Epi

B) Nonmelanotic (hyperchromia)

Der

Mix

b) Variable Epi

Der

1) Congenital

c) General Mix

Epi

Der

2) Acquired

a) Localized Mix

Epi

Der

b) Variable

Mix

Epi

Der

c) General Mix

Epi

Der

Mix

Epi, epidermal; der, dermal; mix, mixed epidermal and dermal.

Table 26.3. Hypo- or achromias (leukoderma). A) Melanotic

B) Nonmelanotic (hypochromia/achromia)

Melanopenia

Melanocytopenia

1) Congenital

2) Acquired

a) Localized

b) Variable

c) General

1) Congenital a) Localized E*

D

M

2) Acquired b) Variable E

D

c) General M

E

D

M

*E, epidermal; D, dermal; M, mixed. Each group can be divided into genetic and nongenetic.

in the innervation of the skin (Koga, 1977). In fact, segmental vitiligo is almost never dermatomal, and the patterns rarely resemble one or even several dermatomes (Hann, 2000; Hann et al., 2000). In everyday parlance, the word “pigment” means a dry substance or chemical used to color or tint an object for printing or for painting pictures. In dermatology, it is well recognized that there is a list of chemicals that affect the color of the skin (Table 26.1). Confusion arises when a nevus flammeus is described as hyperpigmented. The dyes of tattoos are genuine pigments. But tattoos are not considered to be forms of “hyperpigmentation.”

A Perspective for a Consistent Lexicon It is proposed (Table 26.1) that all chemicals and structural items that impart color to the skin be called chromophores. Chromophores can be divided into two types, i.e. melanotic (of or pertaining to melanin) and nonmelanotic (of or pertaining to nonmelanin substances). Words such as pigment, pigmentation, hypopigmented, depigmented, and hyperpigmented should properly be restricted to melanin, melanocytes, disorders, and conditions related to excessive or depleted quantities of melanocytes/melanin. That is, the word pigmentation should be reserved for descriptions of skin color and its abnormalities related to melanocytes and melanin, i.e. the pigmentary system. There are just a few basic categories of pigmentary disorders: those characterized by too much or too little melanin and those related to too few or too many melanocytes. Some neologisms are required to distinguish discolorations

of the skin caused by nonmelanotic components from those caused by the pigmentary system. It is necessary to develop new words because there are no other English lay or scientific words extant that can do the job. Nonmelanotic factors that affect skin color are properly called “chromatics” (Table 26.1). Nonmelanotic abnormalities of skin color can be called dyschromias, and the words hyperchromia (Table 26.2) and hypochromia (Table 26.3) are used to describe abnormally darker or lighter skin respectively. Thus, all nonpigmentary chromophores that alter normal skin color fit into these categories of dyschromias, be they vascular, chemical, deposits, collagen, or others. Leukodermas (i.e. hypochromias or achromias; Table 26.4) can be forms of hypo- or depigmentation produced by partial or total loss of melanin from the epidermis (no dermal variant possible). Alternatively, leukoderma can be a type of hypochromia produced by deposition of collagen in the dermis (scar), decreased blood flow such as in nevus anemicus or Wernoff’s ring, or dermal edema. Most of the more than 200 disorders of skin color described in Part II of this book fit precisely and informatively into this scheme. It is possible to describe normal and abnormal color of skin with these neologisms, definitions, and classifications with ease and clarity once the specific abnormality causing the color abnormality has been determined histologically or by other tests. However, it is likely that, when initially evaluating a patient, the clinician might not be certain about either the etiology of a discoloration observed or the precise diagnosis. In such situations, precise diagnostic terms should be avoided until the abnormality can be determined by various histological or other tests and/or a definitive diagnosis can be made. 501

CHAPTER 26 Table 26.4. A classification of the disorders of skin color.

Table 26.5. Some essential definitions.

A) Hyperchromia I) Melanotic types of hyperchromia (hyperpigmentation) a) Hypermelanosis (increased melanin only and normal population density of melanocytes) 1) Congenital i) Localized, variable, or generalized ii) Epidermal, dermal, or mixed 2) Acquired i) Localized, variable, or generalized ii) Epidermal, dermal, or mixed b) Hypermelanocytosis (increased melanocytes and melanin) 1) Congenital i) Localized, variable, or generalized ii) Epidermal, dermal, or mixed 2) Acquired i) Localized, variable or generalized ii) Epidermal, dermal or mixed II) Nonmelanotic types of hyperchromia 1) Congenital i) Localized, variable, or generalized ii) Epidermal, dermal, or mixed 2) Acquired i) Localized, variable, or generalized ii) Epidermal, dermal, or mixed B) Hypo- or achromia (leukoderma) I) Melanotic types of leukoderma (hypo- or depigmentation) a) Hypomelanosis or amelanosis (decreased melanin only) 1) Congenital i) Localized, variable, or generalized ii) Epidermal, dermal, or mixed 2) Acquired i) Localized, variable, or generalized ii) Epidermal, dermal, or mixed b) Hypo- or amelanocytosis (partial or total absence of melanocytes) 1) Congenital i) Localized, variable, or generalized ii) Epidermal, dermal, or mixed 2) Acquired i) Localized, variable, or generalized ii) Epidermal, dermal, or mixed II) Nonmelanotic hypochromia 1) Congenital i) Localized, variable, or generalized ii) Epidermal, dermal, or mixed 2) Acquired i) Localized, variable, or generalized ii) Epidermal, dermal, or mixed

Achromia: a type of leukoderma; totally white skin from any cause. Amelanocytosis: total absence of all melanocytes in the epidermis that will result in amelanosis. Depigmentation (amelanosis): a type of leukoderma also called amelanosis caused by total absence of melanin in the epidermis from any cause. Dermatomal: following the distribution of a cutaneous sensory nerve. Hyperchromia: skin color that is darker than normal from any cause. Hypermelanocytosis: a higher than normal population density of melanocytes in the skin, in the epidermis, the dermis, or both, resulting typically in hypermelanosis. Hypermelanosis: a type of hyperchromia that results from increased melanin in the skin, in the epidermis, dermis, or both. Hypochromia: a type of leukoderma; skin color that is lighter than normal from any cause. Hypomelanocytosis: a lower than normal population density of melanocytes in the epidermis typically resulting in hypomelanosis. Hypomelanosis: a type of leukoderma caused by decreased melanin in the epidermis. Hypopigmentation (hypomelanosis): a type of leukoderma caused by partial absence of melanin in the epidermis. Leukoderma: skin with a white discoloration from any cause or by any mechanism. Pigmentary system: all melanocytes and their product melanin at all sites within the body. Pigmentation: of or pertaining to melanocytes or melanin. Segmental: one portion of the integument, usually unilateral. Skin color: the color of the skin is determined by two distinct groups of chromophores (cells, structural agents, chemicals that impart color to the skin), those of the pigmentary system (i.e. melanin and melanocytes) and those composed of other elements (chromatics) of the skin such as collagen, blood, carotenes, etc.

Thus, a patient presenting with a darkened skin color should be classified with the clinical descriptive term as having a 3cm brown macule, a type of hyperchromia. When the histological analyses are complete, the disorder can be described more accurately as a hyperpigmentation or a nonmelanotic hyperchromia. Likewise, skin with an abnormally white (light) color should be described as a 3-cm hypochromic or leukodermic macule. The pursuit of an accurate diagnosis is bol502

stered by the use of “neutral” descriptive terminology, rather than words that imply etiology before the precise diagnosis is known. Some essential definitions using newly defined words are necessary (Table 26.5). It is our hope that these words, definitions, and categories become the standard for all those teaching or writing books or articles or for all dermatologists as they discuss disorders of skin color.

References Agner, T. Basal transepidermal water loss, skin thickness, skin blood flow and skin colour in relation to sodium-lauryl-sulphate-induced irritation in normal skin. Contact Dermatitis 25:108–114, 1991. Alaluf, S., D. Atkins, K. Barrett, M. Blount, N. Carter, and A. Heath. The impact of epidermal melanin on objective measurements of human skin colour. Pigment Cell Res. 15:119–126, 2002a. Alaluf, S., U. Heinrich, W. Stahl, H. Tronnier, and S. Wiseman. Dietary carotenoids contribute to normal human skin color and UV photosensitivity. J. Nutr. 132:399–403, 2002b. Aoki, K. Sexual selection as a cause of human skin colour variation: Darwin’s hypothesis revisited. Ann. Hum. Biol. 29:589–608, 2002. Berardesca, E., and H. Maibach. Racial differences in skin pathophysiology. J. Am. Acad. Dermatol. 34:667–672, 1996.

A MORE PRECISE LEXICON FOR PIGMENTATION, PIGMENTARY DISORDERS, AND “CHROMATIC” ABNORMALITIES Berardesca, E., and H. I. Maibach. Contact dermatitis in blacks. Dermatol. Clinics 6:363–368, 1988. Dawson, J. B., D. J. Barker, D. J. Ellis, E. Grassam, J. A. Cotterill, G. W. Fisher, and J. W. Feather. A theoretical and experimental study of light absorption and scattering by in vivo skin. Phys. Med. Biol. 2: 695–709, 1980. Hajizadeh-Saffar, M., J. W. Feather, and J. B. Dawson. An investigation of factors affecting the accuracy of in vivo measurements of skin pigments by reflectance spectrophotometry. Phys. Med. Biol. 35:1301–1315, 1990. Hann, S. K. Clinical features of segmental vitiligo. In: Vitiligo: A Monograph on Basic and Clinical Science. S. H. J. Nordlund (ed.). London: Blackwell Science, 2000, pp. 49–60. Hann, S. K., J. H. Chang, and H. S. Lee. The classification of segmental vitiligo on the face. Yonsei Med. J. 41:209–212, 2000. Jablonski, N. G., and G. Chaplin. The evolution of human skin coloration. J. Hum. Evol. 39:57–106, 2000. Koga, M. Vitiligo: a new classification and therapy. Br. J. Dermatol. 97:255–261, 1977. Lascari, A. D. Carotenemia. A review. Clin. Pediatr. 20:25–29, 1981. Nordlund, J. J., and J.-P. Ortonne. The normal color of human skin. In: Pigmentary System: Physiology and Pathophysiology, J. J. Nordlund, R. E. Boissy, V. J. Hearing, R. A. King, and J.-P. Ortonne (eds). New York: Oxford University Press, 1998, pp. 475–488. Nordlund, J. J., R. E. Boissy, V. J. Hearing, R. A. King, and J.-P. Ortonne (eds). The Pigmentary System: Physiology and Pathophysiology. New York: Oxford University Press, 1998. Ortonne, J.-P., and J. J. Nordlund. Mechanisms that cause abnormal skin color. In: The Pigmentary System: Physiology and Pathophysiology, J. J. Nordlund, R. E. Boissy, V. J. Hearing, R. A. King, and J. P. Ortonne (eds). New York: Oxford University Press, 1998, pp. 489–503. Papier, A. C., R. J. G. Chalmers, J. A. Byrnes, and L. A. Goldsmith.

Framework for improved communication: The Dermatology Lexicon Project. J. Am. Acad. Dermatol. 50:630–634, 2004. Rise, A. L., L. M. Prewitt, and J. J. Johnson. Skin color and ear oximetry. Chest 96:287–290, 1989. Simon, H. The Splendor of Iridescence. New York: Dodd, Mead and Co., 1971. Singh, K. K., C. R. Srinivas, C. Balachandran, et al. Parthenium dermatitis sparing vitiliginous skin. Contact Dermatitis 16:174, 1987. Srinivas, C. R., K. K. Singh, and C. Balachandran. Textile dermatitis sparing nevoid depigmentation. Contact Dermatitis 16:235, 1987. Szabo, G. Tyrosinase in the epidermal melanocytes of white human skin: the histochemical demonstration and the numerical relationship of tyrosine-positive and DOPA-positive cells. Arch. Dermatol. 76:324–329, 1957. Szabo, G. Quantitative histological investigations on the melanocyte system of the human epidermis. In: Pigment Cell Biology, M. Gordon (ed.). New York: Academic Press, 1959, pp. 99–125. Szabo, G. Photobiology of melanogenesis: cytological aspects with special reference to differences in racial coloration. In: Advances in Biology of Skin, vol. 8, W. Montagna and F. Hu (eds). London: Pergamon Press, 1967, pp. 379–396. Valverde, P., E. Healy, I. Jackson, J. L. Rees, and A. J. Thody. Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans [see comments]. Nature Genet. 11:328–330, 1995. van de Kerkhof, P. C., and H. J. Fokkink. Irritancy of dithranol in normally pigmented and depigmented skin of patients with vitiligo. Acta Dermato-Venereol. 69:236–238, 1989. Velangi, S. S., and J. L. Rees. Why are scars pale? An immunohistochemical study indicating preservation of melanocyte number and function in surgical scars. Acta Derm. Venereol. 81:326–328, 2001.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

27

The Normal Color of Human Skin James J. Nordlund and Jean-Paul Ortonne

The world of living objects exhibits a splendiferous array of colors. Plants and animals exhibit every color of the electromagnetic spectrum visible to the human eye. Some fortunate animals such as birds and bees can perceive an exceptionally wide range of colors including some of the ultraviolet spectrum not visible to humans. Because of color, the world is a spectacular environment in which to live. Color adds much to the quality of our lives. In addition to its artistic value, it permits the eye to perceive objects with an enhanced acuity that is over 1000-fold better than that allowed by black and white. Pigment and its color in humans and other mammals have many functions, some of which are outlined below. Many animals are decorated with several or many colors. The most colorful animals come from nonmammalian orders. Within the vertebrate phylum, the birds, amphibians, and other orders are colored striking blues, greens, reds, and yellows (see Chapter 2). Some animals such as birds or butterflies are colored blue not by the production of a pigment but by the formation of feather structures that reflect blue light and absorb red. The animal appears blue because of the physical or reflective properties of their feathers, not because there is a blue pigment within their integument. This phenomenon is called iridescence (Simon, 1971). The mammalian order has a much more restricted range of coloration. Mammalian colors are limited to white, yellows, oranges, browns, or black (see Frontispiece). No mammals display the brilliant yellows, greens, and reds of other vertebrates and nonvertebrates. It is not known why mammals are more limited in their coloration or what true functions the most brilliant yellows and reds serve for the avian and amphibian orders. The pigments responsible for the reds, yellows, and other striking colors are not synthesized in mammals. The latter have a single group of pigments called melanin that has a range of colors varying from pale yellow through orange to browns and blacks.

Human Skin Color The human species exhibits the full range of melanin pigments in the skin, hair, and eyes (Frontispiece A–I). The least pigmented normal human subjects have a very fair complexion, almost white (Frontispiece A–C). Their color can be similar to that of an albino. The darkest humans are deep brown or almost black–brown in color (Frontispiece A, H, and I). The latter are found in a few locations such as some areas of the African continent or among the Australian aborigines. Most peoples of the world are moderate brown or yellow–brown, the latter most commonly observed in Asiatic 504

peoples (Frontispiece D–G). The Caucasian peoples of Europe (Frontispiece B and C), a minority in the world’s population, exhibit a light tan color that can be enhanced by exposure to sunlight, i.e. sun tan. The ability to tan is especially marked in the Mediterranean and Middle Eastern peoples. In contrast, those peoples from the western parts of northern Europe and Great Britain have fair skin, red hair (Frontispiece B), and a tendency to form red–brown freckles after exposure to sunlight. This variation in hair, skin, and eye color seems to be related to mutations in the receptor (MC1R) for amelanocyte-stimulating hormone (MSH) (Abdel-Malek et al., 1999; Kadekaro et al., 2003a; Rana et al., 1999; Rees, 2003). The mutations in the MC1R seem to impute a susceptibility to developing melanoma and skin cancers in Celtic peoples (Abdel-Malek et al., 1999; Kadekaro et al., 2003a; Kennedy et al., 2001; Rana et al., 1999; Rees 2003; Sturm 2002). Many Scandinavians (Frontispiece C) with blonde hair and blue eyes tend to burn in spring but seem to tan unexpectedly well during middle and late summer. It has been suggested that mutations in agouti signaling protein, a natural antagonist for MSH, might result in brown eyes and hair (Kanetsky et al., 2002) (see Chapters 3, 19, and 20). Golger in the nineteenth century postulated his principle (Golger’s rule) that individuals living close to the equator had dark skins, and those living far from the equator had lighter complexions. His observation fits reasonably well with the distribution of skin color for denizens of Europe and Africa. However, it does not describe well the distribution of skin color for the original inhabitants of the American continents, Australia, Tasmania, the Orient, or even the southernmost tip of Africa. In these areas, the color of skin tends to rather homogeneous yellow–brown (Orient) to red–brown (the Americas) or dark brown (southern Africa). Many have attributed the homogeneity of skin color of peoples living in the Asian and American continents to the relative isolation of groups in these areas during the last 20 000 years or so. It is thought that man originated several million years ago in Africa around the Olduvai gorge, whence humans migrated to the area around the Middle East crescent now called Iraq, Iran, and north-eastern Africa. Golger’s rule is attractive and provides a simple explanation for the color variations and tanning of skin as a protective device against sunburn. There are recent proposals suggesting that natural selection was a contributing force for dark skin between the tropics of Cancer and Capricorn (Aoki, 2002; Jablonski and Chaplin, 2000). In contrast, sexual selection or preference has been implicated as the mechanism whereby northern Europeans evolved a lightly pigmented skin. But modern data show that the melanocytes and melanin have many other and more important roles than

THE NORMAL COLOR OF HUMAN SKIN

Fig. 27.1. A schematic drawing of normal Caucasoid and Negroid epidermis illustrating melanin within the epidermis. Melanin is the major epidermal pigment.

Fig. 27.2. An epidermal sheet from an African American removed with a dermatome. The membrane is transparent to the underlying newsprint.

simply synthesizing melanin (Nordlund, 1994, 1995). Some of the other theories and functions of melanin are reviewed later in this chapter, and suggest that Golger’s rule might be an oversimplification of a complex biologic phenomenon (Cripps, 1981; Nordlund, 1994, 1995). The color of the skin is determined by a number of chromophores. The most obvious is the pigment melanin. The type of melanin, which is determined by its chemical structure, and quantity of melanin (Fig. 27.1) are major determinants of skin color (see Section 4). Besides the pigment melanin, there are nonmelanin chromophores that also contribute significantly to skin color. The epidermis is a transparent membrane, and much of the appearance of the skin is due to the constituents of the dermis (Figs 27.2 and 27.3). Nonmelanin chromophores, such as oxygenated and reduced hemoglobin (Plate 27.1, pp. 494–495), passing through the papillary capillaries contribute significantly to the color of skin. Increased blood flow in a nevus flammeus (Fig. 27.4) or decreased flow, such as in a nevus anemicus (Plate 27.3, pp. 494–495), alter the skin color markedly. Other dermal chromophores such as

medications (Chapter 54), chemicals such as carotene or lycopenic acid from foods or even licorice (Anttila et al., 1993), collagen (Velangi and Rees, 2001), and elastic fibers alter the appearance of the skin. Capillary engorgement beneath the areolae of a pubescent girl alter the color from tan to almost a green discoloration (Plate 27.1, pp. 494–495). The green hue disappeared with gentle pressure, indicating that the color was vascular. Thus, it is clear that skin color is determined by many factors, i.e. the pigment melanin and other nonpigment chromophores. In this chapter, we shall use the words pigment and pigmentation to refer exclusively to melanin and chromophores to describe all other chemicals and factors that contribute to skin color (see Chapter 26).

Ethnic Skin Color The size of the pigmentary system within the body, i.e. the total mass of all pigment cells, has been estimated to be about 1.5 g (Rosdahl and Rorsman, 1983). The pigment cells are 505

CHAPTER 27

Fig. 27.3. The histology of the epidermis in Figure 27.2 showing that the epidermis and a small amount of the papillary dermis are in the tissue.

Fig. 27.4. A nevus flammeus, a developmental defect that causes engorgement of the papillary plexus. The skin has an orange to bright red color (see also Plate 27.2, pp. 494–495).

distributed mostly in the epidermis (Fig 27.1) and hair follicles, but are also found in the eyes and ears and meninges overlying the pons. Within the epidermis and follicles, it is the quantity and type of melanin, not the number of melanocytes (Garcia et al., 1977; Seiji et al., 1961; Szabo, 1957, 1959, 1967), that is the primary determinant of color (see Chapter 3). The number of melanocytes in the skin of individuals of all races seems to be similar. The capacity of these cells to synthesize melanin, both in a basal state (called constitutive color) and after stimulation (called facultative color) by sunlight varies enormously. The melanocytes in those with dark skins or with the capacity to tan well have a great capacity to synthesize melanin and transfer it into the surrounding keratinocytes (Quevedo et al., 1975; Quevedo, 506

1979) (Chapter 3). Those with fair skin have a very limited capacity. The chemical structure of melanin found in human skin varies from orange (pheomelanin) to black (eumelanin) (Chapters 10, 14, and 15). Eumelanin and pheomelanin are found in individuals of all skin colors (Thody et al., 1991). Each individual cell produces both types of melanin (Hunt et al., 1995; Tobin et al., 1994), but the ratio varies greatly and determines the hue of the skin, i.e. white, yellow tan (Asiatic), red brown (American natives), dark brown, or black (African or Australian) (Frontispiece A–I). Those with dark skin mostly have eumelanin but smaller amounts of pheomelanin. Those with light skin have small amounts of melanin in the epidermis and larger quantities of pheomelanin and a lesser amount

THE NORMAL COLOR OF HUMAN SKIN

Fig. 27.5. S-100 stain of the epidermis that illustrates dendritic melanocytes in the basal epidermis and dendritic Langerhans cells in the mid-epidermis.

of eumelanin. The melanogenic peptide a-MSH stimulates melanogenesis in skin and hair and seems to shift the synthesis from pheomelanin to eumelanin (Abdel-Malek et al., 1999, 2000; Burchill et al., 1991, 1993; Hunt et al., 1995; Lu et al., 1998; Tobin et al., 1994) (Chapters 3, 14, 15, 19, and 20). The receptor for MSH has been identified and cloned (Cone et al., 1993; Magenis et al., 1994; Mountjoy et al., 1992, 1994). There are at least five receptors for MSH found throughout the body (Chapter 19). Only MC1R and MC2R seem to be found in the skin. The MSH receptor MC1R is mutated in those human (Valverde et al., 1995) and nonhuman mammals (Robbins et al., 1993) that produce large quantities of pheomelanin and have red hair or fur. Other molecules such as agouti signaling protein might also be involved in the regulation of human hair and skin color (Kanetsky et al., 2002) (Chapter 19). Hair color ranges from silver blond to black. Less is known about the number of melanocytes in hair bulbs and the relationship to hair color. It is assumed that the same principle is true for hair color as for skin color, namely that hair color is determined by the quantity and type of melanin transferred to the growing hair and not the number of melanocytes within the follicular bulb (Ortonne and Prota, 1993) (see Chapter 3). Generally, northern Europeans have brown hair, but many have red or auburn hair and freckles on the skin. Freckles probably represent mutations in MC1R (Bastiaens et al., 2001). Those with dark skins generally have dark brown or black hair. But there are notable exceptions. Some groups of Irish people have very fair complexions but black hair and dark eyes. This suggests that there are different genes determining the constitutive skin, hair, and eye color (Brues, 1946, 1975; Kanetsky et al., 2002). Eye color in general correlates with skin and hair color (Chapter 4). Those with dark skins have dark brown to black eyes. Those with fair complexions have blue or hazel-colored

eyes. Many Scandinavians typically have fair skin, an excellent capacity to tan after repeated exposure to sunlight, and blue eyes. Small groups in India and Africa with very dark skin have blue eyes (Brues, 1946). These observations suggest that the constitutive color of the skin, hair, and eyes, i.e. the basal color of these organs, is determined by interactive but different sets of genes. Constitutive skin color is defined as the basal or genetically determined color of an individual’s skin that has not been perturbed by any external factor such as sunlight (Quevedo et al., 1975). Facultative color is that which develops following exposure to a stimulant such as sunlight, i.e. tanning (Chapter 3). A functional classification for skin color has been proposed using these two parameters, basal skin color and response to sunlight (Fitzpatrick, 1988; Fitzpatrick et al., 1974). Six categories have been defined, although many observers condense these into four categories. Type 1 skin always burns from exposure to sunlight and never tans. Type 6 is deeply pigmented and never burns following exposure to sunlight. The definition for each skin type is given in Table 27.1. This classification has been used clinically to divide individuals into groups by constitutive and facultative skin color. However, its usefulness as well as its accuracy have been questioned (Cripps, 1981; Rampen et al., 1988; Westerhof et al., 1990). Several investigators have proposed that the skin color as measured by a chromometer is more accurate and useful in predicting the response of a person to sun exposure or to forms of phototherapy (Amblard et al., 1982; Andreassi et al., 1987). The classification of Fitzpatrick is based on the function considered most important for melanin, namely protection against sunlight. However, the many functions of the pigment system are not well understood, although it is clear that it is an important defense against detrimental effects of sunlight. Thus, a classification based on response to sunlight 507

CHAPTER 27 Table 27.1. Functional skin types (Fitzpatrick classification) (Cripps, 1981). Skin type

History

Examples

I

Always burns, never tans

II

Always burns, tans minimally

III IV

Burns moderately, tans gradually Rarely burns, tans well

Celts, Scots; red hair and freckles (Frontispiece B) Blue-eyed Caucasians, Scandinavians (Frontispiece C) (Frontispiece A)

V

Burns minimally, tans well

VI

Never burns, deeply brown

Darker Caucasians (Frontispiece D and E) Mediterranean peoples, Middle Eastern, Latin American, Orientals (Frontispiece F and G) African peoples, Australian natives (Frontispiece A and H)

might not be the best clinical assessment of skin color (Azizi et al., 1988; Cesarini, 1988). New data suggest that melanocytes can perform a wide array of functions. Better classifications based on a more comprehensive knowledge of melanin and the melanocyte are likely to be proposed in the future.

Functions Proposed for Melanin and Melanocytes Photoprotection The most obvious function for melanin is photoprotection (Chapters 3, 17, and 20). Melanin has been considered a molecule found within all layers of the epidermis that has as its main function the absorption of ultraviolet light. Its absorption supposedly prevents the ultraviolet light from striking functional cells such as the Langerhans cell (Figs 27.1 and 27.5), the basilar keratinocytes, or melanocytes and causing a malignant transformation or alteration in their function. This idea was first proposed formally by Everard Home who covered one hand with a black cloth and exposed his hands to intense sunlight (Chapter 1). He observed that the black cloth did make the covered hand feel warm but protected the covered hand from sunburn. The uncovered hand was cooler but burned. He concluded that melanin both absorbed heat but also prevented sunburn. The conclusion has been considered dogma since. Melanin is a sunscreen. Currently, the efficacy of sunscreens is measured as sun protective factor (SPF). An SPF of 2.0 means that it takes about twofold more exposure to a source of ultraviolet light to cause a sunburn than it takes to burn unprotected skin. An SPF of 20 permits the skin to be exposed 20 times longer to ultraviolet light than unprotected skin before burn occurs. Melanin has an SPF that has been calcu508

lated to be about 1.5 to 2.0, possibly as high as 4, that is, it absorbs about 50–75% of the ultraviolet light striking the skin (Ahene et al., 1995; Cesarini and Msika, 1995; Kobayashi et al., 1995; Nordlund, 1995). There is considerable discussion about the efficacy of melanin as a sunscreen (Kligman, 1995; Morison, 1985, 1995; Nordlund, 1995). That melanin is a sunscreen is not debated. The major controversy is whether it is a sufficiently effective sunscreen or is the only mechanism by which darker-skinned individuals are protected from skin cancer (Plate 27.4, pp. 494–495). The debate is focused on whether dark-skinned individuals are protected from the carcinogenic effects of the sun merely by the presence of large amounts of melanin or by a combination of melanin and other poorly described functions of the pigment system within the skin. It seems likely that melanin alone does prevent some sun damage (Hearing, 2000; Kadekaro et al., 2003b; Smit et al., 2001). It is possible that those with darker skin also have an improved ability to repair UV-induced mutations, possibly in response to MSH (Kadekaro et al., 2005). The protective effects of melanin have been studied (Chapter 17). A group of individuals with light or dark skin (skin types I or II and types III or IV) were evaluated before and after a week-long vacation on a sun-drenched island. Skin thickness, parameters of DNA damage, lymphocyte functions and types were measured. The authors found that those with fair skin had evidence of sun damage similar to those with dark, readily tanned skinned (Plate 27.4, pp. 494–495) (BechThomsen et al., 1993). These data suggest that the screening effects of melanin might not be an exclusive mechanism for sun protection. Melanin production and DNA repair are probably closely linked (Gilchrest et al., 1998; Gilchrest and Eller, 1999). Many skin diseases are caused or aggravated by sunlight, particularly exposure to ultraviolet light. Such disorders include lupus erythematosus (Chapter 37), porphyria cutanea tarda (Chapter 51), xeroderma pigmentosum (Chapter 48), pellagra (Chapter 51), and others. It is notable that photoreactive disorders are as common in blacks as in Caucasians (Westerhof et al., 1990). Dark-skinned individuals are as susceptible to photosensitive disorders as Caucasians. In New York City, the incidence of systemic lupus erythematosus was found to be higher in blacks than in whites (Fotiades et al., 1995; Lim et al., 1994; Siegel and Seelenfreund, 1965). Peoples in Africa, India, or African Americans suffer from disorders such as lupus erythematosus, xeroderma pigmentosum, pellagra, and other disorders. This observation suggests that melanin is only a partial sunscreen and permits enough light to penetrate so that it aggravates these light-aggravated disorders regardless of the person’s skin color. The clinical observations are consistent with the measured SPF for melanin. Kligman (1995) studied the skin of black women and found by histological methods that the facial skin of African Americans exhibits significant evidence of photodamage. The dermis has moderate to dense amounts of elastosis extending deeply into the reticular dermis. There were excessive quantities of glycosaminoglycans, another criterion of photodamage.

THE NORMAL COLOR OF HUMAN SKIN

These data clearly indicate that significant amounts of ultraviolet light are passing through the epidermis despite the melanin and causing dermal damage. The striking observation was that the epidermis was normal in appearance. That suggests that the epidermis has methods other than absorption of light by melanin to prevent or repair damage to keratinocytes and melanocytes. It suggests that melanocytes might have other, unidentified ways of preventing or eradicating photodamage. That melanin absorbs only 50–75% of the ultraviolet light might be ideal. The sun does produce beneficial effects within the epidermis. Some ultraviolet light is necessary for the formation of vitamin D (see below). Excessive amounts of ultraviolet might exceed the capacity of the skin cells to repair the damage to DNA. The proper amount might permit the beneficial effects to occur within the limits of the skin’s ability to eradicate the deleterious effects. Considered from this perspective, an SPF of 2 to 4 seems ideal. It also suggests that other mechanisms are active in the skin of black-skinned individuals that prevent skin cancers and other sun-related disorders. Other controversies are extant about the function of melanin. Most observers consider melanin to be the perfect sunscreen. However, those who have studied melanin have noted that it has some toxic products as well, particularly after it is exposed to ultraviolet light. It seems to degrade into oxygen radicals capable of injuring melanocytes and keratinocytes or other cells (Cesarini, 1988; Hill, 1992; Johnson et al., 1972). Pheomelanin is especially susceptible to photodegradation (Chedekel, 1982; Chedekel et al., 1977, 1978; Koch and Chedekel, 1986, 1987; Liu and Chedekel, 1982; Poh Agin et al., 1980). As an oxygen radical, it might cause mutations in the melanocyte or other cells (Harsanyi et al., 1980). There is melanin in the stria vascularis of the cochlea within the inner ear and in the retinal pigment epithelium and choroid of the eye (Chapter 4). The melanin in these organs has other proposed functions. The melanin might chelate ototoxic (Conlee et al., 1989, 1995; Debing et al., 1988; Eastman et al., 1987; Gratacap et al., 1989; Koneru et al., 1986; Schweitzer, 1993) or oculotoxic drugs (Akeo et al., 1992; Loschmann et al., 1994; Mason, 1977; Zemel et al., 1995) and prevent damage to these organs.

The Melanocyte System and Vitamin D The epidermis is the normal site for the synthesis of vitamin D in the human. The vitamin is derived from dehydrocholesterol stored in the epidermis. It undergoes spontaneous conversion to vitamin D following exposure to ultraviolet light. From the epidermis, the vitamin D is transported first to the liver and then to the kidneys where it is hydroxylated into the active 1,25 dihydroxy vitamin D3 (Anonymous, 1984, 1989; Boyle, 1980; Brazerol et al., 1988; Holick, 1981, 1985; Holick et al., 1981; Neer, 1975; Ranson et al., 1988). It was proposed in the past (Clemens et al., 1982; Loomis, 1967) that melanin was necessary to prevent excessive production of vitamin D in

the skin. Hypervitaminosis D is a toxic condition caused by excessive quantities of the vitamin in the body. Vitamin D determines the rate of absorption of calcium into the blood and metabolism in vital organs. Excessive absorption can be lethal. However, many studies have documented that the production of vitamin D following exposure to standard quantities of ultraviolet light is quantitatively similar in individuals of all colors (Lo et al., 1986; Matsuoka et al., 1990, 1991). That is, the skin color has no influence on the quantity of vitamin D formed in the epidermis and released into the circulation (Holick, 1981; Holick et al., 1981). Unnecessary, excessive quantities of precursors of vitamin D generated following sun exposure are apparently stored in an inactive form in the epidermis until needed, presumably during dark, winter months. Melanin is not needed to protect the individual from excessive vitamin D or absorption of calcium. Nor are darkskinned individuals more prone to disorders of calcium deficiency. African American women have the same bone mass as Caucasian women (Nelson et al., 1988). Osteoporosis is not more prevalent in dark-skinned women than in white women (Nelson et al., 1993). Melanocytes do have receptors for vitamin D3 (AbdelMalek, 1988; Abdel-Malek et al., 1988). It has been shown that vitamin D3 enhances the formation of melanin and that vitamin D3 deficiency can abrogate the ability of the skin to tan in response to sun exposure.

The Melanocyte and Photolysis It has been proposed that melanin has the ability to protect individuals from other deleterious effects of exposure to ultraviolet light. Folic acid is degraded by ultraviolet light (Branda and Eaton, 1978). During times of famine a deficiency of folic acid related to exposure to sunlight would be an evolutionary disadvantage. It has been proposed that melanin minimizes the photolysis of folic acid (Aoki, 2002; Jablonski and Chaplin, 2000).

Melanin and Susceptibility to Environmental Irritants Another hypothesis for the function of melanin is its potential ability to protect against environmental toxins. This proposal has been as controversial as other hypotheses for the importance of melanin (Berardesca and Maibach, 1989). The stratum corneum of black skin has the same number of layers as white skin but seems to be more resistant to tape stripping, an observation that suggests that the stratum corneum of black skin is more compact and adherent (Weigand and Mershon, 1970). Other studies suggest that the electrical impedance of black skin is greater than that of white skin, an indicator of greater hydration (Johson and Corah, 1960). Other physiologic or electrical measures have been noted to differ in black and white skin (Korol and Kane, 1978; Korol et al., 1975) although the significance is not known (Deleo et al., 2002; Taylor, 2002). Studies have been done to determine whether black skin was more resistant to irritation (Weigand and Mershon, 1970) or 509

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contact allergens (Leyden and Kligman, 1977). The results are not consistent (Berardesca, 1994; Berardesca and Maibach, 1988; Rostenberg and Kanof, 1941). Some have found black skin to exhibit a similar response to white skin; others have found a lower sensitivity in black skin. Others have measured transepidermal water loss in normal skin (Wilson et al., 1988) or following application of irritants such as sodium lauryl sulfate, the active ingredient in most soap. Normal black skin seems to be have a twofold greater transepidermal water loss than white skin. The significance of this difference is not known. In another study, black skin showed a much greater water loss than white skin following irritation with this chemical (Berardesca, 1994; Berardesca and Maibach, 1988). Others have found that white skin was more susceptible to irritation (Agner, 1991; Maurice and Greaves, 1983). At this time, probably the only valid conclusion from these various results is that white and black skin do not exhibit identical properties.

Other Theories about Melanin Wasserman in the 1960s suggested that melanin was a mechanism to protect African natives from infections. He observed that melanin could be found in circulating macrophages. The circulation of melanin, he proposed, enhanced the function of the reticuloendothelial system (Wasserman, 1974). He proposed that the augmented phagocytic function of the reticuloendothelial system eliminated a variety of pathogens from the blood.

Other Functions for Melanocytes It is now known that melanocytes can produce many other factors besides melanin. They produce proopiomelanocortin and MSH (Fig. 27.6) (Bhardwaj and Luger, 1994; Schauer et al., 1994). They have also been shown to produce and respond to such cytokines as interleukin (IL)-1, IL-6,

tumor necrosis factor (TNF)a (Swope et al., 1989, 1994), and others (Chapter 21). They also are capable of synthesizing and responding to prostanoids such as prostaglandin E (PGE) and leukotrienes (Abdel-Malek, 1988; Abdel-Malek et al., 1992; Medrano et al., 1993). These data suggest melanocytes might participate in the inflammatory response. Such a role would explain the numerous pigmentary changes observed clinically in skin that has been inflamed by any mechanism (Abdel-Malek et al., 1988; Medrano et al., 1993). Skin substitutes grafted on to nude mice contract very markedly. Those containing melanocytes do not contract, suggesting a role for the melanocyte in healing (Boyce et al., 1993).

Depigmented Skin and Its Function It is well documented that skin devoid of melanocytes, specifically skin affected by vitiligo, has both morphologic and functional defects (Chapter 30). The skin is devoid of one of three main cell types, the melanocyte. Such skin is resistant to irritation and contact sensitization (Plate 27.5, pp. 494–495) (Allende and Reed, 1964; Fregert et al., 1959; Gilhar et al., 1993; Haji-Abrassi et al., 1981; Halter, 1941; Hatchome et al., 1987; Kreibich, 1921; Lisi and Caraffini, 1985; Maurice and Greaves, 1983; Nordlund et al., 1985a; Singh et al., 1987; Srinivas et al., 1987; Uehara et al., 1984) (Plate 27.5, pp. 494–495). In addition, when irritated, the inflammatory response of vitiliginous skin is different histologically from that of normal skin (van de Kerkhof and Fokkink, 1989; Westerhof et al., 1989). These data strongly suggest that the melanocytes have a role in many normal functions of the epidermis and are not merely factories for the production of melanin.

Conclusions It is clear that the melanocyte and melanin are important in protection from the deleterious effects of sunlight. But the role of melanin and the functions of melanocytes are probably

Fig. 27.6. Epidermal sheet showing dendritic melanocytes stained with an anti-MSH antibody. The experiment indicates that MSH or its precursor proopiomelanocortin is synthesized in the epidermis, particularly the melanocytes.

510

THE NORMAL COLOR OF HUMAN SKIN

Fig. 27.7. Graph demonstrating a decrease in the population density of epidermal melanocytes with age.

much more complex than previously thought (see Part I on the physiology of the pigmentary system). What seems most obvious is that melanin is not a simple sunscreen, nor are the melanocyte factories located in the skin merely for the synthesis of melanin. The various ideas about potential roles for melanin and the implications for variations in response of skin of different colors to irritants probably have some validity. But much more work needs to be done to determine correctly the functional differences between heavily melanized skin and lightly melanized skin and the genetic basis responsible for these morphologic and functional differences.

The Effect of Age on Skin Color It is generally thought that skin color is fairly constant throughout the life of the individual. Most observers believe that skin color tends to lighten with age (Fig. 27.7). But, in reality, the color of the skin shows considerable variation throughout the life of the individual (Chapter 3). The changes are not dramatic but are measurable with a chromometer, and are remarkably reproducible in many different populations studied. The importance of these changes is related more to the biology of the pigmentary system than to the appearance of the individual or the popular notions about color.

Premature Infants The pigmentary system is active early in gestation (Chapters 3, 5, and 6). In the retinal pigment epithelium within the eyes (Chapter 4), melanin formation begins a few weeks after conception. Pigment is formed in the skin in small quantities during the middle of the second trimester (Fein and Nordlund, 2003). Virtually all prematurely born babies of African

descent of 24 weeks’ gestational age had an abundance of dark brown to black scalp hair (Heyl, 1986). Much of this falls out and is replaced by hair that is less pigmented. However, the follicular melanocytes must begin producing significant quantities of melanin within the second trimester. Premature infants also exhibit several or many sacral spots (Chapter 52) located mostly over the lumbosacral spine or the buttocks, although similar lesions have been observed on the extremities (Heyl, 1986). It is thought these represent migrating melanocytes in transit from the neural crest to the ultimate home in the epidermis. They are visible as blue dermal macules because there is abundant melanin within the cytoplasm of the cell. The genitalia and perineum are also hyperpigmented in most of these premature babies (Heyl, 1986). The scrotum and penis are both hyperpigmented in most males (Plate 27.6, pp. 494–495), although one or the other can be hypopigmented (Plate 27.6, pp. 494–495). The labia majora, minora (Plate 27.7, pp. 494–495), and clitoris are often pigmented in females of African origin (Plate 27.7). In two sets of dizygotic twins, a set of males and a set of females, one of the twins had albinism. The first manifestation was the complete absence of melanin from the genitalia and perineum (Plate 27.6, pp. 494–495) (Chapter 31). The absence was easily detected by comparison of the child with albinism with the normal twin. About one-quarter of premature infants have hyperpigmentation around the paronychial area of the fingers and toes. This disappears as the child matures (Heyl, 1986). Its etiology and significance are not known. A few premature children had a café-au-lait spot (Heyl, 1986). The cause of café-au-lait spots is not known (Chapter 45). In other studies, preterm babies were studied with a reflectometer. Infants ranged from 25 to 44 weeks’ gestation at the time of birth (Post et al., 1976). At birth of infants less than 32 weeks’ gestation, Caucasian and black children were found to have similar skin color as measured by reflectometry. After 32 weeks’ gestation, black children have a darker skin than whites at birth. Males of both races at all gestational ages consistently had a slightly darker skin than females, although the differences were not statistically significant (Grande et al., 1994; Post et al., 1976; reviewed by Fein, 2003). However, the difference might be real as the same observation has been made by many observers on children and adults, i.e. male skin tends to be slightly darker, as measured by a reflectometer, although the differences are small. These points are discussed below. The forehead of all infants is the most pigmented, the inner arm moderately pigmented, and the skin over the sternum the lightest in color (Grande et al., 1994; Post et al., 1976). Infants of dark-skinned parents born near or at term will develop a fully melanized epidermis within 1–2 weeks of gestation.

Skin Color during Childhood and Adolescence Melanocytes have different capacities to produce melanin during the life of an individual. The number of factors intrinsic 511

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and extrinsic to the individual that are responsible for variations in skin color is large (Jaswal, 1983; Roberts and Kahlon, 1972, 1976). Age itself seems to be one determinant. Melanocytes obtained from infants and from adults undergoing circumcision show very different growth and behavioral patterns in culture (Abdel-Malek et al., 1994). Studies in which color was measured by reflectometry in children and adolescents show that skin color for boys and girls is constant until puberty. Just prior to puberty (9–10 years), for several years, girls tend to become moderately darker than they were as children or will be as adolescents (Frost, 1988; Jaswal, 1983; Kahlon, 1976). This observation suggests that hormonal changes associated with the onset of puberty enhance pigment production (Kahlon, 1976; Kalla, 1969a, 1973, 1974). Males show a similar but less pronounced tendency to darken at the onset of puberty, which is about 2 years later than in females (Jaswal, 1983). After the onset of puberty, the skin of both sexes tends to become lighter and continues to lighten into early adulthood (Frost, 1988; Kalla, 1969b, 1973; Rebato et al., 1993; Tiwari and Calla, 1969). Adults over 20 years are lighter than children or teenagers. These changes are measurable but subtle and rarely noticed by casual observers.

Skin Color in Adults After the age of 20 years, reflectometry studies confirm that adults tend to be lighter in color than younger people (Cartwright, 1975; Wienker, 1979). Histologic studies confirm that younger adults, less than age 50 years, have a higher population density (melanocytes/mm2) of melanocytes than those over age 50 years (Montagna and Carlisle, 1990; Ortonne, 1990; Scheibner et al., 1983) (Fig. 27.7). It has been estimated that, after age 25–30 years, the population density of melanocytes decreases by about 10% per year (Nordlund, 1986; Ortonne, 1990) (Fig. 27.7). Morphologically, the melanocytes of older individuals are larger, stain less intensely with the dopa reaction (a measure of tyrosinase activity), and are more dendritic (Nordlund, 1986). The number of melanosomes in older skin is fewer, another indicator that the capacity of melanocytes to produce melanin is decreased (Montagna and Carlisle, 1990). Some of these changes might represent alterations related to sun exposure, a process labeled photoaging (Chapter 17).

Aging of Melanocytes in Other Locations Hair color also changes. During the later part of the third decade (age 20–30 years), gray or white hair begins to appear in most individuals. The number of hairs that lose some or all pigment increases with each decade. By the seventh decade, many individuals are totally gray or white (Fig. 27.8) (Nordlund, 1986; Ortonne, 1990; Ortonne et al., 1983). There are data to explain the loss of hair color. The hair bulb has two populations of melanocytes, differentiated melanocytes containing melanin in the bulb and a second population of Mitfpositive melanocyte stem cells that contain no melanin found in the niche area (Nishimura et al., 2005; Steingrimsson et al., 512

Fig. 27.8. White hair indicating a complete absence of follicular melanocytes (see also Plate 27.8, pp. 494–495).

2005). The stem cells are responsible for maintaining the differentiated population of cells in the bulb. Transgenic mice that have a nonsense gene for Bcl 2 -/- that inhibits apoptosis begin to gray during their first molt (Nishimura et al., 2005; Veis et al., 1993). The bulbar melanocytes disappear at the first molt. After the second molt, the melanocyte stem cells disappear (Nishimura et al. 2005). Wild-type or Bcl 2 -/+ mice have normal colored hair. Examination of hair bulbs from humans show a progressive loss of melanocyte stem cells from the niche as a function of age. By 70 years of age, all stems cells are absent from human hair follicles. The niche cells and apoptosis might be factors that are involved in normal graying of hair. Nevocellular nevi are derivatives of melanocytes (Chapter 56). At birth, less than 1% of children have a nevus on their skin. However, around 6 months of age, tiny, pinpoint nevi begin to appear. The cause is not known, although most observers attribute this to sun exposure. However, in vitro studies suggest that mitogens such as leukotrienes can produce nevus-like structures (Medrano et al., 1993). Leukotrienes are produced in the epidermis during all forms of inflammation. The origin and cause of nevi remain unresolved. Nevi increase in number from birth to mid-life. They become more prominent at puberty and increase with age in

THE NORMAL COLOR OF HUMAN SKIN

number and size. Around the fifth decade of life, nevi begin to disappear (Nordlund, 1986; Nordlund et al., 1985b; Ortonne, 1990). Nevi within the oral cavity also exhibit a similar pattern of development and disappearance. Nevi become more numerous in the oral cavity from birth through the fourth decade of life, after which they tend to disappear (Buchner and Hansen, 1980). Melanocytes in the retinal pigment epithelium and choroid (Chapter 4) also seem to decrease beginning around the fifth decade of life (Kornzweig, 1979). The loss of cells begins in the periphery of the retina and proceeds toward the center. It rarely interferes with vision. However, macular atrophy, possibly a manifestation of destruction of the retinal pigment epithelium in the foveal area, is responsible for blindness in some younger adults. Melanocytes seem to disappear from the epidermis, nevi, hair follicles, and the eyes around the same time, the fifth decade of life (age 40–50 years). This suggests that melanocytes throughout the body are undergoing senescence and selfdestruction, possibly by apoptosis (Veis et al., 1993), although the actual mechanism has not been determined. Although these changes are subtle and not immediately observable to the individual, they have significance for the biologist trying to understand the function of the pigmentary system.

Sex, Hormones, and Skin Color Skin Color in Males and Females Numerous anthropologic studies have been done to study the origins of groups of people. The skin colors of many small tribes and isolated groups of peoples have been studied with reflectometry. The usual areas assessed are the forehead and the upper, inner surface of the arm. It has been observed repeatedly that females are significantly darker than males just prior to the onset of menarche (Frost, 1988; Jaswal, 1983; Kahlon, 1976). Other than at this time, numerous reports indicate that females are slightly lighter than males (Cartwright, 1975; Clark et al., 1981; Drusini and Tommaseo, 1981; Fregert et al., 1959; Frost, 1988; Jaswal, 1983; Kalla, 1969b, 1973, 1974; Mehrai and Sunderland, 1990; Rebato, 1987; Roberts and Kahlon, 1972; Williams-Blangero and Blangero, 1991). The differences measured by the reflectometry instruments were often minimal and typically not statistically different between the males and females. However, the consistency of the findings suggests that adult females are indeed slightly lighter than males. That this might be a correct observation has been suggested by the tendency of portrait artists to portray the female body with a lighter color than males (Tegner, 1992). However, the latter observation might reflect the propensity of males to consider females with fair complexions more attractive.

Fig. 27.9. Linea nigra, a manifestation of hormonal stimulation of melanogenesis in a pregnant woman (see also Plate 27.9, pp. 494–495).

the vulva, perineum, and anal areas all become darker (Wong and Ellis, 1989). Many women develop chloasma or pregnancy mask (Chapter 53) that typically disappears or lightens following the birth of the infant. The linea nigra (Fig. 27.9), a line from the xiphoid process to the pubic symphysis, darkens in many women, especially those with dark complexion (Esteve et al., 1994) (Fig. 27.9). Nevi (Chapter 56) and freckles (Chapter 50) have also been noted to darken during pregnancy (Wong and Ellis, 1989). Darkening of these same areas has been noted to occur with the onset of menses in some women (Snell and Turner, 1966). These changes have been attributed to the increased secretion of estrogens and progesterone, a conclusion supported by the observation that these changes have been associated with the taking of birth control pills or estrogens for any reason (Abdel-Malek, 1988; Martin and Leal-Khouri, 1992). Receptors for these steroidal hormones have been identified in normal and malignant melanocytes (Ebling, 1986; Jee et al., 1994; McLeod et al., 1994; Stoica et al., 1991).

Skin Color and Pregnancy

Pigmenting Hormones

It is well known that, during pregnancy, women exhibit a darkening of various areas of the skin. The nipples, areolae,

The melanocyte is highly responsive to MSH (Chapters 19 and 20). The role of MSH in human pigmentation is not 513

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known, although all observers consider it to be a major determinant of some changes in skin color in response to various external stimuli. It is synthesized by both melanocytes and keratinocytes and, in mice, by epidermal lymphocytes (Amornsiripanitch and Nordlund, 1989; Bhardwaj and Luger, 1994; Farooqui et al., 1995; Swope et al., 1989). Agouti signaling protein seems to block the effects of MSH and seems to be important in determining skin color. These observations are discussed in great detail in Part I of this book. Melatonin is a pineal agent thought to be responsible for lightening the color of amphibians and other animals such as the weasel. It is secreted by the pineal gland in response to ambient sunlight. During summer, melatonin formation is suppressed; during winter, it is produced and released and alters secretion of a-MSH by the pituitary (Axelrod, 1974; Cassone et al., 1993; Cavallo, 1993; Dubocovich, 1995; Hastings, 1991; Kral, 1994; Maestroni, 1993; Molina Boix et al., 1992; Morgan et al., 1994; Reiter, 1991a, b; Silman, 1992; Webb and Puig-Domingo, 1995). It does not seem to have any effect on basal skin color if administered by mouth over many months (McElhinney et al., 1994). Administration of melatonin to one patient with congenital adrenogenital syndrome seemed to lighten his skin color (Nordlund and Lerner, 1977). These observations suggest that basal skin color might not be affected by pituitary release of a-MSH. However, a pituitary gland releasing excessive amounts of a-MSH because of an adrenal defect might respond to melatonin.

Pigmentary Demarcation Lines The color of skin exhibits variation in hue and intensity at various sites of the body. There are some areas on which pigmentary lines called demarcation lines are observed frequently. The origin and significance of these lines are not known.

Types of Demarcation Lines Six types of demarcation lines (Chapter 47) have been described (James and Carter, 1987). These are defined in Table 27.2. Type A or Futcher’s lines are the best recognized demarcation lines (Plate 27.10, pp. 494–495). Futcher (1938, 1940) first described a sharp, easily discernible line on the upper

aspect of the arm (Plate 27.10, pp. 494–495). They are striking by their precise lineation, the sharp contrast in depth of color between the lateral and medial side of the arm. These lines are most obvious in dark-skinned individuals (Matzumoto, 1913; Miura, 1951; Vollum, 1972). Age and sex seem to have no relationship to the presence or absence of these lines, although the lines have been reported to be most common in Japanese women (Ito, 1965). The lines usually begin at the shoulder and fade just above the condyles of the humerus. They have been observed in up to 38% of blacks and Hispanics (Selmanowitz and Krivo, 1975). Type B lines located on the inner surface of the thigh seem to be more common in Japanese women (Matzumoto, 1913). During pregnancy, they were noted to be markedly enhanced in several Hispanic women (Vazquez et al., 1986). Post partum, the lines faded spontaneously. Type C lines are located over the sternum (Plate 27.11, pp. 494–495). They are hypopigmented lines that can be linear or oval in shape. As many as a third of African Americans were observed to have such hypopigmented macules (Kisch and Nasuhoglu, 1953). These macules have been observed in Japanese (Miura, 1951). Type D lines are pigmented lines located over the posteromedial area of the spine. They have been observed in Japanese but not in other groups (Miura, 1951). Type E lines are common hypopigmented macules scattered along a line that extends from the lateral head of the clavicle to the areola. They are often asymmetric in size, shape, and distribution. Careful examination of the chest will reveal that these are present relatively frequently. They are often confused with tinea versicolor, post-inflammatory hypopigmentation, or early vitiligo. The individual will note that the macules have been present without change for many years. Type F lines are found on the face and were described recently (Chapter 47). None of these lines seems to follow a distribution of sensory nerves (Voigt’s lines). Their etiology or mechanism for their appearance is not known. The mechanisms responsible for their presence might be active in Caucasians. However, it would be most difficult to discern these lines even with a Wood’s lamp in an individual with very fair skin.

Normal Pigmentary Spots Table 27.2. Pigmentary demarcation lines (James and Carter, 1987). A

B C D E F

514

Located on the lateral aspect of the anterior portion of the upper arm (Futcher’s line). Can extend to the pectoral area (Plate 27.10, pp. 494–495). Located on the posteromedial portion of the leg (Fig. 27.10). Hypopigmented lines in the pre- or parasternal region (Plate 27.11, pp. 494–495). Located on the posteromedial area of spine. Hypopigmented macules located on the chest extending from the clavicle to the periareolar skin. Lines on face.

The skin of all individuals is marked by an array of pigmented spots. Because these spots are so common, they could be considered normal. Nevocellular nevi (Chapter 56) are pigmented spots presumably derived from melanoblasts. They are found on virtually all individuals, although members of some kinships seem to exhibit especially large numbers of nevi. It has been noted that individuals of Celtic inheritance are more likely to have a large number of nevi on their integument than those with dark skin (English et al., 1987; Gallagher et al., 1990; Rampen et al., 1986; Sigg and Pelloni, 1989).

THE NORMAL COLOR OF HUMAN SKIN

Fig. 27.11. Freckles (see also Plate 27.12, pp. 494–495).

Fig. 27.10. The Hispanic woman developed this hyperpigmented line on the posterior thigh during a pregnancy, typical of type B demarcation lines.

Fair-skinned individuals (Frontispiece B, C), especially those whose ethnic background is Celtic, are prone to have freckles (Fig. 27.11). These are found only in sun-exposed skin, become darker when exposed to the sun, and fade during the winter months. Other hyperpigmented spots commonly found on the skin are the nevus spilus and the café-au-lait spot (Chapters 45 and 56). The nevus spilus is defined as a tan-colored macule with dark macules and papules scattered over the lesion. The lesion seems to represent a café-au-lait spot with junctional nevi within its confines. They have been observed more frequently in fair-skinned children. Their prevalence has been suggested to be around 2% (Sigg and Pelloni, 1989). Café-au-lait or coffee-colored spots are found normally on many individuals. Their prevalence is not known with certainty. It has been stated that Caucasian children have an average of 0.7 café-aulait spots; blacks have an average of two or three. In one study, 32.5% of children were found to have a café-au-lait spot (Sigg and Pelloni, 1989). Congenital nevi are found on about 1% of all neonates. Most are small but a few are large, and some cover most of

the integument and are called bathing trunk nevi. They can be located on any part of the body. It is of biologic interest and importance that, when these nevi are located on the breasts or genitalia, the melanocytes avoid the areolae and the penis. Similarly, when found on the buttocks overlying a sacral spot, the two types of melanocyte seem to repel each other. It has been suggested that melanocytes are territorial and repel each other to avoid direct contact (Rosdahl and Rorsman, 1983). This unusual clinical phenomenon seems to confirm that normal melanocytes have a tendency to shun each other and can repel nevus cells as well. Hypopigmented macules often called ash-leaf spots are occasionally found on the integument [see Fig. 32.21 (see also Plate 32.13, pp. 494–495) and Fig. 32.22 (see also Plate 32.14, pp. 494–495)]. One or two such lesions have no significance. More might be the first sign of tuberous sclerosis. Some areas of skin have a hypopigmented spot because of decreased vascular flow through the papillary plexus (Chapter 42). Such lesions are called nevus anemicus (Plate 27.3, pp. 494–495). They are thought to be caused by an excessive sensitivity of the capillaries in the papillary dermis to catecholamines. They are typically located on the shoulder area, on either the ventral or the dorsal surface.

The Normal Color of Mucosal Surfaces and Epidermal Cysts The oral cavity is formed by an invagination of the ectoderm. Typically, the number of melanocytes in the mouth is the same as on the skin, about 600–800/mm2. The oral mucosa is pigmented in individuals with dark skin. Although most individuals consider the normal color of the mouth to be pink, that is incorrect. Most of the peoples of the world have type IV to type VI skin. The oral mucosa of most of these individuals has a gray–brown color (Dummett et al., 1980). The vaginal mucosa is also formed from an invagination of the ectoderm during embryogenesis. The labia minora and the vaginal mucosa are typically pigmented in individuals with dark skin. The mucosa of the mouth and genitalia exhibits 515

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various abnormalities described in another chapter (see Chapter 55). Epidermal inclusion cysts are common. Those removed from dark-skinned individuals are often, but not always, moderately to deeply pigmented (Fieselman et al., 1974).

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

28

Mechanisms that Cause Abnormal Skin Color Jean-Paul Ortonne and James J. Nordlund

Introduction Normal human skin color ranges from white or pink to tan, dark brown, or black (Frontispiece A–I; Fig. 28.1). Within any ethnic group, there is a marked variation in skin and hair pigmentation. The components of normal human skin color include brown hues produced by melanins, red and blue related to oxyhemoglobin and deoxyhemoglobin, respectively, and yellow due to carotene (Mosher et al., 1993). Combined variations of cutaneous melanins and hemoglobins account essentially for the entire range of normal human skin colors (Anderson, 1986).

Classification and Pathophysiology From the visual evaluation of patients with disorders of skin color, three clinical alterations have been described: (1) a darkening of the skin; (2) a lightening of skin; or (3) the presence of an unusual skin color. These distinctive changes in skin color may result from abnormally high or low concentrations of major (melanins and hemoglobins) or minor (carotene) normal skin pigments, or from abnormal occurrence of endogenous or exogenous pigments giving an unusual skin color. Much of the skin color comes from the constituents of the dermis, the rest from melanin and the epidermal cells, i.e. stratum corneum and stratum Malpighii. Alterations in the epidermal morphology or the constituents of the dermis cause changes in the color of the skin. The epidermis is more translucent than is commonly recognized. Figure 27.2 in Chapter 27 shows that black epidermis removed with a dermatome is transparent to the underlying newsprint. Histology (Fig. 27.3) confirms that this transparent membrane is composed of epidermis and traces of the dermis. These observations support the concept that the color of the skin is determined by dermal components modified somewhat by epidermal melanin and the morphology of the stratum corneum.

Skin Darkening Darkening of the skin may be the result of four different pathophysiologic processes: (1) abnormal amounts of melanin; (2) abnormal distribution of normal skin pigments such as melanins and hemoglobins; (3) the presence in the skin of endogenous or exogenous pigmented substances abnormally located in the skin; or (4) epidermal thickening (Chapter

26). Only the first two processes involve melanin and are labeled melanin-related disorders. The last two mechanisms alter skin color but do not involve the pigmentary system or melanin.

Melanin-related Disorders An increased amount of melanins in the skin is called hypermelanosis. Two types of hypermelanosis are described according to the clinical color of the skin. Brown hypermelanosis is caused by excessive quantities of melanins within the epidermis. Blue hypermelanosis, which is also called ceruloderma, is produced by large amounts of melanins in the dermis (Fitzpatrick et al., 1981a). Mixed hypermelanosis is characterized by an excess of melanins in both the epidermis and the dermis. The latter conditions exhibit a blue, blue–black, gray, or gray–brown color giving the skin a dirty appearance. Brown Hypermelanosis Brown skin hues result directly from the absorption spectrum of epidermal eumelanins (Kollias, 1995). Most of the visible light backscattered from the skin is due to dermal scattering. This light traverses the epidermis twice, once in penetrating to the dermis and again on returning to the skin’s surface. Visible light is absorbed by the melanins in the epidermis. White light reflected from light-colored skin appears yellow–brown. Black skin absorbs much of the light and appears brown or black. The excess of melanins in brown hypermelanosis may be due to increased melanin production either by an abnormally high number of melanocytes (Fig. 28.2) or by a quantitatively normal melanocyte population of the skin (Fig. 28.3) (Mosher et al., 1993). These abnormalities can result from both acquired and genetic factors. Epidermal Melanocytic Hypermelanosis These lesions are characterized by increased numbers of epidermal melanocytes that are producing excessive quantities of melanin within the epidermis. Commonly, the epidermal morphology is also hyperplastic. Lentigines (Fig. 28.2) (Chapter 46) are the prototype of melanocytic epidermal hypermelanosis accompanied by epidermal hyperplasia. Histologically, they are characterized by an increased number of melanocytes producing an excessive amount of melanin in the epidermis and prolongation of the epidermal rete pegs (Fig. 28.2). Epidermal Melanotic Hypermelanosis In these lesions, the number of epidermal melanocytes is normal. Hypermelanosis 521

CHAPTER 28 Normal epidermis

Hyperpigmentation Negroid

Caucasoid Corneocytes

Normal

Langerhans cells Keratinocytes Melanosomes Melanocytes

Keratinocytes Melanosomes Melanocytes

Basal lamina

Basal lamina

Dermis

Dermis

Fig. 28.1. Normal human epidermis. The number of melanocytes in Caucasoid and Negroid skin is similar. However, the number and size of melanosomes is significantly greater in heavily pigmented epidermis.

Hyperpigmentation

Corneocytes Langerhans cells Keratinocytes Melanosomes Melanocytes Basal lamina Dermis

Fig. 28.2. Epidermal melanocytosis. The hyperpigmented skin exhibits elongation of the rete pegs, a process called acanthosis. As a result, the epidermis has an abnormally high number of melanocytes that are producing an excessive amount of melanins. There is no pigment in the dermis. Clinical examples include lentigines. A drawing of normal skin is provided for comparison.

Hyperpigmentation Normal

Fig. 28.4. Dermal melanosis. This type of hyperpigmentation is caused by melanin within the dermis, between bundles of collagen, or within melanophages (clear cells). The epidermal melanins are normal. Clinical examples include fixed drug eruption, incontinentia pigmenti, lichen planus, and many forms of post-inflammatory hyperpigmentation. A drawing of normal skin is provided for comparison.

Acanthosis

Normal

Epidermal melanosis

Corneocytes Langerhans cells Keratinocytes Melanosomes Melanocytes Basal lamina Dermis

Fig. 28.3. Epidermal melanosis. The hyperpigmented skin shows only excessive quantities of melanins, but a normal number of melanocytes. Clinical examples include freckles, café-au-lait spots, and urticaria pigmentosa. A drawing of normal skin is provided for comparison.

results from increased epidermal melanins secondary either to their increased synthesis or to their decreased degradation and/or elimination (Fig. 28.3). Café-au-lait macules are the typical example of epidermal, melanotic hypermelanosis. The pathogenesis of café-au-lait spots is unknown, but most likely involves different factors regulating melanogenesis. Café-aulait spots are one of the hallmarks of neurofibromatosis type 1 (Chapter 45). Neurofibromatosis type 1 is caused by mutations in the neurofibromatosis type 1 gene. Neurofibromin, the product of this neurofibromatosis type 1 gene, contains a domain that is related to the GTPase activating protein, which 522

Dermal melanosis

Corneocytes Langerhans cells

in turn inactivates ras-GTP complexes (Andersen et al., 1993). Thus, neurofibromin is likely to function as a regulator of cell growth and differentiation (Bollag et al., 1993). Basal keratinocytes express type II neurofibromin, and RNA transcripts have been demonstrated in cultured keratinocytes (Hermonen et al., 1995). This suggests that the pigmented café-au-lait spots may result from a combined dysfunction of both melanocytes and keratinocytes. Giant melanin granules are often present in both melanocytes and keratinocytes of caféau-lait spots in neurofibromatosis type 1, but they are not specific to this disorder. Café-au-lait spots are a prominent feature of polyostotic fibrous dysplasia (McCune–Albright syndrome; Chapter 45). The pattern of these skin lesions follows lines of embryologic ectodermal migration. The obvious clinical genetic transmission suggests that this disorder is caused by a postzygotic lethal mutation that survives by mosaicism. Mutations in the gene encoding the alpha subunit of the stimulatory G protein of adenylate cyclase have been identified in this disorder (Schwindinger et al., 1992). Although the pathogenesis of café-au-lait macules in polyostotic fibrous dysplasia has not been established, it is possible to speculate that an activating Gs alpha mutation arising during embryogenesis would give rise to groups of melanocytes that produce increased amounts of melanins. It is conceivable that the binding of melanocytestimulating hormone (MSH) to the MSH receptors of melanocytes, in which mutations lead to constitutive activation of adenylate cyclase, will result in increased melanin production (Schwindinger and Levine, 1993). Some acquired brown hypermelanoses are induced by UV light, heat, hormones, and chemical compounds. Ceruloderma Blue skin or ceruloderma results from the optical effects of melanins or any other dark substance located in the dermis (Kollias, 1995). Normally, the dermis has no melanin and appears yellow, white, or pink, depending on its blood content. Dermal melanin (see Figs 28.4 and 28.5) has a broad absorption spectrum. Dermal melanin absorbs incident visible

MECHANISMS THAT CAUSE ABNORMAL SKIN COLOR Hyperpigmentation Normal

Dermal melanocytosis

Corneocytes Langerhans cells Keratinocytes Melanosomes Melanocytes Basal lamina Dermis

Fig. 28.5. Dermal melanocytosis. This type of hyperpigmentation is caused by ectopic melanocytes located in the dermis. The epidermal melanin is normal. Such a lesion would have a blue or gray–blue color. Clinical examples include sacral spots, nevus of Ito, and nevus of Ota. A drawing of normal skin is provided for comparison.

Hyperpigmentation Normal

Non-melanin pigmentation

Corneocytes Langerhans cells Keratinocytes Melanosomes Melanocytes Basal lamina Dermis

Fig. 28.7. Hyperpigmented lichen planus. The pathologic features of a biopsy taken from a lesion would resemble Figure 28.4 (see also Plate 28.1, pp. 494–495).

Fig. 28.6. Hyperpigmentation caused by exogenous or endogenous chemicals and metals. This type of hyperpigmentation is caused by deposition of metals, such as iron, hemosiderin, or chemicals in the dermis. The pigmentary system is normal. Clinical examples include pigmented purpuras, tattoos, and many drug eruptions.

light by two mechanisms. It absorbs incident light directly. It also absorbs light based on the mathematical absorption coefficient of the average path length for each wavelength within the tissue. Thus, longer wavelengths such as the “red” region lying between 600 nm and 700 nm are absorbed to a greater degree than the shorter “blue” region lying between about 400 and 500 nm. Therefore, the low reflectance of light in the longer red wavelengths gives the dermal melanin a blue appearance (Fitzpatrick et al., 1981b). Thus, the blue color of the lesion derives not from increased spectral reflectance in the “blue” region but, rather, from decreased spectral reflectance in the “red” region (Findlay, 1970). Blue hypermelanosis or ceruloderma can result from three different mechanisms: pigmentary incontinence (Fig. 28.4); production of melanin by ectopic dermal melanocytes (Fig. 28.5); and deposition of exogenous dark pigments (Fig. 28.6). Dermal Melanosis Melanins can be transferred abnormally to the dermis where they accumulate within melanophages as melanophagolysosomes (Fig. 28.4). This dermal transfer of melanosomes from epidermal cells to the dermis is called pigmentary incontinence. This process is most commonly observed in inflammatory skin disorders, particularly in der-

Fig. 28.8. Hyperpigmented lichen planus. The pathologic features of a biopsy from this lesion would resemble Figure 28.4 (see also Plate 28.2, pp. 494–495).

matoses that involve damage to the epidermal basal cells and/or to the dermo-epidermal junction (Masu and Seiji, 1981). Post-inflammatory hypermelanosis may also occur following trauma or dermabrasion, particularly in those who have darkly pigmented skin. The most important mechanisms causing pigmentary incontinence seem to be interactions between macrophages and injured keratinocytes called Civatte bodies that contain melanosomes (Masu and Seiji, 1981). Typical examples of such disorders are lichen planus (Figs 28.7 and 28.8) or incontinentia pigmenti (Chapter 47). All melanosomes found in melanophages are thought to be derived from either 523

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melanocytes or keratinocytes. The melanosomes transferred into the dermal melanophages disintegrate. Experimental data suggest that the initial change produced within melanophagolysosomes is proteolysis of the melanosomal membrane. Melanin itself seems to be very resistant to digestion, as illustrated by its long persistence in dermal melanophages. The main pathway by which melanins are eliminated is through phagocytosis by the endothelium of lymphatic vessels (Seiji, 1981). The presence of free melanosomes in the dermis is very unusual (Fig. 28.4). This type of ceruloderma has been called dermal melanosis (Fitzpatrick et al., 1981a). Melanosis from metastatic melanoma is the cause of dermal melanosis (Chapter 53) (Plate 53.2, pp. 494–495). This melanin is transferred to the dermis either directly from the melanocyte or indirectly via epidermal keratinocytes in a two-step passage. Cytokines and inflammatory mediators are known to modulate various keratinocyte and melanocyte functions. Some of them are likely to be involved in the process of pigmentary incontinence. Dermal Melanocytosis Melanocytes abnormally located in the dermis (Fig. 28.5) can synthesize melanins. These pigment cells are surrounded by a basement membrane-like structure and do not transfer their melanosomes to keratinocytes or dermal cells. This process is termed dermal melanocytosis. Common examples are the nevus of Ota, nevus of Ito, acquired facial blue macules, and other similar disorders (Chapter 52) (Plate 52.1–52.7, pp. 494–495). Melanocytes migrate during embryogenesis from the neural crest, proliferate as melanoblasts, and subsequently differentiate into melanin-producing melanocytes in the dermis at their final anatomical sites (Chapters 5 and 6). It has been suggested that the ectopic dermal melanocytes have failed to reach their proper location in the epidermis during their migration (Chapter 7). The cellular and molecular events leading to these abnormalities are not well understood. Some of the factors governing the migration, colonization, differentiation, and survival of the pigment cells that could be involved in the pathogenesis of these pigmentary disorders have been identified (Chapters 6 and 29). Their role, if any, in the pathogenesis of dermal melanocytosis is completely unknown. Transgenic mice carrying a metallothionein/ret fusion gene display marked dermal melanosis caused by arrest and proliferation of melanoblasts in the dermis and in various tissues such as the skeletal muscle, the choroid of eyes, the mediastinum, and the peritoneal cavity (Iwamoto et al., 1991). These observations suggest that the ret oncogene may play a role in the differentiation and/or proliferation of melanoblasts (Iwamoto et al., 1992). This or similar genes could be involved in the pathogenesis of dermal melanocytosis such as Ota’s nevus and related disorders.

Hemoglobin-related Disorders Besides melanins, the other major pigment affecting normal skin color is blood content within capillaries of the superficial 524

Fig. 28.9. Pigmented purpura showing deposition of hemosiderin within the dermis (see also Plate 28.3, pp. 494–495).

dermal plexus, which imparts pink or reddish hues or occasionally bluish hues (Plate 27.3, pp. 494–495). Most dermal macromolecules do not absorb light in the longer wavelengths. Thus, normal dermis in vivo appears light pink. Increases in the number or size of blood vessels, from vasodilatation of dermal blood vessels or abnormal blood vessel formation, results in increased quantities of oxyhemoglobin, appearing clinically as red or violaceous colors (Ortonne et al., 1983). This phenomenon explains the color of hemangiomas and the erythema of inflammation. Red–brown discoloration of the skin may be caused by deposition of hemosiderin in the dermis (Figs 28.6 and 28.9) (Chapters 51 and 54). Recurrent extravasation of red blood cells can be inconspicuous. However, extravasated hemoglobin is usually broken down to various other pigments including hemosiderin within a few weeks. The pigmentation resulting from these chemical reactions, called hemosiderosis, is orange–red at first, later fading through ocher and tawny shades. This is called pigmented purpura. A stimulation of melanogenesis also occurs in chronic lesions, producing a coppery or deep brown color.

Exogenous or Endogenous Pigment-related Disorders A number of drugs or accumulation of endogenous pigments such as porphyrins (Chapter 51) may induce brown hypermelanosis by several different mechanisms, including increased photosensitivity and direct stimulation of the melanogenic activity of melanocytes. Dermal hyperpigmentation mimicking ceruloderma may be induced by drugs and heavy metals, after industrial exposure, or as a result of local or systemic medications (Fig. 28.6) (Chapter 53). The slate gray color of the skin and mucous membranes of patients with argyria (Fig. 28.10) results from the deposition of silver granules in the dermis, in relation to the basal lamina of eccrine sweat glands. The skin pigmentation due to excessive administration of gold, called chrysiasis (Fig. 28.11), is blue gray or lilac. Hyperpigmentation of the skin is also observed in patients exposed to mercury or bismuth. The changes in skin color result from the metallic deposits in the skin (Fig. 28.12). They also cause an increased

MECHANISMS THAT CAUSE ABNORMAL SKIN COLOR

Fig. 28.10. Classical state gray color of argyria. See also Plate 54.4, pp. 494–495.

production of melanins. The basic mechanisms by which metal ions stimulate melanogenesis are not known. Metal ions, such as Cu, Zn, Fe, Co, and Ni, have the ability to induce the nonenzymic rearrangement of dopachrome as well as conversion of the 5,6-dihydroxy-indole(s) to melanochrome(s) (Prota, 1988) (Chapter 10). Whether this is the mechanism involved in increased melanogenesis in patients with metal accumulation in the dermis is unknown. Similar increase in melanogenesis is observed in patients with hemochromatosis, a disorder of iron metabolism. Individuals with hemosiderosis have deeply tanned skin from epidermal melanin production. In patients with minocycline-induced hyperpigmentation (Chapter 53), the brown–black deposits within dermal macrophages contain iron and other metal ions, demonstrated by X-ray microanalysis (Sober and Granstein, 1981). Dermal pigmentation may be observed in patients undergoing long-term treatment with chloroquine, a drug known to bind to melanin molecules (Hill, 1992). Ochronosis or alkaptonuria (Fig. 28.13) is an autosomal recessive disorder of homogentisic acid metabolism that results in the accumulation of a dark polymer of homogentisic acid in connective tissue. This nonmelanin type of cerulo-

Fig. 28.11. Lilac discoloration of the skin from chrysiasis caused by gold injections for arthritis. See also Plate 54.7, pp. 494–495.

Fig. 28.12. Lead line on the gingival margins and gums. Courtesy of Dr. Richard Vilter. See also Plate 54.9, pp. 494–495.

derma affects the facial skin, the axillae and genitalia, the dorsum of the hands, and the costochondral junctions. Exogenous localized ochronosis (Chapter 53) (Fig. 28.14) may occur in patients exposed to phenol and hydroquinone (Mosher et al., 1993). 525

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Fig. 28.13. A patient with alkaptonuria (ochrononsis). Note the blue-gray discoloration of the exposed skin. See also Plate 28.4, pp. 494–495.

Fig. 28.14. Ochronosis of the skin caused by excessive application of hydroquinone. See also Plate 59.4, pp. 494–495.

Epidermal Thickening Skin darkening can also be due to alterations in the epidermis and stratum corneum, i.e. ichthyosis, seborrheic keratoses (Chapter 50) (Fig. 28.15). The skin color change results from the thicker epidermis and stratum corneum and the disruption of the translucency of the stratum corneum. The column of melanin is thicker and the amount of light absorbed is greater.

Skin Lightening Leukoderma and hypopigmentation are generic terms. Diminished skin color is most commonly the result of decrease in epidermal melanin content, i.e. melanin related, but may be secondary to a decreased blood supply of the skin. The majority of melanin-related leukodermas and hypopigmentation are caused by defects in melanin formation. The term “pigment dilution” is commonly used to designate a generalized lightening of the skin such as that seen in people with albinism only if the affected individuals are compared with unaffected relatives or controls (Ortonne et al., 1983). The other mechanism for lightening of the skin is absence or loss of melanocytes. This type of leukoderma is found in people with vitiligo or piebaldism. 526

Hyperpigmentation Normal

Epidermal hyperplasia

Corneocytes Langerhans cells Keratinocytes Melanosomes Melanocytes Basal lamina Dermis

Fig. 28.15. Skin darkening caused by epidermal thickening. The epidermis is hyperpigmented because the column of melanin is significantly thickened. Clinical examples include most types of ichthyosis and seborrheic keratoses. A drawing of normal skin is provided for comparison.

Melanin-related Disorders Hypomelanosis is a type of leukoderma characterized by absent or diminished melanin content in the skin. Amelanosis signifies total absence of melanin. Depigmentation implies a complete loss of pre-existing melanin pigmentation. Specific terms are used for hypomelanosis of hair. Poliosis refers to localized hypomelanosis involving only a tuft of hair or a few

MECHANISMS THAT CAUSE ABNORMAL SKIN COLOR Hypopigmentation Normal

Epidermal melanocytopenia

Corneocytes Langerhans cells Keratinocytes Melanosomes Melanocytes Basal lamina Dermis

Fig. 28.16. Melanocytopenic hypomelanosis. This type of hypomelanosis is caused by partial or total absence of melanocytes. Clinical examples include piebaldism, vitiligo, scleroderma, and idiopathic guttate hypomelanosis. A drawing of normal skin is provided for comparison.

hairs in the eyebrows or eyelashes. Canities means a more generalized loss of hair color. Graying of hair is caused by two processes. The first is by an admixture of pigmented and depigmented, i.e. white, hair. The hair appears gray. The second is by the growth of hair that is gray, not white. The latter is the more common mechanism. Whitening of hair is sometimes the endpoint of graying of hair and canities. Often, in families, the hair turns completely white at an early age, before the age of 30 years. Such hair is white and completely devoid of melanin. The mechanism for loss of melanin in the hair is not known but is possibly related to melanocytes failing to migrate from the dermal papilla into the hair follicle. Hypomelanosis may be the result of defects affecting one or several steps in the complex processes involved in the determination of skin color. Melanocytopenic Hypomelanosis In melanocytopenic hypomelanoses, few or no melanocytes are present in the epidermis and/or hair follicles (Fig. 28.16 and Plates 29.4 and 29.5, pp. 494–495). The lack of melanocytes may be the result of several different pathogenic processes operating prenatally in genetic melanocytopenic hypomelanoses (piebaldism, Chapter 31) or postnatally in acquired melanocytopenic hypomelanoses (vitiligo, Chapter 31). Abnormal Embryogenesis of the Melanocyte System Functional melanocytes might fail to colonize the skin and hair follicles because of abnormal migration, differentiation, proliferation, and/or survival of melanoblasts (Chapters 5 and 6). Analysis of murine mutations has led to the identification of several genes controlling migration and differentiation of melanocytes. These genes have a variety of functions including tyrosine kinase receptors, growth factor ligands for these receptors, and lineage-specific transcription factors (Hearing, 1993; Jackson, 1991, 1994). Mutations at the dominant white spotting and steel loci encode the c-kit tyrosine kinase and its ligands, i.e. the mast cell/stem cell growth factor respectively. Mutations in the gene coding for this molecule cause amelanotic patches of skin and coat in mice, other mammals, and humans. The absence of pigment cells is thought to be a failure of colonization of epidermis by melanocytes (Spritz et al.,

1992). Phenotypes associated with similar mutations have been associated with piebaldism in humans (Spritz, 1994; Tomita, 1994). Mouse mutations at the microphthalmia and the splotch loci, which encode a novel basic helix–loop–helix (bHLH) ZIP transcription factor and the paired homeobox protein Pax 3, respectively, result in coat color spotting due to a failure of melanocyte colonization in the skin (Jackson, 1994). Humans with Waardenburg syndrome type 2 (hearing loss, heterochromia irides, white forelock, and early graying) have mutations in the human homolog of the mouse microphthalmia gene called the MITF gene (Tassabehji et al., 1994). The human homolog of the splotch locus, which encodes the HuP2 protein, has been associated with Waardenburg syndrome type 1, yet another amelanotic condition (Tassabehji et al., 1993). Induced and natural mutations of the endothelin-B receptor gene produce megacolon associated with spotted coat color in mice, a disorder known in humans as Waardenburg syndrome type IV (Hosoda et al., 1994) (see Fig. 29.5). These observations demonstrate that the endothelin-B receptor gene is essential for the development of the two neural crest cell lineages, myenteric ganglion neurons and epidermal melanocytes. Further experiments indicate that disruption of the mouse endothelin-3 ligand gene produces a similar phenotype, suggesting that the interaction of endothelin-3 with the endothelin-B receptor plays a critical role in the development of the melanocyte lineage (Greenstein Baynash et al., 1994). Piebaldism and the various types of Waardenburg syndrome have been grouped under the generic name of “neural crest depigmentation syndromes” (Ortonne, 1988). Depending upon the time of action of the affected gene, pigment cells alone or several neural crest derivatives are affected. These concepts explain the monosymptomatic skin involvement in piebaldism and the phenotypic pleiotropy in Waardenburg syndromes. Postnatal Disappearance of Melanocytes Functional melanocytes present at birth disappear during life, due to their destruction (Fig. 28.16). Vitiligo, chemical hypomelanosis (Chapter 35), and hypomelanosis secondary to trauma are examples of this pathomechanism. The factors controlling melanocyte survival and melanocyte cell death may also play a role in the postnatal disappearance of melanocytes. In Bcl-2-deficient mice, the coat becomes markedly hypopigmented after the first hair follicle cycle (Veis et al., 1993). The Bcl-2 gene product inhibits most types of cell death by apoptosis, perhaps by regulating an antioxidant pathway at sites of free radical generation (Hockenbery et al., 1993). Intermediate metabolites of the melanin pathway have potent melanocytotoxic activity. Thus, it has been proposed that a loss of the protective mechanisms against hyperactive chemical species generated during melanogenesis may result in melanocyte damage or melanocyte death. The light mutation, a dominant mutant allele of the brown locus, is probably the result of such a process and leads to premature melanocyte death. In the light mouse, the hair is pigmented only at its tip, 527

CHAPTER 28 Hypopigmentation Normal

Epidermal melanopenia

Corneocytes Langerhans cells Keratinocytes Melanosomes Melanocytes Basal lamina Dermis

Fig. 28.17. Melanopenic hypomelanosis. This type of hypomelanosis is caused by a failure of melanocytes to synthesize normal amounts of melanins. Clinical examples include albinism, nevus depigmentosus, and hypopigmented macules of tuberous sclerosis. A drawing of normal skin is provided for comparison.

but very lightly or not at all pigmented at its base, a phenomenon caused by a disappearance of follicular melanocytes. This phenotype is induced by a missense mutation near the signal sequence in the tyrosinase-related protein 1 (TRP-1) gene (Chapter 14). The mutation probably causes a disruption of the melanosomal membrane and results in the release of toxic intermediate metabolites of melanogenesis in the cytoplasm of pigment cells (Johnson and Jackson, 1992). These mechanisms may help in understanding the molecular mechanisms leading to premature and/or programmed melanocyte death such as that which occurs in vitiligo and in whitening of hair. Melanopenic Hypomelanosis Decreased pigmentation of the skin may also result from abnormalities affecting melanosome synthesis, melanosome transfer, and melanosome degradation (Fig. 28.17). The pathomechanisms involved in melanopenic hypomelanosis are more numerous and more complex. Melanocytes are present in normal numbers but fail to synthesize melanosomes or melanins or to transfer melanosomes to the surrounding keratinocytes. Melanosome degradation may be abnormal. Disorders of melanopenic hypomelanosis include nevus depigmentosus (Chapter 32) and the ash-leaf spot of tuberous sclerosis (Chapter 32). Abnormalities in Melanosome Synthesis and Melanosome Stabilization Pathways Recent studies indicate that melanosomes are specialized lysosomes (Orlow, 1995). A number of mouse mutations affecting these melanosomal components have been identified (Jackson, 1994; Jackson et al., 1994). The pink-eyed locus in the mouse controls the quantity of melanins produced by the melanocytes and influences the shape and size of the melanosomes (Rosemblat et al., 1994). This gene encodes a 110-kDa integral melanosomal membrane protein. Its biological role is unknown but may be related to importing tyrosine into the melanocyte and into the interior of the melanosome where it is used by the melanogenic proteins (Rosemblat et al., 1994). Mutations in the homologous locus in humans are associated with type II oculocutaneous albinism and with the Prader–Willi syndrome and Angelman 528

syndrome (Chapter 31). Mutations at the pallid locus induce a prolonged bleeding time because of platelet granule alterations, a kidney lysosomal secretion defect, and dilute pigmentation of hair and pink eyes as a result of abnormally small melanosomes (White et al., 1992). This observation suggests that pallidin, the product of the pallid gene, is a molecule influencing granulogenesis. These mutant mice may represent the human counterpart of human hypomelanoses associated with abnormal granulogenesis, such as Chediak–Higashi syndrome (Chapter 31). Hermansky–Pudlak syndrome is another disorder associated with abnormalities of melanocytes, platelets, and macrophages. It cannot be explained by a common cell lineage, but suggests that biogenesis of melanosomes, platelets, granules, and lysosomes depends on a common set of genes. The Hermansky–Pudlak syndrome gene maps to chromosome 10q23 (Fukai et al., 1995). Cloning of this gene or of the mouse mutations that produce a Hermansky–Pudlak syndrome-like phenotype is likely to reveal more about the pathways of melanosome morphogenesis (Barsh, 1995). The silver mutation in the mouse is characterized by a reduction in the number of melanin granules in the hair, due to premature death or loss of melanocytes from the hair follicle. Pmel17, the product of the silver locus, contains a putative signal sequence and a transmembrane domain, suggesting its insertion into the melanosomal membrane (Kwon et al., 1994). It may play a role in forming the structure of the melanosome matrix. However, Pmel17 mRNA expression correlates with melanin content, suggesting that this protein may be involved in melanogenesis at distal steps. No human pigmentary disorder has yet been linked to the Pmel17 protein. The gene defective in X-linked ocular albinism type 1 (Nettleshop–Falls type) is probably involved in melanosomal biogenesis. Melanocytes in affected individuals usually contain giant pigment-filled organelles referred to as “macromelanosomes” or “melanin macroglobules.” They are thought to represent an underlying defect of melanosome formation. The OA-1 gene localized to the distal short arm of the X chromosome predicts a 42-kDa amino acid protein with six membrane-spanning domains. It has no similarities to previously identified proteins (Bassi et al., 1994). Abnormalities in Melanosome Melanization Several factors influence the amount and types of melanins produced by melanocytes (Hearing and Tsukamoto, 1991). Tyrosinase is the enzyme most critical to melanin production as it controls the rate of melanogenesis (Chapters 10 and 11). Mutations of the tyrosinase gene are associated with several forms of human oculocutaneous albinism (Chapter 31) (Oetting and King, 1994). Type 3 oculocutaneous albinism is caused by a mutation in the TRP-1 gene. The molecule is responsible for enhancing the production of black rather than brown eumelanin. These individuals have a moderate brown color to their skin and hair (see Plate 27.6, pp. 494–495). No human pigmentary disorders have yet been linked to the other member of the tyrosinase gene

MECHANISMS THAT CAUSE ABNORMAL SKIN COLOR

family, encoding tyrosinase-related protein 2 (TRP-2), identified as DOPAchrome tautomerase (Aroca et al., 1990; Tsukamoto et al., 1992), the enzyme that determines the chemical constituents of melanins (Budd and Jackson, 1995). Inhibition of tyrosinase may result from its interaction with factors present within melanocytes. Cystathionine b synthase deficiency is a genetic disorder resulting in elevated plasma homocyst(e)ine (Chapter 31). Affected patients may have hypopigmentation of the skin and hair that is reversible following treatment to decrease plasma homocyst(e)ine. Experimental data suggest that reversible hypopigmentation in homocystinuria is the result of tyrosinase inhibition by homocysteine. The likely mechanism of this inhibition is the interaction of homocysteine with copper at the active site of tyrosinase (Reish et al., 1995). Tyrosinase and other members of the tyrosinase protein family contain two atoms of copper per molecule. Copper must be transported into the melanocytes and the melanosomes for their function. An X-linked recessive disorder of copper metabolism, called Menke’s disease, is characterized by hypopigmentation of skin and hair that led to the name “Menke’s kinky hair disease” (Chapter 31). The Menke’s gene has been mapped to Xq13.3 and isolated (Vulpe et al., 1993) by nucleotide sequences. The gene is predicted to encode a P-type ATPase with structural similarity to ion transporters from various organisms. It is likely to be involved in copper transport into the cell (Chelly et al., 1993). Hypopigmented hair from these patients contains fewer eumelanins and pheomelanins than normally pigmented hairs. Administration of copper can partially restore the dark color of hair (Tomita et al., 1992). Melanopenic hypopigmentation can be acquired in several ways, for example by exposure to chemicals that inhibit tyrosinase (Chapter 35). Inhibition of tyrosinase has also been suggested to explain hypomelanotic lesions in tinea versicolor (Chapter 36) and in Hansen’s disease (Chapter 36). A substrate deficiency may be involved in the pathogenesis of the pigmentary abnormalities observed in chronic protein deficiency called kwashiorkor (Chapter 34). The relative proportions of pheomelanin and eumelanin are regulated by several factors, the best identified of which is MSH. It acts on its receptor located on the membrane of melanocytes. The product of the agouti gene locus seems to antagonize the action of MSH. Mutations of the agouti or MSH receptor result in coat color changes in mice (Robbins et al., 1993; Siracusa, 1994). These genes have not yet been associated with melanin pigmentary disorders in man, but variants of the MSH hormone receptor gene have been associated with red hair and fair skin in humans (Valverde et al., 1995). Abnormalities in Melanosome Transfer Melanosomes are formed in epidermal and follicular melanocytes. They are secreted from melanocytes into neighboring keratinocytes (Chapters 7 and 8). The mechanisms responsible for movement of the melanosomes to the periphery of the cell are not

well understood. Melanocyte dendrites are crucial for the transfer of melanosomes from melanocytes to keratinocytes, and mutations affecting melanocyte morphology cause hypomelanosis. The DBA strain of mice show a general lightening of the coat despite a normal level of melanin synthesis. The light color is thought to result from a defective transfer of melanin granules by follicular melanocytes. The dilute locus has been shown to encode a myosin-related protein that is involved in the formation of melanocyte dendrites and perhaps in the movement of melanosomes to the periphery of melanocytes (Mercer et al., 1991; Seperack et al., 1995). The human homolog of the “dilute” protein, called myoxin, has been cloned and sequenced (Engle and Kennett, 1994). The skin pigmentary abnormalities in Griscelli–Prunieras syndrome (see also Chapter 31 and Plate 31.6, pp. 494–495) may represent the human counterpart of the dilute mouse mutation. This disorder is characterized by hypomelanosis of hair and skin and combined immunodeficiency resulting in recurrent infections. Melanocytes with few short dendrites are loaded with melanosomes. In contrast, there is an absence of melanin granules in the adjacent keratinocytes (Kanitakis et al., 1991). Several other mouse mutant-causing phenotypes very similar to dilute with adendritic melanocytes have been identified. Their molecular characterization will be most helpful in the understanding of melanocyte dendritogenesis and related disorders (Jackson et al., 1994). Abnormalities in Melanosome Degradation and Melanin Removal Melanin granules are scarce in the Malpighian and horny layers, whereas they are abundant in the epidermal basal layer. This suggests that melanins and melanosomes are degraded during their transit through the epidermis. It has been suggested that the rate of melanosome destruction is a key parameter in the determination of racial differences in skin color, but there are few data to substantiate this hypothesis. Melanosomes are specialized members of the lysosomal lineage of organelles (Orlow, 1995). As lysosomes, their enzyme activities may play a very important role in the degradation of melanin pigments and melanosomal matrix proteins. Hypomelanoses of the feather secondary to melanosomal autophagocytosis have been reported in chickens (Jimbow et al., 1974). Several human hypomelanotic disorders, including Chediak–Higashi syndrome (Chapter 31), are associated with structural abnormalities of melanosomes. However, these disorders most likely result from abnormal melanosome biogenesis rather than accelerated degradation of melanin and/or melanosomes. Rapid turnover of the stratum corneum accelerates melanin removal. This may contribute to the pathogenesis of the light hypomelanosis that occurs in disorders associated with an increased turnover of keratinocytes and of the stratum corneum such as psoriasis (Chapter 37 and Plate 37.3, pp. 494–495) and seborrheic dermatitis. Melanosome transfer may be disturbed by increased keratinocyte turnover and by all processes disturbing the interactions between melanocytes and keratinocytes. Edema of the intercellular spaces might alter melanosomal transfer. 529

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Hemoglobin-Related Disorders Leukodermas without hypomelanosis are due to markedly decreased hemoglobin levels in the skin. Anemic states induce a generalized pallor that is more readily visible on the mucous membranes or in areas such as the palms and soles where there is physiologically less melanin. Vasoconstriction also induces transient decreased levels of circulating oxyhemoglobin and results in localized or generalized skin pallor. It is usually easy to differentiate hypomelanosis from pallor, except for the nevus anemicus (Chapter 42) (see Plate 27.3, pp. 494–495). This “pharmacological nevus” is characterized clinically by one or several areas of pale skin with sharply irregular borders. The disappearance of the lesions under Wood’s light examination demonstrates that the decreased skin color is not related to melanin content. Such lesions, if vigorously rubbed or perturbed by exposure to heat, do not exhibit vasodilatation. This observation demonstrates that the skin lightening is due to permanent vasoconstriction of the dermal blood vessels. The presence of edema fluid in the skin results in decreased absorption of light that renders the skin more pallid than normal.

Unusual Skin Color Endogenous substances and exogenous chemicals may accumulate in the skin, producing an alteration of the normal pigmentation resulting in a very unusual color (Chapter 53). A yellow discoloration of the skin is observed when bilirubin and other bile pigments accumulate in the skin, as in jaundice. An accumulation of lipids induces a yellow–orange discoloration (Plate 54.13, pp. 494–495). Drugs such as atabrine and various chemicals (picric acid, dinitrophenol, santonin) stain the skin yellow (Plate 54.19, pp. 494–495). A yellowish orange discoloration of the skin is also observed in patients with carotenemia. Mepacrine-induced pigmentation produces a yellow color with a greenish hue. A violaceous or gray–blue coloration of the skin may occur in patients taking the medications clofazimine (Plate 54.21, pp. 494–495), amiodarone (Plates 54.14 and 54.15, pp. 494–495), or phenothiazines. In accidental and decorative tattoos, various particles (carbon, cobalt, chrome, cadmium, iron, cinnabar, vegetable dyes) are introduced in the dermis where they produce a wide variety of colors (Plates 54.10–54.12, pp. 494–495).

Diagnosis of Melanin Pigmentary Disorders Clinical History Melanin pigmentary disorders can be acquired, congenital, or genetic in origin. In the last two mechanisms, the abnormalities of skin color are often present at birth. Unfortunately, history can be misleading because the abnormality may not be observed for months or longer. Hypomelanosis is often overlooked or the lesion is almost inapparent. Thus, it is not always easy to determine whether an abnormality of skin color 530

is congenital or acquired. Investigation of the family history is also mandatory to establish that the disorder is hereditary (genetic and/or congenital) and to identify its inheritance pattern. The course of the disorder is a useful parameter in characterizing the disease. Most acquired lesions show progression or regression. Although exceptions are well known, most lesions resulting from hereditary factors are morphologically stable and do not change their shape with time. The past medical history of the patient must be obtained to search for cutaneous or systemic disorders, medications, occupation, or leisure that could be associated with the induction of hyper- or hypomelanotic disorders.

Physical Examination All patients with pigmentary disorders of the skin should be completely undressed and examined fully under both visible (Fig. 28.18) and Wood’s light (Fig. 28.19). The Wood’s lamp enhances the contrast between skin with little or no pigment and epidermis with more or excessive quantities of melanin. One of the most important clinical parameters is the extent of the pigment abnormality. It may be universal or localized to a few spots or areas. Indeed, few disorders are associated with universal hyper- or hypomelanoses. In these patients, the entire skin and mucous membranes are involved, but the melanin pigmentary abnormality may be more easily detected in specific areas of the skin. In universal hypermelanoses such as Addison’s disease (Chapter 50), the pigment darkening is more apparent on sun-exposed areas, on the darker areas of the skin such as axillae, on palmar creases (Plate 28.5), and mucous membranes (Plate 50.27, pp. 494–495). A universal hypermelanosis may pass unnoticed in a very fair-skinned individual, unless the skin and hair are compared with those of an unaffected member of the same family. The majority of melanin pigmentary disorders are circumscribed with identifiable borders between involved skin and normal skin. Circumscribed melanin pigmentary disorders are either localized or generalized. However, even by crossing these three clinical features, the list of disorders in each category is so long that additional parameters should be analyzed to reach a diagnosis. Other features that facilitate diagnosis include distribution, shape, size, color, and number of the lesions, morphology of the borders, involvement of the hair and mucous membranes, additional alterations of involved skin, and associated cutaneous and extracutaneous abnormalities. Several disorders have characteristic areas of involvement. The periorificial lentiginosis of Peutz–Jeghers syndrome (Plate 51.7, pp. 494–495) is a good example. Ephelides and melasma always occur on the most UV-irradiated areas of facial skin. Involvement of bony prominences and sites of trauma is rather suggestive of vitiligo. The central white forelock and the typical distribution of the white macules that involves the anterior surface of the trunk and extremities is typical of piebaldism and Waardenburg syndrome. The clinical characteristics of the skin lesions are the essential elements upon which clinical diagnosis rests. Most pig-

MECHANISMS THAT CAUSE ABNORMAL SKIN COLOR

Fig. 28.18. Skin viewed under standard lighting (courtesy of L’Oréal). It appears relatively normal and free of pigmentary abnormalities.

mentary disorders are macular. The macules may vary considerably in size from a few millimeters to several centimeters. There is, however, a limited number of pigmentary disorders characterized by small macules of less than 1 cm in diameter. These include ephelides and lentigines in the group of hypermelanoses. Idiopathic guttate hypomelanosis, resolved halo nevus, white macules in tuberous sclerosis are localized types of hypomelanoses. The number of lesions may vary from one to 100 or more. Number is not a discriminating feature. Analysis of the morphology of the borders of the lesions may be more informative. Café-au-lait macules (Chapter 45) have very distinct borders, whereas melasma or post-inflammatory hypermelanosis show irregular and illdefined borders with heterogeneity in the degree of hyperpigmentation. Sharp and discrete margins characterize the hypomelanotic macules of vitiligo (Chapter 30) and piebaldism (Chapter 29). Pityriasis alba (Chapter 37), leprosy (Chapter 36), and post-inflammatory hypomelanosis (Chapter 37) exhibit feathered margins. Hyperpigmented borders surrounding a hypomelanotic macule are considered characteris-

Fig. 28.19. The same skin viewed under Wood’s light. Note the large number of hyperpigmented macules, not visible under standard lighting (courtesy of L’Oréal).

tic of vitiligo, but may be seen in other hypomelanotic disorders including piebaldism and tinea versicolor. Erythematous raised borders have been reported in a few cases of inflammatory vitiligo. The presence of a zone of intermediate color between the hypopigmented and the normal skin represents trichrome vitiligo or trichrome halo nevus. Quadrichrome and pentachrome vitiligo including multiple shades of color have been described (Fargnoli and Bolognia, 1995). The pigmentary changes in café-au-lait macules and vitiligo are usually homogeneous. The occurrence of perifollicular, round, pigmented macules within white macules of vitiligo, halo nevus (Chapter 38), chemical or physical hypomelanosis (Chapter 35), tinea versicolor, or postinflammatory hypomelanosis is indicative of spontaneous or induced repigmentation. Hyperpigmented macules of different size and shape within the hypomelanotic macules are a rather typical feature of piebaldism, Waardenburg syndrome (Chapter 29), and related hypomelanotic genodermatoses. Unlike vitiligo, the number and size of these pigmented macules do not change with time. Heterogeneity in color is a constant feature of postinflammatory pigmentary changes. The association of hyper531

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Fig. 28.20. Brown hyperpigmentation caused by indiscriminate application of topical psoralen followed by direct exposure to sunlight. See also Plate 50.14, pp. 494–495.

and hypomelanosis is described as leukomelanoderma. This feature is commonly seen in patients with xeroderma pigmentosum and other uncommon pigmentary genodermatoses. They may be observed in acquired disorders such as vagabond’s disease. The shape of hyper- or hypomelanotic macules may be suggestive of a specific diagnosis. Extensive large, bizarre café-aulait macules with irregular and serrated margins that do not cross the midline strongly suggest the diagnosis of polyostotic fibrous dysplasia. Axillary ephelides are almost only seen in patients with neurofibromatosis NF-1 (Chapter 45) or familial café-au-lait spots (NF-6). The lance-ovate or ash-leaf shape of hypomelanotic macules is fairly characteristic of tuberous sclerosis. The guttate lenticular shape with irregular borders of the white lesions of idiopathic guttate hypomelanosis are diagnostic. A whorled pattern of hyper- or hypomelanosis is characteristic of incontinentia pigmenti or hypomelanosis of Ito, and may be a feature of cutaneous mosaicism. In phytophotodermatosis, the pigmentary changes follow the irregular pattern of the points of contact of the plant stems and leaves with the skin. In Berloque dermatitis, the hypermelanosis follows the pattern formed by the trickle of the droplets of perfume over the skin from their point of application (Fig. 28.20). A segmental distribution of hypomelanosis may be seen in vitiligo, in tuberous sclerosis, and in nevus depigmentosus. Segmental neurofibromatosis may present as café-au-lait macules with a strictly unilateral distribution. Pigmentary abnormalities may appear as linear dermatoses (Harre and Millikan, 1994). Indeed, a linear pattern of hypomelanosis suggests the Koebner phenomenon in vitiligo, post-traumatic hypomelanosis, or post-inflammatory hypomelanosis secondary to an inflammatory linear dermatosis. Abnormalities of hair pigmentation are very common. Although they may be isolated, they are usually associated with involvement of the interfollicular epidermis. Most hyper- or hypomelanotic macules have no other skin abnormalities. Slight scaling is usually present in the white macules of pityriasis alba, tinea versicolor, and post532

inflammatory pigmentary disturbances. Atrophy or sclerosis is observed in hypomelanotic lichen sclerosus and atrophicans and in hyperpigmented morphea respectively. The majority of hyper- or hypomelanotic macules are asymptomatic. However, pruritus is a classic manifestation in macular pigmented amyloidosis and may be present in tinea versicolor and pityriasis alba. It is an uncommon manifestation of vitiligo. Anesthesia or hypesthesia of a hypomelanotic macule is diagnostic of leprosy. Associated dermatological abnormalities, when present, are useful for establishing the diagnosis. Hyper- or hypomelanotic macules in patients with another coexistent dermatosis, such as psoriasis, atopic dermatitis, discoid lupus erythematosus, pityriasis, lichenoides chronica, pemphigus, or Darier’s disease, strongly suggest the diagnosis of post-inflammatory melanin pigmentary abnormalities. The associated skin findings may also be other features of complex disorders such as phacomatoses or multisystem genodermatoses affecting the skin as well as many other organs. Thus, a general physical examination is mandatory and may provide clues to the diagnosis of melanin pigmentary disorders, particularly those of genetic origin.

Special Clinical Procedures Wood’s Light Examination Wood’s light examination should be a part of every evaluation of any pigmentary disorders of the skin, either hypo- and hypermelanosis. This is a very simple technique requiring a Wood’s light with an emission spectrum ranging from 320 to 400 nm (with a peak emission at 365 nm) or any other UV radiation source filtered primarily to emit in the UVA spectrum. The patient should be examined in a darkened room. Skin color derives from the spectral character of light reflected from the skin to the eye. Ultraviolet light from the Wood’s lamp penetrates predominantly in the stratum corneum and epidermis where melanins are distributed in normal skin. Melanin absorbs the visible and ultraviolet light and determines the optical properties of the epidermis. Only the wavelengths reflected back from the skin will reach the eye of the observer. In black skin, in which melanins are abundant and distributed in the more superficial epidermal layers, most of the light is absorbed, and only a small amount of light returns to the eye. The skin appears back. Conversely, the epidermis, which is less pigmented or hypomelanotic, appears whiter when examined under Wood’s light (Figs 28.18 and 28.19). Most ultraviolet light is absorbed in the epidermis, and only a small amount reaches the dermis. Hence, dermal proteins contribute less to the light reflected to the observer. Thus, dermal melanins do not affect the amount of light observed. Therefore, changes in epidermal melanin pigmentation are accentuated by a Wood’s lamp (Figs 28.18 and 28.19). Variations in dermal pigmentation are less apparent under a Wood’s lamp than under visible light (Gilchrest et al., 1977). Wood’s light examination is most useful for detecting hypomelanotic macules on a very fair skin (type I or II) or in

MECHANISMS THAT CAUSE ABNORMAL SKIN COLOR

babies. It is also most helpful in differentiating hypomelanotic macules from leukoderma unrelated to melanins such as the nevus anemicus. Under Wood’s light examination, nevus anemicus becomes inapparent. Under visible light, it is sometimes difficult to distinguish hypomelanosis from amelanosis. The greater the loss of epidermal pigmentation, the more marked the contrast on Wood’s light examination. This explains why vitiligo macules that are completely devoid of melanins are so obvious even in fair-skinned individuals when examined under Wood’s light. It has been suggested that the fluorescence of the hypomelanotic skin in patients with active vitiligo is due to the intraepidermal accumulation of biopterins (Schallreuter et al., 1994). This feature has been proposed as characteristic of vitiligo. However, most observers note that all types of depigmented skin fluoresce under the Wood’s light. All depigmented skin illuminated with a Wood’s lamp fluoresces, giving off a small band of blue light with a spectrum about 412 nm that is emitted by collagen. Wood’s light examination makes evident variations in epidermal pigmentation that are not apparent under visible light, i.e. very lightly pigmented café-au-lait spots or ephelides (Figs 28.18 and 28.19). In addition, it can be used to determine the depth of melanin pigmentation in the skin. The contrast between involved and uninvolved skin is increased in epidermal hypermelanosis. It is decreased in dermal melanosis under Wood’s lamp illumination compared with ambient visible light. Normally, dark skin melanin pigmentation may obscure the detection of abnormal dermal melanins. Therefore, Wood’s light determination of the depth of melanin pigmentation cannot be used reliably in skin type V and VI individuals (Morikawa et al., 1981).

Ultraviolet (UV) and Infrared (IR) Photography The results of Wood’s light examination can be recorded through UV photography (Mustakallio and Korhonen, 1966). This technique allows re-examination and comparative evaluation during the treatment of a pigmentary disorder. It can be very helpful to follow and document therapeutic progress. UV pictures may also be analyzed and evaluated using computerized image analysis systems. Fewer data are available about infrared (IR) photography to evaluate pigmentary disorders (Morikawa et al., 1981). This approach is useless for epidermal hypermelanosis, but is a good tool to reveal dermal hypermelanosis. However, IR photography is not specific for melanin deposits. The drawbacks in using this technique are interferences caused by increased dermal blood vessels and increased dermal collagen.

Epiluminescence Microscopy In vivo epiluminescence microscopy (dermoscopy) using a surface microscope or dermatoscope has recently proved to be a very useful noninvasive technique for the diagnosis of cutaneous pigmented lesions. This method, by employing the enhanced penetration of light with oil immersion, makes sub-

surface structures of the skin visible for in vivo examination. It provides additional criteria for the diagnosis of pigmented skin (Binder et al., 1995). Epiluminescence microscopy, used mostly for the early recognition of thin malignant melanoma, increases the sensitivity of diagnosis but only for formally trained dermatologists. Except for the clinical differential diagnosis between malignant melanoma and benign pigmented nevi, the potential of this technique for the clinical assessment of other pigmentary disorders of the skin has not yet been explored. The magnification power of the dermatoscope can be greatly enhanced by the use of a videomacroscope, a newly developed electronic device with a magnification power up to 1000-fold (Saida et al., 1995).

Reflectance Mode Confocal Microscopy (RCM) RCM allows noninvasive optical sectioning of each layer of the skin in real time with melanin as the main endogenous contrast (Rajadhyaksha et al., 1995). Very few studies testing the applicability of RCM for noninvasive visualization of melanin and melanocytes in vivo have yet been performed. In the human skin, typical dendritic melanocytes are hardly observed. However, supranuclear melanin caps are easily visible. Melanin has a strong refractivity compared with the epidermis. However, the brightness is not homogeneous in the basal layer, suggesting that the distribution of melanin is not uniform and is dependent on the presence of the melanocytes. RCM shows the difference in melanin distribution during the UV-induced pigmentation process in vivo (Yamashita et al., 2005). These observations indicate that RCM can be used to examine the activity of melanocytes by assessing melanin in the epidermal basal layer. Thus, RCM may be a useful tool to address noninvasively the melanin distribution and synthesis in the skin in vivo, and can be useful for studying the pathogenesis and evaluating the treatment of melanin pigmentary disorders.

Chromometry and Reflection Spectrophotometry Quantitation of skin color is a prerequisite in clinical studies. Reflection spectrophotometry has been utilized for studying the differences in skin color. Two types of portable reflectance instruments, tristimulus colorimeters (Chroma Meter CR2000®) and narrow-band spectrophotometers (Dermatospectrometer®), have become available for the quantitation of skin color. One parameter measured by the chromometer, the L value (darkness/lightness), is useful in evaluating and quantifying the tanning response of the skin. It can also be used to quantify the therapeutic value of depigmenting agents. The dermatospectrometer is a narrow-band spectrophotometer designed for measuring specific colors of two major chromophores, hemoglobin and melanins, in human skin (Takiwaki et al., 1994a). The chromometer is easy to use (Andreassi and Flori, 1995) and gives rapid and reproducible results for the measurement of hair color (Gerrard, 1989). In order to improve the power of reflectance spectroscopy, a telespectrophotometric system composed of a CCD camera, a set of 17 interference filters, and a personal computer has been 533

CHAPTER 28

developed for the evaluation of neoplastic and nonneoplastic skin pigmented lesions. The preliminary results suggest that this new system gives useful information and could be utilized for the diagnosis of cutaneous pigmented lesions (Marchesini et al., 1995).

Image Analysis for the Quantitation of Skin Color A method for the quantitative analysis of skin pigmentation on clinical pictures using a videomicroscope interfaced with a computer has been reported (Takiwaki et al., 1994b). This system is suitable for the evaluation of pigmented lesions that are too small or irregular to quantify by conventional methods such as colorimetry and offers excellent interobserver reproducibility. Another study demonstrates that digital images of cutaneous pigmented lesions are as informative as slides (Perednia et al., 1995).

Fig. 28.21. Legend to be supplied.

Light and Electron Microscopy Histologic classification of melanin pigmentary disorders is mainly based on the number and localization of melanocytes and melanin granules in involved skin. Standard stains such as hematoxylin and eosin are simple histologic techniques to assess the amount of melanins in the skin. Melanocytes appear as clear cells having a small, dark-staining nucleus located in the basal layer of epidermis. However, not all clear cells of the epidermal basal layer seen in routine sections are necessarily melanocytes. Thus, more specific melanocyte stains are required to demonstrate pigment cells. Silver nitrate and Fontana–Masson stains indicate the presence of melanins that are both argyrophilic and argentaffin positive, but neither of these staining methods is specific for these melanins. Silver nitrate solutions bind to melanins and, through reduction to silver, stain black. The Fontana–Masson stain is based on the reduction of ammoniated silver nitrate to free black silver by the phenolic compounds present in melanins. Melanin granules accumulate in the cytoplasm of basal keratinocytes in lightly melanized epidermis. Few or no melanin granules are observed in the Malpighian layer. Conversely, in dark skin, melanin granules, present predominantly in the basal layer, are found throughout the entire epidermis including the horny layer. Bleaching of melanins by strong oxidizing agents such as hydrogen peroxide or potassium permanganate may be useful to identify melanins (Pearse, 1972). The most popular histochemical technique for identifying melanocytes is the dihydroxyphenylalanine reaction. This method requires fresh tissue with no or mild fixation. After incubation in a solution of 3,4-dihydroxyphenylalanine (DOPA), tyrosinase melanogenic enzyme present in melanocytes changes this colorless compound into homogeneous black deposits at sites where the enzyme is present. On skin sections, melanocytes appear as dark cells in the epidermal basal layer, with dendrites extending upwards between neighboring keratinocytes of the suprabasal layers (Fig. 28.21). The DOPA stain applied to wholemounts of the split epidermis is the best method of determining the popula534

tion density of epidermal melanocytes. Briefly, the epidermis is separated from the dermis by incubation of the fresh biopsy in a 2 N solution of sodium bromide, then incubated in a DOPA solution, fixed, and finally mounted. The population density of epidermal melanocytes (number of cells per square millimeter of epidermis), their distribution, size, shape, and dendrites are assessed easily (Staricco and Pinkus, 1957). The “intensity” of the DOPA melanin production can be evaluated as a measurement of the melanogenic capacity of the pigment cells. The intensity of DOPA reaction is not used in a quantitative sense but, when several skin biopsies are incubated for the same length of time, those that show a higher level of melanogenesis develop a much darker coloration of DOPA melanin in their melanocytes. This method for detecting melanocytes is based on active melanin synthesis. Thus, a lack of staining does not necessarily indicate that pigment cells are absent and does not rule out the possibility that inactive melanocytes persist in the epidermis. Several monoclonal antibodies directed against different melanocyte antigens unrelated to their melanogenic capacities are now available. Immunocytochemistry with this panel of antibodies reactive to melanocytic cells provides a powerful method of detecting melanocytes, in either normal or reduced amounts, with normal or altered phenotype such as inactive “dormant melanocytes” (Le Poole et al., 1993). Transmission electron microscopy is a powerful tool for studying the subcellular events of melanogenesis. There is no other technique to evaluate the size, shape, structure, maturation, melanization of melanosomes within melanocytes, and the number, distribution, and morphology of melanosomes within epidermal keratinocytes and dermal melanophages. Ultrastructural analysis is also a useful technique to confirm that melanocytes are lacking in the skin. In most patients with melanin pigmentary disorders, a careful evaluation including complete history and physical examination under visible and physical examination are sufficient to reach a clear-cut diagnosis. Light microscopic study of a skin biopsy may be useful in obtaining a clear identifica-

MECHANISMS THAT CAUSE ABNORMAL SKIN COLOR

Thody et al., 1990). More recently, a new method to solubilize differentially pheomelanins and brown-type eumelanins has been also developed (Ozeki et al., 1995).

References

Fig. 28.22. Legend to be supplied.

tion of a pigmented skin lesion for which the clinical differential diagnosis is extremely difficult. The histology can identify the process causing the pigmentary disturbances. In difficult cases, light microscopy including histochemical and immunohistochemical stains is sufficient to evaluate melanocyte numbers very accurately and to distinguish between melanopenic, melanocytopenic, melanotic, and melanocytotic pigmentary disorders. Ultrastructural studies (Fig. 28.22) are performed only for research purposes. They allow detailed characterization of the cellular and subcellular alterations, useful for understanding the pathomechanisms leading to the pigmentary abnormality in most instances. Such studies are of lesser use for establishing the diagnosis, as very few disorders of melanocytes show a characteristic ultrastructural picture. From a theoretical point of view, abnormalities of melanogenesis at the subcellular level result from alterations in: (1) production and structure of melanosome; (2) melanosome melanization; (3) melanosome transfer; and (4) melanosome degradation. However, a clear-cut classification of melanin pigmentary disorders, based on ultrastructural findings, is difficult to establish.

Characterization and Quantification of Melanins in Tissues For basic and clinical research purposes, several methods have been used to analyze melanins in a variety of tissue samples (Ito, 1993). These include a microanalytical high-performance liquid chromatography (HPLC) method to quantify eumelanin and pheomelanin after chemical degradation to pyrrole-2,3,5tricarboxylic acid (PTCA) and aminohydroxyphenylalanine (AHP) respectively (Ito et al., 1984). This method has been used to characterize melanins in human hair of different color and in human interfollicular epidermis (Jimbow et al., 1983;

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MECHANISMS THAT CAUSE ABNORMAL SKIN COLOR Robbins, L. S., J. H. Nadeau, K. R. Johnson, M. A. Kelly, L. RoselliRehfuss, E. Baack, K. G. Mountjoy, and R. D. Cone. Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 72:827–834, 1993. Rosemblat, S., D. Durham-Pierre, J. M. Gardner, Y. Nakatsu, M. H. Brilliant, and S. J. Orlow. Identification of a melanosomal membrane protein encoded by the pink-eyed dilution (type II oculocutaneous albinism) gene. Proc. Natl. Acad. Sci. USA 91:12071–12075, 1994. Saida, T., S. Oguchi, and Y. Ishihara. In vivo observation of magnified features of pigmented lesions on volar skin using video macroscope. Usefulness of epiluminescence techniques in clinical diagnosis. Arch. Dermatol. 131:298–304, 1995. Schallreuter, K. U., J. M. Wood, M. R. Pittelkow, M. Gutlich, K. R. Lemke, W. Rodl, N. N. Swanson, K. Hitzemann, and I. Ziegler. Regulation of melanin biosynthesis in the human epidermis by tetrahydrobiopterin. Science 263:1444–1446, 1994. Schwindinger, W. F., and M. A. Levine. McCune–Albright syndrome. Trends. Endocrinol. Metab. 4:238–242, 1993. Schwindinger, W. F., C. A. Francomano, and M. A. Levine. Identification of a mutation in the gene encoding the alpha subunit of the stimulatory G protein of adenylyl cyclase in McCune–Albright syndrome. Proc. Natl. Acad. Sci. USA 89:5152–5156, 1992. Seiji, M. Biology of dermal melanin. In: Biology and Diseases of Dermal Pigmentation, T. B. Fitzpatrick, A. Kukita, F. Morikawa, M. Seiji, A. J. Sober, and K. Toda (eds). Tokyo: University of Tokyo Press, 1981, pp. 21–38. Seperack, P. K., J. A. Mercer, M. C. Strobel, N. G. Copeland, and N. A. Jenkins. Retroviral sequences located within an intron of the dilute gene alter dilute expression in a tissue-specific manner. EMBO J. 14:2326–2332, 1995. Siracusa, L. D. The agouti gene: turned on to yellow. Trends Genet. 10:423–428, 1994. Sober, A., and R. Granstein. Chemical and pharmacologic agents causing dermal pigmentation. In: Biology and Diseases of Dermal Pigmentation, T. Fitzpatrick, A. Kukita, F. Morikawa, M. Seiji, A. Sober, and K. Toda (eds). Tokyo: University of Tokyo Press, 1981, pp. 117–126. Spritz, R. A. Molecular basis of human piebaldism [review]. J. Invest. Dermatol. 103:137S-140S, 1994. Spritz, R. A., S. A. Holmes, R. Ramesar, J. Greenberg, D. Curtis, and P. Beighton. Mutations of the KIT (mast/stem cell growth factor receptor) proto-oncogene account for a continuous range of phenotypes in human piebaldism. Am. J. Hum. Genet. 51:1058–1065, 1992. Staricco, R. J., and H. Pinkus. Quantitative and qualitative data on pigment cells of adult human epidermis. J. Invest. Dermatol. 28:33–45, 1957.

Takiwaki, H., L. Overgaard, and J. Serup. Comparison of narrowband reflectance spectrophotometric and tristimulus colorimetric measurements of skin color. Skin Pharmacol. 7:217–225, 1994a. Takiwaki, H., S. Shirai, Y. Kanno, Y. Watanabe, and S. Arase. Quantification of erythema and pigmentation using a videomicroscope and a computer. Br. J. Dermatol. 131:85–92, 1994b. Tassabehji, M., A. P. Read, V. E. Newton, M. Patton, P. Gruss, R. Harris, and T. Strachan. Mutations in the PAX3 gene causing Waardenburg syndrome type 1 and type 2. Nature Genet. 3:26–30, 1993. Tassabehji, M., V. E. Newton, and A. P. Read. Waardenburg syndrome type 2 caused by mutations in the human microphthalmia (MITF) gene. Nature Genet. 8:251–255, 1994. Thody, A. J., E. M. Higgins, S. A. Burchill, J. M. Marks, and S. Ito. Epidermal eumelanin and phaeomelanin concentrations in different skin types and in response to PUVA. Br. J. Dermatol. 122:288–289, 1990. Tomita, Y. The molecular genetics of albinism and piebaldism. Arch. Dermatol. 130:355–358, 1994. Tomita, Y., Y. Kondo, S. Ito, M. Hara, T. Yoshimura, H. Igarashi, and H. Tagami. Menkes’ disease: report of a case and determination of eumelanin and pheomelanin in hypopigmented hair. Dermatology 185:66–68, 1992. Tsukamoto, K., I. J. Jackson, K. Urabe, P. Montague, and V. J. Hearing. A second tyrosinase related protein, TRP-2, is a melanogenic enzyme termed dopachrome tautomerase. EMBO J. 11:519–526, 1992. Valverde, P., E. Healy, I. Jackson, J. L. Rees, and A. J. Thody. Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans. Nature Genet. 11:328–330, 1995. Veis, D. J., C. M. Sorenson, J. R. Shutter, and S. J. Korsmeyer. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75:229–240, 1993. Vulpe, C., B. Levinson, S. Whitney, S. Packman, and J. Gitschier. Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nature Genet. 3:7–13, 1993. White, R. A., L. L. Peters, L. R. Adkison, C. Korsgren, C. M. Cohen, and S. E. Lux. The murine pallid mutation is a platelet storage pool disease associated with the protein 4.2 (pallidin) gene. Nature Genet. 2:80–83, 1992. Yamashita, T., T. Kuwahara, S. Gonzalez, and M. Takahashi. Noninvasive visualization of melanin and melanocytes by reflectancemode confocal microscopy. J. Invest. Dermatol. 124:235–240, 2005.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Disorders of Hypopigmentation, Depigmentation and Hypochromia

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Genetic Hypomelanoses: Disorders Characterized by Congenital White Spotting — Piebaldism, Waardenburg Syndrome, and Related Genetic Disorders of Melanocyte Development — Clinical Aspects Richard A. Spritz

Genetic disorders of pigmentation can be grouped into two general categories based on their underlying molecular and biochemical mechanisms. These two general pathogenic categories also correlate well with the characteristic clinical features of these disorders. Genetic disorders of melanocyte function, such as the various types of oculocutaneous albinism (see Chapter 31), are characterized by congenital homogeneously reduced pigmentation due to deficient melanin biosynthesis. In contrast, genetic disorders of melanocyte development (see Chapters 5 and 6), such as piebaldism and the various forms of Waardenburg syndrome, are associated with congenital heterogeneous distribution of pigmentation due to abnormal distribution of melanocytes during embryogenesis. These developmental disorders of melanocyte development stand in contrast to vitiligo (Chapter 30), in which acquired white spotting results from loss of melanocytes from the involved areas. Although the congenital white spotting disorders are individually rare, their characteristic stark patches of white skin (leukoderma) and white hair (poliosis) make them among the most striking of all human disease phenotypes, and therefore these disorders were recognized very early in the history of medicine. In the following discussion of these genetic abnormalities of melanocyte development, MIM numbers for each disorder are from Online Mendelian Inheritance in Man (2004).

Embryology of the Mammalian Pigmentary System Skin melanocytes are highly dendritic cells that originate during embryological development as melanoblasts in the neural crest (Rawles, 1947; see also Chapter 5), whereas melanocytes in the retinal pigment epithelium derive from the optic cup (Chapter 4). The neural crest melanoblasts

subsequently migrate to three principal locations: the epidermal/dermal border of the skin, the hair bulbs which extend into the dermis of the skin, and the uveal tract composed of the iris, choroid, and ciliary body (Bennett, 1991; Fitzpatrick et al., 1981; Hearing and King, 1994; Kawamura et al., 1971; Montagna and Ellis, 1958; Montagna and Parakkal, 1974; Montagna et al., 1991) (see Chapters 4, 5, and 6). The abnormal distribution of skin and hair pigment characteristic of piebaldism and Waardenburg syndrome results from abnormal distribution of melanocytes to the corresponding areas of skin during development, most likely due to defective proliferation of melanoblasts prior to migration to the dermis. In addition to melanocytes, the neural crest is responsible for the development of a great many embryologic structures: connective tissues of the head and neck; components of branchial arch muscles; and nervous tissues, including the sensory and autonomic ganglia and the gut enteric neural plexus. Therefore, developmental abnormalities of pigmentation represent only one of the manifold phenotypic manifestations of so-called “neurocristopathies.” In piebaldism, pigmentary anomalies constitute the only clinically significant feature. However, in the different forms of Waardenburg syndrome, in addition to piebald-like skin depigmentation, there are characteristic abnormalities of other neural crest-derived elements, principally dystopia canthorum (lateral displacement of the inner canthus), deafness, and Hirschsprung disease or megacolon, resulting from defective development of the gut enteric neural plexus.

Piebaldism (MIM #172800) Background and Clinical Features Piebaldism, sometimes incorrectly called “partial albinism,” is 541

CHAPTER 29

Fig. 29.1. A father and daughter with piebaldism resulting from a KIT gene mutation. Note the similar patterns of pigmentary striping. There are areas of depigmentation, and areas of increased and intermediate pigmentation. See also Plate 29.1, pp. 494–495.

an autosomal dominant disorder characterized clinically by congenital patches of white skin and white hair, principally located on the frontal scalp, forehead, ventral chest and abdomen, and extremities (cf. Campbell and Swift, 1962; Comings and Odland, 1966; Cooke, 1952; Froggat, 1951; Geerts-Marchandise and Lachapelle, 1977; Grupper et al., 1970; Jahr and McIntire, 1954; Keeler, 1934; Küster, 1987; Pearson, 1913; Pierard et al., 1978; Smith and Schulz, 1955; Storkan and Hosmer, 1954; Sundfor, 1939; Taylor and Robinson, 1976; Winship et al., 1991). As shown in Figures 29.1–29.3, the regions of leukoderma tend to be generally sharply demarcated and, although they are not precisely symmetric, they tend to be roughly symmetric with respect to the midline, and usually involve only the ventral aspects of the trunk and head. Because of its striking phenotype, piebaldism was known to the ancient Romans (Lucian, 1905; Philostratus, 1917), was probably the first genetic disorder recognized to exhibit autosomal dominant inheritance (Morgan, 1786), and is said to be the first disorder for which a pedigree was presented (reviewed by Froggat, 1951). The leukodermal lesions of piebaldism generally lack melanocytes (Breathnach et al., 1965; Comings and Odland, 1966; Fukai et al., 1989; Jimbow et al., 1975), although scattered melanocytes may sometimes be seen on histologic examination (Barneon and Baldet, 1978; Fukai et al., 1989; Grupper et al., 1970; Hayashibe and Mishima, 1988; Jimbow et al., 1975). Regardless, the population density (cells/mm2) of melanocytes is generally considerably reduced in patches of leukoderma compared with normal. Paradoxically, pigmentation may be increased at the margins of the leukodermal regions, and there are often islands of hyperpigmentation within the patches of leukoderma. These hyperpigmented

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Fig. 29.2. A girl with piebaldism resulting from a KIT gene mutation. Note the white forelock, the depigmentation in the middle of the forehead, and the striped patterns of increased, intermediate, and decreased pigmentation on the trunk. See also Plate 29.2, pp. 494–495.

Fig. 29.3. Depigmentation on the knees in a patient with piebaldism resulting from a KIT gene mutation (see also Plate 29.3, pp. 494–495).

GENETIC HYPOMELANOSES: DISORDERS CHARACTERIZED BY CONGENITAL WHITE SPOTTING

regions appear to contain relatively normal numbers of melanocytes (Breathnach et al., 1965; Jimbow et al., 1975), although their morphology may be aberrant. Café-au-lait spots are not uncommon, and their occurrence has resulted in several patients with piebaldism being misdiagnosed as having neurofibromatosis. In contrast to vitiligo, with which it is sometimes confused, in piebaldism, the leukodermal patches are both congenital and relatively static in shape and size, although limited repigmentation sometimes occurs, especially in milder cases (Davis and Verdol, 1976). Piebaldism is considered to be a lineage-specific developmental disorder of neural crest-derived melanoblasts, most likely involving defective cell proliferation, migration, or survival (Murphy et al., 1992; Steel et al., 1992). Thus, patients with piebaldism typically exhibit only the characteristic pigmentary anomalies of the skin and hair. Pigmentation of the retina and irises is normal, and there are none of the abnormalities of the optic tracts that are characteristic of albinism, and so vision is normal. In contrast, W-mutant mice, the murine homolog to human piebaldism, often manifest both piebald-like “white spotting” and various additional phenotypic features, including gut enteric plexus abnormalities (Huizinga et al., 1995), moderate to severe dyserythropoietic anemia, mast cell deficiency, and germ cell failure (Lyon and Searle, 1989). These findings have been searched for in human patients, but are typically absent. Thus, human patients with piebaldism are typically not anemic (Spritz et al., 1993) or infertile, although dyserythropoietic anemia has been observed in two patients with “piebaldism” who lacked KIT mutations and who both had negative family histories (Costa et al., 2002; Köklü et al., 2002), and who may thus represent a novel syndrome. In general, patients with true piebaldism also do not manifest the various clinical features more typically associated with Waardenburg syndrome. Piebaldism has thus been associated with deafness in only one patient (Spritz and Beighton, 1998), although this may well be mere coincidence. Hirschsprung disease has also been reported in only one patient (Mahakrishnan and Srinivasan, 1980), who in retrospect may actually have had Waardenburg syndrome type 4 (WS4). Piebaldism was reported as being associated with cancer in one family (Gatto et al., 1985); however, the concordance was not perfect, and this again appears most likely to have been coincidence. One patient with apparently homozygous piebaldism has been reported (Hultén et al., 1987), the offspring of firstcousin parents from Pakistan, both affected with typical autosomal dominant piebaldism. Their child had no pigmentation of the skin or hair, blue irides, deafness, severe brachycephaly, hypotonia, and delayed developmental milestones, but no anemia. The phenotype was reminiscent of that of viable W v/W v homozygous mutant mice, and involved abnormalities of multiple neural crest-derived elements. Thus, homozygous piebaldism is clinically much more severe than in the usual heterozygous state, and involves a wider range of defects attributable to aberrant development of the neural crest.

One additional often-discussed but spurious “association” must also be addressed, that between piebaldism and neurofibromatosis type 1 (NF1). This “association” has been reported several times (Angelo et al., 2001; Chang et al., 1993; Riccardi, 1993; Tay, 1998), and several additional case reports have eventually not been published. In fact, the clinical “association” of piebaldism with NF1 is an artifact of misapplication of the minimal clinical diagnostic criteria for NF1, which technically allow the diagnosis of NF1 based on the clinical findings of u 6 café-au-lait spots plus axillary or inguinal freckling. As discussed by Spritz et al. (2004), café-au-lait spots are a common finding in piebaldism, and axillary and/or inguinal freckling also occurs frequently. It is therefore paramount that NF1 should not be misdiagnosed in the context of the more general clinical picture of piebaldism, as the diagnosis of NF1 implies risks (and the need for periodic screening) for serious complications that are not relevant to patients with piebaldism.

Clinical Management Piebaldism is a purely cosmetic defect and, for the great majority of affected individuals, no treatment is sought or required. In some persons, facial hypopigmentation may be significant, although it is usually not overly distressing to Caucasian patients. Treatment of hypomelanotic lesions with either topical 8-methoxypsoralen plus ultraviolet A irradiation (PUVA) (Fukai et al., 1989) or oral methoxsalen plus ultraviolet A (Hayashibe and Mishima, 1988) has not achieved clinically significant repigmentation. However, attempts to repigment localized regions of hypomelanosis by autologous minigrafts of normally pigmented skin have met with some success (Falabella, 1978; Selmanowitz et al., 1977), as have attempts to transplant autologous cultured melanocytes into hypopigmented regions (Lerner et al., 1987). Although these procedures are clearly not applicable to repigmentation of large areas of hypomelanosis, particularly on the face, they are of use in some clinical circumstances.

Incidence and Genetics Piebaldism is relatively rare, with an estimated incidence of perhaps 1 per 100 000. It is inherited as a typical autosomal dominant disorder, with equal frequencies in males and females. As discussed in Chapter 6, classical piebaldism results from mutations in the KIT proto-oncogene, and prenatal diagnosis of piebaldism has been accomplished by DNA-based mutation analysis of the KIT gene (R. A. Spritz, unpublished data). Mutations of the KIT gene are not found in about a quarter of patients with typical piebaldism (Ezoe et al., 1995), suggesting that additional human piebaldism loci may remain to be identified. Thus, deletions involving the SLUG (SNAI2) gene have been described recently in some of these patients who lack mutations in KIT (Sánchez-Martín et al., 2003). A number of patients have been reported in whom sporadic, nonfamilial piebaldism associated with dysmorphic features and usually mental retardation results from chromosomal

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CHAPTER 29

Fig. 29.4. A boy with Waardenburg syndrome type I due to a deletion of distal chromosome 2q that includes the PAX3 gene. This child manifests clinical characteristics of WS1 that include dystopia canthorum, heterochromia irides, small white forelock (not visible here, upper panel), and a large area of thoraco-abdominal leukoderma (lower panel). Upper panel published with permission from Susan Kirkpatrick and Wiley-Liss. Lower panel published with permission from Susan Kirkpatrick. (See also Plate 29.4, pp. 494–495).

deletions and other rearrangements affecting the KIT gene on 4q12 (Fujimoto et al., 1998; Funderburk and Crandall, 1974; Hoo et al., 1986; Lacassie et al., 1977; Ramadevi et al., 2002; Schinzel et al., 1997; Sijmons et al., 1993; Yamamoto et al., 1989); therefore, a high-resolution karyotype and perhaps fluorescent in situ hybridization (FISH) using a KIT probe must be performed in patients who present with such atypical piebaldism phenotypes. Nevertheless, many patients with atypical presentations of piebaldism have no detectable defects of KIT or SLUG (Ezoe et al., 1995), suggesting that these patients may have mutations in other genes that remain to be discovered.

Waardenburg Syndrome Background and Clinical Features Waardenburg syndrome, as first described in 1951 by a Dutch ophthalmologist (Waardenburg, 1951), is an autosomal dom544

inant disorder characterized clinically by piebald-like pigmentary anomalies of the skin and hair, pigmentary abnormalities of the iris (heterochromia irides), lateral displacement of the inner canthi of the eyes (dystopia canthorum), and sensorineural deafness (Arias, 1971; DiGeorge et al., 1960; Waardenburg, 1951) (Fig. 29.4). Waardenburg syndrome is said to account for at least 0.5% of cases of congenital deafness. All the abnormalities in Waardenburg syndrome involve the neural crest, and both Hirschsprung disease (Ariturk et al., 1992; Currie et al., 1986) and neural tube defects (Bergleiter and Harris, 1992; Carezani-Gavin et al., 1992; Chatkupt et al., 1993; Kromberg and Krause, 1993) occur at increased frequencies among affected individuals. Waardenburg syndrome has thus been considered to be a more global disorder of neural crest development than piebaldism. Four principal subtypes of Waardenburg syndrome have been distinguished on clinical grounds (Asher and Friedman, 1990; Farrer et al., 1994). Waardenburg syndrome type I (WS1; MIM #193500) is the classic form. Waardenburg syndrome type II (WS2; MIM #193510) lacks dystopia canthorum (Liu et al., 1995). Waardenburg syndrome type III (WS3; Klein-Waardenburg syndrome; MIM #148820) is associated with limb abnormalities as well as dystopia canthorum (Klein, 1950). Waardenburg syndrome type IV (WS4; WaardenburgShah syndrome; MIM #277580) is similar to WS3, but also includes Hirschsprung disease (Shah et al., 1981). WS1 and WS2 are inherited in an autosomal dominant manner, whereas WS4 is an autosomal recessive condition, and WS3 can be either dominant or recessive, depending on the specific gene mutation. In fact, these clinical designations clearly require revision in view of recent molecular genetic findings that have demonstrated at least nine distinct genetic subtypes of Waardenburg syndrome, several of which correspond to distinct clinical subtypes not recognized under the four classical subtypes. Thus, WS1 and WS3 are allelic, both resulting from abnormalities of the PAX3 gene. A clinically somewhat different disorder, craniofacial–deafness–hand syndrome (MIM #122880), also turns out to be allelic to WS1 and WS3. “WS2” has now been found to result from mutations in at least four different genes: MITF (WS2A), SLUG/SNAI2 (WS2D), and as yet unidentified genes on chromosomes 1p21–p13.3 (WS2B) and 8p23 (WS2C). Similarly, “WS4” has been subdivided based on the finding of mutations in at least three genes, EDN3, EDNRB, and SOX10, each associated with overlapping but clinically distinct phenotypes. These disorders are discussed separately below and in Chapter 6.

Incidence and Genetics Waardenburg syndrome is relatively rare, with an estimated incidence of perhaps 1 per 20 000–40 000 (Delleman and Hageman, 1978). It is usually inherited as a typical autosomal dominant disorder, with equal frequencies in males and females, and new mutations tending to occur in offspring of older fathers (Jones et al., 1975). Autosomal recessive forms of Waardenburg syndrome are relatively rare.

GENETIC HYPOMELANOSES: DISORDERS CHARACTERIZED BY CONGENITAL WHITE SPOTTING

Waardenburg Syndrome Type I (WS1; MIM #193500), Waardenburg Syndrome Type III (WS3; Klein-Waardenburg Syndrome; MIM #148820), and Craniofacial–Deafness–Hand Syndrome Waardenburg syndrome type I, the classic form, is an autosomal dominant disorder characterized clinically by hypopigmented patches, heterochromia irides, dystopia canthorum, and sensorineural deafness (Fig. 29.4). The clinical features of WS1 vs. WS2 have been reviewed in detail (Pardono et al., 2003; Reynolds et al., 1995), and the finding of dystopia canthorum, measured by the W-index, is an important adjunct in the clinical diagnosis of WS1 (Reynolds et al., 1995). The hypopigmented patches in WS1 are generally similar to those in piebaldism, although hypopigmentation in WS1 is typically less severe in degree and extent than in piebaldism. WS3 is similar to WS1, but is additionally associated with musculoskeletal abnormalities. Craniofacial–deafness–hand syndrome is an autosomal dominant disorder with deafness, more severe abnormalities of craniofacial development than WS1, and developmental anomalies of the hands. As discussed in Chapter 6, all three result from mutations in the same gene, PAX3. At least three children with apparently homozygous WS1 have been reported (Ayme and Phillip, 1995; Wollnik et al., 2003; Zlotogora et al., 1995). In each case, the consanguineous heterozygous parents exhibited features of WS1, whereas the homozygous child had a much more severe phenotype, consistent with the diagnosis of WS3, variously including dysmorphic facial features, pigmentary anomalies, flexion contractures, and exencephaly. In the two cases studied, the patients were homozygous for mutations in PAX3, and the parents were each heterozygous for PAX3 mutations. Homozygous WS1 is thus clinically much more severe than in the heterozygous state, and can be considered one cause of WS3.

Fig. 29.5. A mutation of the endothelin B receptor gene that produces megacolon and depigmentation (Waardenburg syndrome type IV). Courtesy of Dr. Susan Mallory. See also Plate 29.5, pp. 494–495.

Waardenburg Syndrome Type II (WS2; MIM #193500)

Waardenburg Syndrome Type IV (WS4; Shah-Waardenburg Syndrome; MIM #277580)

The phenotype of Waardenburg syndrome type II is very similar to that of WS1 except that patients with WS2 lack the clinical feature of dystopia canthorum (Arias, 1971; Farrer et al., 1992), and hypopigmentation in WS2 tends to be rather mild in both degree and extent. In fact, the clinical distinction between WS1 and WS2 can be problematic, and must ultimately depend on molecular findings (Reynolds et al., 1995). As discussed in Chapter 6, WS2A results from mutations in the MITF gene. However, most patients with the phenotype of WS2 do not have mutations in MITF, and mutations in SLUG/SNAI2 have been found in some such patients (Sánchez-Martín et al., 2002). Two additional WS2 loci have been mapped, to chromosomes 1p21–p13.3 (WS2B; Lalwani et al., 1994) and 8p23 (WS2C; Selicorni et al., 2002), but the corresponding genes have not yet been identified.

Waardenburg-Shah syndrome is an autosomal recessive disorder phenotypically similar to WS3, although sometimes with more extreme hypopigmentation (Gross et al., 1995) and involving the additional feature of Hirschsprung disease, the congenital absence of intrinsic ganglion cells of the myenteric and submucosal plexi of the gastrointestinal tract (aganglionic megacolon). There is no deafness (Fig. 29.5) (Gross et al., 1995; Shah et al., 1981). Like melanocytes, gut enteric plexus ganglion cells are derived embryologically from the neural crest, and so it is not surprising that there would be an association between these disorders. In fact, as noted above, Hirschsprung disease has been reported in rare cases of piebaldism (Mahakrishnan and Srinivasan, 1980), WS1 (Ariturk et al., 1992; Currie et al., 1986), and WS2 (van Camp et al., 1995). Similarly, Hirschsprung disease itself has occasionally been associated with pigmentary anomalies, in both humans and mice. At least three distinct clinical subtypes of “WS4” have been found to result from mutations in three dif545

CHAPTER 29

Figs 29.6 and 29.7. Children with large macules of hyperpigmentation, particularly in the inguinal and buttock regions on a hypomelanotic background. [From Ziprowski et al., 1962, with permission.]

ferent genes, and will thus be discussed individually below. As discussed in Chapter 6, classic Waardenburg-Shah syndrome results from mutations in the EDN3 gene.

2002, 2004; Pingault et al., 1998, 2000, 2002), leading to the suggestion of the rather verbose name and pithy acronym (Inoue et al., 2004).

Peripheral Demyelinating Neuropathy, Central Demyelinating Leukodystrophy, Waardenburg Syndrome, Hirschprung Disease (PCWH)

Waardenburg-Hirschprung Disease (HSCR2; MIM #600155)

A subset of “WS4” patients have heterozygous mutations in the SOX10 gene, which encodes a transcriptional activator for a number of genes involved in neural crest development, particularly MITF (Pingault et al., 1998; 2000). Some, but not all, patients with SOX10 mutations exhibit neurological abnormalities (Pingault et al., 2002), probably accounting for most cases of the so-called “Waardenburg-Shah neurologic variant.” Indeed, the range of clinical manifestations associated with SOX10 mutations is quite broad (Inoue et al., 1999, 546

HSCR2 is a rare, autosomal recessive disorder, originally classified as part of Waardenburg–Shah syndrome, in which Hirschsprung disease is frequently associated with white forelock. There is no deafness. The mapping of the HSCR2 locus to the same chromosomal region as the gene encoding the EDN3 receptor (EDNRB; see above) immediately suggested EDNRB as a candidate gene for HSCR2 (Puffenberger et al., 1994). As discussed in Chapter 6, heterozygous mutations of EDNRB have now been identified in a number of patients with HSCR2.

GENETIC HYPOMELANOSES: DISORDERS CHARACTERIZED BY CONGENITAL WHITE SPOTTING

mentation phenotype would surely have been noticed. It may thus be that this disorder is an atypical phenotype of a gene in which most mutations are lethal or result in a different phenotype. Genetic linkage studies and somatic cell hybrid mapping studies have localized the ADFN gene to Xq26.3–q27.1 (Shiloh et al., 1988, 1990). The gene has not yet been identified.

References

Fig. 29.8. Two brothers with Woolf’s syndrome. Both have piebaldism and congenital deafness. [From Woolf et al., 1965, with permission.]

Albinism–Deafness Syndrome (ADFN; Ziprkowski–Margolis Syndrome; Woolf Syndrome; MIM #300700) Several additional human genetic disorders of melanocyte development have been described that have yet to be associated with specific genes. The best defined of these is the “albinism–deafness syndrome.” This X-linked recessive disorder was first described by Margolis (1962) and Ziprkowski (1962) in a large Israeli kindred. Affected males have congenital subtotal neural deafness and exhibit an extreme piebaldlike phenotype, with hypopigmented areas over most of the body and extending well onto the dorsal aspects of the trunk and head (Figs 29.6 and 29.7). Several patients have heterochromia irides. Campbell et al. (1962) and Woolf (Woolf, 1965; Woolf et al., 1965) (Fig. 29.8) described what may have been the same disorder in Hopi Indians. No other cases have been reported, which seems quite surprising in view of the very striking phenotype, leading Zlotogora (1995) to suggest that ADFN might be a rare manifestation of some other X-linked disorder. It is also surprising that no X-linked spotting phenotypes exist in the mouse, in which such an extreme pig-

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piebald trait maps to proximal 4q12. J. Med. Genet. 34:692–695, 1997. Selicorni, A., S. Guerneri, A. Ratti, and A. Pizzuti. Cytogenetic mapping of a novel locus for type II Waardenburg syndrome. Hum. Genet. 110:64–67, 2002. Selmanowitz, V. J., A. D. Rabinowitz, N. Orentriech, and E. Wenk. Pigmentary correction of piebaldism by autografts. J. Dermatol. Surg. Oncol. 3:615–622, 1977. Shah, K. N., S. J. Dalal, M. P. Desai, P. N. Sheth, N. C. Joshi, and L. M. Ambani. White forelock, pigmentary disorder of irides, and long segment Hirschsprung disease; possible variation of Waardenburg syndrome. J. Pediatr. 99:432–435, 1981. Shiloh, Y., L. Sandkuyl, G. Litvak, Y. Ziv, M. Hildesheimer, V. Buchris, and J. Ott. Localization of X-linked albinism–deafness syndrome (ADFN) to the region Xq26–27.1 by linkage analysis. Am. J. Hum. Genet. 43:A158, 1988. Shiloh, Y., G. Litvak, Y. Ziv, T. Lehner, L. Sandkuyl, M. Hildesheimer, V. Buchris, F. P. M. Cremers, P. Szabo, B. N. White, J. J. A. Holden, and J. Ott. Genetic mapping of X-linked albinism–deafness syndrome (ADFN) to Xq26.3–27.1. Am. J. Hum. Genet. 47:20–27, 1990. Sijmons, R. H., U. Kristoffersson, J. H. Tuerlings, R. Ljung, R. Dijkhuis-Stoffelsma, and A. S. Breed. Piebaldism in a mentally retarded girl with rare deletion of the long arm of chromosome 4. Pediatr. Dermatol. 10:235–239, 1993. Smith, N. G., and J. Schulz. Partial albinism. Arch. Dermatol. Syphil. 71:468–470, 1955. Spritz, R. A., and P. Beighton. Piebaldism with deafness: molecular evidence for an expanded syndrome. Am. J. Med. Genet. 75:101–103, 1998. Spritz, R. A., S. A. Holmes, and M. H. Freedman. Lack of mutations of the MGF and KIT genes in Diamond–Blackfan anemia. Blood 81:3165, 1993. Spritz, R. A., P. H. Itin, and D. H. Gutmann. Piebaldism and neurofibromatosis type 1: horses of very different colors. J. Invest. Dermatol. 122: xxxiv–xxxv, 2004. Steel, K. P., D. R. Davidson, and I. J. Jackson. TRP2/DT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor. Development 115:1111–1119, 1992. Storkan, M. A., and J. A. Hosmer. Vitiligo associated with poliosis and ichthyosis. Arch. Dermatol. Syphil. 70:540–541, 1954. Sundfor, H. A pedigree of skin-spotting in man. J. Hered. 30:60–77, 1939. Tay, Y. K. Neurofibromatosis 1 and piebaldism: a case report. Dermatologica 197:401–402. Taylor, D. R., and T. Robinson. Piebaldism. Br. J. Dermatol. 95 (Suppl. 14):43–44, 1976. van Camp, G., M. N. van Thienen, I. Handig, B. Van Roy, V. S. Rao, A. Milunsky, A. P. Read, C. T. Baldwin, L. A. Farrer, M. Bonduelle, L. Standaert, F. Meise, and P. J. Willems. Chromosome 13q deletion with Waardenburg syndrome: further evidence for a gene involved in neural crest function on 13q. J. Med. Genet. 32:531–536, 1995. Waardenburg, P. J. A new syndrome combining developmental anomalies of the eyelids, eyebrows and nose root with pigmentary defects of the iris and head hair and with congenital deafness. Am. J. Hum. Genet. 3:195–253, 1951. Winship, I., K. Young, R. Martell, R. Ramesar, D. Curtis, and P. Beighton. Piebaldism: an autonomous autosomal dominant entity. Clin. Genet. 39:330–337, 1991. Wollnik, B., O. Tukel, O. Uyguner, A. Ghanbari, H. Kayserili, M. Emiroglu, and M. Yuksel-Apak. Homozygous and heterozygous inheritance of PAX3 mutations causes different types of Waardenburg syndrome. Am. J. Med. Genet. A. 122:42–45, 2003.

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CHAPTER 29 Woolf, C. M. Albinism among Indians in Arizona and New Mexico. Am. J. Hum. Genet. 17:23–25, 1965. Woolf, C. M., D. A. Dolowitz, and H. E. Aldous. Congenital deafness associated with piebaldness. Arch. Otolaryngol. 82:244–250, 1965. Yamamoto, Y., H. Nishimoto, and S. Ikemoto. Interstitial deletion of the proximal long arm of chromosome 4 associated with father–child incompatibility within the Gc-system: probable reduced gene dosage effect and partial piebald trait. Am. J. Med. Genet. 32:520–523, 1989.

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Ziprkowski, L., A. Krakowski, A. Adam, H. Costeff, and J. Sade. Partial albinism and deaf-mutism due to a recessive sex-linked gene. Arch. Dermatol. 86:530–539, 1962. Zlotogora, J. X-linked albinism-deafness syndrome and Waardenburg syndrome type II: a hypothesis. Am. J. Med. Genet. 59:386–387, 1995. Zlotogora, J., I. Lerer, S. Bar-David, Z. Ergaz, and D. Abeliovich. Homozygosity for Waardenburg syndrome. Am. J. Hum. Genet. 56:1173–1178, 1995.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Genetic Hypomelanoses: Acquired Depigmentation Sections Rozycki Syndrome Jean L. Bolognia Vitiligo Vulgaris James J. Nordlund, Jean-Paul Ortonne, and I. Caroline Le Poole

Rozycki Syndrome (MIM #221350) Jean L. Bolognia In 1971, Rozycki et al. described two siblings in whom deafness was associated with short stature, vitiligo, muscle wasting, and achalasia. The parents were first cousins and the presumed mode of inheritance was autosomal recessive. A profound congenital sensorineural hearing loss was the presenting feature in both patients. In addition to marked muscle wasting of the hands, legs, and feet, “depigmented areas” were noted on their torsos and necks (Fig. 30.1). Unfortunately, this was the extent of the clinical description of the cutaneous findings. Because of a history of difficulty in swallowing, a barium swallow was done on both individuals (ages 11 and 12 years) and evidence of achalasia was seen. This was confirmed by esophageal pressure and methacholine studies. Although normal muscle fibers and nerves were observed in a muscle biopsy specimen, there was evidence of neuropathy as well as myopathy by electromyography in both children. A literature review failed to reveal additional patients with the same constellation of findings. It is difficult to say with certainty that the disorder of hypopigmentation that these patients had was vitiligo. However, one of the authors (R. Ruben, personal communication) stated that the cutaneous findings were obvious and the diagnosis was straightforward.

References Rozycki, D. L., R. J. Ruben, I. Rapin, and A. J. Spiro. Autosomal recessive deafness associated with short stature, vitiligo, muscle wasting and achalasia. Arch. Otolaryngol. 93:194–197, 1971.

Vitiligo Vulgaris James J. Nordlund, Jean-Paul Ortonne, and I. Caroline Le Poole

Historical Background Vitiligo is a disease that leaves depigmented spots on the skin. The word “vitiligo” seems to have been derived from the Latin word “vitium” meaning a blemish or “vitulum” meaning a small blemish (Mercurialis, 1572; Nair, 1978). It is probable

that, in ancient times, the references to white spots on the skin represented not only vitiligo vulgaris but also other disorders such as leprosy that leave white spots on the skin (Mercurialis, 1572; Singh et al., 1974). It has been only in the last century that the term vitiligo vulgaris has been used specifically for the acquired, progressive disorder characterized by destruction of melanocytes in the skin and other organs. It seems likely that vitiligo was recognized several millennia before Christian times (Hann and Nordlund, 2000). Some of the earliest writings date from around 1500 BC (Nair, 1978). The Ebers Papyrus notes that there are several types of leukoderma, one associated with swellings of the skin, the other macular. The first type might be a description of leprosy, the second a description of vitiligo vulgaris. In the early Vedic scripture Atharvaveda (circa 1500 bc) from India, Kilas or white disease that might represent vitiligo is described. Around 800 bc, “svitra” meaning whiteness is mentioned in the “Charaka samhita,” a medical treatise. In the ancient Japanese book Amarakosa (1200 bc), a collection of Shinto prayers, there is described a disorder called “Shira bito,” which means white man. Whether that refers to albinism, vitiligo, or both is not known. In the Western societies, Hippocrates described white spots on the skin but did not seem to distinguish vitiligo and leprosy or other disorders of depigmentation. In Leviticus, Book 13 of the Old Testament, a variety of disorders characterized by hypo- or depigmentation are described. The Talmud records the association of sudden onset of white hair with vitiligo vulgaris (Goldenhersh, 1992). Therapies were mentioned in these same ancient writings. Some of the recommended herbs included plants now known to contain psoralen. The herbs were administered either orally or topically and the patient exposed to sunlight. The importance of sunlight was emphasized in the Charaka samhita. It seems that psoralens and exposure to ultraviolet light (PUVA) was a common ancient treatment. The plant Ammi majus was first noted in the Arabic book “mofradal Al Adwiya” written by Ibn El Bitar who lived in the thirteenth century ad. Hippocrates described many features of vitiligo that have been emphasized in recent years. He noted that vitiligo was more easily treated when first diagnosed rather than many years after onset. Vaghbhata, in his Indian medical textbook, stated that vitiligo associated with white hairs in the lesion was not easily treated, a concept that will be re-emphasized in 551

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Fig. 30.1. Macule of hypopigmentation on the ankle of a 19-yearold girl with deafness, muscle wasting, and achalasia. [From Rozycki, D. L., R. J. Ruben, I. Rapin, and A. J. Spiro. Autosomal recessive deafness associated with short stature, vitiligo, muscle wasting and achalasia. Arch. Otolaryngol. 93:194–197, copyright 1971, American Medical Association.]

later sections on therapy (this historical review is abstracted from Nair, 1978). Mercurialis attempted to explain the pathogenesis of the depigmentation in his book “De Morbus Cutaneis” (Mercurialis, 1572). He suggested that, if phlegm or “mucous blood” nourished the skin rather than blood, the skin turned white. He distinguished the disease from morphea, which he thought was hyperpigmented. He distinguished several different forms of depigmentation and suggested therapeutic approaches. He also noted that depigmented skin might manifest altered sensation. Gottheil, in his nineteenth century book, called vitiligo vulgaris a form of atrophy of the pigment cells (Gottheil, 1897).

Synonyms Synonyms for vitiligo vulgaris include leukoderma, depigmentation, and acquired depigmentation (Hann and Nordlund, 2000).

Epidemiology and Genetics The prevalence of vitiligo vulgaris is usually said to be about 1 per 100 population, although this estimate may be too high. It has also been assumed that the prevalence is similar throughout the world and in all ethnic groups. However, the data to support this frequency are few. The results of several epidemiologic studies suggest these estimates are too high. Mehta did an extensive survey in India in and around the city of Surat (Mehta et al., 1973). He surveyed households and examined family members in each home for stigmata of vitiligo. He examined 1887 rural inhabitants and 7178 urban citizens. About 60% of the individuals in each area were examined. He found that the prevalence of vitiligo vulgaris was 0.47% (1 per 212) in the rural population and 1.78% in the urban area (1 per 56 individuals). Men and women were 552

affected equally. Many of the geographic areas were occupied almost exclusively by members of one caste or tribe. The prevalence did vary significantly from group to group. Some groups had a prevalence rate of 0%, others up to 3.6%. Although there was grouping of affected individuals within kinships, suggesting a genetic component to the cause, he found that most families had only one affected member, occasionally two. This observation suggested a hereditary predisposition activated by some environmental agent. These conclusions are supported by the results of very recent studies (Majumder et al., 1988, 1993; Nath et al., 1994). On the Island of Bornholm, Denmark, over 47 000 individuals were surveyed for the presence of vitiligo. The subjects were ascertained by announcements in public media such as newspapers and radio and through cooperation with family doctors. The prevalence of vitiligo in this population was 0.38% or 1 per 263 individuals. Men and women were affected equally (Howitz et al., 1977). More recent studies of large groups from Calcutta, India, indicate a prevalence rate of about 1 per 200 (Das et al., 1985a, b). The prevalence was similar for men and women. There was an aggregation within kinships, an observation supporting the concept of a genetic predisposition. That vitiligo clusters within families seems to be confirmed in many studies (Das et al., 1985a, b; Hafez et al., 1983; Majumder et al., 1988; Mayenburg et al., 1976; Mehta et al., 1973; Nath et al., 1994; Salamon et al., 1989). The results from all these studies suggest that vitiligo is not transmitted in a simple Mendelian autosomal dominant or recessive manner. Rather, the transmission seems to be complex and polygenic.

Conclusions From these data, the general prevalence seems to be closer to 1 per 200 individuals. The data from one group should be extrapolated with caution to other populations. That vitiligo vulgaris is clustered within kinships seems to be a consistent finding, an observation supporting the concept that genetic aberrations produce a propensity to develop the disease when aggravated by environmental factors. The genetic susceptibility apparently requires more than one gene, possible three or four genes (Majumder et al., 1988, 1993). Groups of individuals isolated by geography or disposed to marriage within a limited group might have significantly higher prevalence rates than larger populations. Such groups might be ideal for studies on the genetic basis of vitiligo.

Clinical Findings Definition of Vitiligo Vulgaris Vitiligo vulgaris is a disease that begins during postnatal life and, with time, progressively destroys the melanocytes in the skin, mucous membranes and, in some individuals, melanocytes in the eyes and ears (Lerner and Nordlund, 1978a). Although it commonly thought of as just a cosmetic disease, it has a great significance for the patient, the dermatologist and pigment cell biologist (Lerner and Nordlund, 1978b; Nordlund, 1992; Nordlund and Lerner, 1982).

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

Age Vitiligo begins after the birth of an individual, although there are anecdotes that a few infants have been born with vitiligo. Distinguishing congenital vitiligo from piebaldism can be difficult. Infants as young as 4 months have been observed to develop vitiligo. The mean and median age of onset is about 20–23 years (Alkhateeb et al., 2003; Majumder et al., 1988, 1993; Nath et al., 1994; Ortonne et al., 1976a). That is, about half the individuals develop vitiligo vulgaris before the age of 20 years. About a quarter of those who are afflicted by vitiligo will be under 10 years of age when the first manifestations occur (Giam, 1988; Grimes et al., 1986; Jaisankar et al., 1992; Khamaganova et al., 1979; Lacour, 1988; Ortonne et al. 1976a, b; Pinto and Bolognia, 1991). Most individuals develop vitiligo before the age of 40 years, but there are exceptions and elderly patients can be in the ninth decade of life when the first white spots appear (Nordlund and Majumder, 1997). These elderly individuals do not seem to have any underlying disorders that might be considered a predisposing cause for the depigmentation.

Sex The typical population seeking treatment for vitiligo vulgaris is composed of more women than men, usually in a ratio of 2–3:1 (Ortonne et al., 1976a, b). This difference between the sexes might be more apparent than real and might represent the willingness of women to seek medical assistance. Epidemiologic and family studies seem to indicate that males and females are affected equally (Das et al., 1985a, b; Majumder et al. 1988, 1993; Mehta et al. 1973; Nath et al., 1994).

Cutaneous Manifestations There are two commonly recognized forms of vitiligo, i.e. generalized and segmental. The generalized form is characterized by depigmented macules involving both sides of the body in a remarkably symmetrical pattern. For each spot on one side of the body, there is a spot similar in size and location on the other side. This type of vitiligo might be better labeled bilateral, symmetrical vitiligo vulgaris (Fig. 30.2). The other form is segmental vitiligo. It is characterized by unilateral, asymmetrical depigmentation (Fig. 30.3). It could be termed unilateral, asymmetrical vitiligo. Its features are discussed later in this section.

Fig. 30.2. Extensive vitiligo demonstrating its remarkable bilateral, symmetrical distribution (see also Plate 30.1, pp. 494–495).

Bilateral, Symmetrical Vitiligo Generalized or bilateral, symmetrical vitiligo presents as macular depigmentation most commonly observed first on the fingers, hands, and face (Fig. 30.4). The depigmentation is macular, and the epidermis shows no evidence of atrophy or any other changes. The presence of epidermal atrophy should suggest some other disorder. Those with darker skin (type III or IV) might note trichrome or multichrome (Dupre and Christol, 1978; Fargnoli and Bolognia, 1995; Kenney, 1988) vitiligo (Fig. 30.5), i.e. areas of depigmentation, various degrees of hypopigmentation, and normal colored skin. Multicolored vitiligo probably represents epidermis with partial loss

Fig. 30.3. Unilateral, asymmetrical vitiligo usually labeled segmental vitiligo. Note that the depigmentation does not follow a dermatomal pattern (see also Plate 30.2, pp. 494–495).

of melanocytes. These changes are easily seen in individuals with a dark or deeply tanned skin. Depigmentation is not easily seen in individuals with very fair skin, and a Wood’s lamp can assist greatly in defining the areas involved. Some areas such as the palms (Fig. 30.6) and soles (Fig. 30.7) are 553

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Fig. 30.4. Note the bilateral and symmetrical involvement of the fingers and the dorsum of the hands (see also Plate 30.3, pp. 494–495).

Fig. 30.7. Vitiligo of the soles.

Fig. 30.8. Hyperpigmented borders surrounding vitiligo macules (see also Plate 30.6, pp. 494–495). Fig. 30.5. Trichrome vitiligo. Note the partial loss of color as an indicator of partial destruction of melanocytes (see also Plate 30.4, pp. 494–495).

Fig. 30.6. Vitiligo of the palms. The palms and soles must be examined with a Wood’s lamp to see vitiligo in individuals with light-colored skin (see also Plate 30.5, pp. 494–495).

554

light colored in all individuals and are often affected by vitiligo. The depigmentation of this skin is discernible only with a Wood’s lamp. The scalp is also frequently involved but requires a Wood’s lamp to detect the pigment loss. A typical lesion is depigmented in the center with rather discrete borders. Many lesions have a hypopigmented edge called trichrome vitiligo (Dupre and Christol, 1978; Fargnoli and Bolognia, 1995; Kenney, 1988) where partial destruction of melanocytes has occurred. Other lesions will have hyperpigmented borders (Fig. 30.8). The melanocytes in the hair follicles are usually privileged and spared destruction by the melanocytotoxic process(es). The depigmented skin typically has pigmented hairs emanating from the follicles. This is most prominent on the scalp, especially in individuals with dark skin. The interfollicular skin of the scalp can be completely depigmented, but most of the hairs are normally pigmented (Fig. 30.9). Depigmentation spreads in two ways, by enlargement of existing lesions and by development of new macules. Many individuals with vitiligo will note an occasional lesion with a raised red border (Kumakiri et al., 1982; Macmillan and Rook, 1971; Oliveira Neto, 1972; Ortonne et al., 1976b;

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

Fig. 30.9. Vitiligo of the scalp. This is a common area of depigmentation but the scalp is rarely examined.

Fig. 30.11. Contact dermatitis induced by monobenzone, exclusively on depigmented skin (see also Plate 30.8, pp. 494–495).

Fig. 30.10. A patch of vitiligo with raised, erythematous borders (see also Plate 30.7, pp. 494–495).

Fig. 30.12. Vitiligo of the genitalia and perirectal skin.

Watzig, 1974) (Fig. 30.10). Less commonly (Dupre and Christol, 1978; Fargnoli and Bolognia, 1995; Kenney, 1988), one observes numerous inflammatory lesions. These inflammatory lesions show an edematous border and often slight scaliness. As the inflammatory component disappears, the skin becomes depigmented. The significance of one or more inflammatory lesions is not known, although it has been suggested that patients who exhibit this inflammatory pattern are atopic (Macmillan and Rook, 1971). Patients who are treated with the depigmenting compound monobenzone often manifest an inflammatory reaction in the pigmented skin (Nordlund et al., 1985a) (Fig. 30.11). An occasional patient with an inflammatory component has been found to be in contact with depigmenting agents. It is worth questioning patients about exposures to chemical depigmenting agents at home, at work, or in their avocations or hobbies. Patients with vitiligo have been observed who have a blue discoloration on the affected skin. The color is due to dermal melanosis or postinflammatory depigmentation (Ivker et al., 1994).

Recent studies suggest that vitiligo in the patients’ perception begins most commonly on the fingers, hands, face (Chalfin and Putterman, 1980), and feet (Nordlund and Majumder, 1997). Vitiligo can begin on any part of the integument. It typically spreads from the acral sites of onset to involve the central integument on the legs and abdominal skin. The skin of the back is usually the last to develop depigmentation and is the least involved. It is important to note that virtually any pattern of onset and involvement can be seen. It has been suggested that vitiligo begins on or preferentially affects the bodily orifices (Lerner and Nordlund, 1978a, b). Most patients do not note that the umbilicus, the genitalia, or perirectal areas are involved early in the disease, although these areas are involved in most patients later in the disease (Fig. 30.12). Occasionally, vitiligo seems to affect the genitalia almost exclusively (Moss and Stevenson, 1981). The typical progression of the depigmentation from acral sites centripetally suggests that acrofacial vitiligo is an earlier form of generalized vitiligo. 555

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Fig. 30.13. Example of spontaneous repigmentation demonstrating the role of natural sunlight.

The spread of vitiligo can stop at any time leaving the patient spotted. This is the worst outcome for the patient because it is most disfiguring. In a few patients, bilateral vitiligo vulgaris can spread to involve the entire integument. Such completely depigmented patients have a normal appearance, that is their skin is one color albeit very fair. These individuals have a very young appearance because they do not have the pigmented macules associated with aging such as lentigines or pigmented seborrheic keratoses. Most observers will not detect the total loss of color unless the individual had dark skin. From the biologic perspective, this is the most severe outcome. From the patient’s perspective, the outcome is very desirable. Vitiligo can repigment spontaneously (Figs 30.13 and 30.14). The implication of this statement is that the mechanism for stimulation of proliferation and migration of melanocytes into the depigmented skin has not been identified. Although this phenomenon is not observed uncommonly in a few lesions in many patients, it has been reported rarely (Kenney, 1988; Trim and Sequeira, 1931). Three patients have been presented at conferences, all of whom were almost completely depigmented. The three gained back most of their color spontaneously, although the hands, feet, lips, and areas with white terminal hairs did not repigment. The lack of repigmentation in these locations is typical and is probably related to the reservoir for pigment cells (see section on Therapy and the Melanocyte Reservoir in this chapter).

Unilateral, Asymmetrical Vitiligo Segmental vitiligo clinically is a very different disease. In contrast to generalized vitiligo vulgaris, which causes bilateral disease that is very symmetrical, segmental vitiligo is unilateral and asymmetrical. Possibly, it is a form of depigmentation that has a completely different pathophysiology than the generalized form. Few, if any, patients with segmental vitiligo develop the generalized form of the disease. This can be re-

556

Fig. 30.14. Example of spontaneous repigmentation demonstrating the role of natural sunlight (see also Plate 30.9, pp. 494–495).

assuring to the patient, especially if the segment is on a nonexposed patch of skin. Segmental vitiligo seems to affect children more often, although it can appear in adults as well (Barona et al., 1995; Halder et al., 1987; Hann, 2000; Hann et al., 2000). The onset is similar to generalized vitiligo, except the depigmentation is located only in the involved segment of skin. The depigmentation spreads within the segment for a short period of time and then stops. It leaves the area partially or totally depigmented. The involved skin is often considered dermatomal. In an occasional patient, the depigmentation does follow a dermatomal pattern. However, most patients have an affected area that does not conform to any one or group of sensory nerves or autonomic nerves (Fig. 30.15). The patterns of segmental vitiligo do not resemble Blaschko’s lines or developmental metamers. Half of the face, an arm, a leg, a portion of the trunk can be involved (Figs 30.16 and 30.17A, B). In contrast to generalized vitiligo, segmental vitiligo seems to affect the melanocytes in the follicles of about half of those affected (Nelhaus, 1970). The hairs are often completely white, indicating that the pigment cells in the fol-

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

Fig. 30.15. A lady with segmental (nondermatomal) vitiligo that extends onto the buccal mucosa (see Fig. 30.19 and Plate 30.12A, pp. 494–495).

A

Fig. 30.16. Unilateral, asymmetrical (segmental) vitiligo on the face (see also Plate 30.10, pp. 494–495).

licular bulb have been destroyed. This skin is not likely to repigment with any medical therapy. It might be the reason why segmental vitiligo has been observed to be resistant to standard therapies (cf. section on Depigmentation of Hair and Therapy in this chapter). The contrast is not noticeable to others. Segmental vitiligo on the face is as disfiguring as the generalized form.

Dermatoglyphics In several studies, the dermatoglyphics of patients with vitiligo have been compared with those of control subjects. Statistically significant differences between the two groups have been found, such as an increase in the number of whorls, arches, and ridges on the finger tips (Iqbal et al., 1985; Sahasrabuddhe et al., 1975). The significance of the observations is not known but is consistent with a genetic basis for vitiligo.

Depigmentation of the Mucosal Surfaces The epithelial surface least often inspected in patients with

B Fig. 30.17. (A, B) Unilateral vitiligo involving a portion of the trunk and left arm. The pattern is inconsistent with any neural innervation.

vitiligo is the oral cavity. The oral mucous membranes have many melanocytes, and their color in individuals with dark skin is not pink but brown or gray–brown. The melanocytes are affected by a variety of disorders that occur in stratified

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Fig. 30.18. Depigmentation of the oral mucosa. The normal color of the oral mucosa of a dark-skinned individual is gray–brown. See also Plate 30.11, pp. 494–495.

Fig. 30.20. Multiple halo nevi resembling a vitiligo macule. The relationship between halo nevi and vitiligo is not known. Both phenomenon occur most commonly in young people below the age of 20 years.

noted that halo nevi can be observed in almost a third of young patients (Nordlund et al., 1985b). In a case-controlled study, the incidence of halo nevi in patients with vitiligo was higher than in the control population (Barona et al., 1995). Halo nevi are common in teenagers and are generally of little concern as they repigment spontaneously (Fig. 30.20). Whether the association of halo nevi with vitiligo represents a true association or a manifestation of a heightened awareness of depigmented spots on the skin is not known.

Vitiligo and the Pigmentary System of the Eyes Fig. 30.19. Depigmentation of segmental vitiligo involving the chin (see Fig. 30.15) extending onto the buccal mucosa. See also Plate 30.12B, pp. 494–495.

squamous epithelium (see Chapter 56) (Gray, 1978; Har-El et al., 1990; Maize, 1988; Pesce and Toncini, 1983; Spann et al., 1987; Tomich and Zunt, 1990; Weathers et al., 1976). Patchy loss of pigment from the buccal mucosa, gingiva, and gum line is almost invariably observed in patients with vitiligo (Dummett, 1959) (Figs 30.15, 30.18, and 30.19). It is possible that other mucous membranes, such as those covering the vaginal vault, are depigmented just as the genitals are in males (Gaffoor, 1984; Moss and Stevenson, 1981). However, these tissues are rarely examined. The lips of individuals of any skin color, illuminated by a Wood’s lamp in a dark room, can often be noted to have lost color. Depigmentation of the oral mucosa or lips can be used as an early clinical sign that the patient has vitiligo.

Halo Nevi Halo nevi have been associated with vitiligo and are said to represent the same abnormality in a limited form (Lerner and Kirkwood, 1984; Lerner and Nordlund 1978a, b). Others have 558

The eyes have two embryologically distinct layers of pigment cells (Chapter 4). Immediately behind the neuroretina is the retinal pigment epithelium (RPE). It is derived from the primitive forebrain during the first weeks of gestation. This layer is heavily pigmented. Its function is thought to be tropic for the neuroretina. Malignancies of this layer are rarely, if ever, melanomas but rather mesenchymal such as tumors. The second layer is the uveal tract consisting of the choroid, the ciliary body, and the iris. The melanocytes in this layer are derived embryologically from the neural crest later in gestation. The cells in this layer, which is located behind the RPE, are not well studied, and their functions are poorly described. They do contribute to blocking light from penetrating through the eye. Individuals with albinism have a deficiency of pigment in this layer and the RPE. Such individuals have marked photophobia. Recently, it was observed that prostanoids can stimulate the iridial melanocytes to produce melanin even in adults. This observation suggests that the cells are capable of synthesizing melanin when stimulated. Little is known about the melanizing function of these cells. Malignancies from the uvea are usually melanomas. The causes of vitiligo are unknown. However, all the theories currently extant invoke systemic factors. If a systemic factor is involved, one might predict that the eyes might be involved. Most patients with vitiligo have few symptoms

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

Fig. 30.21. A Hispanic gentleman with extensive vitiligo who is blind from uveitis that destroyed the foveae of his eyes. He has the Vogt–Koyanagi–Harada syndrome. See also Plate 30.13, pp. 494–495.

Fig. 30.22. The ocular findings observed in the patient illustrated in Fig. 30.21. There is a typical area of uveitis that has destroyed the neuroretina, the pigment epithelium, and the choroid. See also Plate 30.14, pp. 494–495.

related to the eyes. They might note vague headaches, a slight decrease in night vision, or mild photophobia. A few patients have very severe inflammatory eye problems associated with vitiligo (Figs 30.21 and 30.22). This has been called the Vogt–Koyanagi–Harada (Chapter 39) or the uveomeningoencephalic syndrome. This syndrome is characterized by the association of vitiligo, an inflammatory uveitis and, in some patients, meningeal inflammation and dysacousia. Eye involvement has been described with both bilateral, symmetric vitiligo (Duke-Elder and Perkins, 1966; Friedman and Deutsch-Sokol, 1981; Haye et al., 1973; Kumakiri et al., 1982; Rosenbaum et al., 1979; Rosenthal, 1980; Weber and Kazdan, 1977) and unilateral, asymmetric vitiligo, an association called Allezandrini’s syndrome (Hoffman and Dudley, 1992; Merz et al., 1969; Preste, 1973; Rosenthal, 1980) (Chapter 39). Most of the reports present associations of vitiligo and uveal disease. Other reports present cases of vitiligo associated with destruction of the RPE or retinitis pigmentosa (Albert et al., 1979, 1983; Cowan et al., 1982, 1986; Haye et al., 1973; Rosenbaum et al., 1979).

The meninges, particularly those overlying the brain stem and pons, have a full complement of pigment cells (Goldgeier et al., 1984). It has been suggested that the destructive process killing cutaneous melanocytes also destroys pigment cells in the eyes and meninges (Barnes, 1988). Some patients also have dysacousia. The stria vascularis of the cochlea also has structures formed by melanocytes of neural crest origin (Deol, 1970; Konigsmark, 1972). The dysacousia has been attributed to injury to these cells, although there is no documentation for either supposition about the meninges or the ears. The results of several extensive studies (Albert et al., 1979, 1983) of patients with vitiligo indicate that these individuals have pigment loss in the eyes much more frequently than control populations. Some of the patients had active inflammatory disease of the eyes. Patients with idiopathic uveitis (Wagoner et al., 1983) or iritis (Fig. 30.23) (Nordlund et al., 1981a) had a rather high incidence of vitiligo. Thus, it seems that destruction of cutaneous melanocytes is an indicator for destruction of melanocytes in the eye. Similarly, destruction of 559

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Melanocytes Infundibulum

Sebaceous gland

Shaft

Bulb Fig. 30.23. The pigmentary system of the anterior uvea can be affected in some patients with vitiligo. This patient has iritis. See also Plate 30.15, pp. 494–495.

Melanocytes Fig. 30.24. Drawing illustrating the localization of follicular melanocytes, which is the reservoir for repigmentation. Follicular melanocytes are spared in most patients with vitiligo (Fig. 30.9).

ocular melanocytes seems to be associated with destruction of cutaneous melanocytes. The mechanism for destruction of ocular melanocytes is not known. However, all current theories predict loss of ocular melanocytes because they all propose an intrinsic mechanism that would likely involve melanocytes located in all tissues. The subclinical eye disease is more important for the biologist studying the disease than for the clinician. It does not seem to be necessary to refer patients to an ophthalmologist just because they have vitiligo. It would be necessary only if they had symptoms referable to the ocular pigmentary tract.

Vitiligo and the Pigment Cells in the Inner Ear Within the inner ear is a resident population of pigment cells that form the stria vascularis (see Chapter 1) (Cable et al., 1992; Fukazawa et al., 1994; Gemignani, 1986; Kitamura et al., 1994). The stria vascularis is essential for normal function of the cochlea in which the tissue is found. These pigment cells are of neuroectodermal origin and are absent in congenital hearing disorders such as Waardenburg syndrome (Chapters 6 and 29) (Baldwin et al., 1992; Cable et al., 1994; Tassabehji et al., 1992, 1994). The importance of pigment cells in the inner ear has been noted many years ago (Deol, 1970; Konigsmark, 1972). More recent data confirm that the pigment cells in the stria vascularis are essential for the normal function of the cochlea and provide a pathophysiologic basis for loss of hearing in their absence (Cable et al., 1992; Conlee et al., 1989; Kitamura et al., 1994; Schweitzer, 1993). The Vogt–Koyanagi–Harada syndrome has as one manifestation dysacousia (Cable et al., 1992; Duke-Elder and Perkins, 1966). The association suggests that the melanocytotoxic process(es) causing vitiligo can be active in the ear (Dereymaeker et al., 1989; Escalante-Ugalde et al., 1991; Gemignani, 1986; Konigsmark, 1972; Nitta and Takamori, 1989; Orecchia et al., 1989; Thurmon et al., 1976; Tosti et al., 1986,

560

Fig. 30.25. Scattered depigmented hairs in a child with vitiligo.

1987; Weidauer et al., 1981). Most patients with vitiligo do not have symptoms related to the pigmentary system in the ear and require no evaluation of the auditory system.

Vitiligo and Loss of Melanocytes in the Hair Bulb Melanocytes are in the hair bulb and are responsible for the color of the hair (Fig. 30.24). These pigment cells paradoxically seem to be spared in many or most people with vitiligo (Fig. 30.9). Some follicles are involved, and even young children with vitiligo have a scattering of white hairs on the scalp (Fig. 30.25) (Lerner and Nordlund, 1978a). Depigmentation of the eyebrows and eye lashes (Fig. 30.26) is not uncommon. The white hairs that indicate destruction or loss of all melanocytes in the follicle must be distinguished from gray hairs that seem to be a manifestation of the aging process and represent abnormal melanocytes in the follicle. The interfol-

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

Fig. 30.26. Poliosis of the eyelid in a patient with vitiligo.

However, it has been shown that both pigmented melanocytes in the hair bulb and nonpigmented melanocyte stem cells in the niche are necessary for hairs to be pigmented (Nishimura, 2005; Steingrimsson, 2005). Loss of niche cells causes loss of pigmented melanocytes in the bulb. Apoptosis may be a mechanism by which the stem cells are destroyed (Nishimura, 2005; Steingrimsson, 2005). It has been suggested that apoptosis also is the mechanism by which interfollicular melanocytes are destroyed (Huang et al., 2002). Some individuals develop totally white hair at early ages, before the age of 30 years. Often, the white hair is not associated with cutaneous depigmentation. At times, the hair can turn white very rapidly (Goldenhersh, 1992; Jelinek, 1972), although the mechanism for this phenomenon is not known. It is presumed that the pigmented hairs fall out leaving the appearance of hair turning white suddenly (Chapter 41). This hypothesis is the only possible explanation at this time. Whether total loss of melanocytes in the hair follicles is a form of vitiligo vulgaris or a manifestation of some other pathophysiologic disorder is not known. Most pigment biologists believe that white hair without cutaneous pigment loss represents a disorder different from vitiligo vulgaris.

The Psychologic Impact of Vitiligo

Fig. 30.27. Occasionally terminal hairs are depigmented. This patient has white (not blond) hairs, an indication that the follicular melanocytes are destroyed. This patch of vitiligo has lost its reservoir of melanocytes needed for repigmentation. See also Plate 30.16, pp. 494–495.

licular skin of the scalp is commonly involved in patients with vitiligo, but only a scattering of follicles is involved. The hairs on the other parts of the body can also be involved, although most individuals with the bilateral, symmetrical form of the disease have normally colored hairs on most of their bodies. A few spots might have white hairs (Fig. 30.27), a sign that these areas will not repigment with medical therapies and require transplantation of melanocytes for repigmentation. The assessment of the hair color is most difficult in children with fair skin and blond vellus hairs. At times, one cannot distinguish whether the hairs are depigmented or very blond. More commonly, the hairs in asymmetric, unilateral (segmental) vitiligo are depigmented, an observation that might explain the reputed resistance of this disorder to the standard treatments such as psoralens and ultraviolet light. The mechanism by which hair is caused to turn white is not known.

Vitiligo is important for many reasons (Lerner and Nordlund, 1978b; Nordlund, 1992; Nordlund and Lerner, 1982). For afflicted individuals the cosmetic disfigurement is the most devastating impact of this disease (Hill-Beuf and Porter, 1984; Jancar, 1974; Porter, 1989; Porter and Beuf, 1988, 1991, 1994; Porter et al., 1978, 1986, 1987, 1990; Shuster et al., 1978). The disfigurement from the spotted appearance that typically involves the face, hands, feet, and legs lowers the selfesteem and ability of individuals to cope with the daily stresses (Porter, 1989; Porter and Beuf, 1988, 1991, 1994; Porter et al., 1978, 1986, 1987, 1990). Children also suffer significantly (Hill-Beuf and Porter, 1984). The genitalia are common sites for depigmentation (Moss and Stevenson, 1981). Vitiligo has a deleterious effect on the psychosexual function of many individuals who are embarrassed by the loss of color on the genitals (Porter et al., 1990). Some patients who have occupations that require an attractive appearance have lost jobs or lost opportunities for promotions because of their unsightly image. Models, sales people, business people, trial lawyers, receptionists, and others in public positions have experienced such impacts. Clearly, those with darker colored skin of all ethnic backgrounds are more likely to be affected by bias in a culture that rewards proper appearance. Children can be taunted by classmates in school. Patients often require counseling from their primary physician, the dermatologist or, at times, from a psychiatrist. Children can be assisted by teachers working with the physician. Teachers can be taught about the disease so that they help all students understand that the skin problem is like all other diseases and requires understanding. For those in late grade

561

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Fig. 30.28. Monobenzone-induced depigmentation.

school or high school, they might present a science project on the pigment system and vitiligo for the classmates. The information can often be helpful in soliciting a more empathetic response from their peers.

Precipitating Factors for Vitiligo The causes of vitiligo are unknown. Similarly, the precipitating factors are not well delineated. Some factors, such as melanocytotoxic chemicals and the Koebner phenomenon, also termed the isomorphic response, are well-documented precipitating factors, but their mechanism of action is not understood.

Chemical Melanocytotoxins Depigmentation can be induced by the exposure of some individuals to chemicals that are typically derivatives of hydroquinone. This topic has been reviewed in detail (Cummings and Nordlund, 1995). It seems that not all individuals are equally susceptible to the depigmenting effects of well-known melanocytotoxic chemicals. Whether this depigmentation is vitiligo or depigmentation caused by mechanisms different from those responsible for vitiligo vulgaris is not known. The first chemical to be identified as a melanocytotoxin was monobenzone (Oliver et al., 1940) (Fig. 30.28). Workers were wearing gloves that contained this chemical. The chemical destroyed the melanocytes in the skin, leaving the hands of the workers depigmented. This agent is now used for the treatment of individuals with vitiligo too extensive to repigment (Mosher et al., 1977; Nordlund et al., 1985a). There are many other reports of workers in industrial settings exposed to chemicals with structures similar to monobenzone who have developed depigmentation (Bleehen, 1976a; Bleehen and HallSmith, 1970; Calnan, 1973; Das and Tandon, 1988; Gellin et al., 1970, 1979; James et al., 1977; O’Malley et al., 1988; Romaguera and Grimalt, 1981; Tosti et al., 1991). It is important to question individuals about their occupation and hobbies when they present for evaluation of depigmentation. There are other reports of individuals developing depig562

Fig. 30.29. Red bindhi-induced depigmentation. The bindhi contains monobenzone. See also Plate 30.17, pp. 494–495.

mentation following exposure to items common to everyone’s daily life. These include cosmetics (Catona and Lanzer, 1987), possibly paraphenylenediamine hair dyes (Taylor et al., 1993), cinnamic aldehyde in toothpaste (Mathias et al., 1980), derivatives of hydroquinone in germicides (Bentley-Phillips, 1974; Kahn, 1970), monobenzone in bleaching creams (Dogliotti et al., 1974) and in the rubber seals on swim goggles (Goette, 1984). In India, the red bindhi worn on the forehead by married women can contain monobenzone and produce depigmentation in some individuals (Pandhi and Kumar, 1985) (Fig. 30.29). Recent work with para-tertiary butyl catechol indicates that substituted phenols are toxic for melanocytes. It appears that such agents induce melanocyte apoptosis (Le Poole et al., 1999; Yang and Bossy, 1999; Yang et al., 2000). Apoptosis has been suggested as the mechanism for destruction of melanocytes causing vitiligo (Huang et al., 2002; Van Den Wijngaard et al., 2000).

The Isomorphic Response It is well known that even minor injuries to the skin of patients with vitiligo can leave depigmentation when healed. This is called the isomorphic response (Fig. 30.30). Small cat scratches, abrasions from falling, surgical wounds, and similar injuries have all been observed to cause depigmentation. Many individuals who develop sunburn following excessive sun exposure attribute the depigmentation to the burn. These individuals invariably have very fair skin. It is possible that the isomorphic phenomenon activated by the sunburn is responsible for the depigmentation in susceptible individuals. Another explanation is that the individual burned because the skin was depigmented already and did not have the benefit of the protective effects of the pigment system. It is important for individuals with vitiligo to consider this phenomenon before undergoing elective surgical or cosmetic procedures such as facial peels, electrolysis, dermabrasion, or skin transplants to repigment the skin. The importance of the isomorphic response has been

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

Fig. 30.31. Squamous cell carcinoma occurring in a vitiligo macule. Very few skin cancers have been observed in patients with vitiligo. See also Plate 30.18, pp. 494–495.

Fig. 30.30. Linear depigmentation following surgical procedure. This is called the isomorphic response.

stressed by some who suggest that this phenomenon might explain the onset and distribution of vitiligo (Gauthier, 1995). Repeated mild trauma associated with rubbing, wearing of clothes, gentle pressure on the skin was thought to induce the depigmentation observed in vitiligo. This hypothesis must be substantiated. It does acknowledge the importance of the isomorphic reaction in patients with vitiligo. The mechanism for the isomorphic response is not known. However, it is well established that the clinical normal skin of patients with vitiligo is not normal histologically (Moellmann et al., 1982). Biopsies of normal-appearing skin processed for routine histology appear normal. The routine sections that are embedded in paraffin are too thick for the observer to visualize the abnormalities. The keratinocytes exhibit hydropic changes, there is extracellular debris, and the melanocytes can have autophagosomes (Moellmann et al., 1982). These abnormalities are visible only on thin sections of skin embedded in plastic media or by electron microscopy. The observations suggest that the melanocytes in the normal-appearing skin are susceptible to injury by the inflammatory response that accompanies wound healing.

Stress has also been implicated as a precipitating factor for vitiligo. Almost 40% of individuals confirm that a very stressful event occurred in the 6–12 months preceding the onset of depigmentation. The percentage of individuals giving a positive response can be increased or decreased depending on the interval between the stress event and the onset of vitiligo that the observer considers acceptable. It is also known that a similar percentage of individuals without vitiligo will have experienced a similar traumatic event in a similar time period. The most accurate conclusion about the role of stress in precipitating vitiligo is that it is unknown. That stress can cause other disorders such as headaches, hypertension, and similar disorders is well known. That stress might affect the melanocytes of susceptible individuals is possible, although no mechanism is known. No definitive conclusion can be made from the extant data based on clinical histories.

Vitiligo and Skin Cancer The depigmented skin of individuals with vitiligo has lost its pigmentary system and some of the protection that nature has provided against the carcinogenic effects of sunlight and some chemicals. Melanomas cannot develop because their cell of origin is absent. One would predict that such skin would exhibit a higher than expected susceptibility to the development of skin cancers such as basal cell epitheliomata or squamous cell carcinomas. However, that does not seem to be the case. In the almost 1500 reports reviewed for this chapter, skin cancer is mentioned only three times as a problem in patients with vitiligo. There are a few exceptions (Lisi, 1972; Ortonne et al., 1978a) (Fig. 30.31). One notes the absence of evidence for sun damage in the white skin (Calanchini-Postizzi and Frenk, 1987). There is a remarkable paucity of such reports. The data require confirmation by formal studies. That vitiliginous skin does not exhibit skin cancers can be attributed to many factors. Individuals with vitiligo tend to cover depigmented skin, especially in the summer time. It is also possible that the melanocyte can contribute to the 563

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carcinogenic effects of sunlight. Melanin has been reported to be toxic following exposure to sunlight. The mystery about skin cancers and vitiligo will remain until proper studies confirm the absence of increased susceptibility and other studies work out a mechanism.

The Physiologic Functioning of Depigmented Skin Several studies have indicated that depigmented skin exhibits abnormal autonomic nervous function (Merello et al., 1993). It has been suggested that the sympathetic nervous system is downregulated (Dutta, 1965, 1972; Dutta and Dermat, 1972; Dutta and Mandal, 1972) or that the parasympathetic activity is increased (Chanco-Turner and Lerner, 1965). In the last study, it was observed that the surface temperature of vitiliginous skin was increased, that bleeding time was prolonged, and that sweat production was increased. The investigators concluded that neural factors were responsible for the destruction of the melanocytes. Skin affected by vitiligo is missing one of its three main cell types, the melanocyte. It is possible that such skin might not function normally. However, the results of any studies on the function of the depigmented epidermis must be interpreted in two ways, as a cause of the depigmentation or as an effect of the melanocyte loss.

Depigmentation and the Inflammatory Response The inflammatory response of depigmented epidermis has also been noted to be aberrant. Individuals with both atopic dermatitis and vitiligo (see Fig. 30.11) exhibit a peculiar sparing of the depigmented skin by the dermatitis (Fregert et al., 1959; Halter, 1941; Kreibich, 1921). It also has been reported that dermatitis herpetiformis spares the depigmented skin (Allende and Reed, 1964). There have been many reports over the years that vitiliginous skin does not respond to irritants or allergens with a normal inflammatory response (Haji-Abrassi et al., 1981; Lisi and Caraffini, 1985; Nordlund et al., 1985a; Singh et al., 1987; Srinivas et al., 1987). Several investigators studying this phenomenon have applied anthralin to the normalappearing and depigmented skin of people with vitiligo. Applied frequently enough, the depigmented skin became inflamed, but the histology of the inflammatory infiltrate was different in the white skin compared with the pigmented skin (Maurice and Greaves, 1983; van de Kerkhof and Fokkink, 1989; Westerhof et al., 1989). All the investigators observed that depigmented skin differed in its response from normalappearing skin. Finally, injections of interferon (IFN)-g into vitiliginous skin caused the influx of inflammatory cells, but the number of CD1 cells–ATPase-positive cells (presumably Langerhans cells) was significantly lower than in normal skin. A possible mechanism for this difference in inflammatory reaction has been proposed from data obtained in mice with an inherited form of depigmentation (Nordlund et al., 1995). The mouse has a mutation in the mi locus, analogous to the MITF locus in humans, that results in the postnatal loss of pigmentation (Lamoreux et al., 1992). This depigmented epi564

dermis exhibits a muted capacity to respond to contact irritants or allergens (Rheins et al., 1986). Such skin does not synthesize or express ICAM1 when treated with potent allergens such as dinitrochlorabenzene (DNCB) or when incubated in IFN-g (Nordlund et al., 1995). This inability to express normal amounts of ICAM1 could be the cause of the muted response of the murine epidermis to exhibit a normal response. The low expression of ICAM1 might also reflect the lack of sensitization.

Conclusion The vascular and inflammatory responses of depigmented epidermis differ in many ways from normally pigmented skin. Whether the different responses reflect the cause or an effect of loss of melanocytes is not known.

Associated Disorders Vitiligo vulgaris has been reported in association with many diseases (Table 30.1). If the prevalence of vitiligo is about 1:200 people throughout the world, then about 20 million individuals on the planet currently have vitiligo. That one or more would have vitiligo and some other disorder is expected. Thus, the significance of many of the anecdotal reports is difficult to assess. There are many reports of vitiligo in association with common disorders such as diabetes mellitus or thyroid disease. The prevalence of these latter disorders is high. Diabetes mellitus afflicts about 5% of the population in the United States. The prevalence of thyroid disease depends on the criteria used to make the diagnosis. It has been estimated that thyroid dysfunction affects as many as 10% of the population. It would be expected that groups of patients with vitiligo vulgaris and common disorders would be easily gathered. Thus, reports of associations of vitiligo with common disorders might represent either a significant biologic association or common events occurring together. There are no definitive studies extant that distinguish these two possibilities. The reader must be cautious in interpreting the validity of the reported associations until valid epidemiologic studies have been conducted or molecular biologic techniques demonstrate true associations between common phenomena.

Polyglandular Dysfunction and Vitiligo There have been numerous reports of polyglandular endocrine dysfunction associated with vitiligo (Betterle et al., 1992, 1996; Bloch and Sowers, 1985; Butler et al., 1984; Csaszar and Patakfalvi, 1992; De Rosa et al., 1987; Eisenbarth et al., 1978; Kam et al., 1994; Mandel et al., 1989; Matsumoto et al., 1979; McGregor et al., 1972; Novoa Mogollon et al., 1989; Papadopoulos and Hallengren, 1993; Patier et al., 1992; Peserico et al., 1981; Sharma et al., 1990; Torrelo et al., 1992; Weyermann et al., 1994). The organs or glands that have been involved include the thyroid, parathyroids, islets of Langerhans in the pancreas, the gonads, adrenal glands, and the gastric mucosa. These patients have dysfunction of several or all the endocrine organs noted. Usually, the

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION Table 30.1. Other disorders reported associated with vitiligo. Disorders reported

References

Nail abnormalities Spastic paraparesis Porencephaly, mucoceles, hypertelorism Jaundice Acromegaly Neoplasm Dermatitis herpetiformis Smooth muscle tumor of stomach Psoriasis

Barth et al. (1988) Lison et al. (1981) Tay (1970) Neering (1975) Ryan et al. (1984) Banerjee et al. (1989); Lassus et al. (1972) Olholm-Larsen and Kavli (1980) Zecler et al. (1974) Abraham et al. (1990); Chapman (1973); Dhar and Malakar (1998); Enhamre et al. (1986); Iakovleva and Sen’kin (1984); Koransky and Roenigk (1982); Mancuso and Gasponi (1983); Menter et al. (1989); Moragas and Winkelmann (1970); Powell and Dicken (1983); Roberts (1984) Enhamre et al. (1986) Cruz et al. (1994); Durance and Hamilton (1971); Lamartine de Assis et al. (1983); Shinkai et al. (1985); Tan (1974a, b); Topaktas et al. (1993) Anstey and Marks (1993); Brenner et al. (1979); Cecchi et al. (1994); Melato et al. (2000); Porter et al. (1994); Rubisz-Brzezinska et al. (1996); Saha et al. (1979); Sardana et al. (2002); Tan (1974a, b) Abraham et al. (1993); Alarcon-Segovia (1982); Collen et al. (1979); Durance and Hamilton (1971); Gonggryp and Kalla (1992); Goudie et al. (1979); Ortonne et al. (1976a) Alcalay et al. (1987); Dabski et al. (1985); Todes-Taylor et al. (1983) Aloi et al. (1987); Brown et al. (1977); Brenner et al. (1979); Carter and Jegasothy (1976); Cho et al. (1995); Dhar and Kanwar (1994); Hann et al. (1996); Korkij et al. (1984); Tan (1974b); Thompson et al. (1974) Goudie et al. (1979); Hovdenak (1979); Monroe (1976); Tan (1974b); Thompson et al. (1974) Babanov and Schieferstein (1970); Brenner et al. (1979); De Villiers et al. (1992); Finkelstein et al. (1995); Grosshans and Marot (1990); Khamaganova et al. (1973); Lacour et al. (1992); Melato et al. (2000); Sanchez et al. (1983); Shupen’ko (1980); Thompson et al. (1974); Thurlimann and Harms (1982); Trofimova et al. (1983) Brown et al. (1977); Carter and Jegasothy (1976); Hazini et al. (1988) Cecchi et al. (1994); Hazini et al. (1988) Alexander (1982); Duvic et al. (1987); Ivker et al. (1994); Ruzicka (1981); Tojo et al. (1991)

Parapsoriasis Myasthenia gravis and thymoma Lichen planus

Rheumatoid arthritis Mycosis fungoides, Sezary’s syndrome Alopecia areata

Ulcerative colitis, regional enteritis Scleroderma, morphea

Down’s syndrome Pityriasis rubra pilaris AIDS, Kaposi’s sarcoma

organs fail completely, and the patients have hypothyroidism, hypoparathyroidism, diabetes mellitus, sterility, adrenal insufficiency, or pernicious anemia. In addition, some patients had candidiasis (Ahonen et al., 1990; Evans et al., 1989; Fields et al., 1971; Fisher and Fitzpatrick, 1970; Hertz et al., 1977; Howanitz et al., 1981; Kirkpatrick, 1989; Maggiore et al., 1986; Mandel et al., 1989; Nordlund et al., 1981b; Porter et al., 1992; Schulkind and Ayoub, 1975). Others had gastrointestinal disease (Papadopoulos and Hallengren, 1993), marrow hypoplasia (Mandel et al., 1989), leukemia (De Rosa et al., 1987), or neurologic disorders (Matsumoto et al., 1979). All had vitiligo as well. Polyglandular autoimmune disease is thought to be a genetically determined disorder. The gene has been assigned to chromosome 21 (Aaltonen et al., 1994). One antigen specifically involved in the autoimmune process has been identified and is a cholesterol side-chain cleavage enzyme (Winqvist et al., 1993). The total number of patients with vitiligo and polyglandular failure is small. However, the association of vitiligo vulgaris and such an array of endocrine dysfunction supports a real association very strongly, although definitive proof is

lacking. That many of the endocrine failures seem to be autoimmune in origin suggests that the vitiligo might also have a similar pathophysiology (see section below on Pathophysiology). In addition, the specificity of the antibody response to cellular antigens of endocrine organs suggests that antibodies are also involved in destruction of the melanocytes (Winqvist et al., 1993). These associations, which are very striking, have been used in part to justify the significance of the less convincing association of vitiligo with one or two common endocrine dysfunctions.

Thyroid Dysfunction One of the most common endocrine abnormalities associated with vitiligo is thyroid dysfunction (Fig. 30.32). Patients have been found to have hyperthyroidism, hypothyroidism, or thyroiditis (Alkhateeb et al., 2002, 2003; Anderson et al., 1980; Betterle et al., 1985, 1987; Bickley and Papa, 1989; Bloch and Sowers, 1985; Borisenko, 1971a; Callen, 1984; Cowan et al., 1982; Cunliffe et al., 1968; Curti et al., 1992; Fairfax and Leatham, 1975; Fields et al., 1971; Grimes et al., 1983; Handa and Kaur, 1999; Hegedus et al., 1994; Hirbli et al., 1985; 565

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or other disorder should be evaluated appropriately because of the clinical symptoms rather than as a routine screening test or as a diagnostic test for vitiligo.

Other Endocrine Disorders

Fig. 30.32. Vitiligo and graying of the hair in a patient with hyperthyroidism.

Jacyk et al., 1976; Kemp et al., 2001; Korkij et al., 1984; Lamartine de Assis et al., 1983; Madden et al., 1989; Merello et al., 1993; Midelfart et al., 1983; Mullin and Eastern, 1986; Nishida et al., 1990; Ortonne et al., 1976b; Pal et al., 1980; Papadopoulos and Hallengren, 1993; Ramanathan et al., 1989; Rodermund et al., 1975; Shong and Kim, 1991; Weiss et al., 1996; Zelissen et al., 1995). Many of these citations are case reports. Some provide data on larger groups of patients with vitiligo. The prevalence of thyroid disease in Ortonne et al.’s study is 7.5% of 374 patients (Ortonne et al., 1976a, b). The number is somewhat lower than the prevalence for thyroid dysfunction in the United States, estimated to be about 10–15%. More recent studies have observed a prevalence of thyroid disorders as high as 12% (Alkhateeb et al., 2002, 2003). The low prevalence, whether the same or different from the normal population, suggests that the association is not strongly related to vitiligo in a simple way. Genes for depigmentation linked to others responsible for thyroid dysfunction might be responsible for the association of depigmentation with thyroid dysfunction (Alkhateeb et al., 2002, 2003). Other investigators have studied thyroid functions (Borisenko, 1971a; Kesler, 1974; Kumar et al., 1990; Pal et al., 1980) or assayed the serum of patients with vitiligo for the presence of antithyroid antibodies (Betterle et al., 1976, 1985; Grimes et al., 1983; Korkij et al., 1984; Morgan et al., 1986). Thyroid functions were thought to be depressed in a higher percentage of patients with vitiligo than in control subjects. Antithyroid antibodies were thought to be more prevalent in the sera of patients with vitiligo than in control subjects.

Thomas Addison was the first to describe patients with adrenal insufficiency and vitiligo (Addison, 1855). However, all his patients had a scrofulous, i.e. tuberculous, destruction of the adrenal gland. Others have recorded patients with vitiligo and adrenal insufficiency or anti-adrenal antibodies in the blood (Alkhateeb et al., 2002, 2003; Alper and Garner, 1985; Betterle et al., 1985; Borisenko, 1971a, b; Hofs, 1980; Macaron et al., 1977; Mulligan and Sowers, 1985; Tadzhibaev, 1971), although the number of cases is few except for those with polyglandular insufficiencies as noted above. Diabetes mellitus has been associated with vitiligo (Bloch and Sowers, 1985; Dawber, 1968; Dawber et al., 1971; Fairfax and Leatham, 1975; Gould et al., 1985; Handa and Kaur, 1999; Jacyk et al., 1976; Jaggarao et al., 1989; Macaron et al., 1977; Montagnani et al., 1985; Vijayasingam et al., 1988; Villaverde, 1969, 1973). The data are not from controlled studies. The significance and validity of the association are not known. A small number of patients with pernicious anemia and vitiligo have been reported (Abraham et al., 1993; Grunnet et al., 1970; Gulden, 1990; Held and Kohn, 1990; Matsumoto et al., 1977; Pelosio et al., 1989; Ruzicka, 1981). The association of vitiligo and pernicious anemia was first reported in patients with polyglandular failure. Later, isolated reports of pernicious anemia alone with vitiligo were reported. The significance and validity of the association are not clear.

Other Disorders Vitiligo has been reported in association with a large number of disorders, some of which are recorded in Table 30.1. Most are single case reports. It is important to recall that there are millions of individuals in the world with vitiligo. The fact that isolated patients are reported with other rare disorders complicates the interpretation of the reports. General commentary The authors of most reports present the cases as documentation of an association of vitiligo with another purported immune or autoimmune disorder. They suggest that the association supports the hypothesis that vitiligo has an immune basis.

Depigmentation and Melanoma Commentary Thyroid disease is difficult to assess. It seems to be rather prevalent, affecting as many as 10–15% of the population in the United States and other Western countries (Groves et al., 1990). It is more prevalent in women than in men. More women with vitiligo consult their physician about therapy than men. The presence or significance of autoantibodies is not known. Patients with symptoms or signs of thyroid disease 566

Melanoma is an uncontrolled proliferation of malignant melanocytes. Vitiligo or depigmentation is caused by destruction of benign melanocytes. It is paradoxical that the two processes are sometimes seen together (Fig. 30.33A and B). There are numerous reports of depigmentation developing in patients with melanoma (Adam et al., 1975; Agdal et al., 1983; Alabi et al., 1991; Albert et al., 1978, 1982; Anonymous, 1979; Balabanov et al., 1969; Burdick and

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

A

B

Fig. 30.33. (A, B) An African-American woman who presented with a 3-cm melanoma on the foot with metastases. She survived 2 years and developed striking depigmentation of the face. See also Plates 30.19A and B, pp. 494–495.

Hawk, 1964; Cavallari et al., 1996; Chang et al., 1986; Cohen et al., 1979; Donaldson et al., 1974; Duhra and Ilchyshyn, 1991; Durham-Pierre et al., 1995; Fodor and Bodrogi, 1975; Forrest and Forrest, 1980; Fournier et al., 1984; Gregor, 1976; Hamm and Fiedler, 1979; Happle et al., 1975; Hudson, 1979; Koh et al., 1983; Kossard and Commens, 1990; Kraus and Landthaler, 1980; Milton et al., 1971; Moroc et al., 1980; Nordlund, 1987; Nordlund et al., 1983; Pantoja et al., 1977; Perrot et al., 1976; Rosenberg and White, 1996; Weiss et al., 1996), usually metastatic melanoma (Koh et al., 1983; Milton et al., 1971; Nordlund et al., 1983; Sober and Haynes, 1978). The depigmentation found in humans with melanoma is not identical clinically to vitiligo vulgaris (Chapter 38). The depigmentation begins on the neck, upper back, and chest as a confetti-like depigmentation (Fig. 30.34). It can spread to involve any other areas. Sometimes, the depigmentation is confined to the skin around the primary or metastatic tumors. In most patients with both phenomena, the appearance and distribution of the depigmentation are very different from

Fig. 30.34. A woman with metastatic malignant melanoma who exhibits the typical confetti-like depigmentation located on the chest and shoulders. See also Plate 30.20, pp. 494–495.

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A

B

Fig. 30.35. A young lady with metastatic malignant melanoma who suddenly developed total depigmentation of the scalp (A) and pubic hair (B). See also Plates 30.21A and B, pp. 494–495.

vitiligo vulgaris, which suggests that the depigmentation associated with human melanoma might represent a different mechanism by which the normal melanocytes are destroyed (Fig. 30.35A and B). Others have noted that depigmentation occurs after various forms of therapy such as radiation, cytokines, BCG, or vaccinia vaccination (Burdick and Hawk, 1964; Cohen et al., 1979; Donaldson et al. 1974; Forrest and Forrest, 1980; Friederich, 1967; Harris et al., 1994; Hersey et al., 1986; Matsuzawa et al., 1953; Richards et al., 1992; Roth, 1968; Scheibenbogen et al., 1994; Wolkenstein et al., 1992). More recently, patients with melanoma have been treated with various cytokines such as interleukin (IL)-2. Some of these individuals have developed a vitiligo-like depigmentation (Jager et al., 2000; Juranic et al., 2003; Kawakami et al., 2000; Le Gal et al., 2001; Overwijk et al., 2003; Phan et al., 2001, 2003; Rocha et al., 2000; Rosenberg and White, 1996; Slingluff et al., 2003; Tsao et al., 2002; Wankowicz-Kalinska et al., 2003; Yee et al., 2000). These observations suggest that either cytokines themselves or their alteration of the immune system can destroy melanocytes and produce a vitiligo-like condition. 568

It has been suggested that, despite the presence of metastases, the patients with melanoma and leukoderma have a better prognosis that those with only melanoma (Koh et al., 1983; Nordlund et al., 1983; Sober and Haynes, 1978). There are numerous reports that cytotoxic antibodies or lymphocytes to normal melanocytes cross-react with melanoma cells (Cui et al., 1992, 1993; Fishman et al., 1993; Fritz et al., 1976; Galbraith et al., 1988; Hofs, 1982; James, 1993; Kawakami et al., 2000; Kiniwa et al., 2001; Kirkwood and Robinson, 1990; Le Gal et al., 2001; Lipkin et al., 1985; Merimsky et al., 1994; Mitchell et al., 1980; Mori et al., 1986; Overwijk et al., 1999; Park et al., 1996; Presser et al., 1987; Rocha et al., 2000; Seibert et al., 1977; Steitz et al., 2000; Wankowicz-Kalinska et al., 2003; Yamada et al., 1975). Such antibodies or lymphocytes are thought to be responsible for better survival in patients with melanoma by destroying tumor cells and the melanocytes as innocent bystanders. A chimpanzee immunized with human melanoma cells developed depigmentation on the face. The authors suggested that cytotoxic antibodies produced the depigmentation (Hornug and Krementz, 1975). From such data, it has been suggested that vitiligo be induced in patients

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

Fig. 30.36. Melanocytes are not detectable in depigmented skin by the DOPA reaction on an epidermal sheet.

as a method of improving survival for patients with high-risk melanomas or metastatic disease (Lerner, 1992; Lerner and Kirkwood, 1984; Lerner and Nordlund, 1977). That the association might have biologic significance is suggested by the many animals in which the two disorders, melanoma and depigmentation, are associated, such as the Sinclair swine (Millikan et al., 1973; Misfeldt and Grimm, 1994) and horses (Gebhart and Niebauer, 1977; Lerner and Cage, 1973). However, the data are inconclusive. The pigmentary lesions found in horses are thought to be benign pigmented hamartomas. The lesions in the swine are rarely fatal and regress spontaneously. Whether they are true malignancies is not known. To conclude that human melanoma and its associated depigmentation, regardless of its pathophysiology, are similar to animal models is premature. The significance, importance, and implications for understanding transformation or melanocytotoxicity are still under study.

Fig. 30.37. Transmission electron microscopy. Degenerating melanocytes at the border of a vitiligo macule.

Histology The routine histology of the edge of a spreading lesion is rather unremarkable. When stained by hematoxylin and eosin, the tissue shows few abnormalities. There is usually a sparse sprinkling of mononuclear cells in the dermis (Hann et al., 1992). The epidermis looks normal (Lever and SchaumburgLever, 1991). Border skin stained by the dopa oxidase technique for the presence of tyrosinase-containing melanocytes shows that the depigmented skin is devoid of detectable melanocytes (Fig. 30.36) (Le Poole et al., 1993a). Those remaining in the pigmented skin have been noted to be highly dendritic (Iyengar and Misra, 1988) or have abnormal shapes (Boissy, 2000; Moellmann et al., 1982). Depigmented skin stained for melanin shows an absence of melanin, an observation that is consistent with the absence of melanocytes. The light microscopic findings have been confirmed by electron microscopy. Melanocytes cannot be found in the depigmented skin (Figs 30.37 and 30.38) (Birbeck et al., 1961; Boissy, 2000; Bose, 1994a, b; Hann et al., 1992; Le Poole et al., 1993a; Moellmann et al., 1982; Morohashi et al., 1977; Ortonne

Fig. 30.38. Transmission electron microscopy. Vitiligo skin is devoid of melanosomes, an indication that the melanocytes are not present.

et al., 1978b). The melanocytes also appear to be undergoing a degenerative process (Fig. 30.37). They are vacuolated and the nucleus has a pyknotic appearance. Melanocytes in the normal skin distant from an active lesion are also abnormal (Moellmann et al., 1982). There is an extracellular debris, presumed to be from degenerating cells, either melanocytes or keratinocytes (Bhawan and Bhutani, 1983; Mishima, 1978; Moellmann et al., 1982). It is not clear whether cells have been injured by a direct cytotoxic process or are undergoing apoptosis, although the latter process seems to fit the reported findings and the standard light microscopy. The number of Langerhans cells in depigmented skin has been reported to be increased (Birbeck et al., 1961; Morohashi et al., 1977; Zelickson and Mottaz, 1968), normal, or decreased (Brown et al., 1967; Claudy and Rouchouse, 1984). 569

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The mononuclear cell infiltrate, although sparse, has been typed. The number of (CD3+ CD4+ CD8+) T lymphocytes was increased (Al Badri et al., 1990, 1993). The densest infiltrate was found within 0.6 mm of the edge of the lesion. The authors concluded that lesional rather than circulating lymphocytes were involved in the melanocytotoxic process. Although not intensively studied, the Merkel cell seems to be absent in depigmented skin (Bose, 1994a, b). The significance is not clear. Their absence seems to be responsible for the gaps observed in the basal lamina in patches of vitiligo vulgaris. The histology of inflammatory vitiligo vulgaris is different from that of the non-inflammatory variety. There is a moderate to intense mononuclear cell infiltrate. The epidermis shows marked spongiosis and microvesicles (Macmillan and Rook, 1971; Michaelsson, 1968).

Laboratory Findings There are no routine or diagnostic laboratory findings that assist the clinician in making the diagnosis of vitiligo or in helping him/her to monitor its course. The diagnosis is made clinically from the features of the depigmented skin, the distribution of depigmentation, and other clinical parameters. There are many reports of results of laboratory examinations of the urine or blood (Chowdhury and Banerjee, 1967; Dutta and Mandal, 1969; Dutta et al., 1969; Ghoshal, 1969; Lal et al., 1970; Metzker et al., 1980; Mujahid Ali et al., 1990; Pal et al., 1980, 1981a–d, 1995; Popp, 1979; Retornaz et al., 1976; Sehgal and Dube, 1968; Sharma et al., 1973; Wasfi et al., 1980; Yakura et al., 1976). Most of the studies were done to provide evidence about the etiology of vitiligo vulgaris. Most report some abnormalities but none that are helpful clinically for making the diagnosis of vitiligo vulgaris. They are not carried out currently for the clinical management of patients with this disease. No tests are available to assist the physician in the management of vitiligo or for determining the prognosis or response to therapy.

Criteria for Diagnosis The diagnosis of symmetrical, bilateral vitiligo vulgaris is made from clinical observations. There are no absolute criteria other than the destruction of melanocytes. The clinical manifestations are usually straightforward in a typical case. The patient has depigmentation of the fingers, feet, or face. The examination often requires the use of the Wood’s lamp. Other features include loss of pigment from the lips and the oral mucosa, observations that can be most useful if detectable. The scalp, palms, and soles are sites of depigmentation not often examined but, when involved, can be helpful in making the diagnosis. Occasionally, a child is brought for examination when the loss of pigment is very minimal, i.e. a few hypopigmented patches on the trunk or extremities. Usually, the parent or a sibling has the disease, and the worried parent is having the child examined early with the hope that early detection is useful for therapy. For such patients, no definitive diagnosis can be made. There must be sufficient 570

depigmentation in typical areas such as the fingers, toes, wrists, lips, face, or genitalia for a clinical diagnosis to be made. Not all patches need be completely depigmented. Often some of the patches early in the onset are hypopigmented. The presence of depigmented hair in a site of interfollicular depigmentation strongly suggests a diagnosis of vitiligo vulgaris. Strands of gray hair on the scalp might also be suggestive if found in a child. They can be found in most young adults past the age of 20 years and are therefore not useful clinically for making a diagnosis in adults. The diagnosis of asymmetric, unilateral, segmental vitiligo is usually easier to make. The patient will note the onset of depigmentation in an unusual site, part of the face, an extremity, or the trunk. There are few other diseases that produce unilateral depigmentation without prior skin rashes. Unilateral depigmentation can occur following conditions such as herpes zoster, but the preceding eruption makes the differentiation of vitiligo and post-inflammatory depigmentation easy. The disease progresses within the segment. The patch is easily seen by the physician often without the Wood’s lamp, although the instrument is useful for the examination. Often the involved skin has depigmented vellus or terminal hairs. The determination of depigmentation of vellus hairs can be difficult or impossible in children with blond hair. However, many children will have sufficient pigment in the vellus hairs to make the evaluation of the color of the hairs within a depigmented patch of skin easy. Loss of hair color strongly favors the diagnosis. The distribution of depigmentation in a unilateral distribution involving a segment of the skin is diagnostic of asymmetric, unilateral, segmental vitiligo.

Differential Diagnosis There are a number of other pigmentary disorders that must be distinguished from vitiligo vulgaris.

Piebaldism Piebaldism (Chapters 6 and 29) is a genetic disorder inherited in an autosomal dominant pattern. The family history is usually strongly positive unless the child has the disorder from a new mutation. Often the family knows about the disorder and considers it a family feature. The disorder is characterized by depigmented patches of skin on the ventral surface of the body. There are patches typically found on the forehead, chin, chest, and abdomen. The arms and legs might have depigmented patches. Almost invariably there is a white forelock. The forelock might not be immediately obvious, especially in a very fair child. A Wood’s lamp might identify the depigmented patch at the hairline. Careful inspection usually reveals at least a few, sometime only five or six, depigmented hairs emanating from the white patch. All other depigmented patches of skin will exhibit depigmented hairs. The pigmented skin often has dark areas and an inhomogeneous color. This disorder is present at birth or observed soon after. It is not progressive, although the edges of the depigmentation might show some movement. The family history, the typical distribution of depigmentation on the ventral surface present at

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

or shortly after birth make the diagnosis easy. Molecular testing is available to determine the defect, usually in the c-kit proto-oncogene.

Albinism Albinism (Chapter 31) is a genetic disorder transmitted as an autosomal recessive trait. It is not uncommon for the affected individual to have a new mutation or for the family to be unsuccessful in identifying the trait within the kinship. The child is very fair at birth. The hair is typically white, the skin white, and the eyes gray or blue. The entire integument is involved. The child will have ocular abnormalities such as photophobia, myopia, strabismus, or nystagmus. Usually, an eye examination to assess the fovea and degree of ocular pigmentation will confirm the diagnosis. The fovea is poorly developed, the uveal tract hypopigmented, and the iris transilluminates. The involvement of the entire integument and hair from birth associated with the ocular findings confirms the diagnosis. If desired, testing can confirm the diagnosis and the precise genetic defect.

Tinea Versicolor Tinea versicolor (Chapter 36) is an infection caused by Malassezia furfur, a normal contaminant of the epidermis. The organism invades the stratum corneum and causes hypopigmentation associated with a fine scale. The skin is rarely if ever depigmented. The distribution of the pigment loss is on the trunk, usually the back. It can be very extensive if the infection is not treated. The presence of hypopigmented, scaly patches on the trunk is highly suggestive. A scraping should be examined by microscopy after hydrolysis in potassium hydroxide. The sample will show the typical spores and hyphae confirming the diagnosis.

Pityriasis Alba Pityriasis alba (Chapter 37) is a very common disorder, especially in young people. Young people as a group are also prone to developing vitiligo. The disease is probably a manifestation of mild dermatitis associated with xerosis and postinflammatory hypopigmentation. The patches are typically hypopigmented with borders that are diffuse and difficult to define. Examination with a Wood’s lamp does not accentuate the patch with the same striking features as vitiligo. Vitiligo typically has very discrete borders. There is a subtle scale present in the patches. Typically, pityriasis alba involves the cheeks, the upper parts of the arms, and the thighs. Many of the patients will be atopic or have a family history of atopy and/or dry skin. At times, distinguishing pityriasis alba from vitiligo in its earliest stages is very difficult.

hair follicles are destroyed so that the reservoir for the melanocyte is also absent. Typically, such skin is atrophic and thin. There are other patches of skin that have red plaques typical of active lupus erythematosus. Hair loss, usually of the scarring variety, is common. The tips of the fingers exhibit periungual erythema and dilated capillaries rather than the depigmentation of vitiligo. It is most important not to miss the diagnosis of lupus and treat the patient with ultraviolet light that might aggravate this disease. However, the distinction can be very difficult. At times, it is necessary to obtain a biopsy of the skin to look for features of lupus erythematosus and serologic studies for antinuclear antibodies of various types to insure that the patient has vitiligo and not lupus erythematosus.

Mycosis Fungoides, Sarcoidosis, and Leprosy These three diseases are very different. Mycosis fungoides (Chapter 50) is a lymphoma of the skin; sarcoid (Chapter 37) is a granulomatous inflammation; and leprosy (Chapter 36) is an infection. However, all three can present as macular hypopigmentation. The spots are typically scattered over the trunk and extremities and do not have the distribution on the hands and feet of vitiligo vulgaris. Usually there are other manifestations of each disease, i.e. other lesions that are more typical of the primary disease. Almost invariably, the patches will be hypopigmented rather than depigmented. It is important to obtain a biopsy from hypopigmented lesions that are somewhat atypical in their distribution, degree of color loss, and associated with red plaques. The distinction is not difficult for the observant physician.

Syphilis Secondary syphilis (Chapter 36) can cause depigmentation of the skin. Often the distribution is not typical of vitiligo but not easily distinguished from it. It might produce the classic necklace of Venus, depigmented spots on the neck. The depigmentation or hypopigmentation can produce spots anywhere on the integument. Individuals exhibiting atypical patterns of depigmentation should have a serologic test for syphilis.

Nevus Depigmentosus The title nevus depigmentosus (Chapter 32) is a misnomer. The lesion is hypopigmented and rarely depigmented. This type of lesion might be present at birth or occur at any time after birth. The lesion is unilateral and affects one portion of the integument. The distribution might be confused with that of segmental vitiligo. The hairs in the affected area never lose their color. The skin is hypopigmented, an assessment that can be made by Wood’s lamp examination or with histochemistry on tissue specimens.

Lupus Erythematosus Lupus erythematosus (Chapter 37) is a disorder that has many cutaneous findings. Depigmentation is common, usually because the disease causes destruction of the basilar layer of the epidermis where the melanocyte resides. When the tissue heals, the skin is depigmented. It rarely repigments because the

Hypomelanosis Associated with Mosaicism Hypomelanosis affecting infants with mosaicism (Chapter 32) is present at birth. It is typically stable in distribution. This disorder must be considered for infants thought to have congenital vitiligo, piebaldism, or a nevus depigmentosus. 571

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Typically, the skin is hypopigmented, detectable by Wood’s lamp examination or by histochemistry on tissue specimens. The infant usually has a multiplicity of other anomalies.

Pathogenesis The pathogenesis of vitiligo vulgaris is not known. There are many hypotheses extant, each supported by intriguing data that are currently insufficient to prove the accuracy of the theory. It seems likely that vitiligo vulgaris represents at least one, but more likely several, processes that cause melanocyte destruction. That this is true is suggested by the various clinical presentations. Besides the typical vitiligo vulgaris, there is segmental vitiligo. It seems unlikely that the same mechanism is responsible for both disorders. There are those patients with associated polyglandular failure. This group might represent another mechanism. Individual patients present with atypical features. Occasionally, a patient might have many features of vitiligo vulgaris, such as depigmented patches on the extremities, face, and trunk, but the classic distribution on the fingers, feet, and face is not present. Some individuals show marked loss of pigment from the hair; others show none. These differences might have no importance or significance or might be hints that different mechanisms are involved. The various theories outlined below are intended to summarize current popular hypotheses. A short critique is presented for each about the data that are missing but crucial to the hypothesis. These theories are not all inclusive. They are also not mutually exclusive (Le Poole et al., 1993b). It is possible that several mechanisms are operative to produce melanocyte destruction in a given individual. The Smyth chicken exhibits such a model. The animal has a genetic defect causing destruction of melanocytes. The dead cells elicit an immune response that accelerates the depigmenting process initiated by a genetic defect (Austin et al., 1992; Boissy et al., 1983, 1986, 1988; Erf et al., 1995; Lamont and Smyth, 1981; Lamont et al., 1982; Pardue et al., 1987; Smyth et al., 1981a).

Autoimmune Hypothesis The autoimmune theory is currently the most popular. It has its origins in the observations of Thomas Addison and adrenal failure associated with vitiligo (Addison, 1855). In the 1960s, when the immune system was under intensive study, a role for the immune system in depigmentation was postulated. The association of vitiligo with polyglandular endocrine failure (see section on associated diseases) and subsequently with individual organ failure has been reported frequently. Results of recent epidemiological studies suggest that some of these endocrine failures are occasionally linked to vitiligo. A gene on chromosome 1 was noted to be linked to a gene for thyroid dysfunction on chromosome 6 (Alkhateeb et al., 2003). In addition to the reports of patients with clinical endocrine disease, there are numerous reports of patients with vitiligo having higher than normal prevalence of autoantibodies to endocrine organs without clinical disease (Auer-Grumbach and Stangl, 1993; Betterle et al., 1976, 1977, 572

1985, 1987; Bleifeld and Gehrmann, 1967; Brostoff, 1969; Chan et al., 1994; Chatain et al., 1994; Grimes et al., 1983; Hann et al., 1993a; Hansky, 1974; Korkij et al., 1984; Nordlund et al., 1981b; Soubiran et al., 1985; Vallotton and Forbes, 1966; Van Joost, 1975; Yu et al., 1993). Most of these reports present information about thyroid autoantibodies. Some have information about antibodies to gastric parietal cells, gonadal tissues, the adrenal glands, or pancreas. Other clinical observations have been used to support the autoimmune hypothesis. Vitiligo has been observed in a small group of patients with various lymphomas (Akiyama et al., 1997; Alcalay et al., 1987; Bouloc et al., 2000; Dabski et al., 1985; Demeter et al., 1993; Neumeister et al., 2000; Nordlund and Lerner, 1979; Todes-Taylor et al., 1983; Walker et al., 1993) and thymomas (Kloin, 1985; Shinkai et al., 1985; Tan, 1974). Others have reported patients with AIDS who developed vitiligo (Duvic et al., 1987; Ivker et al., 1994; Tojo et al., 1991). The conclusion of all such reports is that patients with dysfunction of their immune system are prone to vitiligo because it is an autoimmune disease. The first attempts to identify melanocyte-specific antibodies were unsuccessful (Wassermann and Walt, 1973). A patient with mucocutaneous candidiasis and vitiligo was found to have complement deposited on the melanocytes and presumably immunoglobulins (Hertz et al., 1977). However, the antibodies were found in the sera of many patients with mucocutaneous candidiasis who did not have vitiligo (Howanitz et al., 1981; Nordlund et al., 1981b). This suggested that the antibodies were not cytotoxic and the cause of depigmentation, but a marker of the immune dysregulation in this group of patients. The initial attempts to detect anti-melanocyte antibodies utilized immunocytochemical techniques. They were unsuccessful, possibly because they were too insensitive or the quantity of antibodies on the melanocytes was insufficient for detection. In vitro studies using radiolabeled melanoma cells and sera from patients with vitiligo showed positive findings. Immunoglobulin G (IgG) in sera from those who had vitiligo bound to melanoma cells more frequently and in higher titers than immunoglobulins in sera from normal control subjects (Naughton et al., 1983). It has been suggested that the antibodies correlate with the extent of the disease (Bystryn and Naughton, 1985; Bystryn and Pfeffer, 1988; Harning et al., 1991; Naughton et al., 1986a). The same antigens detected on human melanoma cells by antibodies from patients with vitiligo are also identified by antibodies in the sera of various animals with depigmenting syndromes (Bystryn and Naughton, 1985; Bystryn and Pfeffer, 1988; Harning et al., 1991; Naughton et al., 1986b). Subsequently, the studies were extended to use radiolabeled, normal melanocytes growing in culture for the detection of antibodies (Bystryn, 1989; Cui et al., 1992; Harning et al., 1991). These antibodies appear to detect the same or similar antigens on both human melanocytes and melanoma cells (Cui and Bystryn, 1995; Cui et al., 1992, 1995). The antibodies added to cultures of

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

melanocytes cause cytotoxicity, an observation consistent with their having a role in the cause of vitiligo (Cui et al., 1993; Norris et al., 1988). It has been suggested that the antibodies can attack both normal and malignant pigment cells (Cui and Bystryn, 1995; Cui et al., 1992, 1995). These antibodies have been implicated as the cause of depigmentation observed in patients with melanoma. To document the cytotoxic effects of these antibodies, sera from individuals with vitiligo vulgaris were injected twice daily for 4 days into nude mice bearing human skin xenografts. The tissues were biopsied up to 24 hours after injection. The number of dopa oxidase-positive cells was decreased compared with skin on animals injected with normal sera. By electron microscopy, the number of cells seemed to be decreased, and there was evidence of vacuolization of keratinocytes and melanocytes (Gilhar et al., 1995). The antigens identified by most of the identified antibodies are not melanocyte specific (Cui et al., 1992, 1995; Naughton et al., 1984, 1986a). The antibodies also bound to other malignant cell lines not of pigment cell origin. They seem to identify common antigens on cells that are not derived from melanocytes, and one antibody seems to identify portions of the class I major histocompatibility (MHC) complex. Results of recent studies strongly suggest that tyrosinase is the target for antibodies in patients with vitiligo (Song et al., 1994). Sixtyone per cent of patients with vitiligo had such antibodies in their sera. No control subjects had such an antibody, although 12% of individuals with autoimmune endocrine disease did have such antibodies (Song et al., 1994). Such antibodies would be considered melanocyte specific, as the only cells known to contain this enzyme are neural crest melanocytes or forebrainderived retinal pigment epithelial cells.

T-cell-mediated autoimmune responses in vitiligo A role for T cells in progressive depigmentation has long been overlooked, most likely because minute lymphocytic infiltrates are limited to a very narrow margin of actively depigmenting skin. However, lymphocytic infiltrates are consistently present and correlate with progression of depigmentation in both inflammatory and generalized vitiligo (Le Poole et al., 1996; van den Wijngaard et al., 2000). Within these infiltrates the CD4/CD8 ratio is decreased compared to unaffected skin, suggesting that cytotoxic T cells mount a specific immune response to melanocytes during progressive depigmentation (Wankowicz-Kalinska et al., 2003). Indeed, MART-1 reactive CD8+ T cells are more abundant in the circulation of vitiligo patients than controls (Ogg et al., 1998). A causative role for cytotoxic T cells in depigmentation is supported by vitiligo skin-derived T cells with type 1 cytokine profiles demonstrating cytotoxicity towards autologous melanocytes (Wankowicz-Kalinska et al., 2003). In many respects the immune response in vitiligo parallels anti-tumor immunity in melanoma (Das et al., 2000). This is elegantly supported by the observation that mice can develop CD8+ T-cell-mediated progressive depigmentation of the pelage following vaccination against melanoma (van Elsas et al., 2001). Interestingly, the application of stress to the skin is important for inducing

depigmentation in vaccinated mice (Lane et al., 2004). Similarly, human vitiligo patients frequently display a Koebner phenomenon in response to skin damage and evidence is accumulating that melanocyte-derived HSP70, induced in response to stress, will support precipitation of vitiligo by activating dendritic cells (Kroll et al., 2005). In human melanoma patients (Chapter 38) presenting with depigmentation of the skin, T cells expressing the same T-cell receptors are found in the tumor and the depigmenting lesions (Becker et al., 1999). In fact, the development of leukoderma is considered a positive prognostic factor in human melanoma patients (Nordlund et al., 1983). The immunogenicity of melanocytes appears to be defined primarily by the melanosome, targeted by the humoral as well as the cellular response to melanocytic cells. The melanosome is a source of antigens to be presented in the context of MHC class II, and targeting tumor antigens to the melanosome can enhance an immune response mounted against the tumor (Wang et al., 1999). Importantly, T cells derived from vitiligo skin appear to have a higher avidity for their targets than tumor-infiltrating T cells, and it is likely that T cells from vitiligo skin express T-cell receptors with higher affinity for melanosomal antigens than T cells derived from melanoma tumors (Garbelli et al., 2005). It will be of interest to learn whether an inadequate regulatory response in vitiligo patients fails to keep the autoimmune response to melanocytes in check. Commentary The origin of the theory suggesting that vitiligo is an autoimmune disease is not clear. It seems to have arisen during the early 1960s when the immune system was implicated in a large number of diseases. The clinical associations of vitiligo with polyglandular failure have been used to support the hypothesis and might be the strongest clinical data available. That patients with lymphoma develop vitiligo is problematic. Most such patients have immune deficiencies that are the cause of frequent infections. The same problem is raised by those with AIDS who develop vitiligo. It might be suggested that such patients might be protected from vitiligo because their immune system, either humoral or cytotoxic, is impaired. The antibodies to melanocytes have been implicated as definitive data. The antibodies are not melanocyte specific, although they are found most commonly in high titers in patients with vitiligo. Such antibodies could be the markers of the disease rather than the cause of it. That might also explain how melanomas from humans but also many animals share the same antigenic determinants identified by these antibodies (Austin and Boissy, 1995; Song et al., 1994), some of which are cytoplasmic and not membrane molecules. That tyrosinase is identified by the antibodies suggests that the contents of the pigment cells are released into the circulation where they initiate an immune response. That the antibodies can kill melanocytes in vitro suggests that the immune system might be involved in some way in killing of melanocytes, at least in some patients with vitiligo. The plethora of data relating to an autoimmune mechanism 573

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for some individuals with vitiligo is very supportive of this hypothesis. The more recent data about the development of depigmentation during immunotherapy for melanoma (cited above) adds very strongly to the autoimmune hypothesis. It seems very likely that the immune system through activated lymphocytes or immune cytokines is one mechanism by which vitiligo is caused.

The Autocytotoxic Hypothesis The autocytotoxic theory was proposed (Lerner, 1971; Lerner and Nordlund, 1978a) because of the observation that vitiligo seemed to affect hyperpigmented skin more often than normal colored skin. The patients of Thomas Addison were all hyperpigmented because of destruction of their adrenal glands by tuberculosis (Addison, 1855). The skin around the bodily orifices such as the eyes, mouth, nose, umbilicus, and genitalia was considered to be hyperpigmented and thus susceptible to vitiligo. The clinical observations were supported by the observation that hydroquinone derivatives such as monobenzone are capable of causing “chemical leukoderma.” It has been postulated that these chemicals disrupted the normal synthesis of melanin causing excessive production or leakage of toxic intermediates into the cytoplasm of the melanocyte and cytolysis (Cummings and Nordlund, 1995; Mosher et al., 1977). Chemicals with structures similar to melanin intermediates have been added to cultures of melanocytes or melanoma cells. The cells undergo cytolysis (Prezioso et al., 1990; Wick 1987a, b, 1989). It has been suggested that the depigmentation associated with melanoma is related to the high concentrations of melanin precursors in the blood and tissues of these patients. The concentrations are high because most of them have metastatic disease. This theory was the basis for suggesting that vitiligo be induced in patients with melanoma by applications of monobenzone (Lerner and Nordlund, 1977). The compounds have been tried clinically with some success in shrinking melanoma but without changing the ultimate course and/or outcome (Riley, 1967, 1969). Commentary That melanin precursors have the potential to be cytotoxic seems real. The compounds are phenols and quinones and are highly reactive. It seems that some of these compounds have a cytotoxicity specific for melanocytes. The observation that hyperpigmented skin is more involved in vitiligo does not seem verifiable. The skin around the orifices such as the eyes and mouth is darker in some individuals. But that skin is darker related to vascular abnormalities and not melanin, especially around the eyes. The genitalia are darker, but vitiligo seems to affect these tissues late in many patients. The clinical observations are tenuous. The in vitro data are intriguing but remain to be confirmed. It is now known that melanin formation begins in transport vesicles. This observation requires reconsideration of this theory. Like the autoimmune hypothesis, it should not be dismissed. It will require much 574

more study and understanding of how melanin formation occurs in the melanocyte, the opportunities for leakage into vital areas of the cell, and the effects of stimulating melanogenesis on such a leakage for this theory to become a basis for therapy.

Genetic Hypothesis This theory was proposed because of several observations. That vitiligo clusters in families is well documented (Bhatia et al., 1992; Carnevale et al., 1980; Majumder et al., 1988, 1993; see section on epidemiology). In addition, it has been shown that melanocytes from humans with vitiligo do not grow well in tissue culture (Boissy et al., 1991a, b; Medrano and Nordlund, 1990; Puri et al., 1987, 1989; Ramaiah et al., 1989). The cells can be cultured successfully (Medrano and Nordlund, 1990), but special conditions are required compared with those for normal melanocytes. This suggests that the cells have an inherent abnormality that impedes their growth and differentiation in conditions that support normal melanocytes. In the past 5 years, a number of specific genes that might be involved in the vitiligo process have been identified. The first of these is PIG3.V transfected into a line of normal melanocytes (Le Poole et al., 2000). Cytoplasmic features of the transfected cells, i.e. dilated endoplasmic reticulum, resembled those observed by electron microscopy in melanocytes from patients with vitiligo (Boissy et al., 1991a, b; Im et al., 1994). Melanocytes from patients with vitiligo were found to have a mutation in the gene “VIT 1” located at 2p16 (Le Poole et al., 2001). Another group found a linkage between vitiligo and lupus erythematosus and suggested that a gene labeled SLEV1 located at 17p13 might be involved in vitiligo in some patients (Nath et al., 2001). Still other investigators have proposed a defect in the gene. Catalase causing elevated levels of cytoplasmic H2O2 might be a candidate gene in the pathogenesis of vitiligo (Casp et al., 2002). The same group suggest that involvement of the LMP/TAP locus in the MHC might be another gene (Casp et al., 2003). Catechol-O-methyl transferase has been implicated (Tursen et al., 2002). The most recent studies on several large kinships implicated a locus at 1p31, labeled the AlS1 locus that links strongly with vitiligo (Alkhateeb et al., 2002, 2003; Fain et al., 2003). There was a higher than expected association of vitiligo with other endocrine disorders such as thyroid, pancreatic, adrenal glands, or pernicious anemia (Alkhateeb et al., 2003). The same investigators found evidence that loci on chromosomes 1, 7, 8, 11, 19, and 22 had suggestive linkage with vitiligo. Commentary That vitiligo clusters in families could be interpreted to mean that there are environmental melanocytotoxins that affect certain families because of where they live. This theory could easily be implicated in other theories such as the autocytotoxic or autoimmune theories. That the cells have some inherent

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

defect seems inescapable. The nature of the insult that makes the melanocyte susceptible to injury is not clear. It is possible that phenols are one environmental agent, i.e. a version of the autocytotoxic theory. It is also possible that one of the numerous cytokines or chemical mediators of inflammation stimulates the cell and in some way becomes responsible for cellular death. This is a vague theory and can incorporate almost any abnormalities discovered. That individuals inherit a genetic susceptibility to developing vitiligo is strongly supported by the results of many epidemiological investigations and more recently by direct genetic studies. It seems probable that individuals inherit a susceptibility for developing vitiligo. The panoply of genes noted above is not surprising. There might be a large number of genes that could make a person susceptible for developing vitiligo. Various studies have suggested that a person needs three genes to develop vitiligo (Fain et al., 2003; Majumder et al., 1988, 1993; Nath et al., 1994). Thus, inheritance of three of a larger group of genes might expose the individual to developing depigmentation when exposed to the proper environmental inciting agent (Fain et al., 2003; Majumder et al., 1988, 1993; Nath et al., 1994). That many different sets of genes can produce depigmentation might explain the variations in onset, spread, location, and distribution of vitiligo observed clinically. The data supporting an immune hypothesis are strong, as are those underlying the autocytotoxic theory. The two hypotheses are not necessarily mutually exclusive (Le Poole et al., 1993b). It seems likely that individuals might inherit some genes that make the melanocytes more susceptible to injury by various mechanisms. Some of these individuals might have immune genes that are easily activated or overactivated by environmental inciting factors. The combination of three genes results in vitiligo. The clinical presentation of vitiligo seems to be consistent with such a combined theory. If this theory were to be correct, it will take longer to ferret out the various genes involved and their interactions. When the task is completed, our understanding of the melanocyte system and its interaction with the environment and the immune system will be much better understood.

Neural Hypothesis The neural hypothesis is based on superficial data. For example, the melanocyte and the nervous system are both derived from the neural crest. Both cell types use the amino acid tyrosine for their major end-products, melanin and catechols, respectively. Catechols are very similar in structure to some of the intermediates of the melanin pathway. The nervous system is very active in lower vertebrates such as amphibians. It has also been observed that patients who have sympathectomy can develop a hypopigmented iris, an observation suggesting that the melanocyte is innervated (Koga, 1977; Lerner et al., 1966). The depigmented skin exhibits abnormalities of the autonomic nervous system, increased adrenergic tone, and decreased parasympathetic tone (Al’Abadie et al., 1994; Iyengar and Misra, 1988; Kaur and Sarin, 1988; Le Poole et al., 1994; Merello et al., 1993).

Although these data are true, they do not support any hypothesis well. Segmental vitiligo has been one of the strongest clinical signs that suggest a neural origin. It also has been suggested that segmental vitiligo responds to therapy with agents that alter neural function (Koga, 1977). Commentary This theory uses superficial data about melanocytes and neural cells to make an association, i.e. mostly embryological data. That the irides lose color following sympathectomy is not thought to be due to a cytotoxic reaction but rather to loss of stimulation of uveal melanocytes. The distribution of segmental vitiligo is often said to be dermatomal. In actuality, it is not dermatomal, i.e. it does not follow a specific pattern of cutaneous sensory nerves. The terms segmental and dermatomal are often used interchangeably. That is not correct and, as noted above, the distribution of segmental vitiligo is rarely truly dermatomal and does not resemble the distribution of sympathetic or parasympathetic nerves, metameres, Blaschko’s lines, or other developmental patterns. It has been stated that, without implicating the nervous system, it is difficult to explain segmental vitiligo. That might be true but not sufficient for generating a hypothesis. That depigmented skin functions abnormally should not be surprising. That one of three major epidermal cells is absent should suggest that such skin might be abnormal in its functions.

Other Extant Hypotheses Other theories are extant. It has been suggested that melanin synthesis stimulated and altered by melatonin generates radical oxygens causing melanocyte death (Slominski et al., 1989). Others have suggested that a previously unrecognized biochemical pathway for the production of thioredoxin is involved in the death of melanocytes (Schallreuter and Pittelkow, 1988; Schallreuter et al., 1987, 1991). The synthesis of tyrosine in the epidermis and the production of tetrahydrobiopterin have also been implicated (Schallreuter et al., 1994a, b, c). The latter pathway is interconnected with the thioredoxin pathway. It has been suggested that the depigmentation is a result of a blockade of tyrosine synthesis within keratinocytes related to an excess accumulation of 7-tetrahydrobiopterin within the epidermis and catechols in the serum and tissues (Schallreuter et al., 1994a, b, c). The accumulation of the tetrahydrobiopterin is due to a deficiency in the activity of the enzyme 4a-hydroxytetrahydrobiopterin dehydratase that normally recycles the biopterins. The accumulation of 7tetrahydrobiopterin blocks the production of tyrosine from phenylalanine. It is concluded that the melanocytes are deprived of the essential substrate for synthesis of melanin and the skin turns white. It is also stated that vitiliginous skin fluoresces under Wood’s lamp illumination because of the presence of the biopterins. Commentary The role of melatonin in melanocyte physiology is completely unknown at this time. It has an important role in other 575

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metastasize to the lungs and other internal organs. Most of the tumors undergo a spontaneous regression of the melanoma accompanied by loss of normal pigmentation of the skin and hair in about 10% of the animals. Most piglets survive into adult life. Those that depigment appear to have a vitiligo-like condition. Most exhibit destruction of the ocular pigmentation similar to uveitis observed in the Vogt–Koyanagi–Harada syndrome (Feeney-Burns et al., 1985; Richerson et al., 1989). The regression, the development of depigmentation (Cui and Bystryn, 1995), and ocular inflammatory conditions are thought by many to be immune mediated. However, antibodies to ocular melanocytes have not been identified by modern techniques (Feeney-Burns et al., 1985). Fig. 30.39. This C57BL/6mivit/mivit mouse. This animal has a type of depigmentation that resembles, but is not identical to, human vitiligo.

animals including other mammals. It seems to have less effect directly on melanocytes than on the production of melanocytestimulating hormone, at least in other animals. It has not been shown to stimulate free radical formation. The thioredoxin reductase pathway is intriguing. It is present in many, if not most, tissues. Proof of its existence in the skin or melanocytes is lacking. Its role in vitiligo is unknown, but it would be a good candidate mechanism to support a genetic hypothesis. The role of tetrahydrobiopterin remains to be determined. Its basis that the vitiliginous skin fluoresces is not an observation accepted by other clinicians. That melanocytes are present in depigmented skin but incapable of synthesizing melanin due to lack of tyrosine does not correlate well with other data. The histology of the depigmented skin suggests absence of melanocytes. Pigment cells incapable of producing melanin, thereby causing vitiligo, should be observable by electron microscopy just as such cells are easily visible in the skin of albinos who are incapable of synthesizing melanin. The hypothesis does not explain the clinical problem of treating nonhair-bearing skin with psoralen with exposure to ultraviolet light (PUVA). Such skin does not respond for lack of a reservoir (see section on therapy). This hypothesis suggests that all skin should respond to therapy in a similar fashion. More data from future studies might resolve these dilemmas.

Animal Models The Sinclair Swine This breed of swine was observed to have several pigmentary disorders, all thought to be inherited (Amoss et al., 1988; Millikan et al., 1973; Misfeldt and Grimm, 1994; Richerson et al., 1989). The mode of inheritance is complex and probably polygenic (Tissot et al., 1987). The newborn piglets have lesions that clinically and histologically resemble human nevocellular nevi (Amoss et al., 1988; Greene et al., 1994; Millikan et al., 1973). The piglets also have a propensity for developing lesions that resemble human melanoma. The tumors 576

C57BL/6 MivitMivit Mouse This mouse was observed in the breeding colonies of Jackson Laboratories, Bar Harbor, MA, USA. It is a strain of the common C57BL/6 mouse with a spontaneous mutation. It is born with a piebald band on the ventral surface of the thorax visible when the first coat of fur develops. As the mouse goes through successive shedding of fur, it depigments completely over a period of 6–12 months (Fig. 30.39) (Boissy et al., 1987; Lerner et al., 1986). The condition resembles human vitiligo. The animals exhibit a loss of ocular pigment cells (Boissy et al., 1987; Smith, 1995; Smith et al., 1994). The mechanism for destruction of cutaneous and ocular melanocytes is not known but is related to a mutation at the mitf locus (Hodgkinson et al., 1993; Lamoreux et al., 1992). Transplantation of skin from the parental C57BL/6 strain to the mutant mouse does not cause the donor skin to depigment. Reciprocal grafts from the mutant depigment on the parental strain. This suggests that the defect is intrinsic to the melanocyte, a finding consistent with the studies reported on the melanocytes from the mutant mouse in culture (Boissy et al., 1991a, b). The animal, like humans, exhibits a loss of ability to respond to potent contact sensitizers (Rheins et al., 1986). This deficit is not caused by lack of Langerhans cells (Palkowski et al., 1987) or other deficits within the immune system (Amornsiripanitch et al., 1988). It seems to be related to the inability of depigmented skin to produce the adhesion molecule ICAM-1 (Nordlund et al., 1995). The animal is not immune to melanoma. B-16 melanoma cells injected into the mutant animal grow as rapidly as they do in the parental strain (Lerner et al., 1985), an observation that suggests that the mechanism for destruction of the normal melanocytes is intrinsic to the cells and not mediated by an extrinsic factor such as antibodies. The depigmentation in this model is inherited as an autosomal trait. The gene responsible has been identified (Hodgkinson et al., 1993; Lamoreux et al., 1992). It is in the mitf locus and is a transcription factor. The human homolog has been identified and seems to be involved in Waardenburg’s syndrome (Tassabehji et al., 1992). It has been studied in patients with vitiligo and has not been found to be abnormal (Tripathi et al., 1995). The latter observation suggests that this locus is not involved in the human disorder vitiligo vulgaris.

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

Smyth Chicken This chicken was first described at the University of Massachusetts. It was noted to lose the pigment of the feathers during the second molt. In addition, it had a form of uveitis that caused blindness in some birds as well as a form of immune thyroiditis (Boissy et al., 1983, 1988; Fite et al., 1983; Smyth et al., 1981a, b). The animal was found to have a defect in the activity of the tyrosinase enzyme inherited as an autosomal recessive trait (Boissy et al., 1986). The destruction of melanocytes from the inherent defect elicits an immune response to melanocyte-specific molecules such as TRP-1 (Austin et al., 1992; Lamont et al., 1982; Searle et al., 1993). The immune reaction augments the destruction of melanocytes. The histopathology of the lesion shows a dense mononuclear cell infiltrate (Boissy et al., 1983) with a large number of T lymphocytes (Erf et al., 1995), features suggesting an immune attack. Treatment of the birds with immune suppressants delays or retards the amelanosis (Pardue et al., 1987). This animal has depigmentation caused by a complex mechanism, i.e. an intrinsic defect within the melanocyte augmented by an aggressive humoral and lymphocyte attack on the pigment cells in the follicles and eyes. One environmental factor is infection of the chickens with the turkey herpes virus (Erf et al., 2001). Only 10% of DAM line chickens develop amelanosis in the absence of infection, in contrast to about 90% of those that are infected by the virus (Erf et al., 2001).

Other Animals Acquired depigmentation has been described in many species of animals (Boissy and Lamoreux, 1988; Lerner, 1992). These include horses (Lerner and Cage, 1973; Levene, 1972), dogs (Cerundolo et al., 1993; Mahaffey et al., 1978), cats, elephants, whales, and many other animals (Mahaffey et al., 1978; Cerundolo et al., 1993). The mechanism for the loss of pigmentation is not known for any species. It should not be concluded that the destructive mechanisms are identical for all the species nor that any one or several species serve as a perfect model for the study of human vitiligo. Animal depigmentation is probably better termed leukoderma or depigmentation than vitiligo to avoid the implication that the mechanism of melanocytotoxicity is similar in animals and humans. It is worth noting that depigmentation is common throughout the vertebrate species, a suggestion that the melanocytes are susceptible to widespread cytotoxins presumably in foods or similar items.

Treatment of Vitiligo Introduction Vitiligo is a disease that causes the destruction of melanocytes within the epidermis and other tissues and organs. The depigmented epidermis is morphologically and functionally abnormal (see section on histology and function of depigmented skin). In addition, the disfigurement caused by the loss of pigment can be very severe. Therefore, it is appropriate to treat patients with vitiligo to restore the skin to health and the patient to a more normal appearance. Treatment is not always

successful. However, attempts to regain the pigment appropriate for the individual involved should be made when possible. Some areas of depigmentation cannot be repigmented by standard medical therapies such as topical steroids or PUVA. Such limitations should be explained to the patient. Some of these areas are treatable with surgical therapies. However, the problems involved and the cost might make surgical approaches to repigmentation not appropriate at this time. As techniques improve and/or the cost becomes lower, transplantation of melanocytes might be an option for more patients. The best therapeutic options would be a medication that would halt the progression of the depigmentation and preserve melanocytes from prior therapy. No such medication exists currently, although it has been suggested that a short trial of systemic steroids can halt the progression of the disease. Patients have been treated for a period of 4–6 weeks with tapering doses of oral prednisone, the initial dose being 30–40 mg/day. Others have recommended betamethasone given in pulses to minimize toxicity (Pasricha and Khaitan, 1993). The efficacy of these treatments is not known as the natural course of the disease is variable and difficult to assess in a short time period. Clinicians have suggested the use of agents with fewer side-effects such as antioxidants or aspirin. No evidence suggests that such agents work alone or in combination. Most antioxidants such as alpha-tocopherol, ascorbic acid, or beta-carotene are thought to be free from side-effects (Koshevenko Iu, 1989a). However, aspirin has many potential toxic effects in adults and more in children. Its use to halt the progression of the disease should be undertaken with consideration of its experimental nature and toxicity.

The Melanocyte Reservoir The depigmented skin is devoid of melanocytes, at least as can best be determined by current evidence. Depigmented epidermis stained with a variety of melanocyte-specific or nonspecific stains appears to be devoid of melanocytes. Electron microscopy fails to find basilar cells that resemble melanocytes or even indeterminate cells that might represent immature melanocytes. The histologic data seem to be confirmed by the clinical response of depigmented skin to therapy. If the melanocytes are indeed absent, then a source of melanocytes is required to repigment the skin. The source can be the edge of the lesion. However, patients successfully repigmented by a medical therapy such as topical steroids or PUVA rarely exhibit a large migration of pigment from the edges of the lesion into the depigmented skin. For reasons not clear, it seems that melanocytes cannot migrate more than a few millimeters from their origin. The most obvious source is the hair follicle (Fig. 30.24). Several studies have been done that strongly suggest that the melanocytes in the follicle are the source of melanocytes in skin repigmented by a standard therapy (Fig. 30.40) (Arrunategui et al., 1994; Cui et al., 1991; Fukuda, 1979; Ortonne et al., 1978b, 1980). Data suggest that the reservoir resides in the lower third of the follicle (Arrunategui et al., 1994) (Fig. 30.41A and B). The importance of follicles 577

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Fig. 30.40. Repigmentation occurs in a perifollicular pattern. It is one indication that the follicular melanocytes are the reservoir for repigmentation. See also Plate 30.22, pp. 494–495.

as the reservoir of melanocytes is emphasized by the wellknown fact that glabrous (i.e. nonhair-bearing skin) does not respond to therapy. The distal ends of the fingers, the epidermis over the knuckles, the skin below the ankles, the genitalia, and the lips are covered with glabrous skin and do not respond to therapy unless they have some residual pigment. Another observation about the importance of the reservoir is more convincing. Most patches of vitiligo have normally pigmented hairs growing from the follicles. For reasons not known, the melanocyte in the follicle is protected from the injurious processes that affect the melanocytes in the interfollicular skin. Occasionally, in bilateral, symmetrical forms of vitiligo and more often in segmental vitiligo, the follicular melanocytes are involved, and the terminal or even vellus hairs are also depigmented. Such areas will not repigment in response to standard therapies (Fig. 30.42). Areas with white hairs located in a large patch of vitiligo that has mostly pigmented hairs resist treatment while the surrounding skin repigments readily. That such skin or glabrous skin does not repigment strongly suggests that a reservoir of melanocytes is necessary for the affected individual to respond to medical therapies. It also suggests that the reservoir is the follicle, possibly the melanoblasts within the niche area. Glabrous skin or segmental vitiligo responds to transplantation techniques, which are a mechanism for replacing the reservoir of pigment cells. Minigrafts placed into depigmented skin develop islands of pigment after healing that resemble the pigment migrating from the follicle. The islands of pigment coalesce to repigment the treated area. Such grafting techniques in particular suggest the importance of a reservoir. The implications of the need for a reservoir are several. Glabrous skin that is depigmented, i.e. in fact devoid of all melanocytes, cannot respond to therapy. The hands and lips are the most obvious such areas. The genitalia, feet, palms, and soles also cannot respond. The patient must be advised of this limitation before starting therapy as these areas are often of most importance to the person. Skin should be examined very carefully and the color of the hairs assessed. If in fact the 578

A

B Fig. 30.41. Numerous DOPA-positive melanocytes are observed in vitiligo epidermis surrounding the follicular orifice (A) and in the outer root sheath (B).

Fig. 30.42. Striking perifollicular repigmentation emanating from follicles with pigmented hair. In contrast, there is no repigmentation in vitiliginous skin with white terminal hair. See also Plate 30.23, pp. 494–495.

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

hairs are white and not blond or gray, that skin will also not respond to therapy. Depigmentation of the hairs is relatively common in those with segmental vitiligo. It seems probable that segmental vitiligo responds poorly to therapy for this reason. Some patients with bilateral, symmetrical, vitiligo vulgaris will have large areas of depigmented skin with white hairs. These areas will not respond to treatment and none should be undertaken. The patient should be informed of the areas that will not respond before undertaking therapy, especially PUVA, that is costly, time-consuming, and can have significant side-effects. Transplantation techniques are available to repigment such areas, and information about such options should be provided to the patient.

Treatment and Age Vitiligo is a disease of young people. It begins before the age of 20 years in half of those affected and before the age of 10 years in about 25%. Therefore, parents will seek therapies for their children who are disfigured by the disease. Not all therapies are appropriate for children. Generally, systemic PUVA is not indicated before the age of 12 years. Exceptions can be made in special circumstances. These younger people are candidates for topical steroids or for physicians with proper training in topical psoralens. However, they can also suffer significant toxicity from these agents. The parents should be appraised of the toxicity of either therapy before starting them. Depigmentation is sometimes advised for patients with very advanced vitiligo. The decision to depigment has many emotional and biologic implications. It must be considered as a permanent treatment, although pigment can return after applications of melanocytotoxic agents. The child cannot understand the implications and cannot contribute to the decision to undertake such therapy. Thus, children are not good candidates for depigmentation. Usually, the individual should be sufficiently mature to understand the therapy and the implications, around 18 years old. However, there can be situations in which a child of very young age might require depigmentation. Occasionally, the emotional impact of the vitiligo outweighs the objections. After careful consideration by the parents, teachers, and significant others, depigmentation might be undertaken in children. Children often are oblivious to the depigmentation. The parents are distraught. That the parents are upset is normal and understandable. They should be reassured that their feelings are normal. However, the physician must be alert to their propensity to push for standard or experimental therapies that are not appropriate for a child. At times, the parents might need counseling to assist them in bearing the emotional agony of seeing their child disfigured.

Photography The natural cause of vitiligo cannot be predicted. It can exhibit flares, remissions, and spontaneous repigmentation. Therapy takes months. The surrounding skin changes color as the tan of summer fades or is stimulated by exposure to sunlight or

ultraviolet light from artificial sources. These and other factors make the assessment of response to therapy difficult in most patients. It is most useful to take photographs with a simple unit such as a Polaroid camera at the onset and at intervals during therapy. Such photographs are useful only for those with normally dark skin or those with a deep tan. When available, they will document for the physician and for the patient that the therapy is working or is unsuccessful. Such firm data help them make decisions about continuing, changing, or discontinuing the current therapy.

Medical Therapies There is a variety of effective forms of medical therapy for the treatment of vitiligo (Chapters 58–61 and 63). They are applications of topical steroids, immune modulators, and PUVA. All, alone or in combination, can be effective. Often the reason for choosing topical steroids or immunomodulators such as tacrolimus (Grimes et al., 2002; Smith et al., 2002) first is the ease of application, the lower cost, and the possibility of success. If the vitiligo is very widespread, the ease of application is lost. The patient is a better candidate for ultraviolet light, either narrow-band UVB or PUVA, unless they wish to treat limited areas such as the face, neck, and arms. Any of these therapies can be used if the patient is of proper age. Often it is necessary to try one therapy first. If unsuccessful, another should be tried. Trials of therapy should not be indefinite. Steroids or PUVA therapy for a period of 3–4 months is usually sufficient to determine whether the patient is responding. The skin should be examined with a Wood’s lamp to detect perifollicular repigmentation. If present, the trial should continue. If no repigmentation is detectable within this timeframe, the trial should be stopped as unsuccessful. Another therapy can be tried for a similar time period. If no response is noted, the patient should wait 6–12 months before a second series of treatments is instituted. Often a second trial will be successful when the first is not. Topical Steroids and Immunomodulators Topical steroids are the easiest of all therapies and are reported to have a reasonable response rate, possibly up to 50% of those treated (Antoniou and Katsambas, 1992; Antoniou et al., 1989; Bleehen, 1976b; Clayton, 1977; Geraldez and Gutierrez, 1987; Goldstein et al., 1992a, b; Grimes, 1993; Han et al., 1986; Kandil, 1974; Kenney and Grimes, 1983; Kumari, 1984; Liu et al., 1990). Others have used injections of steroids into lesions to achieve repigmentation (Kandil, 1970; Vasistha and Singh, 1979). Whether the intralesional steroids work systemically, locally, or both is not known. There are many topical preparations of steroids available to the physician. Some are extremely potent and others very weak. The physician must recall that he/she will be using the agent for a period of 3–4 months or longer if successful. The agent will be used around the eyes, in crural folds, and over the joints where striae and atrophy might occur. The best topical agent has not been determined. Some recommend short periods of high-potency steroids, 4 weeks or less, followed by 579

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applications of moderate or lower strength steroids. Others recommend moderate-strength steroids from the onset. Some of the preparations in the latter category include desonide 0.05%, hydrocortisone butyrate or valerate 0.01%, triamcinolone acetonide 0.1%, or similar preparations in the class IV or V categories. More potent steroids such as clobetasol proprionate 0.05% have been used successfully. Because they are potent, they are applied once daily for a period of 2 weeks. During the subsequent 2 weeks, the skin can be treated with tacrolimus once daily. The sequence is then repeated over the next 2 months. The patient should be observed for signs of steroid toxicity, although toxicity seems to be rare if a lower strength steroid applied once daily is prescribed or if the potent steroids are limited in their applications. Topical steroids should not be used more than twice each day. If the patient shows evidence of repigmentation, it is appropriate to continue therapy until no further progress is observed. Photographs can be most useful for such an assessment. New medications have become available for the treatment of various cutaneous disorders. Tacrolimus and pecrolimus are two new agents that are immunomodulators similar to ciclosporin. Tacrolimus has been used for vitiligo (Grimes et al., 2002; Smith et al., 2002). The latter was a controlled study, which suggests that tacrolimus is as effective as a single agent as clobetasol proprionate (Smith et al., 2002). Pseudocatalase was proposed as a therapy for vitiligo (Schallreuter et al., 1995, 2000), based on the concept that melanocyte destruction was related to abnormalities in thioredoxin reductase and in tetrahydrobiopterin metabolism (Schallreuter et al., 1994a, b, c, 2000). In uncontrolled trials, about 80% of individuals seemed to improve with applications of pseudocatalase and exposure to ultraviolet light. However, in larger, controlled trials, efficacy for this medication could not be confirmed (Schallreuter et al., 2000). Others have confirmed that this cream was not beneficial (Patel et al., 2002). Narrow-band UVB (308–313 nm) In the last few years, new types of ultraviolet light have become available (Chapters 59 and 60). Ultraviolet B (UVB) is typically emitted by fluorescent lamps in a broad spectrum (290–320 nm). The shorter waves do not penetrate deeply into the skin and produce erythema at lower doses. Newer lamps emitting UVB have a narrow spectrum (308–313 nm), labeled narrow-band UVB or NBUVB. These lamps penetrate deeper into the skin and produce less erythema. They are administered at larger doses, usually 200 mJ or more without causing sunburn. Such lamps have been used successfully for many skin disorders such as psoriasis, but also vitiligo (CastanedoCazares et al., 2003; Dogra and Parsad, 2003; Leone et al., 2003; Menchini et al., 2003; Njoo et al., 2000; Samson Yashar et al., 2003; Scherschun et al., 2001; Tjioe et al., 2002). NBUVB is often combined with applications of topical steroids or tacrolimus (Castanedo-Cazares et al., 2003; Tanghetti, 2003). Success rates are high, probably around 80%. 580

The excimer laser emits a single band of ultraviolet B at 308 nm (Chapter 63). It has been used successfully for treating vitiligo (Baltas et al., 2002; Leone et al., 2003; Spencer et al., 2002; Taneja et al., 2003). The excimer laser is comparable to NBUVB. NBUVB is administered with long (4–6 foot) fluorescent lamps and treats the entire body and both normal and depigmented skin. The excimer laser can be used to spot treat only depigmented skin. It is more costly than NBUVB. Systemic Steroids Systemic steroids have been used (el-Mofty and Nada, 1971, 1974; Farah et al., 1967; Imamura and Tagami, 1976), although their prolonged use and toxicity have made them undesirable. Steroids have been given in pulse doses to minimize toxicity with success (Pasricha and Khaitan, 1993). The results of one study suggest that systemic steroids affect the level and cytotoxicity of anti-melanocyte antibodies (Hann et al., 1993b). The benefits and toxicity of this therapy must be weighed carefully. Psoralens and Ultraviolet Light (PUVA) Psoralens and exposure to ultraviolet light are the standard therapy for treatment of vitiligo in adults and adolescents (see Fig. 30.43 and Chapter 59). Their use dates to the earliest medical writings. Psoralens have been used topically and systemically. Both are effective in many patients, although the principle regarding the need for a reservoir applies to both approaches. During PUVA treatment, the patient can note new areas of depigmentation, although other areas are repigmenting. PUVA does not prevent the onset (Todes-Taylor et al., 1983) or spread of the disease. Those with stable disease or minimally changing disease are the best candidates. Depigmentation can be very rapid. Repigmentation takes months. Rapidly spreading vitiligo is not optimally treated with PUVA. Topical psoralens have been used for many years (Africk and Fulton, 1971; Berakha and Lefkovits, 1985; Halder, 1991; Halder et al., 1987; Khalid et al., 1995; Parrish et al., 1971). They have many advantages such as lack of systemic effects. The patients do not develop nausea, there is no concern about ocular toxicity, and laboratory tests to monitor side-effects are not necessary. They can be used in children of any age. However, they are very potent and tend to cause burns readily. Both topical trimethylpsoralen (Sehgal et al., 1971) and 8methoxypsoralen (Grimes et al., 1982; Halder, 1991) have been used. Currently, only 8-methoxypsoralen is available commercially in the United States. It comes in a 1% concentration which is considered to be too strong (Grimes et al., 1982; Halder, 1991). The preparation is diluted 1:10 or 1:100 in petrolatum or a similar solvent. It is applied in the physician’s office and shortly after the patient is exposed to ultraviolet light. Great care must be taken to prevent overexposure or inadvertent exposure to ultraviolet A in sunlight (Parrish et al., 1971). The dose of ultraviolet A to which the patient is exposed must be monitored with great precision. The starting dose is usually 0.25 joules/cm2 and increased at each session

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

A

B

Fig. 30.43. Excellent repigmentation (B, 25 months later) from PUVA in a patient with extensive vitiligo on the trunk (A). See also Plates 60.1 and 60.2, pp. 494–495.

by 0.1 joule until the treated skin develops a mild pink color after the treatment. Without such precautions, the probability of painful, blistering burns is very high. Such burns often heal with repigmentation but take weeks to do so. Most patients suggest that the toxicity is not tolerable. For the motivated patient able to follow instructions carefully, topical PUVA can be a useful modality of therapy. Systemic psoralens have been widely used for the treatment of vitiligo (Fig. 30.43). Both trimethylpsoralen and 8methoxypsoralen have been used successfully (Abdullah and Keczkes, 1989; Antoniou and Katsambas, 1992; Berakha and Lefkovits, 1985; Bonafe et al., 1983; el-Mofty and Nada, 1974; Gupta and Anderson, 1987; Halder et al., 1987; Kao and Yu, 1992; Kwok et al., 2002; Lindelof and Sigurgeirsson, 1991; Lynch et al., 1977; Ortonne et al., 1979; Tran et al., 2001; Wildfang et al., 1992). Not all patients respond. However, a review of the literature suggests that over half will get a moderate or extensive response (Ortonne et al., 1978c; Pathak et al., 1984). Generally, the 8-methoxypsoralen is used more commonly, although either agent is useful (Parrish et al., 1976). The doses of the two must be adjusted appropriately. The source of ultraviolet A can be a light box made for the treatment of patients with cutaneous diseases (Gupta and Anderson, 1987; Parrish et al., 1976) or the natural sun

(Africk and Fulton, 1971; Pathak, 1984; Theodoridis et al., 1976). The dose of ultraviolet light is more difficult to control for sunlight compared with that from a medical box. The patient takes the drug 1–2 hours before exposure. The dose of 4,5,8-trimethylpsoralen is about 2–3 mg/kg. The dose must be individualized for each patient. The dose of PUVA is adjusted to produce mild erythema. The dose of 8-methoxypsoralen depends on which preparation is used. The older preparation required a higher dose, 0.3–0.5 mg/kg. A newer microsized preparation requires less drug because of better absorption. The recommended dose is 0.2–0.3 mg/kg (el-Mofty et al., 1994). The end-point is erythema in the treated skin. The dose of UVA must be individualized to each patient. The initial amount is determined by the amount of psoralen administered by mouth. For those taking higher doses of 8-methoxypsoralen, 0.3 mg/kg or more, the initial dose is 0.5–1.0 joules, which is increased in similar increments until erythema develops. For those taking smaller doses, 0.2 mg/kg, the first dose of UVA will be about 4 joules, which will increase by 1–2 joules until erythema develops. The final dose of UVA is determined by the development of erythema. Lower doses of psoralen provide a larger margin for error without burn. However, there is some concern about the 581

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carcinogenicity of UVA (Gupta and Anderson, 1987). It has not been established which is the greater carcinogen, UVA alone, psoralen alone, or the two combined. If the number of treatments is monitored so that the patient continues treatment only if repigmentation is occurring, the potential risks for skin cancer, almost invariably squamous cell carcinoma, seem small. Patients are treated two to three times per week. Treatments are never given on successive days. Preferably, a period of 48–72 hours elapses before the next treatment. Therapy is continued for a minimum of 3–4 months. The depigmented skin should be examined with a Wood’s lamp (Siddiqui et al., 1994) to detect the earliest signs of repigmentation. Once repigmentation has started, therapy should be continued until the area is repigmented or progress has stopped. Photographs are invaluable in determining the course of therapy. The patient should wear protective eye wear, NOIR glasses, on the day of therapy until nightfall. Annual eye examinations are recommended. However, there is no evidence to date that PUVA has had deleterious effects on the eyes in humans if done properly (Prystowsky et al., 1992; Stern, 1994). Results of studies indicate that UVA does not penetrate the eyelid (Prystowsky et al., 1992). Studies in patients receiving PUVA for psoriasis have suggested that they have a high incidence of squamous cell carcinomas. In the USA, there is a disturbing trend for patients who have received PUVA to develop melanoma. This has not been confirmed by long-term studies in Europe (Basarab et al., 2000; Hannuksela-Svahn et al., 1999; Lindelof et al., 1999; Stern, 2001). Other Photochemotherapy Patients with vitiligo have been treated with other photosensitizing agents. Khellin, similar to psoralens chemically, has been used systemically and topically with success (AbdelFattah et al., 1982; Jung and Fingerhut, 1988; Morliere et al., 1988; Orecchia and Perfetti, 1992; Vedaldi et al., 1988). The drug has not been approved for use in the United States but is available in other countries. It is reported to cause abnormalities of liver function when used topically and systemically. 5-Methoxypsoralen (5-MOP) is another psoralen that has been used successfully to treat patients with vitiligo (Beretti et al., 1989; Hann et al., 1991; Karppinen et al., 1992; Morliere et al., 1988; Pathak and Fitzpatrick, 1992; Pathak et al., 1992). The drug is less phototoxic and has the capacity to stimulate repigmentation. 5-MOP rarely causes gastric discomfort and is well tolerated by most patients (Hann et al., 1991). The results of a comparative study of the three psoralens available (trimethylpsoralen, 5-MOP, and 8-MOP) suggest that 5-MOP in doses of 0.6–1.2 ng/kg body weight is more effective than 8-MOP and has fewer side-effects (Pathak et al., 1992). In a study of 271 patients, good or excellent repigmentation was obtained in 62% treated with 5-MOP (Karppinen et al., 1992). The addition of phenylalanine to the regimen was thought to improve the results (Beretti et al., 1989). 582

Another agent that has been used is the amino acid phenylalanine (Antoniou et al., 1989; Beretti et al., 1989; Cormane et al., 1985; Kuiters et al., 1986; Schulpis et al., 1989; Siddiqui et al., 1994; Thiele and Steigleder, 1987). The dose of l-phenylalanine is 50 mg/kg body weight. The patients have been exposed to sunlight (Kuiters et al., 1986) or artificial UVA (Siddiqui et al., 1994). Children have been safely and successfully treated with this therapy (Schulpis et al., 1989). Another therapy proposed for the treatment of vitiligo is melaginina. It is a placental extract, and the active ingredient is thought to be a lipoprotein. It is applied topically and the patient is exposed to light. Originally, the activating spectrum was thought to be in the infrared spectrum. Later, the ultraviolet spectrum was thought to be more effective. Most recently, light is no longer considered to be essential for repigmentation. This agent has been alleged to have significant efficacy. However, the data to support the claims are lacking (Nordlund and Halder, 1990). It is considered an unproven therapy at this time. Depigmentation Some patients have vitiligo too extensive for repigmentation therapy. Others have involvement of glabrous skin that cannot respond. Some have significant disfigurement of the face that is partially hidden during the winter months when their tan is gone. These individuals often do not want to undergo PUVA because it accentuates the contrast between involved skin and the depigmented skin. These and other patients often desire to depigment (Chapter 58). Most prefer their normal skin color. All abhor having two skin colors. They accept depigmentation as an acceptable alternative if their normal color cannot be regained. Depigmentation of patients with applications of monobenzone is not a therapy that should be undertaken lightly. It should be considered permanent, although often small islands of pigmentation occur in sun-exposed skin such as the face and arms. These areas must be retreated periodically. At least three patients have been described who had virtually total depigmentation but pigmented hairs on their bodies. These three individuals have repigmented almost their entire body spontaneously, although the glabrous skin remained white. Thus, repigmentation can occur. The depigmented skin will be more photosensitive, and the patient will have to alter his/her perception of himself/herself because they are depigmented. The patient must be given time to understand and accept the change in color. Depigmentation is performed by sparing applications of 20% monobenzone in a cream (Chapter 58). The applications are done twice daily. When starting the treatment, the patient should apply the cream only once daily to a small spot on the arm or trunk. This application is a test area to determine whether the cream is irritating. Some patients develop an allergy to the medication and must stop the applications because of the intense inflammation (Nordlund et al., 1985a). The test spots help to detect the allergy without producing a

GENETIC HYPOMELANOSES: ACQUIRED DEPIGMENTATION

severe reaction in large, exposed areas. If no allergy is detected, the patient continues the application for as long as it takes to depigment. Not all patients respond, but more than half can expect a markedly improved outcome (Mosher et al., 1977). The complications of monobenzone are allergies. It has also been noted to cause a pigment deposit in the cornea but without complications (Hedges et al., 1983). It is preferable to limit depigmentation to adult patients. However, there are instances when the treatment is appropriate for a child.

Surgical Therapies There are many ways to transplant melanocytes from a pigmented patch of skin to a depigmented patch (Chapter 62). The purpose of the transplant is to replace the reservoir of melanocytes usually found in hair follicles. The most common sites treated by transplantation are glabrous skin or vitiliginous skin with white hairs. Transplantation involves two processes. The first is harvesting of pigment cells; the other is removal of the depigmented epidermis from the site to be treated (Chapter 62). Melanocytes can be obtained by many methods. The original method was transfer of skin by punch biopsy in a procedure similar to hair transplants (Beck and Schmidt, 1986; Falabella, 1989; Falabella et al., 1989, 1992, 1995; Suvanprakorn et al., 1985). Sheets of epidermis containing melanocytes can be prepared by suction. Such sheets can be transferred to a recipient site (Agrawal and Agrawal, 1995a, b; Chitale, 1991; Falabella, 1983, 1986, 1988, 1989; Falabella et al., 1992; Kahn and Cohen, 1995; Koga, 1988; Mutalik, 1993; Zachariae, 1994; Zachariae et al., 1993). Others have used cultures of epidermis or melanocytes for transplantation. These methods have also been successful (Brysk et al., 1989; Falabella, 1989; Falabella et al., 1989; Lerner et al., 1987; Lontz et al., 1994; Olsson and Juhlin, 1993, 1995; Olsson et al., 1994; Plott et al., 1989). An adaptation of these techniques has been described that might simplify grafting of melanocytes. The donor skin is removed by dermatome, and the epidermis is made into single-cell suspensions with trypsin. These cells are injected into blisters on the depigmented skin formed by freezing of the skin. About 65% of individuals show good to excellent repigmentation with this method (Gauthier and Surleve-Bazeille, 1992). The recipient site is prepared in several ways. For transfer of punch minigrafts, no special procedures are required (Fig. 30.44). A surgical blade is used to produce puncture sites and the minigraft is inserted. For transfer of sheets of epidermis, the recipient site can be frozen or dermabraded to remove the depigmented epidermis. The sheet of epidermis is applied to the abraded skin and the melanocytes migrate into the healing tissue. Cultured melanocytes are injected into blisters produced by freezing or applications of some vesicant. These techniques produce excellent results (Chapter 62). They are expensive and require special laboratory facilities for culture methods. They are time consuming and not useful for very extensive disease or for children. The isomorphic response might limit the usefulness of these techniques

Fig. 30.44. Note the repigmentation surrounding minigrafts of the neck of a 22-year-old vitiligo patient.

(Hatchome et al., 1990). The long-term survival of melanocytes in this skin or in skin repigmented by medical therapies is not known.

Micropigmentation Tattooing or micropigmentation has been found to have some use in the treatment of vitiligo (Halder et al., 1989; Hegyi et al., 1993). It seems to be especially useful for coloring the mucous membranes of darker colored individuals who have vitiligo. The lips of these individuals become a pink color when affected by vitiligo. The normal color is a gray–brown. Micropigmentation can restore the lips to a more normal appearance, although the color match is not perfect (Chapter 62).

Other Therapies Many other therapies have been suggested (al-Omari et al., 1989; Azambuja, 1992; Bagaeva, 1979; Banerjee and Chowdhury, 1967; Bor, 1973; Borisenko, 1973; Dolecek, 1968; Gupta et al., 1990; Hernandez-Perez, 1979; Karagezian, 1989; Leong, 1990; Montes et al., 1992; Nordlund and Halder, 1990; Orecchia and Perfetti, 1989; Pasricha and Khera, 1994; Renfro and Geronemus, 1992; Shukla, 1981; Srinivas et al., 1990; Tronnier, 1984; Tsuji and Hamada, 1983; Urbanek, 1983; Wakefield et al., 1990). The therapies include B vitamins, vitamins with antioxidant properties, steroids, immunomodulators, extracts, and photosensitizers. Whether they produce a beneficial result is not known. Some have significant toxicity, others minimal or no adverse effects. The physician must evaluate each therapy individually with the consideration that sunlight alone can possibly produce repigmentation. It is important to keep in mind that vitiligo can repigment spontaneously. The benefits and risks must be evaluated carefully before starting a therapy of questionable or unknown benefit.

Counseling Most patients are distressed by vitiligo and would like to have a successful treatment. Some are deeply disturbed, others 583

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concerned, and others not bothered at all (Antoniou and Katsambas, 1992; Boehncke et al., 2002; Grimes, 1993; HillBeuf and Porter, 1984; Kent and Al’Abadie, 1996; Koshevenko Iu, 1989b, c; Papadopoulos et al., 1998; Parsad et al., 2003; Porter and Beuf, 1988, 1991, 1994; Porter et al., 1978, 1986, 1987, 1990; Salzer and Schallreuter, 1995). The physician can care for most of these and needs to be empathetic about the desire for therapy. It is useful for the physician to consider what he/she would do if he/she had vitiligo depigmenting his/her face and hands. There are a few patients who are sufficiently disturbed that formal psychiatric counseling is indicated.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

31

Genetic Hypomelanoses: Generalized Hypopigmentation Sections Oculocutaneous Albinism Richard A. King and William S. Oetting Albinoid Disorders Philippe Bahadoran and Jean-Paul Ortonne Ataxia Telangiectasia Anne-Sophie Gadenne Hallerman–Streiff Syndrome James J. Nordlund Histidinemia Marnie D. Titsch Homocystinuria Allan D. Mineroff Oculocerebral Syndrome with Hypopigmentation Jean L. Bolognia Tietz Syndrome Jean-Paul Ortonne Kappa-Chain Deficiency Jean-Paul Ortonne Menkes’ Kinky Hair Syndrome Tanusin Ploysangam Phenylketonuria Allan D. Mineroff

Oculocutaneous Albinism Richard A. King and William S. Oetting

Historical Perspective Variation in mammalian coat color and pigment patterns has been described throughout human history, dating back to the early cave paintings in Europe. Most of the noted variation has represented normal differences in animal colors and patterns or differences in human color. Altered or abnormal pigment patterns, including the lack of pigment, have also been a long and historical fascination, and descriptions of nonprimates and primates with reduced or absent cutaneous pigmentation have been noted throughout recorded history. Human albinism is perhaps the first observed genetic disease because of this obvious phenotype (Pearson et al., 1911). Pearson et al. (1911) provided the first comprehensive scientific review of albinism, with an extensive monograph that included text, pedigrees, photographs, drawings, and an histologic atlas of individuals and families with albinism from many parts of the world. They noted in a chapter on “early notices of the occurrence of albinism” that Ctesias, the physician to Artaxerxes who lived about 401bc, described two women and five men who had albinism, and Pliny, the Roman naturalist, described eye color, white hair, and photophobia in individuals who likely had albinism. The exploration of large parts of the world by European explorers in the sixteenth and seventeenth centuries provided increasingly frequent observations of individuals and groups who were hypopigmented or who had white hair and skin

(i.e. oculocutaneous albinism) in diverse areas such as north Africa, the Middle East, India, Ceylon, Brazil, and Mexico (Pearson et al., 1911). The advent of scientific medical literature in the eighteenth and nineteenth centuries produced clinical descriptions of individuals with albinism (Hotz, 1907; Spencer, 1977). The rediscovery of Mendelian principles and mechanisms of inheritance at the turn of the twentieth century provided an understanding of the recessive genetic nature of albinism (Davenport and Davenport, 1910), and led to Sir Archibald Garrod including albinism (e.g. oculocutaneous albinism) as one of the “inborn errors of metabolism” in his 1908 Croonian lectures (Garrod, 1908). Garrod based most of his observations of inborn errors on altered urinary excretion of metabolites (e.g. alkaptonuria, cystinuria, and pentosuria), but the obvious lack of melanin in the skin and eyes allowed him to recognize the metabolic nature of albinism and its relationship to reduced synthesis of melanin, and to infer that this resulted from a block in a metabolic pathway because of the lack of the appropriate intracellular enzyme (Garrod, 1908). The development of investigative science and the rediscovery of Mendelian principles also had a profound effect on the study of coat color genetics in nonhuman animals, and early studies on the inheritance of albinism in mice were published by Castle and colleagues as early as 1903 (Castle and Allen, 1903). This important area of science expanded greatly with the development of knowledge about the melanocyte and the melanin biosynthetic pathway (Silvers, 1979). There has been an evolution in our understanding of the complex syndrome of albinism. An individual with no cuta599

CHAPTER 31

neous and ocular pigment has always been easy to recognize as having albinism, and this was called “complete,” “total,” “universal,” or “perfect” albinism or oculocutaneous albinism in the early literature. Individuals with ocular features of albinism and reduced skin and hair pigmentation (i.e. not a total absence of pigment) presented a problem, and a number of confusing terms such as “incomplete” or “partial” or “imperfect” albinism were used. Evidence for genetic heterogeneity was found in the statistical analysis of the frequency of albinism in the general population in comparison to the frequency of consanguinity in families with albinism (Stern, 1973; Waardenburg, 1970). The identification of normally pigmented offspring arising from the mating of two individuals with oculocutaneous albinism provided proof through complementation that genetic heterogeneity must exist (Taylor, 1978; Witkop et al., 1970), and the development of the hair bulb test provided a clinical test for separating types of albinism (Witkop et al., 1961, 1971). This test, in which a freshly plucked hair bulb was incubated in tyrosine or dopa, separated oculocutaneous albinism into “tyrosinase-negative” and “tyrosinase-positive” types based on the absence or presence of melanin in the hair bulb after incubation. A family demonstrating complementation was shown to be a mating between an individual with “ty-neg” and “ty-pos” oculocutaneous albinism, indicating that separate gene loci are associated with these two types of albinism. Subsequent work has shown that albinism can be divided into a number of oculocutaneous and ocular types, and that the most accurate separation comes from direct gene analysis, because there is considerable overlap with the clinical phenotype between the different types.

Definition The term albinism refers to a group of genetic abnormalities of melanin synthesis associated with a normal number and structure of melanocytes in the skin and eyes (King et al., 1995, 2001). Reduced melanin synthesis in the melanocytes of the skin, hair, and eyes is termed oculocutaneous albinism (OCA) whereas hypopigmentation primarily involving the retinal pigment epithelium of the eyes is termed ocular albinism (OA). Cutaneous and ocular hypopigmentation are not sufficient to define albinism, however, because there are conditions with cutaneous or ocular hypopigmentation that are not part of the albinism spectrum. The precise definition of albinism includes ocular and cutaneous hypopigmentation associated with specific changes in the development and function of the eyes and the optic nerves, and the ocular changes are necessary to make this diagnosis (Apkarian et al., 1990; Collewijn et al., 1985; Creel et al., 1974, 1978, 1981, 1990; Guillery, 1974; Guillery et al., 1975; van Dorp, 1987). Changes in the auditory system are also found in the brain in albinism, but produce no clinically apparent problem and are generally not evaluated as part of a clinical evaluation of a child or an adult with albinism (Conlee et al., 1984, 1988, 1989, 1991; Creel et al., 1980, 1983a; Garber et al., 1982). A generalized reduction in skin 600

Table 31.1. Changes in the optic system in albinism. Reduction in iris pigment Iris translucency with globe transillumination Reduction in retinal pigment Misrouting of optic nerves at chiasm Nystagmus Alternating strabismus

pigment without ocular changes should be referred to as cutaneous hypopigmentation rather than albinism or cutaneous albinism, because there is no ocular involvement.

The Optic System Characteristic changes in the development and function of the eye (see Chapter 4) and optic nerves are common to all types of albinism, including OCA and OA, and it has been hypothesized that these changes are related to melanin or a melaninrelated metabolite during development and postnatal life (Donatien and Jeffery, 2002; Ilia and Jeffery, 1999; Jeffery, 1997, 1998). The ocular changes are present only in the individual with albinism and not in the obligate heterozygous parents with autosomal recessive OCA and OA, and a clinical examination of an obligate heterozygote reveals no hypopigmentation or anatomic change in the eye or optic nerve. In contrast to this is X-linked recessive OA in which 80–90% of the obligate female heterozygotes have observable changes in ocular pigment (Charles et al., 1992, 1993; O’Donnell et al., 1976, 1978a, b; Schnur et al., 1994; Shiono et al., 1995; Waardenburg and van den Bosch, 1956). The characteristic changes in the eye and optic system in albinism are listed in Table 31.1.

Iris Pigmentation Iris melanin is normally found in the stromal melanocytes and the posterior pigment epithelium, the latter being a continuation of the ciliary body (Rodrigues et al., 1986). The stromal melanocytes are neural crest in origin and display characteristics similar to those in the skin, whereas the melanocytes on the posterior iris surface epithelium are continuous with those of the ciliary body and retinal pigment epithelium and are neural ectoderm in origin (Mann, 1969; Ozanics and Jakobiec, 1986). The iris has a blue color without the formation of stromal pigment, and melanin in the posterior surface makes the blue iris opaque. The reduction of melanin in the stroma and the posterior epithelial layer in albinism results in a translucent iris that transmits light through the iridial tissue on globe transillumination (Donaldson, 1975; Lee et al., 2001; Summers et al., 1988; Whang et al., 2002; Wirtschafter et al., 1973). The translucency of the iris varies in albinism, depending on the amount of pigment that forms in the stroma and posterior epithelium, and this common feature of albinism may be difficult to demonstrate without a slit-lamp examination (Lee et al., 2001; Whang et al., 2002).

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION

Retinal Pigmentation and Foveal Hypoplasia The pigment epithelium of the retina (RPE) (see Chapter 4) acquires melanin pigment early in embryogenesis and is thought to provide a number of important functions in postnatal life for the proper processing of light into a visual image (Donatien and Jeffery, 2002; Esteve and Jeffery, 1998; Ilia and Jeffery, 1996, 1999, 2000; Jeffery, 1997, 1998; Raymond and Jackson, 1995; Schraermeyer and Heimann, 1999; Sheedlo et al., 1998; Tibber et al., 2004; Zinn and Benjamin-Henkind, 1986). Pigment is also present in the choroid below the retina (Zhao and Overbeek, 2001). The retinal pigment is greatly reduced or absent in albinism, making the retina transparent on ophthalmoscopic examination (Abadi and Cox, 1992; Abadi and Dickinson, 1983; Abadi and Pascal, 1989; Adler, 1910; Fulton et al., 1978; Kinnear et al., 1985; Summers et al., 1988; Usher, 1920). As a result, the choroidal blood vessels are seen below the retina on ophthalmoscopic examination. The electroretinogram is normal (Wack et al., 1989). Significant changes in retinal function develop in the fovea, and this is associated with a moderate to marked reduction in visual acuity (Charles et al., 1993; Edmunds, 1949; Fonda, 1962; Jacobson et al., 1984; Perez-Carpinell et al., 1992; Summers et al., 1988). The fovea in albinism is hypoplastic, and a foveal reflex is usually absent on ophthalmoscopic examination (Abadi and Cox, 1992; Jeffery and Williams, 1994; Pearson et al., 1911; Summers et al. 1988). The result of abnormal foveal development is reduced acuity that cannot be corrected to normal. Visual acuity in albinism ranges from 20/400 to 20/40+ and is usually 20/200–20/100. Many individuals with albinism have myopia, hyperopia, or a large amount of astigmatism, and correction of this can often improve acuity modestly. The presence of foveal hypoplasia does not interfere with color vision which is normal in albinism (Lourenco et al., 1983; Pickford and Taylor, 1968). In animal studies, the rod photoreceptor number in the central retina is reduced, with the amount of reduction correlating directly with the amount of ocular melanin present (Donatien and Jeffery, 2002; Esteve and Jeffery, 1998; Grant et al., 2001). Retinal melanin is located in the RPE, a single cell epithelial layer backing the retina (Jeffery, 1997; Schraermeyer and Heimann, 1999; Zhao and Overbeek, 2001). The RPE melanocytes have the highest concentration of melanin of all tissues and form their pigment before any light exposure, suggesting other roles for the melanin or products of the melanin pathway in eye development and function (Beermann et al., 1992; Drager, 1985). The RPE interacts with the choroid (Sakamoto et al., 1995; Zhao and Overbeek, 2001) as well as producing proteins or other compounds (e.g. l-dopa) that are likely to play an important role in retinal development (Blaszczyk et al., 2004; Fujisawa et al., 1976; Gimenez et al., 2004; Ilia and Jeffery, 1996, 1999, 2000; Petersen et al., 2003; Sheedlo et al., 1998, 2001).

Optic Nerve Development One of the most characteristic changes in the optic system in albinism is the abnormal decussation and misrouting of the

optic fibers at the chiasm. In a mature eye that was pigmented during development, the nerve fibers of the nasal retina cross at the chiasm and terminate in the contralateral lateral geniculate nucleus, whereas the fibers of the temporal retina do not cross and terminate in the ipsilateral lateral geniculate (Creel et al., 1978, 1990; Jeffery, 2001; Leventhal et al., 1985). The ratio of contralateral (crossed) to ipsilateral (uncrossed) fibers in humans is in the range of 55:45, and this correlates with overlapping visual fields, bilateral optic cortex input from each eye, and stereoscopic vision (Collewijn et al., 1985; Creel et al., 1990; Dorey et al., 2003; Lee et al., 2001; Leventhal et al., 1985; van Dorp, 1987). This ratio is greatly altered in albinism, with many ganglion cells in the temporal retina projecting to the contralateral side of the brain rather than remaining uncrossed and projecting to the ipsilateral side (Bouzas et al., 1994; Brecelj and Stirn-Kranjc, 2004; Creel, 1980; Creel et al., 1981; Fitzgerald and Cibis, 1994; Herrera et al, 2003; Jeffery, 2001; Leventhal et al., 1985; Rachel et al., 2002a; Rice et al., 1999; Soong et al., 2000). The effects of the changes on retinal fiber projections are a loss of stereoscopic depth perception and an intermittent and alternating suppression of the vision in one eye, producing an alternating strabismus (Lee et al., 2001; Summers et al., 1996). The misrouting of the optic fibers at the chiasm can be detected clinically by the demonstration of an asymmetrical visual evoked response with a visual evoked potential (VEP) study (Abadi and Pascal, 1989; Akeo et al., 1992; Apkarian et al., 1990; Bouzas et al., 1994; Collewijn et al., 1985; Creel et al., 1990; Dorey et al., 2003; Fitzgerald and Cibis, 1994; Neveu et al., 2003; Shallo-Hoffmann and Apkarian, 1993; Soong et al., 2000; Summers et al., 1991; van Dorp, 1987). The response to flash or pattern onset stimuli is recorded with right and left occipital electrodes, and the first 300 ms of the response indicates normal or abnormal decussation. The asymmetry between the ipsilateral and the contralateral responses is greater at younger ages and tends to be reduced with age (Neveu et al., 2003). In general, the diagnosis of OCA or OA cannot be made without clinical (reduced stereoacuity) or electrophysiological (asymmetric visual evoked potential) evidence of misrouting, but this can usually be obtained on clinical examination by obtaining a history of poor depth perception (often found when trying to obtain a driver’s license) and by observing alternating strabismus, a change that is related to the abnormal development of the optic nerves. Newer methods, including magnetic resonance imaging (MRI) (Hoffmann et al. 2003; Morland et al., 2002; Schmitz et al., 2004) and magnetoencephalography (Ohde et al., 2004), are providing a different and potentially more sensitive approach to demonstrating the optic tract changes in albinism and may become useful in the clinical evaluation of the patient in the future. As described below, the clinical pigmentation spectra for the different types of albinism are large, and the severity of the optic tract abnormalities varies clinically, depending on the amount of melanin present, and MRI may provide a better method for demonstrating gradations in optic tract changes associated with albinism (Schmitz et al., 2004). 601

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The pathophysiologic mechanism of the misrouting is unknown. Restoration of melanin in the RPE in a transgenic mouse model tends to normalize optic fiber projections, with some correlation with the amount of RPE pigmentation that is restored (Beermann et al., 1990, 1992; Cronin et al., 2003; Gimenez et al., 2004; Rachel et al., 2002b; Schiaffino et al., 2002). The timing of RPE tyrosinase expression and melanin formation is important, but the precise mechanism by which RPE melanin influences the axon development of the ganglion cells of the retina remains unknown (Cronin et al., 2003; Gimenez et al., 2004). Several proteins have been identified as playing a role in axon guidance in the developing chiasm, indicating that the process is complex and carefully regulated (Herrera et al., 2003; Marcus et al., 1999; Mason and Sretavan, 1998; Matsunaga et al., 2004; Rachel et al., 2002a; Rice et al., 1999; Shewan et al., 2002).

Nystagmus Nystagmus is found in most but not all individuals with albinism (Abadi and Bjerre, 2002; Castronuovo et al., 1991; Cheong et al., 1992; Dorey et al., 2003; Lee et al., 2001; Preising et al., 2001; Summers et al., 1991; Whang et al., 2002). The nystagmus is often absent at birth and first noticed by the parents in the first few months of life, can be minimal or marked in degree, is often more noticeable when the individual is tired or under stress, and can vary in degree among affected siblings in a family. Individuals with albinism often have a head posture or null point that is associated with its slowing, and this may improve vision. Individuals with albinism are usually not aware of their nystagmus and do not see constant motion in their vision. The mechanism responsible for the nystagmus is not fully understood. The presence of foveal hypoplasia and reduced acuity resulting in poor fixation may be responsible for some of the nystagmus, as is seen in many diseases of the eye that reduce or limit acuity. Abnormal development of the ocular motor system associated with the disorganization of the lateral geniculate nucleus and its projections to the optic cortex are thought to play a role in the development of nystagmus in albinism.

The Auditory System An association between abnormalities of pigmentation and hearing loss has long been observed, suggesting that the pigment cell or melanin may play a role in the development and function of the ear (Cable and Steel, 1991). In humans, pigmented melanocytes are normally present in the stria vascularis of the cochlea and in the vestibular region of the inner ear. No anatomic studies of the ear are present for human albinism, but comparative studies of pigmented and albino mice, guinea pigs, rabbits, and cats show that the number of melanocytes in the stria vascularis is normal in albinism, but the cells contain little or no melanin and have a reduced volume (Conlee et al., 1989). Humans with albinism usually have normal hearing. The only physiologic change that can be demonstrated in human 602

albinism is the prolongation of a temporary threshold shift (TTS) following exposure to noise, but the detection of this requires special testing procedures, and the regular audiogram is normal in an absence of other ear pathology (Creel et al., 1983b; Garber et al., 1982). It should be noted that temporary noise-induced hearing loss, as demonstrated by TTS testing, is a normal process that varies in humans by pigmentation status, with lightly pigmented individuals with sunsensitive skin having the most TTS and well-pigmented individuals with skin that tans easily having the least (Barrenas and Lindgren, 1990). Individuals with albinism and hearing loss have been reported, but the precise mechanism for the hearing loss is unknown (Bassi et al., 1999; Gross et al., 1995; Jacob et al., 1998; Spritz and Beighton, 1998; Tak et al., 2004). X-linked ocular albinism and late-onset hearing loss has been mapped to Xp22.3, which is the same location for X-linked ocular albinism without hearing loss (OA1) (Bassi et al. 1995, 1999; Schnur et al., 1991). The hearing loss associated with this type of albinism may arise from a mutation that involves a gene adjacent to the OA1 gene (Bassi et al., 1999). An additional locus for albinism and hearing loss has been mapped to the long arm of X at q26 (Jacob et al., 1998). Hearing loss is also associated with pigment syndromes that involve defects in melanoblast migration, such as piebaldism, Waardenburg syndrome, and similar syndromes (Amiel et al., 1998; Gross et al. 1995; Spritz and Beighton, 1998; Verheij et al. 2002). These syndromes differ from albinism because the melanocyte is absent in the migration defect syndrome, whereas it is present without normal amounts of melanin in albinism. The melanocyte in the ear may have a structural role that leads to the deafness associated with syndromes in which the melanocyte does not migrate into the ear.

Oculocutaneous Albinism Classification of Albinism The classical description of albinism as recorded in most historical writings through the nineteenth century included a phenotype of white hair, white skin, and blue translucent irides. Garrod in 1908 included this phenotype as one of the original inborn errors of metabolism and indicated that this most likely arose from an absence of melanin pigments in specialized cells in the body because of an enzyme deficiency (Garrod, 1908). It is interesting to note that Garrod was also aware of individuals with OCA who had some cutaneous pigment, but he had no explanation for this phenomenon. The next step was the development of the hair bulb incubation test that demonstrated at least two genetically distinct types of OCA, the tyrosinase-negative type in which a hair bulb incubated in tyrosine or dopa did not form pigment, and the tyrosinasepositive type in which the hair bulb did form pigment (Witkop, 1971). A mating between an individual with tyrosinase-negative and an individual with tyrosinase-positive OCA produced normally pigmented children, the classic genetic complementation test (Witkop, 1971). Clinical and biochemical changes were then used to define a number of

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION Table 31.2. Classification of oculocutaneous and ocular albinism. Type

Subtypes

Gene locus

Includes

Mechanism

OCA1

OCA1A OCA1B

Tyrosinase Tyrosinase

Inactive/missing enzyme Partially active enzyme

OCA2

P

OCA3 OCA4 HPS

TRP-1 MATP HPS

Tyrosinase-negative OCA Minimal pigment OCA Platinum OCA Yellow OCA Temperature-sensitive OCA Autosomal recessive OA (some) Tyrosinase-positive OCA Brown OCA Autosomal recessive OA (some) Rufous OCA Rufous OCA HPS

CHS

CHS

CHS

Unknown

Unknown Unknown Altered vesicular transport Altered vesicular transport

OCA, oculocutaneous albinism; OA, ocular albinism; HPS, Hermansky–Pudlak syndrome; CHS, Chediak–Higashi syndrome.

potentially separate phenotypes (e.g. tyrosinase-negative, minimal pigment, platinum, yellow, autosomal recessive ocular albinism, etc.), but this became confusing and not useful clinically. Molecular studies have now defined 12 or more loci that are associated with the development of albinism, each associated with a broad phenotypic range. Furthermore, the range of many types, as defined by the gene locus involved, overlaps with that of other types, and the precise diagnosis requires molecular studies. Fortunately, the clinical diagnosis of OCA, based on the pigmentation and ophthalmologic examinations, is usually sufficient to care for the affected individual. Precise genotypic identification is useful for family counseling and planning. Albinism classification based on gene locus is given in Table 31.2.

well-characterized allelic series of mutations at the c-locus (tyrosinase locus) in the mouse, each with a distinct phenotype (Silvers, 1979). In general, the different degrees of pigmentation in OCA1 are dependent on the residual enzyme activity associated with the mutant alleles. A useful characteristic of OCA1 is the presence of marked hypopigmentation at birth. Most individuals with OCA1 have white or nearly white scalp hair at birth. The interpretation of “white” varies with each family (this can also be seen in the range of white paint available from most manufacturers), however, and some families that have children with very light yellow hair but lighter than any other hair in family members

Table 31.3. Estimated prevalence of albinism.

Prevalence of OCA Oculocutaneous albinism is the most common inherited disorder of generalized hypopigmentation, with an estimated frequency of 1:20 000 in most populations (King et al., 2001). The estimated prevalence for different albinism types in various populations is given in Table 31.3. OCA has been described in all ethnic groups and in all animal species, making it one of the most widely distributed genetic abnormalities in the animal kingdom. All types of OCA are autosomal recessive in inheritance, except for the rare families that appear to have autosomal dominant OCA, as discussed below.

OCA1. Tyrosinase-related Oculocutaneous Albinism One of the two most common types of albinism is tyrosinaserelated OCA, produced by loss of function of the tyrosinase enzyme resulting from mutations in the tyrosinase gene (see Chapter 11). Classical OCA, with a total absence of melanin in the skin, hair, and eyes, is the most obvious type of OCA1, but there is a wide phenotypic variation associated with tyrosinase gene mutations. This is also seen with the

Population

Type

Prevalence

Reference

World survey United States Caucasian

All

1:10–20 000

Pearson et al. (1911)

All OCA1 OCA2 All OCA1 OCA2

1:18 000 1:39 000 1:36 000 1:10 000 1:28 000 1:10 000

Witkop Witkop Witkop Witkop Witkop Witkop

OCA2 OCA1

1:7900 Rare

OCA2

1:3900

OCA2 OCA2

1:1429 1:2833

Aquaron (1990) Kromberg and Jenkins (1982) Kromberg and Jenkins (1982) Spritz et al. (1995) Kagore and Lund (1995)

African-American

Africa Cameroon South Africa

Tanzania Zimbabwe

et et et et et et

al. al. al. al. al. al.

(1989) (1989) (1989) (1989) (1989) (1989)

OCA, oculocutaneous albinism.

603

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Fig. 31.1 Male with OCA1A. Total absence of melanin. This is the classic “tyrosinase-negative” OCA phenotype. See also Plate 31.1, pp. 494–495.

will call this “white” hair. Some individuals with OCA2 will also have white hair at birth (King et al., 2003a; Passmore et al., 1999), so the clinical sign is not universal but generally useful in the initial evaluation of the individual with albinism. OCA1A Individuals with OCA1A (the classic tyrosinase-negative OCA in older terminology) are unable to synthesize melanin in their skin, hair, or eyes, resulting in a characteristic phenotype, as shown in Fig. 31.1. They are born with white hair and skin and blue eyes, and there is no change as they mature (King et al., 2003a; Kinnear et al., 1985; Pearson et al., 1911). The phenotype is the same in all ethnic groups and at all ages. Scalp hair often has a translucent white appearance early in life that evolves to a denser white as more hair develops, and a slight yellow tint is often seen that appears to result from denaturing of the hair protein with environmental exposure and shampoo use. The irides are translucent on globe transillumination and early in life appear pink and usually turn gray–blue with time. No pigmented lesions develop in the skin, although amelanotic nevi can be present. 604

Fig. 31.2 Female with OCA1B. Note blonde hair and dark eyelashes. See also Plate 31.2, pp. 494–495.

OCA1B Individuals with OCA1B have very little or no hair pigment at birth and develop varying amounts of melanin in the hair and the skin in the first or second decade, as shown in Fig. 31.2. In some cases, the melanin develops within the first year. The hair color changes to light yellow, light blond, or golden blond first, and can eventually turn dark blond or brown in the adolescent and the adult. The original OCA1B phenotype was called yellow albinism because of the color of the pheomelanin that developed in the hair of affected individuals (Hu et al., 1980; Nance et al., 1970), and the amount of cutaneous pigment present in an individual of northern European background can be sufficient to suggest the diagnosis of ocular albinism (Fukai et al., 1995; King et al., 2003a; Morell et al., 1997). The irides can develop light tan or brown pigment, sometimes limited to the inner third of the iris, and iris pigment can be present on globe transillumination. Some degree of iris translucency, as demonstrated by slit-lamp examination, is usually present. Many individuals with OCA1B will tan with sun exposure although it is more common to burn without tanning after sun exposure. Pigmented nevi can develop with time, although most developing nevi are amelanotic. Very few freckles develop.

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION

Temperature-sensitive OCA1 One of the more interesting variations of OCA1B is the temperature-sensitive phenotype (Giebel et al., 1991a; King et al., 1991a). The proband of the originally identified family appeared to have OCA1A through the first years of her life, with white hair and skin, and blue eyes; however, her body hair color changed with puberty. The axillary hair remained white, the scalp hair remained white but developed a slight yellow tint, the arm hair turned light reddish-brown, and the leg hair turned dark brown. The eyes remained blue and the skin remained white without tan. There were three similarly affected sibs in the family, and no other family of this type has been published to date. This type of OCA1B is analogous to the Siamese cat and the Himalayan mouse. Analysis of tyrosinase from scalp and leg hair bulbs of the proband showed that the enzyme was temperature sensitive, losing activity above 35°C. As a result, melanin synthesis would occur in the cooler but not the warmer areas of the body such as the arms and legs, in a pattern similar to the Siamese cat. Autosomal Recessive Ocular Albinism Some years ago, a series of families was described in which children of normally pigmented parents had the ocular features of albinism but did not appear to have significant cutaneous hypopigmentation. This was called autosomal recessive ocular albinism (AROA) because males and females were affected in the sibships in these families (O’Donnell et al., 1978b). It now appears that this designation of AROA may be a misnomer as many of the families with this presentation actually represent individuals with OCA1B or OCA2 who have developed nearly normal cutaneous pigmentation. One family has been described in which the proband with OCA1B was not diagnosed with albinism until mid-life, although she had always been aware of her reduced visual acuity (Summers et al., 1996). Two additional individuals who were initially diagnosed with AROA were found to be compound heterozygotes for mutant tyrosinase alleles (Fukai et al., 1995). Most interestingly, both individuals had one null tyrosinase allele and one allele containing a tyrosinase gene polymorphism that encoded a temperature-sensitive enzyme. The authors suggested that the temperature-sensitive enzyme from the polymorphic mutant allele was insufficient for normal ocular melanin synthesis during embryogenesis when the temperature of the ocular tissues was high, but was associated with normal skin pigment formation after birth because of the cooler skin temperature. Molecular Pathogenesis of OCA1 Analyses of DNA from individuals with OCA1 have shown more than 150 different tyrosinase gene mutations (Breimer et al., 1994; Camand et al., 2001; Fukai et al., 1995; Giebel et al., 1990, 1991a, b, c; King and Oetting, 1992; King et al., 1991b, 2003a; Matsunaga et al., 1996, 1998, 1999; Oetting and King, 1992, 1993; Oetting et al., 1991a, b, 1993, 1994, 1995; Opitz et al., 2004; Park et al., 1993, 1997; Passmore et al., 1999; Schnur et al., 1996; Spritz et al., 1990, 1991,

1997a; Summers et al., 1996; Takeda et al., 1990; Tomita, 1993; Tomita et al., 1989; Tripathi et al., 1992a). A complete list of the mutations is available on the Albinism Database (http://albinismdb.med.umn.edu). Most individuals with OCA1 are compound heterozygotes with different mutant maternal and paternal alleles, and individuals have been identified with all combinations of missense, nonsense, and frameshift mutations. Mutations in both alleles of the tyrosinase gene can be identified in approximately 83% of individuals who meet the clinical criteria of OCA1A and in 37% of those who meet the criteria of OCA1B, whereas 63% of those with OCA1B have an identifiable mutation in only one allele (King et al., 2003a; Matsunaga et al., 1996; Passmore et al., 1999; Spritz et al., 1997a; Tripathi et al., 1993). Mutations of the tyrosinase gene can lead to loss of function by several mechanisms. First, an entire allele can be deleted, but this appears to be an infrequent mutation with this gene (Schnur et al., 1996). Second, it is known that tyrosinase gene missense mutations cluster in and around the copper binding regions of the protein (King et al., 1991b; Oetting and King, 1993; Tripathi et al., 1992a), and this can be associated with a loss of copper binding and enzyme function (Spritz et al., 1997b; Tripathi et al., 1992b). Third, a mutation in a splice domain can produce a partially inactivated splice site that functions intermittently (Matsunaga et al., 1999). Finally, recent studies suggest that the primary effect of a missense mutation is to alter the folding of the nascent protein in the endoplasmic reticulum of the cell, resulting in a protein that is retained in the endoplasmic reticulum and not transported to the melanosome (Berson et al., 2000; Halaban et al., 1997, 2000, 2001, 2002; Toyofuku et al., 2001a, b). It is likely that additional mechanisms will be described and the details of those listed above will be refined in the future.

OCA2. P-related OCA The usual clinical features of OCA2 are the presence of scalp hair pigmentation at birth and iris pigment at birth or shortly thereafter in an individual with albinism. OCA2 can overlap with OCA1B, however, making an accurate clinical diagnosis difficult. There may be some accumulation of pigment in the hair with age, but this is much less pronounced than that found in OCA1B, and many individuals with OCA2 have the same hair color throughout life. OCA2 is the most common type of OCA in the world, primarily because of the high frequency in equatorial Africa (Aquaron, 1990; Durham-Pierre et al., 1994; Jenkins et al., 1990; King et al., 1980; Kromberg and Jenkins, 1982; Ramsay et al., 1992; Spritz et al., 1995). Brown OCA is part of the spectrum of OCA2 (Manga et al., 1995, 2001). In Caucasian individuals, the amount of pigment present in an individual with OCA2 varies from minimal in those of northern European (particularly Scandinavian) ancestry to moderate in those of southern European or Mediterranean ancestry. The hair can be very lightly pigmented at birth, appearing white or having a light yellow or blond color, or more pigmented with a definite blond, golden blond, or even 605

CHAPTER 31

rate genetic susceptibility as these lesions only appear to develop in some OCA2 families and not in others (Bothwell, 1997; Kedda et al., 1994; Kromberg et al., 1989; Ramsay et al., 1992). The presence of ephelides is associated with a lower risk of skin cancer in South African individuals, and may reflect the presence of photoprotective melanin in the skin (Kromberg et al., 1989).

Fig. 31.3 Male with OCA2. This is the classic “tyrosinase-positive” OCA phenotype in a Nigerian individual. See also Plate 31.3, pp. 494–495.

red color (King et al., 1980, 1985, 2003b; Passmore et al., 1999). The skin is creamy white and usually does not tan. The iris color is blue–gray or lighted pigmented, and the degree of translucency correlates with the amount of iris pigment present. With time, pigmented nevi and lentigines may develop, and pigmented freckles are seen in exposed areas with repeated sun exposure. The hair in Caucasian individuals may slowly turn darker through the first two or more decades of life. African-American and African individuals have a distinct phenotype, which may, in part, result from the existence of a single common deletion mutation throughout many parts of subSaharan Africa (Aquaron, 1990; Barnicot, 1952; Garrod, 1908; King et al., 1980; Kromberg and Jenkins, 1982; McCrackin, 1937; Okoro, 1975; Pearson et al., 1911; Stannus, 1913) (Fig. 31.3). The hair is yellow at birth and remains yellow throughout life, although the color may turn darker. The skin is creamy white at birth and changes little with time. Pigmented nevi, lentigines, and freckles develop in some individuals, but there is no tan. The development of lentigines or ephelides, well-demarcated pigmented patches usually on sun-exposed areas of the skin, may reflect a sepa606

Brown OCA The function of the product of the P gene is currently unknown, but studies of human albinism and coat color changes in the mouse suggest that the P gene product has its primary function in the formation of eumelanin (Barsh, 1996). Mutations of the P gene, as seen in the usual OCA2 phenotype, are associated with the development of pheomelanin and a deficiency in eumelanin. Another type of OCA called “brown OCA” has been described in the African and AfricanAmerican populations in which the amount of eumelanin in the skin and hair is reduced but not absent (King and Rich, 1986; King et al., 1980, 1985; Pearson et al., 1911). It is now apparent that this type of albinism is part of the OCA2 spectrum (Manga et al., 1995, 2001). In African and African-American individuals with the “brown OCA” phenotype, the hair and skin color are light brown, and the irides are gray to tan at birth, as shown in Figure 31.4 (King et al., 1980, 1985). With time, there is little change in skin color, but the hair may turn darker and the irides may accumulate more tan pigment. Affected individuals are recognized as having albinism because they have all the ocular features of albinism. The iris has punctate and radial translucency, and moderate retinal pigment is present. The skin may darken with sun exposure. Molecular Pathogenesis of OCA2 Mutations in the human homolog to the mouse pinkeyed dilution (p) gene (see Chapter 7) are associated with OCA2 (Fridman et al., 2003; Gardner et al., 1992; Jenkins et al., 1990; Kedda et al., 1994; Kerr et al., 2000; Lee et al., 1994; Nakatsu et al., 1992; Oetting and King, 1999; Passmore et al., 1999; Ramsay et al., 1992; Saitoh et al., 2000; Schmidt et al., 2001; Spritz et al., 1997c; Suzuki et al., 2003). Mutations and polymorphisms of the P gene identified to date are available in the Albinism Database (http://albinismdb.med. umn.edu). The precise function of the P gene product is unknown, and functional studies of individual mutations are not routinely available (Brilliant, 2001; Manga and Orlow, 1999; Orlow and Brilliant, 1999; Puri et al., 2000; Sviderskaya et al., 1997).

OCA3. Red OCA (ROCA) The human phenotypic variations associated with genetic alterations of the gene that encodes tyrosinase-related protein 1 (TYRP1) have only been explored recently. The first evidence that variations in human pigmentation could be related to mutations in the TYRP1 gene (see Chapter 11) came from the description of an African-American newborn twin boy

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION

1997). The ocular features were not as obvious as with other types of OCA, and many do not have iris translucency, nystagmus, strabismus, or foveal hypoplasia. Furthermore, no misrouting of the optic nerves has been demonstrated by a visual evoked potential, suggesting either that this is not a true type of albinism, as defined in this chapter, or that the hypopigmentation is not sufficient consistently to alter optic nerve development (Kromberg et al., 1990). Molecular Pathogenesis of OCA3 Molecular analysis of the TYRP1 gene in DNA from 19 unrelated South African individuals showed that 19/38 ROCA chromosomes (50%) carried the 368delA mutation and 17/38 ROCA chromosomes carried a separate mutation (S166X) (Manga et al., 1997). Affected individuals from other locations have not been identified.

OCA4. MATP-related OCA

Fig. 31.4 Brown OCA phenotype of OCA2 (see also Plate 31.4, pp. 494–495).

who had light brown skin, light brown hair, and blue/gray irides while his fraternal twin brother had normal pigmentation (Boissy et al., 1996) (see Chapter 27 and Plate 27.6, pp. 494–495). The affected twin had nystagmus, and the clinical presentation was initially felt to be consistent with brown OCA. However, biochemical and molecular studies showed that melanocytes from the affected twin contained no TYRP1 mRNA, produced reduced amounts of insoluble melanin, and appeared brown rather than the black color found in control melanocytes in culture (Boissy et al., 1996). This individual was found to be homozygous for a single base deletion in codon 368 of the TYRP1 gene (368delA) on chromosome 9p, and the authors concluded that mutations of this gene produced a third type of OCA. Subsequent to this report, another type of OCA known as rufous or red OCA (ROCA) was mapped to the TYRP1 locus on chromosome 9p in the South African population, and the same deletion mutation (368delA) of the TYRP1 gene has been found in a substantial number of the study population of individuals with ROCA (Manga et al., 1997, 2001). The phenotype in South African individuals with ROCA includes red or reddish-brown skin, ginger or reddish hair, and hazel or brown irides (Kromberg et al., 1990; Manga et al.,

The human ortholog of the mouse underwhite gene (Lehman et al., 2000; Sweet et al., 1998) encodes a membraneassociated transporter protein (MATP) that localizes to the melanosomal membrane (Costin et al., 2003; Newton et al., 2001). The initial report of the human gene included the description of a single Turkish individual with OCA who was homozygous for a MATP mutation, and this was called OCA4. The clinical features were very similar to those of the more common OCA2 in a Caucasian population (Newton et al., 2001). Subsequent studies have identified additional individuals with OCA4 in Germany (Rundshagen et al., 2004) and Japan (Inagaki et al., 2004). Eighteen Japanese OCA4 individuals were identified, making this the second most common form of OCA in Japan (Inagaki et al., 2004). The pigmentation varied from white to blond to brown scalp hair and blue to brown irides, similar to the range found with OCA1B and OCA2. Seven MATP mutations were identified, including D157N in 14 of 36 OCA4 chromosomes and G188V in 7 of 36 OCA4 chromosomes (Inagaki et al., 2004). It is expected that additional individuals with OCA4 will be identified. The precise prevalence in different populations has not been determined.

Autosomal Dominant OCA Several families have been described with autosomal dominant expression of OCA or cutaneous hypopigmentation, but characterization is incomplete and most do not fit the criteria for albinism (Bergsma and Kaiser-Kupfer, 1974; Fitzpatrick et al., 1974; Frenk and Calame, 1977). In general, affected individuals have light skin that tans, light hair, and reduced iris pigment. One French family had hypopigmentation associated with skin melanocytes containing small melanosomes in three generations, suggesting a primary abnormality of melanosome structure (Frenk and Calame, 1977). Interestingly, one of the individuals with hypopigmentation also had Prader–Willi syndrome (PWS). The abnormal melanosomal size suggests that the hypopigmentation may not be related to the P gene on chromosome 15q, and further information on this family is 607

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not available. A second family included affected parents and children, but the grandparents were normal (Bergsma and Kaiser-Kupfer, 1974). All the features of albinism were present in those affected, and this family is probably an example of pseudodominance. Molecular analysis of OCA1 now suggests that some families that are thought to have a dominant expression of OCA actually have mutations of the tyrosinase gene with affected individuals in several sequential generations. This is classic pseudodominance, and it is expected that many of the families with a “dominant” pattern of albinism will, in fact, have pseudodominance of a more typical recessive OCA. The third family reported does not meet the definition of albinism (i.e. no nystagmus or loss of visual acuity) and does not have OCA (Fitzpatrick et al., 1974). The final characterization of dominant OCA will await the careful evaluation of families with a clear autosomal dominant expression of OCA.

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Albinoid Disorders Philippe Bahadoran and Jean-Paul Ortonne

Genetic Hypomelanoses Related to Defective Melanosome Biogenesis/Transport Hermansky–Pudlak syndrome (HPS), Chediak–Higashi syndrome (CHS), and Griscelli syndrome (GS) are now considered as disorders of the biogenesis (HPS, CHS) or transport (GS) of lysosome-related organelles (LRO). LRO are a newly recognized family of intracellular vesicles, including melanosomes, platelet dense granules, and cytotoxic granules (Marks and Seabra, 2001). This new concept explains why in HPS, CHS, and GS, pigmentary dilution is associated with symptoms reflecting the involvement of other cell types.

Hermansky–Pudlak Syndrome (Table 31.4) Hermansky–Pudlak syndrome (HPS; MIM 203300) is a complex disorder that presents with oculocutaneous albinism (OCA), a bleeding diathesis, and a ceroid–lipofuscin-like lysosomal storage in a variety of tissues. HPS is autosomal recessive in inheritance. Although HPS reflects a process that affects not only melanocytes, but also platelets and other tissues, the OCA is similar to the primary OCA types, and ocular features of HPS are the same as those found with other types of OCA. HPS is rare in most populations, except in the Caribbean island of Puerto Rico, particularly in the northwestern region, where it occurs with an estimated incidence of 1:1800. Consequently, the majority of individuals with HPS are found in Puerto Rico. HPS is not found on other Caribbean islands. HPS is also frequent in one long-isolated mountain village in the Swiss Valais (Huizing et al., 2000; Spritz, 2000). HPS Phenotypes The clinical manifestations of HPS are colored by the ethnic background of affected patients, as well as the causative locus. HPS is a pigmenting type of OCA, in that cutaneous and ocular pigments develop in many affected individuals, and the 613

CHAPTER 31 Table 31.4. Genetic and molecular characteristics of genetic hypomelanoses related to defective melanosome biogenesis/transport. Disease

Gene

Chromosome

Protein characteristics

Mouse homolog

MIM#

10q23 15q15 3q24 22q11.2–q12.2 11p15–p13 10q24.31 6p22.3

Component of BLOC-3 complex Beta3A subunit of AP-3 complex Component of BLOC-2 complex Component of BLOC-3 complex Component of BLOC-2 complex Component of BLOC-2 complex Dysbindin, component of BLOC-1 complex

Pale ear (ep) Pearl (pe) Cocoa (co) Light ear (le) Ruby eye 2 (ru2) Ruby eye (ru) Sandy (sdy)

203300 604982 603401 606118 606682 607521 607522 607145

LYST

1q42.1–42.2

LRO fusion events and/or protein targeting

Beige (be)

214500

MYOVA RAB27A MLPH

15q21 15q21 2q37

Myosin Va, actin-based melanosome transport Rab GTPase, actin-based melanosome transport Melanophilin, actin-based melanosome transport

Dilute (dil) Ashen (ash) Leaden (ln)

160777 607624 606526

Hermansky–Pudlak syndrome Type 1 HPS1 Type 2 AP3B1 Type 3 HPS3 Type 4 HPS4 Type 5 HPS5 Type 6 HPS5 Type 7 DTNBP1 Chediak–Higashi syndrome Griscelli syndrome Type 1 Type 2 Type 3

amount of pigment that forms is quite variable (Fig. 31.5). In Puerto Rico, affected individuals have hair color that varies from white to yellow to brown. Skin color is creamy white and definitely lighter than in normally pigmented individuals in this population. Albinism in HPS is associated with quantitatively reduced and qualitatively abnormal melanosomes in skin and hair melanocytes. Freckles can be present in the sunexposed regions, often coalescing into large areas that look like normal dark skin pigment, but tanning does not occur. Pigmented nevi are common. Iris color varies from blue to brown, and all the ocular features of albinism are present. Typically, HPS patients are legally blind, with a visual acuity of 20/200 or worse, but occasionally a patient has an acuity as high as 20/50. The presence of OCA may not be obvious in a Puerto Rican individual with brown hair, skin pigment in exposed areas, and brown eyes, unless the cutaneous pigmentation is compared with unaffected family members, who are generally darker in pigment, and unless the ocular features of albinism are recognized. Affected individuals have been identified in other populations, and the phenotype shows the same degree of variation in pigmentation as is found in Puerto Ricans. Hair color varies from white to brown, and this correlates somewhat with ethnic group. The skin is white and does not tan. Eye color varies from blue to pigmented (Gahl et al., 1998; Huizing et al., 2000; Spritz, 2000). The bleeding diathesis in HPS usually produces mild hemorrhagic episodes. The first sign in HPS patients usually consists of excess bruising beginning at the time of ambulation. Episodes of epistaxis are frequent but often remit during adolescence. Other events that result in excess bleeding include dental extractions, surgeries, acute colitis, menstrual periods, and childbirth. Fatalities due to bleeding are rare. The cause of bleeding in HPS is absence of platelet dense granules,

614

Fig. 31.5. Puerto Rican male with Hermansky–Pudlak syndrome.

making the disease a platelet storage pool deficiency. Dense granules are intracellular lysosome-related organelles, which contain adenosine diphosphate (ADP), adenosine triphosphate (ATP), serotonin, calcium, and polyphosphates, and disgorge their contents upon stimulation. Released components, ADP in particular, cause aggregation of surrounding platelets, contributing to clot formation. HPS patients have a normal or increased platelet count but an attenuated secondary aggregation response. The bleeding time is often, but not always, prolonged. Absence of dense granules, which confirms the diagnosis of HPS, is demonstrable by wet-mount electron microscopy (Gahl et al., 1998; Huizing et al., 2000; Spritz, 2000). The most severe clinical manifestations of HPS are related to the pulmonary and intestinal changes. Interstitial pul-

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION

monary fibrosis has been described in many individuals with HPS, although the actual prevalence is unknown. The pulmonary fibrosis of HPS begins as restrictive lung disease, manifest by abnormal pulmonary function tests in the late twenties or early thirties. Pulmonary function can plateau for years, and then fall dramatically and result in death in 2 or 3 years. Affected patients typically succumb to extensive fibrotic deterioration of the lungs by the end of the fourth or fifth decade of life. Approximately 15% of HPS patients, whether Puerto Rican or nonPuerto Rican, suffer from a granulomatous colitis. The distal colon is most often involved in this complication, and the colitis of HPS resembles Crohn’s disease. The accumulation of a lipid protein complex called ceroid– lipofuscin in the lysosomes of many cell types is thought to cause the pulmonary fibrosis and granulomatous colitis, but this has not been confirmed. Average survival of HPS patients is 30–50 years, with death usually resulting from restrictive lung disease or, less frequently, hemorrhage or colitis (Gahl et al., 1998; Huizing et al., 2000; Spritz, 2000). Up to now, our understanding of the clinical manifestations of HPS has been strongly influenced by the patients from northwestern Puerto Rico who are homozygous for exactly the same mutation in the HPS1 gene. After the recent identification of six additional HPS genes, some locus-related clinical differences have been reported (see below). Molecular Pathogenesis of HPS HPS can be caused by mutations in any of the seven following genes identified to date: HPS1, HPS2, AP3B1, HPS4, HPS5, HPS6, and DTNBP1. This reflects, in part, the situation in mice where 16 different genes cause hypopigmentation and platelet storage pool deficiency. Consequently, the identification of additional genes in HPS patients is possible. Hermansky–Pudlak Syndrome Type 1 (HPS1; MIM 604982) HPS1 (chromosome 10q23) is the first identified HPS gene. HPS1 mutations are a major cause of HPS in Puerto Rican patients. More precisely, all the northwest Puerto Rico patients, i.e. ~400 people, are homozygous for the same mutation in HPS1. Other HPS1 mutations are the cause of HPS in less than half of nonPuerto-Rican individuals. All patients with HPS1 disease, regardless of the type of mutation involved, are at increased risk of developing pulmonary fibrosis (Gardner et al., 1997). HPS1 codes for a 79.3-kDa protein, HPS1, with no significant homology to any other known proteins. In melanotic cells, HPS1 is contained in two distinct high-molecular-weight complexes distributed between uncoated vesicles, early-stage melanosomes, and the cytosol. Recently, it was shown that HPS1 and HPS4 proteins are associated in a complex called BLOC-3 (Chiang et al., 2003; Martina et al., 2003; Nazarian et al., 2003). It was proposed that HPS1 is involved in the biogenesis of early melanosomes, but its specific role remains to be determined. The mouse pale ear (ep) locus is the homolog to the human HPS1 locus.

Hermansky–Pudlak Syndrome Type 2 (HPS2; MIM 603401) Mutations in AP3B1 (chromosome 15q15), causing HPS2, have been found in four individuals, two of whom are brothers with identical mutations. Patients with HPS2 disease display significant distinguishing features. Besides OCA and bleeding diathesis, they have increased susceptibility to infections and persistent neutropenia (Dell’Angelica et al., 1999; Huizing et al., 2002; Shotelersuk et al., 2000). AP3B1 codes for the beta3A subunit of adaptor complex-3 (AP-3). AP-3 interacts with tyrosinase, and tyrosinase is not targeted properly to melanosomes in AP3B1-deficient melanocytes. These data suggest that, in melanocytes, AP-3 mediates the trafficking of tyrosinase, and presumably other melanosomal proteins, from an intracellular site to melanosomes. In cytotoxic lymphocytes (CTL), AP3B1 deficiency results in loss of microtubule-mediated movement of lytic granules toward the immunological synapse and a profound loss of CTL-mediated killing (Clark et al., 2003). The mouse pearl (pe) locus is the homolog to the human HPS2 locus. Hermansky–Pudlak Syndrome Type 3 (HPS3; MIM 606118) A third HPS-causing gene, HPS3 (chromosome 3q24), was isolated in an enclave of patients with HPS from central Puerto Rico, and thus appears as an important HPS locus in Puerto Rico, although less frequently involved than HPS1. Central Puerto Rico patients had less severe OCA than typical patients with HPS1 and mild bleeding diathesis. They were all homozygous for the same mutation in HPS3 (Anikster et al., 2001). Other HPS3 mutations have also been described in eight nonPuerto Rican patients with HPS. These patients had mild OCA, limited bleeding diathesis, roughly normal pulmonary function, and no colitis. The finding of a common mutation in HPS3 among three Ashkenazi Jews suggests a founder effect and implies that a heightened awareness of the possibility of HPS among this population may be needed. As HPS3 disease associated with severe HPS3 mutations is relatively mild, it cannot be excluded that the diagnosis of HPS3 is overlooked in some patients, and additional studies are needed to assess the exact importance of the HPS3 locus in nonPuerto Rican patients (Huizing et al., 2001). The HPS3 sequence predicts a 113.7-kDa protein without homology to other known proteins. Very recently, it was shown that the HPS3, HPS5, and HPS6 proteins associate in a complex called BLOC-2 (Gautam et al., 2004). It was suggested that the HPS3 protein is required at a late stage of melanosome biogenesis, but its precise function remains to be determined. The mouse pearl (pe) locus is the homolog to the human HPS3 locus. Hermansky–Pudlak Syndrome Type 4 (HPS4; MIM 606682) A fourth HPS-causing gene, HPS4 (chromosome 22q11.2– q12.2), was isolated in a number of nonPuerto Rican individuals, and thus appears potentially to be an important HPS locus in this subset of patients. Based on the small number of

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CHAPTER 31

patients with HPS4 defects, the HPS4 subtype appears to be relatively severe and resembles HPS1 disease in its proclivity for pulmonary involvement (Anderson et al., 2003; Bachli et al., 2004; Suzuki et al., 2002). The HPS4 protein is a 72.7-kDa polypeptide unrelated to any known proteins. HPS4 protein functions in the same pathway of organelle biogenesis as HPS1, but its specific function is currently unknown. The mouse light ear (le) locus is the homolog to the human HPS4 locus. Hermansky–Pudlak Syndrome Type 5 (HPS5; MIM 607521) Mutations in HPS5 (chromosome 11p15.2–p13) were found in a 3-year-old Turkish boy with HPS. The boy had clinically mild OCA and easy bruising. The mouse ruby eye 2 (ru2) locus is the homolog to the human HPS5 locus. The ru2 gene encodes for a protein of 126 kDa, without known homology (Zhang et al., 2003). Hermansky–Pudlak Syndrome Type 6 (HPS6; MIM 607522) Mutations in HPS6 (chromosome 10q24.31) were found in a 39-year-old Belgian woman with HPS. The patient had OCA and easy bruising. She had no pulmonary or gastrointestinal symptoms. The mouse ruby eye (ru) locus is the homolog to the human HPS5 locus. The ru gene encodes for a protein of 88 kDa, without known homology. The ru and ru2 proteins interact in a complex called BLOC-2 (Zhang et al., 2003), which also contains HPS3 (Gautam et al., 2004). Hermansky–Pudlak Syndrome Type 7 (HPS7; MIM 602415) Mutations in DTNBP1 (chromosome 6p22.3) were described in a 48-year-old female patient with OCA, bleeding diathesis, and pulmonary symptoms. DTNBP1 encodes for dysbindin, a 51-kDa protein with no known homology. Dysbindin associates with BLOC-1, a complex involved in melanosome biogenesis, and also with DPC, a complex associated with dystrophin. The role of dysbindin in melanosome biogenesis is still unknown. The mouse sandy (sdy) locus is the homolog for human HPS7 (Li et al., 2003).

Chediak–Higashi Syndrome (Table 31.4) CHS Phenotype Chediak–Higashi syndrome (CHS; MIM 214500) is a rare autosomal recessive disorder characterized by hypopigmentation, severe immunologic defects, bleeding tendency, progressive neurological dysfunction, and the presence of giant peroxidase-positive lysosomal granules in peripheral blood granulocytes. As with HPS, the hypopigmentation is the result of a primary defect that affects many cell types other than the melanocyte. The skin, hair, and eye pigment are reduced or diluted in CHS (Fig. 31.6). The affected individuals often do not have obvious albinism, and the hypopigmentation may only be noted when compared with other family members. Hair color is light brown to blond, and the hair has a metallic silver–gray sheen. The skin is creamy white to slate gray. Iris pigment is present, and nystagmus and photophobia may be present or absent. Histologic studies of the eye in CHS have 616

Fig. 31.6. The skin and hair of this patient with Chediak Higashi syndrome exhibit pigmentary dilution. See also Plate 31.5, pp. 494–495.

shown reduced iris pigment, a marked reduction in retinal pigment granules, and infiltration of the choroid with reticuloendothelial cells. Visual evoked potential studies show misrouting of the optic fibers in a pattern that is similar to individuals with OCA1 and OCA. Children with CHS have many serious medical problems, and the hypopigmentation is usually not a medical concern. Most patients with CHS die young because of the so-called accelerated phase, consisting of a lymphoproliferative syndrome with hemophagocytosis and infiltration of most tissues by activated T lymphocytes, unless they undergo allogenic bone marrow transplantation. About 10–15% of patients exhibit a much milder clinical phenotype and survive to adulthood, but develop progressive and often fatal neurologic dysfunction. Very rare patients exhibit an intermediate adolescent CHS phenotype, presenting with severe infections in early childhood, but a milder course by adolescence, with no accelerated phase (Spritz, 1998). Cells from patients with CHS and from its murine homolog, the beige (be) mouse, contain giant intracellular vesicles that cluster around the nucleus. Affected vesicles are lysosomes and lysosome-related organelles, such as melanosomes, platelet

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION

dense granules, and cytolytic granules. The susceptibility to bacterial infections appears to be the result of the abnormal granules in the neutrophils and other cells. The hypopigmentation also arises from the formation of abnormal granules. Giant melanosomes form in the melanocyte. As a result, the melanosomes (and their contained melanin) stay centralized in the cell and do not transfer to surrounding keratinocytes, with the result being hypopigmentation of the skin and hair. The pigment granules in the hair shaft are large and have an irregular shape when compared with those in normally pigmented hair from an unaffected individual, and this observation has been used to make a prenatal diagnosis of CHS (Spritz, 1998). Molecular Pathogenesis of CHS The mouse beige gene and its human homolog LYST (lysosomal regulator trafficking) gene (chromosome 1q42.1–42.2) have been identified and cloned. Most LYST gene alterations result in a truncated protein because of frameshift or nonsense mutations, suggesting that missense substitutions may result in a milder phenotype. Mutation analysis of 21 unrelated patients with different forms of CHS showed that patients with severe childhood CHS had only functionally null mutant CHS1 alleles, whereas patients with the adolescent and adult forms of CHS also had missense mutant alleles that likely encode CHS1 polypeptides with partial function. These results suggest an allelic genotype–phenotype relationship among the various clinical forms of CHS (Barbosa et al., 1996, 1997; Certain et al., 2000; Karim et al., 2002; Nagle et al., 1996). LYST encodes a 430-kDa acytosolic protein. The function of the LYST protein is still debated. Culture mouse fibroblasts in which the beige protein is overexpressed have smaller than normal lysosomes. In cytotoxic T-lymphocyte (CTL) clones derived from CHS patients, the initial steps of secretory lysosome formation are normal, but these organelles subsequently fuse together during cell maturation to form the giant secretory lysosomes. These data suggest that LYST regulates membrane fusion events (Perou et al., 1997; Stinchcombe et al., 2000). CHS T cells have defective peptide loading and major histocompatibility complex (MHC) class II molecule endosomal sorting, and altered surface expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). These data suggest, on the other hand, that LYST controls membrane targeting of lysosome-related organelle proteins (Barrat et al., 1999; Faigle et al., 1998).

Griscelli Syndrome (Table 31.4) GS Phenotypes Griscelli syndrome (GS) is a rare autosomal recessive disorder characterized by hypopigmentation, severe immune deficiency, and neurologic impairment. Unlike CHS, there are no giant granules in peripheral blood granulocytes (as well as in other cell types), a feature that has been used for differential diagnosis. Pigmentary dilution is found in all GS patients and involves the skin and hair, but not the eye. Cutaneous hypopigmenta-

Fig. 31.7. An Algerian girl with Griscelli syndrome showing light skin and silvery hair. See also Plate 31.6, pp. 494–495.

tion is mild and may only be noted when compared with other family members. The most evident manifestation of pigmentary dilution in GS is a metallic silver–gray sheen of the hair (Fig. 31.7). Iris pigment is present, there are no ocular symptoms such as those found in OCA, and histologic eye studies in murine homologs of GS showed normal retinal pigmentation. So, unlike HPS and CHS, GS does not meet the definition of albinism. The pigmentary phenotype in GS is caused by defective transport of melanin-containing melanosomes to melanocyte dendrites. In the skin, this results in the absence of transfer of melanosomes to keratinocytes. In the hair shaft, the pigment granules have an irregular size and an uneven distribution compared with those in normal hair, and this feature has been used to establish the diagnosis of GS (Griscelli et al., 1978; Klein et al., 1994). The presence of extrapigmentary symptoms in GS is variable and depends on which of the three loci described in the recent years, MYOVA, RAB27A, or MLPH, is involved (see below). The immune deficiency is characterized by early and recurrent infections, and overall by the occurrence during childhood of a so-called accelerated phase, consisting of uncontrolled lymphocyte and macrophage activation, resulting in infiltration and hemophagocytosis in multiple organs, 617

CHAPTER 31

and leading to death in the absence of bone marrow transplantation. The neurologic impairment involves the central nervous system (CNS) and consists of hypotonia, marked motor development delay, and mental retardation (Griscelli et al., 1978; Klein et al., 1994). Molecular Pathogenesis of GS Griscelli Syndrome Type 1 (GS1; MIM 160777) The first gene involved in GS is MYOVA (chromosome 15q21), which encodes for myosin Va. Myosin Va is an actin-based molecular motor that enables the transport and accumulation of melanosomes in melanocyte dendrites. Myosin Va is also required for the transport of smooth endoplasmic reticulum in the dendritic spines of neurons. MYOVA gene mutations cause the GS1 phenotype, e.g. pigmentary dilution with CNS dysfunction. MYOVA mutations have only been reported in a minority of patients with GS, and it has been proposed that the MYOVA locus could also be involved in Elejalde syndrome, but this needs to be confirmed (Bahadoran et al., 2003; Menasche et al., 2003a; Pastural et al., 1997). Griscelli Syndrome Type 2 (GS2; MIM 607624) The second GS gene is RAB27A (chromosome 15q21), which encodes for Rab27a. Rab27a is a small GTPase of the Ras family that serves as a receptor for myosin Va at the surface of melanosomes. Rab27a is also essential in moving the lytic granules from the MTOC to the plasma membrane. RAB27A mutations cause the GS2 phenotype, e.g. pigmentary dilution with immune deficiency (Bahadoran et al., 2003; Haddad et al., 2001; Menasche et al., 2000; Stinchcombe et al., 2001; Wu et al., 2002). Griscelli Syndrome Type 3 (GS3; MIM 606526) Mutation in MLPH (chromosome 2q37), encoding for melanophilin, was described in one patient. Melanophilin is a Rab effector protein that enables the binding of Rab27a to myosin Va. MLPH mutation causes the GS3 phenotype, e.g. pigmentary dilution exclusively. Of note, this phenotype can also be caused by one mutation in the MYOVA gene that specifically abolishes the binding of myosin Va to melanophilin (Menasche et al., 2003b). Other Genetic Hypomelanoses with Immune Deficiency The Chediak–Higashi syndrome (CHS), Griscelli syndrome type 2 (GS2), and Hermansky–Pudlak syndrome type 2 are genetic hypomelanoses with immune deficiency that belong to the newly recognized group of genetic hypomelanoses related to defective melanosome biogenesis/transport and are hence presented above. Partial albinism with immunodeficiency syndrome (PAID, MIM #604228) was reported in 12 patients from eight families (Harfi et al., 1992). Salient clinical features of this syndrome were blond, golden, or silvery gray hair, hypomelanotic skin, recurrent fever, hepatosplenomegaly, pancytopenia, and progressive white matter demyelination. Biological findings were suggestive of defective melanin transport, cellular 618

immune deficiency, and CNS infiltration by immune cells. Taken together, this phenotype is clearly reminiscent of GS2 with secondary CNS involvement as a manifestation of hemophagocytic lymphohistiocytosis. It was stated that gene mapping suggested that PAID syndrome results from mutations in a gene located on 15q21 (Spritz, 1999). Taken together, it is probable that PAID syndrome is allelic to GS2; in this case, PAID should no longer be called an albinism. Confirmation awaits the study of Rab27a in these patients. A syndrome including hypomelanosis of the hair and skin, immunodeficiency, agenesis of the corpus callosum, cleft lip, and cataract (MIM #242840) was first described in two brothers (Vici et al., 1988) and again recently in four additional patients (del Campo et al., 1999). The striking pigmentary changes (including retinal hypomelanosis, which here would justify the appellation of albinism), and the dramatic CNS anomalies, are definitely important objective common traits of this syndrome, as stated by the authors (del Campo et al., 1999). However, given the immunological data, it is tempting to speculate that the recurrent infections are secondary to malnutrition rather than to a primary immune deficiency. Hence, this syndrome may rather be considered as a genetic hypomelanosis with CNS dysfunction, along with Griscelli syndrome type 1 (GS1), Elejalde syndrome (ES), and oculocerebral syndrome with hypomelanosis. There are few histological data available, excepted that the hair shaft was completely depigmented in one patient (del Campo et al., 1999). Finally, it is worth noting that the second patient described by Vici et al. (1988) did not have retinal hypomelanosis, but presented large pigment clusters in the hair shaft, a phenotype that is suggestive of GS1.

References Anderson, P. D., M. Huizing, D. Claassen, et al. Hermansky–Pudlak syndrome type 4 (HPS-4): clinical and molecular characteristics. Hum. Genet. 113:10–17, 2003. Anikster, Y., M. Huizing, J. White, Y. O. Sheuchenko, D. L. Fitzpatrick, J. W. Touchman, J. G. Compton, S. J. Bale, R. T. Swank, W. A. Gahl, and J. R. Toro. Mutations of a new gene causes a unique form of Hermansky–Pudlak syndrome in a genetic isolate of central Puerto Rico. Nature Genet. 28:376–380, 2001. Bachli, E. B., T. Eppler, E. Brack, et al. Hermansky–Pudlak syndrome type 4 in a patient from Sri Lanka with pulmonary fibrosis. Am. J. Med. Genet. 127A(2):201–207, 2004. Bahadoranm P., J. P. Ortonne, and R. Ballotti. Hypomelanosis, immunity, central nervous system: no more “and”, not the “end”. Am. J. Med. Genet. 116:334–337, 2003. Barbosam M. D. F. S., et al. Identification of the homologous beige and Chediak–Higashi syndrome genes. Nature 382:262, 1996. Barbosa, M. D., et al. Identification of mutations in two major isoforms of mRNA of the Chediak–Higashi syndrome gene in humans and mouse. Hum. Mol. Genet. 6:1091, 1997. Barrat, F. J., et al. Defective CTLA-4 cycling pathway in Chediak–Higashi syndrome: a possible mechanism for deregulation of T lymphocyte activation. Proc. Natl Acad. Sci. USA 96:8645, 1999. Certain, S., et al. Protein truncation test of LYST reveals heterogenous mutations in patients with Chediak–Higashi syndrome. Blood 95:979, 2000. Chiang, P. W., N. Oiso, R. Gautam, et al. The Hermansky–Pudlak

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION syndrome 1 (HPS1) and HPS4 proteins are the components of two complexes, BLOC-3 and BLOC-4, involved in the biogenesis of lysosome-related organites. J. Biol. Chem. 278:20332–20337, 2003. Clark, R. H., J. C. Stinchcombe, A. Day, et al. Adaptor protein 3dependent microtubule-mediated movement of lytic granules to the immunological synapse. Nature Immunol. 4:1111–1120, 2003. del Campo, M., B. D. Hall, A. Aeby, M. C. Nassogne, A. Verloes, C. Roche, C. Gonzalez, H. Sanchez, A. Garcia-Alix, F. Cabanas, R. M. Escudero, R. Hernandez, and J. Quero. Albinism and agenesis of the corpus callosum with profound developmental delay: Vici syndrome, evidence for autosomal recessive inheritance. Am. J. Med. Genet. 85:479–485, 1999. Dell’Angelica, E. C., V. Shotelersuk, R. C. Aguilar, et al. Altered trafficking of lysosomal proteins in Hermansky–Pudlak syndrome due to mutations in the beta3A subunit of the AP-3 adaptor. Mol. Cell 3:11–21, 1999. Faigle, W., et al. Deficient peptide loading and MHC class II endosomal sorting in a human genetic immunodeficiency syndrome: the Chediak–Higashi syndrome. J. Cell Biol. 141:1121, 1998. Gahl, W. A., et al. Genetic defects and clinical characteristics of patients with a form of oculocutaneous albinism (Hermansky– Pudlak syndrome). N. Engl. J. Med. 338:1258, 1998. Gardner, J. M., S. C. Wildenberg, N. M. Keiper, et al. The mouse pale ear (ep) mutation is the homologue of human Hermansky–Pudlak syndrome. Proc. Natl Acad. Sci. USA 94:9238–9243, 1997. Gautam, S., R. Chintala, and W. Li. The Hermansky–Pudlak syndrome 3 (cocoa) protein is a component of the biogenesis of lysosome-related organelles complex-2 (BLOC-2). J. Biol. Chem. 279:12935–12942, 2004. Griscelli, C., A. Durandy, D. Guy-Grand, et al. A syndrome associating partial albinism and immunodeficiency. Am. J. Med. 65:691– 702, 1978. Haddad, E., W. Xufeng, J. Hammer, et al. Defective granule exocytosis in Rab27a-deficient lymphocytes from ashen mice. J. Cell Biol. 152:835–841, 2001. Harfi, H. A., J. Brismar, B. Hainau, and R. Sabbah. Partial albinism, immunodeficiency, and progressive white matter disease: a new primary immunodeficiency. Allergy Proc. 13:321–328, 1992. Huizing, M., Y. Anikster, and W. A. Gahl. Hermansky–Pudlak syndrome and related disorders of organelle formation. Traffic 1:823, 2000. Huizing, M., et al. Hermansky–Pudlak syndrome type 3 in Ashkenazi Jews and other non-Puerto Rican patients with hypopigmentation and platelet storage-pool deficiency. Am. J. Hum. Genet. 69:1022, 2001. Huizing, M., C. D. Scher, E. Strovel, et al. Nonsense mutations in ADTB3A cause complete deficiency of the ß3A subunit of adaptor complex-3 and severe Hermansky–Pudlak syndrome type 2. Pediatr. Res. 51:150–158, 2002. Karim, M. A., et al. Apparent genotype–phenotype correlation in childhood, adolescent, and adult Chediak–Higashi syndrome. Am. J. Med. Genet. 108:16, 2002. Klein, C., N. Philippe, F. Le Deist, et al. Partial albinism with immunodeficiency (Griscelli syndrome). J. Pediatr. 125:886–895, 1994. Li, W., Q. Zhang, and N. Oiso. Hermansky–Pudlak syndrome type 7 (HPS-7) results from mutant dysbindin, a member of the biogenesis of lysosome-related organites complex 1 (BLOC-1). Nature Genet. 35:84–89, 2003. Marks, M. S., and M. C. Seabra. The melanosome, membrane dynamics in black and white. Nature Rev. Mol. Cell. Biol. 2:738–748, 2001. Martina, J. A., K. Moriyama, J. S. Bonifacino, et al. BLOC-3, a protein complex containing the Hermansky–Pudlak syndrome gene products HPS1 and HPS4. J. Biol. Chem. 278:29376–29384, 2003.

Menasche, G., E. Pastural, J. Feldmann, et al. Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nature Genet. 25:173–176, 2000. Menasche, G., J. Feldmann, A. Houdusse, et al. Biochemical and functional characterization of Rab27a mutations occurring in Griscelli syndrome patients. Blood 101:2736–2742, 2003a. Menasche, G., C. H. Ho, O. Sanal, et al. Griscelli syndrome restricted to hypopigmentation results from a melanophilin defect (GS3) or a MYO5A F-exon deletion (GS1). J. Clin. Invest. 112:450–456, 2003b. Nagle, D. L., et al. Identification and mutation analysis of the complete gene for Chediak–Higashi syndrome. Nature Genet. 14:307, 1996. Nazarian, R., J. Falcon-Perez, E. C. Dell’Angelica, et al. Biogenesis of lysosome-related organites complex 3 (BLOC-3). A complex containing the Hermansky–Pudlak syndrome (HPS) proteins HPS1 and HPS4. Proc. Natl Acad. Sci. USA 100:8770–8775, 2003. Pastural, E., F. J. Barral, R. Durourcq-Lagelouse, et al. Griscelli disease maps to chromosome15q21 and is associated with mutations in the myosine-Va gene. Nature Genet. 16:289–292, 1997. Perou, C. M. The Beige/Chediak–Higashi protein encodes a widely expressed cytosolic protein. J. Biol. Chem. 272:29790, 1997. Shotelersuk, V., et al. A new variant of Hermansky–Pudlak syndrome due to mutations in a gene responsible for vesicle formation. Am. J. Med. 108:423, 2000. Spritz, R. A. Molecular genetics of Hermansky–Pudlak and Chediak–Higashi syndromes. Platelets 9:21, 1998. Spritz, R. A. Chediak–Higashi syndrome. In: Primary Immunodeficiency Diseases: A Molecular and Genetic Approach. H. D. Ochs, C. I. Smith, and J. M. Puck (eds). New York: Oxford University Press, 1999, pp. 389–396. Spritz, R. A. Hermansky–Pudlak syndrome and pale-ear: melanosome making for the millennium. Pigment Cell Res. 13:15, 2000. Stinchcombe, J. C., L. J. Page, and G. M. Griffiths. Secretory lysosome biogenesis in cytotoxic T lymphocytes from normal and Chediak–Higashi syndrome patients. Traffic 1:435, 2000. Stinchcombe, J. C., D. C. Barral, E. H. Mules, et al. Rab27a is required for regulated secretion in cytotoxic T lymphocytes. J. Cell Biol. 152:825–834, 2001. Suzuki, T., W. Li, Q. Zhang, et al. Hermansky–Pudlak syndrome is caused by mutations in HPS4, the human homolog of the mouse light-ear gene. Nature Genet. 30:321, 2002. Vici, C. D., G. Sabetta, M. Gambarara, F. Vigevano, E. Bertini, R. Boldrini, S. G. Parisi, I. Quinti, F. Aiuti, and M. Fiorilli. Agenesis of the corpus callosum, combined immunodeficiency, bilateral cataract, and hypomelanosis in two brothers. Am. J. Med. Genet. 29:1–8, 1988. Wu, X. S., K. Rao, H. Zhang, et al. Identification of an organelle receptor for myosin-Va. Nature Cell Biol. 4:271–278, 2002. Zhang, Q., B. Zhao, W. Li, et al. Ru2 and Ru encode mouse orthologs of the genes mutated in human Hermansky–Pudlak syndrome types 5 and 6. Nature 33:145–153, 2003.

Angelman Syndrome Angelman syndrome (AS) is a complex developmental disorder with developmental delay and severe mental retardation, microcephaly, neonatal hypotonia, ataxic movements, and inappropriate laughter (Clayton–Smith and Laan, 2003; Williams et al., 1995, 2001). Most AS cases have an interstitial deletion of chromosome 15q11–13, with the remainder having a single gene abnormality, an imprinting defect, or uniparental disomy for the paternal chromosome 15 (Chan et al., 1993; Fang et al., 1999; Jiang et al., 1999, 2004; Nicholls and Knepper 2001). Hypopigmentation, characterized by light 619

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Fig. 31.8. Hypopigmentation in a young girl with Angelman syndrome. See also Plate 31.7, pp. 494–495.

skin and hair, is common in AS (King et al., 1992; Fig. 31.8). There may be a history of nystagmus or strabismus, and iris translucency and reduced retinal pigment may be present (Mah et al., 2000). No analysis of the optic tract organization in an individual who has Angelman syndrome and OCA2 is available (Thompson et al., 1999). Individuals with AS having OCA2 have been described, associated with a paternal P gene missence mutation and a maternal 15q deletion (Fridman et al., 2003; Saitoh et al., 2000). The presence of hypopigmentation in AS correlates with the presence of a 15q deletion (Saitoh et al., 1994; Smith et al., 1996).

Prader–Willi Syndrome Prader–Willi (PWS) and Angelman (AS) syndromes are associated with OCA2 (Butler 1989; Gardner et al., 1992; Hittner et al., 1982; King et al., 1992; Spritz et al., 1997; Wiesner et al., 1987). PWS is a complex developmental syndrome that presents with neonatal hypotonia, hyperphagia and obesity, hypogonadism, small hands and feet, and mental retardation associated with characteristic behavior (Bray et al., 1983; Cassidy, 1984, 1997; Cassidy and Schwartz, 1998; Webb et al., 1995). Approximately 70% of PWS cases have a deletion on the long arm of the paternal chromosome 15 and most cases without a deletion of the paternal chromosome 15 have uniparental disomy for the maternal chromosome 15, whereas a small number have gene mutations in the imprinting center or have chromosomal rearrangements that include the critical region on chromosome 15q (Bittel and Butler, 2005; Buiting et al., 2003; Gunay–Aygun et al., 2001; Horsthemke et al., 1997; Raca et al., 2004; Saitoh et al., 1997). Individuals with PWS may have clinically apparent hypopigmentation but most do not have the complete ocular features of albinism (Butler, 1989; Butler et al., 1986; Creel et al., 1986; Nicholls et al., 1996; Spritz et al., 1997; Wiesner et al., 620

Fig. 31.9. Hypopigmentation in a female with Prader–Willi syndrome.

1987; Fig. 31.9). Several individuals with PWS and OCA have been identified, however, because of the co-occurrence of the paternal 15q deletion and a maternal P gene mutation (Lee et al., 1994; Rinchik et al., 1993). The hypopigmentation in PWS was originally described as cutaneous albinoidism, suggesting only cutaneous without ocular involvement, but subsequent studies demonstrated hypopigmentation of skin, hair, and eyes, consistent with oculocutaneous hypopigmentation. For those without obvious OCA, hair and skin are lighter than unaffected family members, and childhood nystagmus and strabismus are common but often transient. The irides are pigmented with some translucency on globe transillumination, and retinal pigment is reduced in amount. Although foveal hypoplasia is not usually present, the fovea may not appear entirely normal (Creel et al., 1986). Visual evoked potential studies have revealed optic tract misrouting similar to that found in albinism in some, but not all, individuals with PWS and hypopigmentation (Apkarian et al., 1989; Creel et al., 1986). The presence of the optic tract misrouting is an important observation, implying that the hypopigmentation was significant during development and the hypopigmentation in PWS represents a type of albinism. The individuals with PWS

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION

and OCA have not been fully clinically described, but the features of the albinism are typical for OCA2.

Ataxia Telangiectasia

increased incidence of nonlymphoid malignancies observed primarily in older patients (Avet-Loiseau et al., 1992). Women heterozygous for the ataxia telangiectasia gene have a sixfold higher risk of developing skin cancer.

Anne-Sophie Gadenne

Laboratory Findings

Ataxia telangiectasia is a complex neurocutaneous syndrome characterized by progressive ataxia, oculocutaneous telangiectases, lymphoreticular neoplasia, and abnormalities of the immune system. The syndrome was described first in 1926 by Syllaba and Herner and by Louis-Bar in 1941. Boder and Sedgwick more carefully defined the disease in 1957. Almost 10 years later, Hecht noticed the association of ataxia telangiectasia with leukemia. In 1975, Taylor described an enhanced sensitivity of affected individuals to ionizing radiation. In 1991, Gatti reported the location of the responsible gene on chromosome 11 (Gatti, 1991).

The main immunologic abnormalities in patients with ataxia telangiectasia are low serum levels of immunoglobulin A (IgA) and IgE, thymus hypoplasia, impaired T-cell function, and a propensity for recurrent sinopulmonary infections. There may be significant variability in the degree of immune dysfunction among patients with the syndrome. Specific chromosomal abnormalities are characteristic of the syndrome, with a tendency for spontaneous breakage and rearrangement of chromosomes 2, 6, 7, 8, 14, 22, and X. Radioresistant DNA synthesis and increased cellular sensitivity to ionizing radiation and radiomimetic drugs are common findings (Chessa et al., 1992; Willems et al., 1993).

Epidemiology and Genetics

Criteria for Diagnosis

Ataxia telangiectasia, also called Louis-Bar syndrome, is transmitted as an autosomal recessive trait. The incidence of this disorder is between 1/20 000 and 1/100 000 live births (Moghadam et al., 1993). The first clinical manifestations appear by age 7 years, with a slight male predominance.

The diagnosis of ataxia telangiectasia is based on the presence of progressive cerebellar ataxia, cutaneous telangiectases, and oculocutaneous telangiectases. Supporting laboratory tests include elevated a-fetoprotein level in about 95% of patients and characteristic chromosomal translocations most easily detected in circulating lymphocytes.

General Clinical Findings The typical clinical findings include progressive cerebellar degeneration, oculocutaneous telangiectases, immunologic dysfunction, sensitivity to ionizing radiation, neoplasia, elevated serum a-fetoprotein, and premature aging. The first abnormality noticed by parents is truncal cerebellar ataxia, manifested as balance and gait abnormalities (Woods and Taylor, 1992). Intention tremors, choreoathetosis, and dystonia are early findings, followed by generalized hypotonia, loss of muscle power, and peripheral neuropathy in the second decade of life. Other findings include ocular apraxia, nystagmus, dysarthric speech, and exaggerated facial movements. Ocular telangiectasia is seen in almost all patients by the age of 10 years (Gatti, 1995). It is usually present on the lateral bulbar surfaces. Telangiectasia also may be noted over the bridge of the nose, the pinnae of the ears, and less frequently on the extremities, including antecubital, popliteal fossae, and back of the hands. Other dermatologic findings are premature graying of hair, hypertrichosis, and atrophic sclerodermoid plaques. Unusual findings which have been observed in association with ataxia telangiectasia are cutaneous granulomas, ulcerating necrobiosis lipoidica, and intraoral hemangiomas (Gotz et al., 1994; Oetting et al., 1991). Premature aging with senile keratoses, atrophic skin, and canities has been observed in several teenaged cases. Patients with ataxia telangiectasia are unusually susceptible to malignancies. The majority of these are lymphocytic leukemias or lymphomas, with a 70- to 250-fold excess of these cancers, most occurring in childhood (Gatti et al., 1994). Leukemias occur in about 20% of patients. There is an

Differential Diagnosis The differential diagnosis includes other syndromes with chromosomal instability such as Fanconi’s anemia (see Chapter 43), Bloom syndrome, and xeroderma pigmentosum. Several ataxia telangiectasia-like conditions have been reported, such as the Nijmegen breakage syndrome, which has some features of ataxia telangiectasia but not ataxia or telangiectasia, the bases of the syndrome.

Pathogenesis The pathogenesis of ataxia telangiectasia is not well understood (Taylor et al., 1993). The ataxia telangiectasia gene has been localized to chromosome 11q22–q23 (Gatti, 1991; Harnden, 1994). This gene may be involved in regulating several other genes. A number of close genes may cause the clinical heterogeneity of the ataxia telangiectasia phenotype (Bundey, 1994).

Treatment Treatment is symptomatic to control recurrent pulmonary infections. Radiation and chemotherapeutic agents must be used with care for the treatment of malignancies. Prenatal diagnosis may be made by demonstrating increased spontaneous and induced chromosomal breakage in the DNA of cultured lymphocytes from affected fetuses.

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CHAPTER 31 Avet-Loiseau, H., F. Mechinaud-Lacroix, and J. L. Harousseau. L’Ataxie telangiectasie. Arch. Fr. Pediatr. 49:205–210, 1992. Bittel, D. C., and M. G. Butler. Prader–Willi syndrome: clinical genetics, cytogenetics and molecular biology. Expert Rev. Mol. Med. 7:1–20, 2005. Bray, G. A., W. T. Dahms, R. S. Swerdloff, R. H. Fiser, R. L. Atkinson, and R. E. Carrel. The Prader–Willi syndrome: A study of 40 patients and a review of the literature. Medicine 62:59–80, 1983. Buiting, K., S. Gross, C. Lich, G. Gillessen-Kaesbach, O. el Maarri, and B. Horsthemke. Epimutations in Prader–Willi and Angelman syndromes: a molecular study of 136 patients with an imprinting defect. Am. J. Human Genet. 72:571–577, 2003. Bundey, S. Clinical and genetic features of ataxia-telangiectasia. Int. J. Radiat. Biol. 66:S23–S29, 1994. Butler, M. G. Hypopigmentation: a common feature of Prader–Labhart–Willi syndrome. Am. J. Human Genet. 45:140–146, 1989. Butler, M. G., F. J. Meaney, and C. G. Palmer. Clinical and cytogenetic survey of 39 individuals with Prader–Labhart–Willi syndrome. Am. J. Human Genet. 23:793–809, 1986. Cassidy, S. B. Prader–Willi syndrome. Curr. Probl. Pediatr. 14:5–55, 1984. Cassidy, S. B. Prader–Willi syndrome. J. Med. Genet. 34:917–923, 1997. Cassidy, S. B., and S. Schwartz. Prader–Willi and Angelman syndromes: disorders of genomic imprinting. Medicine 77:140–151, 1998. Chan, C. T., J. Clayton-Smith, X. J. Cheng, J. Buxton, T. Webb, M. E. Pembrey, and S. Malcolm. Molecular mechanisms in Angelman syndrome: a survey of 93 patients. J. Med. Genet. 30:895–902, 1993. Chessa, L., P. Petrinelli, A. Antonelli, M. Fiorilli, R. Elli, L. Marcucci, A. Federico, and E. Gandini. Heterogeneity in ataxia telangiectasia: classical phenotype associated with intermediate cellular radiosensitivity. Am. J. Med. Genet. 42:741–746, 1992. Clayton-Smith, J., and L. Laan. Angelman syndrome: a review of the clinical and genetic aspects. J. Med. Genet. 40:87–95, 2003. Creel, D. J., C. M. Bendel, G. L. Wiesner, J. D. Wirtschafter, D. C. Arthur, and R. A. King. Abnormalities of the central visual pathways in Prader–Willi syndrome associate with hypopigmentation. N. Engl. J. Med. 314:1606–1609, 1986. Fang, P., E. Lev-Lehman, T. F. Tsai, T. Matsuura, C. S. Benton, J. S. Sutcliffe, S. L. Christian, T. Kubota, D. J. Halley, H. Meijers– Heijboer, S. Langlois, J. M. Graham Jr., J. Beuten, P. J. Willems, D. H. Ledbetter, and A. L. Beaudet. The spectrum of mutations in UBE3A causing Angelman syndrome. Hum. Mol. Genet. 8:129–135, 1999. Fridman, C., N. Hosomi, M. C. Varela, A. H. Souza, K. Fukai, and C. P. Koiffman. Angelman syndrome associated with oculocutaneous albinism due to an intragenic deletion of the P gene. Am. J. Med. Genet. 119A:180–183, 2003. Gatti, R. A. Ataxia-telangiectasia (group A): localization of ATA gene to chromosome 11q22–23 and pathogenetic implications. Allergol. Immunopathol. 19:42–46, 1991. Gatti, R. A. Ataxia telangiectasia. Dermatol. Clin. 13:1–6, 1995. Gatti, R. A., C. M. McConville, and A. M. Taylor. Sixth International Workshop on Ataxia-Telangiectasia. Cancer Res. 54:6007–6010, 1994. Gotz, A., F. Eckert, and M. Landthaler. Ataxia-telangiectasia (LouisBar syndrome) associated with ulcerating necrobiosis lipoidica. J. Am. Acad. Dermatol. 31:124–125, 1994. Gunay-Aygun, M., S. Schwartz, S. Heeger, M. A. O’Riordan, and S. B. Cassidy. The changing purpose of Prader–Willi syndrome clinical diagnostic criteria and proposed revised criteria. Pediatrics 108:E92, 2001. Harnden, D. G. The nature of ataxia-telangiectasia: problems and perspectives. Int. J. Radiat. Biol. 66:S13–S19, 1994.

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Hittner, H. M., R. A. King, V. M. Riccardi, D. H. Ledbetter, R. P. Borda, R. E. Ferrell, and F. L. Kretzer. Oculocutaneous albinoidism as a manifestation of reduced neural crest derivatives in the Prader– Willi syndrome. Am. J. Ophthalmol. 94:328–337, 1982. Horsthemke, B., B. Dittrich B, and K. Buiting K. Imprinting mutations on chromosome 15 (review). Hum. Mutat. 10:329–337, 1997. Jiang, Y., E. Lev–Lehman, J. Bressler, T. F. Tsai, and A. L. Beaudet. Genetics of Angelman syndrome. Am. J. Human Genet. 65:1–6, 1999. Jiang, Y. H., J. Bressler, and A. L. Beaudet. Epigenetics and human disease. Ann. Rev. Genomics Hum. Genet. 5:479–510, 2004. King, R. A., G. L. Wiesner, D. Townsend, and J. G. White. Hypopigmentation in Angelman syndrome. Am. J. Human Genet. 46:40–44, 1992. Lee, S.-T., R. D. Nicholls, S. Bundey, R. Laxova, M. Musarella, and R. A. Spritz. Mutations of the P gene in oculocutaneous albinism, ocular albinism, and Prader–Willi syndrome plus albinism. N. Engl. J. Med. 330:529–534, 1994. Mah, M. L., D. K. Wallace, and C. M. Powell. Ophthalmic manifestations of Angelman syndrome. J. Am. Assoc. Pediatr. Ophthalmol. Strabismus 4:248–249, 2000. Moghadam, B. K., J. Y. Zadeh, and R. E. Gier. Ataxia-telangiectasia, review of the literature and a case report. Oral Surg. Oral Med. Oral Pathol. 75:791–797, 1993. Nicholls, R. D., and J. L. Knepper. Genome organization, function, and imprinting in Prader–Willi and Angelman syndromes. Annu. Rev. Genomics Hum. Genet. 2:153–175, 2001. Nicholls, R. D., T. Bailin, M. J. Mascari, M. G. Butler, and R. A. Spritz. Hypopigmentation in the Prader–Willi syndrome correlates with P gene deletion but not with haplotype of the hemizygous P allele. Am. J. Human Genet. 59:A39, 1996. Oetting, W. S., H. Y. Handoko, M. M. Mentink, A. S. Paller, J. G. White, and R. A. King. Molecular analysis of an extended family with type IA (tyrosinase negative) oculocutaneous albinism. J. Invest. Dermatol. 97:15–19, 1991. Raca, G., K. Buiting, and S. Das Deletion analysis of the imprinting center region in patients with Angelman syndrome and Prader-Willi syndrome by real-time quantitative PCR. Genet. Tests 8:387–394, 2004. Rinchik, E. M., S. J. Bultman, B. Horsthemke, S. Lee, K. M. Stunk, R. A. Spritz, K. M. Avidano, M. T. C. Jong, and R. D. Nicholls. A gene for the mouse pink-eyed dilution locus and for human type II oculocutaneous albinism. Nature 361:72–76, 1993. Saitoh, S., N. Oiso, T. Wada, O. Narazaki, and K. Fukai. Oculocutaneous albinism type 2 with a P gene missense mutation in a patient with Angelman syndrome (letter). J. Med. Genet. 37:392–394, 2000. Saitoh, S., N. Harada, Y. Jinno, K. Hashimoto, K. Imaizumi, Y. Kuroki, Y. Fukushima, T. Sugimoto, M. Renedo, and J. Wagstaff. Molecular and clinical study of 61 Angelman syndrome patients. Am. J. Human Genet. 52:158–163, 1994. Saitoh, S., K. Buiting, S. B. Cassidy, J. M. Conroy, D. J. Driscoll, J. M. Gabriel, G. Gillessen–Kaesbach, C. C. Glenn, L. R. Greenswag, B. Horsthemke, I. Kondo, K. Kuwajima, N. Niikawa, P. K. Rogan, S. Schwartz, J. Seip, C. A. Williams, and R. D. Nicholls. Clinical spectrum and molecular diagnosis of Angelman and Prader–Willi syndrome patients with an imprinting mutation. Am. J. Human Genet. 68:195–206, 1997. Smith, A., C. Wiles, E. Haan, J. McGill, G. Wallace, J. Dixon, R. Selby, A. Colley, R. Marks, and R. J. Trent. Clinical features in 27 patients with Angelman syndrome resulting from DNA deletion. J. Med. Genet. 33:107–112, 1996. Spritz, R. A., T. Bailin, R. D. Nicholls, S.-T. Lee, S. K. Park, M. J. Mascari, and M. G. Butler. Hypopigmentation in the Prader–Willi syndrome correlates with P gene deletion but not with haplotype of the hemizygous P allele. Am. J. Human Genet. 71:57–62, 1997.

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION Willems, P. J., B. C. Van Roy, W. J. Kleijer, M. Van der Kraan, and J. J. Martin. Atypical clinical presentation of ataxia telangiectasia. Am. J. Med. Genet. 42:777–782, 1993. Williams, C. A., H. Angelman, J. Clayton-Smith, D. J. Driscoll, J. E. Hendrickson, J. H. M. Knoll, R. E. Magenis, A. Schnizel, J. Wagstaff, E. M. Whidden, and R. T. Zori. Angelman syndrome: consensus for diagnostic criteria. Am. J. Human Genet. 56:237–238, 1995. Williams, C. A., A. Lossie A, and D. Driscoll. Angelman syndrome: mimicking conditions and phenotypes. Am. J. Human Genet. 101: 59–64, 2001. Woods, C. G., and A. M. Taylor. Ataxia telangiectasia in the British Isles: the clinical and laboratory features of 70 affected individuals. Q. J. Med. 82:169–179, 1992.

Hallerman–Streiff Syndrome James J. Nordlund

Clinical Description

Fig. 31.10. Typical facies of a boy with Hallerman–Streiff syndrome (see also Plate 31.8, pp. 494–495).

This is a rare syndrome characterized by an unusual bird-like facies, hypoplasia of the mandible and maxilla, dyscephaly (Fig. 31.10), cataracts, microphthalmia, hypotrichosis, atopy of the skin, and short stature (da Fonseca and Mueller, 1994). In addition, many of these children have ocular abnormalities leading to blindness. These include retinitis pigmentosa or choroido-retinal degeneration (Tsukahara et al., 1985). It has been suggested that children with Hallerman–Streiff syndrome have marked pigment dilution of the skin and hair (Fig. 31.11) (Lopez, 1970). This is the only pigmentary disorder, and the mechanism of retarded pigment production is not known. Some children with this syndrome have poor humoral immunity and hypoparathyroidism (Chandra et al., 1978).

References

Fig. 31.11. Hypopigmented eyelashes observed in Hallerman–Streiff syndrome.

Taylor, A. M., N. G. Jaspers, and R. A. Gatti. Fifth International Workshop on Ataxia-Telangiectasia. Cancer Res. 53:438–441, 1993. Thompson, D. A., A. Kriss, S. Cottrell, and D. Taylor. Visual evoked potential evidence of albino-like chiasmal misrouting in a patient with Angelman syndrome with no ocular features of albinism. Dev. Med. Child Neurol. 41:633–638, 1999. Webb, T., D. Clarke, C. A. Hardy, M. W. Lilpatrick, J. Corbett, and M. Dahlitz. A clinical, cytogenetic, and molecular study of 40 adults with the Prader–Willi syndrome. J. Med. Genet. 32:181–185, 1995. Wiesner, G. L., C. M. Bendel, D. P. Olds, J. G. White, D. C. Arthur, D. W. Ball, and R. A. King. Hypopigmentation in the Prader–Willi syndrome. Am. J. Human Genet. 40:431–442, 1987.

Chandra, R. K., S. Joglekar, and Z. Antonio. Deficiency of humoral immunity and hypoparathyroidism associated with the Hallerman–Streiff syndrome. J. Pediatr. 93:892–893, 1978. da Fonseca, M. A., and W. A. Mueller. Hallerman–Streiff syndrome: case report and recommendations for dental care. J. Dent. Child. 61:334–337, 1994. Lopez, B. Trastornos de la pigmentacion. In: Actas del VI Congresso Ibero-Latino Americano de Dermatologia. Barcelona: Editorial Cientifico Medical, 1970, pp. 157–179. Tsukahara, S., M. Sasamoto, I. Watanabe, and C. I. Phillips. Diagnostic survey at Yamanashi School for the Blind: importance of heredity. Jpn. J. Ophthalmol. 29:315–321, 1985.

Histidinemia Marnie D. Titsch Histidinemia is an inborn error of metabolism transmitted as an autosomal recessive trait. It was described first by Ghadimi and coworkers (1961) in two sisters who had a positive ferric chloride and Phenistix test in the urine. These findings suggested phenylketonuria. However, phenylalanine blood levels were normal, but consistently elevated blood and urinary histidine levels suggested an inborn error of histidine metabolism. 623

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Histidinemia affects about 1 in 10 000 infants (Imamura et al., 1984). This figure is based on mass screening programs of newborns to detect histidinemia (Neville and Lilly, 1973). Prior to mass screening, most diagnosed patients were under 23 years of age. There are no known sexual or racial predilections.

Clinical Findings Individuals with histidinemia can be normal or have severe mental retardation (Ghadimi and Partington, 1967; La Du, 1967; Neville and Lilly, 1973). The most frequent clinical findings are mental retardation and speech disturbances (Lott et al., 1970). Speech disturbances are associated with mental retardation in a majority of patients. Some individuals with speech defects have normal intelligence (La Du et al., 1963; Lott et al., 1970). Other clinical findings include cerebellar ataxia (Shaw et al., 1963; Thalhammer et al., 1971), autism (Kotsopoulous and Kutty, 1979), schizophrenia (Lucca et al., 1990), delayed bone age (Cain and Holton, 1968), growth retardation (Andrews et al., 1962; Davies and Robinson, 1963; Ghadimi, 1981), emotional disturbances (Waisman, 1967), convulsions (Duncan et al., 1991), recurrent infections (Raisova and Hyanek, 1986; Rama Rao et al., 1974), anemia (Wadman et al., 1966), learning disorders (Rosenmann et al., 1983; Wilcken and Brown, 1975), atopic dermatitis (Kierland et al., 1968), nystagmus (Dhir et al., 1987), multiple congenital anomalies such as Marfan’s syndrome (Stevens et al., 1975), Joubert’s syndrome (Appleton et al., 1989), and congenital hypoplastic anemia (Gilman and Howell, 1969). No correlation between clinical and biochemical findings has been found (Hyanek and Raisova, 1985; Pieniazek et al., 1985). Pigmentary findings include light-colored hair and eyes and generalized pale skin (Ghadimi and Partington, 1967; Ghadimi et al., 1961), described as congenital universal hypomelanosis. The mechanism for the pigmentary abnormalities is not known.

Laboratory Findings The characteristic biochemical finding that makes the diagnosis of histidinemia is an increased concentration of histidine in blood, urine, and cerebrospinal fluid and increased urinary excretion of transamination products (Auerbach et al., 1962; Imamura et al., 1984; Kuroda et al., 1980; Matsuda et al., 1982).

Diagnosis Diagnosis of histidinemia is based on elevated concentrations of histidine in blood and/or urine, with most cases identified in newborn screening programs (Bradley, 1975; Ito et al., 1981). Prenatal diagnosis of histidinemia is not reliable because histidase activity is detectable in amniotic cells only after 39 weeks’ gestation.

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Differential Diagnosis The differential diagnosis of congenital universal hypomelanosis includes several metabolic disorders, such as Menke’s kinky hair syndrome, phenylketonuria, and homocystinuria. Other genetic diseases characterized by generalized pale skin include tyrosinase-positive and -negative albinism, Chédiak–Higashi syndrome, Cross–McKusick syndrome, and Hermansky–Pudlak syndrome.

Pathogenesis The metabolic abnormality causing histidinemia is a defect in histidase, which is the enzyme responsible for the major degradative pathway of histidine to urocanic acid. The human gene for histidase has been assigned to chromosome 12 by Southern blot analysis of human–mouse somatic cell hybrid DNA (Taylor et al., 1993; Yokoya et al., 1983).

Treatment It is possible to treat infants with histidinemia with a low histidine diet. However, no improvement in clinical symptoms has been observed in several such trials (Gatfield et al., 1969; Wadman et al., 1973).

References Andrews, B. F., P. F. Crosby, and C. R. Angel. Histidinemia: a new metabolic disorder [Abstract]. South. Med. J. 55:1326, 1962. Appleton, R. E., D. Chitayat, J. E. Jan, R. Kennedy, and J. G. Hall. Joubert’s syndrome associated with congenital ocular fibrosis and histidinemia. Arch. Neurol. 46:579–582, 1989. Auerbach, V. H., A. M. DiGeorge, R. C. Baldridge, C. D. Tourtellotte, and M. P. Brigham. Histidinemia, a deficiency in histidase resulting in the urinary excretion of histidine and imidazole pyruvic acid. J. Pediatr. 60:487–497, 1962. Bradley, D. M. Screening for inherited metabolic disease in Wales using urine-impregnated filter paper. Arch Dis. Child. 50:264–268, 1975. Cain, A. R. R., and J. B. Holton. Histidinaemia: a child and his family. Arch. Dis. Child. 43:62–67, 1968. Davies, H. E., and M. J. Robinson. A case of histidinaemia. Arch. Dis. Child. 38:80–82, 1963. Dhir, S. P., M. W. Shisku, and A. Krewi. Ocular involvement in histidinaemia. Ophthalmic Paediatr. Genet. 8:175–176, 1987. Duncan, J. S., P. Brown, and C. D. Marsden. Progressive myoclonus and histidinaemia. Mov. Disord. 6:87–89, 1991. Gatfield, P. D., R. M. Knights, M. Devereux, and J. P. Pozsonyi. Histidinemia: report of four new cases in one family and the effect of low histidine diets. Can. Med. Assoc. J. 101:71–75, 1969. Ghadimi, H. Histidinemia. Biochemistry and behavior. Am. J. Dis. Child. 135:210–213, 1981. Ghadimi, H., and M. W. Partington. Salient features of histidinemia. A. J. Dis. Child. 113:83–87, 1967. Ghadimi, H., M. W. Partington, and A. Hunter. A familial disturbance of histidine metabolism. N. Engl. J. Med. 265:221–224, 1961. Gilman, P. A., and R. R. Howell. The simultaneous occurrence of histidinemia and congenital hypoplastic anemia. J. Pediatr. 75:878–880, 1969. Hyanek, J., and V. Raisova. Speech and language disorders in histidinaemia and other amino acid disturbances. J. Inherit. Metab. Dis. 8 (Suppl. 2):130, 1985. Imamura, I., T. Watanabe, Y. Hase, Y. Sakamoto, Y. Fukuda, H.

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION Yamamoto, T. Tsuruhara, and H. Wada. Histamine metabolism in patients with histidinemia: determination of urinary levels of histamine, N-tau-methylhistamine, imidazole acetic acid, and its conjugate. J. Biochem. 96:1925–1929, 1984. Ito, F., K. Aoki, and Y. Eto. Biochemical parameters for diagnosis. Am. J. Dis. Child. 135:227–229, 1981. Kierland, R. R., H. O. Perry, R. K. Winkelmann, S. A. Muller, and W. M. Sams. Histidinemia and atopic dermatitis. Arch. Dermatol. 98:316–322, 1968. Kotsopoulos, S., and K. M. Kutty. Histidinemia and infantile autism. J. Autism Dev. Disord. 9:55–60, 1979. Kuroda, Y., T. Ogawa, M. Ito, T. Watanabe, E. Takeda, K. Toshima, and M. Miyao. Relationship between skin histidase activity and blood histidine response to histidine intake in patients with histidinemia. J. Pediatr. 97:269–272, 1980. La Du, B. N. Histidinemia: current status. Am. J. Dis. Child. 113:88–92, 1967. La Du, B. N., R. R. Howell, G. A. Jacoby, J. E. Seegmiller, E. K. Sober, V. G. Zannoni, J. P. Canby, and L. K. Ziegler. Clinical and biochemical studies on two cases of histidinemia. Pediatrics 32:216–227, 1963. Lott, I. T., J. A. Wheelden, and H. L. Levy. Speech and histidinemia: methodology and evaluation of four cases. Dev. Med. Child. Neurol. 12:596–603, 1970. Lucca, A., M. Catalano, R. Valsasina, C. Fara, and E. Smeraldi. Biochemical investigation of histidinemia in schizophrenic patients. Biol. Psychiatry 27:67–75, 1990. Matsuda, I., N. Nagata, and F. Endo. Blood histidine levels during course of histidinaemia. Lancet 1:162–163, 1982. Neville, B. G. R., and P. M. Lilly. Histidinaemia: its significance in neonatal screening. Arch. Dis. Child. 48:325–326, 1973. Pieniazek, D., J. Kubalska, E. Pronicka, and E. Stecko. Disturbances in histidine metabolism in children with speech abnormalities. Acta Anthropogenet. 9:117–121, 1985. Raisova, V., and J. Hyanek. Speech disorders associated with histidinemia and other hereditary disorders of amino acid metabolism. Folia Phoniatr. 38:43–48, 1986. Rama Rao, B. S. S., M. N. Subhash, and H. S. Narayanan. Histidinemia. Report of a case. Indian Pediatr. 41:312–313, 1974. Rosenmann, A., C. R. Scriver, C. L. Clow, and H. L. Levy. Histidinaemia. Part II: Impact; a retrospective study. J. Inherit. Metab. Dis. 6:54–57, 1983. Shaw, K. N. F., E. Boder, M. Gutenstein, and E. E. Jacobs. Histidinemia [Abstract]. J. Pediatr. 63:720–721, 1963. Stevens, R., H. E. Cross, and G. Morrow. Histidinemia with features of the Marfan syndrome. J. Pediatr. 86:907–910, 1975. Taylor, R. G., D. Gvieco, G. A. Clarke, R. R. McInnes, and B. A. Taylor. Identification of the mutation in murine histidinemia (his) and genetic mapping of the murine histidase locus (Hal) on chromosome 10. Genomics 16:231–240, 1993. Thalhammer, O., S. Scheibenreiter, and M. Pantlitschko. Histidinemia: detection by routine screening and biochemical observations on three unrelated cases. Z. Kinderheilkd. 109:279–292, 1971. Wadman, S. K., P. K. De Bree, J. P. van Biervliet, and F. J. van Sprang. Dietary correction of histidinemia in older children possible. Clin. Chim. Acta 49:377–382, 1973. Wadman, S. K., F. J. van Sprang, G. J. van Stekelenburg, and P. K. de Bree. Three new cases of histidinemia. Clinical and biochemical data. Z. Gesamte Innere Med. Ihre Grenzgeb. 21:485–492, 1966. Waisman, H. A. Variations in clinical and laboratory findings on histidinemia. Am. J. Dis. Child. 113:93–94, 1967. Wilcken, B., and D. A. Brown. Histidinaemia — evaluation of an improved method for confirmation, and the implications of the diagnosis. Aus. Paediatr. J. 11:126–127, 1975. Yokoya, S., E. Tokuhiro, S. Suwa, and H. Maesaka. Measurement of the skin urocanic acid content in normal and histidinemic infants. Eur. J. Pediatr. 140:330–332, 1983.

Fig. 31.12. Brittle hair in a patient with homocystinuria (see also Plate 31.9, pp. 494–495).

Homocystinuria Allan D. Mineroff Homocystinuria is an inborn error of metabolism transmitted as an autosomal recessive trait. This disorder was identified in 1962 during a study of mentally retarded patients in Ireland (Carson and Neill, 1962). The incidence is thought to be between 1 : 100 000 and 1 : 200 000.

Clinical Findings The phenotype of homocystinuria is variable. About 50% of affected individuals are mentally retarded (Rosenberg, 1991). Dislocation of the ocular lenses, long and thin extremities, pectus excavatum, scoliosis, osteoporosis, and arterial and venous thrombosis are common manifestations. Cutaneous manifestations include malar flush, livedo reticularis of the trunk and limbs, and sparse and brittle hair (Fig. 31.12). The malar flush and the livedo reticularis, which are more apparent after exposure to cold temperatures or after exercise, are believed to be related to vasomotor instability. Abnormal skin crease, nail fold capillaries, and telangiectasias around scars also have been described (Price et al., 1968). The skin, hair, and eyes typically are washed out and light in color. Shelly and Rawnsley (1972) reported a 22-year-old female with homocystinuria whose blond hair blackened after ingesting pyridoxine hydrochloride (vitamin B6). Microscopically, the black hair showed an increased number of melanin granules. It was postulated that the observed darkening resulted from a reduction in the high concentrations of homocysteine that seem to be inhibitory for the melanogenic enzyme tyrosinase. Traboulsi and coworkers (1992) reported a case of a 22month-old girl with cobalamin C complementation type of combined methylmalonic aciduria and homocystinuria. She developed foveal pigment clumping and hypopigmentation of the surrounding retina. They also observed histologically vacuolization of the iris pigment epithelium. 625

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Higginbottom and coworkers (1978) reported a 6-monthold infant with homocystinuria, methylmalonic aciduria, megaloblastic anemia, neurologic abnormalities, and vitamin B12 deficiency. The infant had hyperpigmentation of the extremities. The hyperpigmentation resolved after the baby received parenteral vitamin B12. Thrombotic complications result in premature death in at least 20% of patients. Osteoporosis may worsen with passing years and lead to pathologic fractures. Complications from dislocated lenses may occur at any time.

Pathogenesis A lack of the enzyme cystathionine synthase causes a block in the conversion of homocysteine to cystathionine. As a result of this block, homocysteine accumulates in the blood and urine where it is oxidized to homocystine. Cystathionine synthase has been mapped to chromosome 21q22.3. Deficiencies of 5-methyltetrahydrofolate methyltransferase or 5,10-methylene tetrahydrofolate reductase also may be involved in the disorder.

Therapy About 50% of patients respond favorably to pyridoxine and restriction of dietary protein and methionine.

References Carson, N. A. J., and D. W. Neill. Metabolic abnormalities detected in a survey of mentally backward individuals in Northern Ireland. Arch. Dis. Child. 37:505–513, 1962. Higginbottom, M. C., L. Sweetman, and W. L. Nyhan. A syndrome of methylmalonic aciduria, homocystinuria, megaloblastic anemia and neurologic abnormalities in vitamin B12 deficient breast-fed infant of a strict vegetarian. N. Engl. J. Med. 299:317–323, 1978. Price, J., C. F. H. Vickers, and B. K. Booker. A case of homocystinuria with noteworthy dermatological features. J. Ment. Def. Res. 12:111–118, 1968. Rosenberg, L. Inherited disorders of amino acid metabolism. In: Harrison’s Principles of Internal Medicine, 12th ed., Wilson, J. D., E. Braunwald, K. J. Isselbacher, R. G. Petersdorf, J. B. Martin, A. S. Fauci, and R. K, Root (eds.). New York: McGraw-Hill, 1991, p. 1868–1875. Shelly, W. B., and H. Rawnsley. Pyridoxine-dependent hair pigmentation in association with homocystinuria. Arch. Dermatol. 106: 228–230, 1972. Traboulsi, E. I., J. C. Silva, M. T. Geraghty, I. H. Maumenee, D. Valle, and W. R. Green. Ocular histopathologic characteristics of cobalamin C type vitamin B12 defect with methylmalonic aciduria and homocystinuria. Am. J. Ophthalmol. 113:269–280, 1992.

Oculocerebral Syndrome with Hypopigmentation Jean L. Bolognia

Historical Background In 1967, Cross et al. described three siblings who had severe mental and physical retardation, blindness, spasticity, and athetoid movements in association with hypopigmentation of

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the skin and hair (Cross et al., 1967); a fourth stillborn child was said to have similar ocular anomalies and hypopigmentation. Because of extensive opacification and vascularization of the corneas, it was not possible to determine if the children had pigmentary dilution of the fundi or irides. There was considerable consanguinity among the ancestors of these four children, e.g., the paternal grandmother and the maternal grandfather were first cousins and the parents could be traced back to a common ancestral couple. As a result, the authors suggested that the mode of inheritance was autosomal recessive. During the three decades since the original description, at least 15 cases of oculocerebral syndrome with hypopigmentation (OCSH) have been reported (Castle et al., 1989; Courtens et al., 1989; De Jong and Fryns, 1991; Fryns et al., 1988; Lerone et al., 1992; Ozkan et al., 1991; Passarge and FuchsMecke, 1975; Patton et al., 1987; Preus et al., 1983; Tezcan, 1997; White et al., 1993) (Table 31.5). White et al. (1993) proposed that this group of patients should be subdivided into those with “true” oculocutaneous albinism (i.e., pigmentary dilution of skin, hair, and eyes plus eye findings such as nystagmus) versus those without true albinism. They argued that the individuals who fulfilled the criteria for true albinism (Castle et al., 1989; Patton et al., 1987; White et al., 1993) also lacked spasticity and additional ocular findings such as microphthalmia and corneal anomalies (Table 31.5). A few years earlier, Patton et al. (1987) had come to the conclusion that the syndrome described by Preus et al. (1983) was distinct from that described by Cross et al. (1967), hence the two different Mendelian Inheritance in Man six-digit entries (McKusick, 1994). However, until the genetic mutations responsible for the entire clinical spectrum of OCSH are determined, the need for these distinctions remains uncertain.

Synonyms Cross syndrome (MIM 257800); Kramer syndrome (MIM 257800); oculocerebral hypopigmentation syndrome of Preus (MIM 257790).

Epidemiology The original description of an oculocerebral syndrome with hypopigmentation was in four members of a family from the Old Order Amish of Ohio where there had been several generations of consanguineous marriages (Cross et al., 1967); the surname of the affected family was Kramer (McKusick, 1994). Based upon the pedigree of this family, the presumed mode of inheritance was autosomal recessive. Since then an additional nine patients have been described who had parental consanguinity (De Jong and Fryns, 1991; Fryns et al., 1988; Ozkan et al., 1991; Preus et al., 1983; Tezcan et al., 1997) (Table 31.5). The remainder of the reported cases have been sporadic and no authors have proposed an inheritance pattern other than autosomal recessive. Additional support for an autosomal recessive mode of inheritance comes from the equal number of affected males and females. The racial background

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION

of the patients has varied from Caucasian (Castle et al., 1989; Courtens et al., 1989; Fryns et al., 1988; Lerone et al., 1992; Ozkan et al., 1991; Passarge and Fuchs-Mecke, 1975; Patton et al., 1987; Preus et al., 1983; White et al., 1993) to “Cape Coloured” mixed ancestry (De Jong and Fryns, 1991); countries of origin have included the United States, Canada, the United Kingdom, Belgium, Germany, Italy, Turkey, and South Africa (Castle et al., 1989; Cross et al., 1967; De Jong and Fryns, 1991; Fryns et al., 1988; Lerone et al., 1992; Ozkan et al., 1991; Passarge and Fuchs-Mecke, 1975; Patton et al., 1987; Preus et al., 1983; Tezcan, 1997; White et al., 1993).

Clinical Description In the original description, all three siblings had severe psychomotor retardation, quadriplegic spasticity, and athetosis but notably lacked cerebellar signs or seizures (Cross et al., 1967). These children had severe ocular anomalies including congenital blindness, microphthalmia, spastic ectropion, and corneal opacification (Table 31.5). Course jerky or constant wandering movements of the eyes were observed, but due to extensive opacification and vascularization of the corneas, it was not possible to determine if the children had pigmentary dilution of the fundi or irides. Significant growth retardation was also present with the length and weight of each child falling below the third percentile (Cross et al., 1967). Additional clinical findings included a high-arched palate, abnormal dentition in the form of small, widely spaced teeth, and nonpalpable testes (cryptorchidism versus hypogonadism). In the subsequent reports of OCSH, psychomotor retardation was a consistent finding while spasticity and growth retardation were observed in the majority of patients (Table 31.5). A minority of affected individuals were noted to have ataxia, hypotonia or seizures, neurologic manifestations not observed in the original family (Courtens et al., 1989; De Jong and Fryns, 1991; Fryns et al., 1988; Lerone et al., 1992; Patton et al., 1987; Tezcan, 1997; White et al., 1993). Of the associated ocular anomalies, nystagmus was the one most commonly reported followed by poor vision or blindness. Additional signs or symptoms observed in at least two of the 17 reported patients with OCSH were photophobia, strabismus, deafness, microcephaly, high-arched palate, cerebral atrophy, Dandy– Walker malformation of the posterior fossa, acetabular hypoplasia and generalized osteoporosis (Castle et al., 1989; Courtens et al., 1989; De Jong and Fryns, 1991; Fryns et al., 1988; Lerone et al., 1992; Ozkan et al., 1991; Patton et al., 1987; Preus et al., 1983; Tezcan, 1997; White et al., 1993) (Table 31.5). Although the first two are seen commonly in oculocutaneous albinism, all of these associated findings should be kept in mind in any patient suspected of having OCSH. Whereas all of the patients with OCSH have had hypopigmentation of the skin and/or hair (Fig. 31.13), pigmentary dilution of the ocular fundi and irides has not been a consistent finding (Table 31.5), leading to a debate as to whether or not this disorder should be considered a true form of oculocutaneous albinism (see above). Variation in the color of scalp

Fig. 31.13. A patient with OCSH who has microcephaly and an admixture of white and black hairs. See also plate 31.10, p. XXX. [From DeJong and Gryns JP. Genet. Counsel. 2:151–155, 1991, with permission of the publishers.]

hairs, eyebrows, and eyelashes is one of the most interesting clinical findings in OCSH. Although the oldest child described by Cross et al. (1967) had white hair with a faint yellow tinge, the two younger children were noted to have extremely light hair with rare, darkly pigmented strands. Twenty years later, Fryns et al. (1988) were struck by the patchy distribution of black-gray hairs in the eyebrows of their patients in addition to a tendency toward gray-white hairs medially. At birth, both of the children had gray scalp hairs with a silverblond metallic sheen as did a third sibling who was otherwise normal. Additional patients with an admixture of white and dark scalp hairs have been described (De Jong and Fryns, 1991; Lerone et al., 1992) as have children with eyelashes of different colors (De Jong and Fryns, 1991).

Histology By routine microscopy, skin biopsy specimens have shown no abnormalities (Fryns et al., 1988; Patton et al., 1987) as well as a normal but “depigmented” epidermis (Ozkan et al., 1991; Passarge and Fuchs-Mecke, 1975; Tezcan, 1997). In silverstained sections, Courtens et al. (1989) observed moderate amounts of melanin in the basal layer of the epidermis, but in a discontinuous pattern. When a biopsy specimen of skin was incubated in l-dopa, a normal number of dopa-positive cells was seen (Patton et al., 1987) (Tezan et al., 1997). Electron photomicrographs of the skin from patients with OCSH have demonstrated a decreased number of melanosomes (Courtens et al., 1989) as well as a patchy absence of melanin (Fryns et al., 1988); the latter finding is quite similar to the observations of Courtens et al. (1989) described above. One clinical manifestation of this phenomenon may be the admixture of white and dark scalp and eyebrow hairs in some patients with OCSH. In additional ultrastructural studies, conflicting results were reported — in one study, a normal number of melanocytes with primarily stage II and III melanosomes (suggestive of “tyrosinase-

627

CHAPTER 31 Table 31.5. Summary of clinical findings in reported cases of Cross syndrome.† Cross et al. (1967)

Patient Sex Parental consanguinity Hypopigmentation of skin and/or hair Hypopigmentation of irides Pigmented nevi Mental retardation Spasticity Athetosis Ataxia Seizures Abnormal dentition Nystagmus Fundal appearance of albinism Microphthalmia Corneal anomalies Cataracts Blindness Growth retardation Nonpalpable testes Other

Passarge and Fuchs-Mecke (1975)

Preus et al. (1983)

Fryns et al. (1988)

Courtens et al. (1989)

1 F +

2 M +

3 M +

1 F -

1 F +

2 F +

1 M +

2 F +

1 M -

+

+

+

+

+

+

+

+

+

?*

?*

?*

?

?

?

?

?

+

+ + + + ? + ?*

+ + + + + ?*

? + + + + + ?*

? + + + + + ?

? + + + + +

? ? ? ? ? +

+ + + ? + -‡

? + + ? ? ? -

? + + + + ?*

+ + + + NA Eczematous rash (+Candida); hypertelorism; dysplastic pinnae; scoliosis; enamel hypoplasia

+ + +§§ + + ± NA NA Dolichocephaly; high-arched palate; strabismus; “clawed” fingers; syndactyly; deafness; cerebral atrophy; acetabular hypoplasia

+ + + + + + ?* ?* ?* + + + + + + NA + + Dolichocephaly; high-arched palate

+ NA Strabismus; axial hypotonia; high-arched palate; cerebellar atrophy and cystic malformation of posterior fossa

? + + ±§ + Iris atrophy; photophobia; generalized hypotonia; enamel hypoplasia

+ = Present; - = absent; ? = not commented on in text. †Adapted in part from White et al., Am. J. Med. Genet. 46:592–96, 1993. ‡Albinoid visual evoked potential asymmetry. §Visual evoked potentials—very weak response to flash light. *Could not be visualized because of extensive opacification and vascularization of the corneas. **Optic atrophy. ††Pigmented freckles. §§Severe myopia. Abbreviations: CAL = café-au-lait; NA = not applicable.

positive” oculocutaneous albinism) was seen (Patton et al., 1987) while in a second, scarce stage I and II melanosomes (suggestive of type IB oculocutaneous albinism) were observed (White et al., 1993). Examination of hair shafts has shown a reduction in pigment content (Lerone et al., 1992) as well as near absence of pigmentation (Courtens et al., 1989). Incubation of anagen hair bulbs with l-tyrosine has led to a variety of results ranging from no increase in pigmentation (White et al., 1993) to a slight increase in pigment content (Cross et al., 1967) to a marked increase in pigmentation (Castle et al., 1989). When 628

biopsy specimens containing hair follicles were incubated in l-dopa, a moderately strong reaction was observed (Patton et al., 1987). Electron photomicrographs of hair follicles from the original family showed reduced numbers of melanosomes within melanocytes that were in all stages of development (Witkop, 1971).

Differential Diagnosis The differential diagnosis includes other forms of “tyrosinasepositive” oculocutaneous albinism (see the first section in this chapter), but the associated systemic abnormalities, in partic-

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION Table 31.5. (continued) De Jong and Fryns (1991)

Özkan et al. (1991)

Lerone et al. (1992)

Patton et al. (1987)

Castle et al. (1989)

White et al. (1993)

Tezcan et al. (1997)

1 F +

2 F +

1 M +

2 F +

1 M -

1 M -

1 F -

1 M -

1 M +

+

+

+

+

+

+

+

+

+

-

-

+

?

?

+

+

+

-

? + + + ? + -

? + ± + ? -

? + + + -

? + + ? ? ?

? + + + + + +

+ + + ? + -

+ + + + +

+ + + + +

+ + + ? + ? + ?

+ Microcephaly; high-arched palate; Dandy– Walker malformation of posterior fossa; generalized osteoporosis

+ Photophobia; strabismus; hypotonia; generalized osteoporosis; cerebral atrophy; acetabular hypoplasia

NA Strabismus; photophobia

4/200 + Photophobia; hypotonia; sensorineural deafness; Bartter syndrome

+ ? + ? Strabismus; microcephaly; high-arched palate; Dandy–Walker malformation; sensorineural deafness; uteropelvic obstruction; focal interventricular septal hypertrophy (heart); vacuolization of myeloid series

+ + NA NA CAL macules; hypopigmented macules; microcephaly; cerebellar vermis hypoplasia

? ? ? ?** + ? NA Microcephaly; gingival hypertrophy

ular severe mental retardation, growth retardation, and spasticity, allow one to make a clinical distinction. It is important to exclude patients who have type II oculocutaneous albinism in conjunction with either the Prader-Willi syndrome (PWS) or Angelman syndrome (AS). This occurs when there is a mutation in one copy of the P gene in addition to a deletion of chromosome 15q11–q13 that involves both the P gene and either the PWS gene (paternal chromosome) or the AS gene (maternal chromosome) (Rinchik et al., 1993). Mental retardation is seen in patients with both PWS and AS (McKusick, 1994). Elejalde et al. in 1979 described the association of cutaneous

hypopigmentation (including silver hair) with profound mental retardation and hypotonia in three consanguineous families who had two and possibly four common ancestors (Elejalde et al., 1979). However, with the exception of myopia, there were no ocular abnormalities and the presence of abnormal melanosomes and lysosomes led to the designation “neuroectodermal melanolysosomal disease”. Patients with Griscelli syndrome (type 1) due to mutations in the MYO5A gene also develop severe developmental delay and these two entities may prove to be related. Phenylketonuria (PKU) is also characterized by a combination of hypopigmentation and neurologic dysfunction, but 629

CHAPTER 31

severe ocular anomalies are absent. In patients with OCSH, the lack of evidence for intrauterine infections as well as normal karyotypes and normal levels of plasma and urine amino acids, lysosomal enzymes, and urinary excretion of mucopolysaccharides allow the exclusion of other causes of mental retardation including PKU (Castle et al., 1989; Courtens et al., 1989; Cross et al., 1967; De Jong and Fryns, 1991; Fryns et al., 1988; Lerone et al., 1992; Ozkan et al., 1991; Passarge and Fuchs-Mecke, 1975; Patton et al., 1987; Preus et al., 1983; White et al., 1993). It is also important not to confuse OCSH with the oculocerebrocutaneous syndrome (Delleman-Oorthuys syndrome [164180]; McKusick, 1994). In the latter, patients develop orbital cysts and periorbital skin tags in addition to cerebral malformations.

Pathogenesis Mutations in several genes, e.g., the tyrosinase gene, the P gene, and the tyrosinase-related protein 1 (TYRP1) gene, can result in oculocutaneous albinism (types I, II, and III, respectively) (Spritz, 1994; Wildenberg et al., 1994) (see earlier section on Oculocutaneous Albinism). However, patients with these forms of albinism do not have the systemic abnormalities (e.g., severe mental retardation, spasticity, and ocular anomalies such as microphthalmia) that characterize OCSH. Presumably, the mutation(s) in OCSH affects the development of the central nervous system and eye as well as the pigmentary system. Of note, there is a disorder of hypopigmentation, Waardenburg’s syndrome type 2 (see Chapter 29), where the underlying mutations are in the human homolog of the mouse microphthalmia (mitf) gene (Tassabehji et al., 1994). The protein product is a basic-helix-loop-helix-leucine zipper transcription factor that can regulate melanocyte-specific transcription of the human tyrosinase gene (Yasumoto et al., 1994).

Treatment/Prognosis The goal is early diagnosis so that interventions such as corrective eyewear, special education, and physical therapy can be instituted. Products designed for the visually impaired should be made available to the patient and ophthalmologic surgical procedures may be required. An explanation of the inheritance pattern of autosomal recessive disorders should be provided to the parents via genetic counseling; to date, there are no reports of prenatal diagnosis. Additional information and support can be provided by NOAH, the National Organization for Albinism and Hypopigmentation (www.albinism.com).

De Jong, G., and J.P. Fryns. Oculocerebral syndrome with hypopigmentation (Cross syndrome): the mixed pattern of hair pigmentation as an important diagnostic sign. Genet. Couns. 2:151–155, 1991. Elejalde, B.R., J. Holguin, A. Valencia, E.F. Gilbert, J. Molina, G. Marin, and L.A. Arango. Mutations affecting pigmentation in man: I. Neuroectodermal melanolysosomal disease. Am. J. Med. Genet. 3:65–80, 1979. Fryns, J.P., A.M. Dereymaeker, G. Heremans, J. Marien, J. van Hauwaert, G. Turner, A. Hockey, and H. van den Berghe. Oculocerebral syndrome with hypopigmentation (Cross syndrome). Report of two siblings born to consanguineous parents. Clin. Genet. 34:81–84, 1988. Lerone, M., A. Pessagno, A. Taccone, G. Poggi, G. Romeo, and M.C. Silengo. Oculocerebral syndrome with hypopigmentation (Cross syndrome): report of a new case. Clin. Genet. 41:87–89, 1992. McKusick, V.A. Mendelian Inheritance in Man: Catalogs of Autosomal Dominant, Autosomal Recessive, and X-Linked Phenotypes, 11th edn. Baltimore: Johns Hopkins, 1994. Ozkan, H., E. Unsal, and G. Kose. Oculocerebral hypopigmentation syndrome (Cross syndrome). Turk. J. Pediatr. 33:247–252, 1991. Passarge, E., and S. Fuchs-Mecke. MM — Oculocerebral syndrome with hypopigmentation. Birth Defects Orig. Art. Ser. 11:466–467, 1975. Patton, M.A., M. Baraitser, A.H. Heagerty, and R.A. Eady. An oculocerebral hypopigmentation syndrome: a case report with clinical, histochemical, and ultrastructural findings. J. Med. Genet. 24: 118–122, 1987. Preus, M., F.C. Fraser, and F.W. Wiglesworth. An oculocerebral hypopigmentation syndrome. Journal de Genetique Humaine 31:323–328, 1983. Rinchik, E.M., S.J. Bultman, B. Horsthemke, S.T. Lee, K.M. Strunk, R.A. Spritz, K.M. Avidamo, M.T.C. Jong, and R.D. Nicholls. A gene for the mouse pink-eyed dilution locus and for human type II oculocutaneous albinism. Nature 361:72–76, 1993. Spritz, R.A. Molecular genetics of oculocutaneous albinism. Hum. Mol. Genet. 3:1469–1475, 1994. Tassabehji, M., V.E. Newton, and A.P. Read. Waardenburg syndrome type 2 caused by mutations in the human microphthalmia (MITF) gene. Nat. Genet. 8:251–255, 1994. Tezcan I., E. Demir, E. Asan, G. Kale, S.F. Muftuoglu, and E. Kotiloglu. A new case of oculocerebral hypopigmentation syndrome (Cross syndrome) with additional findings. Clin. Genet. 51:118– 121, 1997. White, C.P., M. Waldron, J.E. Jan, and J.E. Carter. Oculocerebral hypopigmentation syndrome associated with Bartter syndrome. Am. J. Med. Genet. 46:592–596, 1993. Wildenberg, S.C., R.E. Boissy, W.S. Oetting, J.P. Fryer, H. Zhao, and R.A. King. Identification of a mutation in the gene for tyrosinase related protein 1 (TRP1) associated with brown oculocutaneous albinism (OCA3). Am. J. Hum. Genet. 55 (Suppl.):A5(Abstr.), 1994. Witkop, C.J.J. Albinism. In: Advances in Human Genetics, H. Harris, and K. Hirschhorn (eds). New York: Plenum Press, 1971, pp. 61–142. Yasumoto, K.-I., K. Yokoyama, K. Shibata, Y. Tomita, and S. Shibahara. Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene. Mol. Cell. Biol. 14:8058–8070, 1994.

References Castle, D.J., T. Jenkins, and A.A. Shawinsky. The oculocerebral syndrome in association with generalized hypopigmentation. A case report. S. Afr. Med. J. 76:35–36, 1989. Courtens, W., W. Broeckx, M. Ledoux, and E. Vamos. Oculocerebral hypopigmentation syndrome (Cross syndrome) in a Gipsy child. Acta Paediatr. Scand. 78:806–810, 1989. Cross, H.E., V.A. McKusick, and W. Breen. A new oculocerebral syndrome with hypopigmentation. J. Pediatr. 70:398–406, 1967.

630

Tietz Syndrome Jean-Paul Ortonne

Historical Background In 1963, Tietz reported a family whose members had deaf-

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION

mutism, hypoplasia of the eyebrows, blue irides, and cutaneous hypomelanosis, termed by the author as albinism.

Clinical Features These patients exhibited generalized hypomelanosis and had light hair. The irides were blue, but there was no nystagmus, photophobia, or abnormalities of the fundi. Hypoplasia of the eyebrows was not associated with dystopia canthorum. The deafness was of the perceptive type.

Histology Histological study of a biopsy of hypomelanotic skin revealed complete lack of melanin. The pigmentary abnormality was not characterized further.

Differential Diagnosis Since this original report, no other cases have been reported. Reed et al. (1967) were able to re-examine several of these patients. Their clinical observations indicated that (1) the hair of the mother and her four sisters darkened with age; (2) a mother and her sons developed suntan; (3) the sibling had pronounced freckling and one had a black patch in the nuchal area; and (4) cosegregation of light hair and deafness was not observed in a blond female sibling. These findings differed significantly from the observations of Tietz, and Reed’s investigation concluded that the existence of this disorder was doubtful.

Pathogenesis This disorder, described in a six-generation pedigree (68 individuals), was observed in both males (n = 6) and females (n = 8). This suggests an autosomal dominant inheritance and complete penetrance.

References Reed, W. B., V. M. Stone, E. Boder, and L. Ziprkowski. Pigmentary disorders in association with congenital deafness. Arch. Dermatol. 95:176–186, 1967. Tietz, W. A syndrome of deaf-mutism associated with albinism showing dominant autosomal inheritance. Am. J. Hum. Genet. 15:259–264, 1963.

Kappa-Chain Deficiency Jean-Paul Ortonne Kappa-chain deficiency is a rare disorder characterized by a decreased concentration of immunoglobulins specifically with kappa light chain. White hair, extremely long white eyelashes, and very pale skin have been reported in one patient with kappa-chain deficiency with recurrent respiratory infections and diarrhea (Bernier et al., 1972).

Menkes’ Kinky Hair Syndrome Tanusin Ploysangam Menkes’ kinky hair syndrome was described first by Menkes

et al. in 1962. The term “kinky hair disease” was coined by O’Brien and Sampson in 1966. Defective copper absorption was demonstrated by Danks and coworkers (1972). “Steely hair disease,” “kinky hair disease,” and “trichopoliodystrophy” are synonyms for this syndrome. Kinky hair syndrome is an X-linked recessive multisystem disorder caused by inappropriate intracellular copper storage. It is characterized by abnormal hair, puffy facies, seizures, progressive cerebral degeneration, severe mental retardation, skeletal defects, episodic hypothermia, tortuous arteries, arterial rupture, arterial thrombosis, hypopigmentation, and early death.

Epidemiology The disease is rare. Because this is an X-linked disorder, it typically affects males, although a few females with the syndrome have been reported (Gerdes et al., 1990; Kapur et al., 1987). Its incidence is estimated to be approximately 1 in 35 000 to 1 in 254 000 live births (Tonnesen et al., 1991).

Clinical Manifestations Menkes’ disease can be divided into two subgroups: a severe form and a mild form (Danks, 1988; Gerdes et al., 1988). There are many pigmentary abnormalities. These include lightly pigmented hair and generalized or localized decreases in skin pigmentation. At birth, scalp hair may be normal or light-colored and sparse. By about 5 weeks to 5 months of age, the scalp hair becomes sparse and lusterless and acquires a lightly pigmented color. The characteristic hair shaft abnormality is called “pili torti.” It is characterized by wiry, twisted, coarse, kinky, brittle, and short hair shafts. Eyebrows may show similar changes. Other less frequently reported hair abnormalities include monilethrix and trichorrhexis nodosa. Of female carriers, 43% have pili torti, which may be used as a reliable diagnostic feature of the carrier state (Moore and Howell, 1985). The skin is pale, lax, and fat, with a doughy consistency. The face is pudgy-cheeked and lacks expressive movements. Many affected individuals have micrognathia. Others have seborrheic dermatitis of the scalp, face, and other areas. A high-arched palate, a single palmar crease, pectus excavatum, and talipes have been reported. The nails are dystrophic. Some dental abnormalities, such as enamel defects, delayed eruption, and biconical incisors, have been noted (Pallotta et al., 1989). At about 2 months of age, drowsiness and lethargy appear. Focal or generalized seizures are the most common neurologic presentation. Severe progressive neurologic deterioration, e.g., motor and mental retardation, as well as cerebral and cerebellar degeneration, are constant features, as are subdural hematomas. Impaired temperature regulation (hypothermia) and increased susceptibility to infection may be apparent in early infancy. The arteries are grossly tortuous and probably responsible for the subdural hematomas. Skeletal defects include wormian 631

CHAPTER 31

bones of the skull, metaphyseal spurring, and widening. Fractures are common.

Histology Skin biopsy specimens demonstrate lack of pigmentation in the hair papillae, follicles, and shafts. Blood vessels from numerous areas of the body show severe elastic fragmentation with marked intimal thickening. The brain is smaller than expected for age and shows widespread neuronal loss, myelin degeneration, deformities of Purkinje cells in the cerebellum, gliosis, and cysts, as well as a marked decrease in copper levels (Troost et al., 1982).

Pathogenesis Originally, the disease was thought to be caused by copper deficiency. However, it now appears to be a copper-storage disease, with inappropriate copper distribution within cells. Cellular accumulation of metallothionein, a copper-binding protein involved in cytoplasmic copper transport, may be the primary defect in this syndrome (Riordan and JolicoeurPaquet, 1982; Yazaki et al., 1983). Accumulation of copper occurs in fibroblasts and macrophages. Diminished intestinal copper absorption as well as a defect of copper transport and cellular metabolism cause a marked systemic copper deficiency that accounts for all pathologic features. The kinky hair is caused by an excess of free sulfhydryl groups with a decrease in copper-dependent disulfide bonds. Depigmentation of hair is due to low activity of tyrosinase, a copper-containing enzyme. The oxidative deamination of lysyl and hydroxylysyl residues necessary for the cross-linking of collagen and elastin is catalyzed by lysyl oxidase. This enzyme requires copper as a co-factor. It also appears that lysyl oxidase contains copper as an integral part of the active enzyme molecule. Copper deficiency leads to deficiency of lysyl oxidase and results in abnormal synthesis of collagen and elastic fibers (Peltonen et al., 1983; Pinnell and Martin, 1968; Royce and Steinmann, 1990). The conversion of tyrosine to dihydroxyphenylalanine (DOPA) and DOPA to DOPAquinone is catalyzed by a coppercontaining oxidase, i.e., tyrosinase. Decreased cutaneous pigmentation is due to low tyrosinase activity, most likely from a copper deficiency (Holstein et al., 1979). In addition, increased concentration of sulfhydryl groups that can bind copper results in decreased melanogenesis in the hair (Danks et al., 1972). Copper deficiency also results in impairment of the immune system (Prohaska and Lukasewycz, 1990). Female carriers show mild hair involvement and can sometimes be identified on the basis of increased copper uptake by fibroblast cultures. Currently, the defective gene in Menkes’ disease has been localized to chromosome Xql3.3 and predicted to encode a copper-binding P-type adenosine triphosphatase (ATPase) (Tumer et al., 1995; Vulpe et al., 1993). Prenatal diagnosis has been accomplished by detection of increased copper incorporation by cultured skin fibroblasts and amniotic fetal cells (Horn, 1981, 1983). 632

Laboratory Findings and Investigations Microscopically, the hair typically shows pili torti, trichorrhexis nodosa, trichoclasis, and trichoptilosis. Hair analysis reveals a ninefold increase in free sulfhydryl content and low copper levels. Some tissues (e.g., serum, liver, brain, and hair) have markedly decreased copper levels; other tissues (e.g., kidney, duodenum, spleen, pancreas, and fibroblasts) have normal or markedly increased levels of copper. Low plasma ceruloplasmin levels are also a constant finding. Heterozygotes may be detected by abnormal copper metabolism in skin fibroblasts. Radiologic examination reveals long bone metaphyseal spurring and diaphyseal periosteal reaction. Electroencephalographic examination reveals multifocal seizure discharges (White et al., 1993).

Criteria for Diagnosis The combination of hair findings, seizures, severe neurologic degeneration, and bone abnormalities in a male infant with low serum copper and ceruloplasmin levels as well as increased copper uptake in fibroblasts establishes the diagnosis (Tonnesen and Horn, 1989).

Differential Diagnosis The bone changes, psychomotor retardation, and tendency for subdural hematomas might be confused with the “battered child syndrome.” However, the pigmentary changes, low serum copper and ceruloplasmin levels, as well as the characteristic kinky hair are diagnostic for Menkes’ kinky hair syndrome. Hair shaft abnormalities in Menkes’ kinky hair syndrome also are seen in argininosuccinic aciduria, Biornstad’s syndrome, and trichothiodystrophy. However, low copper and ceruloplasmin levels are found only in Menkes’ kinky hair syndrome.

Animal Models Genetically copper-deficient and mottled mutant mice (Camakaris et al., 1983; Levinson et al., 1994; Packman et al., 1984), macular mutant mice (Kumode et al., 1994), and brindled mice (Palida and Ettinger, 1991; Yoshimura et al., 1995) have been used as models for Menkes’ kinky hair syndrome.

Treatment To date, there have been no effective treatments. Copper replacement therapy has not proved useful (Garnica, 1984). Genetic counseling is imperative. Copper histidine therapy may be effective if started early (Sarkar et al., 1993; Sherwood et al., 1989). Oral d-penicillamine may be of benefit (Nadal and Baerlocher, 1988).

Prognosis Those who have the severe form usually die before 5 years of age due to failure to thrive. In the mild form, patients usually survive to adolescence (Gerdes et al., 1988; Sanders et al., 1988) or adulthood (Westman et al., 1988).

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION

References Camakaris, J., M. Phillips, D. M. Danks, R. Brown, and T. Stevenson. Mutations in humans and animals which affect copper metabolism. J. Inherit. Metab. Dis. 6 (Suppl.):44–50, 1983. Danks, D. M. The mild form of Menkes disease: progress report on the original case. Am. J. Med. Genet. 30:859–864, 1988. Danks, D. M., P. E. Campbell, J. Walker-Smith, B. J. Stevens, J. M. Gillespie, J. Blomfield, and B. Tuner. Menkes kinky-hair syndrome. Lancet 1:1100–1103, 1972. Garnica, A. D. The failure of parenteral copper therapy in Menkes kinky hair syndrome. Eur. J. Pediatr. 142:98–102, 1984. Gerdes, A. M., T. Tonnesen, N. Horn, T. Grisar, W. Marg, A. Muller, R. Reinsch, N. W. Barton, P. Guiraud, A. Joannard, M. J. Richard, and F. Guttler. Clinical expression of Menkes syndrome in females. Clin. Genet. 38:452–459, 1990. Gerdes, A. M., T. Tonnesen, E. Pergament, C. Sander, K. E. Baerlocher, R. Wartha, F. Guttler, and N. Horn. Variability in clinical expression of Menkes syndrome. Eur. J. Pediatr. 148:132–135, 1988. Holstein, T. J., R. Q. Fung, W. C. J. Quevedo, and T. C. Bienieki. Effect of altered copper metabolism induced by mottled alleles and diet on mouse tyrosinase. Proc. Soc. Exp. Biol. Med. 162:264–268, 1979. Horn, N. Menkes’ X-linked disease: prenatal diagnosis of hemizygous males and heterozygous females. Prenat. Diagn. 1:107–120, 1981. Horn, N. Menkes’ X-linked disease: prenatal diagnosis and carrier detection. J. Inherit. Metab. Dis. 6 (Suppl.):59–62, 1983. Kapur, S., J. V. Higgins, K. Delp, and B. Rogers. Menkes syndrome in a girl with X-autosome translocation. Am. J. Med. Genet. 26:503–510, 1987. Kumode, M., T. Yamano, and M. Shimada. Histochemical study of mitochondrial enzymes in cerebellar cortex of macular mutant mouse, a model of Menkes kinky hair disease. Acta Neuropathol. (Berl.) 87:313–316, 1994. Levinson, B., C. Vulpe, B. Elder, C. Martin, F. Verley, S. Packman, and J. Gitschier. The mottled gene is the mouse homologue of the Menkes disease gene. Nat. Genet. 6:369–373, 1994. Menkes, J. H., M. Alter, G. K. Steigleder, D. R. Weakley, and J. H. Sung. A sex-linked recessive disorder with retardation of growth, peculiar hair, and focal cerebral and cerebellar degeneration. Pediatrics 29:764–779, 1962. Moore, C. M., and R. R. Howell. Ectodermal manifestations in Menkes disease. Clin. Genet. 28:532–540, 1985. Nadal, D., and K. Baerlocher. Menkes’ disease: long-term treatment with copper and d-penicillamine. Eur. J. Pediatr. 147:621–625, 1988. O’Brien, J. S., and E. L. Sampson. Kinky hair disease: II. Biochemical studies. J. Neuropathol. Exp. Neurol. 25:523–530, 1966. Packman, S., P. Clin, and C. O’Toole. Copper utilization in cultured fibroblasts of the mottled mouse, an animal model for Menkes’ kinky hair syndrome. J. Inherit. Metab. Dis. 7:168–170, 1984. Palida, F. A., and M. J. Ettinger. Identification of proteins involved in intracellular copper metabolism. Low levels of an approximately 48-kDa copper-binding protein in the brindled mouse model of Menkes disease. J. Biol. Chem. 266:4586–4592, 1991. Pallotta, R., F. Del Rossa, S. Domizio, G. Carlone, and A. Petrucci. Enamel defects in a case of Menkes’ syndrome. Acta Stomatol. Belg. 86:33–36, 1989. Pastural, E., F. J. Barrat, R. Dufourtq-Lagelouse, S. Certain, O. Sajal, N. Jabado, R. Seger, C. Griscelli, A. Fischer, and G. de Saint Basile. Griselli disease maps to chromasome 15q21 and is associated with mutations in the Myosin-Va gene. Nature Genet. 16:289, 1997. Peltonen, L., H. Kuivaniemi, A. Palotie, N. Horn, I. Kaitila, and K. I. Kivirikko. Alterations in copper and collagen metabolism in the Menkes syndrome and a new subtype of the Ehlers-Danlos syndrome. Biochemistry 22:6156–6163, 1983. Pinnell, S. R., and G. R. Martin. The cross-linking of collagen and

elastin: enzymatic conversion of lysine in peptide linkage to alphaaminoadipic-delta-semialdehyde (allysne) by an extract from bone. Proc. Natl. Acad. Sci. U. S. A. 61:708–716, 1968. Prohaska, J. R., and O. A. Lukasewycz. Effects of copper deficiency on the immune system. Adv. Exp. Med. Biol. 262:123–143, 1990. Riordan, J. R., and L. Jolicoeur-Paquet. Metallothionein accumulation may account for intracellular copper retention in Menkes’ disease. J. Biol. Chem. 257:4639–4645, 1982. Royce, P. M., and B. Steinmann. Markedly reduced activity of lysyl oxidase in skin and aorta from a patient with Menkes’ disease showing unusually severe connective tissue manifestations. Pediatr. Res. 28:137–141, 1990. Sanders, C., H. Niederhoff, and N. Horn. Life-span and Menkes kinky hair syndrome: report of a 13-year course of this disease. Clin. Genet. 33:228–233, 1988. Sarkar, B., K. Lingertat-Walsh, and J. T. Clarke. Copper-histidine therapy for Menkes disease. J. Pediatr. 123:828–830, 1993. Sherwood, G., B. Sarkar, and A. S. Kortsak. Copper histidinate therapy in Menkes’ disease: prevention of progressive neurodegeneration. J. Inherit. Metab. Dis. 12 (Suppl.):393–396, 1989. Tonnesen, T., and N. Horn. Prenatal and postnatal diagnosis of Menkes disease, an inherited disorder of copper metabolism. J. Inherit. Metab. Dis. 12 (Suppl.):207–214, 1989. Tonnesen, T., W. J. Kleijer, and N. Horn. Incidence of Menkes disease. Hum. Genet. 86:408–410, 1991. Troost, D., A. Van Rossum, W. Straks, and J. Willemse. Menkes’ kinky hair disease. II. A clinicopathological report of three cases. Brain Dev. 4:115–126, 1982. Tumer, Z., B. Vural, T. Tonnesen, J. Chelly, A. P. Monaco, and N. Horn. Characterization of the exon structure of the Menkes disease gene using vectorette PCR. Genomics 26:437–442, 1995. Vulpe, C., B. Levinson, S. Whitney, S. Packman, and J. Gitschier. Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nat. Genet. 3:7–13, 1993. Westman, J. A., D. C. Richardson, O. M. Rennert, and G. Morrow III. Atypical Menkes steely hair disease. Am. J. Med. Genet. 30:853–858, 1988. White, S. R., K. Reese, S. Sato, and S. G. Kaler. Spectrum of EEG findings in Menkes disease. Electroencephalogr. Clin. Neurophysiol. 87:57–61, 1993. Yazaki, M., Y. Wada, Y. Kojima, A. Tanaka, G. Isshiki, N. Kawamura, and M. Hirose. Copper-binding proteins in the liver and kidney from the patients with Menkes’ kinky hair disease. Tohoku J. Exp. Med. 139:97–102, 1983. Yoshimura, N., K. Kida, S. Usutani, and M. Nishimura. Histochemical localization of copper in various organs of brindled mice after copper therapy. Pathol. Int. 45:10–18, 1995.

Phenylketonuria Allan D. Mineroff Phenylketonuria is an autosomal recessive disease resulting from impaired conversion of phenylalanine to tyrosine. It is the most common and clinically important of the hyperphenylalaninemias, affecting one per 18 000 live births (Newbold, 1973). Phenylketonuria is characterized by a virtual absence of phenylalanine hydroxylase activity and an elevation of plasma concentration of the amino acid phenylalanine and its byproducts. Untreated phenylketonuria usually results in mental retardation, oculocutaneous pigmentary dilution (blond hair, blue eyes, fair skin), photosensitivity, dermatitis, unpleasant 633

CHAPTER 31

Fig. 31.14. An infant with phenylketonuria with typical fair skin and blue eyes. See also Plate 31.11, pp. 494–495.

Fig. 31.16. Brittle hair from a child with biopterin deficiency. This Arab child has fair skin and hair, suggestive of phenylketonuria. However, her biochemical defect is a lack of biopterin, which produces a phenotype that resembles the latter disorder. [Courtesy of Dr. Robert Kellum.] See also Plate 31.13, pp. 494–495.

“mousy” body odor, seizures, psychosis, hyperreflexia, and skeletal changes.

Clinical Findings

Fig. 31.15. An Arab child with an inborn error of biopterin synthesis. The child resembles an infant with phenylketonuria but the disorder is not ameliorated by a phenylalanine-free diet. [Courtesy Dr. Robert Kellum.] See also Plate 31.12, pp. 494–495.

634

Of affected individuals, 90% have fair skin and blue eyes (Fig. 31.14). Hair color is usually blond but occasionally dark brown. These changes may be subtle, and comparison of the infant with unaffected family members may be necessary to detect them. Darkening of hair color is noted in patients on low phenylalanine diets or with tyrosine supplementation. The hair becomes lighter when dietary restriction is stopped (Fitzpatrick and Mihm, 1971). Diffuse hypopigmentation of the skin has been described. These patients tend to sunburn easily, though some individuals are capable of tanning. Eczematous dermatitis and indurated scherodermoid have been reported in up to half of patients. Bechelli and coworkers (1978) described two patients with hundreds of pigmented pinpoint or slightly larger patches in addition to fair skin and fair hair. A characteristic musty (sometimes described as mouse-like) odor occurs in these patients and often suggests the diagnosis. The odor is believed to be caused by a block in the conversion of phenylalanine to tyrosine, with resultant excess synthesis of minor phenylalanine by-products. These products (such as phenylacetic acid, phenylpyruvic acid, acetylglutamine, and phenylacetaldehyde) are found in urine and sweat of these patients. Serum phenylalanine levels begin to rise by the third to fourth day of life. Within 3–4 months deterioration of brain function appears and is manifested by bizarre behavior, seizures, and abnormal electroencephalographic findings. If dietary intervention is not incorporated, brain damage progresses. Most patients not given a low phenylalanine diet will have IQ values below 20.

GENETIC HYPOMELANOSES: GENERALIZED HYPOPIGMENTATION

Pathogenesis There are four proposed mechanisms to explain the hypopigmentation in phenylketonuria: 1. Competitive inhibition of the binding of tyrosine to tyrosinase by excess phenylalanine or one of its metabolites (Rosenberg, 1991). 2. Decreased amount or absence of tyrosine, the substrate for the synthesis of melanin (Rosenberg, 1991). 3. Absent or decreased tyrosinase enzyme. Tyrosinase is necessary to convert tyrosine to DOPA and DOPA to DOPAquinone, which is converted to melanin. 4. A primary effect of phenylalanine hydroxylase deficiency (Bolognia and Pawelek, 1988).

Diagnosis Many countries have mandatory newborn screening programs to identify infants with phenylketonuria using the Guthrie inhibition assay test. Prenatal diagnosis is possible in the majority of families carrying the gene for phenylketonuria. Cells from amniotic fluid or the chorionic villi can be used to detect an affected fetus.

Differential Diagnosis Children with an inborn error of biopterin synthesis phenotypically are very similar to children with phenylketonuria

(Fig. 31.15). They have inappropriately fair skin and hair, but urine tests for phenylalanine metabolites are negative. Hair is brittle (Fig. 31.16).

Treatment Prevention of mental retardation in infants afflicted with phenylketonuria can be achieved by restricting dietary intake of phenylalanine.

References Bechelli, L. M., R. P. Goncalves, A. M. Tanaka, and P. M. Pagnano. Cutaneous dyschromia in three cases of phenylketonuria. Ann. Dermatol. Venereol. 105:165–173, 1978. Bolognia, J. L., and J. M. Pawelek. Biology of hypopigmentation. J. Am. Acad. Dermatol. 19:217–255, 1988. Fitzpatrick, T. B., and M. C. Mihm. Abnormalities of the melanin pigmentary system. In: Dermatology in General Medicine, T. B. Fitzpatrick, K. A. Arndt, W. H. Clark, Jr., A. Z. Eisen, E. J. Van Scott, and J. H. Vaughan (eds.). New York: McGraw-Hill, 1971, pp. 1606–1609. Newbold, P. C. H. The skin in genetically-controlled metabolic disorders. J. Med. Genet. 10:101–111, 1973. Rosenberg, L. Inherited disorders of amino acid metabolism. In: Harrison’s Principles of Internal Medicine, 12th ed., J. D. Wilson, E. Braunwald, K. J. Isselbacher, R. G. Petersdorf, J. B. Martin, A. S. Fauci, and R. K. Root (eds.). New York: McGraw-Hill, 1991, pp. 1868–1875.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

32

Genetic Hypomelanoses: Localized Hypopigmentation Sections “Hypomelanosis of Ito” and Mosaicism Wolfgang Küster and Rudolf Happle Focal Dermal Hypoplasia James J. Nordlund Hypomelanosis with Punctate Keratosis of the Palms and Soles Jean L. Bolognia Darier–White Disease (Keratosis Follicularis) Jean L. Bolognia Nevus Depigmentosus Stella D. Calobrisi Tuberous Sclerosis Complex Pranav B. Sheth

“Hypomelanosis of Ito” and Mosaicism Wolfgang Küster and Rudolf Happle

Introduction During the past 25 years, so-called hypomelanosis of Ito (HI) has been described as a distinct multisystem birth defect or, more specifically, as a neurocutaneous syndrome. The main purpose of this chapter is to provide evidence that this syndrome does not exist. Rather, it will be shown that “hypomelanosis of Ito” or “incontinentia pigmenti achromians” represents a cutaneous marker of many different states of genetic mosaicism that may or may not involve, in addition, internal organs such as the brain, the eyes, or the bones. On the cytogenetic level many different chromosomal abnormalities confirm that this pigmentary disorder is caused by genetic mosaicism.

Historical Background In 1952 the Japanese dermatologist Minor Ito described a 21year-old woman who had a widespread, symmetric pattern of depigmented whorls and streaks that had its onset at the age of 5 years. Ito interpreted the anomaly as a case of nevus depigmentosus systematicus bilateralis and called it incontinentia pigmenti achromians, because the distribution of the depigmented lesions was similar to that of the hyperpigmented streaks of incontinentia pigmenti of the Bloch–Sulzberger type. Because the pigmentary lesions are more easily recognizable in darker skin the first cases that were published were of patients of Japanese, African American, and Hispanic origin. Paradoxically, in Europe, HI developed to a more generally recognized “entity” at a time when it became evident, on the basis of cytogenetic studies, that this entity did not exist.

Terminology The terms HI, incontinentia pigmenti achromians, pigmentary dysplasia, and pigmentary mosaicism are synonyms of the same cutaneous symptoms and are used interchangeably in the 636

literature. The name “Ito syndrome” is erroneous and should be avoided, because the term syndrome is defined as a pattern of multiple anomalies being pathogenetically related and reflecting a common cause, and is therefore not applicable to HI. In our opinion the best designation would be “pigmentary mosaicism” or “pigmentary mosaicism of the Ito type,” designating a particular cutaneous phenomenon within the clinical spectrum of pigmentary mosaicism.

Inclusion Criteria In most cases of HI, the skin changes are interpreted as hypopigmented lesions, which are particularly evident when large areas of obviously uninvolved skin are present [see illustrations presented by Bhushan et al. (1989), and by Golden and Kaplan (1986)]. HI is easier to recognize against a background of darker skin, which probably explains why only about one out of five cases have been reported in white people (Moss and Burn, 1988). However, the designation “hypomelanosis of Ito” appears to be less appropriate because it is often difficult to decide whether a hypopigmentation or hyperpigmentation is present (cases 4 and 5 in Ohashi et al., 1992; Metzker et al., 1982; Wulfsberg et al., 1991), especially when the lesions are extensive and intermingled with the normal component of skin. Patients may appear hyperpigmented on clinical grounds, but turn out to be hypopigmented on histopathologic criteria (Happle et al., 1976; Meinecke et al., 1985). It is certainly unjustified to decide merely on clinical examination that the larger area of skin pigmentation should be the normal component of skin. A distinction between hypomelanosis and hypermelanosis appears difficult and somewhat artificial in many cases of HI, and for this reason the neutral terms “pigmentary dysplasia” or “pigmentary mosaicism” appear to be more appropriate. At present unresolved is the question whether skin patterns other than those following Blaschko lines should qualify for the diagnosis of HI (for a classification of such patterns, see Happle, 1993b). Ohashi et al. (1992) presented a patient with a 46,XX/47,XX,+mar mosaic and a phylloid (i.e. leaflike)

GENETIC HYPOMELANOSES: LOCALIZED HYPOPIGMENTATION

32.1

32.2

Figs. 32.1 and 32.2. Systematized linear and patchy depigmented lesions on the trunk, distributed in a pattern following the lines of Blaschko (see also Plate 32.1, pp. 494–495).

pattern and a patient with a 46,XX/47,XX,+14 mosaic and many hypopigmented macules and only occasional streaks. As these patterns likewise appear to represent a state of mosaicism, it seems justified to include these non-Blaschko cases in the group of HI. We propose that only skin patterns following Blaschko lines should qualify for the diagnosis of HI [for a classification of such patterns, see Happle (1993b)]. For example, phylloid hypomelanosis (see Fig. 32.7) is an example of pigmentary mosaicism quite different from HI (Happle, 2000) (Figs 32.1 and 32.2). Cases showing skin changes described as systematized nevus depigmentosus (Fukai et al., 1993), or pigmentary disturbances presenting a “zosteriform” or “dermatomal” pattern (Metzker et al., 1982), or an arrangement in whorls and streaks (Cambazard et al., 1986), should likewise be included into the group of pigmentary mosaicism.

Exclusion Criteria HI should be distinguished from incontinentia pigmenti of the Bloch–Sulzberger type (IP) (see Chapter 47). In the past, the two phenotypes have often been confused (Happle, 1998). IP shares

with HI the streaky pigmentary changes and the frequent occurrence of extracutaneous abnormalities. However, IP is characterized by a dynamic course of distinct cutaneous lesions which appear successively soon after birth. The first stage is characterized by a linear dermatitis with vesicles and erythema, the second stage shows linear verrucous lesions, and the third stage is characterized by hyperpigmented streaks (Carney, 1976; Mosher et al., 1993). As a fourth stage, linear depigmented lesions may develop. In contrast, in HI the hypopigmented areas are either recognized at birth or become visible during the neonatal period or early childhood without preceding inflammatory or verrucous changes (Gordon, 1994). In addition, as described below, the histopathologic features are different in the HI groups and IP. Cases of phylloid hypomelanosis were excluded because the pattern of arrangement of skin lesions differs from the system of Blaschko lines (Happle, 2002). Furthermore, the various states of human chimerism (Findlay and Moores, 1980) should not be categorized as belonging to the group of HI because in a chimera neither the dark nor the light component of skin does represent a cutaneous disease. Conversely, both components should be taken as normal skin. 637

CHAPTER 32

Fig. 32.3. Pigmentary anomalies in a linear distribution on the arm. The depigmented lesions take up a larger area, but they are the pathologic component of the skin.

Fig. 32.4. Close-up view illustrating the swirling pattern of hypopigmentation (see also Plate 32.2, pp. 494–495).

As a further differential diagnosis the hypomelanotic macules of tuberous sclerosis should not be confused with HI. These macules are the earliest manifestation of tuberous sclerosis. They are usually multiple, irregularly bordered and frequently have the configuration of an ash-leaf. Other acquired depigmented lesions caused by endocrine, chemical or nutritional factors, or by post inflammatory lesions and infectious diseases usually do not constitute a problem in differential diagnosis.

Associated Extracutaneous Anomalies

Cutaneous Symptoms The pigmentary lesions in HI are either visible at birth or become recognizable during the neonatal period or early childhood. There are no signs of inflammatory or verrucous changes. The typical phenotype shows hypopigmented areas consisting of bilateral or unilateral streaks corresponding to the lines of Blaschko (Figs 32.1–32.4). However, other types of pigmentary pattern such as a checkerboard arrangement have likewise been described [Metzker et al. (1982), own observation]. In these cases alternating squares of pigmentary anomalies are observed, arranged either unilaterally or bilaterally. 638

HI is often associated with defects of the nervous system, the eyes, and the bones. It has been estimated that about 75% of the patients have extracutaneous abnormalities. Abnormalities of the central nervous system may include seizures, mental and motor retardation, microcephaly, hypotonia, hyperkinesia, ataxia, and deafness (Battistella et al., 1990; Bhushan et al., 1989; Esquivel et al., 1991; Golden and Kaplan, 1986; Jelinek et al., 1973). Ophthalmologic abnormalities consist of ptosis, nonclosure of the upper lid, symblepharon, dacryostenosis, strabismus, nystagmus, corneal opacification, cataracts, myopia, amblyopia, microphthalmia, iridal heterochromia, scleral melanosis, patchy hypopigmented fundi that are increasingly striated in the periphery, and retinal degeneration (Amon et al., 1990; Jelinek et al., 1973; Rott et al., 1990). Furthermore, enamel changes and other dental defects such as hamartomatous dental cusps have been observed (Happle and Vakilzadeh, 1982; Meinecke et al., 1985). Skeletal defects including short stature, pectus carinatum or excavatum, scoliosis, syndactyly, polydactyly, brachydactyly, clinodactyly, asymmetry of limbs, and facial asymmetry have been reported in HI (Jelinek et al., 1973; Ritter et al., 1990; Takematsu et

GENETIC HYPOMELANOSES: LOCALIZED HYPOPIGMENTATION

al., 1983). The most frequent anomalies are scoliosis, thoracic deformity, and finger anomalies. Many reviews have presented the percentages of associated extracutaneous anomalies in so-called HI (Buzas et al., 1981; Griebel et al., 1989; Ruiz-Maldonado et al., 1992; Takematsu et al., 1983). However, such data are meaningless because there is no disease entity that can be considered in this way. Moreover, the interest of the authors’ special field of activity has considerably biased the reports of such associations. For this reason we cannot indicate an “up to 60%” or “approximately 75%” association with, for example muscular hypotonia or scoliosis.

Cytogenetic Evidence for Heterogeneity It is clear from Table 32.1 that in HI karyotypic abnormalities of many different chromosomes are found, which constitutes a conclusive argument against the notion that HI is an entity. Why are so many different chromosomal abnormalities causing a similar phenotype? An explanation may be that skin color is under the control of many different genes. About 60 have been found in the mouse (Silvers, 1979). The cases reviewed in Table 32.1 are characterized by the presence of different cell lines resulting in mosaicism. In some cases, cytogenetic abnormalities of one of the X-chromosomes in female patients are found, which according to the Lyon effect of X-inactivation results in functional X-chromosome mosaicism (Happle, 1993a). Often the state of mosaicism can be demonstrated only in fibroblasts but not in peripheral lymphocytes (Sybert, 1994). Apparently some chromosomal areas within the human genome are more frequently involved than other regions, for example the short arm of the Xchromosome, the short arm of chromosome 12, and chromosome 18 as a trisomy. Additionally, several cases of triploidy were reported (Table 32.1) (Sybert, 1994). In those cases where no mosaicism could be demonstrated by cytogenetic examination, it seems reasonable to assume that either various forms of genetic mosaicism are present at the molecular level, or that a minor cytogenetic aberration may have gone unnoticed. Apparently, the borderlines between the differently pigmented skin areas correspond to division lines between the different clones of a developing embryo. In those cases where skin fibroblasts from dark and light skin were analyzed, the dichotomy of karyotypes did not follow the pigmentation borders, because the distribution of skin fibroblasts does not correspond to the borderlines of pigmentary mosaicism (Donnai et al., 1988; Findlay and Moores, 1980; Flannery, 1990; Flannery et al., 1985; Ohashi et al., 1992; Thomas et al., 1989). There is only one report on chromosomal analysis of keratinocytes, demonstrating in two patients that the dark skin showed one karyotype and light skin contained another one (Moss et al., 1993). So far, the karyotypes of melanocytes have not been investigated in HI due to the difficulties in establishing melanocyte cultures.

Phenotypic Evidence for Heterogeneity Sweat testing in patients with HI revealed in some cases a

Fig. 32.5. Asymmetrically distributed pigmentary lesions with linear depigmentation on the left side of the back (see also Plate 32.3, pp. 494–495).

decreased sweat production, corresponding to the hypopigmented areas (Ito, 1952; Steijlen and Happle, 1990) (Figs 32.5 and 32.6). In other cases sweating was normal. Steijlen and Happle (1990) found hypohidrosis in four, and no disturbance of sweating in two of six patients. Further reports concerning three cases showing normal sweating (De Rijcke et al., 1987; Rubin, 1972), and one case presenting with virtual absence of sweating and absent sweat glands in a skin biopsy (Moss and Burn, 1988) constitute an additional clinical argument in favor of the heterogeneity of HI.

Histopathologic Features of the Skin in HI The histopathologic features of HI are nonspecific but may be of value in the differentiation from incontinentia pigmenti (IP). In HI, there are no changes in the epidermis or dermis in HE stained biopsies (Jelinek et al., 1973), and, in particular, inflammatory changes of the epidermis and melanin deposition in dermal macrophages, representing characteristic features of IP, are lacking. Histopathological changes that are described in some reports on HI are rounded melanocytes with reduced or absent dendrites visible on dopa stain or by electron microscopical examination (Grosshans et al., 1971; 639

CHAPTER 32

Happle et al., 1976; Klug et al., 1982; Meinecke et al., 1985; Nordlund et al., 1977; Saxena et al., 1989), but these findings are by no means a “characteristic feature” of HI. Morohashi et al. (1977) described an abnormally increased number of nerve endings in contact with melanocytes in the skin of patients with vitiligo, Vogt–Koyanagi–Harada syndrome, and HI. The significance of this finding is unclear. Most likely, loss of dendrites represents a nonspecific, stereotypic reaction of melanocytes. Ultrastructural examination has shown a depigmentation including a decrease in the number of melanocytes and a quantitative insufficiency of pigment production. Premelanosomes may be scarce and melanosomes may be small and present in reduced numbers (Stoebner and Grosshans, 1970). These findings do not represent a characteristic criterion and are of limited diagnostic value although they are not observed in other depigmented lesions such as vitiligo or albinism.

Melanin Composition in some Forms of Pigmentary Mosaicism of the Ito Type In one report, the melanin composition has been determined in normal and hypopigmented skin of HI. The content of eumelanin, but not of pheomelanin, was reduced in the hypopigmented areas, suggesting a disturbance in eumelanin synthesis (Fukai et al., 1994).

The Question of Inheritance In the overwhelming number of cases, HI is a sporadic event with no siblings or parents affected. This strongly suggests that these phenotypes arise de novo as a result of a postzygotic

Fig. 32.6. The same patient as in Fig. 32.5 after performing a sweat test using the method described by Minor reveals anhidrosis in the depigmented areas, whereas the hyperpigmented normal component of the skin shows a normal sweating [observation by Steijlen & Happle (1990)] (see also Plate 32.4, pp. 494–495).

Table 32.1. Cytogenetic findings in pigmentary mosaicism of the Ito type. Karyotype

L

46,XX,t(2;8)(q37.2;p21.1) 46,XX,rea(2;10) (L) 46,XX,rea(2;10)/47,XX,+22(SF) 46,XY,del(2)(q32.2;q33.1) 46,XY,9+,2q+ 46,XY,dup(3)(p21.3;pter)/46,XY 46,XX/46,XX,4p+ 46,XY/47,XY,+7 46,XX/47,XX,+7 46,XY/47,XY,+7 45,XY,-7,-15,+der(7)(7;15)t(q34;q13) /46,XY 46,XY/47,XY,+8q-/45,XY,-18/45,X 46,XX,r(10) 46,XY/47,XY,+i(12p) 46,XX/47,XX,+i(12p) 46,XY/47,XY,+i(12p) 46,XX/46,XX,-13, +mar 46,XX/46,XX,del(13)(q11)

+ + + + + +

F

+ +

-

+ + +

-

-

+ + + +

+ + +

-

+ + + + +

-

K

+

+

Reference Miller and Parker (1985) Wertelecki et al. (1986) Glass et al. (1989) Ruiz-Maldonado et al. (1992) Horn et al. (2002) Thomas et al. (1989) Jenkins et al. (1993) Moss et al. (1993) Kivirikko et al. (2002) Pellegrino et al. (1995) Åkefeldt and Gillberg (1991) Sybert et al. (1990) Pallister et al. (1977) Pallister et al. (1977) Sybert et al. (1990) Moss et al. (1993) Sybert et al. (1990) Table continued on following page

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Table 32.1. Continued. Karyotype

L

F

45,XY,der(14q;21q)/46,XY,der(14q;21q),+mar 46,XX,r(14)/45,XX,-14/46,XX,r(14),21ph+/46,XX,-14, der(14;14),21ph+/45,XX,-14, 21ph+/46,XX,21ph+ 46,XX,-14,+i(14)(p11),21s+/46,XX,r(14) (p11q32),21s+/45,XX,-14,21s+/46,XX,21s+ 46,XX,del(15q) 46,XX/46,XX,del15q1 46,XX/46,XX,-15,der(14;15) 46,XY,-15,+r(15q;15q)mat(15q13KcenK15q26) 45,XX,der(15;22)/46,XX 46,XY/47,XY,+18 46,XY/47,XY,+18 46,XX/47XX,+18 46,XY/47,XY,+18 46,XY/47,XY,+18 46,XX/47,XX,+18 46,XY/47,XY,+18 46,XX/47,XX,+18 47,XX,+18(L)46,XX/47,XX,+18/47,XX,+13(SF) 46,XY/92,XXYY/47,XY,+18 46,XX/,47,XX,+mar (18) 46,XX/46,XX,18p46,XX,del(18)(p11.23Kpter)/46,XX,idic(18) (p11.23)/46,XX,-18,+r(18) 46,XY/47,XY,+20 46,XX/47,XX,+20 46,XX/47,XX,+20 46,XY/46,XY,r(22) 46,XX/47,XX,+G 45,X/46,XY 45,X/46,XY 45,X/46,XX 45,X (L); 46,XY/45,X (SF) 46,XX/47,XX,+r(X)(p11;q13) 45,X/46,XX/46,Xr(X) 45,X/46,Xr(X) 45,X/46,X,r(Y) 45,X/46,X,+r 47,XYY/48,XYY,+8 46,X,t(X;9)(p11;q34) 46,X,t(X;9)(p11.21;q33.2) 46,XX,del(X)(q21),inv(9)/45,X,inv(9)(p13q21) 46,X,t(X;10)(p11;q11)mat 46,X,t(X,15) 46,XX,t(X;17)(q13;p13) 46,X,t(X;17)(p11.2;p11.2) 46,X,t(X;18)(p11;q23) 46,X,t(X;22)(q11.2;q13.3) 46,XY/69,XXY 46,XX/69,XXX 46,XX/69,XXX 46,XX/69,XXX 46,XX/69,XXX 46,XX/92,XXXX 46,XX/69,XXX/92,XXXX 46,XX/69,XXX/69,XXX,22p+ 46,XY/47,XY,+r 46,XY/47,XY,+mar 46,XX/47,XX,+mar 46,XX,/47,XX,+mar

+ +

+

+ + + + + + + + + + + + + + + + + + + +

K

Ishikawa et al. (1985) Thomas et al. (1989)

Cantú et al. (1989) Amon et al. (1990) Turleau et al. (1986) Fujimoto et al. (1985) Neri et al. (1983) Sybert et al. (1990) Ohashi et al. (1992) Chemke et al. (1983) Ohashi et al. (1992) Sybert et al. (1990) Chitayat et al. (1990) Murano et al. (1991) Murano et al. (1991) Grazia et al. (1993) Wilson et al. (1983) Atnip and Summitt (1971) Schüler et al.(1992) Thomas et al. (1989) Bocian et al. (1993)

+

+ -

+ + + + + + + + + + + -

+ + +

+

-

+

+ + +

-

+ + + -

+ + + + + +

+ + + + -

+ +

+ + + + + --

+ + + + + + + +

+ -

+

+

+ + + + + +

Reference

-

Warren et al. (2001) Baty et al. (2001), case 1 Baty et al. (2001), case 2 Ritter et al. (1990) Wertelecki et al. (1986) Thomas et al. (1989) Moss et al. (1993) Åkefeldt and Gillberg (1991) Thomas et al. (1989) Koecker-Korus (1996) Rott et al. (1986) Ruiz-Maldonado et al. (1992) Thomas et al. (1989) Sybert et al. (1990) Thomas et al. (1989) Gilgenkrantz et al. (1985) Hodgson et al. (1985) Thomas et al. (1989) Koiffmann et al. (1993) Ruiz-Maldonado et al. (1992) Steichen-Gersdorf et al. (1993) Hodgson et al. (1985) Lungarotti et al. (1991) Sybert et al. (1990) Sybert et al. (1990) Tharapel et al. (1983) Wulfsberg et al. (1991) Donnai et al. (1988), case 1 Donnai et al. (1988), case 2 Leonhard and Tomkins (2002) Vormittag et al. (1992) Daubeney et al. (1993) Rott et al. (1990) Hersh et al.(1983) Ohashi et al. (1992) Du Bois (1992)

L, peripheral blood lymphocytes; F, skin fibroblasts; K, keratinocytes.

641

CHAPTER 32

mutation. The mutation can survive only in a mosaic and is lethal when transmitted to the offspring, because of lacking admixture of normal cell clones (Happle, 1993a). However, one well-documented case of inheritance of HI was reported by Grosshans et al. (1971). Three sisters showed clinical and histopathologic cutaneous features typical of HI, neurological deficit and skeletal dysplasia. At the time of presentation, her mother revealed only noncharacteristic residuals of formerly depigmented areas but was otherwise healthy. Because all affected subjects in this family were females the pedigree would be consistent with an X-linked dominant trait which is lethal for male embryos as has been demonstrated in incontinentia pigmenti (Wiklund and Weston, 1980). Two families have been reported in which males were among the affected members. In one case, two siblings, a boy and a girl, presented with typical features of HI and neurological symptoms. The mother showed similar pigmentary abnormalities but no neurological defect (Patrizi et al., 1987). In another family [family B, cases 3 and 4, reported by Zapella (1993)], dizygotic twins of male and female gender showed depigmentation similar to HI and neurological defects. The father had three small depigmented spots, and the mother showed two depigmented streaks. The couple had experienced three miscarriages and had one other healthy daughter. A histopathological examination of the skin was not performed, making the diagnosis less firm than in the above cases. There is no simple mendelian inheritance pattern that could explain the findings in these two families. A pigmentary mosaicism of the hyperpigmented type distributed along the lines of Blaschko was seen in two halfbrothers (Horn et al., 2002). One of these boys suffering from mental retardation, facial asymmetry, short stature, scoliosis, brachydactyly, and clinodactyly, had a chromosomal mosaic (46, XY, dup (3)(p21.3;pter)/46, XY) which could be found in lymphocytes as well as in fibroblasts. His half-brother presented macrocephaly, facial asymmetry, clinodactyly, and a normal male karyotype in lymphocytes. Hence, only a small subset of cases may be caused by a heritable genetic defect. The majority of cases are sporadic, with no risk for the offspring of affected individuals. Obviously, a search for the genetic defect of HI is in vain. An early attempt was made by one of the authors to assign the locus for HI to chromosome 9 (Happle, 1987), based on a reinterpretation of two cases that had originally been presented as examples of incontinentia pigmenti (Gilgenkrantz et al., 1985; Hodgson et al., 1985). In the light of present knowledge about the genetic heterogeneity of HI, such an attempt can clearly be designated as erroneous. A new concept concerns the action of retrotransposons (Happle, 2002). Retrotransposons or transposable elements are of retroviral origin and are present in high frequencies in the genome of plants and animals. They are also interspersed in large numbers in the human genome. Several studies have also found retrotransposons in several thousands in the human genome (Smit, 1999; Whitelaw and Martin, 2001). These retroviral particles of DNA may cause a methylation or 642

Fig. 32.7. Phylloid hypomelanosis characterized by leaf-like macules reminiscent of an art nouveau ornament. (Horn et al., 1997; Courtesy of Dr. Denise Horn, Berlin, Germany.)

demethylation of neighboring genes, resulting in respective activation or inactivation of the expression of these genes. Furthermore they are able to move within the genome. Normally, retrotransposons are suppressed. When retrotransposons are active during early embryogenesis, they may produce a mosaic pigmentary pattern by silencing or activating a neighboring gene involved in melanin production. Therefore transposable elements may be an explanation for genetic skin disorder which present clinical signs following the lines of Blaschko similar to HI but do not have a mendelian inheritance (Happle, 2002).

Phylloid Hypomelanosis, a Disorder to be Distinguished from “Hypomelanosis of Ito” Recently, phylloid hypomelanosis has been delineated as a distinct neurocutaneous syndrome (Happle, 2000). In contrast to HI, phylloid hypomelanosis is characterized by round or oval macules resembling a floral ornament (Fig. 32.7) or symmetric macules reminiscent of the leaves of Begonia. In most cases of phylloid hypomelanosis, cytogenetic analysis has shown

GENETIC HYPOMELANOSES: LOCALIZED HYPOPIGMENTATION

mosaic trisomy 13 or translocation trisomy 13 (Ohashi et al., 1992; Horn et al., 1997). The patients may have central nervous system defects with mental retardation, absence of corpus callosum, conductive hearing loss, coloboma, craniofacial defects as well as pachydactyly, clinodactyly and camptodactyly (Happle, 2001). Hence, in contrast to hypomelanosis of Ito that is associated with many different forms of genetic mosaicism, phylloid hypomelanosis rather appears to represent a cytogenetically uniform type of pigmentary mosaicism.

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J. Med. Genet. 30:752–755, 1993. (Comment in: J. Med. Genet. 31:260, 1994.) Murano, I., H. Ohashi, M. Tsukahara, H. Tonoki, F. Okino, M. Atsumi, and T. Kajii. Pigmentary dysplasias in long survivors with mosaic trisomy 18: report of two cases. Clin. Genet. 39:68–74, 1991. Neri, G., R. Ricci, A. Pelino, R. Bova, B. Tedeschi, and A. Serra. A boy with ring chromosome 15 derived from a t(15q;15q) Robertsonian translocation in the mother: cytogenetic and biochemical findings. Am. J. Med. Genet. 14:307–314, 1983. Nordlund, J. J., S. N. Klaus, and J. Gino. Hypomelanosis of Ito. Acta Derm. Venereol. 57:261–264, 1977. Ohashi, H., M. Tsukahara, I. Murano, K. Naritomi, K. Nishioka, S. Miyake, and T. Kajii. Pigmentary dysplasias and chromosomal mosaicism: report of 9 cases. Am. J. Med. Genet. 43:716–721, 1992. Pallister, P. D., L. F. Meisner, B. R. Elejalde, U. Francke, J. Herrmann, J. Spranger, W. Tiddy, S. L. Inhorn, and J. M. Opitz. The Pallister mosaic syndrome. Birth Defects Orig. Art. Ser. 13:103–110, 1977. Patrizi, A., M. Masina, C. Varotti, G. Procaccianti, A. Baruzzi, and L. Badiali de Giorgi. Familial hypomelanosis of Ito. Pediatric Dermatology News 6:302–305, 1987. Pellegrino, J. E., R. E. Schnur, R. Kline, E. H. Zackai, and N. B. Spinner. Mosaic loss of 15q11q13 in a patient with hypomelanosis of Ito: is there a role for the P gene? Hum. Genet. 96:485–489, 1995. Ritter, C. L., M. W. Steele, S. L. Wenger, and B. A. Cohen. Chromosome mosaicism in hypomelanosis of Ito. Am. J. Med. Genet. 35:14–17, 1990. Rott, H. D., R. Ulmer, and E. Haneke. Hypomelanosis of Ito and chromosomal mosaicism in fibroblasts. Lancet 2:343, 1986. Rott, H. D., G. E. Lang, W. Huk, and R. A. Pfeiffer. Hypomelanosis of Ito (incontinentia pigmenti achromians). Ophthalmological evidence for somatic mosaicism. Ophthalmic Paediatr. Genet. 11:273– 279, 1990. Rubin, M. B. Incontinentia pigmenti achromians: Multiple cases within a family. Arch. Dermatol. 105:424–425, 1972. Ruiz-Maldonado, R., S. Toussaint, L. Tamayo, A. Laterza, and V. del Castillo. Hypomelanosis of Ito: diagnostic criteria and report of 41 cases. Pediatr. Dermatol. 9:1–10, 1992. Saxena, U., V. Ramesh, B. Iyengar, and R. S. Misra. Hypomelanosis of Ito: histochemical and ultrastructural observations. Australas. J. Dermatol. 30:45–47, 1989. Schüler G., G. Schwanitz, R. Schubert, and K. Zerres. Hypomelanosis of Ito: case report associated with chromosomal abnormalities in skin fibroblasts. 4th Congress of German Society of Human Genetics, Mainz, p. 175. 1992. Silvers, W. K. The Coat Colors of Mice. A Model for Mammalian Gene Action and Interaction. New York: Springer-Verlag, 1979. Smit, A. F. Interspersed repeats and other mementos of transposable elements in mammalian genomes. Curr. Opin. Genet. Dev. 9:657– 663, 1999. Steichen-Gersdorf, E., R. Trawoger, H. C. Duba, U. Mayr, S. Felber, and G. Utermann. Hypomelanosis of Ito in a girl with plexus papilloma and translocation (X;17). Hum. Genet. 90: 611–613, 1993. Steijlen P. M., H. E. Viëtor, M. A. M. van Steensel, and R. Happle. Sweat testing in hypomelanosis of Ito: divergent results reflecting genetic heterogeneity. Eur. J. Dermatol. 9: 217–219, 2000. Stoebner, P., and E. M. Grosshans. Incontinentia pigmenti achromians (Ito): Etude ultrastructurale. Arch. Klin. Exp. Dermatol. 239: 227–244, 1970. Sybert, V. P. Hypomelanosis of Ito: a description, not a diagnosis. J. Invest. Dermatol. 103:141S–143S, 1994. Sybert, V. P., R. A. Pagon, M. Donlan, and C. M. Bradley. Pigmentary abnormalities and mosaicism for chromosomal aberration: association with clinical features similar to hypomelanosis of Ito. J. Pediatr. 116:581–586, 1990. Takematsu, H., S. Sato, M. Igarashi, and M. Seiji. Incontinentia pigmenti achromians (Ito). Arch. Dermatol. 119:391–395, 1983.

GENETIC HYPOMELANOSES: LOCALIZED HYPOPIGMENTATION Tharapel, A. T., R. S. Wilroy, P. R. Martens, J. M. Holbert, and R. L. Summitt. Diploid-triploid mosaicism: delineation of the syndrome. Ann. Genet. 26:229–233, 1983. Thomas, I. T., J. L. Frias, E. S. Cantu, C. Z. Lafer, D. B. Flannery, and J. G. Graham Jr. Association of pigmentary anomalies with chromosomal and genetic mosaicism and chimerism. Am. J. Hum. Genet. 45:193–205, 1989. Turleau, C., F. Taillard, M. Doussau de Bazignan, N. Delepine, J. C. Desbois, and J. de Grouchy. Hypomelanosis of Ito (incontinentia pigmenti achromians) and mosaicism for a microdeletion of 15q1. Hum. Genet. 74:185–187, 1986. Vormittag, W., C. Ensinger, and M. Raff. Cytogenetic and dermatoglyphic findings in a familial case of hypomelanosis of Ito (incontinentia pigmenti achromians). Clin. Genet. 41:309–314, 1992. Warren, N. S., S. Soukup, J. L. King, P. S. Dignan. Prenatal diagnosis of trisomy 20 by chorionic villus sampling (CVS): a case report with long-term outcome. Prenatal. Diagn. 21:1111–1113, 2001. Wertelecki, W., W. R. Breg, J. M. Graham Jr., K. Iinuma, S. M. Puck, and F. R. Sergovich. Trisomy 22 mosaicism syndrome and UllrichTurner stigmata. Am. J. Med. Genet. 23:739–749, 1986. Whitelaw, E., and D. I. Martin. Retrotransposons as epigenetic mediators of phenotypic variation in mammals. Nat. Genet. 27: 361–365, 2001. Wiklund, D. A., and W. L. Weston. Incontinentia pigmenti: A fourgeneration study. Arch. Dermatol. 116:701–703, 1980. Wilson, W. G., M. A. Shires, K. A. Willson, H. E. Wyandt, L. M. Harris, and T. E. Kelly. Trisomy 18/Trisomy 13 mosaicism in an adult with profound mental retardation and multiple malformations. Am. J. Med. Genet. 16:131–136, 1983. Wulfsberg, E. A., W. C. Wassel, and C. A. Polo. Monozygotic twin girls with diploid/triploid chromosome mosaicism and cutaneous pigmentary dysplasia. Clin. Genet. 39:370–375, 1991. Zajac, V., T. Kirchhoff, E. R. Levy, S. W. Horsley, A. Miller, E. Steichen-Gersdorf, and A. P. Monaco. Characterisation of X;17(q12;p13) translocation breakpoints in a female patient with hypomelanosis of Ito and choroid plexus papilloma. Eur. J. Hum. Genet. 5:61–68, 1997. Zapella, M. Autism and hypomelanosis of Ito in twins. Dev. Med. Child Neurol. 35:826–832, 1993.

Focal Dermal Hypoplasia James J. Nordlund

Historical Background In 1970 Goltz and colleagues reported two individuals with linear areas of dermal hypoplasia and herniation of underlying tissue (Goltz et al., 1970). The syndrome is rare but numerous cases have been reported since.

Synonym Goltz’s syndrome.

Clinical Features This syndrome is a disorder of tissue of ectomesodermal origin. There are several typical groups of disorders (Howell and Freeman, 1989). These include: (1) aplasia cutis congenita; (2) linear areas of hypoplasia with nodular outpouchings thought to be lipomatous protrusions; (3) papillomatous growths. About 90% of those involved are females and the disorder is thought to be transmitted as an X-linked trait

(Landa et al., 1993; Skaria et al., 1995; Vabres and Larregue, 1995). Lesions often are linear in pattern of distribution and thought to follow Blaschko lines (see Fig. 32.1) (Happle, 1985; Pujol et al., 1992). It has been suggested that Blaschko lines represent the clinical manifestation of one X gene inactivation, i.e. the Lyon hypothesis. A few males with this syndrome have been reported (Buchner and Itin, 1992; Hall and Terezhalmy, 1983). The pigmentary changes can by either hyper- or hypopigmented (Figs 32.8 and 32.9). The linear areas of atrophy can be hyperpigmented and/or hypopigmented in the same individual. The pigmentation can be reticulated presenting as a form of poikiloderma. In addition the skin can be discolored by yellowish nodules of fatty tissue. Telangiectases can cause a discoloration of the skin, which is related to blood flow and not to deposition of melanin.

Histology The pigmentary changes show increased amounts of epidermal and dermal melanin in the hyperpigmented skin. There is dilution of melanin in the hypopigmented areas. The yellow nodules seem to be caused by heterotopic fat in the dermis rather than herniation of fat from the subcutaneous tissue (Howell and Freeman, 1989). On electron microscopy there seems to be a significant decrease in the amount of collagen. Fibroblasts seem unable to form normal bundles of collagen (Tsuji, 1982). In one study collagen type III, vimentin and fibronectin were detected but no collagen IV in the basal lamina (Buchner and Itin, 1992).

References Buchner, S. A., and P. Itin. Focal dermal hypoplasia syndrome in a male patient. Report of a case and histologic and immunohistochemical studies. Arch. Dermatol. 128:1078–1082, 1992. Goltz, R. W., R. R. Henderson, J. M. Hitch, and J. E. Ott. Focal dermal hypoplasia syndrome. A review of the literature and report of two cases. Arch. Dermatol. 101:1–11, 1970. Hall, E. H., and G. T. Terezhalmy. Focal dermal hypoplasia syndrome. Case report and literature review. J. Am. Acad. Dermatol. 9:443–451, 1983. Happle, R. Lyonization and the lines of Blaschko [review]. Hum. Genet. 70:200–206, 1985. Howell, J. B., and R. G. Freeman. Cutaneous defects of focal dermal hypoplasia: an ectomesodermal dysplasia syndrome. J. Cutan. Pathol. 16:237–258, 1989. Landa, N., J. M. Oleaga, J. A. Raton, J. Gardeazabal, and J. L. Diaz-Perez. Focal dermal hypoplasia (Goltz syndrome): an adult case with multisystemic involvement. J. Am. Acad. Dermatol. 28:86–89, 1993. Pujol, R. M., J. M. Casanova, M. Perez, X. Matias-Guiu, M. Planaguma, and J. M. de Moragas. Focal dermal hypoplasia (Goltz syndrome). Report of two cases with minor cutaneous and extracutaneous manifestations. Pediatr. Dermatol. 9:112–116, 1992. Skaria, A., R. Feldmann, and C. Hauser. The clinical spectrum of focal dermal hypoplasia [German]. Hautarzt 46:779–784, 1995. Tsuji, T. Focal dermal hypoplasia syndrome: An electron microscopical study of the skin lesions. J. Cutan. Pathol. 9:271–281, 1982. Vabres, P., and M. Larregue. X-linked genodermatoses [review] [French]. Ann. Dermatol. Venereol. 122:154–160, 1995.

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32.8

32.9

Figs. 32.8 and 32.9. Hypopigmented and depigmented patches on the face and legs of a patient with focal dermal hypoplasia (see also Plate 32.5, pp. 494–495).

Fig. 32.10. Guttate hypopigmented macules of the trunk and upper extremities in association with hypopigmented keratotic palmar papules. (From Cole, L. A. Hypopigmentation with punctate keratosis of the palms and soles. Arch. Dermatol. 112:998–1000, 1976.)

Hypomelanosis with Punctate Keratosis of the Palms and Soles Jean L. Bolognia The initial description of hypomelanosis with punctate keratosis of the palms and soles was published in 1976 (Cole, 1976). An 18-year-old man had a history of numerous macules of hypopigmentation since age 6 months. The lesions measured 1–15 mm in diameter and were scattered diffusely over the entire skin surface like “spattered paint” (Fig. 32.10). However, there was sparing of the head, neck, nipples, elbows, 646

knees, genitalia, hands, and feet. A decrease, but not total loss, of pigment was noted within the macules and several had 1–2 mm areas of hyperpigmentation within them; the degree of hypopigmentation varied from lesion to lesion. The macules were sharply demarcated from the surrounding skin and had irregular outlines. The second significant cutaneous finding in this patient was multiple flat-topped, keratotic papules on the palms and soles as well as the dorsal aspect of the digits that had been present since age 3 years. These lesions were also hypopigmented and ranged in size from 2 mm to 6 mm (see Fig. 32.10). No abnormalities of the hair, teeth, nails, or the oral mucosa were noted.

GENETIC HYPOMELANOSES: LOCALIZED HYPOPIGMENTATION Table 32.2. Differential diagnosis of multiple guttate hypomelanotic macules. Idiopathic guttate hypomelanosis Confetti-like lesions of tuberous sclerosis and multiple endocrine neoplasia type 1 Pityriasis lichenoides chronica Pityriasis alba Vitiligo ponctué Darier’s disease (admixed with keratotic lesions) (Rowley et al., 1995) Grover’s disease (Rowley et al., 1995) Biphasic amyloidosis (Piamphongsant and Kullavanijaya, 1976) In association with keratosis punctata (Cole, 1976) In association with chromosomal abnormalities (Atnip & Summitt, 1971) Leukoderma punctata (after PUVA therapy) (Falabella et al., 1988) Disseminated hypopigmented keratoses* [flat-topped papules (Morison et al., 1991)] Lichen sclerosus *May be slightly elevated. Adapted from Bolognia, J. L., and P. E. Shapiro. Albinism and other disorders of hypopigmentation. In: Cutaneous Medicine and Surgery: An Integrated Program In Dermatology. K. A. Arndt, P. E. LeBoit, J. K. Robinson, and B. U. Wintrobe, eds. W. B. Saunders, Philadelphia, 1995 with permission from the publisher.

Other than a slight decrease in the melanin content of the epidermis, there were no significant histologic differences between involved and uninvolved skin. Microscopic examination of a palmar papule demonstrated hyperkeratosis overlying a cup-shaped depression, but no atypia of the keratinocytes. According to the patient, six members of his family in three generations had similar clinical findings (hypomelanosis plus keratoses, but neither alone), however, none of these individuals were examined by the author. Based upon the family history, the presumed pattern of inheritance was autosomal dominant. In 2002, Vignale et al. described two siblings (one boy, one girl) with multiple hypopigmented macules and palmoplantar keratoses as having Cole disease. In one child, both types of lesions were present at birth whereas in the other, they appeared during infancy. Although the hypomelanotic lesions spared the acral portions of the body as in the original description by Cole, they did not spare the elbows and knees and more importantly, their size was significantly larger than the guttate lesions depicted in Fig. 32.8. The possibility exists that these children had a different disorder. The differential diagnosis includes other disorders that can present with guttate leukoderma (Table 32.2). Palmoplantar keratoses can also be seen in association with chronic exposure to arsenic, but macules of normally pigmented skin are seen within areas of hyperpigmentation in these patients.

References Atnip, R. L., and R. L. Summitt. Tetraploidy and 18-trisomy in a sixyear-old triple mosaic boy. Cytogenetics 10:305–317, 1971. Cole, L. A. Hypopigmentation with punctate keratosis of the palms and soles. Arch. Dermatol. 112:998–1000, 1976. Falabella, R., C. E. Escobar, E. Carrascal, and J. A. Arroyave. Leukoderma punctata. J. Am. Acad. Dermatol. 18:485–494, 1988. Morison, W. L., B. J. Kerker, W. W. Tunnessen, and E. R. Farmer. Disseminated hypopigmented keratoses. Arch. Dermatol. 127:848– 850, 1991. Piamphongsant, T., and P. Kullavanijaya. Diffuse biphasic amyloidosis. Dermatologica 152:243–248, 1976. Rowley, M. J., L. T. Nesbitt Jr., P. R. Carrington, and C. G. Espinoza. Hypopigmented macules in acantholytic disorders. Int. J. Dermatol. 34:390–392, 1995. Vignale, R., A. Yusin, A. Panuncio, J. Abulafia, Z. Reyno, and A. Vaglio. Cole disease; hypopigmentation with punctate keratosis of the palms and soles. Pediatr. Dermatol. 19:302–306, 2002.

Darier–White Disease (Keratosis Follicularis; 124200) Jean L. Bolognia

Historical Background The initial descriptions of leukoderma in patients with Darier’s disease were published in the Japanese literature in the 1920s (Hidaka, 1924a, b, c) and German literature in the 1940s (Neumann, 1940). It was not until the 1960s that reports of this clinical finding began to appear in the English language (Cattano, 1968; Goodall and Richmond, 1965). Decreased pigmentation in biopsy specimens of keratosis follicularis was appreciated by the 1930s (Périn, 1934).

Epidemiology To date, the vast majority of reports of leukoderma in association with Darier disease have been about individuals with skin types III–VI (Melski et al., 1977), i.e., Asians, Africans, and African Americans (Baba et al., 1984; Berth-Jones and Hutchinson, 1989; Bleiker, 1998; Cattano, 1968; Cornelison et al., 1970; Goodall and Richmond, 1965; Gupta, 2003; Hidaka, 1924a, b, c; Jacyk and Visser, 1992; Ohtake et al., 1994; Peterson, 2001; Rowley et al., 1995). Presumably this is a reflection of easier clinical detection rather than a clinical finding unique to pigmented skin. However, a few authors have suggested that retained melanosomes in the stratum corneum may play a role in determining the color of skin lesions in Darier’s disease (Cattano, 1968; Ohtake et al., 1994) (see Pathogenesis). In several patients, the hypopigmented lesions had been present since childhood (Cornelison et al., 1970; Jacyk and Visser, 1992) and were described as stable in number as well as distribution (Bleiker et al., 1998; Cornelison et al., 1970; Gupta and Shaw, 2003; Peterson et al., 2001).

Clinical Description Darier–White disease (keratosis follicularis) is an autosomal dominant disorder characterized by multiple rough red-brown papules, often accentuated in a “seborrheic” distribution. The 647

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Fig. 32.11. A 48-year-old African-American woman with linear keratosis follicularis involving the arm. The macules of guttate leukoderma (arrow) were admixed with keratotic papules and comedones and all three types of lesions followed the lines of Blaschko. (From Bolognia, J. L., S. J. Orlow, and S. A. Glick. Lines of Blaschko. J. Am. Acad. Dermatol. 31:157–90, 1994 with permission of the publisher.)

papules can coalesce to form plaques, especially in the flexural areas. The diagnosis is confirmed histologically by the presence of acantholytic dyskeratosis (see below). Additional cutaneous findings include palmoplantar keratotic papules, acrokeratosis verruciformis of Hopf-like lesions, V-shaped notches in the distal edge of the nails, longitudinal streaks of red and white in the nails, and “cobblestone” papules of the oral mucosa. Less often, an associated guttate leukoderma is seen in patients with Darier disease. The hypopigmented lesions are usually widely distributed and most commonly involve the trunk followed in frequency by the extremities (Cornelison et al., 1970; Goodall and Richmond, 1965; Jacyk and Visser, 1992). Occasionally, they are also found on the face and neck (Cattano, 1968). The lesions are well demarcated macules that usually measure 1–5 mm in diameter (Fig. 32.11) (Berth-Jones and Hutchinson, 1989; Cornelison et al., 1970; Gupta, 2003; Jacyk and Visser, 1992; Peterson, 2001) and can be either follicular or nonfollicular in distribution (Cornelison et al., 1970). In one patient, there was a striking perifollicular distribution that remained stable over a period of 10 years (BerthJones and Hutchinson, 1989), and in a second patient, the macules coalesced to form thin reticulated strands (Jacyk and Visser, 1992). Gupta and Shaw (2003) described unilateral guttate leukoderma in a patient with unilateral Darier disease. Although the leukoderma was noted in several patients to be admixed with the keratotic papules (see Fig. 32.11) (Cattano, 1968; Ohtake et al., 1994), it also can occur independently, i.e., in areas devoid of red-brown papules (Fig. 32.12) (Berth-Jones and Hutchinson, 1989; Bleiker, 1998; Jacyk and Visser, 1992).

Histology The biopsy specimens of the guttate leukoderma demonstrate 648

Fig. 32.12. Lesions of guttate leukoderma in a patient with Darier’s disease. Note the limited number of keratotic papules.

either (1) the characteristic histologic findings of Darier disease including clefting (lacunae) within the epidermis, acantholysis, and dyskeratosis (e.g., corps ronds and grains) plus decreased pigmentation (Berth-Jones and Hutchinson, 1989; Bleiker, 1998; Gupta, 2003; Ohtake et al., 1994) or (2) only a decrease in the melanin content of the epidermis (Cattano, 1968; Cornelison et al., 1970; Jacyk and Visser, 1992). Both of these patterns can be seen in a single patient when multiple biopsies are performed (Bleiker and Burns, 1998; Gupta and Shaw, 2003; Jacyk and Visser, 1992; Peterson et al., 2001; Rowley et al., 1995). In a histologic study of biopsy specimens of papules from 18 patients with Darier disease, a marked decrease in the pigment content of the basal layer of the epidermis was observed in the central portion of the lesions (Douwes, 1968). Douwes (1968) referred to this central area of decreased pigmentation as “zone III” and stated that it corresponded to the region with the greatest concentration of grains. He also noted an increase in epidermal pigmentation in the lateral portions of the lesions (“zone II”) as compared with uninvolved skin (“zone I”). Cattano (1968) also observed large melanocytes with increased dopa activity and dendricity at the periphery of a papular lesion. In Fontana-Masson and DOPA-stained sections from biopsy specimens of the leukoderma, a marked decrease or absence of epidermal melanin and melanocytes can be seen, respectively (Cattano, 1968). S-100 staining has also revealed an almost complete absence of melanocytes (Berth-Jones and Hutchinson, 1989). Electron microscopic studies in two patients with guttate leukoderma showed no abnormalities in one patient and spherical, irregularly shaped melanosomes in the other (Jacyk and Visser, 1992). In a patient with Grover’s disease and guttate leukoderma (see below), electron photomicrographs demonstrated sparse melanocytes and primarily stage 4 melanosomes in melanocytes and keratinocytes (Rowley et al., 1995).

GENETIC HYPOMELANOSES: LOCALIZED HYPOPIGMENTATION

Differential Diagnosis Guttate leukoderma has also been described in a patient with Grover’s disease (transient acantholytic dyskeratosis) (Rowley et al., 1995). Because this entity, especially the chronic form (Fawcett and Miller, 1983), shares histologic and clinical features with Darier disease, it would be the major entity in the differential diagnosis. Additional causes of guttate leukoderma are listed in Table 32.2 but the clinical setting, additional cutaneous findings, and histologic features should allow their exclusion.

Pathogenesis The controversy is whether the guttate leukoderma is a reflection of the underlying genetic defect (Hidaka, 1924a, b, c) or simply postinflammatory hypopigmentation (Cattano, 1968). In other words, do the guttate lesions arise de novo or are they preceded by keratotic papules? Also, if the lesions are specific for Darier disease, is acantholytic dyskeratosis a prerequisite for their production? Cattano (1968) believed that the leukoderma followed the spontaneous resolution of the papular lesions and was therefore post inflammatory. However, Cornelison et al. (1970) and Jacyk and Visser (1992) found no evidence of preceding papular lesions in any of their seven patients, and with the exception of one patient (two of her three biopsy specimens had evidence of suprabasal clefting), only decreased pigmentation was observed histologically. In the patient described by Berth-Jones and Hutchinson (1989), dyskeratotic acantholysis was present within the guttate lesions and they suggested that the loss of pigmentation was associated with subclinical disease activity. One explanation for the association of acantholysis with hypomelanosis is an interference with the transfer of pigment from melanocytes to keratinocytes (Rowley et al., 1995). Because Ohtake et al. (1994) observed a similar decrease in the epidermal melanin content of both the papular and the hypomelanotic lesions as compared to uninvolved skin, they agreed with Cattano (1968) that the prolonged retention of melanosomes in the thickened corneal layer of the papules (as compared to the thinner stratum corneum of the guttate lesions of leukoderma) was responsible for the darker color of the papular lesions.

Treatment/Prognosis The standard therapy for Darier disease includes emollients, keratolytics, and topical and oral retinoids. There are reports of several patients in which the papular component of the disease improved with either oral vitamin A (150 000– 200 000 U/day) or etretinate (1–1.5 mg/kg/day), but the leukoderma remained unchanged (Cornelison et al., 1970; Jacyk and Visser, 1992). Unfortunately, attempts at repigmentation via phototherapy would be hampered by potential flares of the Darier disease.

References Baba, T., H. Saginoya, M. Sakuma, T. Takase, and K. Uyeno. Two cases of Darier’s disease associated with guttate leukoderma. Nippon Hifuka Gakkai Zasshi 94:1003–1013, 1984.

Berth-Jones, J., and P. E. Hutchinson. Darier’s disease with perifollicular depigmentation. Br. J. Dermatol. 120:827–830, 1989. Bleiker, T. O., and D. A. Burns. Darier’s disease with hypopigmented macules. Br. J. Dermatol. 138:913–914, 1998. Cattano, A. N. An unusual case of keratosis follicularis. Arch. Dermatol. 98:168–174, 1968. Cornelison, R. L., E. B. Smith, and J. M. Knox. Guttate leukoderma in Darier’s disease. Arch. Dermatol. 102:447–450, 1970. Douwes, F. R. Zur Histologie und Histochemie des Morbus Darier. Arch. Klin. Exp. Dermatol. 233:309–322, 1968. Fawcett, H. A., and J. A. Miller. Persistent acantholytic dermatosis related to actinic damage. Br. J. Dermatol. 109:349–354, 1983. Goodall, J. W. D., and Q. M. S. Richmond. A case of Darier’s disease. Br. J. Clin. Pract. 19:475–476, 1965. Gupta S., and J. C. Shaw. Unilateral Darier’s disease with unilateral guttate leukoderma. J. Am. Acad. Dermatol. 48:955–957, 2003. Hidaka, S. Study of Darier’s disease: I — Statistics. Acta Dermatol. (Kyoto) 3:191–236, 1924a. Hidaka, S. Study of Darier’s disease: II — Case reports. Acta Dermatol. (Kyoto) 3:373–412, 1924b. Hidaka, S. Study of Darier’s disease: III — Discussion. Acta Dermatol. (Kyoto) 4:1–59, 1924c. Jacyk, W. K., and A. J. Visser. Leukodermic macules in keratosis follicularis (Darier’s disease). Int. J. Dermatol. 31:715–717, 1992. Melski, J. W., L. Tanenbaum, J. A. Parrish, T. B. Fitzpatrick, H. L. Bleich, and 28 participating investigators. Oral methoxsalen photochemotherapy for the treatment of psoriasis: A cooperative clinical trial. J. Invest. Dermatol. 68:328–335, 1977. Neumann, H. Über die Primärefflorescenz des Morbus Darier. Arch. Dermatol. Syphil. 180:204–207, 1940. Ohtake, N., R. Takano, A. Saitoh, Y. Kubota, S. Shimada, and K. Tamaki. Brown papules and leukoderma in Darier’s disease: clinical and histological features. Dermatology 188:157–159, 1994. Peterson, C. M., J. L. Lesher, Jr., and O. P. Sangueza. A unique variant of Darier’s disease. Int. J. Dermatol. 40: 278–280, 2001. Périn, M. L. Histologie d’une tache pigmentaire au cours de la maladie de Darier. Bull. Soc. Franc. Dermatol. Syphiligr. 41:28–32, 1934. Rowley, M. J., L. T. Nesbitt Jr., P. R. Carrington, and C. G. Espinoza. Hypopigmented macules in acantholytic disorders. Int. J. Dermatol. 34:390–392, 1995.

Nevus Depigmentosus Stella D. Calobrisi

Historical Background Nevus depigmentosus is an uncommon, localized, cutaneous hypomelanosis (Bolognia and Pawelek, 1988) first described by Lesser in 1884.

Synonym This lesion is also known as an achromic nevus.

Epidemiology Nevus depigmentosus occurs equally among males and females and can affect any ethnic group.

Clinical Findings Nevus depigmentosus is typically congenital, although it may not be apparent at birth. Nevus depigmentosus rarely appears first in adults. There is no known inheritance pattern of this lesion, the cause of which is not known. It has been suggested 649

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Fig. 32.13. Nevus depigmentosus which does not appear to cross the midline (see also Plate 32.6, pp. 494–495).

to be a sporadic defect in embryonic development (Pinto and Bolognia, 1991). There appear to be three clinical forms of nevus depigmentosus (Jimbow et al., 1975). The solitary or isolated (circular or rectangular) is the most common. The segmental and the systematized (unilateral whorls or streaks) forms (Coupe, 1967) are much less common. Nevus depigmentosus is characterized as well circumscribed, irregularly shaped, hypopigmented macules or patches whose borders appear feathered or serrated (Jimbow et al., 1975) (Figs 32.13–32.16). Hairs within the macules or patches may also be hypopigmented (Dhar et al., 1993a). The lesion(s) remains stable throughout life although it enlarges proportionately with the growing child. The lesion occurs most commonly on the trunk (Fig 32.17) or proximal extremities, but can also occur on the head and neck (Pinto and Bolognia, 1991). The lesion typically does not cross the midline (Pinto and Bolognia, 1991).

Fig. 32.14. Nevus depigmentosus on the face (see also Plate 32.7, pp. 494–495).

Associated Findings Usually this lesion is not associated with other abnormalities although there have been rare reports of associated seizures and mental retardation (Sugarman and Reed, 1969) and unilateral limb hypertrophy (Berg and Tarnowski, 1974). Furthermore, in a report by Dhar et al., 10% of patients had a history of atopic dermatitis and 28% had a family history of atopy (Dhar et al., 1993b).

Histology Light microscopic findings of lesional skin usually reveal normal or decreased numbers of melanocytes, depending on the series of patients (Jimbow et al., 1975; Takaiwa and Mishima, 1970). Electron microscopic studies of lesional skin typically reveal fewer numbers of melanosome, which appear normal in size, shape, and melanin content, and which appear to aggregate within vacuoles in the melanocytes (Jimbow et al., 1975). Takaiwa and Mishima (1970) report no disturbance in melanosome transfer to keratinocytes. However, 650

Fig. 32.15. Typical nevus depigmentosus on the thigh.

Jimbow et al. (1975) reported abnormal melanosome transfer resulting in a decreased number of melanosomes in the keratinocytes with subsequent hypopigmentation.

Diagnosis The diagnosis is usually based on the clinical examination. The

GENETIC HYPOMELANOSES: LOCALIZED HYPOPIGMENTATION

nevus depigmentosus. Alternatively, one could distinguish the ash leaf macule from nevus depigmentosus by electron microscopy which demonstrates small, poorly melanized melanosomes in the ash leaf spot (Jimbow et al., 1975). To distinguish HI (incontinentia pigmenti achromians – see Chapter 32) from the systematized form of nevus depigmentosus is more difficult. HI is considered to be an autosomal dominant, neurocutaneous disorder which is frequently associated with central nervous system, ocular, and musculoskeletal abnormalities, as well as streaks and whorls of hypopigmentation (Bolognia and Pawelek, 1988). However, there are instances in which there are no other associations, making it nearly impossible to make the distinction between systematized nevus depigmentosus and HI. Fig. 32.16. Congenital hypomelanotic nevus (see also Plate 32.8, pp. 494–495).

Treatment Most patients with nevus depigmentosus do not pursue treatment for their lesion. If, however, the lesion is of cosmetic concern, camouflage makeup is effective in concealing the lesion. If the lesion is small, one could also consider excision. PUVA therapy has not been shown to be beneficial (Berg and Tarnowski, 1974). However, autologous grafting with noncultured melanocytes has been shown to be an effective and fairly simple repigmentation treatment for patients with nevus depigmentosus (Gauthier and Surleve-Bazeille, 1992).

Prognosis

Fig. 32.17. Typical nevus depigmentosus on the flank and breast (see also Plate 32.9, pp. 494–495).

Nevus depigmentosus is a rare, congenital, hypopigmented lesion that remains stable in size and color over time and which typically does not have any systemic associations. The term is a misnomer because it is neither a hamartoma suggested by the word nevus nor is it depigmented but rather hypopigmented. Perhaps macula hypopigmentosus would be a more accurate term for this benign lesion.

References lesion is classically hypopigmented, localized, congenital, and stable in size and color over time. Examination with Wood’s lamp can be useful because the lesion becomes more prominent. Furthermore, stroking the lesion will predictably cause it to flare and it will not disappear with diascopy.

Differential Diagnosis The differential diagnosis of nevus depigmentosus includes nevus anemicus (see Chapter 42) which is also a congenital lesion which disappears with Wood’s lamp examination or diascopy and which does not flare with stroking because it is a localized area of vasoconstriction. A lesion of segmental vitiligo (see Chapter 30) is readily distinguished from nevus depigmentosus in that it is an acquired, depigmented lesion that is devoid of melanocytes when examined microscopically. The ash leaf macule of tuberous sclerosis (see below) easily can be confused with nevus depigmentosus. One must rely on the other cutaneous and systemic signs and symptoms of tuberous sclerosis to differentiate an ash leaf spot from the

Berg, M., and W. Tarnowski. Nevus pigmentosus. Arch. Dermatol. 109:920, 1974. Bolognia, J. L., and J. M. Pawelek. Biology of hypopigmentation. J. Am. Acad. Dermatol. 19:217–255, 1988. Coupe, R. L. Unilateral systematized achromic naevus. Dermatologica 134:19–35, 1967. Dhar, S., A. J. Kanwar, and S. Ghosh. Leucotrichia in nevus depigmentosus [letter]. Pediatr. Dermatol. 10:198–199, 1993a. Dhar, S., A. J. Kanwar, and S. Kaur. Nevus depigmentosus in India: experience with 50 patients [letter]. Pediatr. Dermatol. 10:299–300, 1993b. Gauthier, Y., and J. E. Surleve-Bazeille. Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions. J. Am. Acad. Dermatol. 26(2 Pt 1):191–194, 1992. Jimbow, K., T. B. Fitzpatrick, G. Szabo, and Y. Hori. Congenital circumscribed hypomelanosis: A characterization based on electron microscopic study of tuberous sclerosis, nevus depigmentosus and piebaldism. J. Invest. Dermatol. 64:50–62, 1975. Lesser, E. In: Hanbuchder Hautkrankheiten, 2nd ed., H. V. Ziemssen (eds). Leipzig: Vogel, 1884, p. 183. Pinto, F. J., and J. L. Bolognia. Disorders of hypopigmentation in children. Pediatr. Clin. North Am. 38:991–1017, 1991. Sugarman, G. I., and W. B. Reed. Two unusual neurocutaneous dis-

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CHAPTER 32 orders with facial cutaneous signs. Arch. Neurol. 21:242–247, 1969. Takaiwa, T., and Y. Mishima. Population density of intraepidermal dendritic cells in nevus depigmentosus. Jpn. J. Clin. Electron Microsc. 3:651–652, 1970.

Tuberous Sclerosis Complex Pranav B. Sheth

Historical Background Recognition of the tuberous sclerosis complex (TSC) came in stages over the nineteenth and twentieth centuries. Rayer, a French dermatologist, provided the first illustrations of the fibrovascular papules of TSC in 1835. In 1880, Bourneville, a French neurologist, described “tuberous sclerosis of the cerebral convolutions” on autopsy of a mentally retarded girl with seizures. Although he noted a “confluent vesiculopustular eruption on the nose, cheeks, and forehead”, he did not recognize the facial eruption as part of the disease. “Adenomes sebaces” was both clinically and microscopically reviewed by Pringle in 1890, however, he did not make any association between the cutaneous findings and the intelligence of his patients (which was “decidedly below par”). In 1905, Campbell described the triad of epilepsy, idiocy, and adenoma sebaceum. In 1908, Vogt described the association between cerebral tumors and facial adenoma sebaceum. These were felt to be diagnostic of tuberous sclerosis (Morgan and Wolfert, 1979). It was in 1895 when pigment changes in the skin of patients with tuberous sclerosis were first considered a possible “symptom of a systemic disease” (Morgan and Wolfert, 1979). In 1932, “patches of abnormal whiteness,” resembling “in size and conformation the cafe au lait stains,” were described in 2 of 29 patients with tuberous sclerosis complex (Critchley and Earl, 1932). Further studies of tuberous sclerosis noted “vitiligo, canities (poliosis), and pale freckles” (Butterworth and Wilson, 1941), “depigmented areas (vitiligo) and cafe au lait” (Chao, 1959), poliosis (partial albinism) and café au lait (Nickel and Reed, 1962), and vitiligo (Harris and Moynahan, 1966) in the patients. In 1965, Gold and Freeman proposed that “depigmented nevi” may be the initial cutaneous manifestation of TSC (Gold and Freeman, 1965). Finally, in 1968, Fitzpatrick described the “ash leaf” configuration of the lesions and reviewed the clinical, histologic, and electron microscopic features of the hypopigmented macules of TSC and their differential diagnosis.

Synonyms Bourneville disease, Pringle disease, epiloia [artificial compounding of the words epilepsy and aloia (mindlessness in Greek)]. The term epiloia has also been used to denote the triad of epilepsy, low IQ, and adenoma sebaceum.

Epidemiology Tuberous sclerosis complex (TSC) is a disorder inherited in an autosomal dominant pattern with variable penetrance and is 652

caused by a high rate of spontaneous mutations. Prevalence rates are reported to range from approximately 1 in 10 000 to 1 in 38 000 in the general population (Hunt and Lindenbaum, 1984; Osborne et al., 1991; Umapathy and Johnston, 1989; Wiederholt et al., 1985). The prevalence in newborns is estimated to be 1 in 5800 to 1 in 10 000 (Osborne et al., 1991; Umapathy and Johnston, 1989). It is generally understood that reported prevalence figures underestimate the true rate since mild cases may not be reported. A parent might manifest few signs of tuberous sclerosis but the offspring might be severely affected. The incidence of TSC at institutions for the mentally retarded and epileptic patients was reported to be 1:100 to 1:300 (Kuntz, 1988). Males and females are affected equally in number and severity and there appears to be no racial predilection. Heredity is evident in 25–50% of cases. The rate of spontaneous mutation varies from 66% to 86% depending in part on the completeness of investigation of the extended family (Hunt and Lindenbaum, 1984; Kwiatkowski and Short, 1994; Zvulunov and Esterly, 1995).

Genetics TSC has been determined to result from mutations in two genes, TSC1 mapped to chromosome 9q34 and TSC2 mapped to 16p13.3. The proteins from these genes, hamartin (TSC1) and tuberin (TSC2), modulate cellular growth regulatory function. Hamartin appears to have an interactive function with tuberin. Tuberin has a tumor suppressor function and the gene has an area of homology with the GTPase activating protein rap1GAP (Arbuckle and Morelli, 2000; Jimbow, 1997). Genetic linkage analysis indicates that half of all families manifesting the TSC in which an autosomal dominant inheritance pattern has been documented show genetic linkage to chromosome TSC1 and half to TSC2 (Kwiatkowski and Short, 1994). In addition, TSC1 is located near the locus for dopamine-6-hydroxylase, an enzyme involved in the synthesis of catecholamine neurotransmitters (Jimbow, 1997).

Clinical Manifestations TSC is a disorder of cellular differentiation and proliferation and many of its clinical manifestations result from hamartomas. However, true neoplasms occur, particularly in the kidney and brain. In addition, abnormalities in neuronal migration are felt to play a major role in neurologic dysfunction (Roach and Delgado, 1995). Although characteristically involving the brain, skin, kidneys, and heart, TSC can involve almost any organ system. The muscles and peripheral nervous system appear to be spared (Zvulunov and Esterly, 1995). The most common manifestations of TSC include hypomelanotic macules, facial angiofibromas, dental enamel pitting, infantile spasms, seizures, mental handicap, subependymal glial nodules, cortical tubers, retinal hamartomas, cardiac rhabdomyomas, renal angiolyoma, renal cyst, and psychiatric problems (Ahlsen et al., 1994; Hunt and Lindenbaum, 1984; Osborne et al., 1991; Wiederholt et al., 1985; Zvulunov and Esterly, 1995). Due to the variable penetrance of the TSC gene, a wide range of clin-

GENETIC HYPOMELANOSES: LOCALIZED HYPOPIGMENTATION

Fig. 32.18. Typical facial angiofibromas of tuberous sclerosis (see also Plate 32.10, pp. 494–495).

ical variability and severity is present. In the experience of Gomez, only 29% of patients with TSC had the classic triad of seizures, mental retardation, and adenoma sebaceum (Gomez, 1988). Because the clinical lesions of TSC follow different time courses, clinical presentations further vary depending on the age of the patient. For example, cardiac rhabdomyomas are common in the perinatal period but decline in frequency thereafter. Similarly, the age of onset of hypomelanotic macules, forehead plaques, poliosis (graying of hair), and cortical tubers, is most commonly at birth or in the neonatal period. Seizures, mental retardation, and facial angiofibromas present in infancy and early childhood, subependymal nodules in the first and second decade of life, and multiple ungual fibromas and renal angiomyolipomas are usually late manifestations (Gomez, 1991; Kwiatkowski and Short, 1994). Diagnostic criteria were developed in 1992 to aid in the diagnosis of tuberous sclerosis. They were revised in 1998 in a consensus conference. The criteria are divided into major and minor criteria with a definite diagnosis of TSC occurring when two major or one major plus two minor features are present. A probable diagnosis can be made if one major plus one minor feature are present and a possible diagnosis if either one major or two or more minor features are present. In the new criteria, the first four entities in the major features category are all dermatologic findings. These include facial angiofibromas (Fig. 32.18) or forehead plaque, nontraumatic ungual or periungual fibroma (Fig. 32.19), greater than or equal to three hypomelanotic macules, and the shagreen patch (connective tissue nevus) (Fig. 32.20). Other major criteria include retinal hamartomas, cortical tuber, subependymal nodule or giant cell astrocytoma, cardiac rhabdomyoma, lymphangiomatosis and renal angiomyolipoma. Among the minor features, the only dermatologic finding is confetti hypomelanotic macules. Other minor features include dental enamel pits, rectal polyps, bone cysts, cerebral white matter radial migration lines, gingival fibromas, retinal achromic patch, and multiple renal cysts (Arbuckle and Morelli, 2000).

Fig. 32.19. Periungual fibromas that are very suggestive of tuberous sclerosis (see also Plate 32.11, pp. 494–495).

Fig. 32.20. Shagreen patch (see also Plate 32.12, pp. 494–495).

Pigmentary Disorders in Tuberous Sclerosis Complex The cutaneous manifestations of TSC are hypomelanotic macules, poliosis/leukotrichia, facial angiofibroma, ungual fibroma, forehead plaque, and shagreen patch. The hypomelanotic macule (Fig. 32.21), also referred to as the ash leaf macule (and formerly described as the depigmented macule, vitiligo, depigmented nevus, and partial albinism), is found in 50–100% of patients with TSC (Fitzpatrick et al., 1968; Gold and Freeman, 1965; Harris and Moynahan, 1966; Hunt, 1983; Hurwitz and Braverman, 1970; Roach and Delgado, 1995). Its significance as the earliest cutaneous sign of TSC was first described by Gold and Freeman in 1965. Fitzpatrick in 1968 coined the term “ash leaf” macule for the lanceovate (tapering at one end and round at the other) configuration that was so similar to the “shape of the leaflet from the mountain ash tree” (Fitzpatrick et al., 1968). Although most authors noted the presence of these macules at birth or within the first year of life, identification of the macules is often missed unless a thorough Wood’s lamp examination is performed. The late appearance of hypopigmented maculae, as late as 6 years of 653

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Fig. 32.21. Ash leaf hypopigmented macules that often are the first sign of tuberous sclerosis (see also Plate 32.13, pp. 494–495).

Fig. 32.22. Classical leukotrichia on the scalp in a patient with tuberous sclerosis (see also Plate 32.14, pp. 494–495).

age, has also been described after repeated negative skin examinations (Oppenheimer et al., 1985). Once present, there is no appreciable increase in size or change in shape. The hypomelanotic macules may become less prominent with time and may disappear in adulthood (Jozwiak et al., 1998). The macules are discrete, asymmetrically scattered on the body, and greatest over the trunk and extremities. They are rare on the face. Uncommonly, they may occur along lines of cleavage or follow a dermatomal distribution (Rogers, 1988). The number of macules present varies from 1 to greater than 100. Their shape occurs in three patterns: polygonal (most common), lanceovate (most characteristic), and confetti. The polygonal macule, with the shape of a thumbprint, varies in size from 0.5 cm to 2.0 cm. The lanceovate or ash leaf macule ranges in size from 1 cm to 12 cm. The confetti pattern tiny white macules are rare, range from 1 mm to 3 mm in size, have a predilection for the legs, and are considered to be specific for TSC by some authors (Fitzpatrick, 1991; Fitzpatrick et al., 1968; Gold and Freeman, 1965; Hurwitz and Braverman, 1970). Characteristically, the macules are sharply outlined, and dull white to off-white in color. On Wood’s light examination it is apparent that there is only a partial decrease of pigment in the macules (Fitzpatrick et al., 1968). Perifollicular hyperpigmentation within the macule has also been described (Hurwitz and Braverman, 1970). Poliosis (premature graying of the hair) and leukotrichia (white hair) (Fig. 32.22) may occur in up to 18% of patients (Nickel and Reed, 1962). Often occurring at birth or within the first year of life, small gray and white hair patches may occur overlying a hypopigmented scalp patch. The ash leaf pattern may be seen in the hypopigmented patch of hairs. Poliosis may also occur overlying a forehead plaque or normally pigmented scalp skin (Gomez, 1988; McWilliam and Stevenson, 1978). Hypopigmented iris spots, presumably analogous to the cutaneous hypopigmented macule, have been described in two patients with TSC (Gutman et al., 1982). Hypomelanotic leafshaped macules of the ocular fundus and poliosis circum-

scripta, isolated white eyelashes set among normally pigmented ones, have also been reported (Awan, 1982). The presence of café-au-lait patches, once thought to be significantly associated with tuberous sclerosis, are now felt by most to be an incidental finding. One study revealed that café-au-lait patches occurred with equal frequency in patients with TSC compared with nonaffected individuals (Bell and MacDonald, 1985).

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Pathology The hypopigmented macule shows a normal complement of melanocytes with the dopa reaction, however, the intensity of the reaction is reduced compared to normally pigmented skin. The population density of melanocytes can also be determined with the S100 stain. In addition, the melanocytes contain a perikaryon that is smaller and dendrites that are less developed than those in normal control skin (Jimbow et al., 1975). On electron microscopic examination of hypomelanotic skin and hair, changes are seen in melanogenic organelles, melanosome size and number, rate of synthesis of melanosomes and melanization, and arrangement of melanosomes. No change in shape or suggestions of disturbance in the transfer of melanosomes has been found compared to normal control skin. The number and size of organelles involved in melanosome synthesis (e.g. Golgi apparatus, endoplasmic reticulum, free ribosomes, and mitochondria) in the hypomelanotic skin and hair lesions of patients with TSC is fewer and smaller. There is a great reduction in the number of melanosomes. The melanosomes that are present are mostly in the nonmelanized stage. This suggests a decrease in the rate of synthesis and melanization of melanosomes in both hypomelanotic skin and hair of TSC. The melanosomes maintain the normal ellipsoidal shape with internal striated lamellae but are reduced in size. The number of melanosomes transferred into keratinocytes is greatly decreased. This may be due to the decreased rates of synthesis and melanization of melanosomes. Most of the

GENETIC HYPOMELANOSES: LOCALIZED HYPOPIGMENTATION

melanosomes transferred into the keratinocytes are arranged in complexes in both Negroid and Caucasoid skin. This aggregation of melanosomes is felt to be due to a size-dependent uptake of melanosomes by keratinocytes in which larger melanosomes are arranged in a nonaggregated form in keratinocytes whereas smaller melanosomes aggregate (Jimbow et al., 1975).

Laboratory Findings and Investigations The diagnosis is self evident when the full complement of mental, neurologic, and cutaneous abnormalities is present. Wood’s lamp examination of the skin is important as hypomelanotic macules may be the only visible cutaneous sign early on. Radiologic confirmation of intracranial lesions with X-ray, computed tomography, or magnetic resonance imaging may be useful in suspected or clinically confirmed cases. Electroencephalography is useful for evaluating the seizure disorder. Investigation other than neuroradiological workup should be tailored towards the specific presenting signs and symptoms. In addition, extensive laboratory investigation of parents of affected children is not needed unless the parents have clinical signs suggestive of TSC (Zvulunov and Esterly, 1995).

melanosomes within the melanocytes and a decrease in the number of melanosomes transferred to the keratinocyte. Thus the hypomelanosis of nevus depigmentosus is related to the decreased synthesis of melanosomes and an abnormality in melanosome transfer. Tuberous sclerosis on the other hand shows a decrease in the synthesis, melanization, and size of melanosomes in melanocytes and the formation of melanosome complexes in keratinocytes (Jimbow et al., 1975). Ash leaf hypopigmentation, similar to that found in tuberous sclerosis, has also been reported in the autosomal dominant basal cell nevus syndrome (Gorlin Goltz syndrome) (Breytenbach et al., 1975). In addition, the hypomelanotic macules were reported in a family with multiple members having a small skull, mental deficiency, retarded growth, and hypopigmented and hyperpigmented macules. They may represent a separate neurocutaneous syndrome or be a forme fruste of TSC (Westerhof et al., 1978). Idiopathic hypomelanotic macules are not uncommon and the prevalence of hypopigmented macules in the general population appears to be underestimated. The presence of a few hypopigmented macules on the skin of an otherwise healthy individual without a family history of tuberous sclerosis does not indicate an evaluation to rule out TSC (Vanderhooft et al., 1996).

Differential Diagnosis The differential diagnosis of circumscribed hypomelanotic macules includes hypomelanotic macules of tuberous sclerosis, vitiligo (see Chapter 30), nevus depigmentosus (see above), piebaldism (see Chapter 29), Vogt–Koyanagi–Harada syndrome (see Chapter 39), basal cell nevus syndrome (Gorlin Goltz), and idiopathic hypomelanotic macules. The macules of vitiligo are often pure or milk white in color due to absolute loss of pigment. They often present in a symmetric distribution with preference for periorificial and flexural areas, and they only uncommonly have a lanceovate shape. Dopa staining of mature vitiligo macules shows complete absence of melanocytes. This loss is confirmed by electron microscopy (Fitzpatrick, 1991). In the Vogt– Koyanagi–Harada syndrome there are depigmented macules that resemble vitiligo. The hypomelanotic skin and hair of piebaldism occurs in a distinctive pattern with a triangular forelock on the forehead and scalp and hypomelanotic patches on the chin, trunk, midarm and mid-thigh. Onset is at birth and there are islands of pigmentation within the center of the depigmented macules. Ultrastructurally, there is a loss of functional melanocytes similar to vitiligo (Jimbow et al., 1975). The congenital circumscribed hypomelanotic macules of piebaldism and nevus depigmentosus differ from those in tuberous sclerosis both clinically and ultrastructurally. Nevus depigmentosus may occur in an isolated circular or rectangular area of hypomelanosis, usually on the trunk, in a typical dermatomal pattern or along an atypical dermatomal pattern in a bizarre, sharply angulated streak. Ultrastructurally, there is no obvious change in the size and melanization of melanosomes. There is, however, an aggregation of

Pathogenesis The pathogenesis of TSC is unknown and various hypotheses exist. Defective conversion of phenylalanine to catecholamine neurotransmitters or melanin resulting in skin hypopigmentation was proposed by evidence of linkage studies of the tuberous sclerosis (TS) gene to chromosomes 12q (phenylalanine hydroxylase), 11q (tyrosinase), and 9q (dopamineB hydroxylase) (Fahsold et al., 1991). Other theories, including the disruption of neuronal cell migration, dysregulation of secretion of paracrine growth factors, and a defective glycoprotein encoded on chromosome 9q34 altering neuronal migration, have been proposed (Zvulunov and Esterly, 1995).

Treatment There is no treatment for tuberous sclerosis. Anticonvulsant therapy is used for seizures and adrenocorticotropic hormone is recommended for infantile spasms. Surgical excision of functionally impairing renal tumors is recommended. Excision of tumors of the central nervous system is not recommended unless intracranial hypertension or hydrocephalus occurs or an epileptogenic focus is found. Dermabrasion, electrodesiccation, or laser treatment (argon, CO2, pulsed dye) of cutaneous lesions may be successful at temporarily removing the cutaneous angiofibromas. Hypomelanotic macules need not be treated.

Prognosis Survival curves show a decreased survival for patients with TSC in comparison with that for the general population. Prognosis is often related to the age of onset of seizures and the extent of renal involvement (Zvulunov and Esterly, 1995). 655

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Renal disease secondary to angioleiomyoma was the most common cause of death in one study of 355 patients. In general, causes of death are age related such that in infancy a cardiovascular cause is the most frequent. Similarly, brain tumors are the most common cause of death in the 10–19 age group, status epilepticus in the 20–29 age group, and renal tumors and pulmonary lymphangiomyomatosis in the 40 and above age group. Although the manifestations of tuberous sclerosis might be mild in an adult, his or her offspring might be severely affected. Early detection of TSC allows for early counseling of affected individuals and parents regarding the inheritance risk to their offspring and is the only form of prevention available today (Shepherd et al., 1991).

References Ahlsen, G., I. C. Gillberg, R. Lindblom, and C. Gillberg. Tuberous sclerosis in western Sweden: A population study of cases with early childhood onset. Arch. Neurol. 51:76–81, 1994. Arbuckle, H. A., and J. G. Morelli. Pigmentary disorders: update on neurofibromatosis-1 and tuberous sclerosis. Curr. Opin. Pediatr. 12:354–358, 2000. Awan, K. J. Leaf-shaped lesions of ocular fundus and white eyelashes in tuberous sclerosis. South. Med. J. 75:227–228, 1982. Bell, S. D., and D. M. MacDonald. The presence of cafe-au-lait patches in tuberous sclerosis. Clin. Exp. Dermatol. 10:562–565, 1985. Breytenbach, H. S., G. S. Gericke, C. J. B. Muller, C. J. Nortje, B. C. Shanley, E. Swart, and C. W. Van Wyk. [Clinical characteristics and genetic identity of the basal cell nevus syndrome (Gorlin-Goltz syndrome)] [Afrikaans]. S. Afr. Med. J. 49:544–550, 1975. Butterworth, T., and J. Wilson, M. Dermatologic aspects of tuberous sclerosis. Arch. Dermatol. 43:1–41, 1941. Chao, D. H.-C. Congenital neurocutaneous syndromes in childhood. J. Pediatr. 55:447–459, 1959. Critchley, M., and C. J. C. Earl. Tuberous sclerosis and allied conditions. Brain 55:311–346, 1932. Fahsold, R., H. D. Roff, and P. Lorenz. A third gene locus for tuberous sclerosis is closely linked to the phenylalanine hydroxylase gene locus. Hum. Genet. 88:85–90, 1991. Fitzpatrick, T. B. History and significance of white macules, earliest visible sign of tuberous sclerosis. Ann. N. Y. Acad. Sci. 615:26–35, 1991. Fitzpatrick, T. B., G. Szabo, Y. Hori, A. A. Simone, W. B. Reed, and M. H. Greenberg. White leaf-shaped macules: Earliest visible sign of tuberous sclerosis. Arch. Dermatol. 98:1–6, 1968. Gold, A. P., and J. M. Freeman. Depigmented nevi: The earliest sign of tuberous sclerosis. Pediatrics 35:1003–1005, 1965. Gomez, M. R. Criteria for diagnosis. In: Tuberous Sclerosis, 2nd ed. M. R. Gomez (ed.). New York: Raven Press, Ltd. 1988, pp. 9–19. Gomez, M. R. Phenotypes of the tuberous sclerosis complex with a revision of diagnostic criteria. Ann. N. Y. Acad. Sci. 615:1–7, 1991. Gutman, I., D. Dunn, M. Behrens, A. P. Gold, J. Odel, and M. R. Olarte. Hypopigmented iris spot: an early sign of tuberous sclerosis. Ophthalmology 89:1155–1159, 1982.

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Harris, R., and E. J. Moynahan. Tuberous sclerosis with vitiligo. Br. J. Dermatol. 78:419–420, 1966. Hunt, A. A survey of 97 cases. II: Physical findings. Dev. Med. Child. Neurol. 25:350–352, 1983. Hunt, A., and R. H. Lindenbaum. Tuberous sclerosis: a new estimate of prevalence within the Oxford region. J. Med. Genet. 21:272–277, 1984. Hurwitz, S., and I. M. Braverman. White spots in tuberous sclerosis. J. Pediatr. 77:587–594, 1970. Jimbow, K. Tuberous sclerosis and guttate leukodermas. Semin. Cutan. Med. Surg. 16:30–35, 1997. Jimbow, K., T. B. Fitzpatrick, G. Szabo, and Y. Hori. Congenital circumscribed hypomelanosis: A characterization based on electron microscopic study of tuberous sclerosis, nevus depigmentosus and piebaldism. J. Invest. Dermatol. 64:50–62, 1975. Jozwiak, S., R. A. Schwartz, C. K. Janniger, R. Michalowicz, and J. Chmielik. Skin lesions in children with tuberous sclerosis complex: their prevalence, natural course, and diagnostic significance. Int. J. Dermatol. 37:911–917, 1998. Kuntz, N. Population studies. In: Tuberous Sclerosis, 2nd ed. M. R. Gomez (ed.). New York: Raven Press, Ltd. 1988, pp. 213–215. Kwiatkowski, D. J., and M. P. Short. Tuberous sclerosis. Arch. Dermatol. 130:348–354, 1994. McWilliam, R. C., and J. B. P. Stevenson. Depigmented hair: The earliest sign of tuberous sclerosis. Arch. Dis. Child. 53:961–963, 1978. Morgan, J. E., and F. Wolfert. The early history of tuberous sclerosis. Arch. Dermatol. 115:1317–1319, 1979. Nickel, W. R., and W. B. Reed. Tuberous sclerosis. Arch. Dermatol. 85:89–106, 1962. Oppenheimer, E. Y., N. P. Rosman, and E. C. Dooling. The late appearance of hypopigmented maculae in tuberous sclerosis. Am. J. Dis. Child. 139:408–409, 1985. Osborne, J. P., A. Fryer, and D. Webb. Epidemiology of tuberous sclerosis. Ann. N. Y. Acad. Sci. 615:125–127, 1991. Roach, E. S., and M. R. Delgado. Tuberous sclerosis. Dermatol. Clin. 13:151–161, 1995. Rogers, I. I. I., RS Dermatologic manifestations. In: Tuberous Sclerosis, 2nd ed. M. R. Gomez (ed.). New York: Raven Press, Ltd. 1988, pp. 111–131. Shepherd, C. W., M. R. Gomez, J. T. Lie, and C. S. Crowson. Causes of death in patients with tuberous sclerosis. Mayo Clin. Proc. 66:792–796, 1991. Umapathy, D., and A. W. Johnston. Tuberous sclerosis: prevalence in the Grampian region of Scotland. J. Mental Def. Res. 33:349–355, 1989. Vanderhooft, S. L., J. S. Francis, R. A. Pagon, L. T. Smith, and V. P. Sybert. Prevalence of hypopigmented macules in a healthy population. J. Pediatr. 129:355–361, 1996. Westerhof, W., F. A. Beemer, R. H. Cormane, J. W. Delleman, W. R. Faber, J. G. de Jong, and W. W. van der Schaar. Hereditary congenital hypopigmented, and hyperpigmented macules. Arch. Dermatol. 114:931–936, 1978. Wiederholt, W. C., M. R. Gomez, and L. T. Kurland. Incidence, and prevalence of tuberous sclerosis in Rochester, Minnesota, 1950 through 1992. Neurology 35:600–603, 1985. Zvulunov, A., and N. B. Esterly. Neurocutaneous syndromes associated with pigmentary skin lesions [review]. J. Am. Acad. Dermatol. 32:915–935, 1995.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Genetic Hypomelanoses: Disorders Characterized by Hypopigmentation of Hair Sections Bird-headed Dwarfism (Seckel Syndrome) Stan P. Hill Down Syndrome Rosemary Geary Fisch Syndrome Stan P. Hill Premature Canities James J. Nordlund Mandibulofacial Dysostosis (Treacher Collins Syndrome) Rosemary Geary Myotonic Dystrophy Peggy Tong PHC Syndrome (Böök Syndrome) Stan P. Hill Pierre Robin Syndrome James J. Nordlund Prolidase Deficiency Pranav B. Sheth

Bird-headed Dwarfism (Seckel Syndrome) Stan P. Hill In 1960, Dr. Seckel published a report on two of his patients with dwarfism and reviewed an additional 13 cases dating back to the 1700s that he distinguished from other types of dwarfism. These patients were characterized by “extreme statural shortness, dwarfish size of head and brain, mental retardation short of true idiocy, age proportionateness of body axis and head, a beaklike protrusion of the central face, receding chin and frequent association with multiple congenital malformations” (Seckel, 1960).

Synonym Bird-headed dwarfism.

Epidemiology and Genetics Seckel syndrome appears to follow an autosomal recessive inheritance (McKusick et al., 1967). Both sexes have similar severe clinical manifestations and there is a slight female-tomale predominance of about 11:9 (Majewski and Goecke, 1982).

Clinical Findings Seckel criteria describe the classic findings of this disorder. In 1982, Majewski and Goecke (1982) reviewed 60 reported cases of Seckel syndrome dating back to the 1950s. Of these, more than two-thirds did not meet the diagnostic criteria proposed by Seckel. More recently, the criteria have been revised. The constant features include severe intrauterine and

postnatal growth retardation, severe microcephaly, severe mental retardation, typical face consisting of a prominent birdlike nose, a hypoplastic face with relatively large eyes and teeth, and skeletal defects (Majewski and Goecke, 1982). Pigmentary abnormalities are not mentioned in the original or revised criteria of Seckel syndrome. There are, however, reports of individuals with pigmentary disorders who have some features suggestive of Seckel syndrome. These cases, summarized below, appear to represent rare variant presentations of this equally rare syndrome. In 1974, a report from Malaysia described two Indian sisters, both of whom had numerous findings, including growth and mental retardation, peculiar facies, trident hands, premature canities, peripheral vitiligo, café au lait macules, and multiple lentigines. The facial features were not consistent with those found in bird-headed dwarfism and do not fit neatly under the definition of Seckel syndrome. These sisters may represent a variation of dwarfism in which the typical facial features are not prominent but pigmentary alterations are present (Tay et al., 1974). In 1970, a case was reported from Canada of a male with dwarfism and gradual onset of bird-headed features. He was noted to have male-pattern alopecia and graying of the scalp hair starting at age 18 years. Other cutaneous findings in this individual included palmar skin redundancy characteristic of senile skin (Fitch et al., 1970). This individual, while not meeting all the criteria of Seckel syndrome, appeared to have some variation of bird-headed dwarfism with premature senility and canities (Fitch et al., 1970). This case has been referred to as the “Montreal type” of bird-headed dwarfism (Rook et al., 1992). 657

CHAPTER 33

Differential Diagnosis There are many criteria that classify an individual as having Seckel syndrome. The following syndromes share some characteristics of Seckel syndrome and should be included in the differential diagnosis: Dubowitz syndrome, alcohol embryopathy, Brachmann–de Lange syndrome, trisomy 18, Bloom syndrome, Fanconi pancytopenia, and osteodysplastic primordial dwarfism type II and type III (Majewski et al., 1982a, b; Seckel, 1960).

Pathogenesis The etiology of Seckel syndrome is unknown. It has been suggested that chromosomal instability similar to that seen in Bloom syndrome may result in Seckel syndrome. It has been demonstrated that there are a normal number of chromosomal breaks and low number of sister chromatid exchanges in Seckel syndrome (Cervenka et al., 1979). Hormonal abnormalities have also been suggested as the cause of Seckel syndrome but studies have been unable to detect endocrine abnormalities.

Treatment and Prognosis No specific treatments are known, and there are many individuals with Seckel syndrome who have lived a normal duration of life (McKusick et al., 1967).

References Cervenka, J., H. Tsuchiya, T. Ishiki, M. Suzuki, and H. Mori. Seckel’s dwarfism: analysis of chromosome breakage and sister chromatid exchanges. Am. J. Dis. Child. 133:555–556, 1979. Fitch, N., L. Pinsky, and R. L. Lachance. A form of bird-headed dwarfism with features of premature senility. Am. J. Dis. Child. 120:260–264, 1970. Majewski, F., and T. Goecke. Studies of microcephalic primordial dwarfism I: approach to a delineation of the Seckel’s syndrome. Am. J. Med. Genet. 12:7–21, 1982. Majewski, F., M. Ranke, and A. Schinzel. Studies of microcephalic dwarfism II: the osteodysplastic type II of primordial dwarfism. Am. J. Med. Genet. 12:23–35, 1982a. Majewski, F., M. Stoeckenius, and H. Kemperdick. Studies of microcephalic primordial dwarfism II: an intrauterine dwarf with platyspondyly and anomalies of pelvis and clavicles—osteodysplastic primordial dwarfism type III. Am. J. Med. Genet. 12:37–42, 1982b. McKusick, V. A., M. Mahloudji, M. H. Abbott, R. Lindenberg, and K. Demetrios. Seckel’s bird-headed dwarfism. N. Engl. J. Med. 277:279–286, 1967. Rook, A., D. S. Wilkinson, and F. J. G. Ebling. Textbook of Dermatology, 5th ed. London: Blackwell Scientific Publications, 1992. Seckel, H. P. G. Studies in Developmental Anthropology Including Human Proportions. Springfield, IL: Bannerstone House, 1960. Tay, C. H., E. Rajagopalan, E. McEvoy-Bowe, E. P. C. Tock, and J. L. Da Costa. A recessive disorder with growth and mental retardation, peculiar facies, abnormal pigmentation, hepatic cirrhosis and aminoaciduria. Acta Paediatr. Scand. 63:777–782, 1974.

Down Syndrome

This syndrome is a multisystem disorder characterized by well-defined phenotypic features. Some of the more common characteristics of Down syndrome are mental deficiency, hypotonia, and short stature with an awkward gait. The facial features are flat facies, slanted palpebral fissures, small nose with flat nasal bridge, inner epicanthal folds, and small ears. There is a tendency for the individual to let the mouth hang open and the tongue protrude. The hands have short metacarpals and phalanges, and a single upper palmar crease; the feet have a wide gap between the first and second toes. Cardiac anomalies occur in about 40% of affected patients. There is an increased incidence of leukemia, seizures, and intestinal malformations such as duodenal atresia. Cutaneous manifestations of Down syndrome include loose folds of skin around the posterior neck, cutis marmorata, elastosis perforans serpiginosa, syringomas, atopic dermatitis, cheilitis, onychomycosis, tinea pedis, alopecia areata, and xerosis. There are no pigmentation disorders that are pathognomonic for Down syndrome. The most common pigmentary disorder is vitiligo. Carter and Jegasothy (1976) reported four cases of vitiligo in 214 institutionalized patients with Down syndrome, an incidence of 1.9%. Three of the four patients also had alopecia areata. The prevalence of vitiligo in the world population has been estimated to be between 0.14% and 8.8%. The actual prevalence is likely to be about 1 per 200 individuals (Mosher et al., 1993). The prevalence of vitiligo in those with Down syndrome is about the same as it is in the general population. Premature graying of hair has been noted in the literature, but there are few documented cases. Carter and Jegasothy (1976) noted three patients, ages ranging from 24 to 27 years, with premature graying. Other isolated pigmentary disorders noted in the literature include acral lentiginous melanoma in a 39-year-old female (Nakano et al., 1993). Also noted were a giant, hairy nevus and two café au lait macules (Carter and Jegasothy, 1976). The number of café au lait macules per patient was not mentioned.

Pathogenesis Down syndrome is caused by an extra copy of chromosome 21 (called trisomy 21 syndrome). The most common cause of trisomy 21 is nondisjunction during meiosis of the egg. Faulty chromosome distribution leading to Down syndrome is more likely to occur with increasing maternal age. The incidence is 1 : 1500 live births from mothers who are 15 to 29 years of age; 1 : 800 from those 30 to 34 years; 1 : 270 during 35 to 39 years; 1 : 100 in those 40 to 44 years; and 1 : 50 in those mothers over 45 years of age (Smith and Jones, 1982). Other, less common causes of Down syndrome are chromosomal translocation and mosaicism.

Rosemary Geary

Clinical Findings Down syndrome was originally described in 1866 by Down. 658

References Carter, D. M., and B. Jegasothy. Alopecia areata and Down’s syndrome. Arch. Dermatol. 112:1397–1399, 1976.

GENETIC HYPOMELANOSES: DISORDERS CHARACTERIZED BY HYPOPIGMENTATION OF HAIR Down, J. L. H. Observations on an ethnic classification of idiots. Clinical Lecture Reports, London Hospital 3:259, 1866. Mosher, D. B., T. B. Fitzpatrick, Y. Hori, and J. P. Ortonne. Disorders of melanocytes. In: Dermatology in General Medicine, 4th ed., T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M. Freedberg, and K. F. Austen (eds). New York: McGraw-Hill, 1993, pp. 903–1116. Nakano, J., M. Muto, K. Arikawa, T. Hirota, and C. Asagami. Acral lentiginous melanoma associated with Down’s syndrome. J. Dermatol. 20:59–60, 1993. Smith, D., and K. Jones. Recognizable Patterns of Human Malformation, 3rd ed. Philadelphia: W. B. Saunders, 1982.

Fisch Syndrome Stan P. Hill In 1959, Fisch published the results of an investigation on a hereditary pigmentary syndrome associated with deafness. Fisch noted 13 of the 25 family members with one or more of the following characteristics: early and pronounced graying of the hair, impaired hearing, and partial heterochromia irides. The most prominent sign was the early and extensive premature canities. There usually was total scalp involvement by 20 years of age. No other pigmentary disorders were noted in this family (Fisch, 1959). These features are suggestive of Waardenburg syndrome. Whether Fisch syndrome represents a type of Waardenburg syndrome or is a separate entity has yet to be fully determined (Ortonne, 1988). The differential diagnosis of Fisch syndrome includes Waardenburg syndrome (see Chapter 29), Woolf syndrome (piebaldism with deafness), and Rozycki syndrome (see Chapter 30) (leukoderma with deafness (Mosher et al., 1993).

References Fisch, I. Deafness as part of an hereditary syndrome. J. Laryngol. Otol. 53:355–382, 1959. Mosher, D. B., T. B. Fitzpatrick, Y. Hori, and J. P. Ortonne. Disorders of melanocytes. In: Dermatology in General Medicine, 4th ed., T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M. Freedberg, and K. F. Austen (eds). New York: McGraw-Hill, 1993, pp. 903–1116. Ortonne, J. P. Piebaldism, Waardenburg’s syndrome, and related disorders. Dermatol. Clin. 6:205–216, 1988.

Premature Canities James J. Nordlund That human hair loses its color as an individual ages is of great interest to the cosmetic industry. Many individuals use dyes and bleaches to alter or maintain the color of the hair of their youth. Graying is considered a sign of aging (Kwofh and Qlah, 1965). It is important to differentiate between gray hair and white hair. The latter is devoid of all melanocytes and pigmentation. Gray hair has persisting color but the melanosomes are aberrantly distributed. White hair is often familial. Kinships with striking early whitening of the hair are common. Often the parents have had white hair for as long as the offspring can recall. Photographs of the parents as teenagers or as young adults are the only indication that their hair was nor-

mally colored at one time. By their late twenties or midthirties, their hair is totally white although a few pigmented hairs might be found with very careful inspection. Usually only the scalp is involved. Personal observations indicate that periodically an occasional dark hair will grow. It is striking from family photographs that as siblings reach the beginning of their fourth decade of life, their hair loses its color. About 5% of individuals will have whitening of their hair by the fourth decade of life (Kwofh and Qlah, 1965). The mechanims for loss of hair color are not fully known. Following medical therapies like radiation and chemotherapy, scalp hair commonly falls out. After therapy is complete, white hair is replaced by pigmented hair (Cline, 1988). This observation suggests there is a reservoir of melanocyte precursor. It is known that hairs have two populations of melanocytes. Those in the bulb are pigmented. There is another nonpigmented population of melanocyte stem cells in the niche (Nishimura, 2005; Steingrimsson, 2005). The presence of stem cells seems necessary to prevent loss of the melanocytes in the bulb. Premature loss of the niche stem cells results in graying of hair in mice. In humans, there is a progressive loss of stem cells as individuals age and hair becomes gray (Nishimura, 2005; Steingrimsson, 2005). Gray hair is a function of aging and by age 60 years virtually all individuals will have at least a few gray hairs (Kwofh and Qlah, 1965). Certain disease states like vitiligo predispose individuals to early development of gray hairs (see Figs 30.21 and 30.22, Chapter 30). Lists of genetic and acquired disorders have been published previously (Ortonne et al., 1983). The presence of white hair on all or part of the scalp can be a useful criterion for confirming the diagnosis of a genetic or congenital disorder. It also has been suggested that graying of the hair is an indicator of aging. It has been proposed that early graying (before the age of 40 years) is a predictor of coronary heart disease (Eisenstein and Edelstein, 1982; Schnohr et al., 1995). However, other studies have not confirmed these reports (Glasser, 1991). Several newer syndromes associated with immunodeficiencies have suggested that gray or white hair develop as part of the syndrome. These include ataxia telangiectasia (Cohen et al., 1984), Griscelli’s syndrome (Schneider et al., 1990) or similar unnamed disorders (Harfi et al., 1992). Since the early lists of disorders were published (Ortonne et al., 1983), other causes of loss of hair or skin color have been reported such as pernicious anemia (Noppakun and Swasdikul, 1986). Typically the latter disorder is associated with hyperpigmentation.

References Cline, D. J. Changes in hair color [review]. Dermatol. Clin. 6:295–303, 1988. Cohen, L. E., D. J. Tanner, H. G. Schaefer, and W. R. Levis. Common and uncommon cutaneous findings in patients with ataxia telangiectasia. J. Am. Acad. Dermatol. 10:431–438, 1984. Eisenstein, I., and J. Edelstein. Gray hair in black males a possible risk factor in coronary artery disease. Angiology 33:652–654, 1982. Glasser, M. Is early onset of gray hair a risk factor? Med. Hypotheses 36:404–411, 1991.

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CHAPTER 33 Harfi, H. A., J. Brismar, B. Hainau, and R. Sabbah. Partial albinism, immunodeficiency, and progressive white matter disease: a new primary immunodeficiency. Allergy Proc. 13:321–328, 1992. Kwofh, W. C., and E. J. Qlah. Biology: rate of greying of human hair. Nature 207:877–878, 1965. Nishimura, E. S. G., and D. E. Fisher. Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche. Science 307:720–724, 2005. Noppakun, N., and D. Swasdikul. Reversible hyperpigmentation of skin and nails with white hair due to vitamin B12 deficiency. Arch. Dermatol. 122:896–899, 1986. Ortonne, J. P., D. B. Mosher, and T. B. Fitzpatrick. Approach to the problem of leukoderma. In: Vitiligo and Other Hypomelanoses of the Hair and Skin, J. P. Ortonne, D. B. Mosher, and T. B. Fitzpatrick (eds). New York: Plenum Medical Book Company, 1983, pp. 37–56. Schneider, L. C., R. S. Berman, C. R. Shea, A. R. Perez-Atayde, H. Weinstein, and R. S. Geha. Bone marrow transplantation (BMT) for the syndrome of pigmentary dilution and lymphohistiocytosis (Griscelli’s syndrome). J. Clin. Immunol. 10:146–153, 1990. Schnohr, P., P. Lange, J. Nyboe, M. Appleyard, and G. Jensen. Gray hair, baldness and wrinkles in relation to myocardial infarction: the Copenhagen City Heart Study. Am. Heart J. 130:1003–1010, 1995. Steingrimsson, E. N. C. Melanocyte stem cell maintenance and hair graying. Cell 121:9–12, 2005.

Mandibulofacial Dysostosis (Treacher Collins Syndrome)

1982). Plastic reconstructive surgery can further improve facial features. There are anecdotal reports of premature graying associated with Treacher Collins syndrome, but the literature provides no documentation to support this. Kahana et al. (1986) described a patient with Treacher Collins syndrome who also had neurofibromatosis but no pigmentary changes.

References Collins, T. E. Case with symmetrical congenital notches in the outer part of each lower lid and defective development of the malar bones. Trans. Ophthalmol. Soc. U. K. 20:190–193, 1900. Fazen, L. E., J. Elmore, and H. L. Nadler. Mandibulo-facial dysostosis (Treacher Collins syndrome). Am. J. Dis. Child. 113:405–407, 1967. Franceschetti, A., and D. Klein. The mandibulofacial dysostosis, a new hereditary syndrome. Acta Ophthalmol. (Copenh.) 27:144–146, 1949. Kahana, M., M. Ronnen, M. Schewach-Millet, and A. Feinstein. Treacher Collins syndrome and neurofibromatosis. Int. J. Dermatol. 25:115–116, 1986. Mittman, D. L., and O. G. Rodman. Mandibulofacial dysostosis (Treacher Collins syndrome): a case report. J. Natl. Med. Assoc. 84:1051–1504, 1992. Smith, D., and K. Jones. Recognizable Patterns of Human Malformation, 3rd ed. Philadelphia: W. B. Saunders, 1982. Thomson, A. Notice of several cases of malformation of the external ear, together with experiments on the state of hearing in such persons. Monthly J. Med. Sci. 7:420–424, 1846.

Rosemary Geary

Myotonic Dystrophy This syndrome was first described by Thomson in 1846 but has been associated with Treacher Collins, who described two cases in 1900. Later, Franceschetti and Klein (1949) intensively studied the many variations of this syndrome and renamed it mandibulofacial dysostosis. Mandibulofacial dysostosis, or Treacher Collins syndrome, consists of multiple malformations resulting from the disappearance or abnormal development of the first pharyngeal arch. This aberrant development occurs during the fifth to ninth weeks of embryonic development. The disorder is transmitted as an autosomal dominant trait. It has a spontaneous mutation rate of approximately 60% (Smith and Jones, 1982). Clinical features include antimongoloid slanting, palpebral fissures, malar hypoplasia, mandibular hypoplasia, lower eyelid coloboma, partial to total absence of lower eyelashes, malformation of auricles, external ear canal defects, conductive hearing loss, and projection of scalp hair onto lateral cheeks. Intelligence is usually normal. Some skin findings include skin tags between auricle and angle of the mouth and blind fistulas. One patient with Treacher Collins syndrome also had scarring alopecia and acne keloidalis nuchae (Mittman and Rodman, 1992). The majority of affected individuals have abnormal hearing, and audiology testing should be performed as early as possible to detect hearing loss and to prevent delay in development (Fazen et al., 1967). Hearing may be corrected by hearing aids or surgery. The growth of facial bones during childhood improves appearance of affected infants (Smith and Jones, 660

Peggy Tong Myotonic dystrophy was first recognized and described in 1888 by Dana (Zellweger and Ionasescu, 1973). The syndrome was well characterized by Steinert in 1909 and Curschmann in 1925 and is occasionally referred to as Steinert–Curschmann disease (Zellweger and Ionasescu, 1973).

Synonyms This condition has also been called dystrophia myotonica, myotonia dystrophica, or myotonia atrophica (Zellweger and Ionasescu, 1973).

Epidemiology and Genetics The prevalence of myotonic dystrophy ranges from 1 to 5 per 10 000 general population (Bryant, 1980). Males and females are affected with equal frequency (Zellweger and Ionasescu, 1973). Myotonic dystrophy is transmitted as an autosomaldominant trait with variable phenotypic expression (Zellweger and Ionasescu, 1973). The gene defect has been mapped to chromosome 19 (Pizzuti et al., 1993).

Clinical Findings Onset of the clinical features usually occurs in the second to third decades. The classic presentation includes muscle wasting and weakness, particularly of the sternocleidomastoid, temporal, and facial muscles, that result in the characteristic hatchet facies with flattened cheeks and drooping jaw.

GENETIC HYPOMELANOSES: DISORDERS CHARACTERIZED BY HYPOPIGMENTATION OF HAIR

Myotonia is characterized by delayed relaxation of the muscles following voluntary contraction of muscles. Other features that might be observed include testicular atrophy, mental retardation, ptosis, myotonic cataracts, endocrinopathies such as diabetes mellitus, and skeletal abnormalities especially of the skull. Involvement of cardiac muscle may cause electrocardiographic changes and occasionally a fatal arrhythmia. A cutaneous finding that has been associated with myotonic dystrophy is the occasional occurrence of pilomatricomas that tend to be multiple and may be familial in these patients (Chiaramonti and Gilgor, 1978; Delfino et al., 1985). Individuals with myotonic dystrophy have been described as having a senile appearance resembling patients with Werner syndrome. They often exhibit premature frontal baldness, particularly in males but occasionally in females. Canities (premature graying of the hair) in the second or third decade has also been described (Birnbaum and Baden, 1987; Martin, 1978; Mosher et al., 1993). Myotonic dystrophy is frequently listed in the differential diagnosis of Werner syndrome, which is characterized by features of premature aging, including premature baldness and graying of the hair (Epstein et al., 1966; Martin, 1978; Zucker-Franklin et al., 1968). Myotonic dystrophy has even been said to simulate Werner syndrome in almost every aspect except for the prominent myopathy present in myotonic dystrophy (Zucker-Franklin et al., 1968). Atrophy of the skin, which is a well-known feature of Werner syndrome, has also been described in myotonic dystrophy (Birnbaum and Baden, 1987; Epstein et al., 1966). No treatment is currently available to halt the progression of the disease. A variety of medications, including procainamide, quinine, corticosteroids, and diphenylhydantoin, have been used in an attempt to improve the myotonia (Zellweger and Ionasescu, 1973). Although most patients become confined to a wheelchair 15 to 20 years after the onset of the disease, the prognosis can be extremely variable (Zellweger and Ionasescu, 1973). Life expectancy is often shortened owing to fatal arrhythmia or aspiration pneumonia (Zellweger and Ionasescu, 1973).

pathologic features, genetics and relationship to the natural aging process. Medicine (Baltimore) 45:177–221, 1966. Martin, G. M. Genetic syndromes in man with potential relevance to the pathobiology of aging. In: Genetic Effects on Aging, D. Bergsma and D. Harrison (eds). New York: Alan R. Liss, 1978, pp. 5–39. Mosher, D. B., T. B. Fitzpatrick, Y. Hori, and J. P. Ortonne. Disorders of melanocytes. In: Dermatology in General Medicine, 4th ed., T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M. Freedberg, and K. F. Austen (eds). New York: McGraw-Hill, 1993, pp. 903– 1116. Pizzuti, A., D. L. Friedman, and C. T. Caskey. The myotonic dystrophy gene. Arch. Neurol. 50:1173–1179, 1993. Zellweger, H., and Y. Ionasescu. Myotonic dystrophy and its differential diagnosis. Acta Neurol. Scand. 5(Suppl. 55):5–28, 1973. Zucker-Franklin, D., H. Rifkin, and H. G. Jacobson. Werner’s syndrome: an analysis of ten cases. Geriatrics 23:123–135, 1968.

PHC Syndrome (Böök Syndrome) Stan P. Hill In 1950, Böök published data from a study of 25 patients with an autosomal dominant syndrome that he designated PHC syndrome for premolar area hypodontia, hyperhidrosis of the palms and soles, and premature canities or graying. All 25 cases belonged to one Swedish family that had 172 members. Eighteen of the 25 individuals were examined in detail. The premature whitening of the scalp hair showed a variable expression, with the age of onset from 6 to 23 years of age. In one-third of the cases there was whitening of the axillary, genital, and eyebrow hair. No other hair abnormality was apparent and no pigmentary abnormalities of the skin were noted. These are the only reported cases to date.

Reference Böök, J. A. Clinical and genetic studies of hypodontia. I. Premolar aplasia, hyperhidrosis, and canities prematura; a new hereditary syndrome in man. Am. J. Hum. Genet. 2:240–263, 1950.

Pierre Robin Syndrome James J. Nordlund

Clinical Description Commentary Canities has been described in patients with myotonic dystrophy, primarily in textbooks, but is poorly documented in the literature. This clinical feature has been noted as a “known” fact, but has not been appropriately referenced.

References Birnbaum, P. S., and H. P. Baden. Heritable disorders of hair. Dermatol. Clin. 5:137–153, 1987. Bryant, C. D. Myotonic dystrophy: case report and review of the literature. J. S. C. Med. Assoc. 76:289–291, 1980. Chiaramonti, A., and R. S. Gilgor. Pilomatricomas associated with myotonic dystrophy. Arch. Dermatol. 114:1363–1365, 1978. Delfino, M., G. Monfrecola, F. Ayala, F. Suppa, and A. Piccirillo. Multiple familial pilomatricomas: a cutaneous marker for myotonic dystrophy. Dermatologica 170:128–132, 1985. Epstein, C. J., G. M. Martin, A. L. Schultz, and A. G. Motulsky. Werner’s syndrome: a review of its symptomatology, natural history,

This syndrome is characterized by three groups of defects: micrognathia, cleft palate, and glossoptosis. The latter is responsible for respiratory failure in the neonatal infant (Girard et al., 1989). Many of these children die during the newborn period unless given special care in a neonatal intensive care unit (Couly et al., 1988). In addition, these infants can manifest other autosomal defects such as congenital glaucoma, myopia, and retinal detachment. Microphthalmia has been reported but rarely (Girard et al., 1989). The cause of this syndrome is not known (Poswillo, 1988). There are some cutaneous abnormalities. One child had malformed hands. There was a sclerotic tapering of the fifth digit bilaterally; absence of the palmar flexure folds and the skin ridges; and an extra nail on the large toe (Roger et al., 1986). It has been reported that these children have light colored hair (Porter and Lobitz, 1970). 661

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References Couly, G., G. Cheron, J. de Blic, C. Despres, M. Cloup, and P. Hubert. The Pierre-Robin syndrome: Classification and new therapeutic approach [French]. Arch. Fr. Pediatr. 45:553–559, 1988. Girard, B., F. Topouzis, and H. Saraux. Microphthalmos in Pierre Robin syndrome: Clinical and x-ray computed tomographic study [review] [French]. Bull. Soc. Ophtalmol. France 89:1385–1390, 1989. Porter, P. S., and W. C. Lobitz. Human hair: a genetic marker. Br. J. Dermatol. 83:225–241, 1970. Poswillo, D. The aetiology and pathogenesis of craniofacial deformity [review]. Development 103(Suppl):207–212, 1988. Roger, H., P. Souteyrand, J. P. Collin, G. Vanneuville, and P. Teinturier. Onychoheterotopia with polyonychia associated with Pierre Robin syndrome: apropose of a new case (Trial of classifying nail ectopia) [French]. Ann. Dermatol. Venereol. 113:235–242, 1986.

Prolidase Deficiency Pranav B. Sheth Prolidase deficiency is an inborn error of metabolism characterized by iminodipeptiduria, skin ulcers, mental retardation, characteristic facies, and recurrent infections. The first case of prolidase deficiency was reported by Goodman in 1968. Since then, approximately 24 other cases have been reported (Bissonnette et al., 1993; Freij et al., 1984; Leoni et al., 1987; Milligan et al., 1989).

Cutaneous Manifestations The disease is inherited as an autosomal recessive disorder and shows variability in age of onset (from 3 months to 17 years) and in severity of clinical expression. More than 90% of patients with prolidase deficiency have some skin pathology. Recurrent multiple ulcers of the lower extremities are the most characteristic clinical findings (Freij and Der Kaloustian, 1986). Most patients have maculopapular, purpuric, and eczematous eruptions in the first years of life. Leg ulcerations develop near the time of puberty (Bissonnette et al., 1993). Other reported skin findings include telangiectases, scaly erythematous maculopapular lesions, purpuric lesions, postinflammatory pigmentation, and photosensitivity (Bissonnette et al., 1993; Freij et al., 1984; Milligan et al., 1989). In addition, five of 25 reported patients have shown premature graying of the hair (Arata et al., 1979; Freij et al., 1984; Ogata et al., 1981). The youngest age of onset of gray hair was 7 years. The etiology of the poliosis is unknown.

Other Findings Extracutaneous findings reported include splenomegaly, recurrent infections such as otitis media and sinusitis, hematologic disorders such as anemia and hypergammaglobulinemia, mental retardation, unusual facies, ocular findings, deafness, and short stature (Bissonnette et al., 1993; Freij et al., 1984; Milligan et al., 1989).

Pathogenesis Prolidase is a proline dipeptidase with strict specificity for sub662

strates containing proline or hydroxyproline at the carboxy terminal end of a peptide. Its gene maps to chromosome 19p13.2 (Ledoux et al., 1994). Loss of prolidase activity results in accumulation of dipeptides, which inhibits normal recycling of proline and causes alteration in the metabolism of collagen and other proteins that contain a high content of these amino acids (Leoni et al., 1987). Prolidase activity has been reported to be deficient in erythrocytes, leukocytes, and cultured skin fibroblasts obtained from affected individuals (Freij et al., 1984).

Histology Histologic examination of the skin around the leg ulcers of one patient revealed a deposition of an amorphous substance around the capillaries that stained with Congo red. The collagen fibers showed fragmentation and irregularities in arrangement (Ogata et al., 1981). No histopathology of the gray hairs has been described.

Diagnosis Diagnosis of prolidase deficiency is made by measuring prolidase activity in red blood cells and showing excessive excretion of iminodipeptides in the urine.

Treatment Treatment involves caring for the cutaneous manifestations (leg ulcers, eczematous eruption, etc.) with conventional therapies. Dietary supplementation of manganese, ascorbic acid, and l-proline; topical application of ascorbic acid, glycine, and proline to ulcers; and transfusion of normal erythrocytes have been recommended (Bissonnette et al., 1993).

Prognosis There are no long-term follow-up reports about the prognosis in these patients.

References Arata, J., S. Umemura, Y. Yamamoto, M. Hagiyama, and N. Nohara. Prolidase deficiency: its dermatological manifestations and some additional biochemical studies. Arch. Dermatol. 115:62–67, 1979. Bissonnette, R., D. Friedmann, J. M. Giroux, M. Dolenga, P. Hechtman, V. M. Der Kaloustian, and R. Dubuc. Prolidase deficiency: a multisystemic hereditary disorder. J. Am. Acad. Dermatol. 29:818–821, 1993. Freij, B. J., and V. M. Der Kaloustian. Prolidase deficiency: a metabolic disorder presenting with dermatologic signs. Int. J. Dermatol. 25:431–433, 1986. Freij, B. J., H. L. Levy, G. Dudin, D. Mutasim, M. Deeb, and V. M. Der Kaloustian. Clinical and biochemical characteristics of prolidase deficiency in siblings. Am. J. Med. Genet. 19:561–571, 1984. Goodman, S. I., C. C. Solomons, F. Muschenheim, C. A. McIntyre, B. Miles, and D. O’Brien. A syndrome resembling lathyrism associated with iminodipeptiduria. Am. J. Med. 45:152–159, 1968. Ledoux, P., C. Scriver, and P. Hechtman. Four novel PEPD alleles causing prolidase deficiency. Am. J. Hum. Genet. 54:1014–1021, 1994.

GENETIC HYPOMELANOSES: DISORDERS CHARACTERIZED BY HYPOPIGMENTATION OF HAIR Leoni, A., G. Cetta, R. Tenni, I. Pasquali-Ronchetti, F. Bertolini, D. Guerra, K. Dyne, and A. Castellani. Prolidase deficiency in two siblings with chronic leg ulcerations: clinical, biochemical and morphologic aspects. Arch. Dermatol. 123:493–499, 1987. Milligan, A., R. A. C. Graham-Brown, D. A. Burns, and I. Anderson.

Prolidase deficiency: a case report and literature review. Br. J. Dermatol. 121:405–409, 1989. Ogata, A., S. Tanaka, T. Tomoda, E. Murayama, F. Endo, and I. Kikuchi. Autosomal recessive prolidase deficiency: three patients with recalcitrant leg ulcers. Arch. Dermatol. 117:689–694, 1981.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Metabolic, Nutritional, and Endocrine Disorders Sections Metabolic and Nutritional Hypomelanoses Peter S. Friedmann Hypomelanosis Associated with Endocrine Disorders Peter S. Friedmann

Metabolic and Nutritional Hypomelanoses Peter S. Friedmann

Kwashiorkor Historical Background The first modern description of kwashiorkor is attributed to Williams who made careful long-term observations in malnourished children in the Gold Coast (Ghana) (Williams, 1953).

Epidemiology Kwashiorkor is a disease state seen in children often at the weaning stage. It is associated with protein malnutrition in the presence of adequate caloric intake. This dietary imbalance results in deficiencies of essential amino acids, hypoalbuminemia, edema, anemia, fatty change in the liver, growth retardation, irritability and/or apathy, and various cutaneous manifestations. Kwashiorkor is a significant cause of death in children between 1 and 4 years of age in all underdeveloped countries (Scrimshaw and Behar, 1961). It has been reported in all parts of Africa, the Far East including China, Malaysia, and Indonesia, parts of South America (Williams, 1953) and even the southern states of the USA (Henington et al., 1958; Scrimshaw and Behar, 1961). In 1972 kwashiorkor was reported to affect between 0.5% and 1.5% of the world’s population (Ebrahim, 1972).

Clinical Findings (Table 34.1) In a survey of 1565 cases in Durban, Scragg and Rubidge (1960) observed that cutaneous manifestations of kwashiorkor were present in up to 82% of affected individuals. Skin changes usually begin with hypopigmentation of the face and may precede other clinical features of kwashiorkor (Fig. 34.1). In addition to the facial pallor there may be extensive hyperpigmentation or hypopigmentation of other parts of the integument (Plate 34.2, pp. 494–495). 664

Hyperkeratoses and scaling develop, which are usually hyperpigmented. In areas subject to friction or trauma the scale fractures to produce a “crazy paving stone” dermatitis (Banerjee and Dutta, 1975; Venkatachalam et al., 1954). The scale strips away easily, and where it peels, it leaves erosions that turn into specially pale patches. This appearance is described as resembling “flaky paint” (McLaren, 1994) or “enamel paint” (Scragg and Rubidge, 1960) (Fig. 34.1). There may be angular cheilitis and desquamation at the outer canthi (Williams, 1953). An additional skin rash, often more visible in paler-skinned babies, starts as erythematous patches and superficial erosions that become more extensive and may look like superficial burns. The child may be described as a red baby with a background of fairer skin (Ebrahim, 1972). The erythematous areas darken into reddish, blue-black spots that resemble purpura. They may coalesce and become dry, hard, scaly and raised. When the scale separates, the underlying skin is pale, thin, and smooth (Henington et al., 1958). As healing occurs, hyperpigmentation is common. In a survey of 207 children in Coonoor, India, Venkatachalam et al. (1954) noted crazy paving dermatitis in 18% and purpuralike spots in 3%. The hair is finer and straighter than normal. Typically it is dull, lusterless, brittle, and easily pulled out. This fragility can result in partial, diffuse alopecia of the scalp. Often there also is thinning of eyebrows (Banerjee and Dutta, 1975). The hair may be of normal color but can be hypopigmented (see Plate 34.2, pp. 494–495). The hair changes are more extreme in children of African origin than in those from the Indian subcontinent or Far East (Jelliffe, 1955; Mukherjee and Jelliffe, 1955). The whole head may be involved or there may be a fringe of discoloration around the hair line — a halo effect (Jelliffe, 1955). The hue and intensity of discoloration have little predictive value in relation to either the severity or the chronicity of kwashiorkor (Bradfield and Jelliffe, 1974). Sometimes there are bands of hypopigmentation (the flag sign) corresponding to periods of worse malnutrition (Banerjee and Dutta, 1975; McLaren, 1987, 1994; Miller, 1989). The diameter of the hair shafts is reduced. Sims (1967)

METABOLIC, NUTRITIONAL, AND ENDOCRINE DISORDERS Table 34.1. Clinical features of kwashiorkor. Skin

Hair Nails Mucosa Systemic features

Laboratory findings

“Crazy paving” or “enamel paint” dermatitis that hyperpigments, purpuralike lesions, hypopigmentation. Hypopigmentation, alopecia, hair easily pulled out Thinning, softening, ridges Angular cheilitis, conjunctivitis, xerophthalmia, Bitot spots Growth retardation, diarrhea, muscle wasting, mental changes, irritability, apathy, edema, hepatomegaly Hypoalbuminemia, anemia, hypovitaminosis, fatty change in liver

of five healthy infants. The cellular (Malpighian) layers of epidermis were thinned and the desmosomes were shorter, qualities that may explain the epidermal fragility (Sims, 1968). There is only sparse melanin in the basal layer (Banerjee and Dutta, 1975).

Pathogenesis of Hypomelanosis Most information regarding pathomechanisms of cutaneous hypomelanosis comes from studies on color of hair. The pigment extracted from red hair of kwashiorkor has been shown to be chromatographically different from normal red hair pigment (Nagchaudhuri and Platt, 1954). Chemically it is most similar to hair pigment extracted from hooded rats fed on methionine-deficient diets or from animals exposed to longterm ultraviolet radiation (Nagchaudhuri and Platt, 1954). Normal hair pigment exposed to hydrogen peroxide acquires similar chromatographic properties. Analysis of hair amino acids in kwashiorkor showed reduced cystine levels (Ebrahim, 1972; Narasinga Rao and Gopalan, 1957). In hair showing the flag sign of alternating light and dark bands, the cystine content of the dark bands is higher than that of the light bands but is significantly less than normal (Narasinga Rao and Gopalan, 1957). These findings are compatible with the concept that there is a deficiency of sulfur-containing amino acids. These amino acids can function as antioxidants. A deficiency might result in increased damage to hair melanin from ultraviolet and oxidative mechanisms causing a change of color. There are few data about the origin of the hypomelanosis. It might be speculated that the quantity of tyrosine is insufficient in the epidermis to support the normal production of melanin. Tyrosine might be deficient either due to insufficient intake of tyrosine or its precursor phenylalanine. Phenylalanine is converted to tyrosine through the tetrahydrobiopterin pathway (Schallreuter et al., 1994).

Therapy Fig. 34.1. African child with kwashiorkor showing the “enamel paint” rash on his leg (see also Plate 34.1, pp. 494–495).

Kwashiorkor is treated with protein supplementation. The changes of skin and hair are reversed by proper nutritional treatment.

Other Nutritional Causes of Hypopigmentation reported that the 95% confidence intervals for the long and short diameters of oval hair shafts in 12 healthy Zulu children was 77 m to 39 m. Growth rate was 0.23 mm per day and mean volume of hair was 514 m3 per follicle. In 12 children with kwashiorkor the confidence intervals for shaft diameters were 50 m to 29 m. The growth rate was 0.13 mm per day and mean hair volume was 59 m3.

Histology At the light microscopic level there is dermal edema, separation of collagen fibers, scanty cellular infiltration, and an atrophic epidermis with or without hyperkeratosis and parakeratosis. Sims (1968) compared the histologic features of the epidermis from 10 Zulu babies with kwashiorkor with those

Change in hair color has been recorded in severe protein loss from a variety of causes. Protein loss and malnutrition secondary to bowel disorders has been reported to cause a kwashiorkorlike syndrome (Silverblatt and Brown, 1960). Silverblatt and Brown (1960) observed that a male subject, who for 10 years had diarrhea and hypoalbuminemia following a gastrectomy for duodenal ulceration, developed many manifestations of protein deficiency. These included muscle wasting, ankle edema, and extensive desquamation. He also had various features of vitamin deficiency, endocrine hypofunction, loss of secondary sexual hair, and loss of much scalp hair. The persisting hair was straight, fine and reddish. Tissue obtained by liver biopsy showed moderately severe fatty change. Following protein replenishment with 20 units of 665

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plasma the patient made a good recovery with regrowth of black terminal hair. Mellinkoff (1957) observed reddening of the hair and malnutrition in a 26-year-old white patient with severe ulcerative colitis. With effective treatment and regain of weight, the hair returned to its normal dark-brown color. Petrozzi (1971) observed a 21-month-old black child who developed progressive hypopigmented macules associated with recurrent diarrhea, poor nutrition, failure to thrive, and growth retardation. The loss of pigment began at age 2 months and by 4 months the scalp, palms, soles, and the entire integument except the central face, were affected. There was a speckled or mottled appearance caused by irregular, scattered hypopigmented and hyperpigmented macules. Scalp hair was thin, brittle, and lusterless. The condition remained unchanged until 21 months of age. The author suggested that the pigmentary changes could have been a result of malnutrition but no comment was made on whether full nutrition was associated with reversal of these changes.

Iron Deficiency Anemia Hill (1980) observed two individuals whose hair became prematurely gray and then white. Twenty years later both patients were found to have adult celiac disease and significant iron deficiency anemia. Within weeks of starting a gluten-free diet, the anemia was corrected and there was substantial repigmentation of the hair. Sato et al. (1989) observed segmented heterochromia — irregularly alternating dark and light bands — in the hair of a 15-year-old girl with severe iron deficiency anemia. The authors also cite two other similar cases. The color of eyebrows, lashes, and pubic and axillary hair was normal. The heterochromic bands disappeared and the hair returned to its normal black color after initiation of therapy with ferrous sulfate. Microscopic examination showed that in the white bands there were few or no cortical melanosomes. This finding was confirmed by chemical analysis. The content of eumelanin in the banded hairs was only 34 ng/mg. After repigmentation the melanin content increased to 53 ng/mg.

Vitamin B12 Deficiency (Pernicious Anemia) and Folic Acid Deficiency Pernicious anemia is associated with a variety of autoimmune phenomena, including organ-specific antibodies and with vitiligo (Wintrobe, 1975). Pernicious anemia was also associated with a higher incidence of premature graying (Dawber, 1970). Dawber compared 125 patients with pernicious anemia with 132 control subjects. The prevalence of blond hair and blue eyes was twofold higher in those with pernicious anemia than in control subjects. Also, graying of hair before the age of 20 years was observed in 14 of 125 patients with pernicious anemia compared with 3 of 135 controls. The onset of graying occurred between 20 and 50 years of age in 60 of 125 individuals with pernicious anemia compared with 38 of 135 control subjects. Dawber did not comment on the effect of supplementation with vitamin B12 on the gray hair. 666

Fig. 34.2. Hyperpigmentation of the knuckles of a young patient with folic acid deficiency (see also Plate 34.3, pp. 494–495).

Noppakun and Swasdikul (1986) reported an oriental man with hyperpigmentation of skin and hypopigmentation of the nails and hair. These pigmentary changes returned to normal following vitamin B12 therapy. Carmel (1985) described two Latin American patients with pernicious anemia, one hereditary and one acquired, who had reddish hypopigmentation of the hair. After treatment with vitamin B12 the hair returned to its normal dark-brown color. Patients with severe folic acid deficiency develop hyperpigmentation of gums (Plate 34.4, pp. 494–495) and face (Plate 34.5, pp. 494–495). The knuckles often become hyperpigmented (Fig. 34.2).

References Banerjee, B. N., and A. K. Dutta. Malnutrition in the tropics; dermatoses in nutritional disorders. In: Clinical Tropical Dermatology, O. Canizares (ed.). Oxford: Blackwell Scientific, 1975, pp. 273–277. Bradfield, R. B., and D. B. Jelliffe. Hair-colour changes in kwashiorkor. Lancet 1:461–462, 1974. Carmel, R. Hair and fingernail changes in acquired and congenital pernicious anaemia. Arch. Intern. Med. 145:484–485, 1985. Dawber, R. P. R. Integumentary associations of pernicious anaemia. Br. J. Dermatol. 82:221–223, 1970. Ebrahim, G. J. The skin in malnutrition. In: Essays on Tropical Dermatology, J. Marshall (ed.). Amsterdam: Excerpta Medica, 1972, pp. 124–128. Henington, V. M., E. Caroe, V. Derbes, and B. Kennedy. Kwashiorkor: Report of four cases from Louisiana. Arch. Dermatol. 78:157–170, 1958. Hill, L. S. Reversal of premature hair graying in adult coeliac disease. Br. Med. J. 281:115, 1980. Jelliffe, D. B. Hypochromotrichia and malnutrition in Jamaican infants. J. Trop. Pediatr. 1:25–33, 1955. McLaren, D. S. Skin in protein energy malnutrition. Arch. Dermatol. 123:1674–1676, 1987. McLaren, D. S. Cutaneous changes in nutritional disorders. In: Dermatology in General Medicine, T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M. Freedberg, and K. F. Austen (eds). New York: McGrawHill, 1994, pp. 1601–1613. Mellinkoff, S. M. Temporary redness of the hair in ulcerative colitis. Am. J. Dig. Dis. 2:738–739, 1957.

METABOLIC, NUTRITIONAL, AND ENDOCRINE DISORDERS Miller, S. J. Nutritional deficiency and the skin. J. Am. Acad. Dermatol. 21:1–30, 1989. Mukherjee, K. L., and D. B. Jelliffe. Clinical observations on kwashiorkor in Calcutta. J. Trop. Pediatr. 1:61–66, 1955. Nagchaudhuri, J., and B. S. Platt. A change of hair pigment of malnourished children revealed by paper chromatography. In: Malnutrition in African Mothers, Infants and Young Children. Report of the 2nd Inter-African Conference on Nutrition, 1952, Fajara, Gambia, Inter-African Conference on Nutrition (eds). London: Her Majesty’s Stationery Office, 1954, pp. 215–216. Narasinga Rao, B. S., and C. Gopalan. Some aspects of the hair colour changes in kwashiorkor. Indian J. Med. Res. 45:85–93, 1957. Noppakun, N., and D. Swasdikul. Reversible hyperpigmentation of skin and nails with white hair due to vitamin B12 deficiency. Arch. Dermatol. 122:896–899, 1986. Petrozzi, J. W. Unusual dyschromia in a malnourished infant. Arch. Dermatol. 103:515–519, 1971. Sato, S., K. Jitsukawa, H. Sato, M. Yoshino, S. Seta, S. Ito, Y. Hayashi, and T. Anzai. Segmented heterochromia in black scalp hair associated with iron-deficiency anaemia. Arch. Dermatol. 125:531– 535, 1989. Schallreuter, K. U., J. M. Wood, M. R. Pittelkow, M. Gutlich, K. R. Lemke, W. Rodl, N. N. Swanson, K. Hitzemann, and I. Ziegler. Regulation of melanin biosynthesis in the human epidermis by tetrahydrobiopterin. Science 263:1444–1446, 1994. Scragg, J., and C. Rubidge. Kwashiorkor in African children in Durban. Br. Med. J. 2:1759–1766, 1960. Scrimshaw, N. S., and M. Behar. Protein malnutrition in young children. Science 133:2039–2047, 1961. Silverblatt, C. W., and H. E. Brown. “Kwashiorkor-like” syndrome, associated with burning feet syndrome, in an adult male. Am. J. Med. 28:847–854, 1960. Sims, R. T. Hair growth in kwashiorkor. Arch. Dis. Child. 42: 397–400, 1967. Sims, R. T. The ultrastructure of depigmented skin in kwashiorkor. Br. J. Dermatol. 80:822–832, 1968. Venkatachalam, P. S., S. G. Srikantia, and C. Gopalan. Clinical features of nutritional oedema syndrome in children. Indian J. Med. Res. 42:555–568, 1954. Williams, C. D. Kwashiorkor. J. Am. Med. Assoc. 153:1280–1285, 1953. Wintrobe, M. M. Pernicious anaemia. In: Clinical Haematology, M. M. Wintrobe (ed.). Philadelphia: Lea and Febiger, 1975, pp. 602–620.

Hypomelanosis Associated with Endocrine Disorders Peter S. Friedmann Vitiligo is associated with several of the endocrinopathies which have an autoimmune basis. These include hyperthyroidism, Addison disease, hypoparathyroidism (Fields et al., 1971; Fisher and Fitzpatrick, 1970), and diabetes mellitus. In addition, premature graying of hair may accompany these conditions.

Hypopituitarism People with hypopituitarism are often lightly pigmented and have a “washed out” appearance (Lang, 1981; Sheehan, 1939). The hypomelanosis helps distinguish hypoadrenalism due to Addison disease in which hyperpigmentation occurs, from hypoadrenalism secondary to hypopituitarism. The

pallor is thought to be due to reduced production of melanocyte-stimulating hormone (MSH) and related peptides including adrenocorticotropic hormone (ACTH). Administration of a-MSH has been reported to result in striking repigmentation and even a “tanned hyperpigmentation” (Hadley and Levine, 1993). Interestingly, congenital panhypopituitarism in black dwarfs is not associated with pigmentary changes (Hadley and Levine, 1993). Also, hypophysectomy in normal black individuals does not result in loss of pigment (Hadley and Levine, 1993). The hypomelanosis is most marked in the sexual areas. Also, the tanning response to ultraviolet radiation is reduced (Lang, 1981).

Hypogonadism Castrated human males are characteristically pale (Edwards et al., 1941). They also exhibit an impaired ability to tan in response to ultraviolet radiation. Administration of testosterone restores the skin to a darker and more ruddy hue. Moreover, testosterone restores the capacity to tan after ultraviolet radiation (Edwards et al., 1941). Hamilton and Hubert (1938) reported an interesting effect of testosterone in the case of a castrated man with pale skin who did not tan much during the summer of observation. Some months after the summer sun exposure, he was treated with testosterone and all the previously sun-exposed sites developed a bronzed tan, whereas, sites previously sun shielded by a bathing costume failed to darken. Thus, it appeared that melanin biosynthesis was primed by ultraviolet light but the priming could not be expressed until testosterone was provided.

Cushing Syndrome Pigmentary changes are not a common association of Cushing syndrome. However, Brooks and Richards (1966) reported striking depigmentation in two black West Indian women with Cushing syndrome. In both, the hypopigmentation affected the face and extremities. Following subtotal adrenalectomy, the hypomelanosis returned completely to normal. The authors explain this observation on the presumption that the high cortisol levels had suppressed pituitary production of MSH. They also suggested such hypomelanosis is actually more common than has been suspected, but is underreported because it is hard to observe in white skin.

References Brooks, V. E. H., and R. Richards. Depigmentation in Cushing’s syndrome. Arch. Intern. Med. 117:677–680, 1966. Edwards, E. A., J. B. Hamilton, S. Q. Duntley, and G. Hubert. Cutaneous vascular and pigmentary changes in castrate and eunuchoid men. Endocrinology 28:119–128, 1941. Fields, J. P., L. Fragola, and T. P. Hadley. Hypoparathyroidism, candidiasis, alopecia and vitiligo. Arch. Dermatol. 103:687–688, 1971. Fisher, M., and T. B. Fitzpatrick. Candidiasis, vitiligo, Addison’s disease and hypoparathyroidism. Arch. Dermatol. 102:110–111, 1970. Hadley, M. E., and N. Levine. Hormonal control of melanogenesis. In: Pigmentation and Pigmentary Disorders, N. Levine (ed.). Boca Raton: CRC Press, 1993, pp. 95–114.

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CHAPTER 34 Hamilton, J. B., and G. Hubert. Photographic nature of tanning of the human skin as shown by studies of male hormone therapy. Science 88:481, 1938. Lang, P. G. Pituitary disorders. In: Cutaneous Aspects of Internal

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Disease, J. P. Callen (ed.). Chicago: Year Book Medical Publishers, 1981, pp. 463–471. Sheehan, H. L. Simmonds’ disease due to post-partum necrosis of the anterior pituitary. Q. J. Med. 8:277–309, 1939.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Chemical, Pharmacologic, and Physical Agents Causing Hypomelanoses Sections Chemical and Pharmacologic Agents Causing Hypomelanoses Stefania Briganti, Monica Ottaviani, and Mauro Picardo Physical Agents Causing Hypomelanoses Jean-Phillipe Lacour

Chemical and Pharmacologic Agents Causing Hypomelanoses Stefania Briganti, Monica Ottaviani, and Mauro Picardo

Introduction Acquired Hypomelanosis: Workers Exposed to Chemical Depigmentants Skin pigmentation depends on the production and concentration of melanin in melanosomes, specific lysosome-like organelles located within melanocytes (Seiji et al., 1961) and, subsequently, involves the transfer of melanized melanosomes to surrounding keratinocytes, which, through differentiation and migration towards the stratum corneum, provide the uniform distribution of pigments within the epidermal layers (Seiberg, 2001). In humans, normal levels of pigmentation differ among races and reflect quantitative and qualitative differences in the amount, size, and type of melanins produced by the dendritic melanocytes dispersed at the dermoepidermal junction (Jimbow et al., 1976; see also Chapters 3, 27, and 28). Considering that the number of melanocytes in the skin is almost the same in all skin types (Szabo, 1954), constitutive or basal skin pigmentation is considered to be mainly related to the level of melanogenic activity (Tobin et al., 1994), to the ratio of black–brown eumelanin to red–yellow pheomelanin (Hunt et al., 1997; Maeda et al., 1997), to the melanosome transport, and to the degree of distribution of pigments into the neighboring keratinocytes (Szabo et al., 1969; see also Chapters 3, 12, and 13). The eumelanogenesis pathway involves the activation of melanogenic proteins, tyrosinase, TRP-1 and TRP-2, which are exclusively expressed by melanocytes and located in the melanosome membranes (Hearing and King, 1993; see also Chapters 10 and 11). The intracellular level of thiols, such as cysteine or glutathione, is able to modulate the formation of benzothiazine precursors of pheomelanin (Ito and Prota, 1977). The balance between eumelanin and pheomelanin synthesis is under genetic control (see Chapter 13). Genetic pigmentary disorders, such as albinism (see Chapter 31), are related to mutation in the genes that encode for melanogenic enzymes and pheomelanogenesis is connected to a high content of cysteine and deficit in the functionality of the melanocortin-1 receptor (MC1-R), which is considered as a

key control point for constitutive and abnormal skin pigmentation, both genetically determined (Rees, 2003). Due to its scavenger and filter properties (Barker et al., 1995; Schmitz et al., 1995) and its prevalent localization over the nucleus of keratinocytes (Boissy, 2003), the melanin pigment affords a strong protective effect against the harmful actions of external agents, such as ultraviolet (UV) irradiation (see Chapter 17), leading to prevention of DNA damage induced by both direct UV absorbance and UV-mediated reactive oxygen species (ROS) generation. Consequently, a correlation between low level of melanins and increased risk of skin cancer and UV photo-damage has been reported (Stoll, 1979). Thus, the melanin content and distribution inside the epidermal layers can be considered as a sign of the healthiness of the skin. All the acquired alterations of the skin pigmentation are, in fact, correlated to pathologic conditions, except for the race and the interindividual differences, such as the total amount of pigment or the ratio between eumelanin and pheomelanin, which are under genetic control. A reduction of skin melanins is observed in aged skin, as a marker of melanocyte senescence process (Gilchrest et al., 1979; see also Chapter 27), but a loss of skin pigmentation can also occur in young individuals following exposure to toxic compounds which can induce a destruction of cutaneous melanocytes. Chronic exposure to a large number of chemical and pharmacologic agents causes depigmentation of skin and hair (Cummings and Nordlund, 1995). These phenomena are highly reported in workers who are repeatedly exposed to these agents and they represent occupational diseases. In the majority of individuals such compounds trigger the chemical leukoderma, but in some people, who are genetically predisposed, occupational vitiligo occurs with the extension of skin depigmentation from the initial contact site, leading to the development of generalized vitiligo. Oliver et al. (1939) reported for the first time the appearance of vitiligo-like leukoderma in a high percentage (more than 50%) of workers in a leather company exposed for a long time to monobenzylether of hydroquinone, a substance employed as antioxidant in the gloves. Employing animal models, further studies demonstrated that not only topical exposure but also feeding caused the toxic effect (Denton et al., 1952). Additional data have demonstrated chemical depigmentation in people working in industries which used aromatic or aliphatic derivatives of phenols and catechols in the production 669

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processes (Calnan, 1973; O’Malley et al., 1988). Furthermore skin depigmentation has also been observed following occupational exposure to sulfhydryl compounds (Bleehen et al., 1968). Extensive studies, both in vitro and in vivo, have suggested a selective toxicity against melanocytes of several chemicals (Gellin et al., 1979). An extensive description of all the compounds which are capable of inducing skin depigmentation as a side effect has been recently published (Miyamoto and Taylor, 2000). Interestingly, even if these substances determine a chemical leukoderma in all individuals chronically exposed, large time differences in the appearance of toxic effects have been observed (Boissy and Manga, 2004). People particularly susceptible to the cytotoxic effects of phenolic or catecholic derivatives develop vitiligo-like depigmentation quickly in comparison to more resistant individuals, who require years of contact with the chemicals. Thus, the response to melanotoxic agents seems to be under genetic control. This evidence focuses on how important it is for some people to avoid contact with such toxic compounds. However, due to the fact that these chemicals are present in large amount in convenience goods, such as deodorants, paints, latex gloves, or printing ink, this result is not easy to obtain.

Hypopigmentation as Wanted Therapeutic Effect A lack of skin pigmentation can determine both physiologic and psychologic problems in affected subjects (see Chapter 59), but unwanted hyperpigmentation can also produce significant psychologic stress. Pigmentation plays an important cosmetic role in humans which can be compromised in hyperpigmentary disorders, such as melasma, age spot, or postinflammatory hyperpigmentation (Castanet and Ortonne, 1997; Frenk, 1995). Epidermal and dermal hyperpigmentation can be dependent on either an anomalously high number of melanocytes or increased activity of melanogenic enzymes (Bleehen, 1998; see also Chapter 28). Such pigmentary alterations occur as a result of both genetic and environmental components. Ultraviolet light, chronic inflammation and rubbing of the skin as well as abnormal a-MSH release, are considered triggering factors for these disorders (Im et al., 2002; Kang et al., 2002). Due to the fact that ultraviolet light represents the most important environmental triggering agent, the acquired hyperpigmentations are prevalently localized in photoexposed areas, such as face or neck, leading to an exacerbation of both psychologic and cosmetic implications for these cutaneous disorders. The hyperpigmented spots are more prominent in dark-skinned individuals and they can also take years to fade. Several factors, including specific neuromediators, hormones [a-MSH (see Chapter 19), adrenocorticotropic hormone (ACTH)], cytokines [such as interleukin-1a, IL-6, tumor necrosis factor (TNF)-a], growth factors [such as endothelin-1 or fibroblastic growth factor (FGF) (see Chapters 20 and 21)], and inflammatory mediators (prostaglandins, leukotrienes) are able to modulate the innate melanin pigmentation of skin during a lifetime (Morelli and Norris, 1993; Suziki et al., 1999), and dysregulation of such biologic mediators can induce alteration of skin melanin production and/or distribution. UV exposure 670

induces the stimulation of melanin synthesis, melanosome transfer, melanocyte proliferation, differentiation, and dendricity in a large part through the generation of many of these compounds. The in vitro existence of melanogenic paracrine cytokine networks between various types of skin cells, capable of regulating melanocyte functions, has been demonstrated (see Chapters 20 and 21). The interaction between melanocytes and keratinocytes involves endothelin-1 (Tada et al., 1998), membrane-bound stem cell factor (Hashiya et al., 2001), granulocyte macrophage colony stimulating factor (Imokawa et al., 1996), whereas the melanocyte–fibroblast interaction is mainly regulated by soluble stem cell factor (Imokawa et al., 1998). The upregulation of such networks has been found to be directly involved in the stimulation of proliferation and melanogenesis of melanocytes in vivo in several hyperpigmentary disorders, such as lentigo senilis. Several studies have demonstrated the key role played by endothelin-1 (see Chapter 20) in increased skin pigmentation following UVB exposure and even in the appearance of lentigo senilis. Almost all cutaneous disorders with an inflammatory component, such as acne and eczema, due to the cytokine oversecretion, may result in the accumulation of excessive levels of epidermal pigmentation (Lacz et al., 2004). In the case of inflammation, prostaglandin E produced by keratinocytes also stimulates tyrosinase activity and melanogenesis, probably by binding the G-protein coupled receptors, leading to the production of AMPc as second messenger (Kitawaki et al., 2003; Rhodes et al., 2001). Among inflammatory mediators, histamine may activate protein kinase A via H2 receptors and thus activate melanin synthesis in human epidermal melanocytes (Yoshida et al., 2000). Moreover, a strong association between estrogens plus progestogen agents and hyperpigmentation has been observed in pregnant women or following therapy with birth control pills (Slominski et al., 2004), providing a possible explanation for the higher occurrence of melasma in women than in men. Due to the high psychosocial and socioeconomic impact of alterations of skin pigmentation, it is not surprising that both pharmaceutical and cosmetic companies have made great efforts in screening the effectiveness of a number of whitening agents in reducing melanogenesis. The ideal depigmenting compound should have a potent bleaching effect with a rapid time of onset, have no short- or long-term side effects and lead to a permanent removal of undesired pigment. However, the management of skin hyperpigmentation, and in particular of melasma (see Chapter 54), is still a challenge for dermatologists, as there is no gold standard treatment and recurrences are common. Several strategies have been proposed for the therapy, including: reduction of UV exposure by sunscreen use; inhibition of melanogenic activity inside hyperactivated melanocytes; removal of melanin in excess; and dispersion of melanin granules (see Chapters 59, 62 and 64).

Melanogenic Process and Mechanism of Action of Depigmenting Agents Melanization of the epidermis results from a dynamic process

CHEMICAL, PHARMACOLOGIC, AND PHYSICAL AGENTS CAUSING HYPOMELANOSES

that involves several regulating factors, leading to the peculiar composition of melanin pigments, strictly related to the skin type. Three major steps are involved in epidermal hyperpigmentation: (1) the proliferation of melanocytes (see Chapters 3, 20 and 21); (2) the synthesis and activation of tyrosinase (see Chapters 10 and 11); and (3) the transfer of melanosomes to keratinocytes (Briganti et al., 2003; see also Chapters 8 and 9). Inside the melanosomes, the production of melanin pigment is controlled by a multistep enzymatic process, and thus upregulation and downregulation of melanin synthesis is a consequence of alterations in the activity of one of the enzymes involved in such a pathway (see Chapter 7). The first and rate-limiting step of melanogenesis is the oxidation of the amino acid tyrosine, mediated by tyrosinase, which catalyzes the hydroxylation of tyrosine into 3,4-dihydroxyphenylalanine (dopa) and the consequent oxidation of dopa into dopaquinone, which subsequently serves as substrate of both eumelanin and pheomelanin (see Chapter 10). Less is known about the control of pheomelanin synthesis, although it appears to be less dependent on tyrosinase activity and can proceed even when the activity of tyrosinase is extremely low. The switching between eumelanogenesis and pheomelanogenesis is controlled by several mechanisms, including the different response to a-MSH and concentration of substrate. Recently an inhibitory effect of dopaquinone concentration on the availability of the thiol precursor of cysteinyldopa has been reported (Oyehaugh et al., 2002). Due to the ability of dopaquinone to bind proteins in a covalent manner, it has been suggested that an increment of the intramelanosomal content of dopaquinone might lead to inactivation or interference with a cysteine transporter in the melanosomal membrane. The pigmentation is also determined by the size and the number of melanosomes. Melanosomes are larger in black skin than in white skin and are packaged as a single unit rather than in groups. As a result in high phototype skin a delay in the melanosome degradation inside the keratinocytes can contribute to the higher level of skin pigmentation. The final content of melanin is, in fact, cleared by the degradation of the pigments during the ascent of the keratinocytes toward the outer stratum corneum. Finally, the remaining melanin is shed with the desquamation process. Although following UV exposure, the synthesis of both pheomelanin and eumelanin is stimulated, the degree of tanning only shows a good correlation with the amount of eumelanin (Kadekaro et al., 2003). Thus eumelanogenesis is considered as the main process responsible for the upregulation of skin pigmentation induced by external agents. This evidence can provide a good explanation for the increment in the incidence of acquired hyperpigmentations in dark-skinned individuals. As a consequence, the therapeutic approach to induce depigmentation of the skin should be focused on the decrease of eumelanin synthesis as well as proliferating activity of the melanocytes. The current strategies for developing new compounds controlling the excess in eumelanin pigmentation involve several of these biologic mechanisms. Depigmentation can be

achieved by interfering with: (1) transcription and activity of tyrosinase, TRP-1, and TRP-2; (2) mechanism regulating the uptake and distribution of melanosomes in recipient keratinocytes; (3) melanin and melanosome degradation; (4) turnover of “pigmented” keratinocytes (Fig. 35.1, Table 35.1).

Regulators of Melanogenic Enzymes Due to the key role played by tyrosinase in melanin biosynthesis, the majority of whitening agents act specifically to reduce the synthesis or activity of this enzyme through several different mechanisms: (1) interfering with tyrosinase transcription and glycosylation; (2) inhibiting tyrosinase activity by several modalities (i.e., acting as competitive inhibitor or alternative substrate, interacting with copper at the active site); (3) reducing the tyrosinase products; and (4) promoting degradation of the protein.

Transcriptional Control of Tyrosinase Expression The melanogenic proteins share several structural properties, including a cytoplasmic and a transmembrane domain, a glycosylated luminal domain including the cysteine-rich epidermal growth factor motif and two metal-binding regions involved in the structure of the catalytic site (Furumura et al., 1998). Moreover, tyrosinase, TRP-1, and TRP-2 (see Chapter 11) are encoded by homologous genes mapping to the albino, brown, and slaty loci, respectively. A strong intermolecular association between tyrosinase and TRP-1, resulting in a stabilization of tyrosinase and, thus, in an improvement in functionality of the melanogenic pathway, has been demonstrated (Kobayashi et al., 1998). Transcription of tyrosinase, TRP-1, and TRP-2 genes is under transcriptional control of microphthalmia transcription factor (MITF), which is implicated as a master gene in melanocyte development and in the regulation of melanogenesis, as well as a key transcription factor regulating the expression of major melanogenic proteins (Fang et al., 2002; Tachibana, 2000; Yasumoto et al., 1997; see also Chapter 13). The expression of MITF gene is affected by UV irradiation and mediates its biologic functions through the activation of two different mechanisms: a c-AMP-dependent pathway (Bertolotto et al., 1998) and a transient upregulation of MITF function following the mitogen-activated protein kinase (MAPK)-dependent phosphorylation of MITF itself (Tachibana, 2000). As a consequence, a-MSH and other c-AMP elevating agents transcriptionally upmodulate the expression of MITF through a cAMP-response element (CRE) in the MITF promoter region (Park and Gilchrest, 2002). Substances which are able to inhibit MITF expression and activity, as well as the extracellular signal-regulated kinase (ERK) and serine–threonine kinase (AKT)/protein kinase B (PKB) pathways could represent depigmenting agents (Fang et al., 2002; Kim et al., 2004). Among these, tretinoin (all trans retinoic acid; ATRA), due to binding activity to retinoid-activated transcription factors, interferes with melanocyte development and melanogenesis. ATRA has bimodal effects on melanocytes: it stimulates the differentiation of melanocyte precursors, inducing transcription 671

CHAPTER 35 Table 35.1 Classification of depigmenting agents. Before melanin synthesis Tyrosinase transcription C -Ceramide Tretinoin a-Lipoic acid PKC-b selective inhibitor

Tyrosinase glycosylation PaSSO Ca N-Butyldeoxy-nojirimicin

Tyrosinase inhibition Hydroquinone MEBH 4-Hydroxy-anisole 4-Tertiary-butylphenol 4-S-CAP and derivative a-Arbutin Arbutin Aloesin Esculetin and esculin Miconazole Kojic acid and derivatives Nobiletin Methyl gentisate Resveratrol Oxyresveratrol Gnetol Ellagic acid Azelaic acid

Shift to pheomelanogenesis Cystamine Cysteamine N, N¢dilinoleylcystamine

Peroxidase inhibition Methimazole

Dopachrome tautomerase activation Oxyresveratrol derivative

Redox agents and ROS scavengers Ascorbic acid (AsA) AsA palmitate a-Tocopherol a-Tocopherol-ferulate 6-Hydroxy-3,4-dihydrocoumarins a-Lipoic acid Proanthocyanidins Metallothioneins

2

3

During melanin synthesis

Post-transcriptional tyrosinase control Linoleic acid Phospholipase D2 activation

After melanin synthesis Inhibition of melanosome transfer Serine protease inhibitors Soybean/milk extracts Niacinamide

Melanin dispersion and skin turnover acceleration a-Hydroxyacids Liquiritin

Inhibition of post-inflammatory hyperpigmentation M. chamomilla extracts Glabridin

Fig. 35.1. Possible approaches for counteracting acquired hypermelanosis. M, melanosomes; Tyr, tyrosinase; ROS, reactive oxygen species.

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of tyrosinase by PKC activation and MITF expression, and removes differentiated melanocytes by promoting apoptosis via caspase-3 pathway and bcl-2 downmodulation (Watabe et al., 2002). ATRA does not inhibit melanogenesis in skin equivalents and monolayer culture of murine and human melanocytes (Yoshimura et al., 2001). On the contrary, ATRA enhances the pigmentation of low melanized (S91 murine) melanoma cells, and decreases those of highly pigmented normal human melanocytes (Romero et al., 1994). Topical ATRA stimulates, in vivo, tyrosinase activity only in individuals with low skin type (Talwar, 1993) and improves the irregular hyperpigmentation associated with photoaging or inflammation (Rafal et al., 1992; Bulengo-Ransby et al., 1993). UVB exposure induces MITF promoter activity in vitro in a time and concentration-dependent manner, probably involving the release of a-MSH in the MITF activation pathway (Lin et al., 2002). Due to the evidence that some antioxidants, such as a-lipoic acid, dihydrolipoic acid, and resveratrol, affect MITF promoter activity and reduce both endogenous and induced MITF mRNA and protein levels, whereas other scavenger molecules, such as ascorbic acid or glutathione do not exert any significant effect, the antioxidant activity per se seems to be not involved in the regulation of MITF transcription. Dihydrolipoic acid, however, is able to induce skin lightening in dark-skinned swine and prevent UVB-induced tanning in vivo, possibly targeting other transcription factor elements in the MITF promoter that may cooperate with CRE binding elements in transactivating MITF. C2-Ceramide was found to decrease MITF expression and inhibit cell proliferation and melanogenesis in a mouse melanocyte cell line (Kim et al., 2001; Kim D. S. et al., 2002). The mechanism of action is probably related to interference with AKT/PKB and ERK pathways, with an initial inhibition and a late activation, whereas no significant effect on tyrosinase activity has been found. The existence of an interaction between the PKC pathway and cAMP-dependent protein kinase A pathway has been suggested by the evidence that the PKC inhibitors sphingosine and calphostine also inhibit the melanogenic response dependent on a-MSH (Gordon and Gilchrest, 1989). Recently it has been reported that bisisolylmaleimide GF 109203X (Bis), a selective inhibitor of cPKC, with higher selectivity for PKC-b than for PKC-a and PKC-g, can be considered as a putative new agent for the treatment of unwanted hyperpigmentation (Park et al., 2004). Bis was found to reduce melanin production in human cultured melanocytes, and to decrease both basal and UV-induced skin pigmentation via topical application in guinea pigs.

Glycosylation and Maturation of Melanogenic Enzymes Although the sorting machinery that regulates the post-transcriptional processes of melanogenic proteins must be uncovered, tyrosinase, TRP-1, and TRP-2 appear to be synthesized on ribosomes, transferred through the endoplasmic reticulum, where they mature until they get a stable conformation, which enables them to avoid an accelerated degradation. The mature

polypeptides exit the endoplasmic reticulum, and reach the Golgi apparatus, where they are glycosylated and packaged into vesicles prior to fusion with premelanosomes (Negroiu et al., 2000). Despite numerous studies, it is still unclear whether all the melanogenic enzymes are essential for the pigmentation pathway or whether some are rather involved in other aspects, such as the stability of melanosomes. Even if TRP-1 shows a potential action in melanin synthesis, it seems to play a key role in regulating the stability of tyrosinase and the fully melanized melanosomes (Kobayashi et al., 1998). On the other hand TRP2 was shown to catalyze the last steps of the pathway, but, due to the evidence that melanin may form even in its absence, with the spontaneous conversion of dopachrome to DHICA, it seems clear that the qualities of melanin are regulated not only by the enzymatic activity of TRP-2. Therefore, TRP-2 has been suggested to maintain the cell homeostasis, by controlling the intracellular concentration of 5,6-DHI and avoiding the accumulation of this toxic intermediate of melanin inside the melanocytes (Pawelek and Murray, 1986). The major posttranslational modification of the melanogenic enzymes consists of asparagine-linked glycosylation, which leads to the release of soluble enzyme from the membrane-bound form. During in vivo melanogenesis tyrosinase, TRP-1, TRP-2, and pmel17 are subjected to N-glycosylation, with a mechanism that follows the general metabolic pathway of glycoproteins (Negroiu et al., 1999; Yasumoto et al., 2004). Differences in glycosylation of the melanogenic proteins indicate different accessibility of glycosidase, probably determined by different three-dimensional structures of tyrosinase, TRP-1, and TRP-2 (Negroiu et al., 2003). Inhibition of N-glycosylation by a variety of agents results in reduction of melanosome maturation, inhibition of melanosomal enzyme activities, or suppression of melanogenesis. The modification of the glycosylation pattern makes the protein inactive or less active and affects its intracellular transport or deposition within melanosomes. Negroiu et al. (1999) showed that inhibition of a-glucosidase I, an early step of N-glycosylation inside the endoplasmic reticulum, by Nbutyldeoxynojirimicin is capable of inducing the inactivation of tyrosinase and a dramatic loss of cell pigmentation in B16 melanoma cells, whereas the dopa-oxidase of TRP-1 is only slightly reduced (Negroiu et al., 1999; Petrescu et al., 1997). Thus it seems that TRP-1 is able to bypass the glycosidase blockade and acquire some complex-N-glycans. Calcium d-pantetheine-S-sulfonate (PaSSO3Ca) shows depigmenting activity in normal human melanocytes and melanoma cell lines. Its mechanism of action involves interference with the glycosylation of tyrosine receptor and TRP1, leading to an indirect inhibition of melanogenic enzymes, without alteration of their expression, and a reversible loss of pigmentation (Franchi et al., 2000). Moreover the bleaching activity of PaSSO3Ca could be facilitated by its similarity with the chemical structure of coenzyme A, which plays an important role in the intracellular transport of proteins.

Control of Tyrosinase Activity Tyrosinase activity can be inhibited in different ways, 673

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including: (1) competitive inhibition; (2) noncompetitive inhibition; and (3) chelation of the copper atoms at the active site. The capacity of a chemical compound to act as competitive or noncompetitive tyrosinase inhibitor is defined by inhibition kinetic studies, which are mostly analyzed according to the Lineweaver and Burk (double-reciprocal) plot method that allows determination of Michaelis-Menten constant (Km) and maximum velocity (Vm) and the subsequent calculation of inhibition constant (Ki) and IC50. As general definition, competitive inhibitors are characterized by the capacity to bind active site of the enzyme, leading to an increase of Km without altering Vm, whereas noncompetitive inhibitors can bind both free enzyme and enzyme–substrate complex, inducing a decrease of Vm and not modifying Km. However, tyrosine hydroxylation is the rate limiting step for melanogenesis, but requires l-dopa, the substrate of tyrosinase second activity, as a cofactor, and, thus, tyrosinase activity is not easily examinable through kinetic analyses. In addition, due to the controversial results of the enzyme kinetic experiments, which are performed employing purified mushroom tyrosinase or cell lysates of melanoma cells, it is very difficult to classify tyrosinase inhibitors. The evidence that several tyrosinase inhibitors can work using different mechanisms of action, interacting with both catalytic and regulatory sites of the enzyme or being metabolized by tyrosinase to a product, which, in turn, can act as noncompetitive as well as competitive inhibitor, makes the classification even harder. Competitive and Noncompetitive Inhibition Competitive inhibitors bind the tyrosine site in a reversible manner, avoiding the interaction with the natural substrate. Tyrosinase active site is characterized by: (1) a hydrophobic pocket into which the phenyl ring of the substrate is inserted; (2) high stereospecificity for l-isomers; (3) spatial restriction imposed by the distance between the phenyl ring and the groups on the side-chain. Thus, an effective agent must have a chemical structure which allows interaction with the active site. Most of the competitive inhibitors are phenol/catechol derivatives, structurally similar to tyrosine or dopa, acting as alternative substrates of tyrosinase, and are oxidized by the enzyme without producing melanin pigment (Menter et al., 1981; Passi and Nazzaro-Porro, 1993). Moreover, the quinones generated can react with the sulfhydryl group of melanosomal proteins and/or essential enzymes, interfering with cell growth and proliferation (Menter et al., 1993). The most popular depigmenting agent is hydroquinone (dihydroxybenzene; HQ), which was shown to induce reversible skin depigmentation in humans and animals in 1936 (Oettel, 1936) and was introduced for clinical use in 1961 (Sidi et al., 1961). Several clinical trials have demonstrated the therapeutic efficacy of HQ alone or in association with other compounds (Fisher, 1998; Guevara and Pandya, 2001; Kang et al., 1998; Kauh and Zachian, 1999; Perez-Bernal et al., 2000; Sanchez and Vazquez, 1982). The clinical efficacy depends on HQ concentration, nature of vehicle, and stability of formulation. HQ seems to exert its effects mainly in melanocytes with 674

active tyrosinase activity, such as epidermal hyperactivated melanocytes (Smith et al., 1988) and, due to its strong depigmenting activity, has been considered as a reference standard in evaluating depigmenting agents (Curto et al., 1999; Palumbo et al., 1991). Considering its chemical analogy to melanin precursors, this hydroxyphenolic agent was originally believed to act as an alternative substrate for tyrosinase (IC50 = 4.5 mM) (Prota, 1992), competing for tyrosine and dopa oxidation. Following oxidation HQ generates quinones and ROS, which lead to oxidative damage of membrane lipids and, possibly, to the alteration of membrane-bound proteins, such as tyrosinase. To demonstrate the oxidative process underlying HQ effects, depletion of glutathione, a strong ROS scavenger, contributes to the lightening action of HQ (Bolognia et al., 1995). Other putative mechanisms of interference with pigmentation include the covalent binding to histidine residues at the enzyme active site (Prota, 1992), inhibition of DNA and RNA synthesis, degradation of melanosome formation and melanization extent, alteration of melanosomal structure, an increase in melanosomal degradation rate within keratinocytes, and destruction of melanocytes. Despite the HQ peculiarity to exert its effects mainly in cells with active tyrosinase activity, such as epidermal hyperactivated melanocytes, the appearance of irreversible medium-term side effects, such as persistent hypopigmentation or depigmentation, determined by its melanocyte cytotoxicity, and the hazard of long-term side effects (Gaskell et al., 2005), the use of hydroquinone in cosmetics has been forbidden by European Committee (24th Directive 2000/6/EC). However, it should be taken into account that the use of depigmenting agents with severe side effects, such as a permanent loss of pigmentation, can be useful to obtain a generalized depigmentation in patients with hypopigmentary diseases. HQ monobenzyl ether (MBEH) (see Chapers 30 and 58) is selectively taken up by melanocytes, where it is metabolized to reactive free radicals, causing permanent depigmentation even at sites distant from those of application, and can be useful to obtain generalized depigmentation in patients with hypopigmentary diseases, such as vitiligo (Njoo et al., 1999). 4-Hydroxyphenyl a-d-glucopyranoside (a-arbutin) has been enzymatically synthesized from HQ and saccharides (Kurosu et al., 2002), and shares with HQ the capacity to act as an alternative substrate of mammalian tyrosinase, leading to a decrease of skin pigment generation at noncytotoxic concentrations (Nakajima et al., 1998). The inhibitory activity of a-arbutin against tyrosinases from mushroom, B16 mouse melanoma, and HMV-II human melanoma cells has been examined (Funayama et al., 1995; Sugimoto et al., 2003). A decrement of melanin synthesis directly correlated with the inhibition of cellular tyrosinase activity was found in human melanoma cells and on epidermal equivalents containing melanocytes treated with a-arbutin (Sugimoto et al., 2004). Interestingly, about 70% of the a-arbutin applied to the human skin model was recovered in the medium, whereas HQ was not detected at all after cultivation, suggesting that a-

CHEMICAL, PHARMACOLOGIC, AND PHYSICAL AGENTS CAUSING HYPOMELANOSES

arbutin inhibitory activity was not due to the release of HQ. Also arbutin, an optical isomer of a-arbutin, has been reported to reduce tyrosinase activity on human melanocytes and human melanoma cells at noncytotoxic concentrations (Chakraborty et al., 1998), without any alteration of expression level of tyrosinase mRNA, suggesting an effect at the post-translational level. In addition arbutin was found to inhibit DHICA polymerase activity (pmel17/silver protein), maturation of melanosomes (Chakraborty et al., 1998), and uptake of tyrosine into melanocytes (Bolognia et al., 1991). However, despite encouraging initial results (Cameli et al., 2001), the effectiveness of arbutin as a whitening agent has not been confirmed on intact melanocytes and in clinical trials (Curto et al., 1999). Several phenolic compounds have been screened for evaluating the relationship between chemical structure and tyrosinase inhibitory activity. The essential requirement for an effective action as alternative substrate of tyrosinase was found to be the presence of a hydroxylic group and of an electron donor group in the para position. In para-hydroxylated monophenols, methylation of one hydroxyl favored, in fact, the nucleophilic attack at the active site enhancing the catalytic efficiency (Fenoll et al., 2000). Catechols (ortho-diphenols) bearing electron donor groups in position para are more effective in inhibiting tyrosinase and inducing melanocytotoxic effects than monophenolic derivatives (Thorneby-Andersson, 2000). However, when a derivative with methyl groups in position ortho and meta, such as 2,5,6-trimethyl-para-hydroxymethoxybenzene, is used as substrate of mushroom tyrosinase the oxidation rate is slow and the initial acceleration (lag period) is not observed (Naish-Byfield and Riley, 1998). The presence of methyl groups on the phenolic ring probably prevents the nucleophilic addition of the o-quinone, thus avoiding the formation of an o-diphenol which, in turn, acts as activator of inactive enzyme molecules (Naish-Byfield and Riley, 1998). The presence of thioether side chains in para position on the phenolic ring induces an enhancement of both the rate of oxidation by tyrosinase and cytotoxic effects (Riley et al., 1997). Due to their chemical structures, 4-hydroxyanisole (para-hydroxy-methoxybenzene), 4-tertiary butylphenol, and N-acetyl-4-S-cystaminylphenol (4-SCAP) inhibit melanin synthesis. The presence of lipophilic groups gives these compounds the capacity to penetrate within the epidermis and target pigmented cells. 4-Hydroxyanisole is a good alternative substrate for tyrosinase, being oxidized by the enzyme (Km = 62–80 mM). It exhibits melanocytotoxic effects stronger than those exerted by HQ and a direct relationship between its inhibition of dopa oxidase activity and cytotoxicity has been observed in melanoma cell lines (Rodriguez-Vicente et al., 1997). Clinical effectiveness of 4-hydroxyanisole in skin hyperpigmentation has been demonstrated in combination with tretinoin 0.01% in solar lentigines and related hyperpigmented lesions (Fleicher, 2000). 4-Tertiary butylphenol induces in human melanocytes a competitive inhibition of both tyrosine hydroxylase and dopa oxidase activities of tyrosinase without cytotoxicity (Yang and Boissy, 1999). 4-S-cystaminylphenol

(4-SCAP) and its N-acetylated derivatives have both depigmenting and antimelanoma effects. The property of acting as an alternative substrate of tyrosinase could explain both activities although the existence of a mechanism of nontyrosinasemediated cytotoxicity for phenolic thioether amines has been recently reported (Gili et al., 2000). 4-SCAP seems to be able to induce a marked decrease in the number of functioning melanocytes and melanized melanosomes, a decrease in the number of melanosomes transferred to keratinocytes, and a selective degeneration of melanocytes (Jimbow et al., 1995). Topical treatment in Yucatan pigs induces a stable but reversible depigmentation with slight irritant effects when compared to HQ (Gili et al., 2000; Thomas et al., 1999). Aloesin, a natural hydroxychromone derivative isolated from aloe vera, inhibits mushroom (Okamura et al., 1996; Piao et al., 2002) and melanoma tyrosinase at non-cytotoxic concentration (Jin et al., 1999; Jones et al., 2002). Kinetic studies have reported that aloesin can act either as competitive inhibitor (IC50 = 0.167 mM) or as noncompetitive inhibitor (Ki = 5.3 mM) of tyrosinase activity. Aloesin seems to be a competitive inhibitor on dopa oxidation and an inhibitor, in a noncompetitive fashion, of tyrosine hydroxylase activity. In vivo, aloesin and arbutin cotreatment inhibits UVinduced melanogenesis in a synergistic manner (Choi et al., 2002). A study on the structure–activity relationship of 18 coumarins, which are widely distributed in Umbelliferae and Rutaceae plants, in terms of their inhibitory activity on mushroom tyrosinase has indicated that hydroxylation at the C6 and C7 of the coumarin ring is the most important in increasing the activity but any substitution in the lactone ring is unfavorable to the activity. Among these compounds esculetin exhibited the strongest inhibitory activity (IC50 = 43 mM) on mushroom tyrosinase and it was able to suppress melanin production of B16 melanoma cells and split-epidermal sheets from black guinea pigs. Moreover esculin, a coumarin glucoside, has been reported to have an inhibitory effect in B16 melanoma cell at noncytotoxic concentrations in spite of its low inhibition effect on tyrosinase (Masamoto et al., 2004). Considering that esculin easily penetrates and hydrolyzes to esculetin in melanocytes, it can be assumed that esculetin is responsible for antimelanogenic activity observed following esculin treatment. Recently, miconazole, an antifungal agent extensively employed in the treatment of superficial mycosis, has been found to induce depigmentation in B16 melanoma cells (Mun et al., 2004) by the inhibition of tyrosinase activity and tyrosinase expression as well as by a cytostatic action. Miconazole markedly inhibits a-MSH- or forskolin-stimulated melanogenesis mediated through the activation of the cAMP pathway, suggesting that the depigmenting effect of miconazole may be more effective on hyperactive melanocytes. Kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) is a fungal metabolic product recognized for its effective inhibition of tyrosinase activity. The depigmenting action is attributed to the chelating ability (Battaini et al., 2000), even though 675

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interference with different steps of melanin synthesis (Kahn, 1995) and inhibition of nuclear factor (NF)-kB activation in keratinocytes have been described (Moon et al., 2001). Clinical trials have reported a skin-lightening effect of kojic acid (Lim, 1999). Because of its instability, kojic acid has limited use, especially in solution, and its derivatives have been evaluated. The recently synthesized kojyl-APPA (5-[(3-aminopropyl)phosphinooxyl]2-(hydroxymethyl)-4H-pyran-4-one) showed much stability and greater extension of permeation through skin than kojic acid, and inhibitory effect on tyrosinase in situ, but not on mushroom tyrosinase, suggesting that kojyl-APPA acts as a pro-drug to be converted to kojic acid inside the cells (Kin et al., 2003). Nobiletin, a polymethoxyflavonoid occurring exclusively in Citrus fruit, was identified as a new inhibitor of mushroom tyrosinase (Sasaki and Yoshizaki, 2002), but further studies are required to elucidate its depigmenting effect. The methyl ester of gentisic acid (2,5-dihydroxybenzoic acid; MG), a natural product of Gentiana’s root, inhibits melanin synthesis in murine melanocytes at a concentration unable to induce cytotoxicity (Dooley et al., 1994). MG can act as a prodrug, inhibiting tyrosinase following the intracellular release of HQ, even if kinetic studies have demonstrated a direct effect on the enzyme, possibly through the binding of the copper ions (Curto et al., 1999). Clinical trials have demonstrated the efficacy of the compound (Bocchietto et al., 2001). Most of hydroxystilbene compounds like resveratrol (3,4¢, 5-trihydroxystilbene), naturally contained in herbal medicines, have potent inhibitory effects on mushroom tyrosinase. Resveratrol, one of the ingredients of red wine, possesses several biologic properties, such as free radical scavenging, anti-inflammatory and anticancer activity (Jang et al., 1997; Subbaramaiah et al., 1998). A dose-dependent inhibition on l-tyrosine oxidation by tyrosinase has been described in B16 murine melanoma, without affecting the protein expression and synthesis. Resveratrol is probably metabolized by tyrosinase leading to the formation of an o-hydroxy derivative, which is a competitive inhibitor of the enzyme (Bernard and Berthon, 2000). Kinetics studies revealed that oxyresveratrol, extracted from Morus alba, is a stronger inhibitor (IC50 = 1 mM) on mushroom dopa oxidase activity than resveratrol (IC50 = 54.6 mM) (Y. M. Kim et al., 2002), suggesting that the number and the position of hydroxyl substituents play an important role in the inhibitory potency of hydroxystilbene compounds for tyrosinase activity. Gnetol, a tetrahydroxystilbene extracted from roots of genus Gnetum and chemically related to oxyresveratrol, was recently suggested as a promising candidate as a whitening agent, due to its capacity to induce a strong inhibition of tyrosinase activity and a subsequent suppression of melanin biosynthesis in B16 melanoma cell line (Ohguchi et al., 2003). Ellagic acid (EA), a polyphenol widely distributed in plants, is capable of preventing pigmentation caused by sunburn (Shimogaki et al., 2000). EA inhibits tyrosinase noncompetitively in a dose-dependent manner, through its capacity to chelate copper, though other mechanisms, such as a scavenger 676

effect, have been suggested. Interestingly in brownish guinea pigs, EA-induced a reversible inhibition of melanin synthesis only in UV-activated melanocytes. Compounds able to bind either amino or carboxyl groups may block the access of tyrosine to the active site, behaving as competitive inhibitors. Among these is azelaic acid (AZA), a naturally occurring 9-carbon dicarboxylic acid, isolated from cultures of Pityrosporum ovale. AZA inhibits tyrosinase activity in vitro (Ki = 2.73*10–3 M) (Lemic-Stoicevic et al., 1995) and may also interfere with DNA synthesis and mitochondrial activity in hyperactive and abnormal melanocytes (Nazzaro-Porro, 1987). Topical azelaic acid 15–20% has been reported to be efficacious in improving melasma (Sarker et al., 2002), in post-inflammatory hyperpigmentation (Breathnach, 1996), and in the arrest of the progression of lentigo maligna to melanoma (Nazzaro-Porro et al., 1989). Other Effects Shift to Pheomelanin Synthesis Thiol compounds may react with quinone intermediates of melanin to divert pigment synthesis from eumelanin to pheomelanin (Ito and Prota, 1977). Several data reported that in presence of elevated amount of thiols, including glutathione, a prevalence of pheomelanin and a lack of pigmentation can be observed both in humans and in mice (Benedetto et al., 1982). Due to the nucleophilic addition to dopaquinone, which is much faster than conversion of dopaquinone to dopachrome, cystamine as well as cysteamine, its reduced form, are capable of shunting to pheomelanin synthesis from eumelanin synthesis (Qiu et al., 2000). N, N¢-dilinoleylcystamine, a compound of cystamine and linoleic acid connected by an ester bond, has been recently reported to decrease melanin content in HM3KO melanoma cells (Hwang et al., 2004). This compound has been found to have dual functions: shifting of the melanin biosynthetic pathway in favor of pheomelanin, and reduction of tyrosinase activity, through a downregulation of the level of tyrosinase protein. Dopachrome Tautomerase Activation In the melanogenesis pathway the dopachrome is the precursor of both DHImelanin and DHICA-melanin. The dopachrome, in fact, can spontaneously generate DHI or, conversely, it can be enzymatically converted to DHICA. The spontaneous conversion of dopachrome to DHI is faster than the dopachrome– tautomerase-induced generation of DHICA. When generated, DHICA-eumelanin is able to inhibit the production of DHIeumelanin. The subsequent accumulation of light brown DHICA-eumelanin instead of black DHI-melanin can result in the lightening effect. An oxyresveratrol derivative, with an amide connection chain between the two benzene rings, has been suggested to activate dopachrome tautomerase, thus inhibiting pigmentation (Choi et al., 2004). Post-transcriptional Control of Tyrosinase Substances able to regulate melanin synthesis without affecting the expression of melanogenic proteins are likely to exert

CHEMICAL, PHARMACOLOGIC, AND PHYSICAL AGENTS CAUSING HYPOMELANOSES

a post-transcriptional control of melanogenic enzymes. Unsaturated fatty acids, such as oleic acid (C18:1), linoleic acid (C18:2), or a-linolenic acid (C18:3), suppress pigmentation in vitro, correlated with the degree of unsaturation, whereas saturated fatty acids, such as palmitic acid, increase the rate of melanogenesis (Ando et al., 1998). Linoleic acid in vivo showed the greatest lightening effect in UVB-induced pigmentation, without having any toxic effects on melanocytes. The evidence of no changes in TRP-1 and TRP-2 proteins suggests that tyrosinase is selectively targeted by fatty acids, which seem to act on the degradation of the enzyme during the physiologic proteasome-dependent mechanism, leading to the alteration of tyrosinase protein content in hyperactive melanocytes: linoleic acid accelerates the process, whereas palmitic acid works in an antagonistic manner mimicking protease inhibitors. A similar depigmenting effect has been observed in melanoma cells following treatment with a biphenyl derivative, 2,2¢-dihydroxy-5–5¢-dipropyl-biphenyl (DDB). The mechanism by which DDB induces an early degradation of tyrosinase is still unknown, but an inhibition of correct folding of tyrosinase protein has been suggested (Nakamura et al., 2003). Involvement of phospholipase D2 in regulating pigmentation has been recently suggested (Kageyama et al., 2004). In B16 melanoma cells, the overexpression of phospholipase D2 determined a decrease in melanin content, accompanied by a decrement of amount and activity of tyrosinase, whereas the mRNA level of tyrosinase was unchanged. The treatment with proteasome inhibitors completely blocked the phospholipase D2-induced downregulation of melanogenesis, suggesting the involvement of ubiquitin proteasome-mediated degradation of tyrosinase protein in the whitening effect.

Inhibitors of Peroxidase The involvement of peroxidase in the spontaneous polymerization of melanogenic intermediates has been suggested by the generation in vitro of hydrogen peroxide (H2O2), byproduct of DHI and DHICA oxidation and by the high catalytic efficiency of peroxidase in the oxidation of 5,6-dihydroxyindoles (D’Ischia et al., 1991; Nappi and Vass, 1996). Intracellular H2O2 is generated after UV irradiation or in response to cytokines, such as TNF-a or transforming growth factor (TGF).-b (Lo et al., 1996; Meier et al., 1989; Thannickal and Fanburg, 1995), that causes depigmentation, and can induce a transient reduction of tyrosinase and other melanogenic protein activity, through the downregulation of the MITF transcription factor (Jimenez-Cervantes et al., 2001). However, despite this evidence, the inhibition of peroxidase results in depigmentation, reducing the polymerization rate of eumelanin (Kasraee, 2002a). The major part of melanocytotoxic agents are good substrates of both tyrosinase and peroxidase, leading to the generation of cytotoxic metabolites, and the inhibition of peroxidase could play a role in the mechanism of action of several depigmenting agents, such as glucocorticoids or ascorbic acid, which do not exhibit any effects against the melanogenic enzymes. Methimazole, an

antithyroid agent belonging to the thionamide group, exerts inhibitory action on both mushroom tyrosinase and peroxidase (Kasraee, 2002b) and induces mild to moderate inhibition of melanization in brown guinea pigs, with morphologic changes of melanocytes.

Redox Agents and ROS Scavengers UV-induced ROS can play relevant roles in regulating the proliferation of melanocytes and melanogenesis of melanocytes, while ROS scavengers and inhibitors of ROS production, such as antioxidants, may reduce hyperpigmentation or prevent new UV-induced melanogenesis. Compounds with redox properties can have depigmenting effects by interacting with o-quinones, and thus avoiding the oxidative polymerization during melanin synthesis, or by scavenging ROS, which are generated in the skin by exposure to sunlight and are able to stimulate epidermal melanogenesis either directly, acting as second messengers, or indirectly, through the induction of cytokine production in epidermal and dermal cells (Karg et al., 1993). Moreover, the o-quinone intermediates, formed during the melanogenic pathway, are highly reactive and susceptible to be neutralized by reducing substances. Ascorbic acid (AsA) interferes with different steps of melanization, by interacting with copper ions at the tyrosinase active site and reducing dopaquinone and by blocking DHICA oxidation (Ros et al., 1993). However AsA is highly instable, being quickly oxidized and decomposed in aqueous solution and due to its hydrophilic nature, the degree of penetration in the skin is low. Different esters of AsA, such as magnesium-l-ascorbate-3-phosphate (MAP), stable in water and in alkaline medium and absorbed percutaneously, have been reported to induce a lightening effect in both normal and hyperactive melanocytes (Kameyama et al., 1996). In vitro, a-Toc derivatives inhibit tyrosinase (Shimizu et al., 2001) and melanogenesis in epidermal melanocytes (Ichihashi et al., 1999). The antioxidant properties of a-Toc, which interferes with lipid peroxidation of melanocyte membrane, and increases the intracellular glutathione content, could explain its depigmenting effect (Ochiai et al., 2001; Marmol et al., 1993). Co-treatment with AsA, which regenerates a-Toc from its radicalic form, results in more effective and long-lasting antioxidant response. Topical application of a-Toc and AsA, in vivo, reduced tanning response, through an inhibition of UV-induced melanogenesis and proliferation of melanocytes (Quevedo et al., 2000). An alternative approach is atocopherol ferulate (a-Toc-F), a derivative of a-Toc linked by an ester bond to ferulic acid, which is an antioxidant, and provides a stabilization of a-Toc, similarly to AsA. a-Toc-F inhibits melanin synthesis in normal human melanocytes, without interfering with the tyrosinase pathway. 6-Hydroxy3,4-dihydrocoumarins, novel antioxidants with a-Toc-like chemical structures (Nishiyama et al., 2001), have been recently reported to have antimelanogenic activity in normal human cultured melanocytes at non-cytotoxic concentration without interfering with tyrosinase activity. The acceleration of the biosynthesis of glutathione within melanocytes, leading 677

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to the inhibition of tyrosine transfer to premelanosomes, has been indicated as a mechanism of action (Yamamura et al., 2002). Thioctic acid (a-lipoic acid), a disulfide derivative of octanoic acid, exhibits several biologic effects, which include the quenching of ROS, metal chelation, interaction and regeneration of other antioxidants, redox regulation of protein thiol groups, and effects on gene expression and apoptosis (Packer et al., 1995). Thioctic acid has been reported to prevent UVinduced photo-oxidative damage, mainly through the downmodulation of NF-kB activation and to reduce melanin synthesis with a dual mechanism which involves the modulation of microphthalmia-associated transcription factor (Lin et al., 2002) and the inhibition of tyrosinase activity, probably by chelating the copper ions (Saliou et al., 1999). Oral administration of proanthocyanidin-rich grape seeds has been found to be effective in lightening UV-induced pigmented skin in guinea pigs and in inhibiting tyrosinase activity in B16 melanoma cells (Yamakoshi et al., 2003). The depigmenting effect is suggested to be related to inhibition of melanin synthesis and the ROS-related proliferation of melanocytes. Sasaki et al. (2004) have demonstrated for the first time the expression in melanocytes of metallothioneins, metal-binding proteins with scavenger properties. The induction of these proteins was able to suppress melanogenesis stimulated by nitric oxide as well as other melanogens, such as a-MSH or endothelin-1, suggesting that the suppressive effect is due not only to a scavenging of nitric oxide, but also to a direct inhibition of tyrosinase activity.

Inhibition of Melanosome Transfer Several studies have been focused on the identification of regulatory factors of the melanosome movement in dendrites and of the interaction between keratinocyte and melanocytes during the transfer process (Minwalla et al., 2001; Seiberg, 2001; see also Chapters 8 and 9). The activation of proteaseactivated receptor 2 (PAR-2), a seven trans-membrane Gprotein coupled receptor, which is expressed in keratinocytes and not in melanocytes, was found to activate keratinocyte phagocytosis, enhancing the pigment transfer (Sharlow et al., 2000). Inhibition of PAR-2 cleavage by serine protease inhibitors, such as RWJ-50353, completely avoid the UVBinduced pigmentation of epidermis analogs (Seiberg et al., 2000). In dark-skinned Yucatan microswine, topical treatment with serine protease inhibitors, such as the soybean trypsin inhibitor (STI) and Bowman-Birk protease inhibitor (BBI) in a liposomal delivery system, exerts a significant skin lightening effect without toxic effects on melanocytes. Soybean milk extracts reduced melanin deposition within the swine epidermis and prevented UVB-induced pigmentation in vivo, similar to STI, without the addition of any delivery vehicle. Even niacinamide, the biologically active form of vitamin B3, inhibits melanogenesis, both in vitro and in vivo, probably through the suppression of melanosome transfer (Hakozaki et al., 2002). In fact, purified tyrosinase is not inhibited and pigmentation is reduced in coculture system or in reconstructed epidermis and not in melanocyte monocultures. 678

Melanin Dispersion and Acceleration of Skin Turnover The capacity of several compounds to disperse melanin pigment and/or accelerate epidermal turnover can result in skin lightening. Linoleic acid and retinoic acid exert their depigmenting action in part through the quickening of stratum corneum desquamation. Chemical substances extensively used as exfoliants, such a-hydroxy acids, stimulate cell renewal and facilitate the removal of melanized keratinocyte, leading to melanin pigment loss. Topical application has been shown to reduce the visibility of age spots, without reducing their size or number (Smith, 1999), and can be useful in the treatment of melasma (Jahaveri et al., 2001). Liquiritin, a flavonoid glycoside of liquorice, has been found to induce a significant improvement in hyperpigmentation of patients with bilateral and symmetric idiopathic epidermal melasma (Amer and Metwalli, 2000). The mechanism proposed involves melanin dispersion by means of the pyran ring of its flavonoid nucleus, and acceleration of epidermal renewal.

Inhibitors of Inflammation-induced Melanogenic Response Some mediators produced by keratinocytes after exposure to proinflammatory stimuli or UV exposure, such as IL-1a and endothelin-1, are able to promote melanogenesis. Therefore anti-inflammatory compounds could be useful for the treatment or for the prevention of postinflammation hyperpigmentation endothelin-1 showing unique behavior exerting stimulatory effects both on DNA synthesis and melanization by human melanocytes (Imokawa et al., 1997; see also Chapters 20–22). Activation of epidermal endothelins is determined by the enzymatic cleavage of inactive prepolypeptides by an endopeptidase termed endothelin converting enzyme (ECE), regulated by the pro-inflammatory cytokine IL-1a (Hachiya et al., 2002). Topical application of Matricaria chamomilla extract, an antagonist for endothelin-receptor, inhibits UVB-induced pigmentation, by avoiding endothelin1-induced DNA synthesis, but does not reduce IL-a-induced endothelin-1 production and tyrosinase activation (Imokawa et al., 1997). Among anti-inflammatory drugs, topical corticosteroids have been included in several clinical trials for the treatment of melasma. The possible mechanism is the suppression of cytokines, through the inhibition of NF-kB activation, which may explain their short-lived effect with a transient loss of color. Steroids, however, have been used in different depigmenting formulations to decrease the irritation caused by depigmenting agents (Westerhof and Njoo, 1995). Glabridin, the main component of hydrophobic fraction of liquorice extracts, decreases tyrosinase activity in B16 melanoma cells, without affecting DNA synthesis, and, in guinea pigs, inhibits UVB-induced skin pigmentation, as well as erythema (Yokota et al., 1998). The capability to inhibit superoxide anion production and cyclooxygenase activity suggests that the anti-inflammatory effect involves an interference with the arachidonic acid cascade, and that protection against oxidative stress has a key role in modulating melanogenesis.

CHEMICAL, PHARMACOLOGIC, AND PHYSICAL AGENTS CAUSING HYPOMELANOSES

Variations in the chemical structure modulate the effect, since melanogenesis inhibition is lost by replacing both hydroxyl groups and reduced by substitution at the 4¢ position.

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genesis and morphologic changes by protein kinase A activation via H receptors in human normal melanocytes. J. Invest. Dermatol. 114:334, 2000. Yoshimura, K., K. Tsukamoto, M. Okazaki, V. M. Virador, T. C. Lei, Y. Suzuki, G. Uchida, Y. Kitano, and K. Harii. Effects of all-trans retinoic acid on melanogenesis in pigmented skin equivalents and monolayer culture of melanocytes. J. Dermatol. Sci. 27:68s–75s, 2001. 2

Physical Agents Causing Hypomelanoses Jean-Philippe Lacour Melanocytes are vulnerable to nonspecific trauma. Dark skin or hair may lose pigment in areas exposed to various types of physical injury: thermal burns, freezing, UV radiation, ionizing radiation, and physical trauma.

Thermal and UV-induced Pigmentary Changes Thermal Burns Thermal burns which cause severe epidermal damage may destroy melanocytes to leave a depigmented scar (Fig. 35.2). Skin depigmentation after second- and third-degree burns is a

Fig. 35.2. Depigmentation with some perifollicular repigmentation following thermal burn (see also Plate 35.1, pp. 494–495).

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CHAPTER 35

A

atrophy may follow X-ray therapy. Human hair color changes following X-radiation were reported just after the turn of the last century (Danysz, 1903). Regrowth of white hair has been observed in white or black children following epilating dose of X-ray for the treatment of tinea capitis (Hazen, 1947). Chronic radiodermatitis with pigmentary changes associating hyper- and hypopigmentation has been described following percutaneous coronary interventions (Kawakami et al., 1999). X-ray depigmentation results from a loss of functioning melanocytes. Straile (1964) observed that X-ray irradiation decreases the number of melanocytes in the hair follicles of mice. Other changes included alterations of the length of the dendrites, decrease in the transfer of pigment granules, alterations in the distribution of melanin in the hair, and changes in the color of melanin granules (Straile, 1964). However, the mechanism by which follicular melanocytes seem to disappear after irradiation is unknown.

Ionizing Radiation

B Fig. 35.3. Pigmentary and vascular changes in chromic radiodermatitis (A). The photograph shows the severe vascular changes that are the usual sequelae of radiation burns (B) (see also Plates 35.2A and 35.2B, pp. 494–495).

serious cosmetic problem for dark-skinned people. Because of the destruction of hair follicles in these types of burn, no repigmentation can be observed whether spontaneously or after PUVA. Various grafts (split-thickness skin grafts, epithelial sheet allografts) have been successfully used to treat leukoderma due to burns (Taki et al., 1985). Freezing can be followed by cutaneous depigmentation; this phenomenon can be observed after use of liquid nitrogen in dermatologic practice. However, in contrast, cryotherapy has been reported to help repigmentation of idiopathic guttate hypomelanosis.

Phototoxicity Phototoxicity may lead to pigment loss: a single PUVA (see Chapter 60) (psoralen plus UVA) reaction was observed to lead to depigmentation of an exposed square in a Dutch-belted black rabbit (Ortonne et al., 1983). X-rays have long been known to cause changes in melanin pigmentation. In animals, X-ray exposure causes depigmentation of feathers or hair. In humans, cutaneous depigmentation (Fig. 35.3A, B) with 684

Ionizing radiation could alter the epithelium of the hair follicle with subsequent failure of the induction of follicular melanogenesis or disturbance in the regulation of the differentiation of the secretory melanocytes at the beginning of the hair growth cycle. Chase et al. (1951) observed radiation effects remote from the site of original exposure: depigmentation was delayed until the second growth cycle after irradiation. They concluded that there is an indirect effect of X-rays on melanocytes. On the other hand, melanocytes could be individually and directly killed or inactivated by Xrays. Straile (1964) suggested a more complicated mechanism: X–ray-induced depigmentation could result in part from a complete block in the differentiation of the melanoblasts, possibly related to an injury to the follicular epithelium, and also from direct or indirect destruction or inactivation of the melanocytes on an individual basis. Vitiligo can be induced by radiations in patients predisposed to or having vitiligo (Pajonk et al., 2002). Gamma and neutron radiation from atomic weapons also alters the melanin pigmentation, and depigmentation induced in animals by these radiations has been used for biologic radiation dosimetry (Moshman and Upton, 1954).

Trauma Physical trauma also often leaves depigmentation which is most apparent in dark-skinned people. The reason for the increased sensitivity of melanocytes to a wide variety of injuries is unknown. The low-density, self-perpetuating melanocyte population in the skin with low turnover rate may be responsible.

References Chase, H. B., H. Rauch, and V. W. Smith. Critical stages of hair development and pigmentation in the mouse. Physiol. Zool. 24:1–8, 1951. Danysz, J. De l’action du radium sur les différents tissus. C. R. Acad. Sci. Paris 136:461–464, 1903. Hazen, H. Results of repeated epilation with roentgen rays in tinea tonsurans. Arch. Dermatol. 56:539–540, 1947.

CHEMICAL, PHARMACOLOGIC, AND PHYSICAL AGENTS CAUSING HYPOMELANOSES Kawakami, T., R. Saito, and S. Miyazaki. Chronic radiodermatitis following repeated percutaneous transluminal coronary angioplasty. Br. J. Dermatol. 141:150–153, 1999. Moshman, J., and A. Upton. Depigmentation of hair as a biological radiation dosimeter. Science 119:186–187, 1954. Ortonne, J.-P., D. B. Mosher, and T. B. Fitzpatrick. Topics in Dermatology: Vitiligo and Other Hypomelanoses of Hair and Skin. New York: Plenum Publishing Corporation, 1983. Pajonk, F., C. Weissenberger, and M. Henke. Vitiligo at sites of irra-

diation in a patient with Hodgkin’s disease. Strahlenther. Onkol. 178:159–132, 2002. Straile, W. E. A study of the hair follicle and its melanocytes. Dev. Biol. 10:45–70, 1964. Taki, T., S. Kozuka, Y. Izawa, T. Usuda, M. Hiramatsu, T. Matsuda, K. Yokoo, Y. Fukaya, M. Tsubone, and J. Aoka. Surgical treatment of skin depigmentation caused by burn injuries. J. Dermatol. Surg. Oncol. 11:1218–1221, 1985.

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36

Infectious Hypomelanoses Jean-Philippe Lacour

Treponematoses Nonvenereal treponematoses (yaws, pinta, and bejel) and venereal syphilis are caused by various subspecies of Treponema pallidum (Koff and Rosen, 1993). In all of them, if left untreated, cutaneous depigmented lesions develop at some point in the course of the disease.

Yaws Yaws (framboesia tropica, pian, parangi, paru, buba, bouba) is a treponematosis caused by T. pallidum subspecies pertenue.

Historical Background Yaws is an ancient disease which might have originated around 10 000 bc in the Afro-Asian landmass. The presence of spirochetes in ulcers in patients suffering from parangi in Ceylon was first shown in 1905 by Castellani. The disease is also called “framboesia tropica” because the cutaneous lesions frequently resemble raspberries.

Epidemiology Yaws is prevalent in rural, very warm tropical areas, particularly throughout Africa. Foci of infection persist in Southeast Asia, Pacific Islands, and tropical America. The disease is contracted mostly in childhood with a peak of new cases during the first decade. It is acquired by direct contact with infected carriers, usually through breaks in the skin. Occasional indirect transmission attributed to fomites or insects may occur. Transmission is facilitated by overcrowding and poor hygiene conditions. The current prevalence of yaws is not exactly known. Resurgence has been observed in several tropical countries and millions of children are at risk of contracting the disease (Engelkens et al., 1991a; Sehgal et al., 1994).

Clinical Features Yaws develops in four stages (primary, secondary, latent, and tertiary), but it seems practical to distinguish between an early stage (primary and secondary) in which skin lesions are contagious and a late stage. In the primary stage, initial skin lesions develop at the site of infection after a variable incubation period (9–90 days). They appear as one or more erythematous papules of the lower limbs. They tend to enlarge by extension or by confluence with satellite lesions. They later become crusted, then ulcerate and form papillomatous, raspberry-like lesions. This stage is sometimes accompanied by fever, joint pain, or regional lymphadenopathy. The “mother 686

yaw” usually heals after several weeks, leaving atrophic and hypopigmented scars surrounded by a darker halo. The secondary stage occurs weeks to month later and results from the widespread dissemination of treponemes. Skin lesions resemble the initial lesions except that they are smaller and widespread. Hyperkeratotic plaques occur on palms and soles. Headache, fever, malaise, bone and joint pain, and generalized lymphadenopathy are often present. Lenticular or hypochromic macules can be observed as an uncommon presentation of the second stage. They are considered to be an id reaction and are called “babides,” “framboesides,” or “pianides.” Lesions in the early stage of yaws usually disappear spontaneously, sometimes leaving moderate pigmentary changes. The disease then enters a noninfectious latent period that may last the lifetime of the patient. Tertiary or late yaws develops in approximately 10% of cases. It is characterized by keratodermous subcutaneous nodules with abscess formation that leave keloid scars causing deformities and contractures. At this stage, chronic destructive osteitis leads to curvature of the bones, and exostoses of nasal and adjacent bones (goundou). Neurologic and cardiovascular abnormalities can occur.

Cutaneous Depigmentation in Yaws Leukoderma is observed during the late stage after untreated tertiary yaws. Treatment of the latter prevents development of leukoderma. Hypopigmented macules enlarge until an entire area becomes depigmented. Depigmentation has been found to be uncommon among those with yaws, affecting 686 of 45 035, none of whom were under 9 years and only six of whom were between 10 and 19 years of age. Of those under 30 years of age, 0.11% of males and 0.13% of females had leukoderma as opposed to 4.42% of males and 9.63% of females over 50. It has been suggested the prevalence in females may be related to the fact that females, who are less hairy, may have different follicular melanocytes, which are more susceptible to treponemal toxins (Browne, 1976b). The pattern of depigmentation is characteristic. Lesions are usually symmetric. The most commonly affected areas are the anterior wrists (40%), often having a triangular macule the apex of which points inferiorly. Dorsal hands, and metacarpophalangeal and interphalangeal joints, are involved in 21% and 17% of cases, respectively. Involvement of the feet and legs is much less common. Mottled palmar dyschromia is often associated with typical frambesial hyperkeratosis and contracture, especially of the fourth and fifth digits. Pigment loss over the dorsal hand is

INFECTIOUS HYPOMELANOSES

usually patchy and irregular, although it may be complete over small bony prominences. The olecranon process may be depigmented as may be the fibular head, either side of the tibial crest, anterior ankle, and occasionally large areas of the leg and thigh. Normally deeply pigmented areas, such as female areolae, scrotum, and penis, may be affected if general pigment loss is severe. There are only a few affected sites in any one patient. Cases of generalized achromia of limbs have been reported to follow widespread secondary yaws.

hyperkeratoses. Contrary to yaws, pinta is considered a benign treponematosis because of the absence of extracutaneous involvement.

Cutaneous Depigmentation in Pinta

The causative agent, T. carateum, is microscopically identical to the treponemes of syphilis and yaws. Transmission occurs through direct skin or mucous membrane contact through a minute abrasion or wound. It is prevalent in tropical Central and South America (Engelkens et al., 1991b). The disease shows no sex or racial predilection and occurs essentially in childhood and adolescence. The clinical course of the disease is divided into early and late pinta, but these frequently overlap so that there is also an intermediate stage in which early and late lesions occur simultaneously.

The primary lesion, at the site of inoculation, may be variably pigmented so that hypochromic and achromic areas coexist. The healed lesions are usually hypopigmented. Secondary lesions or pintids show gradations of depigmentation throughout, around the periphery, or in small areas. The presence of alternating macules of erythema, pigmentation, and leukoderma may give the pintids a mottled appearance. Finally, they may become completely depigmented. Pintids may occur at the same site as the primary lesion but extensive dissemination may be observed. Although depigmentation occurs in most cases six months to one year after the appearance of pintids, it may be seen within two or three months. Leukoderma or “white pinta” is characteristic of the late stage of the disease. The occurrence of leukoderma in a patient with pinta reflects the rate of regression of the lesions rather than the duration of the disease. Some patients may develop achromic lesions within a few months (late pinta) whereas others may require many years to develop depigmented macules (intermediate stage). The depigmented macules in late pinta may be localized or generalized and vary in size, shape, and distribution. Near total cutaneous depigmentation may occur. Depigmented macules are seen on the bony prominences such as the knuckles, elbows, knees, and ankles. Involvement of the flexor wrist is common. The contours of the lesions are irregular but well defined and are marked by peripheral brownish hyperpigmentation. The color may be white. Sometimes there is also a mixture of all varieties of dyschromia, hypochromia, and achromia. Usually, this variant is extensive and found on the back and extremities. Sometimes, on the flexor aspect of the upper extremities, especially the forearms, reticulated areas are observed mixed with pinhead-sized depigmented macules and brownish hyperpigmented or bluish puncta. Pea- or beansized hyperpigmented spots are often randomly scattered in areas of depigmentation. Peripheral extension of the depigmented lesions has been observed. In many cases, the skin is otherwise normal. However, in long-standing pinta, atrophic lesions with thinning, wrinkling, alopecia, and dryness of the skin due to suppression of sweating and sebaceous secretion may be observed, particularly on the dorsum of the hands, knees, thighs, and anterior legs.

Clinical Features

Histology

The early stage is characterized by a papule or an erythematosquamous plaque localized on the uncovered parts of the body. The initial lesion disappears spontaneously and, after several months, widespread and smaller lesions appear, comparable to the initial lesion, called “pintids.” Striking changes in skin pigmentation result from these lesions. The main features of tertiary pinta are achromia, skin atrophy, and

In newly developed primary or secondary lesions, there is migration of lymphocytes into an acanthotic and spongiotic epidermis, liquefaction degeneration of the basal layer, and loss of melanin. In the papillary dermis there are numerous melanophages and a moderately dense infiltrate of plasma cells, lymphocytes, and some histiocytes and neutrophils. In these early stages, the pigmentary disturbance is minimal.

Histology and Laboratory Studies In depigmented skin, the epidermis is normal except for flattening of the rete pegs and absence of melanin in the basal layer. No spirochetes can be identified. The serologic tests are positive in early hyperpigmented stages but often negative in the later achromic stages.

Treatment Penicillin is the treatment of choice for patients and contacts by single intramuscular injection of benzathine penicillin. Tetracyclines and erythromycin are recommended in case of allergy. Treatment prior to the tertiary stage prevents the development of leukoderma.

Pinta Pinta (“mal del pinto,” “carate,” “cute,” or “cativa”) is a chronic nonvenereal treponematosis, caused by T. carateum and characterized by partial or complete loss of pigment.

Historical Background Pinta is considered to be the first treponematosis to have occurred in humans. Its causative agent was first thought to be of myotic origin until Armenteros and Triana (1938) and Blanco (1942) identified T. carateum as the causative organism.

Epidemiology

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In old hypochromic or achromic lesions, there is a decrease or absence of melanocytes and of melanin granules in the basal layer of the epidermis. The epidermis is atrophic and there is no inflammatory infiltrate. There is accumulation of melanophages in the hyperchromic areas. Treponemes are demonstrated by silver staining methods in early and recent lesions, but not in old lesions.

primarily in seminomadic populations of the Arabian peninsula and along the southern border of the Sahara desert (Sahel). The prevalence is high: 7.5% of 5–14-year-old children in the African Sahel have clinical lesions and 22% have positive treponemal serology (Engelkens et al., 1991b; Koff and Rosen, 1993).

Clinical Features Mechanism The hypomelanosis in pinta may be post inflammatory. Because hypopigmentation is associated with many treponematoses, it must also be postulated that treponemes may have an inhibitory effect on melanocytes. However, no depigmenting agent of treponemal origin has been isolated so far.

Diagnosis and Differential Diagnosis The diagnosis, which is suggested by epidemiologic and clinical features, may be confirmed by darkfield microscopy from any cutaneous lesion except an old achromic one. Serologic reactions [venereal disease research laboratory (VDRL), fluorescent treponemal antibody-absorption test (FTA-ABS)] become positive from two to three months after the onset of the disease. No serologic reaction can distinguish pinta from other treponematoses. Clinically the lesions may mimic many skin diseases characterized by hypopigmentation such as piebaldism, vitiligo, tinea versicolor, pityriasis alba, and discoid lupus erythematosus. However, the history, the presence of T. carateum, and a positive serologic examination distinguish pinta from other hypomelanoses.

Early lesions are rarely seen, usually appearing as a small papule or ulcer on the oropharyngeal mucosa. The secondary stage is characterized by mucous patches with ulcers on the fauces, tonsils, tongue, lips and buccal mucosa. Laryngitis and regional lymphadenopathy may be seen. Disseminated papules can be observed. Osteoperiostitis of the long bones occurs, causing nocturnal leg pains. Tertiary lesions develop months to years after inoculations as gummas that may progress to chronic ulcers. Gummas tend to resolve with time, leaving atrophic depigmented scars surrounded by hyperpigmentation. Destructive cutaneous, muscular, and osseous lesions can lead to deforming mutilations.

Hypomelanosis in Endemic Syphilis Depigmentation occurs in the late stage of endemic syphilis. Csonka (1953) found pigmentary changes, consisting of depigmentation and pigmentary macules, in 49 of 3507 patients with bejel. The depigmentation in this disease is similar to that of pinta. The hypopigmented macules are usually symmetric and have well-defined borders. The extremities, genitalia, shoulders, areolae, and the trunk are most commonly involved. Pigmented macules may be randomly scattered throughout the hypopigmented ones.

Treatment The current recommended treatment is benzathine penicillin 1.2 million units given intramuscularly. Primary or secondary dyschromic lesions disappear after treatment in four or six months without the occurrence of hypochromia. Rein (1952) observed that penicillin therapy resulted in a complete repigmentation of leukoderma of short duration except for macules over bony prominences. In these areas, there was only a spotty repigmentation or a reduction in the size of the patches of leukoderma. In patients with long-standing achromic lesions, repigmentation is rarely observed.

Diagnosis Epidemiology, other cutaneous features, and skeletal lesions distinguish bejel from pinta, vitiligo, and piebaldism.

Treatment Treatment is with intramuscular penicillin. Early treatment induces satisfactory repigmentation (Csonka, 1953).

Leukoderma in Secondary Syphilis Leukoderma syphiliticum is an occasional clinical finding of secondary syphilis (Fig. 36.1).

Endemic Syphilis Endemic syphilis is a chronic childhood infection of skin, bone, and cartilage.

Epidemiology Endemic syphilis or bejel is transmitted from child to child by close skin contact and fomites such as communal drinking vessels. The main reservoir is in children between 2 and 15 years of age and the infection tends to run in families under conditions of poor hygiene and crowding. There is no sex predilection. The infective agent is T. pallidum subspecies endemicum. Endemic syphilis is prevalent in dry climates. It occurs 688

Clinical Features Leukoderma syphiliticum, found frequently on the lateral neck of women, has been called the “necklace of Venus.” It occurs about three to six months after infection when roseola is fading, but may be seen some time later within the first year. Whereas in some patients this hypopigmentation is easily detected, in most cases it is so subtle that a careful examination, often with Wood’s light, is necessary. The clinical picture is a leukomelanoderma. Small round or oval macules, only slightly hypopigmented, 1–2 cm in diameter, well defined, and not confluent, are randomly scattered on a darker grayish or brownish background. The lesions are

INFECTIOUS HYPOMELANOSES

Leprosy Leprosy is a chronic infectious human disease caused by Mycobacterium leprae that affects the skin and the peripheral nervous system, but which may also involve other organs. Hypopigmentation is one of the main cutaneous features of leprosy.

History

Fig. 36.1. Hypomelanotic macules on the dorsum of the hands in a patient with secondary syphilis.

usually bilateral and nearly symmetrical. The hyperpigmentation is poorly delineated. The combination of the hypopigmented macules and the slightly hyperpigmented background presents a reticular pattern. There is no scaling or itching. Most lesions are on the side of the neck, sometimes with extension to the shoulder, upper trunk, and axillae. The abdomen, inguinal region, and proximal part of the limbs may be involved.

Histology and Mechanism Since this type of leukoderma is always preceded by an erythematous macular syphilide, it is considered to be post inflammatory. However since varying types of hypopigmentation are associated with treponematoses, one may raise the question of a more specific link between the treponemes and pigmentary disturbances. Furthermore, Poulsen et al. (1988) demonstrated the presence of T. pallidum in leukodermal skin around vessels and inside nerve fibers in which the myelin sheaths of the axons showed evidence of degeneration.

Diagnosis and Differential Diagnosis Achromic tinea versicolor or postinflammatory hypopigmentation may be considered in the differential diagnosis. However, diagnosis is generally easily established. History of a primary chancre and other cutaneous findings establish the diagnosis. In association with the skin hypopigmentation, the other clinical features of secondary syphilis (papular hamcolored lesions of the palms and soles; “moth-eaten” alopecia; erosion of the mouth and the tongue; moist, wartlike lesions in the anogenital region; regional or generalized adenopathy) make the diagnosis inevitable. Furthermore, at this stage of the disease, the serology is always positive.

Treatment Treatment is with penicillin. Leukoderma syphiliticum regresses with treatment and usually disappears totally within several months, but up to a year may be required for full resolution.

The disease probably originated in Egypt and other MiddleEastern countries as early as 2400 bc. Since antiquity, depigmented macules have been considered one of the most important clinical signs of leprosy. In 1929, five types of depigmented lesion were described in leprosy. The “perifollicular type” was characterized by pinheadlike hypochromic macules with hyperkeratosis. The second and third types, flat uniformly depigmented macules, were thought to represent further development of the first type. The fourth type represented a transitional stage between the true depigmented and the erythematous lesions, and the fifth or “mottled” type was characterized by perifollicular repigmentation of the white macules. Later, it became evident that hypopigmentation might be observed in all the forms of the disease.

Epidemiology Leprosy is caused by a Gram-positive bacillus, M. leprae, which is an acid alcohol-fast intracellular bacillus. The infection occurs only in humans. Lepromatous leprosy is considered to be the principal source of contagion which spreads from person to person by close contact with an affected individual. The disease is widespread in tropical and subtropical countries and endemic in some temperate zones. It is estimated that the number of leprosy cases has fallen to 5.5 million all over the world. Two-thirds are in Asia and less than a third in Africa. No race has been spared. Lepromatous leprosy is usually twice as frequent in males as in females. The tuberculoid type affects both sexes equally. In endemic areas, leprosy is usually contracted in childhood but exposure in adult life may lead to infection. The incubation period is difficult to calculate and probably varies from three to five years. A considerable proportion of people who are exposed to M. leprae do not develop the disease. Individuals who develop the disease manifest clinical polymorphism.

Classification Several classifications have been proposed, and the current classification of leprosy is based on the degree of host resistance and takes into account clinical, bacteriologic, histopathologic, and immunologic parameters (Sehgal, 1994). The expression may be indeterminate leprosy, a probable prelude to determinate forms. The determinate group forms a continuous spectrum with tuberculoid (TT) and lepromatous leprosy (LL) as polar expressions. These are bridged by borderline groups, namely borderline tuberculoid (BT), borderline borderline (BB), and borderline lepromatous (BL). 689

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a pebbled appearance. The surface of the macules may be slightly scaly, dry, or faintly atrophic. They are asymmetrically distributed on the posterolateral aspects of the extremities, back, buttocks, and face. Alopecia and anhidrosis of the macules is present. The sensations of temperature, touch, and pain are completely lost.

Borderline Tuberculoid Leprosy In BT, hypopigmented and/or erythematous macules and plaques with well-defined yet irregular margins are seen. The surface is dry, bald, and scaly. Sensations are impaired or totally lost. The number of lesions may vary from 3 to 10. Satellite lesions are present. Fig. 36.2. Hypomelanotic macules in leprosy (see also Plate 36.1, pp. 494–495).

Borderline Borderline Leprosy

Lepromatous leprosy is characterized bacteriologically by the presence of abundant M. leprae in lesions, histologically by a characteristic infiltrate of Virchow cells containing globi of bacilli, and immunologically by a negative lepromin test which indicates lowered host resistance. Tuberculoid leprosy is characterized bacteriologically by the rarity or absence of bacilli, histologically by tuberculoid or occasionally sarcoidal structures, and immunologically by a positive lepromin test. Indeterminate leprosy is characterized clinically by involvement of only skin and nerves, bacteriologically by a few or no bacilli, histologically by a nonspecific inflammatory reaction, and immunologically by a lepromin test which may be positive or negative. Tuberculoid and lepromatous leprosy are stable polarized types, whereas indeterminate and borderline leprosy are unstable types.

Borderline Lepromatous Leprosy

Hypopigmentation in Leprosy Hypopigmented lesions can be observed in all forms of leprosy (Fig. 36.2).

Indeterminate Leprosy In this type of leprosy, the macules are usually hypopigmented but may also be erythematous. There is usually a single or few lesions. They are asymmetrically distributed, mostly on the exposed areas, but also on the trunk, limbs, or buttocks. The borders are well- or ill-defined, and not infiltrated; hair is normal. The surface may be mildly scaly or smooth. The macules may be hypoesthetic or anesthetic. These lesions may regress spontaneously or may evolve into one of the more definite types of leprosy.

Tuberculoid Leprosy In TT, involvement can be extensive. The macules have discrete edges and may be quite large, up to 30 cm in diameter. Sometimes there is a central flat macule surrounded by raised borders or the lesions may be uniformly infiltrated or present 690

In BB, the lesions show features of BT as well as LL. They are bilateral but asymmetric.

Morphologic features of BL are similar to BB, but the lesions conforming to LL are predominant as compared with BT morphology. The lesions are numerous and uncountable and tend to be symmetric.

Lepromatous Leprosy Hypopigmented macules may be the earliest expression of LL. The lesions are usually small, multiple, and ill-defined with indistinct borders. They may be difficult to recognize, particularly in light-skinned people. Although the lesions may cover the whole body, they are usually observed symmetrically on the face, extremities, and buttocks. They show little or no anhidrosis or loss of sensation. Later, plaques, nodules, or diffuse infiltration of the skin appear.

Histology Early histologic studies of the hypopigmented macules showed loss of epidermal pigment to be the only significant histologic change. In the epidermis of tuberculoid or lepromatous leprosy, Ishibashi (1964) found extreme irregularity in the distribution of epidermal pigment with a resultant mosaic in which some epidermal cells possess abundant pigment while adjacent cells have little or no pigment and tend to lose pigment with the progression of the dermal granuloma (Fig. 36.3). No inflammatory epidermal changes were described in lepromatous leprosy and there was no correlation between the degree of hypomelanosis and the presence or absence of M. leprae. In tuberculoid leprosy the epidermal depigmentation was related to the intensity of liquefaction degeneration of the basal layer (Ishibashi, 1964). Breathnach et al. (1962) reported a slightly decreased dopa reaction. Nayer and Job (1970) studied split-dopa preparations from 20 patients with macules of hypopigmentation secondary to tuberculoid, indeterminate, and dimorphous types of leprosy. Decreased melanocyte counts were present in 13, to a statistically significant degree in nine. Increased density of melanocytes was noted in six, but in only three cases was

INFECTIOUS HYPOMELANOSES

the space thus provided was filled with a granular material. Folding and replication of the basement membrane were found in areas of contact with several basal melanocytes. Another study also noted that melanosomes were poorly melanized.

Pathogenesis of Hypomelanosis

Fig. 36.3. Areas of hyperpigmentation and hypopigmentation in a patient with leprosy (see also Plate 36.2, pp. 494–495).

the difference statistically significant. The intensity of the dopa reaction was reduced in six cases, increased in five, and unchanged from normal in nine. In 16, there was a slight increase and in one a slight decrease. The length and branching of the dendrites was normal in 14 cases, reduced in two, and increased in four. The authors concluded that the majority of the lesions studied showed a reduction in melanogenic activity, but they were unable to identify the cause of this alteration in the melanocyte (Nayer and Job, 1970).

Electron Microscopy Studies Several ultrastructural studies have been reported (Breathnach et al., 1962; Job et al., 1972; Nayer and Job, 1970). One study showed ultrastructurally normal melanocytes in nine patients with leprosy, whereas in another study of hypopigmented lesions of indeterminate leprosy the number of melanocytes was greatly reduced. Some of these cells showed vacuolization or atrophy, and sometimes a dilated endoplasmic reticulum. In other cells, the number of melanosomes was decreased, the endoplasmic reticulum was reduced, and the Golgi apparatus barely apparent. Retraction of the cytoplasm of some melanocytes from adjacent keratinocytes was observed and

The mechanism of hypopigmentation in leprosy is unknown. Having observed no histologic or ultrastructural alterations in the melanocytes, Breathnach concluded that hypopigmentation in leprosy is probably just a postinflammatory hypomelanosis. This does not explain the histologic and electron microscopic findings reported by Job et al. (1972). The depigmentation of the epidermis could be considered a direct sequela of the invasion of bacilli at the melanocyte level or the peripheral nerve: because of their common neuroectodermal origin, they could show a peculiar susceptibility to M. leprae. Prabhakaran (1971) observed that M. leprae contains an enzyme similar to the tyrosinase of melanocytes. Like plant phenolases and unlike mammalian tyrosinases, this M. leprae enzyme was able to act on catechols and dopa. With fairly purified concentrates of M. leprae from lepromatous nodules, in vitro oxidation of dopa to indole-5,6-quinone was observed. They suggested that the lepra bacillus converts dopa to a quinone, so that dopa is unavailable for melanin production (Prabhakaran, 1971). However, there is no correlation between the degree of hypomelanosis and the presence or absence of M. leprae. The palest areas of skin often have remarkably few bacilli present, whereas lepromatous nodules with many bacilli frequently show no pigmentary changes. Seghal (1973) suggested that, in leprosy, there is a reduction in the cutaneous vascular supply which leads to atrophic changes in the melanocytes and hypomelanosis.

Diagnosis Diagnosis of leprosy is primarily clinical, based on the history (endemic area or familial exposure to leprosy), on the clinical appearance and the demonstration of hypoesthesia of a skin lesion, and by the presence of enlarged, hard, and tender nerve trunks at the sites of predilection or near a hypopigmented macule. M. leprae can be demonstrated in a slit-skin smear stained by the Ziehl–Nielsen method. The diagnosis can be made by biopsy of a skin lesion which may show typical granuloma or M. leprae on Ziehl–Nielsen staining. The lepromin skin test (Mitsuda) has no diagnostic value but is useful for classification.

Differential Diagnosis of Hypomelanosis in Leprosy Hypochromic lesions of leprosy may be mistaken for vitiligo, nevus anemicus, pityriasis alba, tinea versicolor, morphea, postinflammatory leukoderma, pinta and other treponematoses, onchocerciasis depigmentation, and post-kala-azar dermatosis. However, all of these lack hypoesthesia.

Treatment The World Health Organization recommends a multidrug 691

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regimen: rifampicin and dapsone for six months for paucibacillary leprosy and rifampicin, dapsone and clofazamine for two years or until smear negative for multibacillary cases. However, drug resistance, persisters, and relapses are possible (Sehgal, 1994). Treatment usually results in repigmentation of the hypopigmented macules but residual depigmentation can persist.

Tinea Versicolor Tinea versicolor is a common superficial fungus infection which may cause hypopigmentation of the skin (Fig. 36.4). It is due to a lipophilic yeast, Pityrosporum orbiculare (or P. ovale), also known as Malassezia furfur.

A

Histological Background Tinea versicolor was first described in 1846 by Eickstedt and in 1847 by Shuyter. P. ovale was isolated and named by Castellani and Chalmers in 1913 but the fungus had previously been identified in 1855 by Robin and named Microsporon furfur, later termed Malassezia furfur. In the 1950s Gordon isolated spherical and oval yeast strains that he named P. orbiculare (Borelli et al., 1991). Hypopigmentation in tinea versicolor was first described by Ehrmann in 1908. In 1929, Gougerot et al. found the achromia of tinea versicolor to be particularly frequent after sunlight exposure. It was later that the hypochromia shown was a true hypopigmentation and not a pseudoachromia or contrast phenomenon. Several hypotheses were proposed to explain the hypopigmentation. Wertheim, in 1923, studied the role of ultraviolet radiation. In 1927, Kistiakovsky and, in 1936, Lewis and Hopper suggested that hypopigmentation results from obstruction of ultraviolet radiation by the fungus so that delayed tanning does not occur. On the other hand, in 1933 Ruete suggested that the depigmentation in tinea versicolor results from some direct interaction between the fungus and melanocytes (Ortonne et al., 1983).

Epidemiology Tinea versicolor has a worldwide distribution but is more common in tropical climates. In temperate climates, its onset is more frequent during summer. It is found in all races and in both sexes. A 2:1 female to male ratio has been reported. Most cases are observed in adulthood when the sebaceous glands are active. The disease is due to P. ovale, also known as P. orbiculare or M. furfur. They are probably the same polymorphic organism. P. ovale is a lipophilic member of the normal human cutaneous flora. It has also been reported that the skin of neonates could be colonized by P. ovale, however the colonization of the skin usually starts during puberty. In elderly persons the number of yeasts decreases, probably because of a decrease in skin lipids. Although a saprophyte, P. ovale is associated with pityriasis versicolor, pityrosporum folliculitis, seborrheic dermatitis, exacerbation of atopic dermatitis, and Gougerot–Carteaud syndrome. 692

B Fig. 36.4. (A, B) Typical hypopigmented macules on the trunk in two patients with tinea versicolor (see also Plates 36.3A and 36.3B, pp. 494–495).

P. ovale changes from the round blastospore form to the pathogenic mycelial form under the influence of exogenous and endogenous factors: high temperature, humidity, greasy skin, hyperhidrosis, hereditary factors, diabetes, pregnancy, corticosteroids, or immunodeficiency are predisposing conditions.

INFECTIOUS HYPOMELANOSES

Tinea versicolor is usually not a contagious disease. It occurs most frequently on the seborrheic regions of the trunk and can affect the face, particularly in the tropics. Moderate itching is sometimes reported. The lesions start as light brown to red, slightly scaly, nummular plaques that can become white. The diagnosis is made on the basis of the typical clinical picture, the yellow fluorescence under Wood’s lamp examination, and the presence of round and oval budding cells and hyphae in Koll preparation.

Hypopigmentation in Tinea Versicolor The primary lesion is a round or oval hypopigmented macule from several millimeters to one to several centimeters in diameter (Fig. 36.4). Occasionally the lesions appear slightly raised. These macules are tan to dull white so that the involved skin appears moderately hypopigmented. Although the lesions are usually asymptomatic, some patients complain of mild pruritus. While the contrast between the hypopigmented normal skin is striking in dark-skinned patients, Wood’s light examination may be required to locate the lesions in light-skinned people. The borders of macules are sharp to slightly fuzzy. The scaling may not be very apparent but may be obvious when the involved skin is scratched. Depigmentation without any scaling may remain for months after remission of the scaling lesions. The hypopigmented macules are usually distributed over the upper anterior and posterior trunk. However, the proximal part of the limbs, the abdomen, the neck, and the face may be involved. Palms and soles are never affected. Depigmented, scaly, circumscribed macules on the face and gluteal regions of black infants with tinea versicolor have been reported, with islands of depigmentation, extending onto the abdominal wall and the lower back (Jelliffe and Jacobson, 1954). Multiple hypopigmented scaly macules on the penis have been reported in a black man (Blumenthal, 1971). These macules, once established, do not change. In winter they seem to disappear on light-skinned individuals. Following sun exposure, they are again more apparent.

Histology El Gothamy et al. studied melanin formation in the involved skin of tinea versicolor. In seven patients, although there was no hypopigmentation clinically, they observed a decreased density of melanin granules in keratinocytes and a decreased dopa reaction (El Gothamy, 1975). Charles et al. using splitdopa preparations of hypopigmented epidermis showed no change in the density of dopa-positive melanocytes, but the melanocytes appeared larger and more intensively stained than those of uninvolved areas (Charles, 1973).

Electron Microscopy Studies Charles observed that melanosomes were smaller in the involved epidermis than in normal skin. The number of melanosomes in melanocytes was markedly reduced. The dendrites of these cells also showed marked “melanin loading” compared to the dendrites in normal skin. Melanosomes were

irregularly distributed within keratinocytes. While some of these cells, scattered in the basal and spinous layers, were overloaded with melanosomes, others showed little or no melanin uptake. Melanosomes were aggregated rather than dispersed. Dark collapsed cells were also observed scattered throughout the stratum spinulosum. The author suggested that “melanin loaded” cells observed in the basal and spinous layers became the dark collapsed cells of the upper spinous layer and at the same time lost their melanosome complexes. Their interpretation of the cellular and subcellular events that result in hypopigmentation in tinea versicolor was production of abnormally small melanosomes; partial block in melanosome transfer to keratinocytes; and possible increased melanosome degradation in lysosomal complexes resulting in degeneration of keratinocytes. Similar ultrastructural observations were made by Grupper et al. who also found decreased or absent melanin synthesis, aggregation of mature melanosomes, and structural abnormalities of stage I melanosomes (small size, rounded shape, granular matrix). There was a loss of contact between melanocytes and keratinocytes, with an increased intercellular space which contained a granular material and mononuclear cells. Contact between melanocytes and inflammatory cells was observed and no melanosomes were found in those melanocytes close to the inflammatory cells. They also concluded that there was a disturbance in melanin synthesis, in melanosomes, and in melanosome transfer (Grupper, 1975).

Pathogenesis of Hypopigmentation in Tinea Versicolor Ultraviolet radiation seems to be an important etiologic factor since hypopigmented macules often appear after sun exposure. One of the first hypotheses was that the hypopigmentation resulted from filtration of the sun’s rays by the fungus. In 1927, Kistiakovsky concluded that the fungal spores contain a yellowish pigment which strongly refracts light so that the skin affected by fungus does not tan. However, this fails to explain the occurrence of hypopigmentation on covered, unexposed areas. Histochemical and electron microscopic findings indicate, in fact, that there is a disturbance in melanogenesis. Thus it is likely that ultraviolet radiation merely unveils an already present skin condition. Other authors suggested a direct interaction between the fungus and melanocytes, because tinea versicolor hypopigmentation can occur in the absence of a history of sun exposure: the fungus itself may alter the normal mechanism of pigmentation of the skin (Hildick-Smith, 1964; Ruete, 1933). El Gothamy, and Ito and Tanaka, suggested that the fungus may interfere with the metabolism of tyrosine (El Gothamy, 1975; Ito and Tanaka, 1974). Grupper et al. observed close contact between lymphocytes and melanocytes without melanosomes and suggested that hypopigmentation in tinea versicolor may involve cellular immunity. They suggested the following three possibilities: the fungus may secrete a factor toxic to melanocytes and induce modifications in their antigenicity; some factor of the fungus 693

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may absorb specifically at the surface of melanocytes to change their antigenicity; or the fungus may have a cross-antigenicity with melanocytes. The mechanism has been clarified by Nazzaro-Porro and Passi (1979) who have shown that P. ovale was able to liberate azelaic acid, a product capable to interfere with melanogenesis. P. ovale possesses lipoxygenases that act on different unsaturated fats present in skin surface lipids. In culture, the fungus is capable of oxidizing oleic acid to azelaic acid, a saturated 9-carbon atom dicarboxylic acid (COOH-(CH2)7-COOH). It owes its name to the fact that it was obtained from the oxidation of oleic acid by nitric acid (az = azote = nitrogen; elaion = oil). It can be obtained, apart from the oxidation of fatty acids unsaturated in position 9, by fermentation from a variety of microorganisms. Azelaic acid was thought to be a competitive inhibitor of tyrosinase, the key enzyme for melanogenesis, thus it seemed possible that is could be responsible for the hypochromia of pityriasis versicolor (Nazzaro-Porro and Passi, 1979). Furthermore, it is known that some phenolic compounds that act as alternative substrates for tyrosinase have depigmenting properties when applied topically on the skin, and azelaic acid might have a similar effect. However, it soon became evident that the diacid had no depigmenting activity upon normal skin, nor had it any toxic effect. Its cytotoxic effects appear to be limited to the abnormally active and malignant human melanocytes. They are probably due to antimitochondrial activity and interference with nuclear DNA synthesis rather than to competitive inhibition of tyrosinase as originally thought. The hypopigmentation in pityriasis versicolor is more likely due to toxic lipoperoxides formed by the action of P. ovale on the unsaturated lipids of the skin surface (Nazzaro-Porro et al., 1986).

Diagnosis and Differential Diagnosis In typical cases in which hypopigmented macules are associated with pink to brown scaly lesions, a clinical diagnosis can be made easily. In whitish macules, Wood’s light examination shows the skin to be only hypopigmented. In pink to brown macules, the golden-green fluorescence is characteristic. The diagnosis of tinea versicolor may be confirmed by examination of the scrapings of the lesions. Achromic tinea versicolor may be confused with vitiligo, postinflammatory hypopigmentation, progressive macular hypomelanosis of the trunk (see Chapter 40), macular topical hypochromia, or chronic parapsoriasis. However, history, Wood’s light, and examination of the scales with potassium hydroxide readily differentiate the hypopigmentation of tinea versicolor from that of other hypomelanoses.

acid, propylene glycol, ciclopiroxolamine, several imidazoles, and terbinafine are effective topical therapies. Systemic therapy is indicated for extensive lesions, or lesions resistant to topical treatment or for frequent relapse. Ketoconazole, itraconazole, and fluconazole are effective, even with short-term regimens which minimize side effects. All treatments are associated with a relatively high recurrence rate which is due to predisposing factors that can be difficult to eradicate. Consequently, a prophylactic treatment regimen is necessary to avoid recurrence. After appropriate treatment and fungus eradication, hypopigmentation usually fades with time and sunlight exposure. Attempts to accelerate pigmentation are both worthless and disappointing. El Gothamy used meladinine in a single dose two hours before exposure to ultraviolet radiation and after local application of sodium thiosulfate. The incidence of hypopigmentation was 54.5% in 33 patients treated with this combination, whereas it was 56% in 25 treated with sodium thiosulfate and ultraviolet radiation alone and 66.7% in 21 patients treated with sodium thiosulfate only. Hence, the addition of 8methoxypsoralen to the treatment regimen produced minimal improvement. The author suggested that the small dose of 8-methoxypsoralen given to the patients and the fact that the psoralen therapy was administered at the same time as the specific treatment for the fungus may explain these poor results (El Gothamy, 1975). Successful melanogenesis with natural sunlight alone or with psoralen therapy implies that tinea versicolor has been adequately treated.

Onchocerciasis Onchocerciasis or river blindness or blinding filariasis is a parasitic infection caused by Onchocerca volvulus. It is a filariasis transmitted by small biting black flies of the genus Simulian.

Epidemiology An estimated 18 million people are infected and more than 80 million people are at risk of infection. It is endemic throughout Central Africa with small foci in the Middle East and Central and South America. Onchocerciasis usually affects people living near swiftly flowing rivers (Routh and Bhownik, 1976). A study in Sudan showed that the general incidence of the disease in Sudan was 31.41% in men, 21.19% in women, and 300% in children (Browne, 1976a).

Clinical Features Treatment There are numerous ways of treating pityriasis versicolor, topically or systemically. Solutions or shampoos are preferable to creams and ointments that are more difficult to apply to all affected body areas. The whole trunk, neck, arms, and legs should be treated. Selenium sulfide, pyrithione zinc, salicylic

694

The principal site of infection is the skin and pruritus is the first indication of infection. Early skin changes include a papular pruritic inflammatory eruption in the extremities, trunk or face (“gale filarienne” or “craw-craw” or acute papular onchodermatitis). Later on, scattered, flat-topped chronic papules are present on the buttocks, waist area or

INFECTIOUS HYPOMELANOSES

the macules coalesce until complete achromia is present. Then the process usually begins on the contralateral tibia. Later the macules enlarge centrifugally to involve the lateral surface of the lower leg. In the majority of cases the involvement is bilateral. The inguinal region, and Scarpa triangle and the anterosuperior iliac spine may be involved less commonly. In extensive cases, depigmentation can involve the abdomen, chest, back, and genitalia. Finally, islets of normal pigmentation with discrete borders remain. The name “leopard skin” is often used to describe these changes. Should repigmentation occur, these borders become indistinct. It is in this stage that “hanging groin” — senile, atrophic, inelastic, inguinal skin draped over densely fibrotic matted glands — occurs. The following factors correlate with the presence of depigmentation: repeated bites, greater numbers (about five) of palpable onchocercomata, longer duration of onchocercomata, and larger than average size of tumors. Depigmentation of the Scarpa triangle is often associated with the presence of inguinal lymph nodes and in severe cases to multiple chronic nodules. Depigmentation frequently occurs in patients with ocular lesions.

Histology Early Stage Many microfilariae are found close to rete pegs, and nearby melanocytes of the basal layer.

Late Stage Fig. 36.5. Typical white macule on the extensor surfaces of the legs in onchocerciasis (see also Plate 36.4, pp. 494–495).

shoulders, with postinflammatory hyperpigmentation (chronic papular onchodermatitis). With time, lichenified hyperkeratotic plaques, skin atrophy, and depigmentation occur. Dermal nodules (onchocercomas) are the most characteristic lesions. They are localized over the pelvic or pectoral regions but may also be found on the neck and extremities. They contain the encapsulated adult worm. Ocular complications include iritis, posterior synechiae with secondary glaucoma, punctate keratitis, and chorioretinitis. Blindness may occur in 5–10% of cases.

Rete ridges become flattened and microfilariae more numerous. Acanthosis and hyperkeratosis may appear. Cells of rete pegs may be normal or have a shriveled appearance and pyknotic nuclei. Melanocytes may have disappeared or have abnormally shaped melanin granules, or normal granules abnormally distributed. Increased transport of melanin from the basal to the reticular layer is apparent. The only identifiable melanin may be at the tips of the rete ridges.

Final Stage This is marked by the absence of melanin, thinned epidermis, dermal fibrosis with increased elastic tissue and collagen, near or complete absence of microfilariae, total flattening of rete ridges, and a slight lymphohistiocytic infiltrate.

Mechanism Depigmentation in Onchocerciasis In Browne’s study, depigmentation was apparent in 6.2% of male cases, 1.2% of female cases and 0.2% of pediatric cases of onchocerciasis. The age of appearance of depigmentation best correlated with duration of habitation in hyperendemic areas. Depigmentation is first seen as one or more punctate or small, round, yellow-brown islets typically located from the middle third of the tibia medial to the tibial crest (Fig. 36.5). This mottled area enlarges progressively and irregularly and

The mechanism of depigmentation remains unknown. There is no evidence to suggest these patients are more than normally susceptible to other types of depigmentation. A direct effect of the black fly bite is unlikely as the depigmentation commonly occurs in areas not predominantly bitten by the black fly. Furthermore, the depigmentation may occur a long time after the patient has been in the hyperendemic area. It could be due to a hypersensitivity phenomenon by which the adult filaria produces a circulating factor which affects skin, nodes, and eye (Browne, 1976a).

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Diagnosis Diagnosis mostly relies on clinical examination and a history of living in an endemic area of filariasis. Patients should have slit-lamp examination to identify microfilariae in the anterior chamber of the eye. Mazzotti test is positive in about 90% of parasitologically confirmed cases. Swimming microfilariae may be identified on Thiersch skin biopsy suspended in saline on a glass slide. The white cell count shows leukocytosis with eosinophilia.

Treatment The treatment of onchocerciasis relies on diethylcarbamazine or ivermectin. The treatment has been improved significantly since the development of this drug. A single dose results in sustained suppression of microfilarial counts and improvement in skin and reversible eye changes. However, patients must be re-treated after 6–12 months (Routh and Bhownik, 1976). There is little to suggest that leukoderma responds to any treatment except excision of nodules. Browne (1976a) found that in one-third of those in whom complete nodulectomy was performed, repigmentation occurred from the margins of normally pigmented skin. In others repigmentation may fail to occur because a depigmenting agent remains in undetected extranodular or unencapsulated adult filariae or because such a factor has permanently suppressed melanocyte function (Browne, 1976a). Oral or topical 8-methoxypsoralen is generally ineffective in repigmenting onchocerciasis, although Browne did notice slight repigmentation of smaller macules with PUVA.

Post-kala-azar Dermatosis Post-kala-azar dermal leishmaniasis occurs in patients with untreated or incompletely treated chronic kala-azar. The causative agent is the parasite Leishmania donovani, and its close relatives L. infantum and L. chagasi, which infest the skin to produce insidious, nonulcerative granulomas containing the protozoa. L. donovani is transmitted by the sandfly. Post-kala-azar dermatosis is found commonly in India and Bangladesh, less frequently in Sudan and East Africa, rarely in China, and virtually never in Mediterranean countries or South America, despite the fact that kala-azar itself occurs in all these regions. Attempts to eradicate the sandfly have decreased the incidence of post-kala-azar dermatosis in the world.

Hypopigmentation in Post-kala-azar Dermatosis Clinical Features Post-kala-azar dermatosis appears one to two years after the initial infection. Hypopigmentation, which is the initial stage of the process, may precede or occur with dermal nodular lesions. The lesions appear simultaneously on various parts of the body as small pinpoint macules which increase in size but usually do not exceed 1 cm. They are usually singular and 696

discrete but occasionally coalesce to involve large areas such as entire limbs. In these cases, they produce a maplike appearance with irregular but well-defined borders. These hypopigmented macules are bilateral and often symmetrically distributed. They appear more commonly on the chest and back, arms, front of the thighs, and neck and less commonly on the face. The waist-belt area in “dhoti” wearers is conspicuously spared even when the whole body surface is involved. The lesions are hypopigmented and not totally amelanotic. There is no loss of pigment or dyschromia of the hair over the lesions. The depigmentation is not progressive and complete depigmentation does not occur. There is usually no local itching or discomfort and no other epidermal changes. No sensory abnormalities, nerve thickening, or disturbances of sweating are observed (Banerjee and Dutta, 1975). The hypopigmented lesions that constitute the prenodular stage of the disease are initially flat macules and become slightly raised after a period of months or years. Other Clinical Features The two cutaneous features include erythematous macules and yellow to pink nodules. A butterfly erythema of the face involving the nose and cheeks as well as the chin and the perioral skin may also extend to other parts of the body, particularly those areas that typically become hypopigmented. The yellowish pink nodules, often compared to xanthoma and varying from pinhead to plum size, either replace the earlier lesions of hypopigmentation or erythema or may appear de novo. The face, earlobes, trunk, and genitalia are frequently involved. Involvement of palms and soles, as well as mucous membranes, may also be observed. When these nodules disappear, they do not scar or leave pigmentation abnormalities.

Histology In the hypopigmented macular lesions, a slight decrease in basal layer pigmentation is the only epidermal change. In the dermis, there is an infiltrate of histiocytes, lymphocytes, and a few plasma cells in the upper dermis around proliferated blood vessels and deeper around hair follicles, sebaceous glands, and sweat ducts (Sen Gupta and Bhattacharjee, 1953). Leishmania is present in small numbers and is difficult to demonstrate within the histiocytes. A dense bandlike infiltrate in the upper dermis with histiocytes, plasma cells, and lymphocytes has also been reported (Girgla, 1977). Histologic changes are more marked in erythematous and nodular lesions. Pigment loss is more pronounced in erythematous lesions and maximal in nodules, as is the intensity of the dermal cellular infiltrate. L. donovani are more numerous in the erythematous type and most numerous in the nodular type of lesion.

Diagnosis The diagnosis of post-kala-azar dermatosis is based upon a history of treated or untreated kala-azar, clinical features, and demonstration of L. donovani in skin lesions. The parasite

INFECTIOUS HYPOMELANOSES

Fig. 36.6. Residual, unilateral hypopigmentation of the forehead following herpes zoster (see also Plate 36.5, pp. 494–495).

may be demonstrated by split-scraping of the erythematous or nodular lesions but is difficult to find in hypochromic macules. Antibodies to Leishmania species may be demonstrated by various methods. Hypopigmented macules can be differentiated from leprosy, tinea versicolor, pityriasis alba, postinflammatory hypopigmentation, tuberous sclerosis, idiopathic guttate hypomelanosis, and leukodermic syphilis on the basis of associated clinical findings, presence of L. donovani, histology, and the absence of other focal epidermal changes. Absence of anesthesia and M. leprae exclude the diagnosis of leprosy.

Treatment Systemic treatment with antimonials (sodium stibogluconate) is effective. However, it is almost impossible to obtain total repigmentation of hypopigmented areas.

Herpes Zoster Achromic and slightly atrophic macules are frequently observed after herpes zoster (Fig. 36.6). They are located at the site of previous vascular eruption and frequently allow retrospective diagnosis. Their origin is probably a destruction of melanocytes, analogous to scar, rather than postinflammatory hypopigmentation. Rarely, a tuft of white hair can mark the site of herpes zoster of the scalp.

References Banerjee, B. N., and A. K. Dutta. The cutaneous manifestations of kala-azar and post kala-azar dermatosis. In: Clinical Tropical Dermatology, O. Canizares (ed.). Oxford: Blackwell, 1975, pp. 197–205. Blumenthal, H. L. Tinea versicolor of the penis. Arch. Dermatol. 103:461–462, 1971. Borelli, D., P. H. Jacobs, and L. Nall. Tinea versicolor: epidemiologic, clinical, and therapeutic aspects. J. Am. Acad. Dermatol. 25:300–305, 1991. Breathnach, A. S., M. S. Birbeck, and J. D. Everall. Observations on Langerhans cells in leprosy. Br. J. Dermatol. 74:243–253, 1962.

Browne, S. G. Onchocerciasis and the skin. S. Afr. Med. J. 50:301–304, 1976a. Browne, S. G. Treponemal depigmentation, with special reference to yaws. S. Afr. Med. J. 50:442–445, 1976b. Charles, C. R. Hypopigmentation in tinea versicolor: a histochemical and electronmicroscopic study. Int. J. Dermatol. 12:48–58, 1973. Csonka, G. W. Clinical aspects of bejel. Br. J. Vener. Dis. 29:95–103, 1953. El Gothamy, Z. Tinea versicolor hypopigmentation: histochemical and therapeutic studies. Int. J. Dermatol. 14:510–516, 1975. Engelkens, H. J. H., J. Judanaiso, A. P. Oranje, V. D. Vuzevski, P. L. A. Niemel, J. J. Vander Sluis, and E. Stolz. Endemic treponematoses: Part I — Yaws. Int. J. Dermatol. 30:77–83, 1991a. Engelkens, H. J. H., P. L. A. Neimel, J. J. van der Sluis, A. Meheus, and E. Stolz. Endemic treponematoses. Part II. Pinta and endemic syphilis. Int. J. Dermatol. 30:231–238, 1991b. Girgla, H. S. Post-kala-azar dermal leishmaniasis. Br. J. Dermatol. 97:307–311, 1977. Grupper, C. Pityriasis versicolor achromiant: Etude ultrastructurale à propos de 3 cas. Bull. Soc. Franc. Dermatol. Syphiligr. 82:114–117, 1975. Hildick-Smith, G. Fungus Diseases and Their Treatment. London: Churchill, 1964. Ishibashi, Y. Histopathological studies on the skin lesions in leprosy with special reference to the histological changes of pigmentation. Jpn. J. Dermatol. 74:44–60, 1964. Ito, K., and T. Tanaka. Amino acid metabolism of fungi: decomposition of L-tyrosine by fungi. In: Dermatology Proceedings of the XIV International Congress, 1972, F. Flarer (ed.). New York: American Elsevier, 1974, p. 735. Jelliffe, D. B., and F. W. Jacobson. The clinical picture of tinea versicolor in Negro infants. J. Trop. Med. Hygiene 57:290–292, 1954. Job, C. K., A. Nayar, and J. S. Narayanan. Electron microscopic study of hypopigmented lesions in leprosy. A preliminary report. Br. J. Dermatol. 87:200–212, 1972. Koff, A. B., and T. Rosen. Nonvenereal treponematoses: yaws, endemic syphilis, and pinta. J. Am. Acad. Dermatol. 29:519–535, 1993. Nayer, A., and C. K. Job. A study of epidermal melanocytes in hypopigmented patches of leprosy. Indian J. Med. Res. 58:187–193, 1970. Nazzaro-Porro, M., and S. Passi. Identification of tyrosinase inhibitors in cultures of Pityrosporum and their melanocytotoxic effect. In: Pigment Cell: Biologic Basis of Pigmentation: Proceedings of the 10th International Pigment Cell Conference, Cambridge, 1977, vol. 4, S. N. Klaus (ed.). Basel: S. Karger, 1979, pp. 234– 243. Nazzaro-Porro, M., S. Passi, M. Picardo, R. Mercantini, and A. S. Breathnach. Lipoxygenase activity of Pityrosporum in vitro and in vivo. J. Invest. Dermatol. 87:108–112, 1986. Ortonne, J.-P., D. B. Mosher, and T. B. Fitzpatrick. Topics in Dermatology: Vitiligo and Other Hypomelanoses of Hair and Skin. New York: Plenum Publishing Corporation, 1983. Poulsen, A., L. Secher, T. Kobayasi, and K. Weismann. Treponema pallidum in leukoderma syphiliticum demonstrated by electron microscopy. Acta Derm. Venereol. 68:102–106, 1988. Prabhakaran, K. Unusual effects of reducing agents on Odiphenoloxidase of Mycobacterium leprae. J. Bacteriol. 107:787– 789, 1971. Rein, C. R. Repositing penicillin therapy of pinta in the Mexican peasant: A clinical and serologic survey. J. Invest. Dermatol. 18:137–145, 1952. Routh, H. B., and K. R. Bhownik. Filariasis. Dermatol. Clin. 12:719–727, 1976.

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CHAPTER 36 Ruete, A. E. Zur Fraze der depigmenterienden Pityriasis versicolor. Dermatol. Wochenschr. 96:333–336, 1933. Seghal, V. N. Hypopigmented lesions in leprosy (letter). Br. J. Dermatol. 89:99, 1973. Sehgal, V. N. Leprosy. Dermatol. Clin. 12:629–644, 1994.

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Sehgal, V. N., S. Jain, S. N. Bhattachaya, and D. M. Thappa. Yaws control/eradication. Int. J. Dermatol. 33:16–20, 1994. Sen Gupta, P. C., and B. Bhattacharjee. Histopathology of post kalaazar dermal leishmaniasis. J. Trop. Med. Hygiene 56:110–116, 1953.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

37

Inflammatory Hypomelanoses Jean-Philippe Lacour

Pityriasis Alba Pityriasis alba is a common cutaneous disorder, consisting of asymptomatic slightly scaling, hypopigmented patches on the face, neck, upper trunk, and proximal extremities of children and young adults.

History and Synonyms Fox in 1923 described the first reported cases of pityriasis alba manifesting as partial depigmentation of the face of several black children. In 1924, Pardo-Castello and Dominguez described a similar but more extensive dermatosis, “achromia parasitaria,” from which Aspergillus was isolated in six out of 36 cases. Various names have been used to describe the disease: pityriasis streptogenes, pityriasis corporis, pityriasis simplex faciei, erythema streptogenes, pityriasis impetigo furfuracea, chronic impetigo, pityriasis sicca faciei, pityriasis alba faciei, and achromatous pityriasis faciei. The old name “pityriasis alba” is probably the most suitable (O’Farrell, 1956).

Epidemiology Pityriasis alba is probably a very common condition. Bassaly et al. (1963) found that 40% of the children in an Egyptian school health clinic had facial lesions typical of pityriasis alba. The condition occurs in all races, and although it may be encountered in white people, it is considered to be more frequent in black or dark-skinned people, but this increased incidence may be more apparent than real (Wells et al., 1960). Pityriasis alba occurs worldwide; series have been reported from North America, South America, Europe, and Africa. Both sexes are probably equally affected, although some epidemiologic studies have suggested a male predominance (Blessmann Weber et al., 2002; Popescu et al., 1999). Pityriasis alba is a disease of childhood and adolescence. Among 200 patients with pityriasis alba, 90% were between 6 and 12 years and about 10% were between 13 and 16 years of age (Bassaly et al., 1963), i.e., there is a predominance of the condition among children in the prepubertal age group. However, adults may also be affected (Wells et al., 1960). Seasonal and climatic factors have been reported to influence pityriasis alba. Pityriasis alba becomes more apparent in summer and tends to fade in winter; seasonal variation and exposure to the sun may be major etiologic factors (O’Farrell, 1956). Bassaly et al. (1963), in Egypt, observed in most of their cases that the lesions improved or disappeared in May or June only to recur in the former sites in November and December, when the relative humidity is highest, the temperature lowest, and the sunshine least. However, Wells et al.

(1960) noted the effects of the sun exposure to be variable; in some patients sun exposure made the lesions more apparent because the surrounding skin tanned, whereas in others the lesions became less apparent. They concluded that excessive dryness of the skin, whether from winter wind or summer sun, precipitated or aggravated the lesions (Wells et al., 1960). The disorder seems to occur most frequently in atopic patients. Rudzdi et al. (1994) observed that pityriasis alba occurred in 20.8% of 481 patients versus 4% in controls. However, a study from Singapore showed that pityriasis alba was not more frequent among individuals with atopic dermatitis (Tay et al., 2002).

Clinical Description The primary lesion is a pale pink, round, oval, or irregular, scaling macule with ill-defined borders. The lesions usually vary in size from 5 mm to 30 mm, but larger lesions may occur. Usually there are two or three lesions but the number can vary from one to more than 20. The early lesions are erythematous; after a few weeks the erythema fades and the surface becomes whitish and dry, sometimes with slightly raised, pink borders. The scale is characteristically fine or powdery. The face is the most frequent site of involvement. The lesions predominate on the mid-forehead, the cheeks (see Plate 37.1, pp. 494–495), malar ridge, angles of the mouth, or the lateral periorbital areas. Pityriasis alba occurs less commonly on other parts of the body, namely the neck, trunk, back, or limbs (Fig. 37.1). Among 200 cases, Bassaly et al. (1963) noted involvement of the legs in four patients and involvement of the extensor surfaces of the arms and forearms in two patients. In another series of 67 patients, generalized lesions occurred in eight patients, five of whom were postpubescent (Wells et al., 1960). The dermatosis is usually asymptomatic, but rarely patients may complain of itching or burning. Wolf et al. (1992) reported three patients with pityriasis alba whose lesions were confined to the knees and were misdiagnosed as psoriasis. Wood’s light examination demonstrates that the lesions of pityriasis alba are not totally amelanotic. Under Wood’s light, more lesions may become visible. Usually, the patch of hypopigmentation remains the same size and unchanged for several weeks, months, or even years. In most instances, the lesions clear spontaneously at puberty but the fact that typical lesions of pityriasis alba may persist into adulthood suggests that spontaneous remission is not a universal rule. Pigmenting pityriasis alba seems to be a variant of classic pityriasis alba: it is an uncommon, perhaps underreported, condition occurring in childhood (Dhar et al., 1995; du Toit and Jordaan, 1993). du Toit and Jordaan conducted a 699

CHAPTER 37

Fig. 37.1. Extensive pityriasis alba on the legs of a youth (see also Plate 37.2, pp. 494–495).

prospective study over a period of 2 years and found 20 patients with pigmented pityriasis alba. None of the patients were white. The median age was 7.5 years (range 2–19 years) and there were 14 females and six males. No family history or seasonal trends were noted. The lesions were asymptomatic or mildly pruritic. They were usually located on the face with involvement of the forehead and cheeks in 50%. Extrafacial involvement was rare (10%). There were 1–30 lesions (median 7) whose diameter varied from 1 cm to 20 cm; most were approximately 2.5 ¥ 2.5 cm. Lesions had the characteristic feature of a central zone of bluish hyperpigmentation with mild, fine scaling, surrounded by a hypopigmented rim of variable width. They were generally well demarcated, but confluence was occasionally seen. They were mildly elevated or flat. A significant finding was an associated dermatophyte infection in 13 patients (65%), mostly tinea capitis. These patients received griseofulvin resulting in the resolution of pigmenting pityriasis alba in seven children within 4–20 weeks. In patients without dermatophyte infection, the lesions cleared with local 1% hydrocortisone cream. Three patients fulfilled the criteria for a diagnosis of atopic dermatitis. In six patients (30%), classic pityriasis alba was present simultaneously. As stated above, this condition seems to be a variant of classic pityriasis alba showing strong association with dermatophyte infection, especially tinea capitis.

Histology It is usually said that microscopic features of pityriasis alba are of a chronic nonspecific dermatitis. In a study of 39 cases, Vargas-Ocampo (1993) showed that the histology is suggestive, though not pathognomonic, of the diagnosis. The main findings are in the pilary apparatus and consist of follicular plugging, follicular spongiosis, and atrophic sebaceous glands in the early and intermediate stages. In the late stage, the histologic picture is that of a chronic dermatitis with varying degrees of hyperkeratosis and parakeratosis, moderate dilatation of the vessels in the superficial dermis, a slight perivascular lymphocytic infiltrate, and edema of the upper dermis. Silver staining shows reduced pigment in the basal layer of 700

the epidermis which is irregularly pigmented, with normally pigmented or hyperpigmented areas alternating with other hypopigmented areas. In Vargas-Ocampo’s study, three cases were studied with the dopa reaction: two showed dendritic melanocytes filled with melanin whereas one showed a greatly reduced number of dopa-positive melanocytes. In Zaynoun et al.’s (1983) study, split-dopa examination showed a decrease in melanocyte density, and a varying intensity of dopa reaction. Electron microscopic studies have been rarely reported in pityriasis alba. In a study of nine patients, Zaynoun et al. (1983) observed normal melanocytes with normal melanosomes and dendrites laden with mature melanosomes. Adjacent keratinocytes had an adequate number of melanosomes. In only one case the keratinocytes contained very few melanosomes. The shape and degree of melanization of melanosomes was normal. However, a reduction in number of melanosomes was observed in five cases with a decrease in the transverse diameter of melanosomes in six cases. A significant degree of intercellular edema was observed in the epidermis in four patients. Intracytoplasmic lipid droplets were noted in seven cases, and Langerhans cells appeared normal. In pigmenting pityriasis alba biopsy specimens were obtained from two patients in du Toit’s series and in Dhar et al.’s case. They showed similar features, i.e., a subacute dermatitis with variable pigment incontinence. Immunohistochemical studies showed a preponderance of T lymphocytes (Dhar et al., 1995; du Toit and Jordaan, 1993).

Pathogenesis The etiology of pityriasis alba remains unknown. An infectious origin has been suspected: although b hemolytic streptococci, Staphylococcus aureus, and Aspergillus have been occasionally found, no bacteria or fungi can be directly implicated in the pathogenesis of pityriasis alba. Nutritional factors and vitamin deficiencies have been suspected but this is disputed. In a study of 30 cases, Galadari et al. (1992) found the trace elements to be within normal levels, except for serum level of copper, which was significantly reduced in patients with pityriasis alba. The pathogenesis of the primary lesion remains unknown. O’Farrell (1956) considered that pityriasis alba is an eczematous dermatosis of complex etiology with postinflammatory hypopigmentation. Wells et al. (1960) considered pityriasis alba to originate from a nonspecific erythema with a mild dermal inflammatory infiltrate which somehow results in localized hypopigmentation, possibly because of ultraviolet screening by the hyperkeratotic and parakeratotic epidermis. Bassaly et al. (1963) speculated that the hot summer sun stimulated seborrheic and sudoriparous activity and cleared pityriasis alba in some cases. Dryness caused by winter wind and summer sun is an important factor. Finally, pityriasis alba may be a variant of atopic dermatitis.

Diagnosis and Differential Diagnosis The presence of hypomelanotic, slightly scaling macules with poorly defined margins on the face of a child suggests the diag-

INFLAMMATORY HYPOMELANOSES

Fig. 37.2. Distinctive hypopigmented macules in a psoriatic patient with resolved lesions following therapy with ultraviolet light.

nosis of pityriasis alba. The lesions are usually larger than those of tinea versicolor, which can be excluded by a scraping for fungus. Trichrome vitiligo usually has discrete margins and is accompanied by typical amelanotic lesions elsewhere. Postinflammatory hypomelanosis must be excluded by history. When occurring on the trunk or limbs, pityriasis alba may mimic atopic dermatitis.

Treatment No very effective treatment is available. Topical steroids, combined with exposure to ultraviolet radiation which may potentiate the effect of the steroids, may be useful. Emollients seem to be as effective as anything else. Antimicrobial or antifungal therapy is ineffective. As of now, there are no published data on the effect of calcineurin inhibitors.

Fig. 37.3. Classic lesions of psoriasis in a young man undergoing phototherapy. Hypopigmentation persists in lesions that have resolved on the trunk (see also Plate 37.3, pp. 494–495).

Psoriasis Various types of hypopigmentation can be seen in psoriasis: postinflammatory hypomelanosis, vitiligo, and Woronoff ring. Postinflammatory hypopigmentation, which is the most common, appears after healing of the typical psoriatic lesion (Figs 37.2 and 37.3). Increased keratinocyte turnover may interfere with the transfer of melanosomes from melanocytes to keratinocytes, but the mechanisms might be more complex, involving cytokines and leukotrienes. It is also possible that the depigmentation may result from topical steroid therapy when used. Vitiligo has been reported in patients with psoriasis. Both are common problems, but in some patients the vitiligo and the psoriasis involve the same skin surfaces; or different areas may be involved. This problem has been discussed elsewhere in this volume (see Chapter 30). Woronoff ring (Fig. 37.4) is a blanched halo surrounding individual psoriatic lesions after phototherapy or topical treatment (Woronoff, 1926) (see Chapter 37). These rings have an

Fig. 37.4. Woronoff ring. Hypopigmentation surrounding a plaque of psoriasis, frequently observed during phototherapy (see also Plate 37.4, pp. 494–495).

approximately uniform width around the psoriatic plaques, regardless of the size and shape of the psoriatic plaque. The white areas do not develop erythema during phototherapy or topical treatment (Gupta et al., 1986). It is uncertain whether this phenomenon represents vasoconstriction or decreased 701

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of follicular papules, nodules, or ulceration can be seen. It may simulate leprosy. The chronic form may persist for years without evidence of associated disease. The third type clinically resembles the chronic variety but is associated with lymphoma, particularly mycosis fungoides (Gibson et al., 1989).

Hypopigmentation in Follicular Mucinosis

Fig. 37.5. Annular, hypopigmented macule on the check of a young boy with alopecia mucinosa (see also Plate 37.5, pp. 494–495).

melanin pigmentation. Although some experimental work suggested that the Woronoff ring is not a true hypomelanosis but rather a vascular abnormality (Bettman, 1926), Kopton et al. (1971) found normal blood vessels beneath the halo (see Chapter 42), normal vascular reactivity to histamine phosphate and methacholine chloride, and decreased melanin content in both psoriatic and halo epidermis. Thus the Woronoff ring could be a true hypomelanosis. In contrast, it has been suggested that the white ring surrounding ultraviolet (UV)-treated psoriatic plaques results from a local inability to synthesize prostaglandin E2 in response to ultraviolet radiation resulting in reduced erythema because of less vasodilatation (Penneys et al., 1976). However, another mechanism could be responsible since the Woronoff ring has been found around both PUVA and UVB-induced erythema even though the former is not associated with prostaglandin metabolism (Warin and Greaves, 1979). The paleness within the ring could be caused by diffusion of an anti-inflammatory mediator from the psoriatic plaque into its environment (Groebe et al., 1990).

Follicular Mucinosis Follicular mucinosis, or alopecia mucinosa, is an inflammatory disorder characterized clinically by inflammatory plaques with scaling and alopecia and histologically by the accumulation of acid glycosaminoglycans (mucin) in the outer root sheath of the hair follicles.

Clinical Features Follicular mucinosis is divided into three clinical types. The first and largest category consists of skin-colored or erythematous papules and plaques with follicular accentuation (Fig. 37.5). They occur predominantly on the face, scalp, neck, and shoulders. Alopecia is most noticeable on the scalp or eyebrow region. This form is transient and usually clears within two months to two years. The chronic form is characterized by more numerous and more widely distributed lesions. Groups 702

Hypopigmentation is a rarely mentioned clinical manifestation of follicular mucinosis. In 1973, Stern and Krivo reported a 6-year-old black girl with scaly hypopigmented patches on her forehead; a biopsy revealed follicular mucinosis. No followup was mentioned. In 1975, De Graciansky and Taïeb described a 13-year-old boy with multiple nonpruritic hypopigmented eczematoid plaques on the arms, trunk, and head. They regressed over a period of months but hypopigmentation persisted. In 1979, Locker and Duncan reported two adult black men with pruritic follicular papules on the head. They were associated with patches of hypopigmentation. Biopsy from hypopigmented papules disclosed follicular mucinosis. There was minimal mononuclear inflammation around affected hair follicles. Pigment or melanocyte changes were not described and there was no ultrastructural study. Hypopigmented follicular mucinosis appears to be rare and predominates in persons with black skin. Among the reported cases, none was associated with mycosis fungoides. Despite the possibility of follicular mycosis fungoides (Lacour et al., 1993) and hypopigmented mycosis fungoides (Lambroza et al., 1995), hypopigmented follicular mucinosis seems to be distinct from these disorders.

Mechanism The mechanism of the pigment disturbance in follicular mucinosis is not known. It may be post inflammatory.

Treatment There are no clear guidelines for treating follicular mucinosis. Some cases improve spontaneously. Treatment with topical steroids may be of benefit. From the reported cases it seems that hypopigmentation can regress along with follicular mucinosis or persist despite follicular mucinosis regression.

Other Postinflammatory Hypomelanoses Atopic Dermatitis Hypopigmentation is probably frequently observed in atopic dermatitis. It seems to be particularly frequent in individuals of African or Asian descent (Sybert, 1996). However, it has very rarely been studied, whether clinically or histologically. In addition to inflammatory hypomelanosis, Larrègue et al. (1985) observed four cases of vitiligolike depigmentation in severe atopic dermatitis. They reported that achromic macules (Fig. 37.6) had irregular borders that were often hyperpigmented; they were located within the flexural zones that had been involved by atopic dermatitis for several years;

INFLAMMATORY HYPOMELANOSES

Fig. 37.6. Hypopigmentation in resolving lesions of atopic dermatitis (see also Plate 37.6, pp. 494–495).

Fig. 37.7. Characteristic hyperpigmented and hypopigmented lesions on the face of a woman with discoid lupus erythematosus (see also Plate 37.8, pp. 494–495).

lichenification was not a predominant feature in hypopigmented areas. Histologically, they observed the absence of melanocytes in achromic zones. They considered these vitiligoid achromias different from post-inflammatory hypomelanosis of atopic dermatitis and the possible result of the addition of several factors affecting melanogenesis (spongiosis, parakeratosis, lichenification, topical steroids).

Lupus Erythematosus Pigmentary disturbances are common in discoid lupus erythematosus, particularly in dark-skinned people. Hypopigmentation is usually observed in sites of scarring lesions with cutaneous atrophy. It may be interspersed with or surrounded by hyperpigmented lesions. Cutaneous depigmentation may also occur in systemic lupus erythematosus. It was observed in 5–20% of cases. It is usually secondary to inflammatory skin lesions, being localized in the same zones as erythematous and atrophic plaques (Figs 37.7 and 37.8 and Plate 37.7, pp. 494–495) (Tuffanelli and Dubois, 1964). However, true associations of lupus erythematosus with vitiligo have been described (Forestier et al., 1981). Histologically, lupus depigmentation shows epidermal atrophy, degeneration of the basal layer, increase in melanocyte number with pigmentary incontinence in the superficial dermis. In contras, lupus-associated vitiligo is characterized by the absence of melanocytes and no pigmentary incontinence (Forestier et al., 1981). The mechanisms of hypopigmentation in lupus erythematosus are not known. It can be post inflammatory or cicatricial. Depigmentation may regress in recent, nonscarring lesions, whereas it can be definitive in older cicatricial ones. Depigmented inactive lupus lesions have been successfully treated by transferring the roof of suction blister onto dermabraded depigmented area (Gupta, 2001).

Achromic Pityriasis Lichenoides Pityriasis lichenoides is a disease of unknown etiology having

Fig. 37.8. Hypopigmented lesions with scarring in an African patient with discoid lupus erythematosus (see also Plate 37.9, pp. 494–495).

an acute form (or Mucha–Habermann disease) and a chronic form (or guttate parapsoriasis). Postinflammatory hypopigmentation (Fig. 37.9) may occur at sites of healed lesions, but this is not usually a prominent feature. Both acute and chronic lesions of pityriasis lichenoides may be followed by 703

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Fig. 37.9. Hypopigmented macules caused by pityriasis lichenoides (see also Plate 37.10, pp. 494–495).

hypopigmentation. However, in some cases, particularly in dark-skinned people, pityriasis lichenoides chronica may present as a widespread extensive hypopigmented rash (Clayton and Warin, 1979). The distribution of hypopigmented macules is sometimes characteristic, involving the upper arms, the thighs and the axillary folds. In those cases, histologic examination shows a pericapillary lymphocytic infiltrate with overlying epidermal spongiosis and infiltration of the epidermis with inflammatory cells. The diagnosis is facilitated when hypopigmented lesions are associated with typical lesions of pityriasis lichenoides. When hypopigmentation predominates, the lesions may mimic syphilitic leukoderma, eczema, pityriasis versicolor, and vitiligo. The duration of the disease is variable since pityriasis lichenoides may last from several months to years. Since the hypopigmentation occurs in sites involved with papules of guttate parapsoriasis, the mechanism of hypopigmentation is likely post inflammatory. No effective treatment is available. The effect of phototherapy or erythromycin is not known.

References Bassaly, M., A. J. Miale, and A. S. Prasad. Studies on pityriasis alba. Arch. Dermatol. 88:272–275, 1963. Bettman, S. Kapillar mikroskopische Untersuchumgen bei Psoriasis. Dermatol. Wochenschr. 83:1223, 1926. Blessmann Weber, M., L. G. Sponchiado de Avila, R. Albaneze, O. L. Magalhaes de Oliveira, B. D. Sudhaus, and T. F. Cestari. Pityriasis alba: a study of pathogenic factors. J. Eur. Acad. Dermatol. Venereol. 16:463–468, 2002. Clayton, R., and A. Warin. Pityriasis lichenoides chronica presenting as hypopigmentation. Br. J. Dermatol. 100:297–302, 1979. De Graciansky, P., and M. Taïeb. Alopécie mucineuse en plaques multiples d’évolution rapidement favorable. Bull. Soc. Franc. Dermatol. Syphiligr. 82:253–254, 1975. Dhar, S., A. J. Kanwar, and G. Dawn. Pigmenting pityriasis alba [letter]. Pediatr. Dermatol. 12:197–198, 1995. du Toit, M. J., and H. F. Jordaan. Pigmenting pityriasis alba. Pediatr. Dermatol. 10:1–5, 1993. Forestier, J. Y., J. P. Ortonne, J. Thivolet, and P. Souteyrand. [Associ-

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ation of lupus erythematosus and vitiligo (author’s transl)]. Ann. Dermatol. Venereol. 108:33–38, 1981. Fox, H. Partial depigmentation: chiefly on the face in Negro children. Arch. Dermatol. Syphil. 7:268–269, 1923. Galadari, E., M. Helmy, and M. Ahmed. Trace elements in serum of pityriasis alba patients. Int. J. Dermatol. 81:525–526, 1992. Gibson, L. E., S. A. Muller, K. M. Leiferman, and M. S. Peters. Follicular mucinosis: clinical and histopathologic study. J. Am. Acad. Dermatol. 20:441–446, 1989. Groebe, G., W. C. Marsch, and H. Holzmann. Die Woronoff-Ringe im mathematischen Modell. Hautarzt 41:226–228, 1990. Gupta, A. K., C. N. Ellis, M. T. Goldfarb, and J. J. Voorhees. Woronoff ring during anthralin therapy for psoriasis. Arch. Dermatol. 122:248, 1986. Gupta, S. Epidermal grafting for depigmentation due to discoid lupus erythematosus. Dermatology 202:320–323, 2001. Kopton, B. S., K. W. Waterson, and L. Shapiro. The Woronoff ring of psoriasis: Study of a patient. Int. J. Dermatol. 10:233–236, 1971. Lacour, J. P., J. Castanet, C. Perrin, and J. P. Ortonne. Follicular mycosis fungoides. J. Am. Acad. Dermatol. 29:330–334, 1993. Lambroza, E., S. R. Cohen, R. Phelps, M. Lebwohl, I. M. Braverman, and D. DiConstanzo. Hypopigmented variant of mycosis fungoides: Demography, histopathology, and treatment of seven cases. J. Am. Acad. Dermatol. 32:987–993, 1995. Larrègue, M., J. Martin, J. M. Bressieux, C. Canuel, P. de Giacomoni, P. Ramdenée, and P. Babin. Achromies vitiligoides et dermatite atopique grave. Ann. Dermatologie Venereologie 112:589–600, 1985. Locker, E., and W. C. Duncan. Hypopigmentation in alopecia mucinosa. Arch. Dermatol. 115:731–733, 1979. O’Farrell, M. M. Pityriasis alba. Arch. Dermatol. 73:376–377, 1956. Pardo-Castello, V., and M. Dominguez. Achromia parasitaria. Arch. Dermatol. Syphil. 9:82–85, 1924. Penneys, N. S., V. Ziboh, P. Simon, and J. Lord. Pathogenesis of Woronoff ring in psoriasis. Arch. Dermatol. 112:955–957, 1976. Popescu, R., C. M. Popescu, H. C. Williams, and D. Forsea. The prevalence of skin conditions in Romanian school children. Br. J. Dermatol. 140:891–896, 1999. Rudzdi, E., Z. Samochocki, and P. Rabaundel. Frequency and significance of the major and minor features of Hanifin and Rajka among patients with atopic dermatitis. Dermatology 189:41–46, 1994. Stern, W. K., and J. Krivo. Follicular mucinosis. Arch. Dermatol. 107:127, 1973. Sybert, V. P. Skin manifestations in individuals of African or Asian descent. Pediatr. Dermatol. 13:158–168, 1996. Tay, Y. K., K. H. Kong, L. Khoo, C. L. Goh, and Y. C. Giam. The prevalence and descriptive epidemiology of atopic dermatitis in Singapore school children. Br. J. Dermatol. 146:101–106, 2002. Tuffanelli, D. L., and E. L. Dubois. Cutaneous manifestations of systemic lupus erythematosus. Arch. Dermatol. 90:377–386, 1964. Vargas-Ocampo, F. Pityriasis alba: a histologic study. Int. J. Dermatol. 32:870–873, 1993. Warin, A. P., and M. W. Greaves. Woronoff’s ring with PUVA therapy. Arch. Dermatol. 115:105, 1979. Wells, B., J. H. White, and R. R. Kierland. Pityriasis alba: a ten year survey and review of the literature. Arch. Dermatol. 82:183–189, 1960. Wolf, R., D. Wolf, and H. Trau. Pityriasis alba in a psoriatic location. Acta Derm. Venereol. 72:360, 1992. Woronoff, D. L. Die peripheren Veränderunzen der Haut um die Effloreszenzen der Psoriasis vulgaris und Syphilis corymbosa. Dermatol. Wochenschr. 82:249–257, 1926. Zaynoun, S. T., B. G. Aftimos, K. K. Tenekjian, N. Bahuth, and A. K. Kurban. Extensive pityriasis alba: a histological, histochemical and ultrastructural study. Br. J. Dermatol. 108:83–90, 1983.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

38

Hypomelanoses Associated with Melanocytic Neoplasia Lieve Brochez, Barbara Boone and Jean-Marie Naeyaert

Introduction Spontaneous depigmentation of the skin can occur in association with melanoma (see Chapter 25) and pigmented nevi (see Chapter 57). The depigmentation may occur within the lesion, around it, or at a distal site. Loss of pigmentation within the lesion is normally associated with regression of the lesion. It occurs in melanoma, halo nevi, and rarely in congenital nevi. Depigmentation around the lesion is referred to as a leukoderma acquisitum centrifugum. It can be present around various types of pigmented nevi or primary or metastatic melanoma (Kopf et al., 1965). Halo nevus is the specific type of leukoderma acquisitum centrifugum that occurs around a melanocytic nevus (Kopf et al., 1965). Depigmentation distal to a pigmented lesion that resembles vitiligo but is not otherwise explained is referred to as melanoma-associated hypo/depigmentation. It is associated with melanoma. Depigmentation in these different leukodermas probably results from different mechanisms, as it involves the destruction of different types of pigment cell [normal melanocytes, atypical melanocytes, malignant melanocytes (Niles, 1951)], occurs in different locations (together with, adjacent to, or distal to the cells that initiated the process), and is associated with different histologic abnormalities (the presence or absence of a dense cellular infiltrate). Whatever their causes, these leukodermas are experiments of nature that demonstrate that there are mechanisms in humans that can selectively kill pigment cells, including melanoma cells. Understanding these mechanisms may provide clues to the treatment of melanoma.

Halo Nevi Definition and Synonyms Halo nevus is a pigmented nevus, surrounded by a ring of depigmentation, that may progress to spontaneous disappearance of the nevus (Wayte et al., 1968). Synonyms include Sutton nevus, leukopigmentary nevus, perinevoid vitiligo, and perinevoid leukoderma. It is the most common form of leukoderma acquisitum centrifugum (Ortonne et al., 1983). However, leukoderma acquisitum centrifugum is a generic term that applies to other forms of acquired depigmentation around pre-existing pigmented lesions and should not be used to refer specifically to halo nevus.

Historical Evolution of the Disease Concept The first medical description of depigmentation surrounding a pigmented nevus was by Hebra and Kaposi (1874), who believed it was vitiligo. The condition had actually been described previously, in a painting by Grunewald. Sutton is given credit for first recognizing halo nevi as a distinct entity in 1916 and for coining the term “leucoderma acquisitum centrifugum.” The central lesion was first recognized to be an intradermal nevus by Stokes (1923), and to disappear by Gougerot and Carteaud (1934). The term “halo nevi” was coined in 1963 by Feldman and Lashinsky, who mistakenly believed the depigmented halo was a nevus anemicus. More commonly, it was believed the depigmentation was a manifestation of vitiligo (Crawford, 1924; Montpellier, 1922; Petges, 1921; Weber et al., 1924), a belief reflected in use of the terms “perinevoid vitiligo” and “perinevic vitiligo” to refer to this condition. The first suggestion that the cause might be immunological was by Rohdenburg in 1927. At present, the relation between leukoderma acquisitum centrifugum, halo nevi, and vitiligo remains to be fully clarified. Both leukoderma acquisitum centrifugum and vitiligo are similar histologically, share a common terminal event, i.e., the destruction of normal melanocytes, and are associated with immune abnormalities. These common features suggest they share a common pathomechanism at some point in their progression. However, there are enough differences between the two conditions to suggest that the trigger and the exact mechanisms mediating each may be different.

Epidemiology Halo nevi occur in approximately 1% of Caucasians (Larsson and Liden, 1980; Rohdenburg., 1927). There does not appear to be a sex predilection. Peak incidence is during the second decade (Frank and Cohen, 1964; Kopf et al., 1965; Wayte et al., 1968). As many as 85% to 90% of cases occur during the first three decades (Frank and Cohen, 1964; Wayte et al., 1968). There is usually no discernible precipitating factor. The occasional association of onset of halo nevi with sun exposure or sunburn (Kopf et al., 1965; Wayte et al., 1968) is probably due to tanning of normal skin or burning of the nonpigmented halo, bringing attention to the lesion (Ortonne et al., 1983). There are several case reports of halo nevi-like loss of pigmentation due to benoquin (Cornbleet et al., 1958) and guanofuracin (Yamada, 1955). 705

CHAPTER 38

A

B

Fig. 38.1. Halo nevus. (A) There is a sharply outlined ring of depigmentation surrounding an otherwise normal-appearing intradermal nevus. (B) As the lesion progresses, the central nevus begins to disappear.

Halo nevi are not genetically determined. Alhough occasional familial cases occur (Kopf et al., 1965; Ortonne et al., 1983), they are exceptional. In a large study, only 2 of 100 patients with halo nevi had a family history of halo nevi (Wayte et al., 1968). There is an increased prevalence of halo nevi in patients with Turner syndrome (Brazelli et al., 2004). The prevalence of vitiligo in these patients is not increased.

Clinical Features The typical halo nevus consists of a pigmented nevus surrounded by a macule of complete depigmentation, as illustrated in Figures 38.1 and 38.2. The central nevus is usually a typical acquired melanocytic nevus, sometimes a dysplastic nevus, and rarely a congenital nevus. There are usually three phases in the evolution of a halo nevus: appearance of the halo, regression of the central nevus, and disappearance of the halo (Ortonne et al., 1983). Halo nevi typically begin with a circular or oval ring of depigmentation around a pre-existing nevus. The pigment loss occurs in days to weeks, and once fully established does not further progress (Wayte et al., 1968). The pigment loss is asymptomatic, and the patient is otherwise normal. The ring is from 706

0.5 cm to 5 cm in width (Wayte et al., 1968). The underlying skin is normal in appearance, with no scaling or scarring. The margin between depigmented and normally pigmented skin is well defined and regular in outline. There is no relation between the size of the nevus and that of the depigmentation. There may be transient erythema within the depigmented ring of early halo nevi. The erythema is due to vasodilation and not to direct infiltration by inflammatory cells, which are usually absent when the erythema is present (Berman and Herszenson, 1981). In the center of the depigmented halo is a nevus, which is initially normal in appearance. Hairs in the nevus or in the depigmented halo may become gray or white (Kawamura et al., 1963). In a variable proportion of patients, ranging from 3% (Wayte et al., 1968) to over 50% (Frank and Cohen, 1964), the central nevus eventually disappears after several months to many years. Prior to disappearing, the nevus may change in color from brown to brown-red or red-violaceous. Disappearance is asymptomatic. Some patients have localized depigmented macules in other locations that resemble halo nevus in size and color, but without the central pigmented nevus. These may represent halo nevi in which the central

HYPOMELANOSES ASSOCIATED WITH MELANOCYTIC NEOPLASIA

Fig. 38.3. Multiple halo nevi, a very common phenomenon (see also Plate 38.2, pp. 494–495).

Fig. 38.2. Typical halo nevus. The pigmented nevus is in the center surrounded by a white halo (see also Plate 38.1, pp. 494–495).

nevus regressed before they were observed. Rarely, the halo may become repigmented after months to years (Frank and Cohen, 1964; Wayte et al., 1968). More commonly, once depigmentation appears it remains permanent. Halo nevi occur predominantly on the trunk, particularly the upper torso (Ridley, 1974). Their distribution is similar to that of typical melanocytic nevi, but differs from that of melanoma or dysplastic nevi, which often occur on the extremities, genitals, and scalp — areas rarely involved with halo nevi. Multiple halo nevi are present in 25% to over two-thirds (Frank and Cohen, 1964; Kopf et al., 1965) of patients (Fig. 38.3). However, the bulk of pigmented nevi in an affected individual almost invariably remain normally pigmented. Halos of depigmentation may occur around a number of other lesions (Fig. 38.4), including neurofibromas (Kopf et al., 1965; Leszczynski, 1926; Smith and Moseley, 1976), blue nevus (Kopf et al., 1965), Spitz nevus (Kopf et al., 1965; Ortonne et al., 1983), congenital nevus (Figs. 38.5–38.7) (Albert et al., 1992; Brownstein et al., 1977; Garcia and Gano, 1979; Kopf et al., 1965; Tokura et al., 1994), in the choroid of the ocular fundus (Fournier et al., 1984), and warts.

Fig. 38.4. A seborrheic keratosis with a depigmented halo (see also Plate 38.3, pp. 494–495).

Association with Melanoma Individual case reports associate halo nevi with melanoma (Clark, 1975; Epstein et al., 1973; Kopf et al., 1965). How often this occurs is unclear, as there are few studies of the incidence of halo nevi in large populations of melanoma patients. 707

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Leukoderma acquisitum centrifugum, which clinically and histologically resembles the depigmentation around a halo nevus, may be present around primary or metastatic melanoma. The relation of this event to halo nevus is not known. Because the two events look alike, it is reasonable to suspect their cause is similar, particularly since melanoma can also regress in a process that is akin to that of the central nevus in a halo nevus. However, in melanoma it is rare for the tumor to completely resolve.

Relation between Halo Nevus and Vitiligo

Fig. 38.5. Depigmented halo surrounding a congenital nevus (see also Plate 38.4, pp. 494–495).

Fig. 38.6. Spontaneous partial depigmentation of a large, congenital nevus (see also Plate 38.5, pp. 494–495).

Fig. 38.7. Almost total depigmentation of a congenital nevus (see also Plate 38.6, pp. 494–495).

In one, these were present in 6.2% of 64 patients with metastatic melanoma (Laucius and Mastrangelo, 1979), which is higher than in the general population. The halo nevi may appear together with, or following, the removal of a melanoma (Epstein et al., 1973). 708

The relation between halo nevus and vitiligo (see Chapter 30) remains unclear. The two diseases share a common terminal event — the destruction of normal melanocytes — and both appear to be associated with antipigment cell immune responses. However, the association between the two diseases is not as strong as expected if both shared the same initial cause. Between 1% and 48% of patients with halo nevi are reported to have vitiligo (Copeman et al., 1973b; Frank and Cohen, 1964; Gauthier et al., 1975; Kopf et al., 1965; Ortonne et al., 1983; Wayte et al., 1968). This large variation probably reflects that these studies are based on few patients, and that regressed halo nevi can be mistaken for vitiligo, as the depigmented macules in the two conditions are clinically and histologically identical. The incidence of vitiligo in a large study involving 100 patients was only 1% (Wayte et al., 1968) — no different from that in the normal population. Conversely, the incidence of halo nevi in vitiligo is elevated, reported as occurring in as many as 50% of patients (Lerner and Nordlund, 1978). If restricted to five large studies involving at least 100 patients (Barona et al., 1995; Copeman et al., 1973a; El Mofty, 1968; Ortonne, 1974; Perrot et al., 1973), the incidence of halo nevi ranges from 0.5% to 14%. The average incidence of halo nevi among the 1410 patients reported in these five studies was 5.9%, an incidence approximately fivefold greater than that in a normal population. Other observations argue for the trigger for the two conditions to be different. Halo nevi always begin around a nevus, while vitiligo rarely does so. A lymphocytic infiltrate is always present in active nevi surrounded by a halo, but is exceptional in vitiligo. Pigmented nevi can be present in lesions of vitiligo, an unexpected event if the cause of the two diseases is similar. A recent Dutch study (De Vijlder et al., 2004) described human leukocyte antigen (HLA) associations with two types of vitiligo: vitiligo vulgaris (VV) and halo nevi associated with vitiligo (HNV). In the latter group vitiligo is preceded by the appearance of halo nevi and the authors test the hypothesis that a difference in immunopathogenesis might be reflected in an association with different HLA alleles. Seventy-six patients were studied, including 40 with VV and 36 with HNV. The HNV group showed significantly less association with precipitating factors (stress, physical trauma, hormonal changes). Autoimmune disease was almost exclusively found in the VV group. The VV group showed a positive association with HLADR4 and HLA-DR53 and a negative association with HLA-

HYPOMELANOSES ASSOCIATED WITH MELANOCYTIC NEOPLASIA

DR3. In the HNV group there was only a negative association with HLA-DR11. The association of VV with HLA class II points to the involvement of CD4+ T cells that can stimulate both cellular and humoral immune responses.

Pathology and Pathogenesis The histologic findings depend on the stage of evolution at which the biopsy is taken. The most common characteristic is the presence of an intradermal nevus, a dense lymphocytic infiltrate in the nevus, and loss of melanocytes and melanin in the halo. Within the halo, there is decreased or complete absence of melanocytes (Berger and Voorhees, 1971; Cesarini et al., 1968; Hashimoto, 1975; Hori et al., 1976; Mishima et al., 1972; Swanson et al., 1968). Those that remain are in various stages of degeneration (Hashimoto, 1975), with detachment from the basal layer, vacuolization, autophagocytosis of melanosomes, and pyknotic nuclei (Hashimoto et al., 1974). There is a sharp transition between the depigmented halo and the normal adjacent epidermis. In the completely depigmented halo, epidermal melanocytes are absent and there is an increased number of Langerhans’ cells in the basal cell layer (Hashimoto et al., 1975; Mishima et al., 1972; Swanson et al., 1968). The keratinocytes in the halo are usually normal apart from the absence of melanosomes and occasional degenerative changes. The central nevus is usually a compound nevus (Akasu et al., 1994; Wayte et al., 1968), though it may be entirely dermal or junctional (Wayte et al., 1968). There is usually no discernible atypia of nevus cells or of melanocytes (Kopf et al., 1965; Wayte et al., 1968) in early lesions (Akasu et al., 1994). As the regression progresses, mild atypia of nevus cells may become apparent. Atypia occurring at this point is probably a result, rather than a cause of the inflammatory infiltrate. This, and the fact that a hypopigmented halo may develop around dysplastic nevi (Bergman et al., 1985), may account for some reports that mild or moderate atypia is a common feature of nevus cells in halo nevi (Reed et al., 1990). Resolution is characterized by a dense lymphocytic infiltrate that initially appears as a band in the deepest portion of the nevus and proceeds toward the surface (Stegmaier, 1959). Regression of the central nevus has been divided into four stages (Akasu et al., 1994; Jacobs et al., 1975). In stage I (preregression), a moderate number of mononuclear cells surround but do not invade nests of nevus cells. In stage II (early regression), the inflammatory infiltrate is much denser, infiltrates the nests of nevus cells, and is present as a band beneath the nevus. There is degeneration of nevus cells within the dermis, and some inflammation of the junctional nest of nevus cells. In stage III (late regression), there is complete loss of pigmentation, widespread destruction of nevus cells, and damage to melanocytes in the epidermis overlying the nevus. There is also an increased number of Langerhans cells within the epidermis (Akasu et al., 1994). While these antigen-presenting cells are important in initiating and mediating cellular immune responses, their expansion late in the process of nevus regres-

sion suggests that their increasing number is a result rather than a cause of the regression. In stage IV (complete regression), the lesion is fully regressed. The histology resembles that of vitiligo with total absence of nevus cells and melanocytes, few or no inflammatory cells (Jacobs et al., 1975), and some fibrosis. The infiltrate is composed of lymphocytes, plasma cells, and histiocytes. While the density of the infiltrate changes as regression evolves, its composition remains constant (Akasu et al., 1994). Approximately 80% of the cells are T cells. Of these, a relatively high proportion are CD8+ cytotoxicsuppressor cells (Bergman et al., 1985), and approximately 25% are CD4+ cells (Akasu et al., 1994). Increased numbers of activated macrophages appear late in regression (Akasu et al., 1994). Natural killer (NK) cells are rare (Akasu et al., 1994). Similarly, in regressing halo congenital nevi, 60–80% of cells are T cells, with a relative high proportion of CD8+ cells compared to CD4+ cells (Tokura et al., 1994). The expression of class I HLA-A, HLA-B, and HLA-C antigens is markedly upregulated in the vast majority of nevus cells in halo nevi with dense inflammatory infiltrates (Bergman et al., 1985). As the expression of class I HLA antigens is upregulated in primary melanoma and dysplastic nevi (Ruiter et al., 1982; Takei et al., 1984), it was initially thought that this was an indication that the nevus cells were atypical. However, the expression of HLA-A, HLA-B, and HLA-C is upregulated in halo nevi that lack atypia but are close to inflammatory cells, and is normal in nevus cells distant from the infiltrate or in halo nevi with a sparse infiltrate (Bergman et al., 1985). This suggests that HLA upregulation is actually secondary to factors released by the inflammatory cells (Bergman et al., 1985). The expression of class II HLA-Dr antigens is increased occasionally, and then only in the nest of nevus cells that have been infiltrated by T lymphocytes. This suggests that, as with class I HLA, Dr upregulation is induced by adjacent lymphocytes (Bergman et al., 1985). The results of direct immunofluorescence studies are inconsistent. Some are negative (Kopf et al., 1965), others describe deposits of IgM in the intercellular space or dermis in some (Levin, 1933) but not all patients (Berman and Herszenson, 1981), and others report positive findings but do not describe their nature (Uda et al., 1984). Tokura described one patient with regressing congenital halo nevi in which globular clusters of IgM were present at the tip of epidermal rete ridges in what appeared to be nevus cells that had not been destroyed (Tokura et al., 1994). The cellular immune response in halo nevi has been studied in detail by Musette et al. (1999). These authors started from known facts: (1) CD4+ and CD8+ T cells infiltrate halo nevi; (2) T cells are implicated in the destruction of the nevus melanocytes because peripheral T cells of a patient with a halo nevus are able to lyse the melanocytes of a normal nevus; and (3) activated CD8+ T cells are predominant in halo nevi. The authors studied the molecular targets of cytotoxic T cells in halo nevi and the nature and activation of T cells in these lesions. Previous studies had found evidence of a local 709

CHAPTER 38

proliferation of T cell clones in halo nevi by studying the T cell receptor (TCR) (Birck et al., 1997). In this study polymerase chain reaction (PCR)-based technology was used to describe Vb, Jb and CDR3 length usage of the T cells infiltrating 13 halo nevi in eight patients. Basically, two populations of cells are found in halo nevi: (1) nonactivated or polyclonally activated T cells, selected out of the peripheral blood due to cutaneous lymphocyte-associated antigen (CLA) expression and (2) clonally expanded T cells. Interestingly, the b chains of the TCR of the T cells expanded in one halo nevus of a patient were also found to be associated with the T cells expanded in another halo nevus of the same patient but were not found in the blood. Furthermore, the antigen recognition regions of the TCR b chains of expanded T cells shared common amino acid sequences, suggesting that they recognize the same or closely related antigenic peptides. The TCR b chains of the selectively expanded T cell clones do not recognize classic melanoma tumor antigens (MAGE, BAGE, etc). Thus, the nature of the “halo nevus antigens,” likely to be shared by all halo nevi of a given patient, remains unknown. It could be autoantigens, part of a normal differentiation pathway of normal nevi, to which the immune system is normally tolerant. The cause of the halo, and of leukoderma acquisitum centrifugum in general, is difficult to explain solely by either a cellular or humoral mechanism — the former because a cellular infiltrate is absent in the lesions, the latter because it is difficult to explain how a circulating antibody selectively targets only those melanocytes that happen to be around a pigmented lesion. The most reasonable explanation is that leukoderma acquisitum centrifugum is caused by a soluble factor(s) produced in the central neoplasm. The factor(s) could either be a product released from damaged nevus/melanoma cells or a cytokine produced by the inflammatory cells. In either case, it would be consistent with the initial event occurring within the pigmented lesion. Failure of the factor(s) to cause immediate destruction of nevus or melanoma cells can be explained by the greater sensitivity of normal melanocytes to injury (Norris et al., 1988). At this stage it is not possible to differentiate between these two possibilities.

Treatment Patients with benign-appearing, centrally placed nevi within a symmetric halo of depigmentation can be reassured; these halo nevi should not be removed. When observing clinical atypia in the central nevus, eccentric localization of the central nevus and/or presence of an asymmetric halo, histopathologic examination is necessary (Grichnik et al., 2003).

Regression in Melanoma Epidemiology Melanoma (see Chapter 25) is notable for its frequency of spontaneous regression. In a review of all cancer cases with spontaneous regression reported in the literature from 1900 710

to 1987 (n = 741), melanoma was responsible for 12.4% (Challis and Stam, 1990). This is clearly higher than the proportion of melanomas in the total cancer number, where melanoma accounts for 1.3% of all new cancers worldwide up to 2% in developed countries (Parkin et al., 1999). Complete spontaneous regression of the primary tumor — meaning the complete disappearance without treatment or in the “presence of therapy which is considered inadequate to exert a significant influence on neoplastic disease” (Challis and Stam, 1990) — has been documented in over 30 cases (Menzies and McCarthy, 1997). These cases presented with metastatic lymph nodes, had a history of a changing pigmented lesion in an area drained by the affected lymph node(s), and demonstrated an atypical pigmented or depigmented lesion at that site of which histologic features of regression are seen on biopsy (criteria by Smith and Moseley, 1965). Based on these stringent criteria, one can assume that the true prevalence of spontaneous complete regression is underestimated. On the other hand, the observation of melanoma metastases in patients with no detectable primary tumor (cancer with unknown primary) is considered as a possible manifestation of spontaneous regression of the primary. This group is not uncommon, and would constitute up to 5% of all melanoma patients (Nathanson, 1976). Complete spontaneous regression has also been described in melanoma metastases. It has been reported most frequently in cutaneous metastases, but has also been reported in lymph node metastases and visceral metastases including lung, liver, bone, and bowel (King et al., 2001). Reported prevalence rates of histologic regression in primary tumors usually range around 20–30% (Blessing and McLaren, 1992; Brogelli et al., 1992; Fontaine et al., 2003; Kelly et al., 1985; Trau et al., 1983b). Histologic regression is observed more frequently in thinner melanomas, with reported rates from 19% to more than 50% (Blessing and McLaren, 1992; Brogelli et al., 1992; Cooper et al., 1985; Fontaine et al., 2003; Gromet et al., 1978; Kelly et al., 1985; McGovern et al., 1983; Paladagu and Yonemoto, 1983; Ronan et al., 1987; Slingluff et al., 1988; Søndergaard 1985; Trau et al., 1983b). This enormous variation probably reflects in part the different criteria used to define the phenomenon of regression. Histologic regression is less frequent with increasing tumor thickness, and in thick tumors seems to present more often in its late stage (Blessing, 1992). Furthermore, histologic regression seems to be more often observed in an adjacent radial growth phase of melanoma (Trau et al., 1983b; Kelly et al., 1985). Regression is more prevalent in superficial spreading melanomas (Kelly et al., 1985), which could be related to tumor thickness and radial growth phase issues. Several studies reported histologic regression in primary melanoma to predominate in males (Blessing and McLaren, 1992; Kelly et al., 1985; McGovern et al., 1983; Shaw et al., 1987; Søndergaard and Hou-Jensen, 1985) and in melanomas of the trunk (Blessing and McLaren, 1992; Kelly et al., 1985; Søndergaard and Hou-Jensen, 1985; Trau et al., 1983b). In

HYPOMELANOSES ASSOCIATED WITH MELANOCYTIC NEOPLASIA Table 38.1. Different phases of histologic regression in primary melanoma [adapted from Kang et al. (1993)].

Fig. 38.8. Depigmentation within a melanoma. There is an irregular area of partial and complete loss of pigmentation within the tumor. These represent areas of melanoma cell regression. In some cases, the regression can progress to result in complete disappearance of the melanoma (see also Plate 38.7, pp. 494–495).

one study of 844 melanoma patients, patients with regression were on average two years older (Kelly et al., 1985). The authors concluded that this could reflect a longer duration of the tumor, long-standing melanomas having a greater opportunity for host–tumors interactions. Another study reported a trend towards more regression in tumors with higher diameter (Trau et al., 1983b), and another study observed that extensive regression was more often present in larger lesions (Ronan et al., 1987; McLean et al., 1979). Some studies have not been able to confirm an age and/or tumor duration effect (Trau et al., 1983b; Søndergaard and Hou-Jensen, 1985).

Clinical Features The most typical clinical sign of regression in a primary melanoma lesion is the occurrence of a white depigmented, scarlike area within the lesion (Mihm et al., 1971) (Fig. 38.8). The earliest clinical correlate of regression is probably erythema, although this has been documented rarely (Ferradini et al., 1993). It is only when regression evolves further that there is a decrease in the amount of pigmentation, with eventually pink to white areas (Mihm et al., 1971). Sometimes regression may produce bluish areas, or a picture of blue dots observed on surface microscopy, corresponding to the presence of melanophages in the papillary dermis (Mihm et al., 1971; Menzies and McCarthy, 1997; Zalaudek et al., 2004). Clinical regression of part(s) of a tumor may give the impression of satellite metastases or multicentricity (McGovern, 1975).

Pathology Regression in primary melanoma is a dynamic process which is sometimes categorized in three sequential stages: early or active phase, intermediate phase, and late phase (Kang et al., 1993). The early/active phase is characterized by an intense

Early phase

Area(s) of dense lymphocytic infiltration in papillary dermis and possibly in the epidermis Lymphocytes disrupt nests of melanoma cells and are closely opposed to tumor cells (satellitosis) Melanoma cell destruction

Intermediate phase

Area(s) of decreased tumor cell density, increased fibrous tissue and the presence of lymphocytes Variable telangiectasia and melanophages Possible effacement of the epidermal rete pattern

Late phase

Area(s) of extensive dense, horizontally disposed fibrous tissue (scarring) with important reduction in/absence of tumor cells Variable telangiectasia and melanophages Sparse or no lymphocytic infiltration Often effacement of the epidermal rete pattern

inflammatory response with evidence of melanoma cell degeneration, the intermediate phase shows an admixture of inflammation and fibrosis with disruption of the tumour, resulting in areas of extensive fibrosis with minimal presence/absence of tumor cells in the late phase (Table 38.1). Different phases may simultaneously occur in one tumor (McGovern, 1975). Regression can be either focally, partially or rarely completely, but usually is a patchy phenomenon with small segments and skip areas. One study addressing interobserver agreement in the recognition of histologic regression among two expert pathologists using a set of predefined criteria showed concordance rates of 90% up to 96% when examining a predefined area in the slides (Kang et al., 1993). However, when asked to categorize regression observed in the slide as early, intermediate, or late, concordance rates dropped to 66%. Quantification of the extent of regression has been reported in some studies, but may be hampered by the lack of uniform criteria to define regression, the usual multifocality of the phenomenon, and possible interobserver variations. The most accurate, but elaborate assessment used the calculation of the percentage of the area of regression on the total area of the tumor in step-sectioned sections (Ronan et al., 1987). The authors observed a correlation between the extent of regression and prognosis of thin melanomas. Blessing and McLaren (1992) expressed the extent of regression semiquantitatively as 1+ (regression zone <10% of tumor surface area), 2+ (regression zone 20–40% of tumor surface area) and 3+ (regression zone >40% of tumor surface area). Cooper et al. (1985) calculated the percentage of width of all regression zones to the maximal width. Søndergaard and Hou-Jensen (1985) measured both width and thickness of fibrosis in zones with past regression. They observed a tendency towards greater width with increasing tumor thickness in melanomas with Breslow thickness below 1 mm. 711

CHAPTER 38 Table 38.2. Studies on prognostic significance of histologic regression in melanoma. Author

Prognostic significance

Balch et al., 1978 McLean et al., 1979 Day et al., 1982 Paladagu and Yonemoto, 1983 McGovern et al., 1983 Trau et al., 1983b Briggs et al., 1984 Cooper et al., 1985 Kelly et al., 1985 Søndergaard and Hou-Jensen, 1985 Naruns et al., 1986 Ronan et al., 1987 Shaw et al., 1987 Slingluff et al., 1988 Blessing and McLaren, 1992 Brogelli et al., 1992 Massi et al., 1999 Cook et al., 2002 Guitaert et al., 2002 Fontaine et al., 2003 Olah et al., 2003

None None None Adverse None None None None None Adverse Adverse Adverse None Adverse Adverse None Adverse Adverse Adverse None Adverse

“Thin” melanomas

+ + + + +

+ + + +

+ + +

The differential diagnosis of early histologic regression in melanoma includes halo nevus. In case of regression of the intraepidermal component of melanoma, the histological picture can be confused with cutaneous metastasis of melanoma (McGovern, 1975). Regression of one or more tumor areas can create the impression of the presence of microscopic satellites around a primary melanoma (McGovern, 1975). Extensive regression of the dermal component of melanoma can simulate in situ or microinvasive melanoma.

Prognosis As regression may be interpreted as some degree of successful host response against melanoma, one could expect that it would reflect a favorable prognosis. However, the report that Clark’s level II melanomas displaying evidence of regression may occasionally metastasize raised questions about the real prognostic significance of histologic regression in primary melanoma (Reed et al., 1975). Since then several studies have been published on the possible prognostic role of histologic regression in primary melanoma. Most studies have only addressed thin tumors (variably defined as melanoma with Breslow <0.76 mm, <1 mm, and <1.5 mm) (Table 38.2). Only one study evaluated regression clinically (McLean et al., 1979). Two studies reported regression to be a negative prognostic factor where it was extensive. Ronan et al. (1987) found a negative prognostic significance for extensive regression. They evaluated 103 melanomas of 0.76 mm or less with a median follow-up of at least 22.9 months for the presence and extent 712

of histologic regression. Thirty patients (29%) had histologic evidence of regression, with extensive regression (>77%) in seven. Six of 30 patients died of melanoma metastases during the follow-up period. All of these patients showed extensive regression (>77%) and significantly larger lesions. Guitaert et al. (2002) studied 43 patients with metastasizing thin (Breslow <1 mm) melanomas. There was an overrepresentation of axial tumors and extensive regression (defined as 50% or more) in these patients compared to controls without metastasis matched for age, sex, tumor site, and Breslow thickness. There are two recent reports on regression and its value in predicting sentinel lymph node involvement. Olah et al. (2003) found that the occurrence of histologic regression in a primary tumor with Breslow £2 mm conferred around 10-fold higher risk for sentinel node involvement. Of the 14 sentinel node positive patients with regressing tumors, 11 melanomas were thinner than 1 mm. These findings could not be confirmed by Fontaine et al. (2003). The results of these studies may have been influenced by other “confounding” factors such as the presence of ulceration and Clark’s level in thin melanomas (Balch et al., 2001) and the preponderance of histologic regression in males and trunk melanomas, which were not always taken into account upon analysis. Conclusions may also have been influenced by insufficient long-term follow-up, as demonstrated by the study of Gromet et al. where the negative prognostic significance of regression was lost with longer follow-up (Gromet et al., 1978; Kelly et al., 1985). Regression was not mentioned in the prognostic factor analysis of 17 600 melanoma patients (Balch et al., 2001). However from literature, there seem to be indications that histologic regression — especially when extensive — may adversely affect outcome in thin melanomas and that such lesions should be regarded as “undetermined risk.” One hypothesis about the underlying mechanism for this unfavorable impact on prognosis is that Breslow thickness is underestimated in areas of tumor that have regressed, which creates the impression of a thinner melanoma with less metastatic potential. This is supported by the observations by Massi et al. (1999) that the total thickness of tumor and regression zone was one of the two independent predictors of a more aggressive clinical outcome in thin melanomas (Breslow £1.5 mm). Slingluff et al. (1988) found that thin (<0.76 mm) melanomas with regression had a disease-free interval comparable to thicker lesions. Some studies also reported Clark level I / in situ melanomas — theoretically devoid of metastatic potential — with metastasis (Ronan et al., 1987; Guitaert et al., 2002). Some authors on the other hand stated that adding up the thickness of the regression zone to the measured tumor thickness as a measure for the original lesion thickness did not dramatically change thickness in a way that could also influence prognosis (Shaw et al., 1987; Paladagu and Yonemoto, 1983), although scarring fibrosis may lead to underestimation of previous tumor thickness. Another hypothesis is that regression could be an immunoselective phenomenon. The host’s immune response selects

HYPOMELANOSES ASSOCIATED WITH MELANOCYTIC NEOPLASIA

certain clones of cell which undergo attack, while other melanoma cells continue to grow. The surviving tumor clones may have a better capacity to metastasize (Clark, 1990; Prehn, 1996). A third hypothesis suggested by Shaw et al. (1989) is based on the observation that all patients presenting with thin (Breslow <0.76 mm) primary melanoma and concurrent regional lymph node metastasis (n = 28) demonstrated histologic signs of regression in the primary. For patients with thin primary melanoma and metachronous disease recurrence (n = 61) and in patients with thin primary melanoma who remained disease free (n = 735), this was 67% and 61%, respectively (Shaw et al., 1989). They suggested that the spread of melanoma to regional lymph nodes could increase the immune reaction against the primary melanoma. This hypothesis is in line with the finding by Olah et al. (2003) that patients with regression of the primary tumor had more sentinel node involvement.

Melanoma-associated Depigmentation Three different types of hypo/depigmentation are associated with melanoma: leukoderma within the tumor, discussed under “regression,” leukoderma acquisitum centrifugum around the primary tumor/metastases, and depigmentation at distal sites. The latter is often called vitiligo although this condition is not strictly identical to classic vitiligo in terms of epidemiology, clinical appearance, body distribution, histologic features, and underlying mechanisms. We therefore will not use this term when we describe melanoma-associated hypo/depigmentation.

Epidemiology Melanoma-associated depigmentation has been observed by several investigators over the past. A case–control study of 623 melanoma patients in Germany found depigmented lesions in 3.7% of the cases (Shallreuter et al., 1991). The lesions were categorized as symmetric/general (n = 8), segmental (n = 10) and focal (halo-nevuslike with complete absence of central pigmentation) (n = 5). About half of these patients developed depigmented lesions prior to melanoma diagnosis. The other half developed depigmentations after diagnosis of primary and/or metastatic melanoma, usually within the first year. The prevalence of melanoma-associated depigmentation rose in successive risk groups discriminated on the basis of tumor thickness and localization. Since the prevalence of vitiligo in northwestern Europe has been estimated between 0.1% and 2% (Alkhateeb et al., 2003), there seems to be a more than threefold increase in the prevalence of depigmented lesions in melanoma patients based on this study. However, in a group of 386 patients recorded in the Yale melanoma registry the prevalence of pre-existing vitiligo was not higher than expected (1.0%) (Nordlund et al., 1983). In a cohort study investigating the prevalence of melanoma in 1052 patients with “vitiligo,” three melanomas were found (0.3%) (Lindelöf

et al., 1998). In two cases the “vitiligo” developed after diagnosis of melanoma and in one case 32 years before. Based on these studies, it does not seem necessary to have regular follow-up for primary melanoma in patients with vitiligo.

Clinical Features The onset and course of melanoma-associated depigmentation differs from classic vitiligo not associated with melanoma. In classic vitiligo, depigmentation usually starts on the hands, face, and feet, and spreads centripetally to the trunk. In patients with melanoma, depigmentation begins more commonly on the trunk and spreads centrifugally to involve the neck, face, and extremities (Nordlund et al., 1983) (Fig. 38.9; see also Figs 30.33–30.35 in Chapter 30). Koh et al. (1983) studied eight patients and previous case reports with melanoma and widespread leukoderma. The age of onset of melanoma-associated hypopigmentation was between 30 and 61 years with a mean of 49 years. A family history of vitiligo was absent in all the cases. In contrast, classic vitiligo occurs in patients 10–30 years of age with a positive family history in 30%. The distribution of leukoderma appeared to be quite variable and bore no clear relationship to the tumor site. In each of the eight cases the hypopigmentation tended to be extensive, patchy, and not always in the symmetric pattern as seen in vitiligo. Only one patient revealed depigmentation in symmetric sites. All the other patients had hypopigmentation, accentuated by Wood’s light. Different patterns of pigment loss occurred simultaneously in the same patients: three patients showed halo nevi along with widespread leukoderma, whereas four other patients had leukoderma acquisitum centrifugum around surgical sites. A striking clinical observation in the eight cases studied by Koh et al. (1983) was that all patients had either regional or distant metastasis, when hypopigmentation was first noted. The authors stated that this finding was consistent with most of the previously reported cases of leukoderma associated with melanoma. In all cases leukoderma began simultaneously with or some time after the finding of metastasis. In most of the cases a gradual progression of depigmentation was observed over several years after which stabilization occurred. Another striking finding was the presence of ocular abnormalities in patients with melanoma-associated hypopigmentation. Uveitis, chorioretinal scarring, retinal pigment epithelium hypoplasia with and without atrophy, halo nevi, ocular melanoma, central serious retinopathy, and macular hole are eye findings in patients with melanoma and leukoderma (Koh et al., 1983). The association of vitiligo with inflammation of the pigment system of the eye is known in the Vogt– Koyanagi–Harada syndrome (see Chapter 39), a multisystem disorder characterized by uveitis, aseptic meningitis, otic involvement, and vitiligo especially of the head and neck region.

Pathology Few patients with melanoma-associated leukoderma have had 713

CHAPTER 38

A

B

Fig. 38.9. (A, B) Vitiligo-like depigmentation. Note the sharply outlined macule of depigmentation, which resembles vitiligo clinically but occurs in patients with melanoma and has no other explanation.

ultrastructural examination of the hypopigmented skin. Koh et al. (1983) reported the ultrastructural results in eight patients with melanoma-associated hypopigmentation. Splitdopa epidermal preparations of seven patients showed evidence of a complete absence of melanocytes in four, whereas in three hypertrophic macromelanocytes with many long dendrites were present. A repeat biopsy in one of the three patients with macromelanocytes showed the absence of melanocytes. These findings suggest that pathologic destruction of melanocytes first causes compensatory hypertrophy of remaining melanocytes and ultimately results in complete destruction of the cells. Interestingly macromelanocytes have also been described in the active border of classic vitiligo. Electron microscopy of the hypopigmented areas without melanocytes showed no or little pigment throughout the epidermis. The dermis did not exhibit any significant changes. In the hypopigmented areas with macromelanocytes there was a certain amount of pigment present in the basal keratinocytes. The melanocytes contained well-developed cytoplasmatic organelles. The melanosomes were reduced in number in some melanocytes whereas other melanocytes still appeared to possess increased premature melanosomes suggesting impaired 714

melanization. Rarely, spherically granular melanosomes were noted. The number of Langerhans cells in the basal layer of the epidermis was normal. In one patient dermal perivascular infiltrates composed of lymphocytes and a few mast cells were observed. However, epidermal infiltrates were absent in all patients. Within normal looking skin, the most striking change was the vacuolar degeneration of keratinocytes, whereas the melanocytes appeared to be normal. This phenomenon was also present in the normally pigmented skin of vitiligo patients suggesting that both vitiligo and melanoma-associated hypopigmentation affect the whole epidermal melanin unit, the melanocyte, and its corresponding pool of keratinocytes (Koh et al., 1983). The complete absence of epidermal infiltrates as found by Koh et al. was not confirmed by a more recent study. On electron microscopy, Langerhans cells were frequently observed. Also lymphocytes with heavily indented nuclei, frequently associated with acantholytic changes of the adjacent basal keratinocytes were seen. Skin biopsies from five vitiligo patients without melanoma also showed an increased number of Langerhans cells and lymphocytes with heavily indented nuclei. These findings suggest the intervention of a cytotoxic

HYPOMELANOSES ASSOCIATED WITH MELANOCYTIC NEOPLASIA

mechanism both in vitiligo and in melanoma-associated hypopigmentation (Cannavo et al., 1996).

Table 38.3. Immunotherapeutic strategies with reports on depigmentation.

Significance/Prognosis

Passive immunotherapy Monoclonal antibodies: Elsas et al., 1999; Phan et al., 2003; Hara et al., 1995 Adoptive transfer of cytotoxic T cells: Yee et al., 2000; Engelhard et al., 2002

Earlier reports on the prognostic significance of leukoderma in melanoma patients were contradictory with reports of leukoderma being of little importance on disease course (Ortonne et al., 1978), whereas others reported leukoderma to be associated with visceral metastases and a bad prognosis (Laucius and Mastrangelo, 1979). More recent publications have suggested that, although patients with melanoma-associated depigmentation usually have worse prognostic factors than patients with melanoma without leukoderma (primary melanoma arising at sites with poorer prognosis, metastases at least to regional lymph nodes), they have a higher-thanexpected survival according to their prognostic factors (Bystryn et al., 1987; Duhra and Ilchyshyn, 1991; Koh et al., 1983; Nordlund et al., 1983; Rodriguez-Cuevas et al., 1998). Nordlund et al. (1983) studied 51 patients (24 from published reports) with both distant depigmentation and stage I–III melanoma. They found that the 5-year survival among these patients was 67%, whereas this was estimated to be only 50% based on historical controls from publications from other institutions. These results were confirmed by a similar, smaller study where the average 5-year survival of eight patients with both leukoderma and stage II or III melanoma was found to be 60%. Although there is no ideal control group for comparison, the 5-year survival of clinical stage II melanoma patients was estimated at around 30% (Koh et al., 1983). The interpretation of the results of these studies is limited because of the absence of an adequate stratification for the multitude of factors influencing melanoma prognosis and because of the use of historical controls or controls seen in other institutions. A large prospective trial compared the actual with the predicted survival of 46 patients with melanoma and hypopigmentation (Bystryn et al., 1987). The expected 5-year survival was calculated using a multivariate prognostic index based on the most important prognostic factors (Rigel et al., 1985). The predicted 5-year survival in the studied patients was 74.8%, whereas actual 5-year survival was 86.3% (p = 0.03). The better outcome than expected was confirmed when the categories of leukoderma acquisitum centrifugum and hypopigmentation at a distance were taken separately. A possible explanation for the better prognosis in melanoma-associated leukoderma could be its “booster function.” Melanoma cells produce high levels of immunosuppressive cytokines with a profound negative impact on the immune response. This could lead to a gradual suppression of the antitumor immune response or a skewing of functional immunity away from type 1 and towards inefficient type 2 T cell/humoral immunity. In this context, depigmentation observed in melanoma patients can be considered as a booster for restimulation of the antitumor response, the affected skin

Active immunotherapy Tumor cells, hybrid cells: Osanto et al., 2000; Trefzer et al., 2000; Krause et al., 2002 Dendritic cells: Mackensen et al., 2000; Banchereau et al., 2001; Tsao et al., 2002 Genetic immunization: Bronte et al., 2000; Steitz et al., 2000; Weber et al., 1998 Recombinant cytokines (IL-2, IFN, GM-CSF): Le Gal et al., 1996; Asnis et al., 1995; Scheibenbogen et al., 1994; Wolkenstein et al., 1992; Ho et al., 1995; Rosenberg et al., 1996; Slingluff et al., 2003

providing a constant source of autoantigens that are also expressed by melanoma cells. Therefore the role of depigmentation developing during the course of malignant melanoma could be described as adjuvant in nature (WankowiczKalinska et al., 2003).

Therapy-induced Depigmentation For several immunotherapeutic strategies used in the treatment of melanoma, the occurrence of depigmentation has been reported. Table 38.3 gives a nonexhaustive review of studies reporting leukoderma during immunotherapy of melanoma. A simple explanation for the dissociation of antitumor immunity and autoimmune destruction in cancer vaccination studies is the differential level of a target antigen expressed by the tumor (high expression) and normal tissue (low expression). Therefore, the lack of autoimmunity in some cancer vaccine models could reflect a weak vaccine that is able to eliminate a small number of tumor cells under experimental conditions without causing pathology in normal tissues (Lane et al., 2004). Another possibility for uncoupling tumor immunity and autoimmunity lies within the different effector responses (e.g., mediated by antibodies versus cytotoxic T cells) when using two closely related antigen paralogs. Small differences in amino acid sequence seem to lead to qualitative differences in effector responses. Depigmentation induced by TRP-1 vaccination is mediated primarily by antibodies, whereas TRP-2 specific tumor protection depends upon T cell mediated response (Bowne et al., 1999; Bronte et al., 2000). It is likely that TRP-1 antibodies penetrate more easily into the skin resulting in the damage of normal melanocytes whereas access to the skin by autoreactive T cells is extremely limited in the absence of inflammation. Vaccination-induced autoimmune “vitiligo” could also be a consequence of secondary trauma to the skin. When human 715

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TRP-2 DNA was injected in mice intradermally, this resulted in a protective immunity and the development of “vitiligo” in all mice. However, an intramuscular injection with the same substance generated the same protective immunity without development of leukoderma. These observations support the hypothesis that vaccination-induced vitiligo may be a consequence of inflammation in the skin following vaccination, leading to the recruitment of autoreactive T cells into the skin and subsequent depigmentation (Lane et al., 2004). Collela et al. (2000) supported the hypothesis that skin access by effector T cells is required for subsequent destruction of normal melanocytes. Likewise, other studies demonstrated that immunotherapy-induced leukoderma occurred only in the area surrounding the site of tumor inoculation or skin depilation (Bronte et al., 2000; Hara et al., 1995; Overwijk et al., 2003). Engelhard and colleagues observed a small number of cases of localized depigmentation in mice injected with tyrosinase pulsed dendritic cells who subsequently had skin damage as a result of fighting with cagemates. They also found that in the adoptive T cell transfer experiments depigmentation required coadministration of interleukin (IL)-2 as well as sublethal irradiation of the skin. These results suggest that T cell lymphocyte access to the skin is minimized under normal circumstances and increased by skin damage due to injury or irradiation (Engelhard et al., 2002). Even though it is encouraging that control of tumor growth could be dissociated from the induction of vitiligo-like leukoderma, it will be important to ascertain whether this dissociation reflects patterns of T lymphocyte homing, the prevalence of inflammatory stimuli or suppressive mechanisms operating in local niches of the body. In the latter case these mechanisms may also limit the access of T cells to early stage melanomas growing in the skin (Engelhard et al., 2002).

Underlying Mechanisms General Considerations There is strong evidence for a role of the immune system in halo nevi, regression in melanoma, and melanoma-associated depigmentation. Although there have been observations supporting humoral immune responses, growing evidence points to an important role for cellular immune responses. Specific microenvironmental conditions seem to be important in creating an effective immune response. In melanoma, some mechanisms of the tumor cells to surpass immune recognition are known (Curiel-Lewandroski and Demierre, 2000). Possible target antigens in melanoma can be classified in four broad categories: melanocyte differentiation antigens which could be the most important in the context of melanoma-associated leukoderma (melan-A/MART-1, gp100/pmel17, tyrosinase, TRP-1/gp75, TRP-2/DCT); tumorspecific antigens due to mutational events (Nras, CDK4, Caspase 8, b-catenin, MUM-1, MUM-2); the so-called cancer testis tumor antigens expressed in the adult testis and a number of unrelated tumor types (MAGE, BAGE, NY-ESO); 716

and TAA that are overexpressed normal tissue antigens (PRAME, p15, CD63) (Saleh et al., 2001). CD8+ cytotoxic T cells (CTL) could play an important role in melanocyte destruction in halo nevi, regression in melanoma, and melanoma-associated depigmentation. CTLs recognize small peptides exposed on a cell surface in conjunction with major histocompatibility complex (MHC) class I molecules. The presented peptides are derived primarily from the enzymatic degradation of cytosolic proteins by an enzyme complex (LMP1, LMP2, Delta, MB1) after which they are transported from the cytoplasm into the endoplasmic reticulum by adenosine triphosphate-dependent transporters (TAP1 and TAP2). In the endoplasmic reticulum they bind to the MHC class I molecule in complex with b2-microglobulin, upon which this complex is translocated to the Golgi apparatus and eventually to the plasma membrane of the cell where it interacts with the TCR. NK cells can exert MHC-unrestricted cytotoxicity. This cellular cytotoxicity can be stimulated by IL-2 (lymphokineactivated killer activity) or can act in an antibody-dependent way. CD4+ T helper cells (Th) are important in initiating immune responses and helping the effector cells via the production of cytokines. Specialized APCs (Langerhans cells in the skin) present antigenic peptide fragments in an MHC class II restricted manner to the TCR/CD3 complex at the surface of the Th cell. For activation of the T helper cell an additional interaction of costimulating molecules B7 (B7-1 and B7-2) of the antigen-presenting cell (APC) with CD28 on the T cell is required. Absence of this interaction can induce anergy. Whereas Th 1 cells are important in initiating CTL response, Th 2 cells stimulate B cell antibody production. These Th subsets produce different cytokine expression patterns. Th cells can dramatically influence the initiation, maintenance, and termination of immune responses through secretion of cytokines and expression of surface molecules. These signals can increase APC proliferation, chemotaxis, and antigen processing, as well as local MHC class I and II expression (Ridge et al., 1998; Schoenberger et al., 1998). Finally, Th cells can play a determining role in the induction and maintenance of immunologic memory (von Herrath et al., 1996; Walter et al., 1995). Although MHC class I cells could directly present endogenously processed antigenic peptide to CTL, immune responses to less immunogenic proteins tend to display a more pronounced dependency on “help” from CD4+ T cells (Guerder et al., 1992; Tsuji et al., 1997; Wada et al., 1995; Yagi et al., 1992) and the initiation of tumor-specific CTL responses is likely to involve indirect presentation of tumor antigens by specialized APCs (Ossendorp et al., 1998). A growing number of epitopes derived from melanocytic antigens and recognized by CD4+ cells have been defined at the molecular level and several of these T helper epitopes are already entering clinical trials in peptide-based vaccines. Surprisingly, many of the CD4+ cells that have been reported to recognize peptide–MHC class II complexes on the surface of melanoma cells in vitro are directly cytolytic (Wankowicz-

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Kalinska et al., 2003). Even though some authors believe CD4+ cells play a beneficial role in the mechanisms of antitumor immunity, several studies have revealed that in the case of excess IL-2 or IL-12 in the vicinity of a tumor, Th cells show an inhibitory effect on antitumor immunity (Overwijk et al., 1999).

Regression in Melanoma Regression in melanoma is a complex interaction between host and tumor. The possible role of the immune system is supported by a number of observations: ∑ the presence of an inflammatory reaction in early regression which seems to be in close contact with melanoma cells and is associated with tumor cell destruction (see Table 38.1); ∑ the finding that melanoma cells can express both MHC class I and class II molecules, and can stimulate the proliferation of autologous CD4+ T helper and CD8+ cytotoxic T lymphocytes in vitro (Rünger et al., 1994); ∑ the finding that several peptides derived from melanomaassociated antigens that bind specific MHC class I molecules are potent activators of cytotoxic T cells in vitro (Valmori et al., 1998); ∑ the finding that in about half of the patients with melanoma metastases tumor-infiltrating lymphocytes (TIL) recognizing autologous tumor cells in an MHC-restricted way can be extracted (Rosenberg et al., 1996); ∑ the finding that adoptive transfer of melanoma TILs has resulted in tumor remission in patients with metastatic melanoma (Rosenberg et al., 1995), as has vaccination therapy aimed at inducing cytotoxic lymphocytes to melanoma tumorassociated antigens (Nestle et al., 1998). Despite all these observations, the exact underlying mechanisms triggering the induction of regression in melanoma remains to be elucidated (Printz, 2001). Immunohistochemical study of the inflammatory infiltrate in primary melanoma and melanoma skin metastases demonstrated that melanocyte differentiation antigen expression as well as T cell subset distribution were heterogeneous within and between lesions and did not seem to be correlated histologically (Bernsen et al., 2004). PCR analysis of microdissected T cells in four melanoma lesions (one primary lesion and one cutaneous metastasis with signs of regression histologically, and two skin metastases derived from the same patient of which one was clinically regressive and the other clinically progressive) demonstrated limited T cell diversity. There were no major differences in T cell diversity between regressive and nonregressive parts of tumors. Furthermore this study provided evidence for the presence of T cells of clonal origin in two of four lesions (skin metastases with histologic signs of regression and in the clinically progressive skin metastases). These clonal T cells did not show preferential localization to regressive areas of the tumor. The detection of clonal T cell populations in primary regressive melanoma has also been reported by Ferradini et al. (1993). Bernsen et al. (2004) found that both the clinically regressive skin metastasis and the primary melanoma with histologic signs of regression showed

a brisk inflammatory infiltrate with a highly skewed T cell diversity (52% and 38% V2 domain rearrangements, respectively) without evidence for clonality. The fact that there are no consistent antigen and T cell expression patterns in regressive versus nonregressive lesions/areas suggests that there is a functional difference between the T cells most probably controlled by specific microenvironmental conditions. It is presumed that a defective lymphokine secretion could be an essential component in the failure of tumor infiltrating lymphocytes in reaching tumor control (Guilloux et al., 1994; Lüscher et al., 1994). Study of the cytokine mRNA profile in melanomas with clinical and histologic signs of regression (n = 10) showed small but significant increases in granulocyte macrophage colony stimulating factor (GM-CSF), IL-2, and IL-15 transcripts compared with areas showing progression within the same lesions (three tissue samples) and to melanomas with a brisk inflammatory infiltrate (n = 14) (Wagner et al., 1998). There were also elevated IL-10 and transforming growth factor (TGF)-b1 gene expression levels, although not statistically significant. In one tissue sample Th 2 cytokine gene expression levels were twofold (IL-5) and threefold (IL-4) higher at the site of regression compared to the site of progression. Further in situ hybridization study suggested that GM-CSF expression seemed to be derived from macrophages, IL-2 expression was attributed predominantly to CD4+ and CD8+ T cells, and IL15 transcripts were detectable in a variety of cell types including melanoma cells, keratinocytes, fibroblasts, macrophages, and lymphocytes. As IL-15 seems to have a possible role in MHC class I expression in melanoma cells, its production could be important in the induction of an effective CTL response. Saleh et al. (2001) studied the presence of regression in the last primary melanoma from patients with three or more asynchronous primary invasive melanomas and compared this to their first tumor and to matched controls (primary melanoma in a patient with one single melanoma, matched for age, sex, tumor thickness, ulceration, and histologic subtype). They observed a significant increase in the percentage of tumor regression in the last tumor compared to the first, consistent with an “immunization effect.” There was also a significant decrease in immunohistochemical expression of melan-A/MART-1, consistent with MART-1 tumor antigen loss variants. This loss in MART-1 expression was also observed in metastases from patients with occult primary, compared with matched controls. Using an interferon (IFN)-g secreting cytotoxic T lymphocyte assay, the authors demonstrated the presence of MART–1specific cytotoxic T lymphocytes among six of nine HLA-A2 subjects with multiple or occult primary melanoma whereas no MART-1 specific CTLs were found in the matched controls. The presence of MART-1-specific CTL inversely correlated with tumor MART-1 expression. Of note is that these MART1-specific CTLs were present in the blood for a median of 7 years after diagnosis, indicating a T lymphocyte memory response. No tumor antigen loss variants or specific CTLs to gp100 have been demonstrated in this patient group. 717

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There have been several reports on the detection of naturally occurring CD8+ cytotoxic T cells recognizing specific tumor antigens in melanoma patients (Kawakami et al., 1995; Knuth et al., 1989; Van Den Eynde et al., 1989; Wolfel et al., 1989), which have also been the basis for the development of peptide vaccines as an immunotherapeutic strategy (Rosenberg et al., 1998). Zorn and Hercend (1999a) reported on the presence of two antitumor-directed CTL clones in a primary melanoma with histologic signs of spontaneous regression occurring on the lower limb of a female patient. One CTL clone recognized a MAGE-6-encoded peptide (Zorn and Hercend, 1999a) and a second CTL clone targeted a mutated peptide encoded by a new unconventional myosin class I gene (Zorn and Hercend, 1999b). Both clones displayed strong selective antitumor activity. Following a disease-free period of 3 years, the patient eventually developed gastric and lymph node metastasis. Analysis of the metastastic melanoma cells revealed that both the expression of MHC class I and the specific peptides recognized by the CTL clones of the primary tumor were maintained. However, the corresponding CTL could not be detected (Carcelain et al., 1997). Another finding supporting the role of a CTL reaction in regression is the observation of a significantly higher expression of LMP2 and LMP7 and a higher proportion of TAP1staining in lesions with versus without histologic signs of regression, as demonstrated by immunohistochemical study (Dissemond et al., 2003a, b). Deficient expression of these components necessary to establish efficient antigen presentation to CTL could one of the escape mechanisms used by melanoma. There is growing evidence for an important role of CD4+ helper T cells in establishing an efficient antitumor response (Kierstead et al., 2001; Schultz et al., 2000; Toes et al., 1999). Halliday et al. (1995) observed significantly larger numbers of CD4+ T cells in regressing versus nonregressing tumors, which is in contrast with the findings of Bernsen et al. (2004). These CD4+ T cells appeared to be breaking up nests of tumor cells and the authors hypothesized that these CD4+ T cells could induce tumor cell destruction by a yet unknown mechanism. Regressing tumors did not show increased numbers of CD1+ Langerhans cells or CD68+ macrophages, and they did not express MHC class II molecules so that a direct cell-mediated cytotoxicity is unlikely. Denfeld et al. (1995) found expression of B7-1 and B7-2 in all of five primary melanomas displaying spontaneous regression as demonstrated by immunohistochemical and reverse transcriptase (RT)-PCR analysis. In contrast the majority of primary nonregressive melanomas and nevus cells failed to express B7-1 and B7-2 (Denfeld et al., 1995). Melanoma cells in the regression areas expressed both MHC class I and II molecules. Correspondingly CD28 was expressed on both CD4+ and CD8+ cells in the regressive melanomas. These findings could support the importance of Th cells in establishing an effective anti-tumor response, mediated in an MHC class II restricted way. 718

Melanoma-associated Depigmentation Increasing evidence suggests that melanoma-associated leukoderma results from an immune reaction directed against shared antigenic determinants on normal and malignant melanocytes. There is a growing body of evidence that the induction of at least limited autoimmune responses will be required to efficiently eliminate cancer cells that express multiple, (altered) “self”-antigens (Hara et al., 1995; Naftzger et al., 1996; Turk et al., 2002). This implies that immune cells escape the mechanisms of central and peripheral tolerance. The breaking of the immune tolerance to autoantigens may require that atypically high levels of these self-proteins be presented by APCs in the tissue-draining lymph nodes, which may only occur after a tumor has progressed to a substantial size (Wankowicz-Kalinska et al., 2003). Immunization with altered forms of the self-protein may be one strategy to break tolerance (Naftzger et al., 1996; Weber et al., 1998).

Humoral Mechanisms Studies involving passive transfer of TA99 antibodies into mice bearing B16 melanoma tumors demonstrated that antibodies against gp75 can induce B16 tumor rejection and autoimmune depigmentation. Tumor rejection was partially dependent on CD4+ cells and natural killer cells and involved activation of Fc receptors on macrophages (Clynes et al., 1998, 2000; Hara et al., 1995; Takechi et al., 1996). The immune threshold for depigmentation was substantially higher (five times more antibodies required) and the autoimmune melanocyte destruction was not dependent on the presence of NK cells, macrophages, or CD4+ cells (Hara et al., 1995). Active immunization to gp75 in mice induced antibody-dependent immunity as well as patchy depigmentation (Naftzger et al., 1996; Weber et al., 1998). Again — unlike tumor immunity — autoimmunity to gp75 did not require activation of Fcg receptors or the presence of CD4+, CD8+, or NK cells (Clynes et al., 1998; Weber et al., 1998). These studies demonstrate that there may be distinct pathways leading to tumor immunity and autoimmunity. Turk et al. (2002) have shown that passive immunization with TA99 induces depigmentation in mice deficient in FcgR or the C3 component of complement. In contrast, depigmentation does not occur in mice deficient in both of these components, indicating that upon absence of macrophages complement acts as an alternative mechanism for autoimmunity (Turk et al., 2002). Antityrosinase IgG antibodies were found in the sera of patients with diffuse vitiligo, metastatic melanoma, and in sera of patients with melanoma and melanoma-associated depigmentation. The number of antibodies in vitiligo exceeded that in melanoma and melanoma-associated depigmentation (Merimsky et al., 1996, 1998). Screening of a large group of patients for the presence of anti-melanoma antibodies revealed that only 2% of them had spontaneous antibodies to MAGE1 and MAGE-3, and none of them had antibodies to MART1 and tyrosinase even though these last two autoantigens are well characterized targets for both CD4+ and CD8+ T cells

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(Storkus and Zarour, 2000). Humoral immune responses against the cancer testis antigen NY-ESO-1 are frequently observed in patients with NY-ESO-1 expressing tumors. Titers of these anti-NY-ESO-1 antibodies directly correlate with tumor load, which suggests that antigen expression by tumors drives autoantibody production (Jager et al., 1999). Kiniwa et al. (2001) have identified tumor antigens by cDNA expression cloning from patients with melanoma-associated depigmentation. The most frequently isolated clone was named KU-MEL1. Anti-KU-MEL-1 antibodies are frequently detected in the sera of melanoma patients, but there is no clear correlation observed between the development of depigmentation and the presence of these antibodies.

Cellular Mechanisms In humans, by far the largest number of reports of tumor-reactive CD8+ T cells concern melanoma (Bowne et al., 1999; Engelhard et al., 2002; Lee et al., 1999; Lengagne et al., 2004; Valmori et al., 1999). High frequencies of CTL specific for several melanocytic antigens can be detected in the blood and among tumor-infiltrating lymphocytes in melanoma patients. Further analysis of the cytotoxic T cell receptor repertoire has been performed and revealed diverse and nonoverlapping T cell receptor repertoires in response to a self-differentiation tumor antigen (Dietrich et al., 2001; Puisieux et al., 1994; Valmori et al., 2000). Yee et al. (2000) provided the first direct evidence in humans that after adoptive transfer of autologous T cell reactive against MART-1, these T cells target not only antigen-positive tumor cells but also normal tissues expressing the shared tumor antigen. In this study the investigators describe a patient with metastatic melanoma who developed inflammatory lesions after an infusion of MART-1 specific CD8+ T cell clones and IL-2. Analysis of the infiltrating lymphocytes in skin and tumor biopsies demonstrated a localized predominance of MART-1 specific CTL. Skin biopsies demonstrated loss of MART-1 expression, evidence of melanocyte damage and the complete absence of melanocytes in affected regions of the skin (Yee et al., 2000). Le Gal et al. (2001) demonstrated the role of cellular immunity in melanoma-associated leukoderma by expanding infiltrating lymphocytes from fresh frozen biopsy specimens of depigmented patches in melanoma patients. The infiltrating lymphocytes were almost exclusively T lymphocytes and most were CD8+ and expressed the cutaneous homing receptor CLA. Furthermore, the infiltrating lymphocytes had a clonal or oligoclonal T cell receptor profile, possibly reflecting specific antigenic stimulation. Finally, these lymphocytes specifically recognized differentiation antigens shared by normal melanocytes and melanoma cells such as tyrosinase, melan-A and gp100 (Le Gal et al., 2001). Even though CD8+ T cells seem to play an important role in tumor immunity and autoimmunity, Th cells appear more and more essential in the initiation and modulation of an immune reaction. Earlier preclinical experiments performed in murine melanoma models provided strong evidence for the

need of specific Th for the combined destruction of melanoma cells and normal melanocytes in vivo. Overwijk et al. (1999) vaccinated mice with a recombinant vaccinia virus encoding gp75. The investigators noticed absence of both depigmentation and protection against tumor challenge in MHC II knockout mice or mice that were antibody-depleted of CD4+ T lymphocytes. Hawkins et al. (2000) investigated the multiple pathways to tumor immunity and autoimmunity by xenogeneic DNA vaccination of C57BL/6 mice against TYRP-2 and gp100. They observed that the immune response to TYRP-2 was CD4+ T cell dependent. Th cells were required for the immunization phase, but were not necessary for the effector phase. The immune response to gp100 did not require CD4+ T cell, neither during the immunization phase, nor during the effector phase (Hawkins et al., 2000). On the other hand, Nagai et al. (2000) showed that in vivo depletion of CD4+ T cells enhanced the antitumor effect of locally secreted IL-12 on B16 mouse melanoma and induced depigmentation. The investigators hypothesized that depletion of CD4+ T cells may result in elimination of Th 2 cells and induce further activated CD8+ T cells. This study indicates that Th cells can act as immunosuppressive agents in anti-tumor immunity depending on the circumstances.

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Corthals, C. Heirman, K. Thielemans, T. Boon, van der P. Bruggen. A MAGE-A3 peptide presented by HLA-DP4 is recognized on tumor cells by CD4+ cytolytic T lymphocytes. Cancer Res. 60:6272–6275, 2000. Shallreuter, K. U., C. Levening, and J. Berger. Vitiligo and cutaneous melanoma. Dermatologica 183:239–245, 1991. Shaw, H. M., W. H. McCarthy, S. W. McCarthy, and G. W. Milton. Thin malignant melanomas and recurrence potential. Arch. Surg. 122:1147–1150, 1987. Shaw, H. M., S. W. McCarthy, W. H. McCarthy, J. F. Thompson, and G. W. Milton. Thin regressing malignant melanoma: significance of concurrent regional lymph node metastases. Histopathology 15:257–265, 1989. Slingluff, C. L., G. R. Petroni, G. V. Yamshchikov, D. L. Barnd, S. Eastham, H. Galavotti, J. W. Patterson, D. H. Deacon, S. Hibbitts, D. Teates, P. Y. Neese, W. W. Grosh, Chianese-Bullock, K. A., E. M. Woodson, C. J. Wiernasz, P. Merrill, J. Gibson, M. Ross, and V. H. Engelhard. Clinical and immunological results of a randomized phase II trial of vaccination using four melanoma peptides either administered in granulocyte-macrophage colony-stimulating factor in adjuvant or pulsed on dendritic cells. J. Clin. Oncol. 21:4016–4026, 2003. Slingluff, C. L., R. T. Vollmer, D. S. Reintgen, and H. F. Seigler. Lethal “thin” malignant melanoma: identifying patients at risk. Ann. Surg. 208:150–161, 1988. Smith, J., and J. Stehlin. Spontaneous regression of primary malignant melanomas with regional metastases. Cancer 18:1399–1415, 1965. Smith, W. E., and J. C. Moseley. Multiple halo neurofibromas. Arch. Dermatol. 112:987–990, 1976. Søndergaard, K., and K. Hou-Jensen. Partial regression in thin primary cutaneous malignant melanomas clinical stage I. Arch. Virchows (Pathol. Anat.) 408:241–247, 1985. Stegmaier, O. C. Natural regression of the melanocytes nevus. J. Invest. Dermatol. 32:413–421, 1959. Steitz, J., J. Brück, K. Steinbrink, A. Enk, J. Knop, and T. Tüting. Genetic immunization of mice with human tyrosinase related protein2: implications for the immunotherapy of melanoma. J. Int. Cancer 86:89–94, 2000. Storkus, W. J., and H. M. Zarour. Melanoma antigens recognised by CD8+ and CD4+ T cells. Forum (Genova) 10:256–270, 2000. Swanson, J. L., D. M. Wayte, and E. B. Helwig. Ultrastructure of halo nevi. J. Invest. Dermatol. 50:434–450, 1968. Takechi, Y., I. Hara, C. Naftzger, Y. Xu, and A. N. Houghton. A melanosomal membrane protein is a cell surface target for melanoma therapy. Clin. Cancer Res. 2:1837–1842, 1996. Takei, M., Y. Mishima, and H. Uda. Immunopathology of vitiligo vulgaris, Sutton’s leukoderma and melanoma-associated vitiligo in relation to steroid effects. I. Circulating antibodies for cultured melanoma cells. J. Cutan. Pathol. 11:107–113, 1984. Toes, R. E., F. Ossendorp, R. Offringa, and C. J. Melief. CD4 T cells and their role in antitumor immune responses. J. Exp. Med. 189:753–756, 1999. Tokura, Y., K. Yamanaka, H. Wakita, S. Kurokawa, D. Horiguchi, A. Usui, S. Sayam, and M. Takigawa. Halo congenital nevus undergoing spontaneous regression. Arch. Dermatol. 130:1036–1041, 1994. Trau, H., D. S. Rigel, M. N. Harris, A. W. Kopf, R. J. Friedman, S. L. Gumport, R. S. Bart, and R. N. Grier. Metastases of thin melanomas. Cancer 51:553–556, 1983a. Trau, H., A. W. Kopf, D. S. Rigel, J. Levine, G. Rogers, M. Levenstein, R. S. Bart, M. M. Mintzis, and R. J. Friedman. Regression in malignant melanoma. J. Am. Acad. Dermatol. 8:363–368, 1983b. Trefzer, U., G. Weingart, Y. Chen, G. Herberth, K. Adrian, H. Winter, H. Audring, Y. Guo, W. Sterry, and P. Walden. Hybrid cell vaccination for cancer immune therapy: first clinical trial with metastatic melanoma. J. Int. Cancer 85:618–626, 2000. Tsao, H., P. Millman, G. P. Linette, F. S. Hodi, A. J. Sober, M. A.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Miscellaneous Hypomelanoses: Depigmentation Sections Alezzandrini Syndrome Wiete Westerhof, David Njoo, and Henk E. Menke Idiopathic Guttate Hypomelanosis Wiete Westerhof, David Njoo, and Henk E. Menke Leukoderma Punctata Wiete Westerhof, David Njoo, and Henk E. Menke Lichen Sclerosus et Atrophicus Philippe Bahadoran Vagabond Leukomelanoderma Wiete Westerhof, David Njoo, and Henk E. Menke Vogt–Koyanagi–Harada Syndrome Wiete Westerhof, David Njoo, and Henk E. Menke Westerhof Syndrome Wiete Westerhof, David Njoo, Henk E. Menke

In 1959 and 1961 Casala, Alezzandrini, and Cremona described two patients with facial vitiligo, poliosis, deafness and tapetoretinal degeneration with lesions of the macules and specific electroretinograms. In 1964, Alezzandrini encountered three patients with unilateral tapetoretinal degeneration and ipsilateral facial vitiligo and poliosis. Two of these patients also had perceptual deafness. These combinations of symptoms (of which deafness is inconsistently present) have since been known as “Alezzandrini syndrome.” In 1992, Hoffman and Dudley described Alezzandrini syndrome in a patient with juvenile diabetes. The patient also experienced retinal detachment on the same side as the pigmentary defects, a feature which is not commonly reported in this syndrome. Tapetoretinal degeneration, however, could not be observed.

were the first manifestations of the disease. Gradual decrease in visual acuity was the main complaint. Dermatologic features appeared between 3 and 13 years after the onset of the visual abnormalities. These included whitening of the hair of the scalp, eyebrows, and eyelashes and whitening of the skin of the forehead, nose, cheek, upper lip, and chin; all occurred on the same side as the disordered eye (Fig. 39.1). One patient reported having hearing problems in one ear, also on the same side. A second patient developed deafness on both sides. Ophthalmologic investigation of the defective eye revealed a decreased visual acuity, a defect in the visual field, and a depigmented and atrophic iris without any signs of uveitis. Furthermore in the fundi a pale papilla and tapetoretinal degeneration with lesions of the macules were observed. Intraocular pressure was not elevated. Electroretinograms showed specific abnormalities, revealing a dysfunction of the cones. Dermatologic assessment established facial vitiligo-like depigmentation and poliosis, all located ipsilateral to the visual defect. An audiogram confirmed perceptual deafness in two of the three patients.

Synonyms

Associated Disorders

There are no synonyms.

Alezzandrini syndrome is believed to be closely related to the Vogt–Koyanagi–Harada syndrome (VKHS) (see below), in which skin, ocular, and auditory changes are also observed.

Alezzandrini Syndrome Wiete Westerhof, David Njoo, and Henk E. Menke

Historical Background

Epidemiology Alezzandrini syndrome is a very rare disorder. Only a few cases have been reported so far (Alezzandrini, 1964; Casala and Alezzandrini, 1959; Cremona et al., 1961; Hoffman and Dudley, 1992).

Routine Histology, Histochemistry, and/or Immunochemistry

The syndrome has been reported in adolescents and young adults and may occur in both sexes.

In Alezzandrini patients histopathologic examination of the depigmented skin showed normal to atrophic epidermis with an absence of melanocytes in the basal layers, as in vitiligo. The dermis was normal, although in one patient proliferative changes were observed in the connective tissue and in the basal membrane of blood vessels.

Clinical Description

Laboratory Findings and Investigations

In three cases described by Alezzandrini (1964), visual changes

Radiological examination of the cranium and the thorax

General Clinical Findings — Age, Sexual Predilections, Racial Distribution

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prodromal stage, which does not clearly occur in Alezzandrini syndrome. However, some authors believe Alezzandrini syndrome is a segmental or an unilateral variant of VKHS (Shah et al., 1993). The Waardenburg syndrome is also characterized by poliosis, hypopigmented macules, and hypacousia. A typical symptom is the heterochromia irides. Moreover, this syndrome has a genetic inheritance.

Pathogenesis The etiology is unknown. Alezzandrini (1964) postulated that the cause lies in a hypophyso-hypothalamo-retinal disturbance, resulting in a dysregulation of the endocrine, nervous, and optic systems.

Animal Models There are no animal models.

Treatment There is no treatment for Alezzandrini syndrome. The management of vitiligo is discussed in Chapter 30.

Prognosis Alezzandrini syndrome shows a stable course. Spontaneous repigmentation has not been reported.

References

Fig. 39.1. Unilateral facial vitiligo in a patient with Alezzandrini’s syndrome (taken from Alezzandrini, A. A. Manifestations unilaterales de dégénérescence tapetoretinienne de vitiligo, de poliose, des cheveux blancs et d’hypoacousie. Ophthalomologica 147: 409–419. With kind permission of S. A. Karger, Basel, Switzerland; copyright 1964).

showed no pathologic changes. Additional laboratory investigations were not mentioned in the original article by Alezzandrini (1964).

Alezzandrini, A. A. Manifestations unilaterales de dégénérescence tapetoretinienne de vitiligo, de poliose, des cheveux blancs et d’hypoacousie. Ophthalmologica 147:409–419, 1964. Casala, A. M., and A. A. Alezzandrini. Vitiligo y poliosis unilateral con retinitis pigmentaria y hypoacusia. Arch. Argent. Dermatol. 9:449–456, 1959. Cremona, A. C., A. A. Alezzandrini, and A. M. Casala. Vitiligo, poliosis y degeneracion macular unilateral. Arch. Oftalmol. B. Aires 36:102–106, 1961. Hoffman, M. D., and C. Dudley. Suspected Alezzandrini’s syndrome in a diabetic patient with unilateral retinal detachment and ipsilateral vitiligo and poliosis. J. Am. Acad. Dermatol. 26(3 Pt 2): 496–497, 1992. Sheth, A. P., N. Supapannachart, and J. J. Nordlund. Acquired hypomelanotic disorders. In: Pigmentation and Pigmentary Disorders, N. Levine (ed.). Boca Raton, FL: CRC Press, 1993, pp. 337–361.

Criteria for Diagnosis The syndrome includes the combination of unilateral tapetoretinal degeneration with the ipsilateral development, after an interval of months or years, of facial vitiligo and poliosis, inconsistently complicated by unilateral or bilateral perceptual deafness.

Idiopathic Guttate Hypomelanosis Wiete Westerhof, David Njoo, and Henk E. Menke

Historical Background and Synonyms Differential Diagnosis Alezzandrini syndrome has to be distinguished from VKHS and the Waardenburg syndrome (see Chapter 29). The cutaneous and ocular manifestations occur bilaterally in VKHS but unilaterally in Alezzandrini syndrome. Furthermore in VKHS ocular changes also include a panuveitis and retinal detachment, features which are uncommon in Alezzandrini syndrome. VKHS is characterized by a meningo-encephalitic 726

In 1951 Costa first described the disorder under the name “leukopathie symétrique progressive des extremities.” However, he gave credit to Matsumoto, who previously referred to this condition as “leukopathia guttata et reticularis symmetrica.” Arguelles-Casals in 1954 published several cases as “leukodermia lenticular disseminada.” Subsequently, the same manifestations were reported as “macular hypopigmentation of the legs in women” by McDaniel and Richfield (1965).

MISCELLANEOUS HYPOMELANOSES: DEPIGMENTATION

Cummings and Cottel in 1966 finally labeled the leukoderma “idiopathic guttate hypomelanosis” (IGH).

women, 69% in the same age group had lesions. They also found members of the same kinship to be especially prone to acquiring the disorder.

Epidemiology IGH appears to be a very common, benign dermatosis. In a survey of 452 consecutive dermatologic patients (mostly black patients) at Charity Hospital Louisiana, the incidence was found to be 47% (Cummings and Cottel, 1966). Among another 500 dermatologic patients, 68% were found to have the disease (Whitehead, 1966). In a publication by Hamada and Saito (1966) 1595 patients with IGH were reported with a prevalence of about 6% of individuals in the second decade and 70% in the fifth decade.

General Clinical Findings — Age, Sexual Predilections, Racial Distribution IGH has been reported as both more and less common among black people compared with white (Arguelles-Casals, 1969; Cummings, 1966). In a survey by Cummings and Cottel (1966) the incidence was 58% among 123 black males, whereas it was only 20% in 107 white males. The incidence was nearly similar for black and white women, 54% of 117 black and 53% of 105 white patients, respectively. Whitehead et al. (1966), on the other hand, reported an incidence of 74% among 322 Anglo-Saxons, 62% among 81 Northern Europeans, 69% among 36 Southern Europeans, and 30% among 30 blacks and concluded that the skin disorder is most common among Anglo-Saxons and Europeans. That IGH was not found among the six Asians examined does not exclude its occurrence in Eastern or other Far Eastern Asians. Males and females are probably equally affected, as reported by Arguelles-Casals and Gonzales (1969). Some other publications, however, reported a higher prevalence in women than in men. All patients described by McDaniel and Richfield (1965) were females. Cummings and Cottel (1966) found an incidence of 53% in women and 20% in men among white patients, but a quite similar incidence among black patients (54% in women and 58% in men). Among 339 patients, Whitehead et al. (1966) found an incidence of 44.2% in males and 55.8% in females. It is not likely that IGH is more common in females; the apparent increase reported probably results from the increased cosmetic disfigurement perceived by women compared with men (Ortonne et al., 2003). IGH is regarded a disorder of adulthood and senescence and is probably more prevalent with increasing age (ArguellesCasals, 1969). An onset as young as 5 years, however, has also been reported (Whitehead, 1966). McDaniel and Richfield (1965) found the age of patients varying from 14 to 63 years and the peak incidence in the third and fourth decade. Whitehead et al. (1966) encountered only two cases under the age of 10 and found the disease usually manifesting after age 30. The data of Cummings and Cottel (1966) support an increased incidence of the condition with age. Epidemiologic studies performed by Falabella et al. (1987) revealed similar findings; among 400 consecutive patients, 79.4% of the affected male subjects were older than 40 years; among

Clinical Description The characteristic feature of IGH is multiple (several to 10, sometimes more than 50) small, discrete, smooth, depigmented macules of porcelain white color (Figs 39.2 and 39.3). Sometimes small black dots are irregularly distributed in the white macules. The shape of the macules may be round (guttate) or angular. They vary from 0.2 mm to 6 mm, with occasional larger lesions. Once present, these lesions do not change in size. The number of lesions seems to increase with age. There are very sharply defined borders. No peripheral hyperpigmentation can be observed. The macules are not confluent. Typically, primary and secondary skin lines are absent; the surface of the lesions is smooth, not infiltrated and without scarring or atrophy. Sometimes IGH lesions start with a hyperkeratotic scale (Falabella et al., 1987). When this detaches a white guttate lesion becomes visible (Westerhof, 1995, unpublished data). The lesions are most commonly seen on sun-exposed areas of the skin, particularly on the anterior surface of the lower extremities and the lower abdomen. Remarkably, Whitehead et al. (1966) found that male patients had an increased percentage of lesions on the arms. The facial skin can also be affected. Whitehead et al. (1966) found involvement of preauricular skin in some patients and McDaniel and Richfield (1965) observed a depigmented macule around the mouth of one patient. Remarkably, sun-protected skin areas may also be affected. The lesions are totally asymptomatic. Uninvolved skin is normal and patients with this disorder are usually in good health. The cosmetic disfigurement is usually mild.

Associated Disorders There are no associated disorders.

Routine Histology, Histochemistry, and/or Immunochemistry Histopathologic examination shows normal to sometimes atrophic epidermis. An increased thickness of the keratin layer of depigmented macules has also been reported (Ortonne and Perrot, 1980). A decreased dopa reaction in the hypopigmented macules is a consistent finding (Ortonne and Perrot, 1980; Wilson, 1982). There is a variable reduction to complete absence of melanocytes. Ultrastructural studies have shown that two types of melanocyte may be present in the hypomelanotic skin; some cells appear to have normal melanogenic activity and contain numerous melanosomes while the other cells contain only immature melanosomes. The melanosome content of the keratinocyte is generally decreased. Although the number of Langerhans cells has been reported to be normal, a reduction in the number of Birbeck granules has been observed. There are no known changes in the basement membrane constituents (Sheth et al., 1993). 727

CHAPTER 39

Differential Diagnosis IGH must be differentiated from other hypopigmentary disorders such as vitiligo (see Chapter 30), lichen sclerosus et atrophicus (discussed in this chapter), atrophie blanche, scleroderma, leukodermic guttate parapsoriasis (see Chapter 37), tinea versicolor (see Chapter 36), hypopigmentation in sarcoidosis (see Chapter 37), tuberous sclerosis (see Chapter 32), achromic plane warts, pityriasis alba, leprosy, syphilis, chemical depigmentation (see Chapter 35), and postinflammatory hypopigmentation.

Pathogenesis 39.2

39.3 Figs. 39.2 and 39.3. Typical porcelain white macules in idiopathic guttate hypomelanosis (see also Plate 39.1, pp. 494–495).

Occasionally, IGH may be preceded by a hyperkeratotic scale. Electron microscopy of such a lesion shows a slightly increased number of melanocytes which are bulging into the dermis, containing large melanosomes which are singly transferred to the keratinocytes. As a result melanosomes are found even in the stratum corneum, as in the case of dark skin (Westerhof, 1995, unpublished data). Furthermore, there is no dermal abnormality, pigmentary incontinence, or inflammation. When thickening and fibrosis of the papillary dermis have been observed in lesions of IGH, similar changes also have been found in normally pigmented skin (Cummings and Cottel, 1966).

Laboratory Findings and Investigations There are no abnormal laboratory findings known in IGH.

The pathogenesis has not been established. Trauma has been suggested. McDaniel and Richfield (1965) first believed that the disease was induced by shaving with a safety razor which caused localized impairment in the functional ability of the melanocytes. However, many patients reported using only depilatories or electrical shavers. Moreover, this theory could not explain the localization of the disease on the arms, or trunk, areas not commonly shaved, or its occurrence in males. Posttraumatic hypopigmentation has also been considered by Cummings and Cottel (1966), but the ultrastructural features of scars described by Wilson et al. (1982) (such as a flattened dermoepidermal junction and dense, thin, parallel collagen bundles) have not been observed, making this possibility unlikely. Wilson et al. (1982) also found gastric parietal antibodies in three of nine individuals, indicating a possible phenomenon related to autoimmunity. Savall et al. (1980) and Falabella et al. (1987) suggested that a genetic factor should also be considered in the pathogenesis of IGH in view of the family histories of the patients. In a group of 22 Colombian renal transplant patients, HLA-DQ3 was found to be significantly associated with IGH. HLA-DR8 was found in patients without IGH which could play a role as “protective factor” preventing subjects from developing IGH (Arrunategui et al., 2002). Hamada and Saito (1966) who named the skin manifestations “senile depigmented spots” postulated that a senile degeneration of the epidermal-melanin unit is an important etiologic factor. Because there is an increased incidence with age and the lesions mostly occur on sun-exposed areas of the skin, chronic exposure to ultraviolet radiation has been implicated as a major etiologic factor (Cummings and Cottel, 1966; Whitehead, 1966). Most patients indeed appeared to be enthusiastic sunbathers. Furthermore, it must be noted that in spite of the face and neck being among the most exposed areas and where actinic damage is frequently found and sometimes severe, usually no lesions develop (Falabella et al., 1987). Thus, other significant factors are probably involved.

Criteria for Diagnosis The diagnosis can usually be easily established on the basis of typical morphology, distribution, and clinical course of the lesions. The small size, porcelain-white surface, and sharp margins (Figs 39.2 and 39.3) are usually diagnostic (Ortonne et al., 2003). 728

Animal Models There are no animal models.

Treatment The treatment of IGH is a challenging one. Phototherapy is

MISCELLANEOUS HYPOMELANOSES: DEPIGMENTATION

not effective (McDaniel and Richfield, 1965). Falabella et al. (1987) observed that minigrafts of clinically unaffected skin did not result in repigmentation, as observed in other stable leukodermas. On the other hand, careful destruction with trichloracetic acid (25%) or intralesional injection with triamcinolone acetonide (1 to 5 mg/mL; 0.1 mL) produced some improvement. Ploysangam et al. (1990) treated some IGH lesions with liquid nitrogen and observed repigmentation occurring in six to eight weeks. The repigmented lesions contained fewer dopa-positive melanocytes than the normal skin of the control but significantly more than in the untreated lesions. Successful regimentation of achromic spots up to 5 mm diameter has also been reported by Hexsel in 1999. The author used a standard dermabrader with small diamond fraises to induced localized superficial dermabrasion. Advantages of this method are that it is fast, simple, low cost, and safe. Furthermore, it does not require anaesthesia (Hexsel, 1999). Sunbathing should be discouraged and use of topical sunscreens encouraged, since sunlight is an important precipitating factor (Ortonne et al., 2003).

Prognosis IGH lesions are unstable progressive leukodermas. The lesions usually do not increase in size, but their number increases with age. No spontaneous repigmentation has been observed.

References Arguelles-Casals D. Leucodermia lenticular disseminada en la raza negra. Bol. Soc. Cubana Dermatol. Sifilograf. 11: 18–20, 1954. Arguelles-Casals D., and D. Gonzales. La leucodermie lenticulaire disseminée. Ann. Dermatol. Syphiligr. (Paris) 96: 283–286, 1969. Arrunategui A., R. A. Trujillo, M. P. Marulanda, F. Sandoval, A. Wagner, A. Alzate, and R. Falabella. HLA-DQ3 is associated with idiopathic guttate hypomelanosis, whereas HLA-DR8 is not, in a group of renal transplant patients. Int. J Dermatol. 41: 744–747, 2002. Costa O. G. Leucopathie symétrique progressive des extrémités. Ann. Dermatol. 78: 452–454, 1951. Cummings K. I., and W. I. Cottel. Idiopathic guttate hypomelanosis. Arch. Dermatol. 93: 184–186, 1966. Falabella R., C. Escobar, N. Giraldo, P. Rovetto, J. Gil, M. I. Barona, F. Acosta, and A. Alzate. On the pathogenesis of idiopathic guttate hypomelanosis. J. Am. Acad. Dermatol. 16: 35–44, 1987. Hamada T., and T. Saito. Senile depigmented spots (idiopathic guttate hypomelanosis). Arch. Dermatol. 95: 279–281, 1966. Hexsel D. M. Treatment of idiopathic guttate hypomelanosis by localized superficial dermabrasion. Dermatol. Surg. 25: 917–918, 1999. McDaniel W. E., and D. F. Richfield. Macular hypopigmentation in legs of women. Dermatol. Dig. 4: 59–66, 1965. Ortonne J. P., P. Bahadoran, T. B. Fitzpatrick, D. B. Mosher, and Y. Hori. Hypomelanoses and hypermelanoses, disorders of melanocytes. In: Fitzpatrick’s Dermatology in General Medicine, 6th ed. I. M. Freedberg, A. Z. Eisen, K. Wolff, K. F. Austen, L. A. Goldschmidt, and S. I. Katz (eds). New York: McGrawHill, 2003, pp. 860–861. Ortonne J. P., and H. Perrot. Idiopathic guttate hypomelanosis. Arch. Dermatol. 116: 664–668, 1980. Ploysangam T., S. Dee-Ananlap, and P. Suvanprakorn. Treatment of idiopathic guttate hypomelanosis with liquid nitrogen: light and

electron microscopic studies. J. Am. Acad. Dermatol. 23: 681–684, 1990. Savall R., C. Ferrandiz, I. Ferrer, and J. Peyri. Idiopathic guttate hypomelanosis. Br. J. Dermatol. 103: 635–642, 1980. Sheth A. P., N. Supapannachart, and J. J. Nordlund. Acquired hypomelanotic disorders. In: Pigmentation and Pigmentary Disorders, N. Levine (ed.). Boca Raton: CRC Press, 1993, pp. 337– 365. Whitehead W. J., D. G. Moyer, and D. E. Van der Ploeg. Idiopathic guttate hypomelanosis. Arch. Dermatol. 94: 279–281, 1966. Wilson P. D., R. M. Lavker, and A. M. Kligman. On the nature of idiopathic guttate hypomelanosis. Acta Derm. Venereol. (Stockh). 62: 301–306, 1982.

Leukoderma Punctata Wiete Westerhof, David Njoo, and Henk E. Menke

Historical Background In 1988, Falabella et al. presented 13 patients with vitiligo who developed numerous punctate hypopigmented and achromic spots, during and after treatment with PUVASOL. Most of the patients were girls receiving relatively low systemic methoxsalen doses followed by solar exposure, over a period varying from 5 months to 36 months. One patient even received intermittent therapy for several months for 10 years. The spots were descriptively given the name “leukoderma punctata.”

Synonyms There are no synonyms.

Epidemiology Falabella et al. (1988) did not give any estimate regarding the incidence of leukoderma punctata in long-term PUVA-treated vitiligo patients.

General Clinical Findings — Age, Sexual Predilections, Racial Distribution The 13 patients described by Falabella et al. (1988) were aged 7–38 and most of them were girls. Remarkably, a more rapid course and widespread leukodermic elements were observed in children.

Clinical Description Falabella et al. (1988) described the pigmentary abnormalities as follows. Before initiation of the PUVASOL therapy (see Chapter 60), no punctiform hypopigmented lesions were present. After several months of treatment vitiligo lesions in all patients underwent variable degrees of repigmentation, ranging from 0% to 100% of the previously involved areas. During treatment (nine patients), or a few months after cessation of the PUVASOL therapy (four patients), all patients developed multiple punctiform, white hypopigmented and achromic spots predominantly on sun-exposed areas: on the upper and lower extremities and occasionally on the face, gluteal regions, and upper aspect of the back or chest areas (Figs 39.4 and 39.5). A few patients also showed lesions in 729

CHAPTER 39

Routine Histology, Histochemistry, and/or Immunochemistry

39.4

Fontana-Masson stain and dopa reaction of achromic lesions revealed decreased numbers but not an absence of functional melanocytes and melanin granules. Only one patient had a moderate “verruciform-like” hyperkeratosis in a leukodermic lesion. Besides a slight flattening of the rete pegs of both “normal” and achromic specimens and a mild diffuse or perivascular mononuclear dermal infiltrate in the achromic and “normal” skin of most patients, no other abnormalities were found. Ultrastructural studies showed varying degrees of cellular damage within keratinocytes as well as in melanocytes, both in normal and affected skin. The damage included intracellular edema and intracytoplasmic vacuolar degeneration of both keratinocytes and melanocytes, although the pigmentary cells were less affected. These abnormalities have also been previously observed in the borders of a vitiligo lesion (Moellmann et al., 1982). In one patient an intracytoplasmic “myelin figure” was visualized within a degenerated keratinocyte, in a section of “normal” adjacent skin. Myelin figures are believed to derive from plasma as well as organelle membranes after cellular injury caused by different noxa (Robbins et al., 1984). A control specimen taken from the sun-protected arm area of one patient revealed moderate vacuolar degeneration, whereas in another patient these changes were minimal.

Laboratory Findings and Investigations No abnormal laboratory findings are known in this disorder. 39.5

Criteria for Diagnosis

Figs. 39.4 and 39.5. Leukodermic spots, not larger than 1.5 mm, on the thigh of a patient with generalized vitiligo (taken from: Falabella R., C. E. Escobar., E. Carrascal, J. A. Arroyave. Leukoderma punctata. J. Am. Acad. Dermatol. 18: 485–494, 1988. With kind permission of Mosby Year Book, St. Louis, USA; copyright 1988).

The appearance of new punctiform hypopigmented and achromic spots in a long-term PUVA or PUVASOL treated patient in combination with characteristic histologic and ultrastructural findings may suggest the diagnosis.

some protected, non–sun-exposed regions. The lesions were usually numerous, distinct, round or oval macules of sharply defined margins, varying in diameter from 0.5 mm to 1.5 mm. Remarkably, no achromic and hypopigmented lesions were observed on the antecubital fossae, popliteal regions, elbows, or knees. Five patients also presented a few very small punctiform and subtle spots on the dorsa of the hands and feet. Two patients reported that the leukodermic spots improved and decreased in number during the first and second years following discontinuation of their treatment, respectively. The other patients, however, experienced no changes in their clinical picture. None of the treated patients developed signs of phototoxicity at any time on the normal skin during PUVASOL therapy.

The lesions of leukoderma punctata can be clinically and histopathologically distinguished from other leukodermic conditions, such as idiopathic guttate hypomelanosis, vitiligo and lichen nitidus. Idiopathic guttate hypomelanosis occurs predominantly with advancing age, and when children become affected, only a few lesions are observed. Furthermore, larger lesions (more than 1.5 mm) are found in idiopathic guttate hypomelanosis. Also in this condition no lesions are found on the face. Moreover, in idiopathic guttate hypomelanosis, the ultrastructural vacuolar degeneration of keratinocytes and melanocytes is not visualized. Like leukoderma punctata, however, the dopa reaction within the leukodermic defect shows a reduction and not a total absence of melanocytes. Typical vitiligo lesions are achromic rather than hypopigmented. Punctate spots have not been reported before in vitiligo patients. Vitiligo macules are usually larger (between 0.5 cm and 2.0 cm in diameter) and have less defined borders. Furthermore, histologic examination of a typical vitiligo lesion shows a total absence of melanocytes. Nevertheless, an

Associated Disorders Falabella et al. (1988) suggest that there is probably a relation between leukoderma punctata, idiopathic guttate hypomelanosis (see above), and vitiligo (see Chapter 30). 730

Differential Diagnosis

MISCELLANEOUS HYPOMELANOSES: DEPIGMENTATION

interesting observation was that, as in leukoderma punctata, degenerative cellular changes were visualized by electron microscopy. The only condition in which the clinical picture resembles that of leukoderma punctata is lichen nitidus. This, however, is a papular dermatosis with well-defined histopathologic findings, revealing subepidermal patchy mononuclear infiltrates.

Pathogenesis Falabella et al. (1988) postulated that leukoderma punctata probably is a complication of long-term exposure to psoralen photochemotherapy. The sequence of PUVASOL therapy followed by the development of these punctate hypopigmented spots have made this possibility very likely. Although PUVA and PUVASOL have different ultraviolet photobiologic action spectra on the skin, the finding of hypopigmented lesions, described by several investigators, during PUVA treatment (Gschnait et al., 1980; Kanerva et al., 1981, 1983; TodesTaylor et al., 1983) indicates cellular damage to keratinocytes and melanocytes, which also may occur with PUVASOL therapy (Falabella et al., 1988). Two patients experienced spontaneous partial repigmentation, so melanocyte photodamage may not be the exclusive etiologic event acting in the achromic defect. Other factors in the affected skin may also play a role in this pigmentary alteration and may be responsible for the lack of repigmentation as also seems to occur in IGH (Falabella et al., 1988).

Animal Models The harmful effects of photochemotherapy to human skin have been shown in several experimental models. Nordlund et al. (1981) observed in mice that pigment cells are susceptible to ultraviolet injury and can be killed by large doses of PUVA. In experiments performed by Pathak et al. (1986) PUVA was found to produce deoxyribonucleic acid (DNA)psoralen photoadducts in mammalian skin, indicating nuclear damage of both keratinocytes and melanocytes, although the latter are less vulnerable to photodamage.

Treatment Because children seem to be more susceptible to develop leukoderma punctata, Falabella et al. (1988) do not recommend psoralen photochemotherapy before adolescence as a treatment for vitiligo, unless widespread lesions cause major cosmetic problems. They suggest that small areas may be better treated by local therapy such as topical methoxsalen, topical steroids and 5-fluorouracil. Segmental vitiligo which is not responding to psoralen photochemotherapy and 5fluorouracil may be successfully repigmented by autologous minigrafting. When indicated, this procedure may be postponed until adulthood.

Prognosis Two of the patients described by Falabella et al. (1988) experienced a spontaneous reduction of the leukodermic defect, whereas the remaining patients showed a rather stable

clinical course. In some cases, however, the lesions had also gradually increased numerically, even after PUVASOL therapy was stopped.

References Falabella, R., C. E. Escobar, E. Carrascal, and J. A. Arroyave. Leukoderma punctata. J. Am. Acad. Dermatol. 18:485–494, 1988. Gschnait, F., K. Wolff, H. Honigsmann, G. Stingl, W. Brenner, E. Jaschke, and K. Konrad. Long-term photochemotherapy: histopathological and immunofluorescence observations in 243 patients. Br. J. Dermatol. 103:11–22, 1980. Kanerva, L., K. M. Niemi, and A. Lassus. Hyperpigmentation and hypopigmentation of the skin after long-term PUVA therapy. J. Cutan. Pathol. 8:199–213, 1981. Kanerva, L., J. Lauharanta, K. M. Niemi, T. Juvakoski, and A. Lassus. Persistent ashen gray maculae and freckles induced by long-term PUVA treatment. Dermatologica 166:281–286, 1983. Moellmann, G., S. Klein-Angerer, D. Scollay, J. J. Nordlund, and A. B. Lerner. Extracellular granular material and degeneration of keratinocytes in the normally pigmented epidermis of patients with vitiligo. J. Invest. Dermatol. 79:321–330, 1982. Nordlund, J. J., A. E. Ackles, and F. F. Traynor. The proliferative and toxic effects of ultraviolet light and inflammation on epidermal pigment cells. J. Invest. Dermatol. 77:361–368, 1981. Pathak, M. A., Z. Zarebska, and M. C. Mihm. Detection of DNApsoralen photoadducts in mammalian skin. J. Invest. Dermatol. 86:308, 1986. Robbins, S. L., R. S. Cotran, and V. Kumar. Cellular injury and adaptation. In: Pathologic Basis of Disease, 3rd ed., S. L. Robbins, R. S. Cotran, and V. Kumar (eds). Philadelphia: WB Saunders, 1984, pp. 26–27. Todes-Taylor, N., E. A. Abel, and A. J. Cox. The occurrence of vitiligo after psoralens and ultraviolet A therapy. J. Am. Acad. Dermatol. 9:526–532, 1983.

Lichen Sclerosus et Atrophicus Philippe Bahadoran Lichen sclerosus, usually reported in the literature as lichen sclerosus et atrophicus, is an inflammatory disease of unknown origin. It is markedly commoner in women and has a predilection for the genital area, but it may also occur in men and in all regions of the body (Fig. 39.6). Lichen sclerosus typically begins as white polygonal papules that coalesce into plaques. Lesions of the vulva (Fig. 39.7) and the perineum are often very pruritic and consequently lichenified as a result of itching (see Chapter 56). At the late stage, genital lichen sclerosus in women leads to anatomical changes such as obliteration of the labia minora and periclitoral structures (see Chapter 56). The histologic features of lichen sclerosus are characteristic and remarkably consistent whatever the lesion location. A compact hyperkeratosis contrasts with a thinned underlying epidermis. The papillary dermis is edematous and homogenized and later sclerotic and hyalinized, and there is a lower dermal lichenoid mononuclear infiltrate. Ultrastructural studies have shown that papillary dermis changes consisted both of elastin degeneration and collagen changes (Meffert et al., 1995). Unfortunately, there are no details about histologic 731

CHAPTER 39

Fig. 39.6. Depigmented lichen sclerosus et atrophicus of the trunk. (Courtesy of Dr. Gharby) (see also Plate 39.2, pp. 494–495).

Recently, in a study aimed at determining what the mechanism of hypopigmentation is in lichen sclerosus, Carlson et al. (2002a) examined samples from cases of lichen sclerosus for alterations in melanin content and melanocyte number and compared these findings with those in controls of normal skin, acute scars, vitiligo, and lichen planus. Based on their analysis, they proposed that several mechanisms operate in the production of leukoderma in lichen sclerosus: (1) decreased melanin production; (2) block in transfer of melanosomes to keratinocytes; and (3) partial melanocyte loss. They also found regional areas of melanocytic hyperplasia which rarely result in clinically pigmented melanocytic proliferations arising in lesions of lichen sclerosus. The clinicopathologic features of these melanocytic proliferations associated with lichen sclerosus were assessed in two recent studies (Carlson et al., 2002b; El Shabrawi-Caelen et al., 2004). An “activated” melanocytic phenotype is seen in lichen sclerosus melanocytic proliferations, implicating a stromal-induced change. As a result, genital lentigines and melanocytic nevi with associated lichen sclerosus may show features that mimic malignant melanoma. In the absence of a well-defined cause for lichen sclerosus, various hypotheses have been proposed such as infection especially with Borrelia burgdorferi, decreased testosterone metabolism in genital skin, local trauma, and autoimmunity (Meffert et al., 1995).

References Carlson J. A., R. Grabowski, X. C. Mu, A. Del Rosario, J. Malfetano, and A. Slominski. Possible mechanisms of hypopigmentation in lichen sclerosus. Am. J. Dermatopathol. 24:97–107, 2002a. Carlson J. A., X. C. Mu, A. Slominski, K. Weismann, A. N. Crowson, J. Malfetano, V. G. Prieto, ad M. C. Mihm, Jr. Melanocytic proliferations associated with lichen sclerosus. Arch. Dermatol. 138: 77–87, 2002b. El Shabrawi-Caelen L., H. P. Soyer, H. Schaeppi, L. Cerroni, C. G. Schirren, C. Rudolph, and H. Kerl. Genital lentigines and melanocytic nevi with superimposed lichen sclerosus: a diagnostic challenge. J. Am. Acad. Dermatol. 50: 690–694, 2004. Mann, P. R., and M. A. Cowan. Ultrastructural changes in four cases of lichen sclerosus and atrophicus. Br. J. Dermatol. 89:223–231, 1973. Meffert, J. J., B. M. Davis, and R. E. Grimwood. Lichen sclerosus. J. Am. Acad. Dermatol. 39:393–416, 1995. Wang, J. B., H. Yan, H. Y. Yang, J. Y. Si, L. P. Jia, K. Li, and J. Yu. Histopathological and ultrastructural changes of lichen sclerosus and atrophicus on the vulva. Chin. Med. J. (Engl.) 104:868–871, 1991. Fig. 39.7. Early depigmented lichen sclerosus et atrophicus in a child (Courtesy of Dr. Gharby) (see also Plate 39.3, pp. 494–495).

Vagabond Leukomelanoderma Wiete Westerhof, David Njoo, and Henk E. Menke

or ultrastructural pigmentary changes in most reports of lichen sclerosus. In an ultrastructural study of four cases of lichen sclerosus, Mann and Cowan (1973) found an absence of melanocytes in active lesions and a reduced number of melanocytes with degenerative changes in resolving lesions. In their report of 82 cases of vulvar lichen sclerosus, Wang et al. (1991) mentioned a decrease or a total disappearance of melanotic granules in the lesions. 732

Historical Background Vagabond leukomelanoderma is a skin disorder which classically occurs in those who are unkempt in personal habits, have pediculosis corporis, and give a history of infrequent bathing or changing of clothes, dietary deficiency, and chronic alcoholism (Ortonne et al., 1983). The description matches with that of a typical “beggar” or “vagabond.” The disorder was

MISCELLANEOUS HYPOMELANOSES: DEPIGMENTATION

first described in the late nineteenth century (Bondet, 1892; Greenhow, 1876). In 1902, Marie and Guillain discussed the similarities in the skin manifestations of vagabond disease and Addison disease. Pautrier summarized the characteristics of the disease in 1925 and 1927 (Pautrier and Levy, 1925; Pautrier et al., 1927). In 1965, Thiers et al. reported two more cases and investigated the role of adrenal dysfunction in the pathogenesis of the disease.

undergo degenerative changes with autophagic vacuoles (Table 39.1). The keratinocytes contain fewer melanosomes than normal. The melanosomes are normal in size and shape. There are no signs of defective transfer of melanosomes from melanocytes to keratinocytes. The number of Langerhans cells

Synonyms Both terms “vagabond leukomelanoderma” and “vagabond disease” are used to describe the skin disorder.

Epidemiology This is probably a rare disorder, more common among people living in poor socioeconomic and hygienic conditions.

General Clinical Findings — Age, Sexual Predilections, Racial Distribution Vagabond disease occurs in older, undernourished people with poor personal hygiene and heavy infestation with lice.

Clinical Description Typical lesions include macules and patches of diffuse lightbrown hyperpigmentation especially seen around the wrists and groin, between the thighs, in the axillae, over the back of the neck and particularly the upper back and chest (Fig. 39.8). The face and the extremities usually appear less pigmented than the trunk (Jeghers, 1944). Diffuse hyperpigmentation of the oral mucosa has also been seen in these patients (Greenhow, 1876; Lanzerberg, 1932). Co-existing areas of depigmentation are probably postinflammatory and associated with scratching of pediculous infestations (Habermann, 1933; Pautrier et al., 1927).

Associated Disorders Owing to the addisonianlike pattern of the pigmentation, vagabond disease is said to be related to Addison disease.

Routine Histology, Histochemistry, and/or Immunochemistry Grosshans (1972), in ultrastructural studies, found a decreased number of dopa-positive melanocytes in the depigmented macules. The melanocytes do not disappear but

Fig. 39.8. Clinical picture of a patient with vagabond leukomelanoderma (taken from: Grosshans E. La leucomélanodermie des vagabonds. Étude anatomopathologique et ultrastructurale. Ann. Dermatol. Syphiligr. (Paris) 99: 141–166, 1972. With kind permission of Masson, Paris, France; copyright 1972).

Table 39.1. Vagabond leukomelanoderma: clinical description, histopathology and other investigations. Clinical description

Histopathology of the hypo/depigmented skin

Other investigations

Diffuse light brown hyperpigmented and hypopigmented macules on protected non–sun-exposed areas mostly on the upper back; diffuse hyperpigmentation of oral mucosa

EM: Melanocytes: decreased numbers, degenerative changes with autophagic vacuoles; decreased melanosome content in keratinocytes, Langerhans cells, and Schwann cells containing melanin

No abnormalities

EM, electron microscopy.

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CHAPTER 39

is markedly reduced and some of those remaining contain melanosomes. There are abundant pigment deposits in the upper dermis and few Schwann cells contain melanin granules. Thus, depigmentation occurring in vagabond disease probably results from a combination of pigment incontinence with phagocytosis of the melanin granules, loss of melanocytes, and loss of melanosomes in autophagic vacuoles within melanocytes and phagocytosis of melanosomes by Langerhans cells, and resorption of melanosomes by Schwann cells.

Laboratory Findings and Investigations In most cases adrenal function tests are normal (Thiers et al., 1965).

Criteria for Diagnosis The finding of a characteristic addisonianlike distribution of hyperpigmented and hypopigmented lesions in a patient with poor hygiene and nutritional status, and with pediculosis corporis is suggestive of the diagnosis.

Jeghers, H. Pigmentation of the skin. N. Engl. J. Med. 231:88–100, 1944. Lanzerberg, M. P. Maladie des vagabonds, avec pigmentation de la mugueuses buceale. Opposition avec un autre malade atteint de phitiriase et d’un autre type mélanodermie. Bull. Soc. Franc. Dermatol. Syphiligr. 39:293–295, 1932. Marie, P., and G. Guillain. Mélanodermie de cause incertaine (maladie d’Addison ou maladie des vagabonds). Bull. Mem. Soc. Med. Hop. Paris 19:198–203, 1902. Ortonne, J.-P., D. B. Mosher, and T. B. Fitzpatrick. Topics in Dermatology: Vitiligo and Other Hypomelanoses of Hair and Skin. New York: Plenum Medical Book Company, 1983, pp. 613–672. Pautrier, L. M., and G. Levy. Maladie des vagabonds et mélanodermie généralisée. Bull. Soc. Franc. Dermatol. Syphiligr. 32:172–175, 1925. Pautrier, L. M., G. Levy, and Stoimovici. Maladie des vagabonds et mélanodermie généralisée (contribution l’étude des mélanodermies). Bull. Soc. Franc. Dermatol. Syphiligr. 34:249–253, 1927. Thiers, H., D. Colomb, and B. Durand. Deux cas de mélanodermie des vagabonds: Étude des épreuves de functionnement surrénal. Bull. Soc. Franc. Dermatol. Syphiligr. 72:82–83, 1965.

Vogt–Koyanagi–Harada Syndrome Differential Diagnosis

Wiete Westerhof, David Njoo, and Henk E. Menke

The disorder must be distinguished from Addison disease. Additional laboratory tests can be helpful.

Historical Background

Pathogenesis The pathogenesis is unclear. Vagabond disease has variously been attributed to physical factors, to nutritional deficiency (Jeghers, 1944), or to addisonianlike changes (Greenhow, 1876; Marie and Guillain, 1902; Pautrier et al., 1927). However, adrenal function tests reveal normal findings in most patients (Thiers et al., 1965). The leukomelanoderma is most probably postinflammatory, related to multiple ectoparasitic infections.

Animal Models There are no animal models.

Treatment The pigmentary abnormalities usually improve with bathing, disinfection, clean clothes, good diet and vitamin supplements.

Prognosis The prognosis is good.

References Becker, S. W., and M. Obermayer. Modern Dermatology and Syphilology. Philadelphia: Lippincott, 1940, pp. 3–7. Bondet, M. Mélanodermic parasitaire. Lyon Med. 70: 262–264, 1892. Greenhow, E. H. A case of vagabond’s discoloration simulating the bronzed skin of Addison’s disease. Trans. Clin. Soc. Lond. 9:44–47, 1876. Grosshans, E. La leucomélanodermie des vagabonds: Étude anatomopathologique et ultrastructurale. Ann. Dermatol. Syphiligr. (Paris) 99:141–166, 1972. Habermann, R. Paratypische pigmenatanomalien. In: Toxicodermien. Handbuch der Haut-und Geschlechtskrankheiten, IV 2, E. Guttmann (ed.). Berlin: Springer-Verlag, 1933, pp. 854–856.

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In 1906, Vogt presented a patient with idiopathic atraumatic uveitis, poliosis and alopecia, a complex of symptoms which would later be known as “Vogt–Koyanagi–Harada syndrome” (VKHS). However, descriptions of this syndrome had been made even earlier. The twelfth-century physician Mohammed al Ghafagi had regarded eyelash poliosis as a disease. Schenkl in 1873 reported a case of silvery-white eyelashes associated with sympathetic ophthalmia. Jacobi (1874) observed a similar patient with canities of the upper and lower eyelids one year later. Similar cases were reported by Tay, Bock, and Nettleship. They considered the occurrence of white eyelashes and sympathetic ophthalmia no mere coincidence (Bock, 1890; Nettleship, 1884; Tay, 1892). In 1892, in the course of the presentation of a case of eyelash poliosis and severe deafness, Hutchinson (1892–1893) referred to another patient with atraumatic destructive uveitis, partial hair loss, and poliosis of scalp and axillary hair. In 1910, Gilbert observed a patient who developed the concomitant onset of vitiligo and bilateral optic neuritis, which resulted in iridocyclitis. He attributed this to ocular herpes; 20 years later he implicated endocrine factors. Komoto (1912) reported that seven years earlier at an ophthalmologic meeting he had presented a case of sympathetic ophthalmia with circumscribed vitiligo of the forehead. Erdmann (1911) described a case consistent with VKHS characterized by vitiligo, poliosis, and chronic atraumatic choroidal inflammation. He suggested this was a type of heterochromic inflammation and attributed the vitiligo to a similar process. In 1912, Peters encountered six patients with sympathetic ophthalmia associated with deafness. Cramer, in 1913, presented a patient with VKHS associated with extraction of a luxated lens. In 1926, Harada described five cases of bilateral posterior uveitis and retinal detachment. Severe

MISCELLANEOUS HYPOMELANOSES: DEPIGMENTATION

headaches and cerebrospinal fluid (CSF) pleocytosis were also frequently observed. A similar disorder was also reported by Tagami in 1931. In 1929, Koyanagi presented 16 cases of a syndrome characterized by headache, fever, dysacousia, vitiligo, poliosis, alopecia, and bilateral anterior uveitis with occasional exudative retinal detachment. Parker in 1931 published the first review in the English literature. He suggested the syndrome was associated with both idiopathic uveitis and sympathetic ophthalmia.

Synonyms In 1934, Ogawa used the names of Vogt and Koyanagi to refer to the syndrome of “idiopathic uveitis with poliosis and leukoderma.” It was Babel, however, in 1939 who first labeled the syndrome the Vogt–Koyanagi syndrome. Other terms have also been used to describe the syndrome such as uveoencephalitis (Cowper, 1951), oculocutaneous syndrome (Yuge, 1957), idiopathic neuraxitis (Kissel, 1963), and uveomeningoencephalitis (Riehl, 1966).

Epidemiology The worldwide incidence of uveitis has been estimated to be approximately 15 cases per 100 000 population (Friedman, 1982). The incidence of VKHS has been reported to be between 4% and 7% of patients with uveitis. The incidence of dermatologic features in this syndrome varies remarkably. Rosen (1945) encountered poliosis in 90%, alopecia in 73%, and vitiligo in 63%. In Japan, poliosis or alopecia was found in 60% and vitiligo in 25% of the patients (Beniz et al., 1991). In the USA, 2–13% of the cases were reported to have developed poliosis, vitiligo, and alopecia (Ohno et al., 1977). The most recent nationwide survey on the occurrence of VKHS in Japan revealed an estimated annual prevalence of 15.5 per million and an estimated incidence of 6.5 per million. Approximately 1800 patients were treated for this disease annually and there were about 800 new cases each year (Murakami et al., 1994).

General Clinical Findings — Age, Sexual Predilections, Racial Distribution VKHS mostly occurs in dark-skinned people (Asians, black people, Hispanics, and American Indians) or white people with heavy pigmentation (Hammer et al., 1993; Perry and Font, 1977; Rubsamen et al., 1991). Males and females are probably equally affected. The syndrome occurs most frequently among young people in the second or third decade, or between the ages of 30 and 40 (Carrasquillo, 1942). Age of onset varying from 10 to 57 years has also been reported (Murakami et al., 1994; Perry and Font, 1977). Furthermore, VKHS also appears to occur among children (Weber and Kazdan, 1977). Social and hereditary factors do not seem to be important (Carrasquillo, 1942; Parker, 1940). Only one familial case (Flynn, 1952) and only one case of consanguinity (Zentmayer, 1942) have been reported.

Clinical Description VKHS appears in three stages. The first stage is characterized

by meningoencephalitic signs and symptoms. This prodromal phase may include malaise, fever, severe headache, and nausea and vomiting. Papilledema and nystagmus may be present. Nuchal rigidity may be the first signs of meningeal irritation. In the acute phase, the patient may be mildly confused, delirious or frankly psychotic. EEG shows signs of diffuse encephalitis (Cowper, 1951). Other neurologic features reported are hemiparesis, paraplegia, loss of sphincter control, generalized muscle weakness, slight aphasia, syncope, and unilateral convergent squint. Abnormalities in the cerebrospinal fluid that are present at this phase include an increase of both the protein concentration and the number of white blood cells. The histopathology reveals an adhesive arachnoiditis (Leyva, 1959). The degree of central nervous system (CNS) involvement is highly variable. Permanent defects may develop, particularly long tract signs, paresis, expressive aphasias, and personality disorders (Alajouanine, 1953; Carrasquillo, 1942; Reed, 1958). The second stage may appear shortly after the first and is dominated by ophthalmic and auditory involvement. It is of gradual or fulminant onset and may last 10 years or more. Beginning of uveitis is characterized by rapidly decreasing visual acuity, photophobia and eye irritation (see Figs 30.21 and 30.22 in Chapter 30). Blurring, solely, may be the initial symptom (Perry and Font, 1977). Beside progression to blindness, this stage is feared for other ophthalmic complications such as retinal detachment, glaucoma, optic neuritis, and retinal edema. Although initially only one eye may be inflamed, the contralateral eye will become affected in most cases (Beniz et al., 1991). The auditory manifestations of the second stage of the VKHS are marked by a transitional, bilateral dysacousia, and occur in 50–89% of the patients (Carrasquillo, 1942; Perry and Font, 1977). They usually disappear over weeks to months. Noise hyperesthesia, tinnitus, and partial deafness are common features after the onset of the disease. The eighth nerve defect, which is of variable degree, is in the high pitch range and may not be noticed by the patient. Spontaneous recovery may occur. The third stage, the convalescent stage, occurs as the uveitis subsides and is mainly dermatologic. The clinical manifestations are vitiligo, poliosis (Fig. 39.9; see also Figs 30.21 and 30.22 in Chapter 30), and alopecia which are present three to nine months after the onset of the disease. The first case of leukoderma in patients with VKHS was observed by Gilbert in 1910. Poliosis may appear abruptly a few weeks after the onset of the disease; involvement of the eyebrows, eyelashes, scalp, beard, and body hair, particularly in the sacral area, may be patchy or diffuse (Pattison, 1965). Usually, depigmentation of the eyebrows and eyelashes is accompanied by ipsilateral depigmentation of the iris. The cutaneous depigmentation consist of macules which are symmetrically distributed as in vitiligo (Johnson, 1963). Parker (1940) observed depigmentation most commonly on the head, shoulders, nape of the neck, and eyelids. As in vitiligo, partial or complete repigmentation may develop. Also halo nevi have 735

CHAPTER 39

C

A

B

D

Fig. 39.9. (A–D) Vitiligo and poliosis in a patient with Vogt–Koyanagi–Harada syndrome (taken from: Mosher D. B., T. B. Fitzpatrick, J. P. Ortonne, and Y. Hori. Disorders of Pigmentation: Dermatology in General Medicine, 3rd ed. Fitzpatrick T. B., A. Z. Eisen, K. Wolff, I. M. Freedberg, K. F. Austen. (eds). New York: McGraw-Hill Book Company, 1987, pp. 794–877. With kind permission of McGraw Hill Inc., New York, USA; copyright 1987).

been reported as the first cutaneous manifestations of the syndrome, but they are more commonly seen in the population at large (Nordlund et al., 1980). Vitiligo has been observed to occur up to five years after the appearance of eye symptoms of VKHS (Johnson, 1963). Alopecia areata may present after the apparent onset of the disease. Commonly the alopecia manifests as discrete patches of small areas of hair loss, but gradually the entire scalp may be involved. Regrowing hair may be gray and thinner, shorter, and more pointed than the original hair. Regrown hair may remain white to gray (Johnson, 1963). Some general thinning of the scalp hair may also be noticed. In some cases, the cutaneous features may precede the uveitis (Johnson, 1963).

Associated Disorders VKHS has been reported associated with adiposogenital syndrome (Jess, 1932); mandibulofacial dysostosis (Cambraggi 736

1958; Ludwig, 1932); progressive facial hemiatrophy (Carrasquillo, 1942); pituitary dysfunction (Ludwig, 1950); Fröhlich syndrome (Pinero, 1952); Klippel-Feil syndrome with ear agenesis (Schwartz, 1955); a hypothalamic syndrome with hyperglycemia, hypercholesterolemia, nocturia, polyuria, and diabetes insipidus (Walsh, 1957); facial hemiatrophy and alopecia (Franchetti, 1958), and insomnia, amenorrhea, polyuria, and hypertension (Kastnelson, 1958).

Routine Histology, Histochemistry, and/or Immunochemistry The Eye Histology The characteristic lesion is a granulomatous uveitis but nongranulomatous uveitis has also been reported (Perry and Font, 1977). Nodular eye lesions show typically epithelioid cells containing melanin granules surrounded by a wall of plasma

MISCELLANEOUS HYPOMELANOSES: DEPIGMENTATION

cells and lymphocytes in the choroid. Frequently, formation of Dalin-Fuchs nodules can be observed. This and the infiltrate in the iris are findings similar to those in sympathetic ophthalmoplegia (Hager, 1957; Kissel, 1963; Yuge, 1957). In a later study of the eyes of five patients with VKHS most changes were observed in the choroid and the ciliary body. Histologic examination showed proliferation of melanocytes, surrounded by lymphocytes and plasma cells. Melanocytes became swollen and lost their cytoplasmic processes. Gradually, melanocytes showed conversion to epithelioid cells, which were polygonal or spindle shaped and which had a variable number of melanin pigment granules and a small amount of chromatin in their acidophilic cytoplasm (Matsuda, 1970). In smear preparations of uveal tissues, epithelioid cells are rounded and show a wide range of morphologic abnormalities. Some pigment granules are barely visible whereas others are closely attached to one another to form a coarse clump of pigment (Ikui and Mimura, 1970). Electron Microscopy Matsuda (1970) performed ultrastructural studies on the limbal corneal epithelium and uveal tissue of eight patients with VKHS and of three patients with sympathetic ophthalmia. In the early stage of the disease, he found that melanocytes were in close contact with lymphocytes. Later, melanocytes were observed to lose cytoplasmic organelles engaged in the melanogenesis. Melanocytes were also seen transformed into epithelioid cells in the uveal tract and into dendritic cells of the limbal corneal epithelium. The observer found no morphologic differences between VKHS and sympathetic ophthalmia at the electron microscopic level. Furthermore, Matsuda stated that the close apposition of lymphocytes to melanocytes is significant in the pathogenesis of the disease. The “metamorphosis” of melanocytes into epithelioid cells seen on light microscopy was confirmed on electron microscopy (Ikui and Mimura, 1970).

The Depigmented Skin Histology The epidermis and the dermis usually show normal features. Melanocytes are absent. However, in the dermis of involved depigmented skin, Garza Toba (1964) observed edema, vasodilatation, vessel wall abnormalities, lymphocytic infiltrate around adnexa, particularly sebaceous glands, and pigment incontinence with melanophages around vessels, and in the papillary dermis. On the contrary, Howsden (1973) found no other abnormalities than the absence of melanocytes. Histologic examination by Ikui and Mimura (1970) showed that the melanocytes surrounding the involved hair bulbs had become swollen, had assumed the form of epithelioid cells and were surrounded by an infiltrate of lymphocytes and plasma cells. The histologic picture of the depigmented skin appeared to be similar to that of the uveal tract.

Electron Microscopy Findings identical to those of the uveal tract in which melanocytes lose their organelles have been observed at the electron microscopy level (Bazex, 1976). Morohashi et al. (1977) reported melanocytes with cytoplasmic vacuolization, melanosome aggregation, autophagic vacuoles, fatty degeneration, and pyknosis or homogeneous cytoplasmic degeneration. Melanocytes appeared to be replaced by Langerhans cells and indeterminate dendritic cells, as in vitiligo (Kumakiri, 1982). Nerve connections to normal and degenerated melanocytes were also observed. Immunofluorescence Indirect immunofluorescence was found negative in one patient with VKHS and psoriasis. Direct immunofluorescence was faintly positive with IgG, which was confined to epidermal intercellular space. Both IgM and C3 staining revealed negative findings. Sections from skin with psoriasis and vitiligo showed not only these abnormalities but also yellowgreen fluorescence beneath the dermal–epidermal junction. Immunofluorescence studies of normal or control skin were negative (Howsden, 1973).

Laboratory Findings and Investigations Characteristic laboratory findings are usually observed during the meningo-encephalitic stage. Cerebrospinal fluid analysis shows an increase of both the protein concentration and the number of white blood cells. Diffuse encephalitis may be seen on electroencephalography (EEG). Furthermore, additional ophthalmologic investigations, such as fluorescein angiography and echography have proved to be useful in the diagnosis and management of VKHS (Rubsamen and Gass, 1991).

Criteria for Diagnosis According to the International Committee on Vogt– Koyanagi–Harada Disease Nomenclature, the following criteria should be present to establish the full diagnosis of VKHS (Read et al., 2001): 1 no history of ocular trauma or surgery preceding the initial onset of uveitis; 2 no clinical or laboratory evidence suggestive of ocular disease entities; 3 bilateral ocular involvement: (a) early signs: diffuse choroiditis; (b) late signs: ocular depigmentation; 4 neurologic/auditory findings: meningismus, tinnitus, and cerebrospinal fluid pleocytosis; and 5 integumentary findings: alopecia, vitiligo, and poliosis. VKHS is thus a disease that is usually first diagnosed by ophthalmologists because the symptoms of uveitis develop initially.

Differential Diagnosis As the occurrence of vitiligo alone is common in patients with VKHS, a careful history and investigation of abnormal ocular, auditory and neurologic symptoms is required to differentiate 737

CHAPTER 39 Table 39.2. Comparison of clinical and histologic features of Vogt–Koyanagi–Harada syndrome with some other similar leukodermas. Inheritance

Pigment abnormalities of skin and hair

Natural course leukodermic defects

Ocular and auditory features

Histology depigmented maculae

Vogt–Koyanagi–Harada syndrome

Acquired

Progressive

Uveitis; bilateral dysacousia

Melanocytes absent

Alezzandrini syndrome

Acquired

Bilateral symmetrically distributed depigmented maculae; poliosis (and alopecia areata) Unilateral facial vitiligo, ipsilateral poliosis

Stable

Melanocytes absent

Piebaldism (see Chapter 29)

Autosomal dominant

Stable

Waardenburg syndrome (see Chapter 29) Segmental vitiligo (see Chapter 30) Vitiligo vulgaris (see Chapter 30)

Autosomal dominant

Bilateral symmetrically distributed depigmented maculae; hyperpigmented macules within white maculae; white forelock Bilateral symmetrically distributed depigmented maculae; white forelock Unilateral (dermatomal) depigmented maculae Bilateral symmetrically distributed depigmented maculae

Tapetoretinal degeneration; dysacousia Dysacousia (Woolf syndrome)

Acquired Acquired

the patient with VKHS from one with vitiligo (see Chapter 30). Alezzandrini syndrome (see Chapter 39) can be distinguished by the presence of unilateral tapetoretinal degeneration and the ipsilateral occurrence of facial vitiligo and poliosis, sometimes with bilateral perceptual deafness (Table 39.2). Most of the other syndromes of cutaneous depigmentation and ocular and auditory involvement are hereditary and therefore can be readily excluded.

Pathogenesis The etiology of VKHS has not been established, although several theories have been suggested. These include endocrine disturbances (Koyanagi, 1942), vitamin deficiency (Gilbert, 1910), bacterial infection (Demaria, 1917), brucellosis (Riehl and Andrews, 1966), neurotrophic disturbance, viral infection, and autoimmune disturbance. At present, only two models seem to be important, the viral and the autoimmune hypotheses.

Viral Hypothesis Gilbert (1910) postulated herpes zoster as the cause of VKHS. Others have thought herpes to be a neurotrophic virus (Bunge, 1934; Davies, 1935; Magitot, 1958). The viral hypothesis has been supported by many observations. Occurrence and recurrence after paranasal sinusitis are compatible with a viral (or at least infectious) etiology. Mohan and Gupta (1967) proposed that the meningeal symptoms occurring in the initial phase support an infectious, e.g. viral, agent. The presence of pleocytosis in the final stage of the meningeal phase has been interpreted in favor of this theory. The apparent involvement of the hypothalamus could be implicated in the pathogenesis 738

Melanocytes absent; in hyperpigmented macules: autophagolysosomes Melanocytes absent

Stable

Heterochromia irides; dysacousia

Stable

None

Melanocytes absent

Progressive

Hypopigmented retinae

Melanocytes absent; lymphocytes in active areas

of the pigmentary defect. Although virus infection has been thought to trigger the disease no specific virus has been implicated and investigators have been unable to isolate or culture a virus from patients (Sheth, 1993) (see section on “Animal models”).

(Auto)immune Hypothesis The first authors to suggest that an allergy was responsible for uveitis associated with pigmentary abnormalities were Pusey in 1903 and Elschnig in 1910. The latter considered sympathetic ophthalmia to result from release of damaged uveal pigment which had antigenic properties and resulted in formation of organ-specific antibodies. Thus anaphylactic pigment destruction was the result. Peters (1912) suggested that uveitis caused destruction of choroid pigment and dissemination of breakdown products which led to sensitization and allergic reactions in other body pigmentary structures. There are clinical similarities between sympathetic ophthalmia and VKHS. Sympathetic ophthalmia has been associated with alopecia, poliosis, and deafness (Cramer, 1913) and VKHS has been reported after eye surgery (Schwartz, 1955). The general concept of sympathetic ophthalmia is that of a severe traumatic reaction. Subsequent uveal degradation is followed by release of antigens which stimulate antibody formation. Once adequate titers are reached, sympathetic uveitis occurs in the untraumatized eye. A similar mechanism may play a role in VKHS. Both humoral and cellular hypersensitivity to uveal and retinal pigment antigens have been demonstrated (Hammer, 1993) and a close association has been found between VKHS and class II human leukocyte antigen (HLA) antigens, which

MISCELLANEOUS HYPOMELANOSES: DEPIGMENTATION

suggested the possibility of a disease susceptibility gene (Martinez et al., 1992). More recent studies in patients with melanoma, vitiligo, and VKHS have shown melanocytic antigens which are capable of inducing both T cellular and humoral responses. Some of the potential “VKHS-specific antigens” are MART-1 (Sugita et al., 1996), the tyrosinase family proteins (Yamaki et al., 2000), and KU-MEL 1 (Kiniwa et al., 2001). However, more studies are needed to confirm the exact role of these antigens in inducing autoimmunity in VKHS. So VKHS is suggested to be the result of an (auto)immune mechanism, which may be influenced by genetic factors. Some authors even believe that the VKHS may represent one end of the spectrum of vitiligo. In vitiligo, the epidermal melanocytes are destroyed. In addition, 40% of vitiligo patients have asymptomatic pigment cell destruction in the epidermal pigment cell epithelium. It is conceivable that whatever process (immunologic or otherwise) causes destruction of the pigment cell in the epidermis can also damage the same cell in the various layers of the eyes, ears, and meninges. The patients with VKHS therefore manifest their complex of symptoms and signs of destruction of pigment cells in the skin, leptomeninges, inner ear, and uvea. A patient who presents with the classic triphasic symptom complex as in VKHS might be considered to have a more inflammatory and aggressive form of vitiligo which is on the opposite end of the spectrum from a patient who has only vitiligo and asymptomatic chorioretinitis (Sheth, 1993).

the stage in which the virus was present and identifiable had passed when most of these unsuccessful transfer experiments were performed. So a viral genesis still cannot be excluded. Various experimental studies support the (auto)immune theory. Foreign protein injected into rabbit eyes produced sensitizing reactions in the contralateral eye (Riehm, 1933), but only in pigmented and not in albino rabbits. Friedenwald (1938) observed that the tissue reaction after intracutaneous injection of uveal pigment was identical to sympathetic ophthalmia and found a delayed hypersensitivity response. Other studies showed similar results with VKHS and sympathetic ophthalmia (Fine, 1957; McPherson and Wood, 1948). Hammer (1977) found uveal pigment to stimulate lymphocyte cultures from patients with sympathetic ophthalmia or VKHS. Furthermore, Tagawa (1978) found that lymphocytes from patients with VKHS may show microcytotoxicity against allogenic melanoma cells in vitro.

Animal Models

Prognosis

Several animal experiments have been performed to study the viral and/or autoimmune genesis of the VKHS. Takahashi (1930) inoculated rabbit cisterna with sterile vitreous from an enucleated eye afflicted with Harada disease and observed optic neuritis and generalized uveitis in two of five rabbits. Brain material from one of these, injected into normal rabbit vitreous, also caused uveitis and optic neuritis; similar injection of cerebrospinal fluid from a VKHS patient gave similar results. Tagami (1931) transferred the disease through three rabbit generations. Similar transference results were obtained by others (Friedenwald, 1934; Yoshida, 1957). Friedenwald and MacKee (1938) succeeded in producing the typical lesions and passed the agent via cerebrospinal fluid serially through rabbits, cats, and dogs. Several investigators have isolated a virus from the cerebrospinal fluid (Erbakan, 1962) and from the eyes (Sugiura, 1953) from patients with VKHS. In the serum of another patient with VKHS, Sugiura (1953) managed to show a positive neutralization test against the virus. However, neither isolation nor culture of a virus from the skin of patients with VKHS has been accomplished. Bruno and McPherson (1949) found that injection of subretinal fluid from affected patients into the brains of mice did not induce the disease. In other patients, repeated egg embryo, mouse brain, and rabbit eye inoculations of VKHS subretinal or spinal fluid have failed. Thus, neither the transfer nor the isolation experiments have been completely reproducible. It is possible that because the diagnosis usually was not made early,

The uveitis is usually self-limiting but may take over a year to recover. Most patients lose some degree of visual acuity, and a few develop total blindness. There are usually no permanent auditory defects. The vitiligo has a stable course (Sheth, 1993).

Treatment Treatment is mainly directed towards the uveitis and consists of high-dose systemic corticosteroids or ciclosporin or a combination therapy of ciclosporin and (low-dose) corticosteroids (Rubsamen and Gass, 1991; Hammer, 1993). The vitiliginous lesions in VKHS patients seem to respond well to the more established phototherapeutic and autologous transplantation methods that are used in idiopathic vitiligo (see also Chapter 30).

References Alajouanine T. Sur une méningoencéphalite avec uvéite unilatérale et décollement de la rétine, faisant suite à rechute; persistance depuis six mois d’un syndrome confusionnel avec dilatation ventriculaire et alterations de liquide céphalorachidien. Rev. Neurol. (Paris) 89: 281–285, 1953. Babel J. Syndrom de Vogt-Koyanagi, uveite bilaterale, poliosis, alopecie, vitiligo et dysacousie. Schweiz. Med. Wochenschr. 69: 1136–1140, 1939. Bazex A. Syndrome de Vogt–Koyanagi–Harada. XVeme Congres de l’Association des Dermatologistes et Syphiligraphes de Langue Française, Ajaccio, 1976. Bazex A. Vogt–Koyanagi–Harada disease. About two observations. Ann. Dermatol. Venereol. 104: 49–53, 1977. Beniz J., D. J. Forster, J. S. Lean, R. E. Smith, and N. A. Rao. Variations in clinical features of the Vogt–Koyanagi–Harada syndrome. Retina 11: 275–280, 1991. Bock E. Ueber frühzeitiges Ergrauen der Wimpern. Klin. Monatsbl. Augenheilkd. 28: 484–492, 1890. Bruno M. G., and S. D. McPherson Jr. Harada’s disease. Am. J. Ophthalmol. 32: 513–522, 1949. Bunge E. Über doppelseitige Iridozyklitis und Poliosis. Arch. Augenehilkd. 108: 212–225, 1934. Cambraggi A. Hallerman-Streiff syndrome (mandibulo-facial dysostosis) with Fuch’s heterochromia. Boll. Oculist. 37: 365–374, 1958.

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CHAPTER 39 Carrasquillo H. R. Uveitis with poliosis, vitiligo, alopecia and dysacousia (Vogt-Koyanagi syndrome). Arch. Ophthamol. 28: 385–414, 1942. Cowper A. R. Harada’s disease and Vogt-Koyanagi syndrome: uveoencephalitis. Arch. Ophthalmol. 45: 367–376, 1951. Cramer E. Zur Frage der anaphylaktischen Entstehung der sympathischen Entzündung. Klin. Monatsbl. Augenheilkd. 51: 205–206, 1913. Davies W. S. Uveitis with associated alopecia, poliosis, vitiligo and deafness. Arch. Ophthalmol. 14: 239–243, 1935. Demaria E. B. Consideraçiones sobre dos casos de iridoçiclocoroiditis doble y grave en sujetos con vitiligo. Arch. Oftalmol. Hisp. Am. 17: 355–362, 1917. Erbakan S. Harada’s disease. The first case reported in Turkey. Am. J. Ophthalmol. 21: 723–738, 1938. Erdmann P. Zur Frage eienes Zusammenhanges zwischen Vitiligo und Augenleiden. Klin. Monatsbl. Augenheilkd. 49: 129–138, 1911. Fine B. S., and J. H. Gilligan. The Vogt-Koyanagi syndrome. Am. J. Ophthalmol. 43: 433–440, 1957. Flynn G. E. Bilateral uveitis, poliosis and vitiligo with an hereditary factor. Am. J. Ophthalmol. 35: 568–572, 1952. Franceschetti A, and G. Maeder. Facial hemiatrophy with alopecia (Romberg’s syndrome) associated with heterochromic cyclitis (Fuch’s syndrome). J. Genet. Hum. 7: 116–120, 1958. Friedenwald J. S. Notes on the allergic theory of sympathetic ophthalmia. Am. J. Ophthalmol. 17: 1008–1018, 1934. Friedenwald J. S., and MacKee C. M. Filter passing agent as a cause of endophthalmitis. Am. J. Ophthalmol. 21: 723–738, 1938. Friedman A. H., M. Luntz, and W. L. Henley. Diagnosis and management of uveitis: an atlas approach. Baltimore: Williams and Wilkins, 1982. Garza Toba M. Un caso clinico de sindrome de Vogt-Koyanagi. Medicina Revisita Mexicana 44: 73–79, 1964. Gilbert J. A., E. S. Pollack, and C. V. Pollack. Jr. Vogt–Koyanagi– Harada syndrome: case report and review. J. Emerg. Med. 12: 615–619, 1994. Gilbert W. Vitiligo und Auge, ein Betrag zur Kenntnisder herpetischen Augenerkrankungen. Klin. Monatsbl. Augenheilkd. 48: 24–31, 1910. Hager G. Sympathetic ophthalmitis. Vogt-Koyanagi’s syndrome and Harada’s disease. Klin. Monatsbl. Augeinheilkd. 113: 89–100, 1957. Hammer H. Lymphocytic transformation in sympathetic ophthalmitis and the Vogt–Koyanagi–Harada syndrome. Br. J. Ophthalmol. 55: 850–852, 1977. Hammer H., M. Janaky, and I. Suveges. Vogt–Koyanagi–Harada syndrome. Acta Ophthalmol. 71: 711–713, 1993. Harada Y. Beitrag zur klinischen Kenntnis von viechteitriger Choroiditis (Choroiditis diffusa acuta). Nippon Gankai Zasshi 30: 356–378, 1926. Howsden S. M. Vogt–Koyanagi–Harada syndrome and psoriasis. Arch. Dermatol. 108: 395–398, 1973. Hutchinson J. A case of blanched eyelashes. Arch. Surg. (Lond.) 4: 357–358, 1892–1893. Ikui H., and M. Mimura. Cytological analysis of ocular and cutaneous lesions in Vogt–Koyanagi–Harada syndrome. Acta Soc. Ophthalmol. Jpn. 74: 1100–1106, 1970. Jacobi J. Vorzeitige und acute Entfärbung der Wimpern beschränkt auf die Lider eines sympathisch erkrankten Auges. Klin. Monatsbl. Augenheilkd. 12: 153–161, 1874. Jess A. Poliosis, Vitiligo und Doppelseitige schwerste Iridocyclitic bei Dystrophia adiposo genetalis. Ber. Dtsch. Ophthalmol. Ges. 49: 469–473, 1932. Johnson W. C. Vogt–Koyanagi–Harada syndrome. Arch. Dermatol. 88: 146–149, 1963.

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Kastnelson A. B. Harada’s disease: uveomeningeal syndrome. Zh. Oftalmol 4: 229–234, 1958. Kiniwa Y., T. Fujita, M. Akada, K. Ito, T. Shofuda, Y. Suzuki, A. Yamamoto, T. Saida, and Y. Kawakami. Tumor antigens isolated from a patient with vitiligo and T-cell-infiltrated melanoma. Cancer Res. 61: 7900–7907, 2001. Kissel P. Les uveo-neuraxites virales ou présumées telles. Rev. Neurol. (Paris) 108: 267–294, 1963. Komoto J. Ein Betrag zur Taubheit bei sympathischer. Ophthalmic. Klin. Monatsbl. Augenheilkd. 50: 129–135, 1911. Koyanagi Y. Alopecia and vitiligo accompanied with uveitis. Acta Soc. Ophthalmol. Jpn. 39: 118, 1935. Quoted in Yuge T. The relation between Vogt-Koyanagi syndrome and sympathetic ophthalmia. Am. J. Ophthalmol. 43: 735–744, 1957. Koyanagi Y. Dysakusis, Alopecia und Poliosis bei schwerer Uveitis nicht traumatischen Ursprunges. Klin. Monatsbl. Augenheilkd. 82: 194–211, 1929. Kumakiri M., T. Kimura, and Y. Miura. Vitiligo with an inflammatory erythema in Vogt–Koyanagi–Harada disease. J. Cutan. Pathol. 9: 258, 1982. Leyva A. V. Uveomeningitis, uveoencephalitis; Vogt–Koyanagi– Harada syndrome. Report of three cases. Arch. Hosp. Univ. (Habana) 11: 215–231, 1959. Ludwig A., and G. Korting. Vogt-Koyanagi-like syndrome and mandibulofacial dystosis (Franceschetti-Zwahlen syndrome). Arch. Dermatol. Syphilol. (Berlin) 190: 307–316, 1950. Magitot A., and A. Dubois-Poulsen. Un cas de syndrome de Harada, uvéite grave avec décollements bilateraux et encephalite. Bull. Soc. Ophthalmol. Fr. 51: 223, 1939. Quoted in Kastnelson A. B. Harada’s disease: uveomeningeal syndrome. Zh. Oftalmol. 4: 229–234, 1958. Matsuda H. Electrom microscopic studies on Vogt–Koyanagi–Harada syndrome and sympathetic ophthalmia with special reference to the melanocyte. Acta Soc. Ophthalmol. Jpn. 74: 1107–1112, 1970. McPherson S. D., and A. C. Wood. The significance of the intracutaneous test for hypersensitivity to uveal pigment. Am. J. Ophthalmol. 31: 35–45, 1948. Mohan H., and A. N. Gupta. Vogt-Koyanagi syndrome. J. All India Ophthalmol. Soc. 15: 117–119, 1967. Morohashi M. Ultrastructural studies of vitiligo, Vogt-Koyanagi syndrome and incontinentia pigmenti achromians. Arch. Dermatol. 113: 755–766, 1977. Murakami S., Y. Inaba, M. Mochizuki, A. Nakajima, and A. Urayama. A nationwide survey on the occurrence of Vogt–Koyanagi–Harada syndrome in Japan. Jpn. J. Ophthalmol. 38; 208–13, 1994. Nettleship E. Remarks on sympathetic ophthalmitis with whitening of the eyelashes. Trans. Ophthal. Soc. UK. 4: 83–84, 1883– 1884. Nordlund J. J., D. Albert, B. Forget, and A. B. Lerner. Halo nevi and Vogt–Koyanagi–Harada syndrome; manifestations of vitiligo. Arch. Dermatol. 116: 690, 1980. Ogawa, U. Histologic findings in idiopathic uveitis. Acta. Soc. Ophthalmol. Jpn. 38: 1005, 1934. Quoted in Yuge T. The relation between Vogt-Koyanagi syndrome and sympathetic ophthalmia. Am. J. Ophthalmol. 43: 735–744, 1957. Ohno S., D. H. Char, S. J. Kimura, and O’Connor R. Vogt– Koyanagi–Harada syndrome. Am. J. Ophthalmol. 83: 735–740, 1977. Parker W. R. Severe uveitis with associated alopecia, poliosis, vitiligo and deafness. A second review of the published records. Arch. Ophthalmol. 24: 439–446, 1940. Parker W. R. Uveitis associated with alopecia, poliosis, vitiligo and deafness: report of two cases. Arch. Ophthalmol. 14: 577–581, 1931. Pattison E. M. Uveomeningoencephalitic syndrome (Vogt– Koyanagi–Harada). Arch. Neurol. 12: 197–205, 1965.

MISCELLANEOUS HYPOMELANOSES: DEPIGMENTATION Perry H. D., and R. L. Font. Clinical and histopathological observations in severe Vogt–Koyanagi–Harada syndrome. Am. J. Ophthalmol. 83: 242–254, 1977. Peters A. Sympatische Ophthalmie und Gehörstörungen. Klin. Monatsbl. Augenheilkd. 502: 433–439, 1912. Pinero y Carrion A., and R. Jimenez Munoz. Sindrome de VogtKoyanagi (estudio de un caso en un tipo constituciónal de Froelich). Arch. Soc. Oftalmol Hisp. Am. 12: 1402–1406, 1952. Pusey B. Cytotoxins and sympathetic ophthalmia. Arch. Ophthalmol (old series) 32: 334–338, 1903. Read R.W., G. N. Holland, N. A. Rao, K. F. Tabbara, S. Ohno, L. Arrellanes-Garcia, P. Pivetti-Pezzi, H. H. Tessler, and M. Usui. Revised diagnostic criteria for Vogt–Koyanagi–Harada disease: Report of an international committee on nomenclature. Am. J. Opthalmol. 131: 647–652, 2001. Reed H. The uveoencephalitic syndrome of Vogt–Koyanagi–Harada disease. Can. Med. Assoc. J. 79: 451–458, 1958. Riehl J. L., and J. M. Andrews. The uveomeningoencephalitic syndrome. Neurology 16: 603–609, 1966. Riehm W. Die neue Theorie von J. Meller über die tuberkulöse Aetiologie der sympathischen Ophthalmie und die Schiecksche Eigenblutinjektion in die vordere Augenkammer. Klin. Monatsbl. Augenheilkd. 90: 477–485, 1933. Rosen E. Uveitis with poliosis, vitiligo, alopecia and dysacousia (VogtKoyanagi syndrome). Arch. Ophthamol. 33: 281–292, 1945. Rubsamen P. E., and J. D. Gass. Vogt–Koyanagi–Harada syndrome. Clinical course, therapy and long term visual outcome. Arch. Ophthalmol. 109: 682–687, 1991. Schenkl A. Ein Fall von plötzlich aufgetrener Poliosis circumscripta der Wimpern. Arch. Dermatol. Syphilol. (Berlin) 5: 137–139, 1873. Schwartz E. A. Vogt-Koyanagi syndrome precipitated by lens extraction. Report of a case. Am. J. Ophthalmol. 39: 488–499, 1955. Sheth A. P., N. Supapannachart, and J. J. Nordlund. Acquired hypomelanotic disorders. In: Pigmentation and Pigmentary Disorders. N. Levine (ed.). Boca Raton: CRC Press, 1993, pp. 337–365. Sugita S, M. Sagawa, S. Mochizuki, S. Shichijo, and K. Itoh. Melanocyte lysis by cytotoxic T lymphocytes recognizing the MART-1 melanoma antigen in HLA-A2 patients with Vogt– Koyanagi–Harada disease. Int. Immunol. 8: 799–803, 1996. Sugiura S. The viral nature of Harada’s and Vogt-Koyanagi syndrome (abstr.) Acta Soc. Ophthalmol. Jpn. 57: 117, 1953. Tagami K. Beitrage zur Kenntnis der acuten nicht eitrigen diffusen exsudativen Choroiditis (sig Haradesche Krankheit) aus dem Resultat des Tierversuches mit subretinalem Exsudat. Acta Soc. Ophthalmol. Jpn. 35: 1289–1337, 1931. Tagawa Y. Lymphocyte-mediated cytotoxicity against melanocyte antigens in Vogt–Koyanagi–Harada disease. Jpn. J. Ophthalmol. 22: 36–39, 1978. Takahashi M. Klinische und experimentelle Studien über das Wesen von sog idiopathischer Doppelseitiger schwerer Uveitis. Acta Soc. Ophthalmol. Jpn. 34: 506–549, 1930. Tay W. Diseases of the eyelids. 1. A case of symmetrical whitening of the eyelashes and eyebrows in connection with sympathetic ophthalmitis. Trans. Ophthal. Soc. UK 12: 29–30, 1891–1892. Vogt A. Frühseitiges Ergrauen der Zilien und Bemerkungen über den sogenannten plötzlichen Eintritt dieser Veränderung. Klin. Monatsbl. Augenheilkd. 44: 228–242, 1906. Walsh F. B. (ed.). Clinical Neuro-ophthalmology, 2nd ed. Baltimore: Williams and Wilkins, 1957. Weber S. W., and J. J. Kazdan. The Vogt–Koyanagi–Harada syndrome in children. J. Pediatr. Ophthalmol. 14: 96–99, 1977. Yamaki K., K. Gocho, K. Hayakawa, I. Kondo, and S. Sakuragi. Tyrosinase family proteins are antigens specific to Vogt– Koyanagi–Harada disease. J. Immunol. 165: 7323–7329, 2000.

Yoshida E. Experimental studies in pathogenesis of idiopathic uveitis (uveoencephalitis) II. Acta Soc. Ophthalmol. Jpn. 61: 1211–1220, 1957. Yuge T. The relation between Vogt-Koyanagi syndrome and sympathetic ophthalmia. Am. J. Ophthalmol. 43: 735–744, 1957. Zentmayer W. Severe uveitis with associated alopecia, poliosis, vitiligo and deafness. A second review of the published records. Arch. Ophthalmol. 27: 342–344, 1942.

Westerhof Syndrome Wiete Westerhof, David Njoo, and Henk E. Menke

Historical Background In 1978, Westerhof et al. presented three generations of a family of Indian origin with congenital hypomelanotic and hypermelanotic macules, which suggested autosomal dominant inheritance. Some affected members also showed retarded growth and mental deficiency. Based upon clinical, histologic, and ultrastructural findings they suggested that this complex of symptoms represented a new neurocutaneous syndrome different to tuberous sclerosis.

Synonyms Westerhof et al. (1978) published the syndrome under the name “hereditary congenital hypopigmented and hyperpigmented macules.” The syndrome is also known as “Westerhof syndrome.”

Epidemiology The true prevalence of this syndrome in the population is unknown.

General Clinical Findings — Age, Sexual Predilections, Racial Distribution Pedigree study of the family suggested the syndrome is probably autosomal dominant inherited, so males and female are equally affected (Westerhof et al., 1978). Lesions are probably present from birth.

Clinical Description Typically, patients with this syndrome present with asymptomatic hypopigmented and hyperpigmented macules (Fig. 39.10). Solitary as well as multiple lesions (up to 20) can be seen, varying from 1 cm to 15 cm in diameter. They may occur on any part of the body, but mostly on the arms, legs, and trunk. One patient examined by Westerhof et al. (1978) had a lesion on the chin. The lesions are well defined, asymmetrical, and not confined to any dermatome. Remarkably, the mother had discrete lesions, of which she was hardly aware. In contrast, the majority of her children had skin lesions all over their bodies. Mucous membranes are spared. Mostly, patients are dark skinned. Male subjects are somewhat retarded in growth. Microcephaly is a characteristic finding. Patients with this syndrome may have a low IQ. Westerhof et al. (1978) presented one patient with signs of an organic brain

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disease resulting in a certain degree of sensory aphasia. Sexual development is normal.

Routine Histology, Histochemistry, and/or Immunochemistry Melanocyte Counts

Associated Disorders There are no associated disorders.

There are no substantial differences between the number of dopa-positive cells found in hypopigmented and hyperpigmented macules and those found in the normal skin. In the dark and light patches, as well as in normal skin, the melanocytes are of comparable size and are mixed bipolar and stellate in appearance with well developed branching dendrites. No giant melanosomes are present. The dopa reaction is equally intense in normal and pathologically affected skin. In hyperpigmented macules the background staining of keratinocytes is pronounced. In normal control skin it is moderate, whereas in hypopigmented macules it is negligible.

Electron Microscopy

Fig. 39.10. Hypopigmented and hyperpigmented macules of a patient with Westerhof syndrome (with kind permission of the American Medical Association, Chicago, USA; copyright 1978).

No degenerative changes are present in the normal skin or in the hyperpigmented and hypopigmented macules. There are neither signs of inflammation in the epidermis nor in the dermis. A few melanophages can be seen. The melanocytes bulge toward the dermis and contain premelanosomes and melanosomes in stages 1–4. Melanosomes do not aggregate inside the melanocytes and are ellipsoidal and lamellar. The dispersion of melanosomes in the cytoplasm of the melanocytes (perinuclear and dendrites) is equal. The transfer of melanosomes from the melanocytes to the keratinocytes in abnormal and normal skin is not disturbed. Melanosomes occurring in melanocytes and keratinocytes of hyperpigmented macules are large (average transverse diameter 0.6 mm). The melanosomes are distributed exclusively as single units in the keratinocytes. On the other hand, the melanosomes in melanocytes and keratinocytes of hypopigmented macules are twice as small (average transverse diameter 0.3 mm) as compared with the hyperpigmented areas, and their distribution pattern inside the keratinocytes is mostly in complexes. In normal skin the average transverse diameter of the melanosomes is 0.4 mm, whereas the melanosomes are distributed in keratinocytes singly as well as in complexes. In the patients studied by Westerhof et al. (1978) it was striking that differences in melanosome size and distribution

Table 39.3. Westerhof syndrome: clinical description, histopathology and other investigations. Clinical description

Histopathology of the hypo/hyperpigmented lesions

Other investigations

Multiple hypopigmented and hyperpigmented macules; 1 to 15 cm in diameter; well defined, asymmetrical, not confined to any dermatome; growth retardation; mental retardation; microcephaly

LM: melanocytes normal EM hyperpigmented lesions: large melanosomes (diameter 0.6 mm); distributed as singles into keratinocytes EM hypopigmented lesions: small melanosomes (diameter 0.3 mm) distributed as complexes into keratinocytes

No abnormalities

LM, light microscopy; EM, electron microscopy.

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Café au lait and hypopigmented patches: sebaceous adenomas; shagreen skin, periungual fibromas Decreased Small melanosomes with low electron density in keratinocytes

Skin

Glial proliferation in brain, epilepsy, mental retardation, muscular spasm

CNS

Autosomal dominant

Inheritance

Eye

Renal tumors, rhabdomyoma of heart, interstitial fibrosis of lungs Retinal hamartomas, optic disc hamartomas, cataract

Internal organs

Hormonal status

Thickened calvaria

Skeleton

DOPA activity Electron microscopy

Tuberous sclerosis

Characteristics

Optic nerve glioma, hyperpigmented spots in iris and choroid, exophthalmus, glaucoma Autosomal dominant

Associated with Addison disease, hyperparathyroidism, pheochromocytoma Gastrointestinal tumors

Spina bifida, kyphosis, scoliosis, subperiosteal hyperostosis Neurilemmoma of nerve stem, epilepsy, oligophrenia

Normal Giant melanosomes in melanocytes and keratinocytes

Café au lait spots, cutaneous neurofibromas, axillary freckles

Recklinghausen disease

Unilateral exophthalmus, papilledema, optic nerve atrophy, visual field defect Autosomal dominant

Pubertas praecox

Polyostosis cysts (fibrous dysplasia)

Normal Giant melanosomes in melanocytes and keratinocytes

Café au lait spots

Albright disease

Table 39.4. Comparison of characteristics of Westerhof syndrome with five similar diseases.

Decreased or normal Melanosome aggregation in melanocytes

Hypopigmented macules

Nevus depigmentosus

Hyperpigmented spots within hypomelanotic lesions not containing functional melanocytes Increased Abnormal (spherical and granular) and normal (ellipsoidal and lamellar) melanosomes in hyperpigmented areas

Piebaldness

Autosomal dominant

Cervical rib, small skull, small sella turcica Mental retardation

Normal Big melanosomes, single distribution; small melanosomes, complex distribution

Hypopigmented and hyperpigmented macules

Westerhof syndrome

CHAPTER 39

pattern in keratinocytes of hypomelanotic macules, normal control skin, and hypermelanotic macules, resemble those typical for white, oriental, and black skin, respectively. There is no difference in electron density between melanosomes of normal skin and that of hyperpigmented and hypopigmented macules.

Laboratory Findings and Investigations Liver and kidney function tests show normal findings. Also amino acid chromatography of the urine shows normal findings. One of the studied cases, a 16-year-old boy born prematurely, had increased lactic dehydrogenase levels (fractions III and IV). Amino acid chromatography of his urine indicated an increase in 1-methylhistidine, but dietary causes could not be excluded. Feces benzidine tests show negative findings. ECG is normal. Skeletal X-ray films show no abnormalities. X-ray studies of the prematurely born child, however, showed a small sella turcica and a veiled right sinus maxillaris; one cervical rib on the right but no abnormal calcification of cysts; a normal thorax. Ophthalmologic examination showed no abnormalities. Westerhof et al. (1978) encountered one male patient, who complained of seeing things unclearly. His visual acuity was 4/5 without correction. There was hyperesthesia of the cornea and subscapular opacities of the lens in both eyes, but the fundi were normal. The visual field showed fluctuating findings, but electroretinography and visually evoked responses revealed no abnormalities. Neurologic examination mostly shows normal results. EEG is normal.

Criteria for Diagnosis The presence of hyperpigmented and hypopigmented macules in members of one family, with or without signs of retarded growth and mental deficiency, are typical of “Westerhof syndrome.” Light/electron microscopy of affected skin is helpful.

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Differential Diagnosis Westerhof syndrome can be readily distinguished from tuberous sclerosis (see Chapter 32), Recklinghausen disease (neurofibromatosis) (see Chapter 45), Albright disease (see Chapter 45), nevus depigmentosus, and piebaldness (see Chapter 29) by the clinical and histologic picture (see Tables 39.3 and 39.4). The pigmentation abnormalities of Westerhof syndrome mostly resemble those of tuberous sclerosis. The patients described by Westerhof et al. (1978) however, do not demonstrate major symptoms of tuberous sclerosis, such as leafshaped light macules, shagreen skin, subungual fibromas, sebaceous adenoma, and other skin tumors, as well as seizures and muscular spasms. Only a few minor symptoms (small skull, mental deficiency, and retarded growth), which are also prevalent in many other neurocutaneous syndromes, are present.

Pathogenesis This syndrome is most likely inherited in an autosomal dominant way. The precise genetic defect is not clear.

Animal Models There are no animal models.

Treatment Genetic counseling is indicated, because some affected children may experience learning disabilities and mental retardation.

Prognosis The pigmentary disorder shows a stable course.

Reference Westerhof, W., F. A. Beemer, R. H. Cormane, J. W. Delleman, W. R. Faber, J. G. de Jong, and W. W. van der Schaar. Hereditary congenital hypopigmented and hyperpigmented macules. Arch. Dermatol. 114:931–936, 1978.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Miscellaneous Hypomelanoses: Hypopigmentation Sections Disseminated Hypopigmented Keratoses Wiete Westerhof, David Njoo, and Henk E. Menke Hypermelanocytic Punctata et Guttata Hypomelanosis Wiete Westerhof, David Njoo, and Henk E. Menke Progressive Macular Hypomelanosis Henk E. Menke, Germaine Relyveld, David Njoo, and Wiete Westerhof Sarcoidosis Wiete Westerhof, David Njoo, and Henk E. Menke

Disseminated Hypopigmented Keratoses Wiete Westerhof, David Njoo, and Henk E. Menke

Historical Background In 1991, Morison et al. presented two cases of asymptomatic, widespread keratotic eruptions in young female patients. Based upon typical clinical and histologic findings they suggested this was a new recognized dermatologic entity. The skin eruption was given the name “disseminated hypopigmented keratoses.”

Associated Disorders The disorder is manifestations.

not

associated

with

any

systemic

Routine Histology, Histochemistry, and/or Immunochemistry Skin biopsy specimens of one of Morison’s patients revealed hyperorthokeratosis and papillomatosis. A Fontana–Masson stain showed apparently normal content of melanin in the basal cell layer. No sign of inflammation was present and no melanophages were observed. Immunoperoxidase staining for human papilloma virus virion antigen was negative.

Synonyms There are no synonyms.

Laboratory Findings and Investigations

Epidemiology

There are no abnormal laboratory findings known in this disorder.

The lesions of disseminated hypopigmented keratoses are both inconspicuous and asymptomatic. Therefore Morison et al. (1991) assumed that this skin disorder is probably more prevalent than their two cases suggest.

General Clinical Findings – Age, Sexual Predilections, Racial Distribution

Criteria for Diagnosis The diagnosis is based on the typical clinical and histologic findings. Individuals receiving therapeutic ultraviolet (UV) irradiation are regarded as having a high risk for developing the disease.

Disseminated hypopigmented keratoses was found in two young white females; one was 19 years of age, the other was 5 years old. Morison et al. (1991) suggested that the disorder mostly affects young people. Both patients reported that none of the other members of their families had similar eruptions. Additional epidemiologic studies have not been performed so far.

Differential Diagnosis

Clinical Description of Disease

Pathogenesis

Asymptomatic, nonfollicular, 1–2 mm, slightly raised, hypopigmented round papules that seem to be “stuck on” to the surrounding skin are characteristic (Fig. 40.1). The lesions are uniformly and symmetrically distributed on the extremities and trunk, and are best seen when viewed with side lighting. The face, palms, and soles are spared. Usually no oral lesions are present.

The exact etiology is unclear. One factor that seems to be involved in the pathogenesis of this condition is exposure to UV irradiation. Morison’s patients (1991) had a history of exposure to therapeutic UV irradiation. However, only one patient was receiving treatment at the time of onset, and continuation of this treatment in both patients has not influenced the progression of the disease. A genetic factor seems to be

Disseminated hypopigmented keratoses can be distinguished from other keratotic eruptions by its specific clinical and histologic features. The differential diagnosis includes stucco keratoses, lichen nitidus, verruca plana, Darier’s disease, epidermodysplasia verruciformis, and keratosis in patients with psoriasis.

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Fig. 40.2. Hypomelanotic macules in a patient with hypermelanotic punctata et guttata hypomelanosis (see also Plate 40.1, pp. 494–495).

Fig. 40.1. Hypopigmented round papules on the arm of a patient with disseminated hypopigmented keratoses. (Taken from: Morison W. L., B. J. Kerker, W. W. Tunnessen, and E. R. Farmer. Disseminated hypopigmented keratoses. Arch. Dermatol. 127: 848–850, 1991. With kind permission of the American Medical Association, Chicago, USA; copyright 1991.)

unlikely; the disorder was not found among relatives of the patients.

Animal Models There are no animal models.

Treatment One patient was treated with tretinoin and propylene glycol for several weeks without any effect. Application of 6% salicylic acid gel under occlusion made the lesions slightly less obvious.

Prognosis Disseminated hypopigmented keratoses appears to be a benign and slowly progressive leukodermatosis.

sented a case report of two sisters, 30 and 27 years of age, with hypopigmented eruptions morphologically resembling lesions of idiopathic guttate hypomelanosis (IGH) and leukoderma punctata (LP). Histopathologic examination of a hypopigmented macula showed that the melanin content was decreased, while remarkably, the number of melanocytes was increased compared with that in normal skin. In the period 1992–94, they encountered three more patients with these characteristic clinical and histologic profiles. The pigmentary disorder was descriptively given the name “hypermelanocytic punctata et guttata hypomelanosis” (HPGH) (Hulsmans et al., 1991, 1993).

Synonyms There are no synonyms.

Epidemiology The incidence of HPGH is unknown.

General Clinical Findings — Age, Sexual Predilections, Racial Distribution So far, this disorder has been found among three females and two males. HPGH probably affects both sexes equally. There is also probably no age predisposition, since the disorder was found in children and among older people as well.

Clinical Description of Disease

Wiete Westerhof, David Njoo, and Henk E. Menke

HPGH consists of discrete and grouped, round and oval, punctate and guttate, hypopigmented macules, which vary in diameter from 2 mm to 5 mm (Fig. 40.2). Mostly, a unilateral (segmental) distribution can be seen, though in one patient a disseminated form was observed. The lesions are located on the thorax, moderately sharp demarcated in the midline and (unilaterally) spreading to the extensor areas of the upper and/or lower extremities. Facial and neck regions are usually not affected. The lesions are not itchy or scaly.

Historical Background

Associated Disorders

In 1986 Westerhof and Hulsmans from the Netherlands pre-

There are no associated disorders.

Reference Morison, W. L., B. J. Kerker, W. W. Tunnessen, and E. R. Farmer. Disseminated hypopigmented keratoses. Arch. Dermatol. 127:848– 850, 1991.

Hypermelanocytic Punctata et Guttata Hypomelanosis

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MISCELLANEOUS HYPOMELANOSES: HYPOPIGMENTATION

Routine Histology, Histochemistry, and/or Immunochemistry Histologic examination with Masson–Fontana staining of lesional skin shows a marked decrease of pigmentation in the epidermal basal layer and an overall linear increase in the number of melanocytes. The epidermis does not show rete ridges. Electron microscopic examination of lesional skin shows a linear increase of morphologically and cytologically normal melanocytes to such an extent that, in most areas, they even predominate in the basal layer (Fig. 40.3). Although the cell bodies of the basal keratinocytes are ousted from their basal position by the crowding melanocytes, thin extensions of these keratinocytes circumvent the melanocytes to occupy the whole stretch of the basal lamina, leaving the melanocytes almost completely out of touch with it. The intercellular spaces in the

basal and suprabasal layers of the epidermis swarm with long winding melanocytic dendrites (Westerhof et al., 2004). The morphology of the organelles is mostly normal. Besides a well-developed Golgi apparatus and endoplasmic reticulum, they contain melanosomes in all stages of maturation (albeit in small numbers). In addition, the melanocytic dendrites contain immature as well as mature melanosomes. There is no autophagocytosis of melanosomes inside the melanocytes. The number of melanosomes in the keratinocytes is generally low. When present, they are concentrated in membrane-bound vesicles as usually seen in white skin.

Laboratory Findings and Investigations Microscopic mycologic examination of lesional skin scrapings is negative. Examination with Wood’s light may accentuate the lesions from the surrounding healthy skin.

Criteria for Diagnosis The diagnosis of HPGH is based on clinical and especially on histologic examination.

Differential Diagnosis Clinically the lesions have to be differentiated from lesions of IGH, usually found in older people, LP seen in patients with vitiligo treated by PUVASOL or PUVA therapy (see Chapter 60), and from hypopigmented guttate macules (HGM) or “white freckles” or “confetti-like spots” reported in patients with tuberous sclerosis (Table 40.1). Both IGH and LP are histologically characterized by a (focal) decrease of melanocytes, which in IGH appear cytologically normal but in LP may be edematous and vacuolated. Hypomelanotic macules as seen in patients with tuberous sclerosis show a normal number of melanocytes with poorly developed dendrites (Table 40.2).

Fig. 40.3. Electron microscopy showing a linear increase of melanocytes in the basal layers of the epidermis.

Table 40.1. Clinical features of disorders with punctate and guttate hypopigmented macules.

Distribution Number Diameter (mm)

IGH

LP

HGM

HPGH

Disseminated Innumerable 0.2–6.0

Disseminated Innumerable 0.5–1.5

Localized 1–3 2–3

Disseminated or segmental Innumerable 2–5

Abbreviations: IGH, idiopathic guttata hypomelanosis; LP: leukoderma punctata; HGM, hypopigmented guttate macules (as seen in patients with tuberous sclerosis); HPGH, hypermelanocytic punctata et guttata hypomelanosis. Table 40.2. Histologic and electron microscopic features of disorders with punctate and guttate hypopigmented macules. IGH

LP

HGM

HPGH

Normal

Normal

Atrophy

Pigmentation Melanocytes

Normal; sometimes: atrophy, loss of rete ridges Decreased Decreased normal aspect

Decreased Normal aspect

Decreased or absent Increased normal aspect

Melanosomes

Incomplete melanization

Decreased or absent Focal cytoplasmic vacuolar degeneration Aberrant melanosomes

Small stage II/III melanosomes

Normal or decreased

Epidermis

For abbreviations see Table 40.1.

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CHAPTER 40

Pathogenesis The exact pathogenesis is not clear. Although the increase in the number of melanocytes in the hypomelanotic lesions of patients with HPGH appear paradoxical, the hypomelanosis may be explained by a disturbed contact of melanocytes with keratinocytes. The intricate conglomerate of melanocytic dendrites and keratinocyte extensions in the spaces between keratinocytes and melanocytes is highly suggestive of a strong attempt of these cells to communicate with each other. Failure may result in a deficient melanosome transfer. Furthermore it looks as if the melanocytes are competing with the keratinocytes for a place along the basal lamina. The keratinocytes prevail with their long extensions, so that there is almost no attachment of the melanocytes to the basement membrane. Whether this failure leads to a compensatory increase of the number of melanocytes is not known (Westerhof et al., 2004). Genetic factors may be involved, since the disorder was found in two sisters.

Animal Models There are no animal models.

Treatment There is no treatment for HPGH.

Prognosis The hypopigmented macules in HPGH appear to show a stable course. Spontaneous repigmentation has not been reported.

References Hulsmans, R. F. H. J., W. Westerhof, K. P. Dingemans, A. H. M. Vermeulen, and A. M. J. van der Kleij. Segmental hypermelanocytary punctate and guttate hypomelanosis and the disseminated variety in two sisters: A hitherto unreported cutaneous pigmentary anomaly. Third Congress of the European Academy of Dermatology and Venereology, Copenhagen, Denmark, September 26–30, 1993. Hulsmans, R. F. J. H., W. Westerhof, K. P. Dingemans, A. H. M. Vermeulen, and J. van der Kleij. Segmental hypermelanocytary punctate and guttate hypomelanosis and the disseminated variety in two sisters: A hitherto unreported cutaneous pigmentary anomaly. Third Annual Meeting of the European Society for Pigment Cell Research, Amsterdam, September, 1991. Westerhof W., K. P. Dingemans, and R. F. H. J. Hulsmans. Hypermelanocytic guttate and macular segmental hypomelanosis. Br. J. Dermatol. 151:701–705, 2004.

Progressive Macular Hypomelanosis Henk E. Menke, Germaine Relyveld, David Njoo, and Wiete Westerhof

Historical Background Progressive macular hypomelanosis is a syndrome of hypopigmentation described in 1988 by Guillet et al. in France and Martinique. This syndrome was observed in young women in the French West Indies and occasionally in France among 748

immigrants from that area. Borelli, from Venezuela, published in 1987 in a Venezuelan journal a new syndrome which he named “cutis trunci variata.” He claimed in a letter in the Annals of Dermatology and Venereology (Borelli, 1992) that this syndrome was identical to that described by Guillet et al. in 1988. In the Netherlands, patients with an identical clinical picture have been identified since 1985 and clinical facts were presented at several conferences on pigment cells and dermatology (Menke et al., 1989, 1990, 1993, 1997).

Synonyms The French named the syndrome “progressive macular hypomelanosis of the trunk” (Guillet et al., 1988). Borelli (1987) from Venezuela described it as “cutis trunci variata.” Menke et al. used the descriptive term “nummular and confluent hypomelanosis of the trunk.” It is also named creole dyschromia in the French West Indies (Lesueur et al., 1994) and idiopathic multiple large-macule hypomelanosis in the USA (Sober and FitzPatrick, 1996). There are reasons to believe that these syndromes, published by different groups, are identical.

Epidemiology The true prevalence of progressive macular hypomelanosis of the trunk is unknown.

General Clinical Findings — Age, Sexual Predilections, Racial Distribution According to Guillet et al. (1988) the syndrome mainly occurs in women between 18 and 25 years of age, especially in those of mixed Caucasoid and Negroid racial origin. This was based on observations in the French West Indies (Martinique) and in France among immigrants. In the Netherlands, we found an age distribution of 16–45 years and a male:female ratio of approximately 1 : 4. Furthermore the disorder was not confined to people of mixed racial origin, as was observed by the French. Although the syndrome mainly occurred in people from the West Indies and the northern part of South America, it was also observed in Caucasian patients of Dutch and other European origin and also in people originating from Asia (Indonesia, East India).

Clinical Description of Disease Progressive macular hypomelanosis is not preceded by any other (inflammatory) disorder. It is characterized by ill-defined nummular hypopigmented spots (1–3 cm in diameter) symmetric and localized on the trunk. In the majority of patients a confluent hypopigmented area can be recognized on the front as well as on the back of the trunk. The width of this confluent region varies from 5 cm to 20 cm (Fig. 40.4). In the lateral regions of the trunk, more or less round solitary macules can be recognized. There is no scaling, nor also after traction on the skin. The lesions are not itchy. The spots are more clearly visible under Wood’s light. The disorder is mostly seen on the trunk, but lesions on the face, the neck, and the

MISCELLANEOUS HYPOMELANOSES: HYPOPIGMENTATION

mentation is probably caused by the alteration in melanosome size and distribution.

Laboratory Findings and Investigations

Fig. 40.4. Progressive macular hypomelanosis of the trunk: a typical lesion on the back of a 21-year-old Caribbean Hindustani woman living in the Netherlands (see also Plate 40.2, pp. 494–495).

upper extremities have been observed. According to Guillet et al. (1988), the hypopigmentation is absent in the dorsolumbar line, however, this feature is not confirmed in the group of patients seen in the Netherlands (Menke et al., 1997). Also according to the French group the disorder disappears after 3–5 years, however in the Dutch series this was not confirmed; the disorder appeared to be more or less stable during the observation period of up to 10 years.

Associated Disorders There are no associated disorders.

Routine Histology, Histochemistry, and/or Immunochemistry Routine histologic examination shows a decrease of melanin content only in the epidermis of the hypopigmented spots compared with that in the adjacent normal skin. There are no abnormalities in the dermis. Immunohistochemical studies have not been performed so far. Electron microscopy of macular lesions performed by Guillet et al. (1988) showed a switch from stage IV single melanosomes (Negroid) in the normal-looking skin to small type I–III aggregated melanosomes (Caucasoid) in the hypopigmented spots. These electron microscopic findings were confirmed by the Dutch investigators (Relyveld et al., to be published). The hypopig-

The French group has not published any other additional laboratory investigations (Guillet et al., 1988). Borelli (1987) only reported that the potassium hydroxide (KOH) preparation in all of his patients was negative. Menke et al. (1997) performed several laboratory investigations. Borelli’s findings were confirmed that in KOH preparation of scrapings from epidermal material from the hypopigmented spots hyphae and spores (the microscopic picture of pityriasis versicolor) were not seen. However, Pityrosporum cultures from the hypopigmented spots of the trunk, were positive in 17/20 patients (85%). This percentage, however, is not significantly different from the percentage (70%) of positive Pityrosporum cultures from the trunk of healthy control patients. Because of the resemblance with pityriasis alba, a disorder that is classified in the eczema group by many authorities, allergy tests were performed in a limited number of patients with progressive macular hypomelanosis of the trunk. Patch tests with the European standard test panel resulted in a positive reaction to nickel in 14/20 patients (70%). Only two of these 14 patients suffered from eczema that was related to contact with nickel objects. The occurrence of a positive nickel patch test in 70% of these patients was much higher than was expected. The significance of this remarkable finding is not yet clear and certainly needs to be confirmed and further investigated. The total IgE level was elevated in 4/10 patients. These four patients also showed a positive RAST against one or more atopic inhalation allergens. The serum levels of antibodies IgE, IgG, IgM, and IgA against Pityrosporum yeast extracts were not different from a healthy control group in all six patients tested. Intracutaneous tests with Pityrosporum yeast extract (immediate as well as delayed-type reactions) were negative in these six patients. Blood analysis including erythrocyte sedimentation rate, leukocyte total and differential count, hemoglobin level, liver and kidney function tests, antinuclear antibody (ANA), and syphilis tests also showed no abnormalities in these six patients. Westerhof et al. (2004) observed the presence of Propionibacterium acnes in the hypopigmented spots (see Pathogenesis).

Criteria for Diagnosis Progressive macular hypomelanosis is a clinical diagnosis. Diagnosis is based on the typical clinical morphology with solitary and coalescing, nonscaly, non-itchy, hypopigmented spots on the skin. In a dark room under Wood’s lamp a red follicular fluorescence is present in the hypopigmented spots, which is absent in the adjacent normal skin.

Differential Diagnosis This “new” disorder [progressive macular hypomelanosis (PMH)] should be differentiated from all other similar disorders of hypopigmentation. The most important ones, 749

CHAPTER 40 Table 40.3. Differential diagnosis. Disorder

Clinical picture

Histology

Laboratory examination

Progressive macular hypomelanosis

Symmetric, hypopigmented, ill-defined spots, mainly on the trunk, sometimes on the face, neck and the upper extremities, nonscaling, non-itchy Irregular hypopigmented, sharply defined, often asymmetric spots, scaling, mostly on neck and trunk, sometimes elsewhere

Loss of epidermal pigment, no dermal abnormalities, Grampositive bacteria

Microbiologic cultures show Propionibacterium acnes bacteria

Hypopigmentation of the epidermis, spores and hyphae in the upper layers of the epidermis, slight inflammation in the dermis Discrete eczematous changes in epidermis and dermis

Positive KOH-preparations, cultures Pityrosporon ovale

Pityriasis versicolor

Pityriasis alba

Hypopigmented, sometimes slightly scaling macules, localization anywhere on the body is possible (predilection for face)

however, are pityriasis versicolor (see Chapter 36) and pityriasis alba (see Chapter 37). The differences between these two disorders and progressive macular hypomelanosis are listed in Table 40.3.

Pathogenesis Until recently the etiology of PMH was unknown. A few hypotheses have been postulated but none has been proved. Guillet et al. (1988) hypothesized that the disorder was caused by a “melting” of genes of white and black parents based on their observation of this disorder only in people of mixed racial (black and white) origin, and, furthermore, on the ultrastructural finding of stage IV single melanosomes (these are the types of melanosome normally seen in black skin) in healthylooking skin and small stage I–III aggregated melanosomes (these are the types of melanosome normally seen in white skin) in the hypopigmented spots. Borelli (1992) considered it to be a genodermatosis based on the fact that the disease was mainly observed in siblings. Although Sober and Fitzpatrick (1996) did not study a large number of patients to determine the histopathology, they suggested that the hypopigmentation probably resulted from a transfer block, caused by a fungal infection, with the pigment changes remaining long after the infection had disappeared. Recently Westerhof et al. (2004) discovered follicular red fluorescence restricted to the hypopigmented spots. They suspected a relation with a porphyrin-producing bacteria residing in the sebum of the pilosebaceous duct. All biopsy specimens taken from lesional skin of eight women demonstrated Gram-positive bacteria in the pilosebaceous duct. In all but one patient, P. acnes was demonstrated in cultured biopsy specimens taken from follicular lesional skin. Healthy follicular skin did not show bacteria in histologic sections, and cultures did not yield anaerobic bacteria. They proposed that a factor is produced by these strains of P. acnes, which interferes with melanogenesis and subsequently causes the hypopigmentation.

Animal Models There are no animal models. 750

Sometimes tests positive for atopy, associated to the disorder

Treatment According to Guillet et al. (1988) there is no specific treatment. Menke et al. (1997) have attempted several treatment modalities including topical and systemic antifungal agents, topical steroids and PUVA. The hypopigmented spots disappeared only with PUVA (after six weeks of treatment), however, about six weeks after the cessation of PUVA treatment, the induced repigmentation cleared and the hypopigmented spots appeared again. Based on the hypothesis of Westerhof et al. (2004) we are now investigating the effect of an antibacterial treatment in combination with UVAphototherapy. The results are promising.

Prognosis Guillet et al. (1988) suggested that the disorder spontaneously disappeared within 3–5 years. In the Dutch series (Menke et al., 1997) this spontaneous regression was not confirmed. On the contrary, the disorder appeared to be stable or showed a slight increase of hypopigmentation in patients who were followed for up to 10 years. Spontaneous repigmentation was never seen.

References Borelli, D. Cutis trunci variata: Nueva genodermatosis. Med. Cutanea Ibero-Lat-Am. 15:317–319, 1987. Borelli, D. [Concerning the article: Confluent progressive hypomelanosis of the melanodermic junction. (Ann. Dermatol. Venereol. 119:19–24, 1992) (letter)]. Ann. Dermatol. Venereol. 119:680, 1992. Guillet, G., R. Helenon, Y. Gauthier, J. E. Surleve-Bazeille, P. Plantin, and B. Sassolas. Progressive macular hypomelanosis of the trunk: primary acquired hypopigmentation. J. Cutan. Pathol. 15:286–289, 1988. Lesueur, A. V., N. R. Garcia-Granel, N. Helenon, and D. Cales-Quist. Macular confluent progressive hypomelanosis in black patients of mixed ethnic origin: an epidemiological study in 511 patients. Ann. Dermatol. Venereol. 121:880–883, 1994. Menke, H. E., S. Doornweerd, J. Zaal, C. Roggeveen, K. P. Dingermans, W. Bergh-Weerman, and W. Westerhof. Acquired hypomelanosis of the trunk. Second Meeting of the European Society for Pigment Cell Research, Uppsala, Sweden, June, 1989. Menke, H. E., S. Dekker, J. Zaal, H. Koemans, K. P. Dingermans, M. Bergh-Weerman, and W. Westerhof. Acquired hypomelanosis of

MISCELLANEOUS HYPOMELANOSES: HYPOPIGMENTATION the trunk. XIVth International Conference Center, Kobe (ICCK), Kobe, Japan, November, 1990. Menke, H. E., M. C. J. van Praag, R. Ossekoppele, V. NoordhoekHegt, S. Dekker, V. Vuzevski, and W. Westerhof. A new syndrome of hypopigmentation of the trunk. Third E. A. D. V. Congress, Copenhagen, Denmark, September, 1993. Menke, H. E., W. R. Ossekoppele, S. K. Dekker, and W. Westerhof. Nummulaire en confluerende hypomelanosis van de romp. Ned. Tijdsch. Dermatol. Venereol. 1997;7:117–122. Sober, A., J. and T. B. Fitzpatrick. Yearbook of Dermatology. St Louis, MO: Mosby-Year Book; 1996, pp. 416–417. Westerhof W., G. N. Relyveld, M. M. Kingswijk, P. de Man, and H. E. Menke. Propionibacterium acnes and the pathogenesis of progressive macular hypomelanosis. Arch. Dermatol. 140:210–214, 2004.

Sarcoidosis Wiete Westerhof, David Njoo, and Henk E. Menke

Historical Background Sarcoidosis is an idiopathic systemic granulomatous disorder which often presents with cutaneous manifestations. Leukoderma is one of the less commonly observed features. Sarcoidosis was first described in 1875 by Hutchinson. However, leukoderma was not recognized as a feature until 1963 (Maycock et al., 1963). It was later reported by Harmon and Peterson (1972) in a patient with asthma and generalized lymphadenopathy, Kotler and Zwi (1967) in a review from South Africa, and Cornelius et al. (1973) in four additional cases. A series of eight patients was reviewed by Clayton et al. (1977), who also performed electron microscopy. Since then only a few other cases have been described (Alexis, 1994; Hall et al., 1984; Meyrick et al., 1981; Mitchell et al., 1986; Shaw, 1982).

Synonyms The earliest description of a case which would now be categorized as sarcoidosis was reported in 1889 by Besnier. He described reddish-blue lesions in the face and nose associated with swelling of the fingers. The name “lupus pernio” was given to reflect his opinion that this might be a variant of lupus vulgaris. In 1898, two more cases with a skin eruption, probably sarcoidosis, were reported by Hutchinson. He named it “Mortimer’s malady” after one of his patients. In 1899, Boeck recorded his “multiple benign sarkoid of the skin.” The current term “sarcoidosis” in fact stems from his misinterpretation of the histopathologic features. It was Boeck who first developed the concept of a generalized disease involving both skin and internal organs, a concept which was taken further by Schaumann (1917). Since then the multisystemic disease has also been known as “morbus Besnier–Boeck– Schaumann.”

Epidemiology In general, sarcoidosis mostly occurs in developed countries. Estimations vary between 10 and 200 cases per 100 000 population (James, 1988; James and Brett, 1964). In 20–30% of cases of systemic sarcoidosis, skin lesions have been observed. On the other hand, cutaneous sarcoidosis may occur without

a past history of systemic sarcoidosis (Hanno et al., 1981; Kerdel and Moschella, 1984; Veien et al., 1987). The true incidence of leukoderma as a cutaneous manifestation of sarcoidosis is unknown. In the series reported by Maycock et al. (1963), hypopigmentation was observed in 6% of the cases. However, other reports did not mention hypopigmentation (Ferguson and Paris, 1958; Longcope and Freeman, 1952; McCort, 1947; Reisner, 1944; Ricker and Clark, 1949).

General Clinical Findings — Age, Sexual Predilections, Racial Distribution Sarcoidosis in general is predominant in black people, but is not uncommon among white people especially from the southeastern parts of the USA and Scandinavia. Peak incidence is in the third or fourth decade. Females are usually more affected than males. Analyses of the cases of hypopigmentation summarized by several authors (Clayton et al., 1977; Cornelius et al., 1973; Hall et al., 1984; Maycock et al., 1963; Meyrick et al., 1981; Shaw, 1982) show that the epidemiologic features are quite similar to that of sarcoidosis in general; the high-risk group includes black females between 30 and 40 years old. However, there has been a single report of hypopigmented lesions in a 25-year-old white male patient with proven sarcoidosis (Mitchell et al., 1986).

Clinical Description of Disease Sarcoidosis may present with systemic and cutaneous symptoms. Hypopigmentation is a rare skin manifestation. Maycock et al. (1963) observed hypopigmentation in 8 of 145 patients with sarcoidosis (Fig. 40.5A, B); all the cases occurred among black patients. However, no details were given of the clinical or histologic features of the lesions. Cornelius et al. (1973) reported hypopigmented macules, papules, and nodules in four black patients with sarcoidosis. Rhodes (1975) characterized the hypopigmented lesions in seven patients. All patients were between 30 and 40 years old when first seen; in two of five, hypopigmented macules were the first presenting complaint, and in another two, hypopigmented macules appeared within two years after the diagnosis of sarcoidosis was established. All patients were black, and the female to male ratio was 5 : 2. The lesions are described as circumscribed or poorly marginated macules or plaques that vary from 1 cm to 3 cm in diameter with or without induration. Two patients had papules within the white macules and one had raised borders with a central violaceous hue. Lesions were mostly located on the arms and legs but were also seen on the face in two patients and on the trunk in one. The lesions had no epidermal component other than hypopigmentation and were nonsymptomatic. Spontaneous repigmentation occurred in one case (Rhodes, 1975). In 1977, Clayton et al. described a series of eight black patients with hypopigmented lesions of sarcoidosis. The female:male ratio was 5 : 3. Six patients showed dermal nodules surrounded by hypopigmentation and four had hypopigmented macules, both mostly located on the thighs and upper arms. The nodules were dark red–brown in the center and light tan in the periphery. Most were 1–6 cm in diameter. Margins were 751

CHAPTER 40

A

otic macules. Alexis (1994) also reported a sarcoid patient with hypopigmented areas but with repeatedly negative skin biopsies. Hypopigmented cutaneous lesions lacking sarcoidal granulomas seem to be more commonly macules (Clayton et al., 1977; Hall et al., 1984); the presence of induration, papules, and nodules appears to be related to the presence of granulomas (Alexis, 1994). Furthermore, Cornelius et al. (1973) observed dopa-positive melanocytes in the basal layer in two patients. The melanocyte counts were normal, which was confirmed by Hubler (1977). The epidermal turnover rates of normal and abnormal skin were the same: 23 days (Cornelius et al., 1973). Thus, the histologic picture of the sarcoid granuloma in a macule of leukoderma shows no major differences when compared with a granuloma from normal pigmented skin.

Electron Microscopy

B

Electron microscopic examinations were first performed by Clayton et al. (1977). Some melanocytes had normal features, others showed cytoplasmic vacuolation, marked dilatation of rough endoplasmic reticulum and degenerative changes of mitochondria. Melanosomes showed normal morphology. The number of melanosomes in keratinocytes was decreased; those observed were packaged as singles. These findings suggest that a disturbance in the transfer of melanosomes may be responsible for the leukodermic defect. Changes in the dermis include the presence of melanin-laden macrophages, granulomatous infiltration, mast cells, and some Schwann cells containing ingested melanosomes.

Fig. 40.5. (A, B) Hypopigmented macules with erythema typical of sarcoidosis (see also Plate 40.3, pp. 494–495).

Laboratory Findings and Investigations

indiscrete, sometimes with perifollicular macules of pigmentation. Some nodules were tender. The behavior of hypopigmented lesions in patients with sarcoidosis does not seem to correlate with disease activity.

Associated Disorders No disorder has been found to have a possible association with the appearance of hypopigmented lesions in sarcoidosis. Diseases associated with sarcoidosis in general include infections, immunologically mediated conditions, Cushing’s syndrome, diabetes insipidus, vasculitis, malignancy, necrobiosis lipoidica, granuloma annulare, psoriasis, gout, pyoderma gangrenosum, primary biliary cirrhosis, and porphyria cutanea tarda (Savin, 1992).

Routine Histology, Histochemistry, and/or Immunochemistry Histopathology Cornelius et al. (1973) found noncaseating granulomas in the hypomelanotic macules in all of the patients but no granulomas in the surrounding normal-appearing skin. Granulomas were also found in the hypopigmented sarcoid lesions studied by Clayton et al. (1977) but only in one of two hypomelan752

Certain laboratory examinations have to be done to establish the diagnosis of sarcoidosis, especially in a patient with hypopigmented cutaneous lesions as a primary manifestation of the disease. Blood analysis may reveal hypochromic and microcytic anemia, eosinophilia, leukopenia, and hypergammaglobulinemia. The erythrocyte sedimentation rate is elevated in two-thirds of patients and, if present with erythema nodosum, may rise and fall as the erythema nodosum exacerbates and remits. Alkaline phosphatase levels may increase with or without clinical granulomatous invasion of bone and liver. Hypercalcemia, hypercalciuria and increased hydroxyproline excretion may be observed in some patients. Serum levels of angiotensin-converting enzyme are raised in about 60% of cases. IgA may be markedly elevated and IgG somewhat elevated. There is often an impaired delayed hypersensitivity. A positive Kveim test has been observed in 75% of the patients with sarcoidosis (Anderson, 1963). A chest X-ray may show hilar lymphadenopathy. Hand X-rays show cystic changes in chronic disease only and usually when there are clinical abnormalities in the fingers. Sputum should be examined and cultured for acid-fast bacilli, and a weak or negative Mantoux test may add weight to the diagnosis of sarcoidosis. In difficult cases, biopsy may be taken from lymph nodes, liver, muscles, nerves, or bronchial mucosa (Savin, 1992).

MISCELLANEOUS HYPOMELANOSES: HYPOPIGMENTATION

Criteria for Diagnosis The diagnosis of sarcoidosis should be suspected when hypopigmented macules on arms and legs are associated with somewhat indurated plaques, especially in a black female patient with uveitis, hilar or peripheral adenopathy, or pulmonary abnormalities. Additional laboratory investigations may be helpful.

Differential Diagnosis The differential diagnosis includes leprosy (see Chapter 36), postinflammatory hypopigmentation (see Chapter 37), idiopathic guttate hypomelanosis, mycosis fungoides (see Chapter 50), pinta, pityriasis versicolor (see Chapter 36), and pityriasis lichenoides chronica (see Chapter 37) (Meyrick et al., 1981; Savin, 1992).

Pathogenesis The mechanism of hypopigmentation is unknown. According to histopathologic findings, it is obviously not related to decreased melanocyte count or to altered keratinocyte turnover. Ultrastructural studies indicate that a disturbance of the melanosome transfer may be responsible. Since the granulomatous process is not in direct contact with the basal melanocyte-containing layer, some other dermal–epidermal interaction must be responsible for the disturbance in the melanogenesis (Ortonne et al., 1983). A factor secreted by histiocytes may be involved. Also, the histiocytes may have an indirect effect, such as a vascular-mediated influence on epidermal melanocytes (Clayton et al., 1977).

Animal Models There are no animal models.

Treatment None of the lesions described by Clayton et al. (1977) repigmented with topical, oral, or intralesional corticosteroids. One patient with an immune defect was reported to show improvement in macular lesions after treatment with transfer factor (Ortonne et al., 1983). Also repigmentation with PUVA has been reported (Mosher et al., 1987).

Prognosis The presence of hypopigmented macules appears to be of no prognostic value in the development of the disease sarcoidosis. Spontaneous repigmentation seems to be unusual; only one case has been reported (Clayton et al., 1977).

References Alexis, J. B. Sarcoidosis presenting as cutaneous hypopigmentation with repeatedly negative skin biopsies. Int. J. Dermatol. 33:44–45, 1994. Anderson, R. The Kveim test in sarcoidosis. Lancet 21:650–653, 1963. Besnier, E. Lupus pernio de la face: synovites fongueuses symmetriques des extremites superieures. Ann. Dermatol. Syphiligr. (Paris) 10: 333–336, 1889. Boeck, C. Multiple benign sarcoid of the skin. J. Cutan. Genito-Urin. Dis. 17:543–550, 1899.

Clayton, R., A. Breathnach, B. Martin, and M. Feiwel. Hypopigmented sarcoidosis in the Negro: Report of eight cases with ultrastructural observations. Br. J. Dermatol. 96:119–125, 1977. Cornelius, C. E., K. M. Stein, W. J. Hanshaw, and D. A. Spott. Hypopigmentation and sarcoidosis. Arch. Dermatol. 108:249–251, 1973. Ferguson, R. H., and J. Paris. Sarcoidosis: study of 29 cases, with a review of splenic, hepatic, mucous membrane, retinal, joint manifestation. Arch. Intern. Med. 101:1065–1084, 1958. Hall, R. S., J. F. Floro, and L. E. King. Hypopigmented lesions in sarcoidosis. J. Am. Acad. Dermatol. 11:1163–1164, 1984. Hanno, R., A. Needelman, and R. A. Eiferman. Cutaneous sarcoidal granulomas and the development of systemic sarcoidosis. Arch. Dermatol. 117:202–207, 1981. Harmon, R., and P. Peterson. Cutaneous sarcoidosis with hypopigmentation and asthma. Rocky Mountain Med. J. 69:70–72, 1972. Hubler, W. R. Hypomelanotic canopy of sarcoidosis. Cutis 19:86–88, 1977. Hutchinson, J. A. Illustrations of Clinical Surgery. London: Churchill, 1875, p. 42. James, D. G. Sarcoidosis around the world. Postgrad. Med. J. 64:177–179, 1988. James, D. G., and G. Z. Brett. Prevalence of intrathoracic sarcoidosis in Britain. Acta Med. Scand. Suppl. 425:115–117, 1964. Kerdel, F. A., and S. L. Moschella. Sarcoidosis: an updated review. J. Am. Acad. Dermatol. 11:1–19, 1984. Kotler, M. N., and S. Zwi. Sarcoidosis in South Africa: clinical manifestations. S. Afr. Med. J. 41:615–624, 1967. Longcope, W. T., and D. G. Freeman. A study of sarcoidosis based on combined investigation of 160 cases including 30 autopsies from Johns Hopkins Hospital and Massachusetts General Hospital. Medicine (Baltimore) 31:1–132, 1952. Maycock, R. L., P. Bertrand, C. E. Morrison, and J. H. Scott. Manifestations of sarcoidosis: analysis of 145 patients with a review of nine series selected from the literature. Am. J. Med. 35:67–89, 1963. McCort, J. J. Sarcoidosis: a clinical and roentgenologic study of 28 proven cases. Arch. Intern. Med. 80:293–321, 1947. Meyrick, T., P. H. McKee, and M. M. Black. Hypopigmented sarcoidosis. J. R. Soc. Med. 74:921–923, 1981. Mitchell, I. C., M. C. Sweatman, M. H. A. Rustin, and R. Wilson. Ulcerative and hypopigmented sarcoidosis. J. Am. Acad. Dermatol. 15:1062–1065, 1986. Mosher, D. B., T. B. Fitzpatrick, J.-P. Ortonne, and Y. Hori. Disorders of pigmentation. In: Dermatology in General Medicine, 3rd ed., vol. 1, T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M. Freedberg, and K. F. Austen (eds). New York: McGraw-Hill, Inc.; Section 14. Disorders of Melanocytes, 1987, pp. 794–876. Ortonne, J.-P., D. B. Mosher, and T. B. Fitzpatrick. Topics in Dermatology: Vitiligo and Other Hypomelanoses of Hair and Skin. New York: Plenum Medical Book Company, 1983, pp. 613–672. Reisner, D. Boeck’s sarcoid and systemic sarcoidosis: a study of 35 cases. Am. Rev. Tuberc. 49:289–437, 1944. Rhodes, A. 1975. Case presentation #4: New England Dermatology Society, 12 November. Quoted in Ricker, W., and M. Clark. Sarcoidosis: a clinicopathologic review of 300 cases including 22 autopsies. Am. J. Clin. Pathol. 19:725–749, 1949. Savin, J. A. Sarcoidosis. In: Textbook of Dermatology, 5th ed., R. H. Champion, J. L. Burton, and F. J. G. Ebling (eds). Oxford: Blackwell Scientific Publications, 1992, pp. 2383–2406. Schaumann, J. Etude sur le lupus pernio et ses rapports avec les sarcoides et la tuberculose. Ann. Dermatol. Syphiligr. (Paris) 6:357–373, 1917. Shaw, M. Sarcoidosis presenting with hypopigmentation. Clin. Exp. Dermatol. 7:115–118, 1982. Veien, N. K., D. Stahl, and H. Brodthagen. Cutaneous sarcoidosis in Caucasians. J. Am. Acad. Dermatol. 16:534–540, 1987.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Miscellaneous Hypomelanoses: Extracutaneous Loss of Pigmentation Sections Alopecia Areata Wiete Westerhof Heterochromia Irides Wiete Westerhof, David Njoo, and Henk E. Menke Senile Canities Wiete Westerhof, David Njoo, and Henk E. Menke Sudden Whitening of Hair Wiete Westerhof, David Njoo, and Henk E. Menke

Alopecia Areata

tion, and racial distribution of the pigmentary phenomena associated with alopecia areata.

Wiete Westerhof

Historical Background Alopecia areata is a hair disease which is often associated with various conditions of hypopigmentation and depigmentation affecting both skin and hair. There is an association with vitiligo (see Chapter 30) and the Vogt–Koyanagi–Harada syndrome (see Chapter 39). Lubowe (1958) reported that in a large majority of his patients with alopecia totalis, hypomelanosis of the scalp and face was present. With corticosteroid-corticotropin therapy repigmentation could be achieved. The patients described by Demis and Weiner (1963) suffered from “alopecia universalis onychodystrophy and total vitiligo.” However, the vitiligo was more of a “generalized pallor” than an actual vitiligo and one of the patients, still kept the ability to tan. “Mysterious” historical reports on sudden whitening of the hair are probably caused by an acute selective shedding of pigmented hairs in combination with the selective sparing of nonpigmented hairs in the course of a diffuse alopecia areata (see section on “Sudden whitening of hair”). During the regrowth stage of alopecia areata, new hairs are initially nonpigmented. This amelanosis is usually temporary and the amelanotic hairs are eventually replaced by normal pigmented hairs (Ortonne et al., 1983).

Epidemiology The incidence of alopecia areata is reported to be approximately 0.05–0.1% in the Caucasian population. Among dermatologic patients visiting outpatient clinics the incidence is reported to vary from 0.9% to nearly 4% (Gollnick and Orfanos, 1990). Alopecia areata occurs in 4% of patients with vitiligo (Muller and Winkelman, 1963). About 70% of the patients diagnosed as having Vogt–Koyanagi–Harada syndrome develop alopecia areata (Rosen, 1945) (Chapter 39). White hairs, during the regrowth phase of alopecia areata, are seen in almost every patient (Gollnick and Orfanos, 1990). There is no information available about age, sexual predilec754

General Clinical Findings — Age, Sexual Predilections, Racial Distribution The majority of recently published reports indicate a higher incidence of alopecia areata in females (Friedmann, 1985; Gollnick and Orfanos, 1990). More males are affected according to Bastos Araujo and Poiares Baptista (1967) (male:female = 3:1). Muller and Winkelman (1963) reported an equal distribution among men and women. According to Dawber et al. (1993) the onset of disease may occur at any age with a peak decade lying at some point between the ages of 20 and 50 years. Muller (1963) reported onset before age 2 in 2% of 736 cases studied in North America. There is some genetic predisposition. Familial incidence is often reported to be high, varying between 5% and 25% (Gollnick and Orfanos, 1990).

Clinical Description of Disease Regrowing hairs in alopecia areata lesions are initially thin and often nonpigmented (Fig. 41.1). New hairs are often seen in the center of the lesions, expanding in a ringlike pattern. Within several weeks these hairs regain their original thickness and color. In some cases regrowth of white hair may be observed over a long period of time. Also regrowth in one region of the scalp may occur while hair loss is progressing in others (Gollnick and Orfanos, 1990). (For the clinical description of vitiligo and Vogt-Koyanagi-Harada syndrome see Chapters 30 and 39, respectively.)

Routine Histology, Histochemistry, and/or Immunochemistry Messenger and Bleehen (1984) performed light and electron microscopic studies on regrowing white hair follicles of alopecia areata patients. They found that the number of melanocytes and their melanization were decreased. Other observations such as a decreased number of melanosomes within keratinocytes and autophagocytosis of melanosomes

MISCELLANEOUS HYPOMELANOSES: EXTRACUTANEOUS LOSS OF PIGMENTATION

of lichen ruber, folliculitis decalvans, alopecia parvimaculata atrophicans, and all types of “pseudopelade” with mild scarring. Other inflammatory conditions which show scalp involvement with circumscribed but clearly scarring lesions can be easily excluded: morphea, discrete lesions of discoid lupus erythematosus, rare conditions such as incontinentia pigmenti and atypical porokeratosis of Mibelli and bullous disorders such as cicatricial pemphigoid. Less frequent are diseases such as lichen sclerosus et atrophicus or necrobiosis lipoidica of the scalp (Gollnick and Orfanos, 1990).

Pathogenesis

In general, routine laboratory investigations are not useful (Gollnick and Orfanos, 1990).

The exact mechanisms behind the occurrence of hypopigmentation and depigmentation of skin and hair in patients with alopecia areata are unknown. Based on light and electron microscopic findings Messenger and Bleehen (1984) suggested that the pigmentary defect in the regrowing white hair is probably postinflammatory and also included the possibility that the pigmentary system is of primary importance in the disease process of alopecia areata. Tobin and colleagues (1990) were able to visualize morphological degenerative changes in melanocytes in hair bulbs of acute alopecia areata and postulated that hair bulb melanocytes may be actively involved in the pathogenesis of alopecia areata. Nutbrown et al. (1995) confirmed these findings on electron microscopy, but also found abnormalities in the ultrastructure of melanocytes and the outer root sheath of clinically normal hair follicles from alopecia areata scalps indicating a subclinical condition in alopecia areata. Paus et al. (1993) attempted to clarify the mechanisms behind the selective attack to anagen hair follicles. Based on immunologic studies, they hypothesized an autoimmune condition in which T-cytotoxic lymphocytes attack melanogenesis-related proteins (MRP-DP), which are only generated during anagen and which are exposed by abnormal major histocompatibility (MHC)-I expression in the anagen hair bulb. However, fundamental proof for an autoimmune mechanism is still lacking. Moreover, Khoury and Price (1995) failed to demonstrate cell-surface-reactive antibodies against cultured autologous melanocytes in alopecia areata sera. These findings, however, do not give a full explanation for the development of white hairs during the regrowth phase in alopecia areata.

Criteria for Diagnosis

Animal Models

Alopecia areata is a diagnosis made on clinical examination. “Exclamation mark” hairs are pathognomic but are not always present. A trichogram may give useful information (Gollnick and Orfanos, 1990).

There have been no animal models to further clarify the association between hypopigmentation and depigmentation and alopecia areata.

Fig. 41.1. Regrowing depigmented hairs in a patient with alopecia areata (see also Plate 41.1, pp. 494–495).

within melanocytes suggested a defective pigment transfer. Tobin et al. (1990) performed ultrastructural studies on hair bulbs from patients with acute alopecia areata and observed degenerative changes in the melanocytes. The main features of the affected melanocytes, both at the dermal papilla/matrix junction and in the inner matrix, were nuclear and cytoplasmic condensation, indicative of impending cell death. Increased numbers of bizarre melanosomes were found in affected melanocytes compared with normal ones. These dystrophic melanosomes showed incomplete or “aborted” melanization, resulting in poor pigment deposition, and were disrupted, enlarged, and rounded, with loss of normal ellipsoidal shape. Furthermore, an unusual outer root sheath distribution of hair bulb melanocytes was observed. Other, but atypical, melanosome changes included marked pigment displacement into peribulbar and dermal papillary melanophages. In the dermal papilla clumped melanin granules formed giant spherical complexes within discernible limiting membranes, which were sometimes associated with lymphocytes.

Laboratory Findings and Investigations

Treatment Differential Diagnosis Alopecia areata can be easily diagnosed on clinical grounds. The rare diffuse type, alopecia areata in the elderly, or the simultaneous occurrence with other types of hair loss may give diagnostic difficulties. Circumscribed alopecic lesions also occur in alopecia mucinosa. Further, alopecia areata has to be differentiated from syphilitic alopecia, the small macular type

It has been reported that systemic steroid treatment of alopecia areata may lead to immediate repigmentation of regrowing hairs (Gollnick and Orfanos, 1990).

Prognosis Regrowing hairs are initially white and thin with variations in caliber. Pigmentation does not seem to occur until the hair has 755

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grown more than 1–2 mm above the scalp. It begins in the medulla of the hair shaft and full melanization of the hair cortex develops later (Gollnick and Orfanos, 1990).

References Bastos Araujo, A., and A. Poiares Baptista. Algunas consideracions sobre 300 casos de pelada. Trab. Soc. Portuges. Dermatol. Venereol. 15:135–139, 1967. Dawber, R. P. R., F. J. G. Ebling, and F. T. Wojnarowska. Disorders of hair. In: Textbook of Dermatology, 5th ed., R. H. Champion, J. L. Burton, and F. J. G. Ebling (eds). Oxford: Blackwell Scientific Publications, 1993, pp. 2586–2595. Demis, D. J., and M. A. Weiner. Alopecia universalis, onychodystrophy and total vitiligo. Arch. Dermatol. 88:195–201, 1963. Friedmann, P. S. Clinical and immunologic associations of alopecia areata. Semin. Dermatol. 4:9–24, 1985. Gollnick, H., and C. E. Orfanos. Alopecia areata. In: Pathogenesis and Clinical Picture: Hair and Hair Diseases, C. E. Orfanos, and R. Happle (eds). Berlin: Springer-Verlag, 1990, pp. 529–569. Khoury, E. L., and V. H. Price. No cell-surface-reactive antibodies against cultured autologous melanocytes found in alopecia areata sera. J. Invest. Dermatol. 104(5 Suppl):24S–25S, 1995. Lubowe, I. I. Pigmentation and alopecia totalis. Arch. Dermatol. 77:593–594, 1958. Messenger, A. G., and S. S. Bleehen. Alopecia areata: Light and electron microscopic pathology of the regrowing white hair. Br. J. Dermatol. 110:155–162, 1984. Muller, S. A., and R. K. Winkelman. Alopecia areata. Arch. Dermatol. 88:290, 1963. Nutbrown, M., S. P. MacDonald Hull, W. J. Cunliffe, and V. A. Randall. Abnormalities in the ultrastructure of melanocytes and the outer root sheath of clinically normal hair follicles from alopecia areata scalps. J. Invest. Dermatol. 104:12S–13S, 1995. Ortonne, J.-P., D. B. Mosher, and T. B. Fitzpatrick. Topics in Dermatology: Vitiligo and Other Hypomelanoses of Hair and Skin. New York: Plenum Medical Book Company, 1983, pp. 613–672. Paus, R., A. Slominski, and B. M. Czarnetski. Is alopecia areata an autoimmune-response against melanogenesis-related proteins, exposed by abnormal MHC class I expression in the anagen hair bulb? Yale J. Biol. Med. 66:541–554, 1993. Rosen, E. Uveitis with poliosis, vitiligo, alopecia and dysacousia (Vogt-Koyanagi syndrome). Arch. Ophthalmol. 33:281–292, 1945. Tobin, D. J., D. A. Fenton, and M. D. Kendall. Ultrastructural observations on the hair bulb melanocytes and melanosomes in acute alopecia areata. J. Invest. Dermatol. 94:803–807, 1990.

Heterochromia Irides Wiete Westerhof, David Njoo, and Henk E. Menke

Historical Background In medical textbooks heterochromia irides is described as a congenital or an acquired symptom. It may be a harmless sign, but may also represent a serious underlying disease (see “Clinical description”). Many authors have given Hutchinson (1869) credit for first describing a difference in the color of the irides, which was associated with cataract. However, Calhoun, in his classic thesis (1919), noted it was probably Lawrence (1853) who first reported irregularities in the color of the iris in two cases: in one of slowly changing color and another of changed color of the iris with cataract. Lawrence in his article also quoted 756

Wilde who in 1848 mentioned the fact that heredity plays an important role in iris abnormalities. This section discusses the relationship of heterochromia irides with Horner syndrome. In 1875, Horner first drew attention to a case in which a light iris occurred on the same side with a ptosis of sympathetic origin. It was Mayou in 1910 who first accurately described the classic signs of the syndrome which include ptosis, miosis, anhidrosis, and facial hemiatrophy (Mayou, 1910). Later, other observers, including Bistis (1912), added heterochromia irides as a symptom to Horner syndrome.

Synonyms Calhoun (1919) found that several terms have been used in the past to describe the inequality in the color of the irides, such as “heteroglaucus” in ancient times. Weill (1904) used the term “heterophthalmus;” “anisoiridochromia” was mentioned by Scalinci (1915). Malgat in 1895 suggested the name “chromoheteropia” and in his opinion the term “heterochromia” should be applied to those cases in which parts of the same iris have different colors. Butler (1911) used the name “heterochromic cyclitis” in cases where a cyclitis accompanied the iris decoloration whereas Fuchs (1906) used the term “chronic cyclitis with decoloration of the iris” to describe a similar condition. Bocock (1919) also mentioned the word “irideterochromia.” According to Calhoun the current term “heterochromia irides” exactly expresses and implies the clinical picture from its derivation, “xteroo crwma,” which means “other color.”

Epidemiology The true incidence of heterochromia irides is unknown, but is assumed to be rare. It appeared difficult to assess the incidence because slight differences in eye color are often overlooked, especially in light-eyed individuals. According to a review article by Fields and Barker (1992) there have been numerous case reports on Horner syndrome, but estimations of the prevalence in the population have not been given. Weinstein et al. (1980) estimated that no more than 5% of cases with Horner syndrome are of congenital origin.

General Clinical Findings — Age, Sexual Predilections, Racial Distribution It is well known that heterochromia irides may be genetically inherited. Acquired forms may occur at any age and are often associated with ocular diseases. Horner syndrome is mostly acquired and may occur at any age. Both sexes may be equally affected. The congenital type is rare (Weinstein et al., 1980).

Clinical Description of Disease Heterochromia irides is an asymmetry of iris pigmentation and structure (Chapter 4). There are two forms of heterochromia irides: a hypopigmentation of the iris with iris hypoplasia or a hyperpigmentation with iris hyperplasia. Both eyes or only one eye may be involved (Figs 41.2 and 41.3). There may be a partial or complete defect. Horner syndrome may be congenital or acquired. The essen-

MISCELLANEOUS HYPOMELANOSES: EXTRACUTANEOUS LOSS OF PIGMENTATION

Fig. 41.2. Heterochromia irides affecting both eyes (see also Plate 41.2, pp. 494–495).

Fig. 41.4. Heterochromia irides in a patient with Waardenburg syndrome (see also Plate 41.4, pp. 494–495). Fig. 41.3. Unilateral, partial heterochromia irides (see also Plate 41.3, pp. 494–495).

tial characteristics are a moderate miosis, a minimal ptosis, and narrowing of the palpebral fissure. Other typical clinical findings are anhidrosis on the affected side of the face, an “upside down” ptosis (elevation of the lower lid), conjunctival congestion, and transient ocular hypotonia. Heterochromia irides is almost only seen in patients with the congenital form of Horner syndrome and is transmitted as an irregular dominant trait. Several familial cases have been reported (Calhoun, 1919; Durham, 1958; Luxenberg, 1994). Acquired heterochromia irides in Horner syndrome is rare and is often associated with a (traumatic) lesion in the ocular sympathetic pathways (Diesenhouse et al., 1992).

Associated Disorders According to Gladstone (1969) heterochromia irides may be associated with the following disorders: ∑ The “simple type” heterochromia irides may be familial and benign, but may also occur in diseases such as Waardenburg syndrome (Fig. 41.4; see also Chapters 6 and 29), piebaldism,

status dysraphicus (Bremer syndrome), Sturge–Weber syndrome, Romberg syndrome, and vitiligo (“vitiligo irides”). ∑ The “complicated type” heterochromia is associated with ocular disease. “Complicated heterochromia of the hypopigmented type” can be observed in Fuchs heterochromic cyclitis, and Posner–Schlossmann syndrome, and is associated with diffuse unilateral iris atrophy or with infiltration of the iris by an amelanotic tumor. ∑ “Complicated heterochromia of the hyperpigmented type” may follow a childhood eye injury, hyphema, iron foreign body, vascular abnormalities, melanosis bulbi, or malignant melanoma of the iris. ∑ Horner syndrome represents the “sympathetic type” of heterochromia irides. This syndrome is associated with a neurogenic lesion of the sympathetic nerve supply of the iris which may be either congenital or acquired. Heterochromia irides may also occur in patients with vitiligo. It has long been assumed, however, that no matter how extensive the pigment loss and how long the duration of loss, under ordinary observation, the color of the irides does not change in vitiligo. Albert et al. (1979), who have specially 757

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drawn attention to ocular abnormalities occurring with vitiligo, presumed that the few cases of depigmentation of the iris in vitiligo resulted from iris inflammation (iritis, uveitis) due to an immunologically mediated destruction of iris melanocytes (Albert et al., 1979; Lerner et al., 1977). In a survey of 151 patients with vitiligo, Cowan et al. (1986) found the occurrence of iris transillumination defects to be significantly less in black patients than in white patients with vitiligo (5 of 91 and 14 of 60, respectively). Moynahan (1961) described a patient with virtually complete loss of pigment from his skin and who developed eye color changes in one eye, from brown to blue.

Routine Histology, Histochemistry, and/or Immunochemistry, and Animal Models Histopathologic findings of heterochromia irides have mostly been based upon animal models. Calhoun (1919) quoted the microscopic results performed on rabbit eyes by Bistis (1912) which showed great reduction of the pigment in the anterior layer and thickening of vessel walls. There were no melanocytes detected in the iris stroma, instead, large numbers of unidentified “nuclei” were observed. These nuclei were round or oval and were situated in a basement substance composed of fibrillary connective tissue. He also found deposits in the posterior surface of the cornea. These findings, together with a cloudiness of the aqueous body and the vessel wall changes, were in his opinion suggestive of an inflammation process. Calhoun (1919) by his own microscopic studies on rabbits as well, could confirm most of Bistis’ findings but was unable to detect signs of inflammation. Biochemical and electron microscopic studies have been performed to investigate the relationship between the sympathetic nerve system and ocular melanocytes. The existence of such a connection is conceivable since cells of the sympathetic ganglia and iris melanocytes are both neural crest derivatives and thus may share biochemical similarities. In rabbit models, Laties and Lerner (Laties, 1974; Laties and Lerner, 1975) have demonstrated a rapid decrease in tyrosinase activity of ocular melanocytes that correlated with a change in iris pigmentation after interruption of the preganglionic and postganglionic sympathetic pathways. In addition, Mukuno and Witmer (1977) have shown evidence of synaptic contact between human ocular melanocytes and adrenergic (and possibly cholinergic) nerve terminals. Other ultrastructural studies of iris melanocytes in animals showed similar findings which further favors the concept of a neural regulation of ocular melanocytes (Ehinger and Falck, 1970; Ringvold, 1975). McCartney et al. (1992) were one of the few investigators who were able to perform electron microscopic studies on a human iris with heterochromia from a patient with congenital Horner syndrome. Comparison of his normal brown iris with the depigmented blue iris showed depletion of anterior border cells and absence of sympathetic nerve fibers. Stromal melanocytes were also diminished but melanosome numbers within the remaining cells were not significantly different. 758

Laboratory Findings and Investigations By application of cocaine 4% one can demonstrate the disturbance in the sympathetic innervation in Horner syndrome. A healthy pupil dilates after 30–45 minutes while a “Horner” pupil does not. When the lesion is postganglionic (i.e., superior cervical neuron), the affected pupil does not react to hydroxyamphetamine (1%) but shows supersensitivity to phenylephrine (1%) when this is given after cocaine. Routine laboratory investigations are not useful.

Criteria for Diagnosis Heterochromia irides is a clinical symptom and one must look for other existing signs of an underlying disease (see “Associated disorders”). To diagnose “Horner syndrome” in a patient with heterochromia irides the examiner must find typical features such as miosis, ptosis, and anhidrosis on the affected side. Heterochromia irides is found mostly in the congenital form of Horner syndrome.

Differential Diagnosis Regarding the symptom heterochromia irides as such, there should be no differential diagnostic problems. However, regarding the underlying diseases associated with this phenomenon there are several differential diagnostic possibilities (see “Associated disorders”).

Pathogenesis It is generally accepted that heterochromia irides may occur as a genetically inherited feature without any sign of development of a pathologic lesion. Much has become clear about the role of the sympathetic innervation to the iris in the pathogenesis of heterochromia irides. The possible mechanisms of a defect in the sympathetic nerve system causing iris discoloration will be discussed, using Horner syndrome as a model. In a review of the literature by Fields and Barker (1992) it was noted that acquired Horner syndrome has been attributed to many different causes including birth trauma (which was also mentioned by Calhoun in 1919), inflammation of the cavernous sinus with sixth nerve palsy, lesions of the neck including gunshot wounds through the superior cervical ganglion, injudiciously placed intercostal drains, tumors of the apex of the lung (Pancoast tumor), schwannomas of the superior cervical ganglion, migraine headaches, lesion of the midbrain, orbital neuroblastoma, local anesthesia in the area of the sympathetic pathway, Lyme disease, and others. All these causes indicate a neural genesis of the syndrome. Heterochromia irides is frequently seen in congenital Horner syndrome. On the other hand heterochromia in acquired Horner syndrome is rare. Calhoun had remarked on this rare incidence in his thesis in 1919. In his series of experiments on rabbits he was able to show that the depletion of iris pigmentation was related to the length of survival after sympathectomy. Bistis (1912) reported a 38-year-old woman in whom heterochromia developed one year after the patient was diagnosed with Horner syndrome after injury to the sympathetic nerves during a cervical operation. Makley and

MISCELLANEOUS HYPOMELANOSES: EXTRACUTANEOUS LOSS OF PIGMENTATION

Abbott (1955) described a 35-year-old man who developed heterochromia two years after the onset of Horner syndrome secondary to a right superior cervical ganglionectomy. Laties (1974) noted heterochromia in a woman 15 years after Horner syndrome had developed after removal of a neck tumor at the carotid bifurcation at the age of 14. Miller (1985) reported heterochromia in two subjects with acquired Horner syndrome. In two subjects (one a 12-year-old boy), the Horner syndrome followed an internal carotid artery cutdown, and in the second patient, it followed thyroidectomy. In 1992, Diesenhouse et al. presented two cases: a 48-year-old man developing heterochromia 15 years after Horner syndrome was diagnosed after a reparative surgery of a gunshot wound on his right lower neck and shoulder, and a 65-year-old woman who had Horner syndrome after removal of a neurofibroma of her left lower neck and who developed heterochromia five years after this operation. Above reports show that a traumatic sympathetic lesion may lead to an acquired form of Horner syndrome while a heterochromia may develop, sometimes many years after the syndrome has been diagnosed. So, what is the link between a sympathetic paralysis and the development of a pigmentary disorder of the iris? Calhoun (1919) in his thesis emphasized that “in a large percentage of the cases, paralysis of the cervical sympathetic nerves can cause heterochromia irides and that the paralysis could be inherited. The defect may occur as early as in fetal life. The pigmentary changes in the iris can only be explained by nutritional disturbances of trophic origin; first a vasodilatation, then a hyaline degeneration of the adventitia, and finally a contraction of the vessel lumen.” According to McCartney et al. (1992) we can learn much about the pathogenesis of heterochromia irides by simply viewing the embryogenesis of the iris. Pigmentation of the iris depends predominantly on the distribution of pigment within melanocytic cells on the anterior border and within the stroma. These cells, like the sympathetic ganglia cells, are derived from the neural crest and migrate into the human iris late in gestation and postnatally, so that full pigmentation is not normally present until nine months after birth. Both migration and maintenance of neural crest-derived, iris pigment-containing cells appear to be under sympathetic control. The concept of a neural regulation of iris melanocytes has been supported by Laties (1974) who demonstrated a decrease in tyrosinase activity in iris melanocytes of rabbits after sympathectomy, and by the ultrastructural studies of Mukuno and Witmer (1977) which demonstrated synaptic contact between human iris melanocytes and adrenergic nerve terminals. Based on the findings of Laties (1974) and Mukuno and Witmer (1977), Weinstein et al. (1980) postulated that probably a “transsynaptic degeneration” causing dysfunction of one or more sympathetic neurons is responsible for the development of congenital heterochromia irides. In addition, the neural lesion may be followed by loss of normal postnatal migration of cells into the iris, as decreased numbers of melanocytes can be observed in the iris stroma (McCartney et al., 1992). Diesenhouse et al. (1992) agreed with the theories of Weinstein. They explained the development of acquired heterochromia irides by the theory that a

similar trans-synaptic degeneration may reduce the neuronal innervation of iris melanocytes, resulting in decreased tyrosinase activity. The iris melanocytes in early life are more likely to be responsive to these trophic influences and this responsiveness is probably lost in the large majority of “older” melanocytes, thus probably causing delayed or even absent development of heterochromia irides in the adult subject.

Treatment There is no treatment for heterochromia irides.

Prognosis Spontaneous or induced repigmentation of the affected iris has not been reported.

References Albert, D. M., J. J. Nordlund, and A. B. Lerner. Ocular abnormalities occurring with vitiligo. Ophthalmology 86:1145–1158, 1979. Bistis, J. La paralysis du sympathetique dans l’etiologie de l’heterochromie. Arch. d’Ophthalmol. 32:578, 1912. Calhoun, F. P. Causes of heterochromia irides with special reference to paralysis of the cervical sympathetic. Am. J. Ophthalmol. 2:255–269, 1919. Cowan, C. L., Jr., R. M. Halder, P. E. Grimes, S. G. Chakrabarti, and J. A. Kenney Jr. Ocular disturbances in vitiligo. J. Am. Acad. Dermatol. 15:17–24, 1986. Diesenhouse, M. C., D. A. Palay, N. J. Newman, K. To, and D. M. Albert. Acquired heterochromia with Horner syndrome in two adults. Ophthalmology 99:1815–1817, 1992. Durham, D. G. Congenital hereditary Horner’s syndrome. Arch. Ophthalmol. 60:939–940, 1958. Ehinger, B., and B. Falck. Innervation of iridic melanophores. Z. Zellforsch. Mikroskop. Anat. 105:538–542, 1970. Fields, C. R., and F. M. Barker. Review of Horner’s syndrome and a case report. Optom. Vis. Sci. 69:481–485, 1992. Gladstone, R. M. Development and significance of heterochromia of the iris. Arch. Neurol. 21:184–192, 1969. Hutchinson, J. Notes of miscellaneous cases. Royal. London Oph. Hospital Reports VI:44, 1869. Laties, A. Ocular melanin and the adrenergic innervation to the eye. Tran. Am. Ophthalmol. Soc. 72:560–605, 1974. Laties, A. M., and A. B. Lerner. Iris color and relationship of tyrosinase activity to adrenergic innervation. Nature 255:152–153, 1975. Lerner, A. B., J. J. Nordlund, and D. M. Albert. Pigment cells in the eyes of people with vitiligo. N. Engl. J. Med. 296:232, 1977. Luxenberg, M. N. Congenital bilateral heterochromia of the choroid and iris. Arch. Ophthalmol. 112:1247–1248, 1994. Makley, T. A., and K. Abbot. Neurogenic heterochromia: report of an interesting case. Am. J. Ophthalmol. 59:927–928, 1965. Mayou, M. S. Heterochromia irides associated with paralysis of the sympathetic in early life. Trans. Ophthalmol. Soc. U. K. 30:196–197, 1910. McCartney, A., P. Riordan-Eva, R. Howes, and D. J. Spalton. Horner’s syndrome: an electron microscopic study of a human iris. Br. J. Ophthalmol. 76:746–749, 1992. Miller, N. R. Walsh and Hoyt’s Clinical Neuro-Ophthalmology, 4th ed., vol. 2. Baltimore: Williams and Wilkins, 1985, pp. 500–511. Moynahan, E. J. Acquired albinism with intraocular depigmentation. Proc. R. Soc. Med. 54:696–697, 1961. Mukuno, K., and R. Witmer. Innervation of melanocytes in human iris: An electron microscopic study. Graefes Arch. Klin. Exp. Ophthalmol. 203:1–8, 1977.

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CHAPTER 41 Ringvold, A. An electron microscopic study of the iris stroma in monkey and rabbit with particular reference to intercellular contacts and sympathetic innervation of anterior layer cells. Exp. Eye Res. 20:349–365, 1975. Weinstein, J. M., T. J. Zweifel, and H. S. Thompson. Congenital Horner’s syndrome. Arch. Ophthalmol. 98:1074–1078, 1980.

Senile Canities Wiete Westerhof, David Njoo, and Henk E. Menke

Historical Background Graying of hair is one of the most obvious manifestations of the process of aging in man (Chapters 27 and 30). Compared with other cutaneous features of aging, this physiologic phenomenon has received relatively little attention in research. In a French scientific journal, Metchnikoff (1901) suggested that the natural graying of hair probably results from the phagocytosis of melanin. In 1921, Bloch, a German investigator, demonstrated a decreased to even an absent 3,4-dihydroxyphenylalanine (dopa) reactivity in gray and white hairs, respectively. In 1955, using autoradiography, Kukita and Fitzpatrick found an absence of tyrosinase activity in the lower hair bulb of white hairs. Electron microscopic studies performed by Orfanos (1970) showed that many melanocytes are still present at their normal anatomical position in gray hairs but contain large cytoplasmic vacuoles and incompletely melanized melanosomes. In addition, another ultrastructural study by Herzberg and Gusek in the same year revealed a decrease in the number of melanocytes in white hairs; those remaining showed abnormal structural changes. In white hairs melanocytes were found to be absent. Thus, there is strong evidence of progressive reduction in melanocyte numbers and activity in the graying hair, but the underlying mechanisms behind this defect are not clearly understood. Do melanocytes possess their own genetically determined internal “clock” for development and resolution as suggested by Ortonne et al. in 1983? Are natural aging processes in melanocytes strictly irreversible or can they be influenced artificially?

but more fair-haired than dark-haired persons become completely gray. The possible explanation for this apparent contradiction is that the first signs of graying are seen more directly against a dark background than against a fair background. Graying may begin in the second or third decade, but is usually first visible in the late forties or early fifties (Fitzpatrick et al., 1965). Both sexes are affected. In an Australian population, Keogh and Walsh (1965) found no significant difference in the age of graying between men and women. They found 22.29% of 2574 men and 23.35% of 698 women between 24 and 35 years of age had some graying and 0.25% of men and 0.15% of women in these same populations showed complete graying. Burch (1971) reported some gray hair in dark-haired men between 20 and 24 years of age. Onset as early as the second decade or as late as the ninth decade is also possible. Keogh and Walsh (1965) showed that at 50 years of age, regardless of sex, 50% of people have at least 50% white hairs. Senile canities affects all races, but is probably less common in black people. The age of onset has been observed to vary among the races. Boas and Michelson (1932) found that, in Caucasoid races, white hairs first appear at the age of 34 ± 9.6 years. In black people the onset is at 43.9 ± 10.3 years and in the Japanese between 35 and 39 years in females and between 30 and 34 in males (Straile, 1964). In Bantus, graying of hair is said to be uncommon before the age of 40 to 50 (Wade, 1970).

Clinical Description of Disease Graying first appears at the temples, extends to the vertex and over a period of years slowly spreads to the crown and later to the occipital area. The rate of progression is extremely variable. Beard and body hair are usually involved later. Chest, pubic, and axillary hairs may keep their pigment even in old age (Ortonne et al., 1983). Fitzpatrick et al. (1965) observed no hypomelanotic hairs in the axillae, presternum, and pubis.

Associated Disorders

Senile canities is regarded as a normal physiologic process and occurs to varying extents in almost all individuals.

It is very likely that premature graying has a genetic basis. Occasionally it occurs as an autosomal dominant condition in rare diseases and syndromes. Pathologic graying of hair is believed to be associated with autoimmunity (Bleehen et al., 1992). Graying of hair certainly has an association with the autoimmune disease pernicious anemia (Dawber, 1970; Klaus, 1980). In dystrophia myotonica, the onset of canities may precede the myotonia and muscle wasting. Premature canities is an inconsistent feature of the Rothmund–Thomson syndrome; when present it typically starts in adolescence. A third of the patients with chromosome 5p syndrome (cri-du-chat syndrome) have prematurely graying hair.

General Clinical Findings — Age, Sexual Predilections, Racial Distribution

Routine Histology, Histochemistry, and/or Immunochemistry

The age of onset of physiologic canities is primarily heredity determined, though acquired factors may play a role. Graying appears earlier in dark-haired than in light-haired individuals,

Bloch found a gradual reduction of the dopa oxidase reaction in the hair bulbs of six persons over 57 years of age. Dopa reactivity was weak in the gray hair bulbs and it was

Synonyms “Canities” or “graying of hair” is the term used to describe the replacement of pigmented by hypopigmented hairs. It could be due to physiologic (= “senile canities”) as well as pathologic (= “premature graying”) processes. “Gray hairs” do not virtually exist but are in fact a mixture of white hairs and normally pigmented hairs (black, brown, blond, etc.).

Epidemiology

760

MISCELLANEOUS HYPOMELANOSES: EXTRACUTANEOUS LOSS OF PIGMENTATION

absent in the white hair bulbs. Bloch stated that hairs that were originally pigmented were replaced by white hairs and that hairs did not undergo “depigmentation.” In some of his subjects, he found clumps of melanin in the dermis, but he was unable to establish whether the melanin originated from intact melanocytes or came from disintegrated melanocytes, granules of which were phagocytized by macrophages in the papillae. Bloch finally concluded that physiologic graying of scalp and beard hair was due to a gradual loss of tyrosinase activity. Using autoradiographic techniques Kukita and Fitzpatrick (1955) were unable to detect the presence of tyrosinase in the hair bulb of eight individuals with gray hair. In another study by Orfanos (1970) large quantities of pigment debris and an occasional intact melanin granule were found in white hair. They suggested that melanocytes in old people produce only a few imperfect melanin granules which disintegrate in the hair shaft. According to Herzberg and Gusek (1970) the number of active melanocytes in gray hair was greatly reduced or even totally absent. Pigment cells were markedly vacuolated; fewer melanosomes were present and those observed showed abnormal structural changes. However, pigment transfer appeared to be unaffected. Kukita et al. (1971) studied the dendritic cells present in the matrix of human senile white and gray hair. They found no melanocytes in white hair but there were Langerhans cells and cells closely resembling indeterminate cells. In senile gray hair, melanocytes and Langerhans cells were observed, but indeterminate cells could not be visualized. Ultrastructural studies disclosed identical dendritic cell populations in both senile white hairs and white hairs from vitiliginous macules. Indeterminate cells were also observed by Sato and coworkers (1973); their significance, however, is still obscure. Electron microscopic studies performed by Westerhof and Nanninga (1993) showed that melanocytes were still present in white hair follicles (Fig. 41.5). Their morphology had changed; they were large and highly dendritic, had convoluted and indented nuclei, showed vacuoles and widened rough endoplasmic reticulum and contained many bizarre melanosomes. In some hair follicles no melanocytes were seen at all (Fig. 41.6). No indeterminate cells or Langerhans cells could be observed. Unlike Herzberg (1970), Westerhof and Nanninga (1993) found the keratinocytes containing only a few or even no melanosomes at all, indicating a defect in the pigment transfer (Fig. 41.6). Interestingly, recent ultrastructural studies have shown melanocytes undergoing apoptotic changes (unpublished data).

Fig. 41.5. Electron microscopic view of a whitening hair follicle; melanocytes are found on the basal lamina and suprabasally. Note the vacuolization of the melanocytes. Pigment is seen in the dermal papilla; the keratinocytes are almost devoid of melanosomes (¥588).

Fig. 41.6. Melanocyte in a whitening hair follicle; indented nucleus, widened rough endoplasmic reticulum and vacuolization (¥4437) (with kind permission of Parthenon Publishing Group, Carnforth, United Kingdom; copyright 1993).

Criteria for Diagnosis Laboratory Findings and Investigations In individuals with physiologic canities no abnormalities are found in laboratory investigations. Recently, an a-MSH binding assay has been thought to be a useful tool to investigate the presence of a-MSH binding sites on melanocytes. Westerhof and Nanninga (1993) found that these binding sites seem to be present only on melanin-synthesizing melanocytes. They were unable to demonstrate these binding sites on follicles of senile white hairs.

“Senile canities” is a “diagnosis” based on clinical examination. Premature graying of hair has been defined as onset of graying before 20 years in white people and before 30 years in black people.

Differential Diagnosis Physiologic or senile canities has to be distinguished from pathologic and/or premature graying. The latter occurs as an isolated autosomal dominant condition or may be associated 761

CHAPTER 41

with autoimmune disorders, such as pernicious anemia and hyperthyroidism and some rare diseases and syndromes.

Pathogenesis There are several theories about the mechanism of senile graying (Chapters 27 and 30). Metchnikoff (1901) stated that phagocytosis of melanin is the cause of natural graying of hair. Yosikawa (1937) compared the copper content of white and normally pigmented human hairs from aged subjects. In all cases, the copper content of white hair was lower than of colored hair. However, according to Ortonne et al. (1983) it is unlikely that such a common event as senile canities is related to a disturbance in copper metabolism, which by itself is a rare condition. Burch and Jackson (1966) suggested that age-specific prevalence of senile graying may indicate some disturbance of immune tolerance. It is a fact that premature graying is related to autoimmune disorders, but the “immune tolerance” hypothesis for physiologic graying is without firm foundation (Ortonne et al., 1983). Perhaps the innervation of the follicle may also play a role; after hemisympathectomy, the denervated site, compared with the normal site, showed fewer white hairs (Lerner, 1966). Orfanos (1970) suggested that senile graying is due not to corpuscular color but due to a constellation of impressions arising from the actual color of the hair keratin and from the reflection of light on the boundary surfaces and islets of infundibular matrix. Ultrastructural studies showed that senile graying is probably related to a decreased number of melanocytes and a decreased melanogenic activity of the remaining melanocytes (Fitzpatrick et al., 1958; Herzberg and Gusek, 1970; Kukita, 1971; Lloyd et al., 1987; Orfanos, 1970; Takada et al., 1992; Westerhof and Nanninga, 1993). The mechanisms underlying this decrease has not been established and it could be due to a disappearance of melanocytes or lack of tyrosinase activity. According to Ortonne et al. (1983) melanocytes in the hair probably have their own genetically determined internal clock for development and resolution. The same process seems to occur in the epidermal melanocytes. The number of dopapositive melanocytes in human skin decreases with age by about 10–20% per decade (Quevedo et al., 1969; Snell and Bischitz, 1963). Thus, at the end of their programmed life, functioning melanocytes are irretrievably lost. Electron microscopic studies by Westerhof and Nanninga (1993) revealed that, in white hair bulbs, melanocytes are still present, but unable to transfer their melanosomes to the adjacent keratinocytes. How long these melanocytes remain present in the follicle is not yet clear. Stimulation of this hampered transfer could restore the hair color. This recovery of transfer mechanism could explain the reversible nature of the hair color in certain conditions, including infections of the scalp. This might be due to an expression of the gene products (receptors to hormones or cytokines or other mediators) on the melanocyte cell membrane responsible for melanocyte– keratinocyte interaction. Furthermore, due to a defect in either 762

melanocytes or keratinocytes the transfer from melanosomes to the latter could be reduced and finally stopped, resulting in unpigmented hairs. In the melanocytes, melanin synthesis goes on and toxic intermediates and precursors of melanin accumulate, which damage and finally destroy the cells. In the meantime the melanocytes get overloaded with melanosomes which partially leak to the dermal papilla where they are phagocytized by macrophages. These processes may well be genetically regulated by an “internal clock” of melanocytes, as previously postulated by Ortonne et al. in 1983. At the end of their programmed life, melanocytes undergo apoptotic changes finally resulting in cell death. The concept of apoptosis occurring in terminal melanocytes is, however, still preliminary. a-MSH binding sites, which are normally present in pigmented human scalp hair follicles, are also found to be absent in senile white hair follicles. This indicates that repigmentation as found sometimes in completely white hair is probably not achieved by the stimulatory effects of a-MSH, since no binding sites for a-MSH could be demonstrated on white hair follicles. The inactive melanocytes in these hair follicles may have lost their binding sites due to a blockade of the genes encoding for the expression of these binding sites (Westerhof and Nanninga, 1993). Human hair graying and/or whitening with age is caused by defective cell maintenance of melanocyte stem cells. In melanocytes-tagged transgenic mice this process is accelerated dramatically with Bcl2 deficiency, which causes selective melanocyte apoptosis. Pax3 and Mitf have also been identified as key molecules that help regulate the balance between melanocyte stem cell maintenance and differentiation (Nishimura et al., 2005; Steingrimsson et al., 2005).

Animal Models Experiments with animals suggest that heredity plays an important role in senile graying. Kirby (1974) found agerelated graying in a population of wild-type Australian mice. This progressive loss of hair pigmentation with aging was inherited as an autosomal dominant trait and influenced by unspecified maternal effects. Similar genetic age-related graying has also been observed in black and brown rats, guinea pigs, rabbits, dogs, sheep, and horses (Searle, 1967; Silvers, 1979). Morse et al. (1985) described graying with age in mice infected endogenously or exogenously with murine leukemia virus. In an ultrastructural study demonstrating that amelanotic hairs contain clear cells resembling the melanocytes present in hair bulbs of albino mice, they suggested that the appearance of graying with age was the result of melanocyte dysfunction rather than loss. The possibility that melanocytes are susceptible to virus infection at a critical stage of differentiation and that virus expression could interfere with cellular functions at some later stage of melanocyte development was also raised.

Treatment There is no causative treatment for this condition. Several

MISCELLANEOUS HYPOMELANOSES: EXTRACUTANEOUS LOSS OF PIGMENTATION

cases have been described of a reversible darkening of hair after exposure to toxic agents and after the use of certain drugs. Darkening of gray hair may occur following large doses of para-aminobenzoic acid; Sieve (1941) gave 100 mg three times per day orally to 460 gray-haired people and noted a response in 82% of the subjects. Pigmentation was obvious within two to four months of starting treatment. Unfortunately, the hairs regained their gray color two to four weeks after cessation of the trial. Mechanisms responsible for spontaneous and drug-induced repigmentation of senile white hairs are still unknown and need to be further investigated.

Prognosis In general, graying of hair is progressive and permanent and is usually regarded as a normal manifestation of the aging process. The reported repigmentation of white hair in association with Addisonian hypoadrenalism may result from a mechanism similar to that in alopecia areata or vitiligo, in view of the known association between these diseases (Rook and Dawber, 1991). The mechanisms of repigmentation of white hair and persistence of well-pigmented hair into old age associated with hypothyroidism remain unclear (Morris and Wright, 1986). There have been a number of reports of repigmentation of senile white hairs, giving hope that graying is not necessarily irreversible (Comaish, 1972; Verbov, 1981). Ronchese (1972) reported gradual repigmentation of white hair in a male patient over the course of a long terminal illness. The patient had shown graying at the age of 38. Several hairs plucked from his scalp showed a remarkable loss of pigment distally and normally pigmented hair and roots proximally; the transition from black to white was gradual over a length of nearly 6 mm. Spontaneous partial reversal of canities has also been reported in a 22-year-old healthy Chinese man. Ultrastructural studies of repigmented hairs revealed “intermittent melanogenesis,” indicating gradual recovery in some hair follicles (Tobin and Cargnello, 1993).

References Bleehen, S. S., F. J. Ebling, and R. H. Champion. Disorders of skin color. In: Rook/Wilkinson/Ebling Textbook of Dermatology, 5th ed., vol. 3, R. H. Champion, J. L. Burton, and F. J. G. Ebling (eds). Oxford: Blackwell Scientific Publications, 1992, pp. 1602–1615. Bloch, B. Ueber die Entwicklung des Haut- und Haarpigmentes beim meschlichen Embryo und ueber das Erloeschen der Pigmentbildung im ergrauenden Haar. Arch. Dermatol. Syphil. 135:77–108, 1921. Boas, F., and N. Michelson. The greying of hair. Am. J. Phys. Anthropol. 17:213–228, 1932. Burch, P. R. J. The age-prevalence of arcus senilis, greying of hair and baldness: etiological considerations. J. Gerontol. 26:364–372, 1971. Burch, P. R. J., and D. Jackson. The greying of hair and the loss of permanent teeth considered in relation to an autoimmune theory of aging. J. Gerontol. 21:522–528, 1966. Comaish, S. White scalp hairs turning black: an unusual reversal of the aging process. Br. J. Dermatol. 86:513–514, 1972. Dawber, R. P. R. Integumentary associations of pernicious anaemia. Br. J. Dermatol. 82:221–223, 1970. Fitzpatrick, T. B., P. C. J. Brunet, and A. Kukita. The nature of hair

pigment. In: The Biology of Hair Growth, W. Montagna (ed.). New York: Academic Press, 1958, pp. 255–303. Fitzpatrick, T. B., G. Szabo, and R. Mitchell. Age change in the human melanocyte system. In: Advances in the Biology of the Skin, W. Montagna (ed.). Oxford: Pergamon Press, 1965, pp. 35–50. Herzberg, J., and W. Gusek. Das Ergrauen des Kopfhaares. Arch. Klin. Exp. Dermatol. 236:368–384, 1970. Keogh, E. V., and R. J. Walsh. Rate of greying of human hair. Nature 207:877–878, 1965. Kirby, G. C. Greying with age: a coat-color variant in wild Australian populations of mice. J. Hered. 65:126–128, 1974. Klaus, S. N. Acquired pigment dilution of the skin and hair; a sign of pancreatic disease in the tropics. Int. J. Dermatol. 19:508–511, 1980. Kukita, A. The electron microscopic study on dendritic cells in the hair matrix of human white and grey hair. Jpn. J. Dermatol. [B] 81:326–334, 1971. Kukita, A., and T. B. Fitzpatrick. The demonstration of tyrosinase in melanocytes of the human hair matrix by autoradiography. Science 121:893–904, 1955. Lerner, A. B. Grey hair and sympathectomy. Arch. Dermatol. 93:235–236, 1966. Lloyd, T., F. L. Garry, E. K. Manders, and J. G. J. Marks. The effect of age and hair colour on human hairbulb tyrosinase activity. Br. J. Dermatol. 116:485–489, 1987. Metchnikoff, E. Sur le blanchissement des cheveux at des poils. Ann. Inst. Pasteur Lille 15:215, 1901. Morris, D. E., and F. K. Wright. Hair turning grey to black in a patient with autoimmune hypothyroidism. Br. Med. J. 292:1430, 1986. Morse, H. C., R. A. Yetter, J. H. Stimpfling, O. M. Pitts, T. N. Fredrickson, and J. W. Hartley. Greying with age in mice: Relation to expression of murine leukemia viruses. Cell 41:439–448, 1985. Nishimura, E. K., S. R. Granter, and D. E. Fisher. Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche. Science 307:720–724, 2005. Orfanos, C. Das weisse Haar älterer Menschen. Arch. Klin. Exp. Dermatol. 236:395–405, 1970. Ortonne, J.-P., D. B. Mosher, and T. B. Fitzpatrick. Topics in Dermatology: Vitiligo and Other Hypomelanoses of Hair and Skin. New York: Plenum Medical Book Company, 1983, pp. 613–672. Quevedo, W. C., Jr., G. Szabó, and J. Virks. Influence of age and UV on the population of DOPA-positive melanocytes in human skin. J. Invest. Dermatol. 52:287–290, 1969. Ronchese, F. A comment on even whitening of senile hair. Int. J. Dermatol. 11:84–85, 1972. Rook, A., and R. P. R. Dawber. Colour of the hair. In: Diseases of the Hair and Scalp, A. Rook, and R. P. R. Dawber (eds). Oxford: Blackwell Scientific Publications, 1991. Sato, S., A. Kukita, and K. Jimbow. Electron microscopic studies of dendritic cells in the human grey and white hair matrix during anagen. In: Pigment Cell: Mechanisms in Pigmentation, vol. 1, V. J. McGovern, P. Russell, and V. Riley (eds). Basel: S. Karger, 1973, pp. 20–26. Searle, A. G. Comparative Genetics of Coat Colour in Mammals. London: Academic Press, Inc., 1967, p. 308. Sieve, B. F. Darkening of hair following para-aminobenzoic acid. Science 95:257, 1941. Silvers, W. K. The Coat Colors of Mice. A Model for Mammalian Gene Action and Interaction. New York: Springer-Verlag, 1979, p. 332. Snell, R. S., and P. G. Bischitz. The melanocytes and melanin in human abdominal wall skin. A survey made at different ages in both sexes and in pregnancy. J. Anat. 97:361–376, 1963. Steingrimsson, E., N. G. Copeland, and N. A. Jenkins. Melanocyte stem cell maintenance and hair graying. Cell 121:9–12, 2005. Straile, W. E. A study of the hair follicle and its melanocytes. Dev. Biol. 10:45–70, 1964.

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CHAPTER 41 Takada, K., K. Sugiyama, I. Yamamoto, K. Oba, and T. Takeuchi. Presence of amelanotic melanocytes within the outer root sheath in senile white hair. J. Invest. Dermatol. 99:629–633, 1992. Tobin, D. J., and J. A. Cargnello. Partial reversal of canities in a 22-year-old normal Chinese male [letter]. Arch. Dermatol. 129:789–791, 1993. Verbov, J. Erosive candidiasis of the scalp, followed by reappearance of black hair after 40 years. Br. J. Dermatol. 105:595–598, 1981. Wade, W. G. A study of two families with multiple autoimmune disease. Ir. J. Med. Sci. 3:463–473, 1970. Westerhof, W., and P. B. Nanninga. Graying of hair in humans: morphological and biological data. In: Dermatology: Progress and Perspectives. The Proceedings of the 18th World Congress of Dermatology, W. H. C. Burgdorff, and S. I. Katz (eds). New York: The Parthenon Publishing Group, 1993, pp. 508–512. Yosikawa, H. Studies in biochemistry of copper; copper in keratinous appendages of skin. Jpn. J. Med. Sci. II. Biochem. 3:267–272, 1937.

Sudden Whitening of Hair Wiete Westerhof, David Njoo, and Henk E. Menke

Historical Background “Sudden whitening” refers to the abrupt appearance of totally depigmented hair in contradistinction to graying, which is a gradually developing admixture of pigmented and hypopigmented hairs. For centuries the mysterious sudden appearance of white hair as a response to fear or grief has fascinated the literary medical and anthropological worlds. Many reports have been overdramatized, but it certainly occurs. The first case was reported in the year 83 bc in the Talmud (Babylon). A young Jewish scribe experienced sudden whitening of his hair during an overnight scientific experiment (Goldenhersh, 1992). In the year 1300, Louis II, the Duke of Bayern, executed his wife after she had betrayed him. Within three days he discovered his hair had turned white. Scholar Guareno of Verona (1370–1460) had sent numerous Greek manuscripts to Italy by ship; when he was told they had been lost on the way his hair rapidly became white. After Henry of Navarre escaped from the horrors of the Massacre of St. Bartholomew in 1572, his hair turned white overnight. Shakespeare referred to sudden whitening in King Henry IV, when Sir John Falstaff states to Hotspur: “Worchester is stolen away tonight. Thy father’s beard is turned white with the news.” In the “Prisoner of Chillon,” Byron wrote: “My hair is gray, but not with years, Nor grew it white, In a single night, As men’s have grown from sudden fears.” A young officer in the regiment of Touraine (1781) is said to have experienced violent spasms of the body and whitening of the beard and hair after a night with a mulatto. Sir Thomas Moore, Chancellor of England and author of Utopia, was held imprisoned in the Tower of London for his failure to place the will of the king above that of the Pope and the Church. When he was sentenced to be beheaded for high treason, Sir Thomas’s scalp hair and beard became white the night before his execution. As Marie Antoinette mounted the scaffold to be beheaded, 764

her characteristic auburn hair had turned to white. Some, however, had observed her to have a fully whitened head of hair some nine months earlier, during her flight to Varennes in 1791 (Sainte-Beuve, 1851). Smiley (1851) reported a gambler who staked $1000 on a single card and experienced sudden whitening of his hair. General Charles Gordon had been noted to have dignified streaks of gray hair two years before his death. During the long siege of Khartoum by the Mahdi in 1885, Gordon became so anxious that he developed snow-white hair. Jacques Orsario, a Spanish nobleman, was unfortunate enough to fall in love with a lady in the court of King Ferdinand of Spain. A barking dog revealed his hiding place when he was climbing a tree in the royal garden. Imprisoned and sentenced to be beheaded, he felt so tormented that he became as white haired as a man of 80. The Moghul ruler, Shah Jahan, is said to have been so grieved at the death of his favorite wife (who bore him 14 children) that he developed white hair within two weeks. John Libeny, convicted of the attempted assassination of Emperor Franz Josef of Austria, and E. S. Stokes, murderer of financier and former associate Jim Fiske, both experienced whitening of their hair. In 1940, McCarthy wrote in his textbook Diseases of the Hair that rape may be a cause of sudden whitening in young females. Jelinek (1972) remarked that whereas the commoner who experienced sudden whitening was considered punished for sexual indulgence, heresy, or anti-Calvinistic behavior, the patrician developed white locks only out of valor. Recent reports of sudden whitening of the hair are of those occurring under stress. The hair of a Viennese man became white one day after his mother was killed in an air raid (Menninger-Lerchental, 1948). Seven weeks after an accident, a motorcyclist noticed whitening of his hair (MacLeod, 1937). Ephraim (1959) remarked he could find four cases of whitening associated with the horrors of World War I but none related to World War II. Holzgraefe (1947) reported a 58-year-old woman whose husband had suddenly turned white-haired when his house caught fire and who herself turned white-haired overnight after her two sons were poisoned by gas.

Synonyms There are no synonyms for this phenomenon.

Epidemiology The true incidence of sudden whitening is unknown, but the curiosity surrounding its occurrence suggests it is indeed rare.

General Clinical Findings — Age, Sexual Predilections, Racial Distribution There are no underlying predisposing factors concerning age, sex, race, or family history.

Clinical Description of the Disease In the literature the phenomenon of sudden whitening of previously colored hair is reported to involve scalp, beard, mus-

MISCELLANEOUS HYPOMELANOSES: EXTRACUTANEOUS LOSS OF PIGMENTATION

Fig. 41.7. Normal hair color. (Taken from: Dawber R. P. R., D. Van Neste. Alopecia areata. In: R. P. R. Dawber (ed.) Hair and scalp disorders. Martin Dunitz Limited, London, UK, 1992, pp. 169–190. With kind permission of Dr. R. P. R. Dawber; copyright 1992.)

Fig. 41.9. The tips of the hairs are normally pigmented. The pigmentation of the hair shaft abruptly stopped associated with sudden whitening of the patient’s hair (this patient is seen in Fig. 30.35 in Chapter 30 on vitiligo).

nomenon and found sudden whitening of the hair in patients following hemiplegia, brain tumor, meningitis, ataxia, encephalitis, delirium tremens, and schizophrenia.

Routine Histology, Histochemistry, and/or Immunochemistry

Fig. 41.8. Rapid whitening of hair (two weeks later). (Taken from: Dawber R. P. R., D. Van Neste. Alopecia areata. In: R. P. R. Dawber (ed.) Hair and scalp disorders. Martin Dunitz Limited, London, UK, 1992, pp. 169–190. With kind permission of Dr. R. P. R. Dawber; copyright 1992.)

tache, and eyebrow hairs (Figs 41.7, 41.8, and 41.9). The extent to which it occurs seems variable, from “white locks” to “fully whitened scalp and facial hairs.” Some hair loss may be noticed by the patient. On the scalp some “exclamation point” hairs may be seen. There may also be some regression of the frontotemporal hair line. Furthermore, an overall thinning of hair may also be observed. The clinical picture may resemble that of alopecia areata diffusa. A “trichogram” and a histologic examination may confirm the diagnosis of alopecia areata (Plinck et al., 1993).

Information regarding light and electron microscopic findings of hair follicles from patients with sudden whitening of the hair is unfortunately not available. Guin et al. (1981) performed immunofluorescence microscopy of biopsy material taken from a patient with rapid whitening of scalp hair which was associated with a diffuse, subtotal alopecia areata. They observed marked deposits of IgG and IgM in a granular pattern in the epithelium of the lower portions of hair follicles. This is not found in physiologically whitened hair follicles. Whether these findings are of etiologic significance is not clear (Guin et al., 1981).

Laboratory Findings and Investigations Abnormalities in laboratory examinations have not been reported.

Criteria for Diagnosis The sudden or rapid appearance of white hairs in an individual with previously “normal” colored hair may suggest this phenomenon.

Differential Diagnosis Sudden whitening of the hair is not a diagnostic entity, but is nowadays mostly regarded as a symptomatic phenomenon associated with alopecia areata (diffusa) and vitiligo.

Pathogenesis Associated Disorders Many cases of sudden whitening of the hair are found to be associated with neurologic and psychiatric disorders. Ephraim (1959) reviewed 150 years of medical literature on this phe-

Many authors have regarded sudden whitening as fictional and not compatible with known anatomic and physiologic phenomena. von Stieda (1910) even proposed that sudden whitening appeared because neurologically compromised or 765

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grief-stricken patients were no longer able to dye their hair. Ephraim (1959) quoted Hebra and Kaposi who have also doubted this cannot be other than evolutionary type of natural replacement of normally pigmented hairs by white hairs. Observers cannot agree whether hair turns white throughout the entire shaft simultaneously (if at all), from the root or from the distal tip. Ephraim (1959) discussed this with hairdressers who stated they never noted short white hairs during physiologic growing, only long ones. Brown-Sequard (1869) for some weeks plucked all the white hairs from his beard yet found white ones of equal length present the next morning. Given the observation that hairs grow only between 0.32 mm and 0.39 mm daily, he could not accept these as newly grown hairs and concluded established pigmented hairs had depigmented overnight. Other theories have been suggested. Ephraim (1959) quoted Vauquelin who considered the existence of a peculiar substance, which was secreted under mental stress and which probably dissolved the pigment. Metchnikoff (1901) suggested pigment was phagocytized and carried down the hair bulb to be deposited in connective tissue. The phage cells were termed “pigmentophages.” No such cells, however, have ever been visualized. Landois (1866) suggested and others have observed, that the presence of air in the hair bulbs reversibly alters the refractive index so that the hairs appear white. This mechanism is responsible for the paler segments of banded hair of pili annulati. No one has explained, however, how a biologically inactive tissue like white hair can be altered to admit air. According to Ortonne et al. (1983) it is certainly possible that pigmented hairs fall out and leave depigmented hairs remaining. Some patients indeed noticed some hair loss. Alopecia areata, either focal or diffuse, has often been shown to follow psychic stimuli and nearly exclusively to affect pigmented hairs, resulting in the loss of pigmented hairs and the remaining of depigmented ones. When hair regrows in areas of alopecia, it is initially amelanotic. In a patient previously white, with even 10% of scalp remaining pigmented, loss of many pigmented hairs could lead to the impression of sudden whitening of the hair. Many authors support this theory (Burton, 1970; Damste, 1968; Helm and Milgrom, 1970; Klingmüller, 1958; Lehnert, 1971; Lerner, 1972; Montgomery, 1967). Plinck et al. (1993) reported a female patient with sudden whitening of hair in whom later alopecia areata diffusa was diagnosed. Goldenhersch (1992) explained the phenomenon on the basis of a two-step process. The first step involves the actual abrupt development of white hair due to vitiligo or alopecia areata. The second step involves the apparent sudden whitening of the scalp hair, due to either simultaneous lengthening of the white hair or selective loss of the dark hair. In fact, the hair that is perceived as suddenly whitening is already white. However, the selective sparing of senile white hairs in alopecia areata is still a mystery (see Chapters 27 and 30).

Animal Models There are no animal models. 766

Treatment No therapeutic attempts have been reported to repigment white hairs.

Prognosis Although suddenly whitened hairs are presumed to be irreversible, spontaneous repigmentation has been reported (Vignolo-Lutati, 1918).

References Brown-Sequard, M. Expérience démontrent que les poils pouvent passer rapidement du noir au blanc chez l’homme. Arch. Physiol. 2:442–443, 1869. Burton, J. L. Canities of rapid onset due to diffuse alopecia. Practitioner 205:655–656, 1970. Damste, T. J. A case of alopecia with acute pigment loss. Dermatologica 136:440, 1968. Ephraim, A. I. On sudden or rapid whitening of the hair. Arch. Dermatol. 79:228–235, 1959. Goldenhersh, M. A. Rapid whitening of the hair first reported in the Talmud. Possible mechanisms of this intriguing phenomenon. Am. J. Dermatopathol. 14:367–368, 1992. Guin, J. D., V. Kumar, and B. H. Petersen. Immunofluorescence findings in rapid whitening of scalp hair. Arch. Dermatol. 117:576–578, 1981. Helm, F., and H. Milgrom. Can scalp hair suddenly turn white? Arch. Dermatol. 102:102–103, 1970. Holzgraefe, A. Alopecie als Symptom neurohormonaler Erkrankungen. Nervenarzt 18:134–138, 1947. Jelinek, J. E. Sudden whitening of the hair. Bull. N. Y. Acad. Med. 48:1003–1013, 1972. Klingmüller, G. Über plötzliches Weisswerden und psychische Traumen bei der Alopecia areata. Dermatologica 117:84–92, 1958. Landois, L. Das plötzliche Ergrauener Haupthaare. Arch. Pathol. Anat. Physiol. 35:575–599, 1866. Lehnert, W. Beitrag zur Frage des “plötzlichen Weisswerdens” von Körperhaaren. Dtsch. Gesundheit. 26:740–743, 1971. Lerner, A. B. Vitiligo. Prog. Dermatol. 6:1–6, 1972. MacLeod, J. M. H. A case of leukodermia and leukotrichia following motor accident. Br. J. Dermatol. 49:437–438, 1937. McCarthy, L. Diseases of the Hair. St. Louis: Mosby, 1940. Makley, T. A., and K. Abbott. Neurogenic heterochromia: report of an interesting case. Am. J. Ophthalmol. 59:927–928, 1965. Menninger-Lerchental, E. Plötzliches Ergrauen der Haare durch Schreck. Wien. Klin. Wochenschr. 60:295–296, 1948. Metchnikoff, E. Sur le blanchissement des cheveux at des poils. Ann. Inst. Pasteur Lille 15:215, 1901. Montgomery, P. R. White overnight? (letter). Br. Med. J. 1:300, 1967. Orfanos, C. Das Weisse Haar älterer Menschen. Arch. Klin. Exp. Dermatol. 236:395–405, 1970. Ortonne, J.-P., D. B. Mosher, and T. B. Fitzpatrick. Topics in Dermatology: Vitiligo and Other Hypomelanoses of Hair and Skin. New York: Plenum Medical Book Company, 1983, pp. 613–672. Plinck, E. P., J. D. Peereboom-Wynia, V. D. Vuzevski, W. Westerhof, and E. Stolz. Grijs worden in een nacht, kan dat? Ned. Tijdschr. Geneeskd. 137:1207–1210, 1993. Sainte-Beuve, C. A. Marie-Antoinette. In: Causeries du Lundi, 3rd ed., vol. IV, C. A. Sainte-Beuve (ed.). Paris: Garnier Freres, 1851, pp. 330–346. Smiley, E. R. Blanched hair from sudden emotions. Boston Med. Surg. J. 44:438–440, 1851. Vignolo-Lutati, C. Canizie precoce e psicopatie di guerra. Policlinico 25:680–685, 1918. von Stieda, L. Ist plötzliches Ergrauen des Haupthaares möglich? Wien. Med. Wochenschr. 36:1484–1487, 1910.

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Hypochromia without Hypomelanosis Jean-Philippe Lacour

Nevus anemicus is a congenital anomaly characterized by macules of varying size and shape, which appear paler than the surrounding skin.

heat or cold fails to induce erythema in the nevus area in contrast with normal surrounding skin which shows reactive hyperemia. Nevus oligemicus is a variant characterized by a lower skin surface temperature within the lesion, which is less prominent than nevus anemicus (Plantin et al., 1992).

Histological Background and Synonyms

Associated Disorders

Nevus anemicus was first described by Vörner (1906). He reported four patients with hypochromic skin lesions and described the differences between nevus anemicus and partial albinism, with which the patients had been erroneously diagnosed. He suggested that nevus anemicus was a vascular malformation caused by aplasia of the cutaneous vessels. In 1929, Weber proposed to use the term nevus ischemicus but the term nevus anemicus is usually preferred.

There are reports of an association with neurofibromatosis (Fleisher and Zeligman, 1969; see also Chapter 45), but the most frequently associated condition is port wine stain. Katugampala and Lanigan (1996) reported that 12 of the 146 patients (8%) with a port wine stain attending a laser clinic had a nevus anemicus. In such cases, the nevus anemicus is usually located within or near the port wine stain, resulting in a striking contrast between the two lesions. Association with Sturge–Weber syndrome or with port wine stain and hypoplasia of a leg is possible (Katugampala and Lanigan, 1996).

Nevus Anemicus

Epidemiology The prevalence of nevus anemicus is not known. Piorkowski (1944) observed only seven cases in 19 years. However, as the disease is asymptomatic and causes no cosmetic disfigurement, it is likely that few affected people seek medical counseling. Katugampala and Lanigan (1996) recorded 15 cases within a period of 42 months, during which they had specifically looked for nevus anemicus. It is more frequent in women (Plantin et al., 1992), and the lesions are present at birth.

Histology Histologic examination of the affected areas reveals normal papillary and subpapillary vasculature, indistinguishable from uninvolved skin (Daniel et al., 1977; Fleisher and Zeligman, 1969). Electron microscopy of involved skin is usually normal (Greaves et al., 1970). However, in some instances, light and electron microscopy have shown closely apposed vessel walls (Mountcastle et al., 1986).

Clinical Description Nevus anemicus is present at birth but may be discovered later. It appears as an area of pale skin with sharply outlined irregular borders (Fig. 42.1). Sometimes it appears as a number of small, grouped macules “arranged like the fronds of a maidenhair fern.” Nevus anemicus is usually unilateral and located on the trunk, most commonly on the chest, neck, and upper arms. However, the face or extremities may be involved. A patient with a linear nevus anemicus on one arm has been described (Piorkowski, 1944). The epidermis appears otherwise normal. Nevus anemicus is usually asymptomatic and there are no local sensory abnormalities. Simple tests facilitate the diagnosis: on examination under Wood’s light the lesions of nevus anemicus are unchanged or less apparent, testifying that the lighter color of the involved skin is not secondary to decreased melanin content. Pressure at the edge of the lesion with a glass slide renders the area of the nevus indistinguishable from that of the blanched surrounding skin. Rubbing or application of

Diagnosis and Differential Diagnosis The diagnosis of nevus anemicus is easily established clinically. Wood’s light examination, local reactivity after stroking, and hot or cold exposure distinguish nevus anemicus from leukoderma related to melanin abnormalities such as vitiligo, tuberous sclerosis, nevus depigmentosus, and Hansen disease. Histologic and ultrastructural studies are usually not necessary.

Pathogenesis Nevus anemicus is considered to be a pharmacologic nevus rather than a histologic anomaly (Greaves et al., 1970). Pharmacologic studies have shown that the sustained vasoconstriction in nevus anemicus could be due to increased local hypersensitivity of a-adrenergic receptor of cutaneous blood vessels to catecholamines (Daniel et al., 1977; Greaves et al., 1970). A sympathetic nerve block renders the lesion indistinguishable from normal skin. Transplantation studies have 767

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Anemia The skin color results from a mixture of various pigments, including hemoglobin in the cutaneous vasculature (Chapter 27). Decreased levels of circulating oxyhemoglobin in anemia result in a pallor which is proportional to the severity of anemia.

Edema of the Skin

Fig. 42.1. Nevus anemicus appearing as a hypopigmented macule (see also Plate 42.1, pp. 494–495).

Cutaneous edema gives an appearance of pallor. Several mechanisms might be responsible for this leukoderma which is not a hypomelanosis: decreased absorption of light, reduced capillary blood flow, increase in dermal thickness.

References demonstrated maintenance of pale coloration when skin from a nevus was grafted at another site on the body (Daniel et al., 1977).

Treatment No effective treatment is available. When nevus anemicus is associated with port wine stain, laser treatment of the latter can markedly decrease the contrast between these two conditions. Nevus anemicus results in only slight cosmetic disfigurement.

Woronoff Ring It is not clear whether Woronoff ring (see Chapter 37, Fig. 37.4, and Plate 37.4, pp. 494–495) corresponds to true hypomelanosis (Chapters 26 and 28).

768

Daniel, R. H., W. R. Hubler, J. E. Wolf, and W. R. Helder. Nevus anemicus. Arch. Dermatol. 113:53–56, 1977. Fleisher, T. L., and I. Zeligman. Nevus anemicus. Arch. Dermatol. 100:750–755, 1969. Greaves, M. W., D. Birkett, and C. Johnson. Nevus anemicus: a unique catecholamine-dependent nevus. Arch. Dermatol. 102:172– 176, 1970. Katugampala, G. A., and S. W. Lanigan. The clinical spectrum of naevus anemicus and its association with port wine stains: report of 15 cases and a review of the literature. Br. J. Dermatol. 134:292–295, 1996. Mountcastle, E. A., M. R. Diestelmeier, and G. P. Lupton. Nevus anemicus. J. Am. Acad. Dermatol. 14:628–632, 1986. Piorkowski, F. O. Naevus anaemicus (Vörner). Arch. Dermatol. Syphil. 50:374–377, 1944. Plantin, P., J. P. Leroy, and G. Guillet. Nevus oligemicus: a new case. J. Am. Acad. Dermatol. 26:268–269, 1992. Vörner, H. Über naevus anaemicus. Arch. Dermatol. Syphil. (Berlin) 82:391–398, 1906. Weber, F. P. Case of nevus anemicus. Br. J. Dermatol. 41:119–120, 1929.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Disorders of Hyperpigmentation and Hyperchromia

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Genetic Epidermal Syndromes: Disorders Characterized by Generalized Hyperpigmentation Sections Adrenoleukodystrophy Sheila S. Galbraith and Nancy Burton Esterly Familial Progressive Hyperpigmentation Nancy Burton Esterly, Eulalia Baselga, Beth A. Drolet, Susan Bayliss Mallory, and Sharon A. Foley Fanconi Anemia Sheila S. Galbraith and Nancy Burton Esterly Gaucher Disease Sheila S. Galbraith and Nancy Burton Esterly

Adrenoleukodystrophy Sheila S. Galbraith and Nancy Burton Esterly

Historical Background In 1923, Siemerling and Creutzfeldt described “bronzed sclerosing encephalomyelitis” in a 7-year-old boy whose skin became darker at 3 years of age, and who three years later developed gait abnormalities with spasticity and paralysis.

Synonyms Siemerling–Creutzfeldt disease; Bronze–Schilder disease; melanodermic leukodystrophy.

Genetics Fanconi et al. (1964) suggested an X-linked recessive inheritance on the basis of pedigree analysis of reported patients.

Clinical Findings X-linked adrenoleukodystrophy (ALD) (OMIM 300100) is a peroxisomal disease in which very long chain fatty acids (VLCFA) accumulate in blood and tissues causing progressive dysfunction of the adrenal cortex and nervous system white matter. ALD is the most frequent genetic cause of Addison disease, and the estimated incidence is between 1:20 000 and 1:50 000 (Moser et al., 2001). Knowledge about the disease came from studies of Powers et al. demonstrating abnormal cytoplasmic inclusions containing cholesterol esters with an excess of VLCFAs in the cells of the brain and adrenal cortex (Powers and Schamburg, 1974). The clinical manifestations are protean and six distinct phenotypes have been recognized based on age of onset, rate of progression, and site of most severe involvement (Moser et al., 1984, 1987, 1992, 1995). Different phenotypes may be noted within the same kindred. In the childhood cerebral form, the most frequent phenotype (40%), neurologic symptoms begin between 3 and 10

years of age with hyperactivity, learning difficulties, behavioral disturbance, impaired auditory discrimination, impaired vision, and seizures (Aubourg et al., 1982; Moser et al., 1995; Schaumburg et al., 1975). In 86% of the boys with this childhood form, neurologic symptoms precede symptoms of adrenal insufficiency. At the time of onset of the neurologic symptoms, 22% have clinically evident Addison disease and 85% have impaired cortisol response to adrenocorticotropic hormone (ACTH) stimulation (Moser et al., 1984, 1987, 1991). Neurologic deterioration (inflammatory demyelination) progresses rapidly (mean 1.9 ± 2 years) resulting in paralysis and a vegetative state in which patients are bedridden, unable to speak, eat, or see. In the adolescent cerebral form of ALD the clinical manifestations and rate of progression are similar to the childhood form but neurologic symptoms develop later in life — between 11 and 21 years of age. Adrenomyeloneuropathy (AMN), which represents 25–40% of the cases, first becomes manifest in adulthood (between 20 and 40 years of age) as a demyelinating process affecting mostly the spinal cord, brain stem, and peripheral nerves (Budka et al., 1976; Griffin et al., 1977). The main neurologic symptoms are spasticity, impaired vibration sense, and other sensory deficits. One-third of patients will develop cerebral demyelination, however, which often has a prognosis similar to that of the childhood cerebral form (Aubourg and Chaussain, 2003). Bladder and sexual function are almost always impaired. Progression is usually slow. Overt symptoms of Addison disease may precede, follow, or occur simultaneously with the neurologic symptoms but at the time AMN is diagnosed ACTH levels are elevated in most patients. In some instances skin pigmentation may appear in early childhood with relatively normal adrenal function due to the high ACTH levels (Griffin et al., 1977). The adult form of cerebral ALD, affecting 3% of the patients, presents after 21 years of age causing cerebral 771

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symptoms without spinal involvement. Early diagnosis may be difficult because symptoms resemble a psychotic disturbance, dementia, multiple sclerosis, or a focal neurologic deficit. Adrenal insufficiency, which is almost always present, is an important feature for diagnosis. Progression to a vegetative state is also rapid. Up to 15% of the patients have an Addison–disease-only phenotype (Moser et al., 1984; Sadeghi-Nejad and Senior, 1990). In countries where tuberculosis is rare, up to 40% of male patients with Addison disease have the biochemical defect of ALD (Moser et al., 1991). Some of the patients labeled as adrenal insufficiency only will later develop neurologic manifestations of AMN and most of them are found to have slight neurologic involvement after a careful neurologic examination, neuropsychologic testing, or radioimaging. Fifty-five percent of obligate heterozygous women may have some neurologic abnormalities resembling AMN and multiple sclerosis after 30–40 years of age (Aubourg and Chaussain, 2003). Adrenal function, with a few exceptions (Abarbanel et al., 1987; Dumic et al., 1992; Heffungs et al., 1980; Hewitt, 1957; Penman, 1960; Pilz and Schiener, 1973), is normal. Melanoderma in X-linked ALD is not well described. However, it can be assumed to be indistinguishable from that seen in Addison disease (Braverman, 1981). Hyperpigmentation ranges from brown to black and is diffuse with accentuation on sun-exposed parts of the body. Pigmentation is usually more intense in dark-skinned patients. The face, areola, genitalia, knees, knuckles, beltline, palmar creases, lips, gums, tongue, buccal mucosa, and areas of friction are the sites of maximum pigmentation. Scars, pigmented nevi, and hair may become darker. The oral pigmentation is discrete and is especially evident on the gums, along the alveolar ridge, and on the tongue. Patients with the AMN phenotype may become hyperpigmented many years before developing any neurologic symptoms or overt adrenal insufficiency. In the childhood phenotype, although hyperpigmentation is not a frequent presenting complaint, it is often recognized in retrospect. Other reported dermatologic features in ALD include sparse scalp and body hair (Dumic et al., 1992; Papini et al., 1994) with hair shaft abnormalities consisting of irregular swellings, incomplete breakages, and twisting (Calvieri et al., 1992; Papini et al., 1994). Amino acid analysis of the hair has demonstrated an increase in aspartic acid and a reduction of serine levels (Calvieri et al., 1992; Papini et al., 1994). Other cutaneous findings that might be coincidental, because they have been reported in a single patient, include areas of alopecia areata like hair loss, intense seborrhea on the face, scalp, and chest, pseudoacanthosis nigricans, and mild ichthyosiform dermatitis (Papini et al., 1994).

Histology Routine histologic studies reveal increased melanin in the basal keratinocytes without an obvious increase in the number of melanocytes. 772

Laboratory Investigations Hyperpigmentation in ALD, as in any state of ACTH excess, is thought to be secondary to the stimulation of melanocytes by ACTH, a-melanocyte stimulating hormone (MSH), and bMSH which share a common precursor molecule, proopiomelanocortin (Abdel-Malek, 1988). Serum levels of ACTH and MSH are elevated.

Diagnosis Diagnosis of ALD is based on demonstration of elevated levels of VLCFA in plasma, circulating blood cells, or cultured skin fibroblasts. Obligate heterozygotes can generally be detected with these tests. Prenatal diagnosis is feasible with cultured amniotic cells or cultured chorionic villus samples. Computed tomography (CT) scan and magnetic resonance imaging (MRI) show characteristic periventricular white matter lesions. Radioimaging is important to assess the progression of the disease.

Differential Diagnosis Differential diagnosis is extensive as the neurologic manifestation may resemble an attention deficit disorder, Schilder disease, metachromatic leukodystrophy, seizure disorder, brain tumor, multiple sclerosis, schizophrenia, or dementia. Recognition of a clinical or subclinical primary adrenal insufficiency may be helpful in these cases. More difficult is differentiation from other childhood neurologic diseases with adrenal insufficiency such as X-linked glycerol kinase deficiency, triple A syndrome, central pontine myelinosis, hypoglycemic or anoxic damage associated with Addison disease (Chapter 50), and glucocorticoid deficiency with achalasia. Biochemical analysis is diagnostic in those cases (Moser et al., 1987). Peroxisomal biogenesis disorders (PBD) and singleenzyme deficiencies (SED) in the peroxisomal b-oxidation pathway (acyl CoA oxidase deficiency and bifunctional protein deficiency) also accumulate VLCFA; however, these disorders typically present during the neonatal period and patients die during the first months to years of life. Corzo and associates (2002) described three neonates with symptoms clinically consistent with PBD or SED; however, they were diagnosed as having X-linked ALD on a biochemical basis (Corzo et al., 2002).

Pathogenesis The biochemical abnormality in X-linked ALD is deficient activity of the b-oxidative peroxisomal enzyme very– long-chain acyl-CoA synthetase which results in the accumulation of VLCFAs in blood and tissues (Lazo et al., 1988; Wanders et al., 1988). The gene for X-linked ALD (ABCD1) has been mapped to Xq28 (Migeon et al., 1981). This gene encodes a 75 kD peroxisomal membrane ALD protein (ALDP) and belongs to the ATP-binding cassette (ABC) superfamily of transporters (Mosser et al., 1993, 1994) that are believed to function as heterodimers with other half-transporters to regulate the import of VLCFA into the peroxisome or as a cofactor in VLCFA metabolism (Aubourg et al., 1993; Storr et

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY GENERALIZED HYPERPIGMENTATION

al., 2002). Although it is known that X-linked ALD peroxisomes have reduced very–long-chain acyl-CoA synthetase activity, it has been shown that ALDP does not function as a synthetase and is not required for transport of the enzyme into the peroxisome (Steinberg et al., 1999).

Treatment Replacement therapy is of foremost importance in patients with adrenal insufficiency as it may be life saving. Patients who do not have clinical signs of adrenal insufficiency or elevated ACTH levels should be tested for adrenal function. Hyperpigmentation reverses rapidly after initiation of glucocorticoid therapy. Attempts to improve neurologic function or to halt progression of the disease have been disappointing (Moser et al., 1992; Poulos et al., 1994). Dietary therapy combining restriction of VLCFA and increased intake of glycerol esters of oleic and erucic acids (Lorenzo oil) normalizes or significantly decreases the plasma levels of VLCFA (Rizzo et al., 1989), but does not alter the rate of disease progression (Aubourg et al., 1993). The effect of this diet in preventing neurologic involvement in asymptomatic patients is unknown. Bone marrow transplantation has shown encouraging results in patients with early neurologic involvement (Aubourg et al., 1990; Shapiro et al., 2000), but may accelerate deterioration in patients with advanced disease (Moser et al., 1984; Weinberg et al., 1988). An early diagnosis is unlikely in boys who already have symptoms due to the rapidly progressive nature of the disease. It has been suggested, however, that young asymptomatic boys diagnosed with ALD because of a family history should be monitored with serial MRI scans for early evidence of demyelination and endrocrinologic tests to evaluate for adrenal insufficiency so that they can undergo bone marrow transplantation as soon as possible (Peters et al., 2003). Other potentially promising treatments include androgens, HMGCoA reductase inhibitors, and intravenous arginine butyrate; however, further studies need to be done to establish efficacy (McGovern et al., 2003; Petroni, 2002; Weinhofer et al., 2002).

References Abarbanel, J. M., A. Osimani, Z. Shorer, Y. Lichtenfeld, S. Fischer, and Y. Herishanu. Adrenomyeloneuropathic syndrome in a woman, associated with morphologic abnormalities of muscle mitochondria. Neurology 37:1563, 1987. Abdel-Malek, Z. A. Endocrine factors as effectors of integumental pigmentation. In: Dermatologic Clinics, vol. 6, J. Nordlund (ed.). Philadelphia: W. B. Saunders Company, 1988, pp. 175–183. Aubourg, P., J. L. Chaussain, O. Dulac, and M. Arthuis. Adrenoleukodystrophy in children: Apropos of 20 cases. Arch. Fr. Pediatr. 39:663–669, 1982. Aubourg, P., and J. L. Chaussain. Adrenoleukodystrophy: the most frequent genetic cause of Addison’s disease. Hormone Res. 59 (Suppl 1):104–105, 2003. Aubourg, P., S. Blanche, I. Jambaque, F. Rocchiccioli, G. Kalifa, C. Naud-Saudreau, M. O. Rolland, M. Debre, J. L. Chaussain, C. Griscelli, A. Fischer, and P. F. Bougneres. Reversal of early neurologic and neuroradiologic manifestations of X-linked adrenoleukodystrophy by bone marrow transplantation. N. Engl. J. Med. 322:1860–1866, 1990.

Aubourg, P., C. Adamsbaum, M. C. Lavallard-Rousseau, F. Rocchicciolo, N. Cartier, I. Jambaque, C. Jakobezak, A. Lamaitre, F. Boreau, C. Wolf, and P. F. Bougneres. A two-year trial of oleic and erucic acids. N. Engl. J. Med. 329:745–752, 1993. Braverman, I. M. Skin Signs of Systemic Disease, 2nd ed. Philadelphia: Saunders, 1981, p. 366. Budka, H., E. Sluga, and W. D. Heiss. Spastic paraplegia associated with Addison’s disease: Adult variant of adrenoleukodystrophy. J. Neurol. 213:237–250, 1976. Calvieri, S., S. Giustini, B. Amorosi, and A. Rossi. Adrenoleukodystrophy: Hair studies. Abstracts of the 6th International Congress of Pediatric Dermatology, Toronto (Ontario), Canada, 8–11th June 1992. Pediatr. Dermatol. 9:210, 1992. Corzo, D., W. Gibson, K. Johnson, G. Mitchell, G. LePage, G. F. Cox, R. Casey, C. Zeiss, H. Tyson, G. R. Cutting, G. V. Raymond, K. D. Smith, P. A. Watkins, A. B. Moser, H. W. Moser, and S. J. Stienberg. Contiguous deletion of the X-linked adrenoleukodystrophy gene (ABCD1) and DXS1357E: a novel neonatal phenotype similar to peroxisomal biogenesis disorders. Am. J. Human Genet. 70:1520–1531, 2002. Dumic, M., N. Gubarev, N. Sikic, A. Roscher, V. Plavsic, and B. Filipovic-Grcic. Sparse hair and multiple endocrine disorders in two women heterozygous for adrenoleukodystrophy. Am. J. Med. Genet. 43:829–832, 1992. Fanconi, A., A. Prader, W. Isle, F. Luthy, and R. E. Siebenmann. Morbus Addison mit Hirnsklerose im Kindesalter. Ein hereditaeres Syndrom mit X-chromosomaler Vererbung? Helv. Paediatr. Acta 18:480–501, 1964. Griffin, J. W., E. Goren, H. Shaumburg, W. K. Engel, and L. Loriaux. Adrenomyeloneuropathy: A probable variant of adrenoleukodystrophy. Neurology 27:1107–1113, 1977. Heffungs, W., H. Hameisier, and H. H. Ropers. Addison disease and cerebral sclerosis in an apparently heterozygous girl: Evidence of inactivation of the adrenoleukodystrophy locus. Clin. Genet. 18:184–188, 1980. Hewitt, P. H. Addison’s disease occuring in sisters. Br. Med. J. 2:1530–1531, 1957. Lazo, O., M. Contreras, M. Hashmi, W. Stanley, C. Irazu, and I. Singh. Peroxisomal lignoceroyl-CoA ligase deficiency in childhood adrenoleukodystrophy and adrenomyeloneuropathy. Proc. Natl. Acad. Sci. U. S. A. 85:7647–7651, 1988. McGovern, M. M., M. P. Wasserstein, A. Aron, and S. P. Perrine. Biochemical effect of intravenous arginine butyrate in X-linked adrenoleukodystrophy. J. Pediatr. 142:709–713, 2003. Migeon, B. R., H. W. Moser, A. B. Moser, J. Axelman, D. Sillence, and R. A. Norum. Adrenoleukodystrophy: evidence for X-linkage inactivation, and selection favoring the mutant allele in heterozygous cells. Proc. Natl. Acad. Sci. U. S. A. 78:5066–5070, 1981. Moser, H. W., A. B. Moser, I. Singh, and B. P. O’Neil. Adrenoleukodystrophy: Survey of 303 cases: Biochemistry, diagnosis, and therapy. Ann. Neurol. 12:628–641, 1984. Moser, H. W., S. Naidu, A. J. Kumar, and A. E. Rosenbaum. The adrenoleukodystrophies. Crit. Rev. Neurobiol. 3:29–88, 1987. Moser, H. W., A. Bergin, S. Naidu, and P. W. Ladenson. Adrenoleukodystrophy: New aspects of adrenal cortical disease. Endocrinol. Metab. Clin. No. Am. 20:297–318, 1991. Moser, H. W., A. B. Moser, K. D. Smith, A. Bergin, J. Borel, J. Shankroff, O. C. Stine, C. Merette, J. Otti, W. Krivit, and E. Shapiro. Adrenoleukodystrophy: Phenotypic variability and implications for therapy. J. Inherit. Metab. Dis. 15:645–664, 1992. Moser, H. W., K. D. Smith, and A. B. Moser. X-linked adrenoleukodystrophy. In: The Metabolic Basis of Inherited Disease, 7th ed., C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle (eds). New York: McGraw-Hill, 1995, pp. 2325–2349. Moser, H. W., K. D. Smith, P. A. Watkins, J. Powers, and A. B. Moser. X-linked adrenoleukodystrophy. In: The Metabolic and Molecular Basis of Inherited Disease, 8th ed., C. R. Scriver, A. L. Beaudet,

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CHAPTER 43 W. S. Sly, and D. Valle (eds). New York: McGraw-Hill, 2001, pp. 3257–3301. Mosser, J., A. M. Douar, C. O. Sarde, P. Kioschis, R. Feil, H. Moser, A. M. Poustka, J. L. Mandel, and P. Aubourg. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature 361:726–730, 1993. Mosser, J., Y. Lutz, M. E. Stoeckel, C. O. Sarde, C. Kretz, A. M. Douar, J. Lopez, and P. Aubourg. The gene responsible for adrenoleukodystrophy encodes a peroxisomal membrane protein. Hum. Mol. Genet. 3:265–271, 1994. Papini, M., P. Calandra, S. Calvieri, S. Lauren, and G. Casucci. Adrenoleucodystrophy: Dermatological findings and skin surface lipid study. Dermatology 188:25–27, 1994. Penman, R. W. B. Addison’s disease in association with spastic paraplegia. Br. Med. J. 1:402, 1960. Peters, C., C. G. Steward, and National Marrow Donor Program, International Bone Marrow Transplant Registry, Working Party on Inborn Errors, and European Bone Marrow Transplant Group. Hematopoietic cell transplantation for inherited metabolic diseases: an overview of outcomes and practice guidelines. Bone Marrow Transplantation 31:229–239, 2003. Petroni, A. Androgens and fatty acid metabolism in X-linked adrenoleukodystrophy. Prostaglandins Leukot. Essent. Fatty Acids 67:137–139, 2002. Pilz, P., and P. Schiener. Kombination von Morbus Addison und Morbus Schilder bei einer 43 Jahrigen Frau. Acta Neuropathol. (Berl.) 26:357–360, 1973. Poulos, A., R. Gibson, P. Sharp, K. Beckman, and P. Grattan-Smith. Very long chain fatty acids in X-linked adrenoleukodystrophy brain after treatment with Lorenzo’s oil. Ann. Neurol. 36:741–746, 1994. Powers, J. M., and H. H. Schamburg. Adrenoleukodystrophy (sexlinked Schilder’s disease): A pathogenic hypothesis based on ultrastructural lesions in adrenal cortex, peripheral nerve and testis. Am. J. Pathol. 76:481–500, 1974. Rizzo, W. B., R. T. Leshner, A. Ordone, A. L. Dammann, D. A. Craft, M. E. Jensone, S. S. Jennings, S. Davis, R. Jaitly, and J. A. Sgro. Dietary erucic acid therapy for X-linked adrenoleukodystrophy. Neurology 39:1415–1422, 1989. Sadeghi-Nejad, A., and B. Senior. Adrenomyeloneuropathy presenting as Addison’s disease in childhood. N. Engl. J. Med. 322:13–16, 1990. Schaumburg, H. H., J. M. Powers, C. S. Raine, K. Suzuki, and E. P. Richardson. Adrenoleukodystrophy: A clinical and pathological study of 17 cases. Arch. Neurol. 32:577–591, 1975. Shapiro, E., W. Krivit, L. Lockman, et al. Long-term effect of bonemarrow transplantation for childhood-onset cerebral X-linked adrenoleukodystrophy. Lancet 356:713–718, 2000. Siemerling, E., and H. G. Creutzfeldt. Bonzekrankheit und sklerosierende Encephalomyelitis. Arch. Psychiatr. Nervenkr. 68: 217–244, 1923. Steinberg, S. J., S. Kemp, L. Braiterman, and P. A. Watkins. Role of very-long-chain acyl-coenzyme A synthetase in X-linked adrenoleukodystrophy. Ann. Neurol. 46:409–412, 1999. Storr, H. L., M. O. Savage, and A. J. Clark. Advances in the understanding of the genetic basis of adrenal insufficiency. J. Pediatr. Endocrinol. Metab. 15(Suppl 5):1323–1328, 2002. Wanders, R. J. A., C. W. T. van Roermund, M. J. A. van Wijland, R. B. H. Scutgens, H. van den Bosch, A. W. Schram, and J. M. Tager. Direct demonstration that the deficient oxidation of very long chain fatty acids in X-linked adrenoleukodystrophy is due to an impaired ability of peroxisomes to activate very long fatty acids. Biochem. Biophys. Res. Commun. 153:618–624, 1988. Weinberg, K., A. Moser, P. Watkins, C. Lenarsky, S. Winter, H. Moser, and R. Parkman. Bone marrow transplantation (BMT) for adrenoleukodystrophy (ALD). Pediatr. Res. 23:334A, 1988.

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Weinhofer, I., S. Forss-Petter, M. Zigman, and J. Berger. Cholesterol regulates ABCD2 expression: implications for the therapy of Xlinked adrenoleukodystrophy. Hum. Mol. Genet. 11:2701–2708, 2002.

Familial Progressive Hyperpigmentation Nancy Burton Esterly, Eulalia Baselga, Beth A. Drolet, Susan Bayliss Mallory, and Sharon A. Foley

Historical Background This rare dermatosis was first reported under the rubric of familial progressive hyperpigmentation by Chernosky in 1971. Wende and Baukus described a brother and sister with an unusual generalized nevoid pigmentary change in 1919. Later reports by Leber (1936), Pegum (1955), and Siemens (1967) describe individuals affected by the same condition.

Synonyms Alternative terms that have been used for this entity include familial universal or diffuse melanosis (Leber, 1936; Pegum, 1955; Wende and Baukus, 1919), generalized nevoid pigmentation (Siemens, 1967), melanosis diffusa congenita (BraunFalco et al., 1980; Kint et al., 1987), melanosis universalis hereditaria (Rebora and Parodi, 1989), and universal acquired melanosis (Ruiz-Maldonado, 1988; Ruiz-Maldonado et al., 1978).

Epidemiology This disorder has been described in black, white, Hispanic, and Oriental individuals. Both sexes are affected. In two large kindreds (Debao and Ting, 1991; Rebora and Parodi, 1989) and two smaller ones (Betts et al., 1994; Chernosky et al., 1971) the disorder appears to be inherited in an autosomal dominant manner. However, two of the early reports document this syndrome affecting siblings but no other family members (Pegum, 1955; Wende and Baukus, 1919). The latter cases might represent instances of autosomal recessive inheritance or germ-line mosaicism.

Clinical Findings The salient clinical finding is intense pigmentation that is variably described as jet black, dark brown, or bronze. Patchy hyperpigmentation occurs at birth or develops in early infancy (Fulk, 1984; Furuya and Mishima, 1962) and progresses relentlessly during the first few years until it encompasses the entire body. The initial site of involvement is usually the face or groin. Generally the pigment becomes diffusely distributed due to coalescence of the pigmented macules. However, segmental patterns consisting of streaks and whorls located mainly on the torso have also been described (Chernosky et al., 1971). An occasional patient has had small hypopigmented macules interspersed with the pigmentation (Pegum, 1955; Rebora and Parodi, 1989; Wende and Baukus, 1919) and/or superimposed lentigines (Betts et al., 1994). The pigmentation involves the palms, soles, lips, oral mucosa, and the

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY GENERALIZED HYPERPIGMENTATION

conjunctivae. The cornea may be affected as well (Chernosky et al., 1971). The mucous membranes have a characteristic mottled appearance. The hair is unaffected. Longitudinal pigmented bands were described in the nails of one patient (Ruiz-Maldonado et al., 1978). With time, the color may become less uniform, with blotchy or reticulated patterns and even white spots (Moon-Adams and Slatkin, 1955; Rebora and Parodi, 1989; Wende and Baukus, 1919). In adult life, the pigmentation has a tendency to fade resulting in a bronze color or mottled appearance. Apart from the pigmentary disorder, these patients have no other medical problems. Development and mentation are normal.

Histology Histopathologic findings in biopsy specimens from the hyperpigmented skin show a striking increase in melanin throughout the epidermis including the stratum corneum. This distribution of melanin may explain the complaint by these patients that their skin stains light-colored clothing. Pigmentation is most pronounced in the basal layer keratinocytes which are engorged with melanin. Epidermal melanocytes are normal in size and number, a finding that has been confirmed by 3,4-dihydroxyphenylalanine (dopa) stains (Chernosky et al., 1971; Ruiz-Maldonado et al., 1978). Melanin macroglobules (giant melanosomes) are seen in small numbers (Betts et al., 1994). The dermis is normal with no changes in blood vessels, nerves, or skin appendages. Occasionally, dermal melanophages are present in small numbers, but no evidence for tyrosinase activity in the dermis has been found with dopa staining. In sections of buccal mucosa, melanin granules were most dense in the basal cell layer but were identified in the more superficial epithelial layers as well. Electron microscopy has confirmed the presence of melanin in all layers of the epidermis. The melanocytes are filled with large numbers of heavily melanized melanosomes that are normal in shape, size, and structure. Keratinocytes are packed with mature melanosomes about half of which are dispersed individually, even in patients who are not biologically black. Separate mature melanosomes are evident in every layer of epidermis including the stratum corneum, and rare melanophages are detectable in the dermis. The basal lamina is normal ultrastructurally (Betts et al., 1994; Rebora and Parodi, 1989; Ruiz-Maldonado et al., 1978).

Laboratory Investigations Numerous laboratory studies have been performed on these patients and no abnormalities have been identified. Investigations have included measurements of serum concentrations of ACTH, a- and b-MSH, T3, T4, thyroid stimulating hormone (TSH), and cortisol levels. Karyotypes, X-rays of the skull, sella turcica, and bones, neurologic and ophthalmologic evaluations, serum and urinary amino acids, as well as findings of routine hemogram and blood chemistry are all normal.

Diagnosis The diagnosis is apparent from the extent and depth of the hyperpigmentation combined with the absence of associated abnormalities. The characteristic histologic findings of markedly increased amounts of melanin throughout the epidermis with heavy concentration in the basal layer and absence of pigment in the dermis are confirmatory features.

Differential Diagnosis The differential diagnosis includes the Naegeli–Franceschetti– Jadassohn syndrome in which there are abnormalities of other organ systems. Dyschromatosis symmetrica universalis (see below), reticulated pigmentation of Kitamura, and acropigmentation symmetrica of Dohi all have more localized, less intense pigment deposition. Furthermore, additional features typify these conditions such as the presence of hypopigmented and depigmented areas in acropigmentation of Dohi, and pits and breaks in the palmar epidermal ridge patterns in Kitamura disease. In dermatopathia pigmentosa reticularis there is associated alopecia and onychodystrophy.

Treatment There is no known effective treatment. The prognosis is excellent for good health. However, the cosmetic aspects of the disorder are problematic. Some patients have experienced lightening of the pigmentation during adulthood usually resulting in a reticulated or mottled appearance (Pegum, 1955; Ruiz-Maldonado, 1988).

References Betts, C. M., F. Bardazzi, P. A. Fanti, A. Tosti, and C. Varotti. Progressive hyperpigmentation: case report with a clinical, histological, and ultrastructural investigation. Dermatology 189:384–391, 1994. Braun-Falco, O., G. Burg, D. Selzle, and C. Schmoeckel. Melanosis diffusa congenita. Hautarzt 31:324–327, 1980. Chernosky, M. E., D. E. Anderson, J. P. Chang, M. W. Shaw, and M. M. Romsdahl. Familial progressive hyperpigmentation. Arch. Dermatol. 103:581–591, 1971. Debao, L., and L. Ting. Familial progressive hyperpigmenttion: a family study in China. Br. J. Dermatol. 125:607, 1991. Fulk, C. S. Primary disorders of hyperpigmentation. J. Am. Acad. Dermatol. 10:1–16, 1984. Furuya, T., and Y. Mishima. Progressive pigmentary disorder in Japanese child. Arch. Dermatol. 86:412–418, 1962. Kint, A., C. Oomen, M. L. Geerts, and F. Breuillard. Melanose diffuse congenitale. Ann. Dermatol. Venereol. 114:11–16, 1987. Leber, R. Uber eine Familie mit erblichem universellem Melanismus. Z. Kinderheilkd. 58:142–147, 1936. Moon-Adams, D., and M. H. Slatkin. Familial pigmentation with dystrophy of the nails. Arch. Dermatol. 71:591–598, 1955. Pegum, J. S. Diffuse pigmentation in brothers. Proc. R. Soc. Med. 48:179–180, 1955. Rebora, A., and A. Parodi. Universal inherited melanodyschromatosis: a case of melanosis universalis hereditaria. Arch. Dermatol. 125:1442–1443, 1989. Ruiz-Maldonado, R. Universal acquired melanosis: A 13-year followup. J. Int. Dermatol. (Barcelona) 1:128–129, 1988. Ruiz-Maldonado, R., L. Tamayo, and J. Fernandez-Diez. Universal acquired melanosis. Arch. Dermatol. 114:775–778, 1978.

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CHAPTER 43 Siemens, H. W. Die naevusahnliche Melanose (Melanosis naeviformis, alias Naevus pigmentosus tardus). Hautarzt 18:299–303, 1967. Wende, G. W., and H. H. Baukus. A hitherto undescribed generalized pigmentation of the skin appearing in infancy in a brother and sister. J. Cutan. Dis. 37:685–701, 1919.

Fanconi Anemia Sheila S. Galbraith and Nancy Burton Esterly

Historical Background In 1927 Fanconi described three brothers with aplastic anemia, skeletal malformations, genital hypoplasia, retarded growth and pigmented macules on the trunk (Fanconi, 1927, 1967). This constellation has been reported in over 900 additional patients and is known as Fanconi anemia.

Epidemiology and Genetics Fanconi anemia (OMIM 227650), a rare autosomal recessive disorder, is phenotypically and genotypically a heterogeneous syndrome in which patients manifest various congenital abnormalities, bone marrow failure, and a predisposition to malignancy. The inheritance is autosomal recessive and the disease is found in all races with an incidence of 1–5 per million and a carrier frequency of 1/300 (Joenje and Patel, 2001; Schroeder et al., 1976; Swift, 1971). It has been suggested that at least 0.5% of the general population is heterozygous for the gene responsible for Fanconi anemia. Eight complementation groups have been identified (FA-A, B, C, D1, D2, E, F, G) and seven genes found on various chromosomes have been cloned including FANCA, C, D2, E, F, G, and BRCA-2 (FA-D1 and possibly FA-B are now thought to be associated with BCRA-2). FA-A, C, and G are the most common complementation groups (Tischkowitz and Hodgson, 2003).

Clinical Findings Extensive clinical heterogeneity has been observed in Fanconi anemia and reflects the genetic heterogeneity. It is characterized by multiple congenital abnormalities, bone marrow failure, and a susceptibility to cancer. A third of cases have no congenital abnormalities, however, and are diagnosed only when they develop hematologic problems. Cutaneous abnormalities are present in almost 80% of the patients. The most consistent cutaneous finding is diffuse olive brown hyperpigmentation most prominent on the trunk (Fig. 43.1). Hyperpigmentation develops at birth or during early childhood and may precede or follow the anemia. In most patients hyperpigmentation is diffuse being most prominent on skin not exposed to sunlight, especially the lower trunk, neck, axillae, groin, popliteal fossae, and antecubital fossae. There may be dark-brown guttate macules superimposed upon this generalized hyperpigmentation. The oral mucosa is not involved. Café-au-lait macules and guttate achromic macules are also present. Following repeated blood transfusions, hyperpigmentation due to iron overloading may develop. 776

Fig. 43.1. Diffuse olive-brown hyperpigmentation of the perineum and inner thigh in a young woman with multiple squamous cell carcinomas of the vulva and who had Fanconi anemia (see also Plate 43.1, pp. 494–495).

Of the systemic manifestations of Fanconi anemia several cause severe and often fatal complications. The onset of pancytopenia occurs during childhood, usually before 10 years of age. Most patients present with overt infection or a bleeding diathesis. Of 129 patients with Fanconi anemia, 66% had skeletal malformations which included aplasia or hypoplasia of the skeletal axis, metacarpals or radii, hip dislocations or scoliosis (Gmyrek and Syllm-Rapoport, 1964). Radial ray defects are the most common skeletal defect. Short stature with slender build has been reported in 60% of patients and most have low birth weight. Malformations of other organ systems have also been observed. About a fourth of patients have renal deformities including renal aplasia and horseshoe kidney. About 21% have ocular abnormalities including strabismus and microphthalmia. Fewer than 20% of patients have been observed to have central nervous system abnormalities such as hyperreflexia and mild mental retardation. Deformities of the ear cause deafness in about 10% of patients. Heart defects, including patent ductus arteriosus, aortic stenosis, and atrial septal defects have been observed in eight patients. Hypogonadism is found in 20% of affected individuals. These patients also have a very high incidence of malignancies such as acute myelogenous leukemia and myelodysplastic syndrome, with an estimated risk of 50% by the age of 40 years (Butturini et al., 1994). Individuals have a 10% risk for developing various solid tumors such as squamous cell carcinoma and hepatocellular carcinoma, with the risk increasing rapidly over the age of 20 (Alter, 2003). Relatives have an increased incidence of hematologic malignancies.

Histology There are no well-documented histologic studies of the diffuse hyperpigmentation. Johansson et al. (1982) took biopsies from the darker, discrete macules and found increased epider-

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY GENERALIZED HYPERPIGMENTATION

mal melanin in the basal cell layer. They also noted the presence of dermal macrophages. There was loss of nuclear organization and the nuclei of the epidermal keratinocytes were hyperchromatic. Ultrastructural studies revealed increased numbers of compound melanosomes in both melanocytes and keratinocytes.

sible functions include roles in DNA repair, cell cycle control, regulation of oxidative stress, survival signaling pathways of hematopoietic stem cells, and maintenance of apoptosis and telomeres (Tischkowitz and Hodgson, 2003). It has also been proposed that several Fanconi anemia gene products form complexes with each other and interact with other proteins to carry out their functions.

Laboratory Findings Clinical laboratory abnormalities reflect bone marrow failure and include thrombocytopenia, anemia, and leukopenia. This disorder is associated with a high frequency of spontaneous chromosomal abnormalities (Auerbach and Wolman, 1979). Cells from those with Fanconi anemia show chromosomal fragility, slow growth in culture, and increased sensitivity to DNA cross-linking agents. Prenatal diagnosis can be made by culturing amniotic fluid cells obtained from the fetus in the presence of diepoxybutane. This induces chromosomal aberrations in fetal cells of those afflicted with Fanconi anemia (Auerbach et al., 1989).

Diagnosis The diagnosis is usually suspected in a youngster with hypogonadism, pigmentary changes, and aplastic anemia. Although the primary defect has not been identified the diagnosis can be confirmed by diepoxybutane or mitomycin C induced chromosomal breakage (Auerbach, 1995). This cellular characteristic can also be used as a diagnostic tool in the pre-anemic patient. Prenatal diagnosis is available by detecting chromosomal fragility in cells from the amniotic fluid (Auerbach et al., 1985). Flow cytometry can be used to detect characteristic arrest within the G2 phase of the cell cycle in Fanconi anemia cells in the presence of cross-linkers and is less timeconsuming than chromosome studies. Retroviruses expressing FANCA, FANCC, or FANCG cDNA can also be used to determine complementation groups accurately and rapidly (Tischkowitz and Hodgson, 2003).

Differential Diagnosis Dyskeratosis congenita (Chapter 49) is often confused with Fanconi anemia. Dyskeratosis congenita is an X-linked recessive disorder characterized by ulceration and leukokeratotic plaques of the oral mucosa, nail and dental abnormalities, poikiloderma, and anemia. The pigmentary changes start on the face and neck and are hypopigmented as well as hyperpigmented rather than the generalized hyperpigmentation seen in Fanconi anemia. In addition, the hematologic abnormalities generally occur later than those observed in Fanconi anemia. VATER/VACTERL, thrombocytopenia-absent radii (TAR) syndrome, Diamond–Blackfan anemia, and Nijmengen breakage syndrome can also clinically be confused with Fanconi anemia; however, there is no hyperpigmentation in these conditions.

Pathogenesis Most of the Fanconi genes have been cloned; however, the exact function of their protein products remains unclear. Pos-

Prognosis The prognosis is usually poor due to aplastic anemia with death within 3 years following the onset of anemia. Anabolic steroids have been shown to improve the survival of these patients. Bone marrow or cord blood transplants can be curative (Alter, 1992). The average life expectancy of a patient with Fanconi anemia is 20 years (Joenje and Patel, 2001).

Treatment Many patients who develop bone marrow failure initially respond to supportive measures such as blood transfusions, androgens, and granulocyte colony stimulating factor (GCSF); however, definitive treatment is bone marrow transplant, which offers a 2-year survival rate of 70% for related donor transplants (Guardiola et al., 1998). Clinical trials in gene transfer therapy are under way (Galimi et al., 2002).

References Alter, B. P. Fanconi anemia. Current concept. Am. J. Pediatr. Hematol. Oncol. 14:170–176, 1992. Alter, B. P. Cancer in Fanconi anemia. Cancer 97:425–440, 2003. Auerbach, A. D. Fanconi anemia. (Review). Dermatol. Clin. 13:41– 49, 1995. Auerbach, A. D., and S. R. Wolman. Carcinogen induced chromosome breakage in chromosome instability syndromes. Cancer Genet. Cytogenet. 1:21–28, 1979. Auerbach, A. D., M. Sagi, and B. Adler. Fanconi anemia: prenatal diagnosis in 30 fetuses at risk. Pediatrics 76:794–800, 1985. Auerbach, A. D., A. Rogatko, and T. M. Schroeder-Kurth. International Fanconi Anemia Registry. Relation of clinical symptoms to diepoxy butane sensitivity. Blood 73:391–396, 1989. Butturini, A., R. P. Gale, P. C. Verlander, B. Adler-Brecher, A. Gillio, and A. D. Auerbach. Hematologic abnormlities in Fanconi anemia. An International Fanconi Anemia Registry study. Blood 84:1650m– 1655m, 1994. Fanconi, G. Familial constitutional panmyelocytopathy, Fanconi’s anemia (FA) 1: Clinical aspects. Semin. Hematol. 4:233–240, 1967. Fanconi, G. Familiarere infantile perniziosaartage Anaemie (pernizioeses Blutbild und Konstitution). Jahrbuch Kinderheild 117:257–80, 1927. Galimi, F., M. Noll, Y. Kanazawa, T. Lax, C. Chen, M. Grompe, and I. M. Verma. Gene therapy of Fanconi anaemia by lentivectors. Blood 100:2732–2736, 2002. Gmyrek, D., and I. Syllm-Rapoport. Fanconi anemia: Analysis of 129 cases. Z. Kinderheilkd 91:297–337, 1964. Guardiola P., G. Socie, R. Pasquini, I. Dokal, J. J. Ortega, M. van Weel-Sipman, J. Marsh, F. Locatelli, G. Souillet, J. Y. Cahn, P. Ljungman, R. Miniero, J. Shaw, C. Vermylen, E. Archimbaud, A. N. Bekassy, G. Krivan, B. Diartolomeo P, A. Bacigalupo, and E. Gluckman. Allogeneic stem cell transplantation for Fanconi anaemia. Severe Aplastic Anaemia Working Party of the EBMT and EUFAR. European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 21(suppl 2):S24–27, 1998.

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CHAPTER 43 Joenje, H., and K. J. Patel. The emerging genetic and molecular basis of Fanconi anaemia. Nat. Rev. Genet. 2:446–459, 2001. Johansson, E., K. M. Niemi, M. Siiemes, and S. Pyrhonen. Fanconi’s anemia. Tumor-like warts, hyperpigmentation associated with deranged keratinocytes, and depressed cell-mediated immunity. Arch. Dermatol. 118:249–252, 1982. Schroeder, T. M., D. Tilgen, J. Kriger, and F. Vogel. Formal genetics of Fanconi’s anemia. Hum. Genet. 32:257–288, 1976. Strathdee, C. A., and M. Buchwald. Molecular and cellular biology of Fanconi anemia. Am. J. Pediatr. Hematol. Oncol. 14:177–185, 1992. Swift, M. Fanconi anemia in the genetics of neoplasia. Nature 230:370–373, 1971. Tischkowtiz, M. D., and S. V. Hodgson. Fanconi anaemia. J. Med. Genet. 40:1–10, 2003.

Gaucher Disease Sheila S. Galbraith and Nancy Burton Esterly

Historical Background Gaucher disease was originally described in 1882 by Phillippe Charles Ernest Gaucher who reported the case of a patient who had massive hepatosplenomegaly and findings suggestive of leukemia but did not become ill or die. In 1924, Epstein described the presence of the lipid, cerebroside, in the cells of patients with this disease and declared it to be a lipid storage disorder (Mankin et al., 2001).

Genetics Gaucher disease is inherited as an autosomal recessive trait and has been mapped to chromosome 1q21 (Ginns et al., 1985).

Clinical Findings Gaucher disease (OMIM 230800) is a rare inborn error of metabolism characterized by the accumulation of lipidlike substances in the reticuloendothelial system (Peters et al., 1977). Clinically it is a heterogeneous group of disorders with variable clinical expression which is inherited as an autosomal recessive trait. The disease is caused by a deficiency of acid bglucocerebrosidase. Many different mutations have been found in this enzyme accounting for the phenotypic variation. There are three clinical subtypes of Gaucher disease: type I (adult type) is by far the most common (99% of cases) and the only type to display cutaneous findings. The adult type of Gaucher disease is the most common lysosomal storage disease, with a prevalence of 1:50 000 in the general population. It is most common in people of Ashkenazi-Jewish ancestry, occurring in 1:400 to 1:865 people, and its defining feature is lack of neurologic involvement (non-neuropathic) (de Fost et al., 2003). The signs and symptoms develop gradually with accumulation of Gaucher cells (lipidladen macrophages) in the liver, spleen, bone marrow and, to a lesser extent, in the lung. The median age of diagnosis is 33 years although some patients may have very few manifestations and are diagnosed much later in life. An early onset form has also been identified which is associated with a poor prog778

nosis. Hepatosplenomegaly is often a presenting feature, and there is progressive weakness, dull bone pain, and diffuse hyperpigmentation. The skin manifestations can be separated into two broad categories, i.e., those that are caused by a primary enzyme deficiency in the skin and those that are secondary changes from systemic disease. The pigmentary changes caused by the enzymic deficiency are manifested by a generalized, diffuse yellow-brown pigmentation and these patients report easy tanning even during the winter months (Goldblatt and Beighton, 1984). Peripheral “streaky pigmentation” has been reported although not observed in the largest series of patients reported (Bloem et al., 1936). Thirty percent of the patients have nonspecific, transient hyperpigmented macules. Caféau-lait macules are also reported. The secondary changes due to liver and bone marrow involvement include spider telangiectases, ecchymoses, purpura and jaundice (Wechsler and Gustafson, 1936). Anemia/thrombocytopenia, bleeding tendencies, and osteoporosis with debilitating bony deformities are end-stage systemic features. The prognosis for patients with the adult type is good and survival to old age is not uncommon. There is an increased risk of hematologic malignancies and gammopathies (Cox et al., 2001). There have also been several reports of early onset parkinsonism associated with type I Gaucher disease; however, the link between Gaucher disease and Parkinson syndrome is unclear (Varkonyi et al., 2003). Types II and III have a much more acute course with severe neurologic involvement and death in early childhood.

Diagnosis The diagnosis is usually made by bone marrow biopsy, but assays of leukocyte, fibroblast, and urine b-glucocerebrosidase activity as well as DNA-mutation analysis are widely available (Beutler, 1991; de Fost, et al., 2003). Acid phosphatase, angiotensin-converting enzyme levels, ferritin, hexosaminidase, and the lysosomal hydrolase chitotriosidase are elevated. Studies have shown that chitotriosidase levels are closely associated with the total body burden of Gaucher cells and are a good marker to monitor disease progression and response to therapy (Aerts and Hollak, 1997).

Pathogenesis Gaucher disease is caused by mutations in a 7.5 kb gene located on chromosome 1q21. About 150 mutations have been described, but a missense mutation of the N370S allele is the most frequent mutation in the USA (especially in Ashkenazi Jews) and is associated with milder disease (Cox, 2001; Mankin et al., 2001). The gene encodes a-glucocerebrosidase which catalyzes the cleavage of glucose and ceramide from glucocerebroside (abundant in cell membranes). Defects in glucocerebrosidase cause accumulation of glucocerebroside in macrophages and subsequent activation of macrophages. Macrophage release of cytokines such as interleukins (IL-1, IL-6, IL-10) and tumor necrosis factor (TNF-a) is thought to play a role in organ disease (Altarescu et al., 2003).

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY GENERALIZED HYPERPIGMENTATION

Treatment Until recently, the treatment of Gaucher disease was purely symptomatic. However, enzyme replacement using a modified enzyme from placental tissue [alglucerase (Ceredase)] and an enzyme produced by recombinant techniques [(imiglucerase (Cerezyme)] is now available and has shown a remarkable clinical response including correction of hematologic abnormalities, organomegaly, liver dysfunction, pulmonary manifestations, and alleviation of bone pain (de Fost et al., 2003). However, Rudzki et al. (2003) found that enzyme replacement therapy may accelerate osteopenia in some patients. Substrate reduction with OGT 918, an inhibitor of the enzyme which catalyzes the first step in the synthesis of most glycosphingolipids (glucosylceramide synthase), is also a possible therapy for patients with mild disease (de Fost et al., 2003). Partial splenectomy and corrective orthopedic procedures are also of benefit. Experimental studies with gene transfer are currently under way.

References Aerts, J. M., and Hollak, C. E. Plasma and metabolic abnormalities in Gaucher disease. Baillieres Clin. Haematol. 10:691–709, 1997. Altarescu, G., M. Phillips, A. J. Foldes, D. Elstein, A. Zimran, and M. Mates. The interleukin-6 promoter polymorphism in Gaucher disease: a new modifier gene? Q. J. Med. 96:575–578, 2003. Beutler, E. Gaucher’s disease. N. Engl. J. Med. 325:1354–1360, 1991.

Bloem, T. F., J. Groen, and C. Postma. Gaucher’s disease. Q. J. Med. 5:517, 1936. Cox, T. M. Gaucher disease: understanding the molecular pathogenesis of sphingolipidoses. J. Inherited Metab. Dis. 24(Suppl 2):106–121, 2001. de Fost, M., J. M. F. G. Aerts, and C. E. M Hollak. Gaucher disease: from fundamental research to effective therapeutic interventions. Netherlands J. Med. 61:3–8, 2003. Ginns, E. I., P. V. Choudary, S. Tsuji, B. Martin, B. Stubblefield, J. Sawyer, J. Hozier, and J. A Barranger. Gene mapping and leader polypeptide sequence of human glucocerebrosidase: implications for Gaucher disease. Proc. Natl. Acad. Sci. U. S. A. 82:7101–7105, 1985. Goldblatt, J., and P. Beighton. Cutaneous manifestations of Gaucher disease. Br. J. Dermatol. 111:331–334, 1984. Mankin, H. J., D. I. Rosenthal, and R. Xavier. Gaucher disease. New approaches to an ancient disease. J. Bone Joint Surg. –(Am.) 83A:748–762, 2001. Peters, S. P., R. E. Lee, and R. H. Glew. Gaucher’s disease: a review. Medicine 56:425–442, 1977. Rudzki, Z., K., Okon, M. Machaczka, M. Rucinska, B. Papla, and A. B. Skotnicki. Enzyme replacement therapy reduced Gaucher cell burden but may accelerate osteopenia in patients with type I disease — a histological study. Eur. J. Haematol. 70:273–281, 2003. Varkonyi, J., H. Rosenbaum, N. Baumann, J. J. MacKenzie, Z. Simon, J. Aharon-Peretz, J. Walker, N. Tayebi, and E. Sidransky. Gaucher disease associated with parkinsonism: four further case reports. Am. J. Med. Genet. 116A:348–351, 2003. Wechsler, H. F., and E. Gustafson. Gaucher’s disease associated with multiple telangiectases in an elderly woman. N. Y. State J. Med. 40:517, 1936.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Genetic Epidermal Syndromes: Disorders Characterized by Reticulated Hyperpigmentation Sections Berlin Syndrome Eulalia Baselga and Nancy Burton Esterly Cantu Syndrome Eulalia Baselga and Nancy Burton Esterly Kindler Syndrome Eulalia Baselga and Nancy Burton Esterly Dermatopathia Pigmentosa Reticularis Eulalia Baselga and Nancy Burton Esterly Dyschromatosis Universalis Hereditaria Eulalia Baselga and Nancy Burton Esterly Epidermolysis Bullosa with Mottled Pigmentation Eulalia Baselga and Nancy Burton Esterly Familial Mandibuloacral Dysplasia Eulalia Baselga and Nancy Burton Esterly Hereditary Acrokeratotic Poikiloderma Eulalia Baselga and Nancy Burton Esterly Hereditary Sclerosing Poikiloderma Eulalia Baselga and Nancy Burton Esterly Mendes Da Costa Disease Eulalia Baselga and Nancy Burton Esterly Naegeli–Franceschetti–Jadassohn Syndrome Eulalia Baselga and Nancy Burton Esterly Reticulated Acropigmentation of Dohi (Dyschromatosis Symmetrica Hereditaria) Eulalia Baselga and Nancy Burton Esterly Reticulate Acropigmentation of Kitamura Eulalia Baselga and Nancy Burton Esterly Rothmund–Thomson Syndrome Eulalia Baselga and Nancy Burton Esterly

Berlin Syndrome Eulalia Baselga and Nancy Burton Esterly

Historical Background In 1961, Berlin described four siblings with a syndrome of melanoleukoderma, hypodontia, hypotrichosis, short stature, and mental retardation (Berlin, 1961). These children were from a sibship of 12 children born to consanguineous parents of Iranian descent. Both sexes were affected. There are no further descriptions of this entity. Epidemiological data are unavailable.

Clinical Findings The pigmentary anomaly was absent at birth having its onset in infancy or early childhood. It consisted of a generalized mosaiclike dyschromia in various shades of yellow, brown, gray, and white. These changes were present over almost the entire body but were most pronounced on the limbs where well-defined brown macules were interspersed with white or ivory ones. The macules ranged from 1 mm to 10 mm, were of an irregular shape, and formed a retiform pattern in some 780

areas. There was no associated atrophy even in areas of pigment loss. The scalp and nails were normal as were the mucous membranes. The lips had telangiectases on the vermilion border. Fine telangiectases over the elbows, knees, and some phalangeal joints created a poikilodermatous effect. The skin overall was dry and smooth but the palms and soles were hyperkeratotic. Sweating was normal. The scalp hair was normal as were the eyelashes. However, the outer two-thirds of the eyebrows were missing. Body hair was also absent as was sexual hair in the males but not in the females. There was a tendency to premature graying. Additional features included a characteristic facies with a flat, saddle-shaped nose, thick lips, and deep furrows about the eyes and mouth. The affected individuals had short stature with an asthenic body habitus, delayed eruption of teeth with hypodontia, hypogonadism in the males, and mental retardation.

Histology Only one skin biopsy from an arm is described. The Malpighian layer was atrophic. Excess pigment, sometimes arranged in a cuplike configuration over the nuclei of the basal

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION

cells, was present. There was pigment in the upper epidermis in some areas and total absence of pigment in others. A few melanophages were detected within the dermis. No hair follicles or sebaceous glands were seen.

Diagnosis The diagnosis is made by the presence of the characteristic pigmentary change with the above-mentioned associated features.

Differential Diagnosis Differential diagnosis with Naegeli–Franceschetti–Jadassohn syndrome may be difficult as they share reticulated hyperpigmentation, hypodontia, and hyperkeratosis of the palms and soles. However, patients with Berlin syndrome lack hypohidrosis and inheritance is more consistent with an autosomal recessive disorder. Other differential diagnostic considerations include ectodermal dysplasias, dyskeratosis congenita, dermatopathia pigmentosa reticularis, and dyschromatosis universalis hereditaria. However, none of these entities have the specific cluster of abnormalities found in Berlin syndrome.

Pathogenesis and Prognosis The pathogenesis of this disorder is not understood. There is no known treatment. One affected woman gave birth to a normal child but the men were believed to be incapable of fathering children because of hypogonadism.

Reference Berlin, C. Congenital, generalized melanoleucoderma associated with hypodontia, hypotnchosis, stunted growth and mental retardation occurring in two brothers and two sisters. Dermatologica 123:227–243, 1961.

Cantu Syndrome Eulalia Baselga and Nancy Burton Esterly

Historical Background Cantu syndrome is an autosomal dominant disorder first described by Cantu et al. in 1978. The same group of authors reported a second patient in 1993, but no further cases have been described (Figuera et al., 1993). This pigmentary disorder should not be confused with another syndrome described by Cantu characterized by congenital hypertrichosis, osteochondrodysplasia, and cardiomegaly.

Clinical Findings Cantu syndrome is characterized by hyperpigmented macules on the face, arms, hands, legs, and feet and hyperkeratotic papules on the palmoplantar surfaces. The onset is in adolescence and the pigmentation is progressive. The pigmentary changes consist of multiple 12 mm brown macules that coalesce into larger patches. The pigmentation is diffuse but is most prominent on the face and arms. The palms and soles have thick, irregular hyperkeratotic papules and scattered brown macules. Histologically there is increased pigmentation

of the basal layer of the epidermis and subtle hypergranulosis (Cantu et al., 1978). The disorder constitutes a cosmetic problem primarily and is not associated with any systemic abnormalities. The entity can be distinguished from Peutz–Jeghers syndrome by the absence of melanosis oris and intestinal polyposis. LEOPARD syndrome features cardiac abnormalities, deafness, and other anomalies, which distinguishes it from Cantu syndrome. The pathogenesis is unknown and there is no known treatment.

References Cantu, J. M., J. Sanchez-Corona, R. Fragoso, E. Macotela-Ruiz, and D. Garcia-Cruz. A “new” autosomal dominant genodermatosis characterized by hyperpigmented spots and palmoplantar hyperkeratosis. Clin. Genet. 14:165–168, 1978. Figuera, L. E., M. A. Rodriguez-Catellanos, A. Gonzalez-Mendoza, and J. M. Canut. Hyperkeratosis-hyperpigmentation syndrome: a confirmative case. Clin. Genet. 43:73–75, 1993.

Kindler Syndrome Eulalia Baselga and Nancy Burton Esterly

Historical Background In 1954, Theresa Kindler described an unusual clinical syndrome characterized by congenital poikiloderma with traumatic bullae formation and progressive cutaneous atrophy. She thought this constellation of findings represented the association of epidermolysis bullosa and poikiloderma congenitale. Similar cases were reported subsequently (Al Aboud et al., 2002; Alper et al., 1978; Binder et al., 2002; BlanchetBardon et al., 1985, 1990; Bordas et al., 1982; Chimenos et al., 2003; Forman et al., 1989; Haber and Hanna, 1996; Hacham-Zadeh and Garfunkel, 1985; Hovnanian et al., 1989; Krunic et al., 1997; Patrizi et al., 1996; Person and Perry, 1979; Ricketts et al., 1997; Salamon et al., 1966; Suga et al., 2000; Wiebe et al., 1996, 2003) and it became clear that this was a separate disease from epidermolysis bullosa. For many years, the genetic basis of Kindler syndrome remained a mystery. However, in 2003, two groups identified mutations in a novel protein, named kindlin-1, encoded by the gene KIND1 on short arm of chromosome 20 (Ashton et al., 2004; Jobard et al., 2003; Siegel et al., 2003).

Synonyms Congenital poikiloderma with blisters of Kindler-Salamon; Kindler disease; congenital poikiloderma with traumatic bullae formation and progressive cutaneous atrophy.

Clinical Findings The mode of inheritance is unknown, however, analysis of the pedigrees of two reported families (Hacham-Zadeh and Garfunkel, 1985) and the history of consanguinity in three reported cases (Blanchet-Bardon et al., 1990; Pinol-Aguade et al., 1973; Verret et al., 1984) suggest an autosomal recessive inheritance. The salient clinical features are: (1) traumatic or 781

CHAPTER 44

Fig. 44.1. Reticulated erythematous, brownish network with irregular hypopigmented islands interspersed within the mottled pigmentation on the lateral aspects of the neck (see also Plate 44.1, pp. 494–495).

spontaneous blistering in the neonatal period and early childhood; (2) poikiloderma with progressive atrophy; (3) photosensitivity; and (4) palmoplantar hyperkeratosis (Ashton, 2004). The distinction between this entity and the hereditary acrokeratotic poikiloderma described by Weary is a subject of controversy and both disorders may represent clinical variants of the same disease process (Draznin et al., 1978; Forman et al., 1989; Larregue et al., 1981; Wallach et al., 1981). Blisters are present at birth or appear in early infancy and seem to be provoked by trauma, resembling epidermolysis bullosa. The blisters, which are located mainly on acral parts of the extremities, heal without milia or scar formation. Mucosal blisters may occasionally form (Alper et al., 1978; Pinol-Aguade et al., 1973). Formation of blisters ameliorates in late childhood and signs of progressive poikiloderma become evident on sun-exposed skin (Fig. 44.1). Poikiloderma starts as pigmentary changes with reticulated erythematous, brownish network with irregular hypopigmented islands interspersed within the mottled hyperpigmentation. These changes appear during early infancy but can be present at birth and persist into adult life (Alper et al., 1978; Draznin et al., 1978). The initial pigmentary changes are found on sun-exposed areas and progress centripetally to covered areas of the chest and abdomen and the extensor aspects of arms and thighs. The flexural surfaces also may be involved (Verret et al., 1984). In the original patient described by Kindler (1954), the hyperpigmentation was preceded by a reticulated erythema. Telangiectases are also seen. Kindler’s patient had telangiectases in the oral mucosa as well as on the skin. Marked photosensitivity manifested as an early erythema after minimal sun exposure is usually present but also tends to improve with age (Alper et al., 1978; Blanchet-Bardon et al., 1985; Bordas et al., 1982; Hacham-Zadeh and Garfunkel, 1985; Hovnanian et al., 1989; Kindler, 1954; Verret et al., 1984).

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Fig. 44.2. Diffuse atrophy with pigmentary changes on the dorsum of the hands (see also Plate 44.2, pp. 494–495).

A progressive, marked, diffuse atrophy of the epidermis characterized by a cigarette-paper wrinkling follows the onset of pigmentation (Fig. 44.2). Atrophy is more pronounced on the dorsum of hands, feet, knees, and elbows and is not confined to the areas of pigmentary change. Punctate keratosis of the palms and soles (Draznin et al., 1978; Person and Perry, 1979; Pinol-Aguade et al., 1973; Verret et al., 1984) or diffuse hyperkeratosis is usually present (Blanchet-Bardon et al., 1985; Bordas et al., 1982; Hovnanian et al., 1989; Kindler, 1954; Pinol-Aguade et al., 1973). Other common findings include mild webbing of the digits (Kindler, 1954; Shimizu et al., 1997), onycholysis, and nail dystrophy (Alper et al., 1978; Bordas et al., 1982; Kindler, 1954) and less frequently associated findings include pseudoainhum of the fingers (Verret et al., 1984) and anhidrosis (Person and Perry, 1979). Early and severe periodontitis with spontaneous gingival bleeding and desquamative gingivitis are characteristic features (Chimenos et al., 2003; Ricketts et al., 1997; Wiebe et al., 2003). Teeth may be dystrophic or may be lost early. Other mucosal involvement includes urethral (Alper et al., 1978; Blanchet-Bardon et al., 1985, 1990; Person and Perry, 1979; Verret et al., 1984), vaginal, esophageal (Alper et al., 1978; Verret et al., 1984) and anal (Person and Perry, 1979). Some patients may develop oral commissure stenosis as well as ectropion of the eyelids (Alper et al., 1978; Bordas et al., 1982; Kindler, 1954; Ricketts et al., 1997).

Histology Histopathologic examination of the hyperpigmented areas shows poikilodermatous changes such as epidermal atrophy, focal vacuolization of the basal layer, vascular ectasia, and melanophages in the papillary dermis. Increased melanin pigmentation in the epidermis is mentioned only in the original report (Kindler, 1954). Rare colloid bodies and dermoepidermal clefts may occasionally be observed (Blanchet-Bardon et al., 1985; Hovnanian et al., 1989; Kindler, 1954; Verret et al., 1984). Biopsy of the vesicles and bullae show an intraepider-

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION Table 44.1. Genetic poikilodermas. Disease

Inheritance*

Bullae

Poikiloderma

Acral keratoses

Other

Hereditary acrokeratotic poikiloderma Kindler syndrome

AD

Acral

Yes

Eczema

AR

Acral

Spares face except eyelids; increased in flexures Widespread

+/-

AD

No

AR

Light-exposed skin

Marked skin atrophy: webbing of the digits. Mutation in KIND1 gene Clubbing; linear sclerotic bands in flexures Photosensitivity; cataracts; alopecia; nail and dental defects; warty keratoses over bony prominences

Hereditary sclerosing poikiloderma Rothmund–Thomson

Spares chest, scalp, face except eyelids Photodistributed

No +/-

* AD, autosomal dominant; AR, autosomal recessive.

mal or subepidermal cleavage plane (Blanchet-Bardon et al., 1985; Kindler, 1954; Verret et al., 1984). Biopsy specimens of the palmar and plantar keratoses demonstrate acanthosis and orthokeratosis (Verret et al., 1984). Electron microscopic studies of poikilodermic and atrophic skin demonstrate large numbers of melanosomes within the basal keratinocytes and increased phagocytosis of melanosomes by melanophages in the upper dermis. There is extensive reduplication of the lamina densa which is a distinguishing feature from dystrophic epidermolysis bullosa (Hovnanian et al., 1989; Patrizi et al., 1996; Shimizu et al., 1997).Within a single biopsy specimen there might be intraepidermal clefts with disruption of basal keratinocytes, clefts in the lamina lucida, and others in the dermis beneath the lamina densa (Blanchet-Bardon et al., 1985, 1990; Hovnanian et al., 1989; Patrizi et al., 1996; Shimizu et al., 1997; Verret et al., 1984). Hemidesmosomes, subhemidesmosomes, dense plaques, anchoring filaments and fibers, and collagen fibers are normal. Immunofluorescence and antigen mapping studies of the basement membrane zone does not reveal absence or reduction of any hemidesmosomal component. There is normal linear labeling with antibodies against a6 and b4 integrins, BP 180, BP 230, and uncein. However, there is broad reticular staining with antibodies against laminin 5, and type IV and VII collagen (Shimizu et al., 1997; Wiebe and Larjava, 1999). Immunostaining with a novel anti-kindlin-1 polyclonal antibody shows a marked reduction or complete absence of staining in the epidermis and dermo-epidermal junction (Siegel et al., 2003).

Pathogenesis The molecular pathogenesis leading to Kindler syndrome has been recently shown to involve a loss-of-function mutation in a novel gene KIND-1 on chromosome 20p, encoding for kindlin-1 (Ashton et al., 2004; Has and Bruckner-Tuderman, 2004; Jobard et al., 2003; Siegel et al., 2003). This protein is a component of the actin cytoskeleton present mainly in basal

keratinocytes and plays a role in the attachment of the actin cytoskeleton via focal contacts to the extracellular matrix. Therefore in contrast to the skin fragility secondary to keratin intermediate filament-extracellular matrix linkage seen in epidermolysis bullosa, in Kindler syndrome the skin fragility is due to abnormal linkage between actin microfilamentextracellular matrix linkage.

Differential Diagnosis Differential diagnosis includes other hereditary poikilodermas that are characterized by formation of blisters such as Rothmund–Thomson syndrome, dyskeratosis congenita, Mendes da Costa syndrome, and epidermolysis bullosa simplex with mottled pigmentation (Table 44.1). Hereditary sclerosing poikiloderma might also be considered although, in that disorder, bullae are absent and sclerosing bands and clubbing of the fingers are characteristic. Hereditary acrokeratotic poikiloderma of Weary may be impossible to differentiate from this syndrome. However, an autosomal dominant mode of inheritance, the presence of keratotic verrucalike keratoses, absence of photosensitivity, and less pronounced atrophy are more characteristic of Weary disease.

Treatment There is no known treatment for this condition.

References Al Aboud, K., K. Al Hawsawi, D. Al Aboud, and A. Al Githami. Kindler syndrome in a Saudi kindred. Clin. Exp. Dermatol. 27:673–676, 2002. Alper, J. C., H. P. Baden, and L. A. Goldsmith. Kindler’s syndrome. Arch. Dermatol. 114:457, 1978. Ashton, G. H. Kindler syndrome. Clin. Exp. Dermatol. 29:116–121, 2004. Ashton, G. H., W. H. McLean, A. P. South, N. Oyama, F. J. Smith, R. Al Suwaid, A. Al Ismaily, D. J. Atherton, C. A. Harwood, I. M. Leigh, C. Moss, B. Didona, G. Zambruno, A. Patrizi, R. A. Eady, and J. A. McGrath. Recurrent mutations in kindlin-1, a novel keratinocyte focal contact protein, in the autosomal recessive skin

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CHAPTER 44 fragility and photosensitivity disorder, Kindler syndrome. J. Invest. Dermatol. 122:78–83, 2004. Binder, B., D. Metze, and J. Smolle. Kongenitale bullose Poikilodermie (Kindler-Syndrom). Hautarzt 53:546–549, 2002. Blanchet-Bardon, C., N. Nazzaro, and C. Mimoz. Poikilodermie congenitale avec formation de bulles traumatiques et atrophie cutanee progressive: poikilodermie de Theresa Kindler. Ann. Dermatol. Venereol. 112:703–704, 1985. Blanchet-Bardon, C., M. Paille, C. Mimoz, F. Seon, and L. Dubertret. Poikilodermie de Theresa Kindler de l’adulte. Ann. Dermatol. Venereol. 117:787–789, 1990. Bordas, X., J. Palou, J. M. Capdevila, and J. M. Mascaro. Kindler’s syndrome. Report of a case. J. Am. Acad. Dermatol. 6:263–265, 1982. Chimenos, K. E., F. R. Fernandez, L. J. Lopez, and C. E. Rodriguez de Rivera. Kindler syndrome: a clinical case. Med. Oral. 8:38–44, 2003. Draznin, M. B., N. B. Esterly, and D. F. Fretzin. Congenital poikiloderma with features of hereditary acrokeratotic poikiloderma. Arch. Dermatol. 114:1207–1210, 1978. Forman, A. B., J. S. Prendiville, N. B. Esterly, A. A. Hebert, M. Devic, Y. Horiguchi, and J.-D. Fine. Kindler syndrome: Report of two cases and review of the literature. Pediatr. Dermatol. 6:91–101, 1989. Haber, R. M., and W. M. Hanna. Kindler syndrome. Clinical and ultrastructural findings. Arch. Dermatol. 132:1487–1490, 1996. Hacham-Zadeh, S., and A. A. Garfunkel. Kindler syndrome in two related Kurdish families. Am. J. Med. Genet. 20:43–48, 1985. Has, C., and L. Bruckner-Tuderman. A novel nonsense mutation in Kindler syndrome. J. Invest. Dermatol. 122:84–86, 2004. Hovnanian, A., C. Blanchet-Bardon, and Y. de Prost. Poikiloderma of Theresa Kindler: Report of a case with ultrastructural study and review of the literature. Pediatr. Dermatol. 6:82–90, 1989. Jobard, F., B. Bouadjar, F. Caux, C. Blanchet-Bardon, S. Emre, J. Weissenbach, M. Ozguc, M. Lathrop, J. F. Prud’homme, and J. Fischer. Identification of mutations in a new gene encoding a FERM family protein with a pleckstrin homology domain in Kindler syndrome. Hum. Mol. Genet. 12:925–935, 2003. Kindler, T. Congenital poikiloderma with traumatic bulla formation and progressive cutaneous atrophy. Br. J. Dermatol. 66:106–111, 1954. Krunic, A. L., M. Ljiljana, A. Novak, G. Carlos, and R. E. Clark. Hereditary bullous acrokeratotic poikiloderma of Weary-Kindler associated with pseudoainhum and sclerotic bands. Int. J. Dermatol. 36:529–533, 1997. Larregue, M., F. Prigent, G. Lorrete, C. Canuel, and P. Ramdenee. Acrokeratose poikilodermique bulleuse et hereditaire de WearyKindler. Ann. Dermatol. Venereol. 108:69–76, 1981. Patrizi, A., P. Pauluzzi, I. Neri, G. Trevisan, L. B. De Giorgi, and G. Pasquinelli. Kindler syndrome: report of a case with ultrastructural study and review of the literature. Pediatr. Dermatol. 13:397–402, 1996. Person, J. R., and H. O. Perry. Congenital poikiloderma with traumatic bulla formation, anhidrosis, and keratoderma. Acta Derm. Venereol. 59:347–351, 1979. Pinol-Aguade, J., J. Peyri-Rey, and C. Herrero. Poiquilodermia congenita con ampollas, tipo Kindler-Salamon. Med. Cutanea IberoLat-Am. 3:221–225, 1973. Ricketts, D. N., C. L. Morgan, J. M. McGregor, and P R Morgan, Kindler syndrome: a rare cause of desquamative lesions of the gingiva. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 84:488–491, 1997. Salamon, T., B. Bogdanovic, and O. Lazovic. Uber einen Fall von Thomson Sindrom und Epidermolysis bullosa dystrophica artigen Hautveranderungen. Arch. Klin. Exp. Dermatol. 225:194–200, 1966. Shimizu, H., M. Sato, M. Ban, Y. Kitajima, S. Ishizaki, T. Harada, L. Bruckner-Tuderman, J. D. Fine, R. Burgeson, A. Kon, J. A.

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McGrath, A. M. Christiano, J. Uitto, and T. Nishikawa. Immunohistochemical, ultrastructural, and molecular features of Kindler syndrome distinguish it from dystrophic epidermolysis bullosa. Arch. Dermatol. 133:1111–1117, 1997. Siegel, D. H., G. H. Ashton, H. G. Penagos, J. V. Lee, H. S. Feiler, K. C. Wilhelmsen, A. P. South, F. J. Smith, A. R. Prescott, V. Wessagowit, N. Oyama, M. Akiyama, D. Al Aboud, K. Al Aboud, A. Al Githami, K. Al Hawsawi, A. Al Ismaily, R. Al-Suwaid, D. J. Atherton, R. Caputo, J. D. Fine, I. J. Frieden, E. Fuchs, R. M. Haber, T. Harada, Y. Kitajima, S. B. Mallory, H. Ogawa, S. Sahin, H. Shimizu, Y. Suga, G. Tadini, K. Tsuchiya, C. B. Wiebe, F. Wojnarowska, A. B. Zaghloul, T. Hamada, R. Mallipeddi, R. A. Eady, McLean WH, McGrath JA, and E. H. Epstein. Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellularmatrix linker protein UNC-112, causes Kindler syndrome. Am. J. Hum. Genet. 73:174–187, 2003. Suga, Y., R. Tsuboi, Y. Hashimoto, H. Yaguchi, and H. Ogawa, A. Japanese case of Kindler syndrome. Int. J. Dermatol. 39:284–286, 2000. Verret, J. L., M. Avenel, M. Larregue, and C. Panigel-Nguyen. Syndrome de Kindler; un cas avec etude ultrastructurale. Ann. Dermatol. Venereol. 111:259–269, 1984. Wallach, D., D. O. Vignon-Pierini, and F. Cottenot. Poikilodermie congenitale avec bulles, type Weary. Ann. Dermatol. Venereol. 108:79–83, 1981. Wiebe, C. B., and H. S. Larjava. Abnormal deposition of type VII collagen in Kindler syndrome. Arch. Dermatol. Res. 291:6–13, 1999. Wiebe, C. B., J. G. Silver, H. S. Larjava. Early-onset periodontitis associated with Weary-Kindler syndrome: a case report. J Periodontol. 67:1004–1010, 1996. Wiebe, C. B., H. Penagos, N. Luong, J. Slots, E. Epstein, Jr, D. Siegel, L. Hakkinen, E. E. Putnins, H. S. Larjava. Clinical and microbiologic study of periodontitis associated with Kindler syndrome. J. Periodontol. 74:25–31, 2003.

Dermatopathia Pigmentosa Reticularis Eulalia Baselga and Nancy Burton Esterly

Historical Background Dermatopathia pigmentosa reticularis (DPR) is a reticulate pigmentary disorder first reported by Hauss and Oberste-Lehn in 1958. The original report described two sisters who developed asymptomatic brown macules on the neck and trunk at approximately two years of age. Two years later a brother and sister with the same condition were reported (Flegel, 1960). Since then, several cases have been documented (Arthur et al., 1995; Bu et al., 1997; Gahlen, 1964; Heimer et al., 1992; Lunder and Fettich, 1973; Maso et al., 1990; Rycroft et al., 1977; Van der Lugt, 1970; Verbov, 1980)

Epidemiology and Genetics Very few cases have been reported since the original reports. Based on the available data, the inheritance pattern appears to be autosomal dominant (Griffiths, 1984; Heimer et al., 1992; Maso et al., 1990). Linkage studies favor a linkage to a region in chromosome 17 within the interval where the putative gene for the Naegeli–Franceschetti–Jadassohn syndrome resides (Sprecher et al., 2002).

Clinical Findings Classic clinical findings include reticulate pigmentation that

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION Table 44.2. Genetic poikilodermas. Disease

Inheritance*

Bullae

Poikiloderma

Acral keratoses

Other

Hereditary acrokeratotic poikiloderma Kindler syndrome

AD

Acral

Yes

Eczema

AR

Acral

Spares face except eyelids, increased in flexures Widespread

+/-

AD

No

AR

Light-exposed skin

Marked skin atrophy, webbing of the digits Clubbing, linear sclerotic bands in flexures Photosensitivity, cataracts, alopecia, nail and dental defects, warty keratoses over bony prominences

Hereditary sclerosing poikiloderma Rothmund–Thomson syndrome

Spares chest, scalp, face except eyelids Photodistributed

No +/-

* AD, autosomal dominant; AR, autosomal recessive.

begins in early childhood, noncicatricial alopecia of the scalp, eyebrows, and axillae, and onychodystrophy. The pigmentary disorder begins early in life, often at birth but usually before 2 years of age. The hyperpigmentation is generalized but most prominent on the trunk and proximal extremities. There are small, 1–3 mm round or confettilike macules that show a tendency to coalesce forming a reticulate pattern. There is no scale or erythema and the lesions are usually asymptomatic. Hypopigmented macules may be seen among the hyperpigmented areas (Bu et al., 1997; Van der Lugt, 1970). The breasts of the females are heavily pigmented and the nipples of both sexes typically are almost black. Two patients had brown macules on the oral and/or conjunctival mucosa. Sparse hair of scalp, eyelashes, and eyebrows with progressive and diffuse hair loss is characteristic of this syndrome. Axillary hair also tends to be sparse. Thick wiry hair was described in the original patient (Hauss and Oberste-Lehn, 1958). Onychodystrophy consists in hyperpigmented, soft, grooved and easily broken nails. Toenails are more often affected than fingernails. Loss of nails, with pterygia formation often during the second year of life, has been noted. Other cutaneous findings, in addition to the diagnostic triad of reticulated hyperpigmentation, noncicatricial alopecia and onychodystrophy, include hypohidrosis or hyperhidrosis, punctate palmoplantar keratoses, absent dermatoglyphics, and nonscarring acral blisters. Fine punctate spots on the cornea and brown pigmentation of the bulbar conjunctiva have also been described (Van der Lugt, 1970). In the first reported case melanin pigmentation was present in the optic fundi (Hauss and Oberste-Lehn, 1958). Systemic symptoms and signs have not been identified.

Histology The epidermis is normally pigmented. Histologic examination of the pigmented macules has demonstrated marked pig-

mentary incontinence with aggregates of melanin-filled melanophages within the papillary dermis (Bu et al., 1997; Rycroft et al., 1977). In addition to these findings, two patients had liquefaction degeneration of the basal epidermal layer as well as a diffuse hyalinization of the dermal collagen (Flegel, 1964; Maso et al., 1990).

Criteria for Diagnosis Criteria for diagnosis of dermatopathia pigmentosa reticularis include reticulate hyperpigmentation that does not fade with age, noncicatricial alopecia, and onychodystrophy.

Differential Diagnosis The differential diagnosis of heritable or congenital pigmentary disorders includes several entities that have overlapping features (Table 44.2) (Griffiths, 1984; Schnur and Heymann, 1997). Naegeli–Franceschetti–Jadassohn syndrome is an autosomal dominant closely related entity with an overlapping phenotype. It shares with DPR mottled hyperpigmentation, absence of dermatoglyphics, heat intolerance, palmoplantar keratoderma, and punctuate keratosis. The major discriminatory features between both entities are that in Naegeli– Franceschetti–Jadassohn syndrome the pigmentary changes tend to fade with age and enamel defects are an essential feature. Linkage studies have shown that the putative gene for both entities is within the same area, and it has been suggested that they are allelic diseases. Congenital diffuse mottling of the skin can be ruled out by histologic examination which shows elongated, often club-shaped, epidermal rete ridges with hyperpigmentation of the basal layer without pigmentary incontinence. Dyschromatosis universalis hereditaria is manifested by a mottled hyperpigmentation, small stature, and high tone deafness but does not appear to be inherited (Griffiths, 1984). Dyskeratosis congenita is also manifested by reticulate hyperpigmentation, that usually develops later, onychodystrophy 785

CHAPTER 44

and palmoplantar hyperkeratosis, but it lacks noncicatricial alopecia. Furthermore dyskeratosis congenita is usually transmitted as an X-linked recessive gene, and leukoplakia and hematologic abnormalities are highly characteristic (Griffiths, 1984). Other disorders include acromelanosis, reticulate acropigmentation of Dohi, reticulate acral pigmentation of Kitamura, heterochromia extremitarum, and reticulate pigmented anomaly of the flexures (Griffiths, 1984). Most of these disorders do not have nail dystrophy and can be ruled out by the distribution of pigmentation.

Pathogenesis The pathogenesis of this rare disorder is not well understood. However, clinical features point to a defect in a structural protein expressed in all affected ectodermal-derived tissues or from an abnormal regulatory protein controlling ectodermal differentiation, cell adhesion, or intercellular signaling (Spretcher et al., 2002).

Prognosis The patients are asymptomatic except for occasional heat intolerance. Their life span is normal.

Treatment There is no known treatment for the pigmentary disorder. Associated keratoderma has been successfully treated with etretinate (Arthur et al., 1995).

References Arthur, E. A., B. A. Brod, and G. R. Kantor. Palmoplantar keratoderma associated with dermatopathia pigmentosa reticularis: successful treatment with etretinate. Int. J. Dermatol. 34:645–646, 1995. Bu, T. S., Y. K. Kim, and K. U. Whang. A case of dermatopathia pigmentosa reticularis. J. Dermatol. 24:266–269, 1997. Flegel, H. Dermatopatia pigmentosa reticularis. Hautarzt 6:262–265, 1960. Flegel, H. Dermatopathia pigmentosa reticularis hypohidrotic et atrophica. Dermatol. Wochenschr. 150:193–198, 1964. Gahlen, W. Dermatopatia pigmentosa reticularis hypohidrotica et atrophica. Dermatol. Wochenschr. 150:193–198, 1964. Griffiths, W. A. D. Reticulate pigmentary disorders — a review. Clin. Exp. Dermatol. 9:439–450, 1984. Hauss, H., and H. Oberste-Lehn. Dermatopathia pigmentosa reticularis. Dermatol. Wochenschr. 138:1337–1341, 1958. Heimer, W. L., I. I., G. Brauner, and W. D. James. Dermatopathia pigmentosa reticularis: a report of a family demonstrating autosomal dominant inheritance. J. Am. Acad. Dermatol. 26:298–301, 1992. Lunder, M., and J. Fettich. Beitrag zum Begriff der Dermatopatia Pigmentosa Reticularis. Zeitschrift fur Hautkrankheiten 48:857–863, 1973. Maso, M. J., R. A. Schwartz, and W. C. Lambert. Dermatopathia pigmentosa reticularis. Arch. Dermatol. 126:935–939, 1990. Rycroft, R. J. G., C. D. Calanan, and C. F. Allenby. Dermatopatia pigmentosa reticularis. Clin. Exp. Dermatol. 2:39–44, 1977. Schnur, R. E., and W. R. Heymann. Reticulate hyperpigmentation. Semin. Cutan. Med. Surg. 16:72–80, 1997. Sprecher, E., P. Itin, N. V. Whittock, J. A. McGrath, R. Meyer, J. J. DiGiovanna, S. J. Bale, J. Uitto, and G. Richard. Refined mapping of Naegeli-Franceschetti-Jadassohn syndrome to a 6 cM interval on

786

chromosome 17q11.2-q21 and investigation of candidate genes. J. Invest. Dermatol. 119:692–698, 2002. Van der Lugt, L. Dermatopatia pigmentosa reticularis. Dermatologica 140:294–302, 1970. Verbov, J. Hereditary diffuse hyperpigmentation. Clin. Exp. Dermatol. 5:227–234, 1980.

Dyschromatosis Universalis Hereditaria Eulalia Baselga and Nancy Burton Esterly

Historical Background Dyschromatosis universalis hereditaria was first described by Ichikawa and Higawa in 1933. Dyschromatosis universalis symmetrica hereditaria is classified in the group of dyschromatoses which also includes a localized acral form, dyschromatosis symmetrica hereditaria (reticulated acropigmentation of Dohi).

Synonyms Dyschromatosis hereditaria.

symmetrica

hereditaria,

dyschromatosis

Epidemiology Dyschromatosis universalis hereditaria has been reported mostly in patients from Japan and India (Gharpuray et al., 1994). It has an autosomal dominant pattern of inheritance (Al Hawsawi et al., 2002; Gharpuray et al., 1994; Nuber et al., 2004; Schoenlaub et al., 1998; Sethuraman et al., 2000; Suenaga, 1952).

General Clinical Findings The pigmentary features of dyschromatosis universalis hereditaria generally appear during infancy or early childhood. They consist of pinpoint to “pea-sized” hyperpigmented macules mingled with similar hypopigmented lesions (Findlay and Whiting, 1971). The pigmentary anomaly usually starts on the hands but progresses to affect the entire integument especially on the trunk, abdomen, and extremities (Figs 44.3–44.6). The face may be involved (Sethuraman et al., 2000). The hyperpigmented macules display various colors from light to dark brown and are irregular in size and shape. Sunlight seems not to be an aggravating factor. Involvement of the palm and soles has been described (Gharpuray, 1994; Sethuraman et al., 2002), but dermatoglyphics are present. Thin, hyperpigmented dystrophic nails with pterygium formation have been described rarely (Sethuraman et al., 2002). Mottled pigmentation of the tongue and oral mucosa also occur rarely (Sethuraman et al., 2002).

Associated Disorders Association of dyschromatosis universalis hereditaria with coxa valgae and nerve compression sign has been reported (Suenaga, 1952). Other associated findings mostly in single case reports include: small stature, high-tone deafness (Rycroft et al., 1977); photosensitivity, neurosensory hearing defect

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION

44.4

44.3

44.5

Fig.s 44.3–44.5. Hyperpigmented and hypopigmented macules affecting the entire integument (see also Plates 44.3–44.5, pp. 494–495).

(Shono and Toda, 1990); epilepsy (Pavithran, 1991); and Xlinked ocular albinism (Yang and Wong, 1991).

Pathology The epidermis and dermis are normal apart from pigmentary changes. There are increased amounts of melanin in the basal cells within the hyperpigmented lesions with secondary pigmentary incontinence. In contrast, in hypopigmented macules, there is a decreased quantity of melanin, although normal numbers of morphologically normal melanocytes are usually present (Kim et al., 1997; Nuber et al., 2004; Schoenlaub et al., 1998). With electron microscopy, melanocytes and keratinocytes within hyperpigmented areas appear laden with increased numbers of fully melanized melanosomes (Yang and Wong, 1991; Nuber et al., 2004). Keratinocytes from hypopigmented areas may show absent to considerable numbers of melanosomes (Kim et al., 1997; Nuber et al., 2004).

Jadassohn syndrome and dyskeratosis congenita. Reticulated acropigmentation of Kitamura and dyschromatosis symmetrica hereditaria (acropigmentation of Dohi) are distinguished by its localized features.

Pathogenesis The gene effect of dyschromatosis universalis hereditaria is not known but linkage studies have mapped it to chromosome 1q. The pathogenesis remains elusive although a defect in the rate of melanosome synthesis has been suggested (Nuber et al., 2004).

Animal Models None.

Treatment No specific treatment has been established.

Differential Diagnosis Differential diagnosis includes other diseases with generalized reticulated hyperpigmentation and hypopigmentation such as dermatopathia pigmentosa reticularis, Naegli–Franceschetti–

Prognosis Dyschromatosis universalis hereditaria is not progressive and spontaneous regression has not been reported. 787

CHAPTER 44 Sethuraman, G., C. R. Srinivas, M. D’Souza, D. M. Thappa, and L. Smiles. Dyschromatosis universalis hereditaria. Clin. Exp. Dermatol. 27:477–479, 2002. Sethuraman, G., D. M. Thappa, J. Vijaikumar, and S. Srinivasan. Dyschromatosis universalis hereditaria: a unique disorder. Pediatr. Dermatol. 17:70–72, 2000. Suenaga, M. Dyschromatosis universalis hereditaria in 5 generations. Tohoku J. Exp. Med. 55:373–376, 1952. Yang, J. H., and C. K. Wong. Dyschromatosis universalis with Xlinked ocular albinism. Clin. Exp. Dermatol. 16:436–440, 1991.

Epidermolysis Bullosa with Mottled Pigmentation Eulalia Baselga and Nancy Burton Esterly

Historical Background A distinct subtype of epidermolysis bullosa simplex (EBS) with the additional feature of irregular, mottled, and reticulated pigmentation was first decribed in 1979 by Fischer and Gedde-Dahl. Subsequently a number of kindreds have been reported (Boss et al., 1981; Bruckner-Tuderman et al., 1989; Coleman et al., 1993; Combemale and Kanitakis, 1994; Irvine et al., 1997; Moog et al., 1999).

Epidemiology and Genetics

Fig. 44.6. Hyper- and hypopigmented macules (1 mm–1 cm diam.) over the entire integument in a patient with dyschromatosis universalis hereditaria (courtesy of Dr. Adrian Pierini) (see also Plate 44.6, pp. 494–495).

The mode of inheritance, as in all forms of EBS, is autosomal dominant and there is no race or sex predilection. All major subtypes of EBS are caused by mutations of the basal keratin genes, keratin 5 or keratin 14. In EBS with mottled pigmentation a single and recurrent heterozygous mutation in the nonhelical V1 domain of keratin 5 gene has been identified in unrelated families and sporadic cases (Hamada et al., 2004; Irvine et al., 1997, 2001; Moog et al., 1999; Uttam et al., 1996).

References

Clinical Findings

Findlay, G. H., and D. A. Whiting. Universal dyschromatosis. Br. J. Dermatol. 85:66–70, 1971. Gharpuray, M. B., S. N. Tolat, and S. P. Patwardham. Dyschromatosis: its occurrence in two Indian families with unusual features. Int. J. Dermatol. 33:391–392, 1994. Ichikawa, T., and Y. Hiraga. A previously undescribed anomaly of pigmentation dyschromatosis universalis hereditaria. Jpn. J. Dermatol. 34:360–364, 1933. Kim, N. S., S. Im, and S. C. Kim. Dyschromatosis universalis hereditaria: an electron microscopic examination. J. Dermatol. 24:161–164, 1997. Nuber, U. A., S. Tinschert, S. Mundlos, and I. Hauber. Dyschromatosis universalis hereditaria: familial case and ultrastructural skin investigation. Am. J. Med. Genet. 125A:261–266, 2004. Pavithran, K. Dyschromatosis universalis hereditaria with epilepsy. Indian J. Dermatol. Venereol. Leprol. 57:102–103, 1991. Rycroft, R. J. G., C. D. Calnan, and R. S. Wells. Universal dyschromatosis, small stature and high-tone deafness. Clin. Exp. Dermatol. 2:45–48, 1977. Shono, S., and K. Toda. Universal dyschromatosis associated with photosensitivity and neurosensory hearing defect. Arch. Dermatol. 128:1659–1660, 1990. Schoenlaub, P., J. P. Leroy, D. Dupre, P. Schoenlaub, J. P. Leroy, D. Dupre, C. Chaboche, and P. Plantin. Dyschromatose universelle: une observation familiale. Ann. Dermatol. Venereol. 125:700–704, 1998.

Typically these patients have, as in other types of epidermolysis bullosa simplex, mechanically induced, nonscarring, hemorrhagic blisters. The feet, hands, and knees are most commonly affected and there is marked seasonal variance in severity. Mucous membranes are generally spared. The blisters heal with thin, atrophic “cigarette-paperlike” skin, without milia or scarring. Blistering generally improves with age. The hyperpigmentation usually arises in infancy and it is not preceded by blistering. Areas of predilection are the neck, axilla, inguinal folds, lower abdomen, and extremities (Fig. 44.7). It may affect palmar aspects (Irvine et al., 2001). The lesions are well-delineated, coalescent, brown macules measuring 2–5 mm in diameter arranged in a reticular pattern which gives the skin a dirty or mottled appearance. There may be areas of hypopigmentation as well, giving it a poikilodermatous appearance. The dyspigmentation may fade with age (Boss et al., 1981; Bruckner-Tuderman et al., 1989; Coleman et al., 1993; Combemale and Kanitakis, 1994). Other associated findings include photosensitivity (Moog et al., 1999), palmo-plantar hyperkeratosis (Boss et al., 1981; Moog et al., 1999), and carious teeth. Telangiectasia

788

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION

on the cheeks and extensor surfaces of the extremities has been noted in one family (Moog et al., 1999). The nails may be dystrophic and hyperconvex (Moog et al., 1999). The warty hyperkeratotic, punctate palmoplantar lesions develop later in childhood and can be exquisitely painful. Pyloric stenosis has been reported in one case (Coleman et al., 1993).

Histology Histologic examination of the pigmented areas reveals marked epidermal atrophy with flattened rete ridges and melanin deposition in the basal layer of the epidermis. There may be subtle pigmentary incontinence. The blisters are due to cleav-

age within the basal layer of the epidermis. There is vacuolation of the keratinocytes and colloid body formation (Coleman et al., 1993). Immunofluorescence studies reveal normal distribution of the bullous pemphigoid antigen, laminin, collagen IV, and collagen VII at the floor of the blister on salt-split skin. Ultrastructural studies suggest degeneration of the basal keratinocytes as the primary event leading to blisters at the basal cell layer as in other types of EB. The hemidesmosomes, anchoring fibrils, lamina lucida, and lamina densa are all normal in appearance. In the pigmented areas there are abundant mature melanosomes in the basal keratinocytes.

Differential Diagnosis

Fig. 44.7. Hyperpigmented macules of the extremities in a child with epidermolysis bullosa and mottled hyperpigmentation (see also Plate 44.7, pp. 494–495).

The differential diagnosis includes epidermolysis bullosa Dowling-Meara type, the Mendes da Costa form of EBS, Weary-Kindler syndrome and Naegeli–Franceschetti– Jadassohn syndrome (Table 44.3). EBS Dowling-Meara shares blistering and some patients may also develop a similar type of hyperpigmentation and palmoplantar hyperkeratosis (Medenica-Mojsilovic et al., 1986; Tay, 1999; Tay and Weston 1996). Blisters are usually more prominent with herpetiform clustering. Ultrastructurally there is characteristic clumping of tonofilaments. Despite these differences, there is considerable clinical and ultrastructural overlap of EBS Dowling-Meara with mottled pigmentation and EBS with mottled pigmentation. Molecular studies support separation of both entities as in EBS Dowling-Meara with mottled pigmentation a mutation in keratin 14 gene instead of keratin 5 has been found (Ning et al., 2001).

Table 44.3. Genetic diseases with reticulated epidermal hyperpigmentation. Disease

Inheritance*

Distribution

Special features

Associated findings

Acropigmentation of Kitamura Acropigmentation of Dohi Dermatopathia pigmentosa reticularis

AD AD AD

Acral Acral Generalized

Lesions are atrophic Hypopigmented macules Hypopigmented macules

Anonychia with flexural pigmentation

AD

Flexural

Hypopigmented macules

Mendes da Costa syndrome Dowling–Degos syndrome

XLR

Blistering early, hypopigmented macules Onset delayed

Naegeli–Franceschetti– Jadassohn syndrome

AD

Generalized face and limbs Flexural, then generalized Generalized

Palmar pits, breakage of epidermal ridges Predominant in males Sweating disorders, decreased dermatoglyphics, alopecia, onychodystrophy, palmoplantar keratoses Sparse hair, hypohidrosis, decreased dermatoglyphics, absent/rudimentary nails Microcephaly, mental retardation, atrichia, short conical fingers Perioral pits, keratoses, epidermal cysts

Epidermolysis bullosa with mottled pigmentation

AD

Trunk, proximal limbs

Blistering disorder

AD

Keratoderma, enamel hypoplasia, hyperhidrosis, nail dystrophy Keratoderma of palmoplantar surface, carious teeth, photosensitivity

* AD, autosomal dominant; XLR, X-linked recessive.

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CHAPTER 44

The pigmentation observed in Mendes da Costa EBS is generally more acrally located and this entity is associated with other severe malformations such as dwarfism, atrichia, and mental retardation. Furthermore Mendes da Costa is an Xlinked recessive disorder and thus it only affects boys. Weary–Kindler syndrome (hereditary acrokeratotic poikilodermia) shares many of the clinical manifestations such as acral blistering, skin atrophy, hypo/hyperpigmentation, keratotic papules on hands and feet, mild photosensitivity, and nail dystrophy. The main distinguishing feature is that the predominant feature in this latter disorder is the progressive poikiloderma. Naegeli–Franceschetti–Jadassohn syndrome also shares many features: acral blisters, pigmentation in the same areas of predilection, palmo-plantar keratosis, and nail dystrophy. In this disease, however, there is hypohidrosis, dental abnormalities, and loss of dermatoglyphics which point to an ectodermal dysplasia. Siemens syndrome is also characterized by mottled pigmentation, blister formation during the first years of life, and focal hyperkeratosis of the soles. Blistering in this entity is much limited and there is no atrophy of the skin. Differential diagnosis, however, may be impossible in some cases (Westerhoff and Dingeman, 2004).

Pathogenesis Blistering at basal layers is explained by abnormal binding of keratin 5 to desmoplakin I. The pathogenesis of hyperpigmentation is unknown but it has been suggested that the abnormal keratin perturbates normal melanosome trafficking (Uttam et al., 1996).

Prognosis The blistering and hyperpigmentation in these patients tends to improve with age and they have a normal life span (Boss et al., 1981). No treatment is currently available.

References Boss, J. M., C. N. A. Matthews, R. D. Peachy, and R. Summerly. Speckled hyperpigmentation, palmoplantar punctate keratosis and childhood blistering: A clinical triad, with variable associations. Br. J. Dermatol. 105:579–585, 1981. Bruckner-Tuderman, L., A. Vogel, S. Ruegger, B. Odermatt, O. Tonz, and U. W. Schnyder. Epidermolysis bullosa simplex with mottled pigmentation. J. Am. Acad. Dermatol. 21:425–432, 1989. Coleman, R., J. I. Harper, and B. D. Lake. Epidermolysis bullosa simplex with mottled pigmentation. Br. J. Dermatol. 128:679–685, 1993. Combemale, P., and J. Kanitakis. Epidermolysis bullosa simplex with mottled pigmentation. Case report and review of the literature. Dermatology 189:173–178, 1994. Fischer, T., and T. Gedde-Dahl Jr. Epidermolysis bullosa simplex and mottled pigment: A new dominantly inherited syndrome. Clin. Genet. 15:228–238, 1979. Hamada, T., N. Ishii, Y. Kawano, Y. Takahashi, M. Inoue, S. Yasumoto, and T. Hashimoto. The P25L mutation in the KRT5 gene in a Japanese family with epidermolysis bullosa simplex with mottled pigmentation. Br. J. Dermatol. 150:609–611, 2004. Irvine, A. D., K. E. McKenna, H. Jenkinson, and A. E. Hughes. A mutation in the V1 domain of keratin 5 causes epidermolysis

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bullosa simplex with mottled pigmentation. J. Invest. Dermatol. 108:809–810, 1997. Irvine, A. D., E. L. Rugg, E. B. Lane, S. Hoare, C. Peret, A. E. Hughes, and A. H. Heagerty. Molecular confirmation of the unique phenotype of epidermolysis bullosa simplex with mottled pigmentation. Br J. Dermatol. 144:40–45, 2001. Moog, U., C. E. de Die-Smulders, H. Scheffer, P. van der Vlies, C. J. Henquet, and M. F. Jonkman. Epidermolysis bullosa simplex with mottled pigmentation: clinical aspects and confirmation of the P24L mutation in the KRT5 gene in further patients. Am. J. Med. Genet. 86:376–379, 1999. Medenica-Mojsilovic, L., N. A. Fenske, and C. G. Espinoza. Epidermolysis bullosa herpetiformis with mottled pigmentation and an unusual punctate keratoderma. Arch. Dermatol. 122:900–908, 1986. Ning, C. C., S. C. Chao, J. Uitto, C. C. Shieh, and J. Y. Lee. Mutation analysis in the family of a Taiwanese boy with epidermolysis bullosa simplex Dowling-Meara. J. Formos. Med. Assoc. 100:407– 411, 2001. Tay, Y. K. An additional case of epidermolysis bullosa simplex herpetiformis of Dowling-Meara with mottled pigmentation. Pediatr. Dermatol. 16:490–491, 1999. Tay, Y. K., and W. L. Weston. Epidermolysis bullosa simplex herpetiformis of Dowling-Meara with mottled pigmentation: the relationship between EBS herpetiformis and EBS with mottled pigmentation. Pediatr. Dermatol.13:306–309, 1996. Uttam, J., E. Hutton, P. A. Coulombe, I. Anton-Lamprecht, Q. C. Yu, T. Gedde-Dahl Jr., J. D. Fine, and E. Fuchs. The genetic basis of epidermolysis bullosa simplex with mottled pigmentation. Proc. Natl. Acad. Sci. U. S. A. 93:9079–9084, 1996. Westerhoff, W., and K. P. Dingemans. Generalized mottled pigmentation with postnatal skin blistering in three generations. J. Am. Acad. Dermatol. 50:S63–S69, 2004.

Familial Mandibuloacral Dysplasia Eulalia Baselga and Nancy Burton Esterly

Historical Background Mandibuloacral dysplasia was identified as a new syndrome by Young et al. in 1971. Since then very few additional cases have been described (Caux et al., 2003; Cutler, 1991; Danks et al., 1974; Freidenberg et al., 1992; Fryburg et al., 1995; Hoeffel, 2000; Ng and Stratakis, 2000; Novelli et al., 2002; Pallotta and Morgese, 1984; Pedagogos et al., 1995; Prasad et al., 1998; Schrander-Stumpel et al., 1992; Simha and Garg, 2002; Tenconi et al., 1986; Tudisco et al., 2000; Welsh, 1975; Zina et al., 1981). Most patients are from Italy.

Synonym Craniomandibular dermatodysostosis.

Epidemiology and Genetics Mandibuloacral dysplasia is a rare autosomal recessive disorder (Prasad et al., 1998; Tenconi et al., 1986; Zina et al., 1981). Most of the reported cases are from Italy, suggesting the existence of a founder effect. Recently mutations on the LMNA gene on chromosome 1q21, encoding two nuclear envelop proteins (lamin A and C), have been detected (Caux et al., 2003; Novelli et al., 2002).

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION

Clinical Findings

Pathogenesis

The main features of the syndrome are mandibular and clavicular hypoplasia, delayed closure of the cranial sutures, acroosteolysis of the hands and feet, joint stiffness, short stature, and lipodystrophy. The first signs appear between 1 and 7 years and consist of shortening of distal phalanges and receding of the chin (Schrander-Stumpel et al., 1992).The mandibular hypoplasia may cause premature loss of teeth, dental overcrowding, and an “Andy Gump” appearance. The nose is thin and pointed and the eyes prominent. The acro-osteolysis causes short, club-shaped terminal phalanges. Progressive lipodystrophy and metabolic complications of insulin resistance such as diabetes, hypertriglyceridemia, and acanthosis nigricans may be noted. Type A lipodystrophy with loss of subcutaneous fat in the extremities and fat accumulation in the upper trunk, face, submental region, and occiput is more common. A different pattern of lipodystrophy is type B in which generalized loss of subcutaneous fat involving the face, trunk, and extremities may also be seen (Simha and Garg, 2002). Insulin resistance has been increasingly recognized in these patients (Caux et al., 2003; Freidenberg et al., 1992; Novelli et al., 2002; Simha et al., 2002). Less commonly associated features are hypogonadism, delayed puberty, and infantile uterus (Prasad et al., 1998), and sensorineural deafness (Freidenberg et al., 1992; Ng and Stratakis, 2000; Simha et al., 2002). Cutaneous manifestations include thin, atrophic, and taut skin on acral regions, with poikilodermatous appearance. Mottled hyperpigmentation may develop on the hands and feet and also on the trunk and proximal limbs (Novelly et al., 2002; Schrander-Stumpel et al., 1992; Zina et al., 1981). Acanthosis nigricans in the axilla and groin may develop. Keratotic papules over joints, back, palms, and soles as a manifestation of calcinosis cutis have been described (Danks et al., 1974; Prasad et al., 1998; Schrander-Stumpel et al., 1992). Sometimes there is discharge of cheesy material (Prasad et al., 1998). The scalp hair is sparse and lusterless and progressive alopecia ensues by the second or third decade of life. The nails may be broad, brittle, and curved downward.

Familial mandibuloacral dysplasia is now included in the laminopathies. This group includes seven different diseases with skeletal and/or cardiac muscle or peripheral nerve involvement due to mutations in lamin or lamin-associated protein genes. Lamins are structural proteins of the nuclear lamina, a structure near the inner nuclear membrane and the peripheral chromatin. The pathophysiology of this group of diseases, however, remains elusive.

Histology Examination of skin biopsy specimens shows an atrophic epidermis with a normal dermis and absence of subcutaneous tissue (Welsh, 1975; Zina et al., 1981). Biopsy of keratotic papules reveals calcified deposits in the mid and lower dermis which stain positive with Von Kossa.

Laboratory Investigations Hyperinsulinemia is often present both basally and after a glucose load, due to insulin resistance. Elevated serum triglycerides with low high-density lipoprotein cholesterol may be found. X-ray findings include hypoplastic phalanges with acro-osteolysis, widened cranial sutures, wormian bones, and hypoplastic mandible and clavicles.

Diagnosis and Differential Diagnosis Diagnosis can be suspected after recognition of the progressive mandibular hypoplasia and acro-osteolysis. Differential diagnosis includes other progeroid syndromes (especially acrogeria and Werner syndrome) (Table 44.4) with many overlapping features, cleidocranial dysostosis, and the Hallermann–Streiff syndrome. Hereditary sclerosing poikiloderma also shares many of the clinical findings.

References Caux, F., E. Dubosclard, O. Lascols, B. Buendia, O. Chazouilleres, A. Cohen, J. C. Courvalin, L. Laroche, J. Capeau, C. Vigouroux, and S. Christin-Maitre. A new clinical condition linked to a novel mutation in lamins A and C with generalized lipoatrophy, insulinresistant diabetes, disseminated leukomelanodermic papules, liver steatosis, and cardiomyopathy. J. Clin. Endocrinol. Metab 88: 1006–1013, 2003. Cutler D. L., S. Kaufmann, and G. R. Freidenberg. Insulin-resistant diabetes mellitus and hypermetabolism in mandibuloacral dysplasia: a newly recognized form of partial lipodystrophy. J. Clin. Endocrinol Metab. 73:1056–1061, 1992. Danks, D. M., V. Mayne, H. N. B. Wettenhal, and R. K. Hall. Craniomandibular dermatodysostosis. Birth Defects Orig. Art. Ser. X:99–105, 1974. Freidenberg, G. R., D. L. Cutler, M. C. Jones, B. Hall, R. J. Mier, F. Culler, K. L. Jones, C. Lozzio, and S. Kaufmann. Severe insulin resistance and diabetes mellitus in mandibuloacral dysplasia. Am. J. Dis. Child. 146:93–99, 1992. Fryburg, J. S., and N. Sidhu-Malik. Long-term follow-up of cutaneous changes in siblings with mandibuloacral dysplasia who were originally considered to have hereditary sclerosing poikiloderma. J. Am. Acad. Dermatol. 33:900–902, 1995. Hoeffel, J. C., L. Mainard, P. Chastagner, and C. C. Hoeffel. Mandibuloacral dysplasia. Skeletal. Radiol. 29:668–671, 2000. Ng, D., and C. A. Stratakis. Premature adrenal cortical dysfunction in mandibuloacral dysplasia: a progeroid-like syndrome. Am. J. Med. Genet. 95:293–295, 2000. Novelli, G., A. Muchir, F. Sangiuolo, Helbling-A. Leclerc, D’M. R. Apice, C. Massart, F. Capon, P. Sbraccia, M. Federici, R. Lauro, C. Tudisco, R. Pallotta, G. Scarano, B. Dallapiccola, L. Merlini, and G. Bonne. Mandibuloacral dysplasia is caused by a mutation in LMNAencoding lamin A/C. Am. J. Hum. Genet. 71:426–431, 2002. Pallotta, R., and G. Morgese. Mandibuloacral dysplasia: a rare progeroid syndrome: two brothers confirms autosomal recessive inheritance. Clin. Genet. 26:133–138, 1984. Pedagogos, E., G. Flanagan, D. M. Francis, G. J. Becker, D. M. Danks, and R. G. Walker. A case of craniomandibular dermatodysostosis associated with focal glomerulosclerosis. Pediatr. Nephrol. 9:354– 356, 1995. Prasad, P. V., L. Padmavathy, and S. Sethurajan. Familial mandibuloacral dysplasia — a report of four cases. Int. J. Dermatol. 37: 614–616, 1998. Schrander-Stumpel, C., A. Spaepen, J. P. Fryns, and J. Dumon. A

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CHAPTER 44 Table 44.4. Progeroid syndromes with hyperpigmentation. Werner

Progeria

Acrogeria

Metageria

Mandibuloacral dysplasia

Age of onset Inheritance

Adolescence AR

Childhood AR

Beaked nose

Pinched face

“Andy Gump”-like; loss of teeth or overcrowded teeth

Skin

Atrophy; calluses; ulcers; diffuse or mottled hyperpigmentation Cataracts Premature graying

Prominent eyes Alopecia

Acral atrophy; mottled hyperpigmentation and telangiectasia on trunk; ulcers Prominent eyes Fine and thin

Acral atrophy; mottled Hyperpigmentation

Eyes Hair Stature Sexual development Systemic findings

Small Absent

Small Absent

Birth Sporadic, AD?, AR? Pinched face, beaked nose, micrognathia, wrinkled Acral atrophy, wrinkling, mottled hyperpigmentation; prominent veins on trunk Normal Normal; recession of hair line Normal Normal

Birth ? possibly AR

Facies

Infancy ? AR and AD reported Plucked bird; prominent scalp veins; midfacial cyanosis Sclerodermalike; mottled hyperpigmentation

Atherosclerosis; diabetes Reduced

Atherosclerosis

No

Reduced

Normal

Life span

Tall Normal Diabetes; atherosclerosis Reduced

Prominent eyes Alopecia Small Normal/hypogonadism/ delayed puberty No; insulin resistance? Normal

AD, autosomal dominant; AR, autosomal recessive.

severe case of mandibuloacral dysplasia in a girl. Am. J. Med. Genet. 43:877–881, 1992. Simha, V., and A. Garg. Body fat distribution and metabolic derangements in patients with familial partial lipodystrophy associated with mandibuloacral dysplasia. J. Clin. Endocrinol. Metab. 87:776–785, 2002. Tenconi, R., F. Miotti, A. Miotti, G. Audino, R. Ferro, and M. Clementi. Another Italian family with mandibuloacral dysplasia: Why does it seem more frequent in Italy? Am. J. Med. Genet. 24:357–364, 1986. Tudisco, C., G. Canepa, G. Novelli, and B. Dallapiccola. Familial mandibuloacral dysplasia: report of an additional Italian patient. Am. J. Med. Genet. 94:237–241, 2000. Welsh, O. Study of a family with a new progeroid syndrome. Birth Defects Orig. Art. Ser. XI:25–38, 1975. Young, L. W., J. F. Radebaugh, P. Rubin, J. A. Sensenbrenner, and G. Fiorelli. A new syndrome manifested by mandibular hypoplasia, acroosteolysis, stiff joints and cutaneous atrophy (mandibuloacral dysplasia) in two unrelated boys. Birth Defects Orig. Art. Ser. VII:291–297, 1971. Zina, A. M., A. Cravario, and S. Bundino. Familial mandibuloacral dysplasia. Br. J. Dermatol. 105:719–723, 1981.

Wallach et al., 1981) or different names such as congenital poikiloderma with vesicles and bullae (Pinol-Aguade et al., 1972), incontinentia pigmenti with a poikilodermatous state, or hereditary diffuse hyperpigmentation (Verbov, 1980). Clinical overlap with Kindler syndrome has often caused confusion in making a clear diagnosis, and some authors consider hereditary acrokeratotic poikiloderma described by Weary to be a variant of poikiloderma of Kindler (Draznin et al., 1978; Forman et al., 1989) and refer to both entities as poikiloderma of Weary–Kindler (Larregue et al., 1981; Malleville et al., 1982). Different mode of inheritance was one of the major distinguishing features. As the genetic basis of Kindler syndrome has recently been elucidated, separation of both diseases seems reasonable until genetic studies are performed in acrokeratotic poikiloderma of Weary.

Synonyms Acrokeratotic poikiloderma of Weary; congenital poikiloderma with bullous lesions “Weary type”; Weary–Kindler disease.

Hereditary Acrokeratotic Poikiloderma Eulalia Baselga and Nancy Burton Esterly

Historical Background In 1971 Weary et al. described 10 members of a single family with an autosomal dominant trait that resembled hereditary acrokeratotic poikiloderma of Kindler. Similar cases have been reported under the same (Draznin et al., 1978; Larregue et al., 1981; Malleville et al., 1982; Pierini et al., 1979; Pons, 1997; 792

Clinical Findings Weary et al. (1971) reported 10 members of a family with autosomal dominant trait that they called hereditary acrokeratotic poikiloderma. They described the following types of abnormalities: (1) vesicopustule formation confined to the hands and feet — the blistering began around 1–3 months of age and resolved in late childhood; (2) widespread eczematous dermatitis somewhat resembling atopic eczema that started

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION

between the ages of 3 and 6 months and completely resolved by 5 years of age; (3) the gradual appearance of a diffuse poikiloderma with striate and reticulate atrophy that spared only the face, scalp, and ears and persisted into adult life (Fig. 44.8); and (4) the development of keratotic papules on the hands (Fig. 44.9), feet, elbows, and knees that first appear prior to 5 years of age and persist indefinitely. The pigmentary changes usually appear during the first year of life but rarely are present at birth (Weary et al., 1971). They progress gradually during childhood and thereafter persist or slowly fade during adulthood. The pigmentary abnormality consists of a widespread, mottled, stellate, and reticulated hyperpigmentation which is more pronounced in the flexural areas such as the neck, axillae, antecubital and popliteal fossae, and inguinal region. The abdomen, buttocks, lumbar area, and eyelids may also be affected but the remainder of the face is usually spared. The skin in the hyperpigmented areas may show other signs of poikiloderma such as atrophy and telangiectases although never as prominently as the pigmentary changes. The formation of vesicles seems to be the initial event appearing between 5 weeks and 6 months and gradually improving with age. These lesions develop without apparent triggers such as trauma, heat, or sun exposure and usually are limited to the hands and feet. The vesicles resolve without scarring or milia formation. The eczematous dermatitis is an inconstant feature and may be indistinguishable from atopic dermatitis except for a tendency to spare the face. Acrokeratoses are an almost constant feature in the affected individuals. The acrokeratoses are 15 mm lichenoid papules that appear on hands, feet, elbows, and knees and resemble flat warts. Those on the palms and soles may look like small pits or resemble arsenical keratoses. Similar lesions were observed in some family members without the other features of the syndrome, suggesting a forme fruste variant of the disorder. These acrokeratoses tend to appear later than the other skin changes characteristic of the syndrome. Nails, teeth, eyes, and mucous membranes are not affected and there is no associated photosensitivity except in the patient reported by Malleville (1982). Three individuals in the original kindred had elevated IgG levels. Porphyrin levels were normal.

Fig. 44.8. Diffuse poikiloderma and reticulate atrophy on the flank (see also Plate 44.8, pp. 494–495).

Fig. 44.9. Diffuse poikiloderma with pigmentary changes on the dorsum of he hands of a patient with hereditary acrokeratotic poikiloderma (see also Plate 44.9, pp. 494–495).

degeneration of the basal layer with a lichenoid inflammatory infiltrate obliterating the dermoepidermal junction (Weary et al., 1971). Other authors noted a relative decrease in elastic fibers (Draznin et al., 1978; Kindler, 1954).

Diagnosis The diagnosis depends on recognition of the cutaneous findings of poikiloderma, acrokeratoses, and transient blistering.

Histology Skin biopsy specimens obtained from the poikilodermatous skin demonstrate slight epidermal atrophy, irregular distribution of melanin pigment along the lower epidermis and rare vacuolization of the basal cell layer (Larregue et al., 1981; Malleville et al., 1982; Wallach et al., 1981; Weary et al., 1971). In the papillary dermis there are melanophages and rare IgM colloid bodies (Blanchet-Bardon et al., 1985; Malleville et al., 1982; Verret et al., 1984). Biopsies of the bullae show an intraepidermal blister with cytolysis of basal cells (Larregue et al., 1981; Pinol-Aguade et al., 1972; Verbov, 1980). Acrokeratotic lesions have hyperkeratosis, hypergranulosis, and irregular epidermal hyperplasia (Weary et al., 1971). Weary observed, in early vesicles, extensive hydropic

Differential Diagnosis Differential diagnosis includes other hereditary bullous poikilodermas such as Rothmund–Thomson syndrome, dyskeratosis congenita, Mendes da Costa syndrome, epidermolysis bullosa simplex with mottled pigmentation (Table 44.5). Hereditary sclerosing poikiloderma (Weary et al., 1969), dermatopathia pigmentosa reticularis, Naegeli–Franceschetti–Jadassohn syndrome, and Degos–Touraine syndrome may bear some resemblance to hereditary acrokeratotic poikiloderma. However, the absence of bullae or keratotic papules and the presence of sclerosing bands and clubbing of the fingers in the former disorder distinguishes hereditary sclerosing poikiloderma from hereditary acrokeratotic poikiloderma. Hereditary acrokeratotic 793

CHAPTER 44 Table 44.5. Genetic poikilodermas. Disease

Inheritance*

Bullae

Poikiloderma

Acral keratoses

Other

Hereditary acrokeratotic poikiloderma

AD

Acral

Yes

Eczema

Kindler syndrome

AR

Acral

Spares face except eyelids; increased in flexures Widespread

+/-

Hereditary sclerosing poikiloderma

AD

No

Marked skin atrophy: webbing of the digits Clubbing; linear sclerotic bands in flexures

Rothmund–Thomson

AR

Light-exposed skin

Spares chest, most prominent in flexures Photodistributed

No

+/-

Photosensitivity; cataracts; alopecia; nail and dental defects; warty keratoses over bony prominences

* AD, autosomal dominant; AR, autosomal recessive.

poikiloderma may be impossible to differentiate from epidermolysis bullosa with mottled pigmentation and from the poikiloderma of Kindler because they share acral bullae in infancy and early childhood and generalized progressive poikiloderma. Based on a review of the literature (Passwell et al., 1973; Person and Perry, 1979; Pierini et al., 1979; Wallach et al., 1981) several authors (Draznin et al., 1978; Forman et al., 1989; Malleville et al., 1982; Verret et al., 1984) have suggested that the Kindler syndrome and Weary hereditary acrokeratotic poikiloderma are variants of the same disease. In Kindler syndrome, however, there is photosensitivity, the face tends to be affected, and epidermal atrophy is more prominent. Ultrastructural differences have also been emphasized (Hovnanian et al., 1989). With identification of kindlin-1 as the target protein in Kindler syndrome, immunofluorescence studies with anti-kindlin-1 antibody may help in the differential diagnosis (Siegel et al., 2003).

Pathogenesis and Treatment The pathogenesis remains unknown. Treatment for this condition has not been reported.

References Alper, J. C., H. P. Baden, and L. A. Goldsmith. Kindler’s syndrome. Arch. Dermatol. 114:457, 1978. Blanchet-Bardon, C., N. Nazzaro, and C. Mimoz. Poikilodermie congenitale avec formation de bulles traumatiques et atrophie cutanee progressive: poikiledermie de Theresa Kindler. Ann. Dermatol. Venereol. 112:703–704, 1985. Draznin, M. B., N. B. Esterly, and D. F. Fretzin. Congenital poikiloderma with features of hereditary acrokeratotic poikiloderma. Arch. Dermatol. 114:1207–1210, 1978. Forman, A. B., J. S. Prendiville, N. B. Esterly, A. A. Hebert, M. Devic, Y. Horiguchi, and J.-D. Fine. Kindler syndrome: Report of two cases and review of the literature. Pediatr. Dermatol. 6:91–101, 1989. Hovnanian, A., C. Blanchet-Bardon, and Y. de Prost. Poikiloderma of Theresa Kindler: Report of a case with ultrastructural study and review of the literature. Pediatr. Dermatol. 6:82–90, 1989. Kindler, T. Congenital poikiloderma with traumatic bulla formation and progressive cutaneous atrophy. Br. J. Dermatol. 66:106–111, 1954. Larregue, M., F. Prigent, G. Lorrete, C. Canuel, and P. Ramdenee.

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Acrokeratose poikilodermique bulleuse et hereditaire de WearyKindler. Ann. Dermatol. Venereol. 108:69–76, 1981. Malleville, J., Y. Cavaroc, G. Boiron, J. M. Tamisier, J. E. SurleveBazeille, M. Larregue, P. H. Sarrat, and G. Guillet. Poikilodermie diffuse avec acrokeratose et precession de lesions bulleuses: maladie de Weary-Kindler. Ann. Dermatol. Venereol. 109:949–959, 1982. Passwell, J., L. Zipperkowski, and D. Katznelson. A syndrome characterized by congenital ichthyosis with atrophy, mental retardation, dwarfism and generalized aminoaciduria. J. Pediatr. 82:466–471, 1973. Person, J. R., and H. O. Perry. Congenital poikiloderma with traumatic bulla formation, anhidrosis, and keratoderma. Acta Derm. Venereol. 59:347–351, 1979. Pierini, A. M., D. O. Pierini, and J. Abulafia. Poiquilodermis acroqueratosica hereditaria. Arch. Argent. Dermatol. 29:243–256, 1979. Pinol-Aguade, J., C. Herrero, T. Castel, A. Castells, and F. Grimalt. Poiquilodermia congenita con lesiones vesiculoampollosas. Med. Cutanea Ibero-Lat-Am. 6:427–436, 1972. Pinol-Aguade, J., J. Peyri-Rey, and C. Herrero. Poiquilodermia congenita con ampollas, tipo Kindler-Salamon. Med. Cutanea IberoLat-Am. 3:221–225, 1973. Siegel, D. H., G. H. Ashton, H. G. Penagos, J. V. Lee, H. S. Feiler, K. C. Wilhelmsen, A. P. South, F. J. Smith, A. R. Prescott, V. Wessagowit, N. Oyama, M. Akiyama, D. Al Aboud, K. Al Aboud, A. Al Githami, K. Al Hawsawi, A. Al Ismaily, R. Al-Suwaid, D. J. Atherton, R. Caputo, J. D. Fine, I. J. Frieden, E. Fuchs, R. M. Haber, T. Harada, Y. Kitajima, S. B. Mallory, H. Ogawa, S. Sahin, H. Shimizu, Y. Suga, G. Tadini, K. Tsuchiya, C. B. Wiebe, F. Wojnarowska, A. B. Zaghloul, T. Hamada, R. Mallipeddi, R. A. Eady, W. H. McLean, J. A. McGrath, and E. H. Epstein. Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actinextracellular-matrix linker protein UNC-112, causes Kindler syndrome. Am. J. Hum. Genet. 73:174–187, 2003. Verbov, J. Hereditary diffuse hyperpigmentation. Clin. Exp. Dermatol. 5:227–234, 1980. Verret, J. L., M. Avenel, M. Larregue, and C. Panigel-Nguyen. Syndrome de Kindler; un cas avec etude ultrastructurale. Ann. Dermatol. Venereol. 111:259–269, 1984. Wallach, D., D. O. Vignon-Pierini, and F. Cottenot. Poikilodermie congenitale avec bulles, type Weary. Ann. Dermatol. Venereol. 108:79–83, 1981. Weary, P. E., Y. T. Hsu, D. R. Richardson, C. M. Caravati, and B. T. Wood. Hereditary sclerosing poikiloderma: Report of two families with an unusual and distinctive genodermatosis. Arch. Dermatol. 100:413–422, 1969.

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION Weary, P. E., W. F. Manley, and G. F. Graham. Hereditary acrokeratotic poikiloderma. Arch. Dermatol. 103:409–422, 1971.

Hereditary Sclerosing Poikiloderma Eulalia Baselga and Nancy Burton Esterly

Historical Background This disorder was first described by Weary et al. in 1969 in seven members of two unrelated black families. A subsequent report in 1978 by Greer et al. provided information on three other members of the same families. In 1995, Fazio et al. reported the first case in a white person, and a second family with four affected members has been reported (Grau et al., 1999).

Genetics

Fig. 44.10. Hyper- and hypopigmented macules on the extremities of a patient with hereditary sclerosing poikiloderma (see also Plate 44.10, pp. 494–495).

From the inheritance pattern in one of the kindreds with six affected members, it was proposed that this condition is transmitted as an autosomal dominant trait.

Clinical Findings The pigmentary changes are absent at birth but develop gradually over the first few years of life and are usually apparent by the age of 4 years. Irregular hyperpigmented, hypopigmented, and depigmented macules intermingle resulting in a mottled appearance of the involved skin. The lesions are small, measuring up to 1 cm in diameter. They are widespread but usually spare the upper chest. Atrophy and telangiectases are characteristic in some areas of pigmentary change, particularly over the elbows, knees, and interphalangeal joints and in some flexures. The combination of atrophy, telangiectases, and pigmentary changes produce a poikilodermatous appearance to the skin. An additional striking cutaneous feature is the presence of reticulated or linear sclerotic and hyperkeratotic bands located primarily in the flexural areas. They appear somewhat later in life than the abnormal pigmentation. These bands may have a beaded appearance, are oriented perpendicular to the flexure, and increase in number with time. They may traverse the flexure in parallel lines or form a reticulated pattern in the axillae, antecubital, and popliteal fossae (Fig. 44.10). The hands appear sclerodermiform and Raynaud phenomena may be present (Grau et al., 1999). Diffuse pebbly sclerosis as well as punctate keratoderma and mottled hyperpigmentation of the palms and soles are almost constant features (Fig. 44.11), and at times cause mild contractures of some of the digits (Greer et al., 1978; Grau et al., 1999). Another characteristic finding is clubbing of the fingers, the significance of which is unknown. Calcinosis cutis was a late manifestation in one individual. Several patients have had heart murmurs, and in at least one family, were secondary to valvular disease and the leading cause of death (Grau et al., 1999). No other organs are affected (Greer et al., 1978; Weary et al., 1969).

Fig. 44.11. Diffuse pebbly sclerosis and mottled pigmentation on the feet (see also Plate 44.11, pp. 494–495).

Histology The histopathologic changes are variable. The sclerotic bands show foci of marked hyperkeratosis beneath which the collagen is dense and homogeneous and almost devoid of elastic tissue. No deposits of extraneous material are seen. Hyperpigmented areas show a poikilodermatous appearance. Melanin pigment is irregularly distributed in the basal layer and lower epidermis. The quantity is variable ranging from total absence to heavy deposition. Pigment-containing macrophages are encased in the dense collagen of the upper dermis underlying the areas of epidermis completely lacking pigment. There is focal homogenization of the collagen around blood vessels in the mid dermis although the dermis is not thickened. Appendageal structures are normal (Weary et al., 1969).

Diagnosis The diagnosis is made by recognizing the particular constellation of findings characteristic of this disorder as there are no helpful laboratory studies and the skin biopsy findings are not pathognomonic. 795

CHAPTER 44 Table 44.6. Genetic poikilodermas. Disease

Inheritance*

Bullae

Poikiloderma

Acral keratoses

Other

Hereditary acrokeratotic poikiloderma

AD

Acral

Yes

Eczema

Kindler syndrome

AR

Acral

Spares face except eyelids; increased in flexures Widespread

+/-

Hereditary sclerosing poikiloderma

AD

No

Marked skin atrophy: webbing of the digits Clubbing; linear sclerotic bands in flexures

Rothmund–Thomson

AR

Light-exposed skin

Spares chest, most prominent in flexures Photodistributed

No

+/-

Photosensitivity; cataracts; alopecia; nail and dental defects; warty keratoses over bony prominences

* AD, autosomal dominant; AR, autosomal recessive.

Differential Diagnosis Differential diagnosis includes other poikilodermas such as hereditary acrokeratotic poikiloderma (Weary–Kindler syndrome), Rothmund–Thomson syndrome (Table 44.6), and dyskeratosis congenita as well as pigmentary disorders such as dyschromatosis universalis. None, however, have these unique linear and sclerotic bands with clubbing of the fingers. A more difficult problem is distinguishing hereditary sclerosing poikiloderma from mandibuloacral dysplasia (MAD syndrome). Re-examination of two siblings described in the original reports of hereditary sclerosing poikiloderma (family 2) disclosed features of both disorders (Fryburg and Sidhu-Malik, 1995). MAD syndrome is characterized by an abnormal facies, alopecia, a hypoplastic mandible, crowding and loss of the lower teeth, delayed closure of the cranial sutures, dysplastic clavicles, clubbed terminal phalanges, acroosteolysis, and atrophy of the skin on the hands and feet (Fryburg and Sidhu-Malik, 1995; Pallotta and Morgese, 1984; Zina et al., 1981). Some of the patients reported by Weary had many of these features but also had the sclerotic bands unique to hereditary sclerosing poikiloderma. Mandibuloacral dysplasia is an autosomal recessive disorder whereas hereditary sclerosing poikiloderma seems to be autosomal dominant. Nevertheless, Fryburg et al. suggest that the original diagnosis might have been incorrect, exemplifying the clinical difficulties in separating these diseases.

Pathogenesis and Treatment The pathogenesis is completely unknown. These patients are in good health otherwise and laboratory studies have been within normal limits. There is no known treatment.

References Fryburg, J. S., and N. Sidhu-Malik. Long-term follow-up of cutaneous changes in siblings with mandibuloacral dysplasia who were originally considered to have hereditary sclerosing poikiloderma. J. Am. Acad. Dermatol. 33:900–902, 1995. Fazio, M., S. Lisi, A. Amantea, A. Maini, G. Menaguale, G. Sacerdoti, and L. Balus. Poikilodermie sclerosante hereditaire de Weary. Ann. Dermatol. Venereol. 122:618–620, 1995.

796

Grau, S. C., V. Pont, J. R. Cors, and A. Aliaga. Hereditary sclerosing poikiloderma of Weary: report of a new case. Br. J. Dermatol. 140:366–368, 1999. Greer, K. E., P. E. Weary, R. Nagy, and M. Robinow. Hereditary sclerosing poikiloderma. Int. J. Dermatol. 17:316–322, 1978. Pallotta, R., and G. Morgese. Mandibuloacral dysplasia: a rare progeroid syndrome: two brothers confirms autosomal recessive inheritance. Clin. Genet. 26:133–138, 1984. Tenconi, R., F. Miotti, A. Miotti, G. Audino, R. Ferro, and M. Clementi. Another Italian family with mandibuloacral dysplasia: Why does it seem more frequent in Italy? Am. J. Med. Genet. 24:357–364, 1986. Weary, P. E., Y. T. Hsu, D. R. Richardson, C. M. Caravati, and B. T. Wood. Hereditary sclerosing poikiloderma: Report of two families with an unusual and distinctive genodermatosis. Arch. Dermatol. 100:413–422, 1969. Zina, A. M., A. Cravario, and S. Bundino. Familial mandibuloacral dysplasia. Br. J. Dermatol. 105:719–723, 1981.

Mendes Da Costa Disease Eulalia Baselga and Nancy Burton Esterly

Historical Background In 1908 Mendes da Costa described a genodermatosis in two brothers from a family in Amsterdam under the rubric of dystrophia bullosa hereditaria, typus maculatus (Mendes da Costa and van der Valk, 1908). Several additional cases were then described in subsequent generations of the same family (Carol and Kooij, 1937; Hassing and Doeglas, 1980). Recently a second family with Mendes da Costa disease has been reported in Italy (Lungarotte et al., 1994). Analysis of both pedigrees shows that only males are affected and the obligate carrier females are healthy. These data support an X-linked mode on genetic transmission. Because some clinical features arte similar to those observed in epidermolysis bullosa, Mendes da Costa disease was originally considered a subtype of epidermolysis bullosa simplex (Fine et al., 1991), but it has became clear that is not a true mechanobullous disorder (Fine et al., 2000) but a poikilodermatous disorder.

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION Table 44.7. Genetic diseases with reticulated epidermal hyperpigmentation. Disease

Inheritance*

Distribution

Special features

Associated findings

Acropigmentation of Kitamura

AD

Acral

Lesions are atrophic

Acropigmentation of Dohi Dermatopathia pigmentosa reticularis

AD AD

Acral Generalized

Hypopigmented macules Hypopigmented macules

Anonychia with flexural pigmentation

AD

Flexural

Hypopigmented macules

Mendes da Costa disease

XLR

Generalized, face and limbs

Dowling–Degos disease

AD

Naegeli–Franceschetti–Jadassohn syndrome Epidermolysis bullosa with mottled pigmentation

AD

Flexural, then generalized Generalized

Blistering early; hypopigmented macules Onset delayed

Palmar pits; breakage of epidermal ridges Predominant in males Sweating disorders; decreased dermatoglyphics; alopecia; onychodystrophy; palmar/plantar keratoses Sparse hair; hypohidrosis; decreased dermatoglyphics; absent/ rudimentary nails Microcephaly; mental retardation; atrichia; short conical fingers

AD

Trunk, proximal limbs

Blistering disorder

Perioral pits; keratoses; epidermal cysts Keratoderma; enamel hypoplasia; hyperhidrosis; nail dystrophy Keratoderma; palmar/plantar; carious teeth; photosensitivity

* AD, autosomal dominant; XLR, X-linked recessive.

Epidemiology and Genetics Mendes da Costa disease has been mapped by linkage analysis of the two reported families to Xq27-q28 region (Wijker et al., 1995).

Synonyms Mendes da Costa disease; dystrophia bullosa hereditaria, typus maculatus of Mendes da Costa; epidermolysis bullosa simplex, Mendes da Costa variant; hereditary bullous dystrophy macular type.

pedigree reported by Carol (1937) increased melanin in the epidermis was noted in a skin biopsy from one member. Electron microscopy studies of blistered skin demonstrate disruption of the plasma membrane of basal keratinocytes with normal desmosomes, hemidesmosomes, and anchoring fibrils (Geerts et al., 1978).

Pathogenesis The pathogenesis is unknown.

Differential Diagnosis Clinical Findings Mendes da Costa disease is characterized by spontaneous blistering during early childhood, reticulated hyperpigmentation and hypopigmentation on the face and extremities, and atrichia. Other inconstant features are microcephaly, growth retardation, and short conical fingers. The formation of bullae ceases after the third year of life. Blistering is not provoked by trauma. The blisters heal without scarring or milia formation. Mucosae are not affected. The pigmentary abnormality is progressive although in the reported cases the age of onset is not mentioned. It is described as a reticular red to brown pigmentation with intermingled lenticular hypopigmentation most prominent on the face and extremities.

Histology Histopathologic examination of biopsy specimens of blisters demonstrates an intraepidermal cleavage plane. Hassing and Doegles (1980) also describe vacuolization of the basal layer with “pigment” and colloid bodies in the upper dermis. In the

The differential diagnosis includes other blistering diseases with reticular pigmentation such as Kindler disease, hereditary acrokeratotic poikiloderma of Weary, and epidermolysis bullosa with mottled hyperpigmentation. In Kindler disease and hereditary acrokeratotic poikilodermia the skin changes are more poikilodermatous, there is no atrichia and photosensitivity and acral keratotic papules, present, respectively, in these two diseases, are not a feature of Mendes da Costa syndrome (Table 44.7). Rothmund–Thomson syndrome may also be considered but the characteristic appearance and pattern of appearance of the rash, with an acute erythematous phase, allows differentiation.

References Carol, W. L. L., and R. Kooij. Typus maculatus der bullosen hereditaren dystrophie. Acta Derm. Venereol. 18:265–283, 1937. Fine, J. D., E. A. Bauer, R. A. Briggaman, D. M. Carter, R. A. J. Eady, N. B. Esterly, K. A. Holbrook, S. Hurwitz, L. Johnson, A. Lin, R. Pearson, and V. P. Sybert. Revised clinical and laboratory criteria for subtypes of inherited epidermolysis bullosa. J. Am. Acad. Dermatol. 24:119–135, 1991.

797

CHAPTER 44 Fine, J. D., R. A. Eady, E. A. Bauer, R. A. Briggaman, L. BrucknerTuderman, A. Christiano, A. Heagerty, H. Hintner, M. F. Jonkman, J. McGrath, J. McGuire, A. Moshell, H. Shimizu, G. Tadini, and J. Uitto. Revised classification system for inherited epidermolysis bullosa: Report of the Second International Consensus Meeting on diagnosis and classification of epidermolysis bullosa. J. Am. Acad. Dermatol. 42:1051–1066, 2000. Geerts, M. L., J. Overbeke, A. Kint, and R. H. Cormane. Comparative electron microscopic study between Mendes da Costa’s disease and recessive epidermolysis bullosa dystrophica. Br. J. Dermatol. 98:529–536, 1978. Hassing, J. H., and H. M. G. Doeglas. Dystrophia bullosa hereditaria, typus maculatus (Mendes da Costa-van der Valk): a rare genodermatosis. Br. J. Dermatol. 102:474–476, 1980. Lungarotte, M. S., C. Martello, G. Barboni, D. Mezzetti, G. Mariotti, and A. Calabro. X-linked mental retardation, microcephalaly, and growth delay associated with hereditary bullous dystrophy macular type: report of a second family. Am. J. Med. Genet. 51:587–601, 1994. Mendes da Costa, S., and J. W. van der Valk. Typhus maculatus der bullosen hereditaren dystrophie. Arch. Dermatol. Syphil. 91:1–8, 1908. Wijker M., M. J. Ligtenberg, F. Schoute, J. C. Defesche, G. Pals, P. A. Bolhuis, H. H. Ropers, T. J. Hulsebos, F. H. Menko, and van Oost, B. A. The gene for hereditary bullous dystrophy, X-linked macular type, maps to the Xq27.3-qter region. Am. J. Hum. Genet. 56:1096–1100, 1995.

Naegeli–Franceschetti– Jadassohn Syndrome

Clinical Features The pigmentation changes include a reticulated brown and gray brown pigmentation of the periocular/orbital regions and the abdomen with more variable involvement of the skin of the neck, upper trunk, axilla, groin, and/or flexures. This discoloration develops early in childhood, at ages ranging from 3 months to 5–6 years (Itin et al., 1993). The pigmentation gradually increases during the first 10 years of life but begins to fade around puberty (Itin et al., 1993). Of the three oldest patients in the original Swiss family who had pigmentation changes documented by earlier reports, no typical pigmentation changes were found when re-examined by Itin (1993). Other characteristics of Naegeli–Franceschetti–Jadassohn syndrome include a diffuse and punctate palmoplantar keratoderma, hypohidrosis, and poor heat tolerance, absence of dermatoglyphics (Itin et al., 1993; Papini, 1978; Sparrow et al., 1976), dental anomalies (yellowish discoloration of dental enamel, abnormally shaped teeth and supernumerary teeth), and onychodystrophy (Sparrow et al., 1976). Malalignment of great toenails was described in one family (Itin et al., 1993). Bullous lesions on the hands and feet shortly after birth, which resolved without sequelae, were noted in Sparrow’s patients (Sparrow et al., 1976) and one of the patients seen by Itin (1993). The patients have normal mental development. The family reported by Tzermias et al. (1995) had extensive flexural milia.

Eulalia Baselga and Nancy Burton Esterly

Historical Background

Histology

Synonyms

Histologic findings include patchy hyperpigmentation of the epidermis and marked pigmentary incontinence with numerous melanophages in the upper dermis. Eccrine glands are present and do not show any abnormality (Sparrow et al., 1976). In an electron microscopic study of skin from two members of the original family, pigment incontinence and variable amounts of colloid amyloid bodies were found in the superficial dermis (Frenk et al., 1993). The colloid amyloid bodies could also be found around sweat glands in the reticular dermis (Frenk et al., 1993).

Naegeli syndrome, NFJ syndrome, Franceschetti–Jadassohn syndrome.

Criteria for Diagnosis

The disorder currently labeled the Naegeli–Franceschetti– Jadassohn syndrome was first described in a Swiss family by Naegeli in 1927. It was originally confused with incontinentia pigmenti. In 1954 Franceschetti and Jadassohn reexamined the original family and found the mode of inheritance to be autosomal dominant. This feature differentiated it from incontinentia pigmenti, which is an X-linked disorder.

Epidemiology and Genetics Naegeli–Franceschetti–Jadassohn syndrome has been well described only in three families. The first was the Swiss family reported by Naegeli that later was studied by Franceschetti and Jadassohn. This same family was studied again by Itin (1993) expanding the pedigree. The second and third family were described by Sparrow (1976) and Papini (1978, 1994), respectively. There are other reports of possible cases of Naegeli–Franceschetti–Jadassohn syndrome but most of these individuals lack one or more key features (Greither and Haensch, 1970; Kitamura and Hirako, 1955; Kudo et al., 1995; Levi et al., 1971; Tzermias et al., 1995; Verbov, 1975; Whiting, 1971). Linkage studies in the original family have mapped the putative gene to a 6 cM region on chromosome 17 (Sprecher et al., 2002; Whittock et al., 2000). 798

Criteria for the diagnosis of Naegeli–Franceschetti–Jadassohn syndrome include reticulated pigmentation that fades after puberty, palmoplantar keratoderma, hypohidrosis, enamel defects, and adermatoglyphia.

Differential Diagnosis The differential diagnosis for Naegeli–Franceschetti– Jadassohn syndrome includes other reticulated pigmentary disorders. In the literature this syndrome has often been confused with incontinentia pigmenti although they are quite distinct, both phenotypically and genetically. Incontinentia pigmenti is an X-linked disorder and pigmentary changes are preceded by inflammation. Hereditary and bullous acrokeratotic poikiloderma of Weary and Kindler has some similarities to Naegeli–Franceschetti–Jadassohn syndrome but lacks hypohidrosis and has atrophy of the skin (Itin et al., 1993). Der-

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION

matopathia pigmentosa reticularis has many of the features of Naegeli–Franceschetti–Jadassohn syndrome: pigmentary changes, heat intolerance, absent dermatoglyphics, keratoderma, and onychodystrophy. However in dermatopathia pigmentosa reticularis the discoloration does not fade over time and there is a characteristic noncicatricial alopecia of the scalp, eyebrows, and eyelashes not found in Naegeli– Franceschetti–Jadassohn syndrome.

Pathogenesis Naegeli–Franceschetti–Jadassohn syndrome is thought to be a subtype of ectodermal dysplasia. This would explain the changes seen in the teeth, nails, and sweat glands. Naegeli– Franceschetti–Jadassohn syndrome is likely to result from an abnormality in a structural protein expressed in all affected ectodermal derived tissues or from an abnormal regulatory protein controlling ectodermal differentiation, cell adhesion, or intercellular signaling (Sprecher et al., 2002).

Prognosis The major problem patients with Naegeli–Franceschetti– Jadassohn syndrome experience is hypohidrosis. Some are unable to tolerate hot environments or physical activity. All affected patients show normal intelligence and are otherwise in good health (Itin et al., 1993).

References Franceschetti, A., and W. Jadassohn. A propos de l’incontinentia pigmenti delimitation de deux syndromes differents figurant sous le meme terme. Dermatologica 108:1–28, 1954. Frenk, E., B. Mevorah, and D. Hohl. The Nageli-FranceschettiJadassohn syndrome: a hereditary ectodermal defect leading to colloid-amyloid formation in the dermis. Dermatology 187:169– 173, 1993. Greither, A., and R. Haensch. Anhidrotische retikuäre pigmentdermatose mit blasig-erythematösem anfangsstadium. Schweiz. Med. Wochenschr. 100:228–233, 1970. Itin, P. H., S. Lautenschlager, R. Meyer, B. Mevorah, and T. Rufli. Natural history of the Naegeli-Franceschetti-Jadassohn syndrome and further delineation of its clinical manifestations. J. Am. Acad. Dermatol. 28:942–950, 1993. Kitamura, K., and T. Hirako. Ueber zwei japanische fälle einer eigenartigen retikulären pigmentierung: zur frage der dermatose pigmentaire reticulée (Franceschetti-Jadassohn). Dermatologica 110:97–107, 1955. Kudo, Y., S. Fujiwara, S. Takayasu, H. Ooki, and A. Ogawa. Reticulate pigmentary dermatosis associated with hypohydrosis and short stature: a variant of Naegeli-Franceschetti-Jadassohn syndrome? Int. J. Dermatol. 34:30–31, 1995. Levi, L., G. Galbiati, and G. Ghislanzoni. La dermatite pigmentaria reticolare o sindrome di Franceschetti-Jadassohn: osservazioni su di un caso. G. Ital. Dermatol. Minerva Dermatol. 46:319–322, 1971. Naegeli, O. Familiärer chromatophorennâvus. Schweiz. Med. Wochenschr. 8:48, 1927. Papini, M. Sindrome di Naegeli-Franceschetti-Jadassohn. Ann. It. Derm. Clin. Sper. 32:281–292, 1978. Papini, M. Natural history of the Naegeli-Franceschetti-Jadassohn syndrome [letter]. J. Am. Acad. Dermatol. 31(5 Pt 1):830, 1994. Sparrow, G. P., P. D. Sammam, and R. S. Wells. Hyperpigmentation and hypohidrosis (the Naegeli-Franceschetti-Jadassohn syndrome):

report of a family and review of the literature. Clin. Exp. Dermatol. 1:127–140, 1976. Sprecher E., P. Itin, N. V. Whittock, J. A. McGrath, R. Meyer, J. J. DiGiovanna, S. J. Bale, J. Uitto, and G. Richard. Refined mapping of Naegeli-Franceschetti-Jadassohn syndrome to a 6 cM interval on chromosome 17q11.2-q21 and investigation of candidate genes. J. Invest. Dermatol. 119:692–698, 2002. Tzermias, C., A. Zioga, and I. Hatzis. Reticular pigmented genodermatosis with milia – a special form of Naegeli-FranceschettiJadassohn syndrome or a new entity? Clin. Exp. Dermatol. 20:331–335, 1995. Verbov, J. Anonychia with bizarre flexural pigmentation – an autosomal dominant dermatosis. Br. J. Dermatol. 92:469–474, 1975. Whiting, D. A. Naegeli’s reticular pigmented dermatosis. Br. J. Dermatol. 85(Suppl 7):71–72, 1971. Whittock, N. V., C. M. Coleman, W. H. McLean, G. H. Ashton, K. M. Acland, R. A. Eady, and J. A. McGrath. The gene for Naegeli-Franceschetti-Jadassohn syndrome maps to 17q21. J. Invest. Dermatol.115:694–698, 2000.

Reticulated Acropigmentation of Dohi (Dyschromatosis Symmetrica Hereditaria) Eulalia Baselga and Nancy Burton Esterly

Historical Background Reticulated acropigmentation of Dohi is an autosomal dominant genodermatosis hyperpigmented and hypopigmented macules on the extremities and freckle-like pigmented macules on the face that was first described under the name leukopathia punctata et reticularis symmetrica by Matsumoto in 1924. The name of acropigmentation of Dohi was first introduced by Komaya, who in 1924 published 12 cases seen by Professor Dohi. Toyama (1929) was the first to introduce the term dyschromatosis symmetrica hereditaria.

Synonyms Acropigmentation symmetrica of Dohi (Koyama, 1924), dyschromatosis symmetrica hereditaria (Toyama, 1929), acromelanosis albopunctata (Siemens, 1964), leukopathia punctata et reticularis symmetrica (Matsumoto, 1924), symmetrical dyschromatosis of the extremities (Koyama, 1924).

Epidemiology This entity is rare. It is most common in Asian populations (Gharpuray et al., 1994; He et al., 2004; Oyama et al., 1999). It has an autosomal dominant pattern of inheritance although an autosomal recessive pattern of inheritance has been described (Alfadley, 2000) as well as sporadic cases. No difference in the incidence in males and females has been observed.

General Clinical Findings Dyschromatosis symmetrica hereditaria generally appears during infancy or early childhood (He et al., 2004). Bilaterally symmetric pinpoint to pea sized (3–5 mm) hyperpigmented and hypopigmented macules are localized on the back 799

CHAPTER 44

44.12

Fig. 44.14. Hyperpigmented and hypopigmented macules on the knee (see also Plate 44.14, pp. 494–495).

pathic torsion dystonia has been reported (Patrizi et al., 1994). A family with associated ichthyosis vulgaris has also been described (He et al., 2004).

Pathology

44.13 Fig.s 44.12 and 44.13. Typical pigmentary changes on the dorsum of hands (Fig. 44.12) and feet (Fig. 44.13) (see also Plates 44.12 and 44.13, pp. 494–495).

The epidermis and dermis look normal other than pigmentary changes. Increased melanin pigmentation in the basal keratinocytes and melanocytes are noted. Secondary pigmentary incontinence has been reported. Increased number of melanosomes in the melanocytes and keratinocytes of the hyperpigmented macules has been described. The hypopigmented macules seem to have a low density of the dopapositive melanocytes (Hata and Yokomi, 1985). Melanocyte count may be decreased in the entire involved skin area irrespective of its color changes (Sheu and Yu, 1985). In some areas there are no visible melanocytes.

Laboratory Findings of the hands and feet (Figs 44.12 and 44.13) and sometimes on the proximal portions of the limbs (knees and elbows) (Fig. 44.14). In Chinese patients extension to the neck and chest has also been described (He et al., 2004). Intermingled hyperpigmented and hypopigmented macules form the obvious reticulated pattern of demarcation. No atrophy of the macules is apparent and the lesions are asymptomatic. Palms and soles are usually spared. Scattered small frecklelike macules can be found on the face, without intermingled hypopigmented macules. Hyperpigmented macules have various colors from light to dark brown. Nails, hair, teeth, and mucosal membrane are not involved. Sun exposure may accentuate the pigmentation (Satoh and Yoshida, 1980). The number of lesions is progressive during childhood but usually stabilizes with age. A segmental form of dyschromatosis referred to as unilateral dermatomal pigmentary dermatosis has been described and may be a variant of acropigmentation of Dohi (Trattner and Metzker, 1993).

Associated Disorders One patient who was also affected by neurologic signs of idio800

There are no specific laboratory abnormalities.

Criteria for Diagnosis Hypopigmented and hyperpigmented macules on the dorsum of hands and freckles on the face.

Differential Diagnosis The assessment of DNA repair levels might be necessary to exclude the mild form of xeroderma pigmentosum (Nishigori et al., 1986). Reticulate pigmented anomaly of the flexures (Dowling–Degos disease) should be excluded by the location on the flexures, the presence of dark comedo-like lesions, and pitted perioral acneiform scars. Histopathologic characteristics are also different with epithelial buds with basilar hyperpigmentation in an antlerlike pattern. Reticulated acropigmentation of Kitamura should be excluded by the atrophic aspect of the pigmented macules on the dorsum of the hands and palmoplantar pits. Overlapping features of Dowling–Degos disease, reticulated hyperpigmentation of Kitamura, and acropigmentation of Dohi have been noted (Thami et al., 1998) (Table 44.8). Pigmentary changes in epi-

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION Table 44.8. Genetic diseases with reticulated epidermal hyperpigmentation. Disease

Inheritance* Distribution

Special features

Associated findings

Acropigmentation of Kitamura Acropigmentation of Dohi Dermatopathia pigmentosa reticularis

AD AD AD

Acral Acral Generalized

Lesions are atrophic Hypopigmented macules Hypopigmented macules intermingled

Dermatopathia universalis hereditaria Anonychia with flexural pigmentation Mendes da Costa disease

AD

Generalized

AD

Flexural

Hypopigmented macules intermingled Hypopigmented macules

Palmar pits; breakage of epidermal ridges Frecklelike macules on the face Sweating disorders; decreased dermatoglyphics; alopecia; onychodystrophy; palmar/plantar keratoses None

XLR

Generalized, face and limbs

Dowling–Degos disease

AD

Naegeli–Franceschetti– Jadassohn syndrome Epidermolysis bullosa with mottled pigmentation

AD

Flexural, then generalized Generalized

AD

Trunk, proximal limbs

Blistering early; hypopigmented macules Onset delayed Fading of pigmentation with age Blistering disorder

Sparse hair; hypohidrosis; decreased dermatoglyphics; absent/rudimentary nails Microcephaly; mental retardation; atrichia; short conical fingers Perioral pits; keratoses; epidermal cysts Keratoderma; enamel hypoplasia; hyperhidrosis; nail dystrophy Keratoderma; palmar/plantar; carious teeth; photosensitivity

* AD, autosomal dominant; XLR, X-linked recessive.

dermolysis bullosa with mottled pigmentation may be clinically similar, although lesions are more widespread, and there is also bullae formation.

Pathogenesis The molecular pathogenesis of acropigmentation of Dohi is still unknown. However, linkage studies have mapped the disease locus to chromosome 1q11–21 within a very wide region (He et al., 2004; Miyamura et al., 2003; Zhang et al., 2003). Mutations of the RNA-specific adenosine deaminase gene (DSRAD) have been detected both in Chinese and Japanese patients (Liu et al., 2004; Miyamura et al., 2003).

Animal Models None.

Treatment No specific treatment has been established.

Prognosis Dyschromatosis symmetrica hereditaria is not progressive and no spontaneous regression has been reported. It does not affect the general health of the patients.

References Alfadley, A., A. Al Ajlan, B. Hainau, K. T. Pedersen, and I. Al Hoqail. Reticulate acropigmentation of Dohi: a case report of autosomal recessive inheritance. J. Am. Acad. Dermatol. 43:113–117, 2000. Gharpuray, M. B., S. N. Tolat, and S. P. Patwardham. Dyschromatosis: its occurrence in two Indian families with unusual features. Int. J. Dermatol. 33: 391–392, 1994. Hata, S., and I. Yokomi. Density of dopa-positive melanocytes in dyschromatosis symmetrica hereditaria. Dermatologica 171:27–29, 1985.

He, P. P., C. D. He, Y. Cui, S. Yang, H. H. Zu, M. Li, W. T. Yuan, M. Gao, Y. H. Liang, C. R. Xu, S. J. Chen, H. D. Chen, W. Huang, and X. J. Zhang. Refined localization of dyschromatosis symmetrica hereditaria gene to a 9.4-cM region at 1q 21–22 and a literature review of 136 cases reported in China. Br. J. Dermatol. 150:491–496, 1999. Liu, Q., W. Liu, L. Jiang, M. Sun, Y. Ao, X. Zhao, Y. Song, Y. Luo, W. H. Lo, and X. Zhang. Novel mutations of the RNA-specific adenosine deaminase gene (DSRAD) in Chinese families with dyschromatosis symmetrica hereditaria. J. Invest. Dermatol. 122: 896–899, 2004. Koyama, G. Symmetrische Pigmentanomalie der Extremitäten. Arch. Dermatol. Syphil. 147:389–393, 1924. Matsumoto, S. Leucopathia punctata et reticularis symmetrica. Acta Dermatol. 2: 191 7, 1923. Miyamura, Y., T. Suzuki, M. Kono, K. Inagaki, S. Ito, N. Suzuki, and Y. Tomita. Mutations of the RNA-specific adenosine deaminase gene (DSRAD) are involved in dyschromatosis symmetrica hereditaria. Am. J. Hum. Genet. 73:693–699, 2003. Nishigori, C., Y. Miyachi, H. Takebe, and S. Imamura. A case of xeroderma pigmentosum with clinical appearance of dyschromatosis symmetrica hereditaria. Pediatr. Dermatol. 3:410–413, 1986. Omaya, M., H. Shimizu, Y. Ohata, S. Tajima, and T. Nishikama. Dyschromatosis symmetrica hereditaria (reticulate acropigmentation of Dohi): report of a Japanese family with the condition and a literature review of 185 cases. Br. J. Dermatol. 140:491–496, 1999. Patrizi, A., V. Manneschi, A. Pini, E. Baioni, and P. Ghetti. Dyschromatosis symmetrica hereditaria associated with idiopathic torsion dystonia. A case report. Acta Derm. Venereol. 74:135–137, 1994. Satoh, Y., and M. Yoshida. Clinical and photobiological differences between dyschromatosis symmetrica hereditaria and xeroderma pigmentosum. J. Dermatol. 7:317–322, 1980. Satoh, Y., and M. Yoshida. Clinical and photobiologic difference between dyschromatosis symmetrica hereditaria and xeroderma pigmentosum. J. Dermatol. 7:317–322, 1980. Sheu, H. M., and H. S. Yu. Dyschromatosis symmetrica hereditaria – A histochemical and ultrastructural study. J. Formosan Med. Assoc. 84: 238–249, 1985.

801

CHAPTER 44 Siemens, H. W. Acromelanosis albo-puntata. Dermatologica 128:86– 87, 1964. Thami, G. P., R. Jaswal, A. J. Kanwar, B. D. Radotra, and I. P. Singh. Overlap of reticulate acropigmentation of Kitamura, acropigmentation of Dohi and Dowling-Degos disease in four generations. Dermatology 196:350–351, 1998. Toyama, I. Dyschromatosis symmetrica hereditaria. Jpn. J. Dermatol. 29:95–96, 1929. Trattner, A., and A. Metzker. Unilateral dermatomal pigmentary dermatosis: a variant dyschromatosis? J. Am. Acad. Dermatol. 29:1060, 1993. Zhang, X. J., M. Gao, M. Li, M. Li, C. R. Li, Y. Cui, P. P. He, S. J. Xu, X. Y. Xiong, Z. X. Wang, W. T. Yuan, S. Yang, and W. Huang. Identification of a locus for dyschromatosis symmetrica hereditaria at chromosome 1q11–1q21. J. Invest. Dermatol. 120:776–780, 2003. 44.15

Reticulate Acropigmentation of Kitamura Eulalia Baselga and Nancy Burton Esterly

Historical Background Reticulate acropigmentation of Kitamura was first described in 1943 (one case) and subsequently in 1953 (two cases).

Epidemiology and Genetics The original three patients were of Japanese origin. In 1976, Griffiths reported seven cases from five ethnically diverse families from Great Britain, India, Ghana, and Iran. This report demonstrated that the condition was not confined to individuals of Japanese heritage. The first well-accepted American case was reported in 1979 by Woodley et al., 1979. These authors cite a 1939 report of two American siblings and an affected father. However, the distribution of the hyperpigmented macules in those individuals was somewhat atypical and they might represent some other pigmentary disorder. Although the first three Japanese patients reported were males, the subsequent reports indicate that there is no sex predilection for reticulate acropigmentation of Kitamura. Many cases are familial and the disorder is believed to be inherited as an autosomal dominant trait (Bajaj and Gupta, 1984; Griffiths, 1976; Kanwar et al., 1990; Sharma et al., 1989).

44.16 Figs 44.15 and 44.16. Typical hyperpigmented macules on the dorsum of the feet (Fig. 44.15) and hands (Fig. 44.16) (see also Plates 44.15 and 44.16, pp. 494–495). (Courtesy of Dr. Yoon Key Park and Dr. Sungbin Im.)

Clinical Findings The cutaneous lesions are asymptomatic, angular, slightly depressed, sharply demarcated brown macules 15 mm in length that develop on the dorsum of the hands and feet usually during the first decade of life. They may darken after onset, particularly after sun exposure. The spots often increase in number and spread centripetally with age, so that the extensor aspects of the limbs, neck, upper trunk, lower face, and eyelids eventually become involved. Flexures may occasionally be involved (Al Hawsawi et al., 2002). The macules are asymptomatic and characteristically coalesce to form a reticulated pattern (Figs 44.15–44.17). Rarely they also appear on the palms and soles (Lestringant et al., 1994). The palms and soles are also the site of small pits that form breaks in the epi802

Figs. 44.17. Sharply demarcated brown macules on the dorsum of the hands of a patient with reticulate acropigmentation of Kitamura (see also Plate 44.17, pp. 494–495). (Courtesy of Dr. Yoon Kee Park and Dr. Sungbin Im.)

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION Table 44.9. Genetic diseases with reticulated epidermal hyperpigmentation. Disease

Inheritance* Distribution

Special features

Associated findings

Acropigmentation of Kitamura Acropigmentation of Dohi Dermatopathia pigmentosa reticularis

AD AD AD

Acral Acral Generalized

Lesions are atrophic Hypopigmented macules Hypopigmented macules intermingled

Dermatopathia universalis hereditaria Anonychia with flexural pigmentation Mendes da Costa disease

AD

Generalized

AD

Flexural

Hypopigmented macules intermingled Hypopigmented macules

Palmar pits; breakage of epidermal ridges Frecklelike macules on the face Sweating disorders; decreased dermatoglyphics; alopecia; onychodystrophy; palmar/plantar keratoses None

XLR

Dowling–Degos disease

AD

Naegeli–Franceschetti– Jadassohn syndrome Epidermolysis bullosa with mottled pigmentation

AD

Generalized, face and limbs Flexural, then generalized Generalized

AD

Trunk, proximal limbs

Sparse hair; hypohidrosis; decreased dermatoglyphics; absent/rudimentary nails Blistering early; Microcephaly; mental retardation; atrichia; hypopigmented macules short conical fingers Onset delayed Perioral pits; keratoses; epidermal cysts Fading of pigmentation with age Blistering disorder

Keratoderma; enamel hypoplasia; hyperhidrosis; nail dystrophy Keratoderma; palmar/plantar; carious teeth; photosensitivity

* AD, autosomal dominant; XLR, X-linked recessive.

dermal ridge patterns (Griffiths, 1976; Woodley et al., 1979), a requisite finding for the diagnosis. Pits have also been observed on the dorsum of the digits (Griffiths, 1976; Lestringant et al., 1994; Sharma and Chandra, 2000). These patients are usually otherwise in good health. A single case with associated localized congenital noncicatricial alopecia has been reported (Wallis and Mallory, 1991). Absence of terminal phalanges of the toes has been reported in a single patient (el Hoshy and Hashimoto, 1996).

Histology Histopathologic examination of biopsies from the hyperpigmented lesions shows epidermal atrophy, elongation and melanization of the rete ridges, and an increased number of dopa-positive melanocytes in the basilar layer of the epidermis (Kameyama et al., 1992; Mizoguchi and Kukita, 1985). With immunohistochemical stains, Kameyama showed that the clear cells were dopa-positive and contained tyrosinase and tyrosinase-related protein. Some keratinocytes also stain with these antibodies (Kameyama et al., 1992). Staining of the keratinocytes has been attributed to transfer of the enzymes with the melanosomes (Kameyama et al., 1992). Ultrastructural studies have demonstrated numerous melanosomes and melanosome complexes within keratinocytes. There are also melanosome complexes within the melanocyte cytoplasm as well as within dendrites (Kameyama et al., 1992; Mizoguchi and Kukita, 1985). The dermis is usually unaltered and neither inflammatory infiltrate nor incontinence of pigment is seen.

Diagnosis The diagnosis is made on clinical and histologic grounds. There are no helpful laboratory studies.

Differential Diagnosis Reticulate acropigmentation of Kitamura must be differentiated from several other causes of macular pigmentation (Table 44.9). The pigmented lesions can be differentiated from ephelides by their atrophic appearance and reticulate pattern. Dermatopathia pigmentosa reticularis is more generalized, has hypopigmented as well as hyperpigmented lesions and lacks the atrophy and distinctive palmar/plantar pitting and epidermal ridge breaks. Acropigmentation of Dohi is characterized by hypopigmented and hyperpigmented lesions but has neither atrophy nor epidermal ridge breaks. However, there may be clinical overlap with Dowling–Degos disease (Dhar et al., 1994; Lestringant et al., 1997; Thami et al., 1998). Patients with either dyskeratosis congenita or Franceschetti–Jadassohn–Naigeli syndrome have more generalized reticulated pigmentation and other associated abnormalities (Salmon and Frieden, 1995). The most difficult disorder to differentiate from acropigmentation of Kitamura is Dowling–Degos disease, also called reticulated pigmented anomaly of the flexures. It has been suggested that these two disorders represent different clinical expressions of the same disease process (Al Hawsawi et al., 2002; Cox and Long, 1991; Crovato and Rebora, 1986; Dhar et al., 1994; Gatti and Nimi, 1992; Ostlere and Holden, 1994; Rebora and Crovato, 1984). However, in classic cases of Dowling–Degos disease, the pigmented lesions are confined to the intertriginous areas, there is no epidermal atrophy and pitted scars and comedolike lesions appear on the face and neck. Moreover, the characteristic histopathology of the skin lesions differs considerably from that of reticulate acropigmentation of Kitamura (Kanwar et al., 1990; Salmon and Frieden, 1995). 803

CHAPTER 44

Pathogenesis The pathogenesis is unknown. It has been postulated that an increased number of activated melanocytes and accelerated transfer of melanosomes to keratinocytes is responsible for the pigmented lesions but the origin of the epidermal atrophy is unclear (Mizoguchi and Kukita, 1985). The immunohistochemical findings also suggest metabolic activation of melanocytes in patients with reticulate acropigmentation of Kitamura.

Treatment and Prognosis There is almost no information in the literature about therapy for this condition. Treatment with 20% azelaic acid resulted in remarkable reduction of hyperpigmentation in one patient (Kameyama et al., 1992) but failed to produce any change in another (Gatti and Nimi, 1992). However, in the former patient, the pigment returned when the azelaic acid preparation was discontinued. These patients are otherwise healthy and have a normal life span.

References Al Hawsawi, K., K. Al Aboud, A. Alfadley, and D. Al Aboud. Reticulate acropigmentation of Kitamura-Dowling Degos disease overlap: a case report. Int. J. Dermatol. 41:518–520, 2002. Bajaj, A. K., and S. C. Gupta. Reticulate acropigmentation of Kitamura: A report of two families. Dermatologica 168:247–249, 1984. Cox, N. H., and E. Long. Dowling-Degos disease and Kitamura’s reticulate acropigmentation: support for the concept of a single disease. Br. J. Dermatol. 125:169–171, 1991. Crovato, F., and A. Rebora. Reticulate pigmented anomaly of the flexures associating reticulate acropigmentation: one single entity. J. Am. Acad. Dermatol. 14:359–361, 1986. Dhar, S., A. J. Kanwar, R. Jebraili, G. Dawn, and A. Das. Spectrum of reticulate flexural and acral pigmentary disorders in northern India. J. Dermatol. 21:598–603, 1994. el Hoshy, K., and K. Hashimoto. Bony anomalies in a patient with reticulate acropigmentation of Kitamura. J. Dermatol. 23:713–715, 1996. Gatti, S., and G. Nimi. Treatment of reticulate acropigmentation of Kitamura with azelaic acid. J. Am. Acad. Dermatol. 29:666–667, 1992. Griffiths, W. A. D. Reticulate acropigmentation of Kitamura. Br. J. Dermatol. 95:437–443, 1976. Kameyama, K., M. Morita, K. Sugaya, S. Nishiyama, and V. J. Hearing. Treatment of reticulate acropigmentation of Kitamura with azelaic acid – an immunohistochemical and electron microscopic study. J. Am. Acad. Dermatol. 26:817–820, 1992. Kanwar, A. J., S. Kaur, and M. Rajagopalan. Reticulate acropigmentation of Kitamura. Int. J. Dermatol. 29:217–219, 1990. Lestringant, G. G., G. A. Aal, P. M. Frossard, and K. I. Qayed. Reticulate acropigmentation of Kitamura: pigment specks and pits in unusual locations. Br. J. Dermatol. 131:137–139, 1994. Lestringant, G. G., I. Masouye, P. M. Frossard, E. Adeghate, and I. H. Galadari. Co-existence of leukoderma with features of Dowling-Degos disease: reticulate acropigmentation of Kitamura spectrum in five unrelated patients. Dermatology 195:337–343, 1997. Mizoguchi, M., and A. Kukita. Behavior of melanocytes in reticulate acropigmentation of Kitamura. Arch. Dermatol. 121:659–661, 1985. Ostlere, L., and C. A. Holden. Dowling-Degos disease associated with Kitamura’s reticulate acropigmentations. Clin. Exp. Dermatol. 19:492–495, 1994.

804

Rebora, A., and F. Crovato. The spectrum of Dowling-Degos disease. Br. J. Dermatol. 110:627–630, 1984. Salmon, J. K., and I. J. Frieden. Congenital and genetic disorders of hyperpigmentation. Curr. Probl. Dermatol. 7:145–196, 1995. Sharma, R., S. C. Sharma, B. D. Radotra, and S. Kaur. Reticulate acropigmentation of Kitamura. Clin. Exp. Dermatol. 14:302–303, 1989. Sharma, R., and Chandra, M. Pigmentation and pits at uncommon sites in a case with reticulate acropigmentation of Kitamura. Dermatology 200:57–58, 2000. Thami, G. P., R. Jaswal, A. J. Kanwar, B. D. Radotra, and I. P. Singh. Overlap of reticulate acropigmentation of Kitamura, acropigmentation of Dohi and Dowling-Degos disease in four generations. Dermatology 196:350–351, 1998. Wallis, M. S., and S. B. Mallory. Reticulate acropigmentation of Kitamura with localized alopecia. J. Am. Acad. Dermatol. 25(1 Pt 1):114–116, 1991. Woodley, D. T., I. Caro, and C. E. Wheeler, Jr. Reticulate acropigmentation of Kitamura. Arch. Dermatol. 115:760–761, 1979.

Rothmund–Thomson Syndrome Eulalia Baselga and Nancy Burton Esterly

Historical Background In 1887, Rothmund, a German ophthalmologist, described a group of individuals from an isolated Bavarian village with cataracts and a peculiar poikiloderma developing during childhood. Many years later three similar patients were reported by a British dermatologist (Thomson, 1923, 1936) who named the disorder poikiloderma congenitale. However, Thomson’s patients did not have cataracts. Carlton in 1943 and Sexton in 1954 suggested that Rothmund’s and Thomson’s patients might have the same disorder. Taylor (1957) established the two syndromes as a single entity. Although there is still some controversy over this concept, the term Rothmund–Thomson syndrome has become firmly entrenched in the modern literature.

Synonyms Rothmund–Thomson syndrome; poikiloderma congenitale; poikiloderma atrophicans.

Epidemiology and Genetics Rothmund–Thomson syndrome affects patients from all ethnic groups (Vennos et al., 1992). Although a female predominance is often cited in the literature (Moss, 1990), male predominance (Wang et al., 2001) and an equal sex distribution (Vennos et al., 1992) have been found in recent reviews. Genetic analysis of pedigree data is consistent with autosomal recessive inheritance. Consanguinity has been documented in 16–27% of unaffected parents or other ancestors (Starr et al., 1985; Vennos et al., 1992). Karyotyping studies of affected individuals have disclosed no consistent chromosomal abnormalities. Recently, mutations in the DNA helicase gene RECQL4 on chromosome 8q24.3 have been found in some patients (Balraj et al., 2002; Beghini et al., 2003; Kitao et al., 1999; Lindor et al., 2000; Wang et al., 2000).

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION

Fig. 44.19. Similar pigmentary changes and severe atrophy with deformities of the forearm and hands (see also Plate 44.19, pp. 494–495).

Fig. 44.18. Poikiloderma of the face with hyper- and hypopigmentation (see also Plate 44.18, pp. 494–495).

Clinical Findings Rothmund–Thomson syndrome is characterized by poikiloderma, short stature, cataracts, skeletal abnormalities and predisposition to certain malignancies. The poikiloderma of the face (Fig. 44.18) and extremities is a constant feature. The skin changes usually begin as a photosensitive erythematous or blistering eruption on the face between 3 and 6 months of age although an occasional patient is affected at birth or acquires the cutaneous eruption as late as 2 years of age (Wang et al., 2001). Within months there is prominent, reticulated erythema on the cheeks that spreads to other areas of the face such as the forehead and ears, to the neck, buttocks, and the extensor surfaces of the extremities. This acute erythematous phase may last for months or years and is followed by a chronic and persistent phase with all the characteristic features of poikiloderma, i.e., atrophy, dyspigmentation (both hyperpigmentation and hypopigmentation), and telangiectasia. The hyperpigmentation may have a slight gray discoloration. Photosensitivity at this stage is less common. Patients with Rothmund–Thomson syndrome have a typical facies with frontal bossing, saddle nose, and prognathism. Warty hyperkeratoses, which usually develop after puberty, may appear as early as 2 years of age and are located over the extensor limbs and bony prominences. They are found in

approximately 25% of affected persons (Haneke and Gutschmidt, 1976; Moss, 1990; Vennos and James, 1995). Sparse brows and eyelashes is another very constant feature. Less frequently scalp hair is sparse with variable degrees of alopecia that progresses with age (Vennos et al., 1992; Wang et al., 2001). A third of patients have dystrophic or thin nails. Dental abnormalities occur in 40% of patients and most commonly consist of hypodontia, microdontia, and a tendency to develop caries (Hall et al., 1980; Vennos and James, 1995). The most typical dental finding is microdontia with conical teeth. The extracutaneous findings include low birthweight and short stature or dwarfism of unknown etiology (Drouin et al., 1993; Hall et al., 1980; Wang et al., 2001). Many patients have skeletal abnormalities (Fig. 44.19) such as skeletal dysplasias (abnormal trabeculation and/or irregular metaphysis), osteoporosis, delayed bone formation, radial ray defects with hypoplastic or absent radii, absence of the metacarpals and phalanges of the thumbs, bifid thumb, thumb appendages, and small hands and feet (Drouin et al., 1993; Wang et al., 2001). Hypogonadism, delayed sexual development, and infertility have been reported, although several pregnancies have been reported in women with this syndrome. Ophthalmologic abnormalities are found in 50–75% of patients. The most common ophthalmologic finding is bilateral subcapsular cataracts that arise suddenly and progress rapidly causing loss of vision if untreated (Dollfus et al., 2003). Cataracts may be unilateral (Wang et al., 2001) and usually develop before the age of 7 years (Collins et al., 1991). Several other ocular anomalies have been detected in a small percentage of patients (Collins et al., 1991; Moss, 1990; Dollfus et al., 2003). Patients with Rothmund–Thomson syndrome are at increased risk of malignancy particularly osteosarcoma (Drouin et al., 1993; Haneke and Gutschmidt, 1979; Judge et al., 1993; Starr et al., 1985; Wang et al., 2001, 2003). Squamous cell carcinomas of the skin and tongue have also been reported (Martin-Bertolin et al., 1998; Piquero-Casals et al., 805

CHAPTER 44

2002; Vennos and James, 1995; Wang et al., 2001). Malignant eccrine poroma and malignant fibrous histiocytoma have been rarely described (Ilhan et al., 1995; Van Hees et al., 1996). Gastrointestinal abnormalities may occur. Some patients experience feeding difficulties that usually improve with age, but are severe enough to require the placement of a feeding tube (Guler et al., 1998; Wang et al., 2001). Hematologic abnormalities may also occur, mainly myelodysplasia, neutropenia, and aplastic anemia (Knoell et al., 1999; Narayan et al., 2001; Pianigiani et al., 2001; Porter et al., 1999; Starr et al., 1985). Intelligence and immunologic function are usually normal.

Histology The histologic features of involved skin are those of a poikiloderma, namely, hyperkeratosis, flattening of the epidermis, vacuolization of the basal layer, and edema of the papillary and reticular dermis with dilatation of the capillaries. There is incontinence of pigment and numerous melanophages in the upper dermis (Berg et al., 1987; Collins et al., 1991; Drouin et al., 1993; Roth et al., 1989). At a later stage there is hyperpigmentation of the basal layer, fragmentation of the elastic tissue, and depletion of the dermal appendages (Vennos and James, 1995). In one case melanosome complexes were found within mast cells in the dermis. These melanosomes were located in large mast cell granules, in phagocytic vacuoles and others lying free within the cytoplasm. All of the melanosomes were stage 4 suggesting that they were formed outside the mast cells (Nazzaro et al., 1987). The histology of the warty hyperkeratoses is characterized by massive orthokeratosis, acanthosis with budding of the basal cell layer, and a prominent granular layer with bizarre dyskeratotic cells containing abnormally large keratohyalin granules (Shuttleworth and Marks, 1987). Ultrastructural studies have confirmed the light microscopic findings and show loss of cellular polarity, apoptosis, and accumulations of glycogen in the cells of the spinous and granular layers. Autoradiographic studies demonstrated an elevated basal cell labeling index of 18% suggesting that the verrucous lesions may be predisposed to malignant change (Shuttleworth and Marks, 1987).

Laboratory Investigations These patients have no consistent or identifying laboratory abnormalities. Screening tests for metabolic disorders have always been negative. There are rare reports of defective immune function (Haneke and Gutschmidt, 1976; Kubota et al., 1993). Endocrinologic studies have generally been normal (Hall et al., 1980; Vennos and James, 1995). Results of phototesting have been reported only occasionally but are usually normal (Berg et al., 1987; Kubota et al., 1993; Shuttleworth and Marks, 1987) whereas the results of studies of DNA repair have been inconsistent and do not support a defective DNA repair (Vennos and James, 1995). Karyotyping studies have demonstrated trisomy 8 806

mosaicism in some patients either in peripheral lymphocytes or in skin fibroblasts and lymphocytes (Der Kaloustian et al., 1990; Ying et al., 1990). Other complex mosaics and ring chromosomes have been described (Wang et al., 2001). Hematologic abnormalities may be seen in a subset of patients.

Diagnosis Diagnosis is made on clinical grounds based on the characteristic appearance and pattern of development of the cutaneous eruption. The other clinical features are inconstant and there is considerable clinical heterogeneity. A diagnosis of probable Rothmund–Thomson can be made if the rash is atypical and two other features are present such as a family history, skeletal changes, cataracts, sparse hair, and short stature (Wang et al., 2001). Karyotyping studies and DNA excision repair diagnosis are not useful for diagnosis as they are normal in many patients. Molecular diagnosis may be done on a research basis and may be useful for prenatal diagnosis in families with known mutations.

Differential Diagnosis Rothmund–Thomson syndrome shares many features with the syndromes of premature aging such as acrogeria, progeria, and Cockayne syndromes. Acrogeria may be distinguished by the characteristic lipoatrophy that is not a feature of Rothmund–Thomson syndrome. In progeria there is no true poikiloderma. Cockayne syndrome can be differentiated by characteristic features such as mental retardation or retinal degeneration. Syndromes that feature congenital poikiloderma such as Kindler syndrome, hereditary sclerosing poikiloderma, and hereditary acrokeratotic poikiloderma may be also considered in the differential diagnosis but they lack the extracutaneous manifestations of Rothmund–Thomson syndrome (Table 44.10). However, as poikiloderma is usually the first manifestation of Rothmund–Thomson, differentiation from other forms of poikiloderma may be more difficult early on disease evolution. Acrokeratotic poikiloderma of Weary usually spares the face. In Kindler syndrome the poikilodermatous changes are more prominent on the dorsum of the hands, photosensitivity is a more constant feature, and blistering is more prominent and not photo induced. In hereditary sclerosing poikilodermia there are sclerotic bands over flexures. Bloom syndrome and Werner syndrome, the two other known diseases due to RECQ helicase DNA gene mutation, share many of the clinical features such as growth retardation, predisposition to malignancy, juvenile cataracts, and premature aging of the skin (Lindor et al., 2000). In Bloom syndrome severe dwarfism is a more constant feature and skin changes are not truly poikilodermatous. Increase in sister chromatid exchange may help in diagnosis as there is a dramatic increase only in Bloom syndrome. In Werner syndrome, the cutaneous changes are mainly sclerodermiform and onset of symptoms is more delayed. Additional considerations are Hallermann–

GENETIC EPIDERMAL SYNDROMES: DISORDERS CHARACTERIZED BY RETICULATED HYPERPIGMENTATION Table 44.10. Genetic poikilodermas. Disease

Inheritance*

Bullae

Poikiloderma

Acral keratoses

Other

Hereditary acrokeratotic poikiloderma

AD

Acral

Yes

Eczema

Kindler syndrome

AR

Acral

Spares face except eyelids; increased in flexures Widespread

+/-

Hereditary sclerosing poikiloderma

AD

No

Marked skin atrophy: webbing of the digits Clubbing; linear sclerotic bands in flexures

Rothmund–Thomson

AR

Light exposed skin

Spares chest, scalp, face except eyelids Photodistributed

No

+/-

Photosensitivity; cataracts; alopecia; nail and dental defects; warty keratoses over bony prominences

* AD, autosomal dominant; AR, autosomal recessive.

Streiff syndrome, xeroderma pigmentosum, and the ectodermal dysplasias. None, however, have the identical spectrum of defects as Rothmund–Thomson syndrome (Roth et al., 1989; Vennos et al., 1992).

Pathogenesis There is evidence that some cases of Rothmund–Thomson syndrome are caused by mutations in the DNA helicase gene RECQL4 on chromosome 8q24.3 (Balraj et al., 2002; Beghini et al., 2003; Kitao et al., 1999; Lindor et al., 2000; Wang et al., 2000). This gene protein belongs to the RecQ family of DNA helicases, which includes proteins encoded by genes that are disrupted in Bloom syndrome and Werner syndrome. However, there is genetic heterogeneity even within families which explains the variable clinical presentation and suggests that a second locus may be involved. Those patients having a truncating mutation of the RECQL4 gene have a higher predisposition to the development of osteosarcomas (Wang et al., 2003).

Management and Treatment There is no treatment for the disorder. Patients should be instructed to use sunscreens, to avoid sun exposure, and to have yearly dermatologic examinations to detect precancerous lesions. Baseline long bone radiologic survey at around 5 years of age is recommended and they should be repeated whenever there is clinical suspicion of osteosarcoma. Parents should be informed about the possibility of developing malignancies and instructed about the clinical signs or symptoms of malignancy such as bone pain or swelling. Periodic blood counts are also recommended to detect hematologic abnormalities. Annual eye examinations to detect cataract are warranted as they may develop later in life. The poikilodermatous component of the syndrome may be significantly improved by treatment with the flashlamp pulse dye laser (Houwing et al., 1991; Potozkin and Geronemus, 1991). Hyperkeratotic lesions have improved with use of etretinate, vitamin A, keratolytics and topical DNCB. Prenatal testing may be possible in families with a known mutation.

Prognosis The prognosis is determined by the particular manifestations of the syndrome in a given individual. In the absence of malignancy life span is probably normal.

References Balraj, P., P. Concannon, R. Jamal, A. Beghini, T. S. Hoe, A. S. Khoo, and L. Volpi. An unusual mutation in RECQ4 gene leading to Rothmund-Thomson syndrome. Mutat. Res. 508:99–105, 2002. Beghini, A., P. Castorina, G. Roversi, P. Modiano, and L. Larizza. RNA processing defects of the helicase gene RECQL4 in a compound heterozygous Rothmund-Thomson patient. Am. J. Med. Genet. 120:395–399, 2003. Berg, E., T.-Y. Chuang, and D. Cripps. Rothmund-Thomson syndrome. A case report, phototesting, and literature. J. Am. Acad. Dermatol. 17:332–338, 1987. Carlton, A. Skin diseases and cataract. Br. J. Dermatol. 55:83, 1943. Collins, P., L. Barnes, and M. McCabe. Poikiloderma congenitale: Case report and review of the literature. Pediatr. Dermatol. 8:58–60, 1991. Der Kaloustian, V. M., J. J. McGill, M. Vekemans, and H. R. Kopelman. Clonal lines of aneuploid cells in Rothmund-Thomson syndrome. Am. J. Med. Genet. 37:336–339, 1990. Dollfus, H., F. Porto, P. Caussade, C. Speeg-Schatz, J. Sahel, E. Grosshans, J. Flament, and A. Sarasin. Ocular manifestations in the inherited DNA repair disorders. Surv. Ophthalmol. 48:107–122, 2003. Drouin, C. A., E. Mongrain, D. Sasseville, H. L. Bouchard, and M. Drouin. Rothmund-Thomson syndrome with osteosarcoma. J. Am. Acad. Dermatol. 28:301–305, 1993. Guler, O., M. Aydin, S. Ugras, E. Kisli, and A. Metin. Rothmund Thomson syndrome associated with esophageal stenosis: report of a case. Surg. Today 28:839–842, 1998. Hall, J. G., R. A. Pagon, and K. M. Wilson. Rothmund-Thomson syndrome with severe dwarfism. Am. J. Dis. Child. 134:165–169, 1980. Haneke, E., and E. Gutschmidt. Warty hyperkeratoses, cellular immune defect, and tapetoretinal degeneration in poikiloderma congenitale. Dermatologica 152:331–336, 1976. Haneke, E., and E. Gutschmidt. Premature multiple Bowen’s disease in poikiloderma congenitale with warty hyperkeratosis. Dermatologica 158:384–389, 1979. Houwing, R. H., R. F. Oosterkamp, M. Berghuis, F. A. Beemer, and W. A. Van Vloten. Rothmund-Thomson syndrome. Br. J. Dermatol. 125:279–280, 1991.

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CHAPTER 44 Ilhan, I., U. Arikan, and M. Buyukpamukcu. Rothmund-Thomson syndrome and malignant fibrous histiocytoma: a case report. Pediatr. Hematol. Oncol. 12:103–105, 1995. Judge, M. R., A. Kilby, and J. I. Harper. Rothmund-Thomson syndrome with osteosarcoma. Br. J. Dermatol. 129:723–725, 1993. Kitao, S., A. Shimamoto, M. Goto, R. W. Miller, W. A. Smithson, N. M. Lindor, and Y. Furuichi. Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome. Nat. Genet. 22:82–84, 1999. Knoell, K. A., N. K. Sidhu-Malik, and R. K. Malik. Aplastic anemia in a patient with Rothmund-Thomson syndrome. J. Pediatr. Hematol. Oncol. 21:444–446, 1999. Kubota, M., M. Yasunaga, H. Hashimoto, H. Kimata, H. Mikawa, A. Shinya, and C. Nishigori. IgG4 deficiency with RothmundThomson syndrome: A case report. Eur. J. Pediatr. 152:406–408, 1993. Lindor N. M., Y. Furuichi, S. Kitao, A. Shimamoto, C. Arndt, and S. Jalal. Rothmund-Thomson syndrome due to RECQ4 helicase mutations: report and clinical and molecular comparisons with Bloom syndrome and Werner syndrome. Am. J. Med. Genet. 90:223–228, 2000. Marin-Bertolin, S., J. Amorrortu-Velayos, and B. A. Aliaga. Squamous cell carcinoma of the tongue in a patient with Rothmund-Thomson syndrome. Br. J. Plast. Surg. 51:646–648, 1998. Moss, C. Rothmund-Thomson syndrome: A report of two patients and a review of the literature. Br. J. Dermatol. 122:821–829, 1990. Narayan, S., C. Fleming, A. H. Trainer, and J. A. Craig. RothmundThomson syndrome with myelodysplasia. Pediatr. Dermatol. 18:210–212, 2001. Nazzaro, V., P. Y. Venencie, and C. Blanchet-Bardon. Mast cell phagocytosis of melanosomes in a case of Rothmund-Thomson’s congenital poikiloderma. Clin. Exp. Dermatol. 12:366–369, 1987. Pianigiani, E., G. De Aloe, A. Andreassi, P. Rubegni, and M. Fimiani. Rothmund-Thomson syndrome (Thomson-type) and myelodysplasia. Pediatr. Dermatol. 18:422–425, 2001. Piquero-Casals, J., A. Y. Okubo, and M. M. Nico. RothmundThomson syndrome in three siblings and development of cutaneous squamous cell carcinoma. Pediatr. Dermatol. 19:312–316, 2002. Porter, W. M., C. M. Hardman, S. H. Abdalla, and A. V. Ponles. Haematological disease in siblings with Rothmund-Thomson syndrome. Clin. Exp. Dermatol. 24:452–454, 1999. Potozkin, J. R., and R. G. Geronemus. Treatment of the poikilodermatous component of the Rothmund-Thomson syndrome with the

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flashlamp-pumped pulsed dye laser: A case report. Pediatr. Dermatol. 8:162–165, 1991. Roth, D. E., L. C. Campisano, J. P. Callen, J. H. Hersh, and J. W. Yusk. Rothmund-Thomson syndrome: a case report. Pediatr. Dermatol. 6:321–324, 1989. Rothmund, A. Uber kataract in verbindung mit einer eugntumliche haut degeneration. Arch. Ophthalmol. 4:159, 1887. Sexton, G. B. Thomson’s syndrome. Can. Med. Assoc. J. 70:662–665, 1954. Shuttleworth, D., and R. Marks. Epidermal dysplasia and skeletal deformity in congenital poikiloderma (Rothmund-Thomson syndrome). Br. J. Dermatol. 117:377–384, 1987. Starr, D. G., J. P. McClure, and J. M. Connor. Non-dermatological complications and genetic aspects of the Rothmund-Thomson syndrome. Clin. Genet. 27:102–104, 1985. Taylor, W. B. Rothmund’s syndrome: Thomson’s syndrome; congenital poikiloderma with or without juvenile cataracts. Am. Med. Assoc. Arch. Dermatol. 75:263–244, 1957. Thomson, M. S. A hitherto undescribed familial disease. Br. J. Dermatol. 35:455–462, 1923. Thomson, M. S. Poikiloderma congenitale. Br. J. Dermatol. 48:221, 1936. Van Hees, C. L., Van C. M. Duinen, J. A. Bruijin, and B. J. Vermeer. Malignant eccrine poroma in a patient with Rothmund-Thomson syndrome. Br. J. Dermatol. 134:813–815, 1996. Vennos, E. M., and W. D. James. Rothmund-Thomson syndrome. Dermatol. Clin. 13:143–150, 1995. Vennos, E. M., M. Collins, and W. D. James. Rothmund-Thomson syndrome: Review of the world literature. J. Am. Acad. Dermatol. 27:750–762, 1992. Wang L. L., M. L. Levy, R. A. Lewis, A. Gannavarapu, D. Stockton, D. Lev, C. M. Cunniff, and S. E. Plon. Evidence for genetic heterogeneity in Rothmund-Thomson syndrome. Am. J. Hum. Genet. 67 Suppl 2:376, 2000. Wang, L. L., M. L. Levy, R. A. Lewis, M. M. Chintagumpala, D. Lev, M. Rogers, and S. E. Plon. Clinical manifestations in a cohort of 41 Rothmund-Thomson syndrome patients. Am. J. Med. Genet. 102:11–17, 2001. Wang, L. L., A. Gannavarapu, C. A. Kozinetz, M. L. Levy, R. A. Lewis, M. M. Chintagumpala, R. Ruiz-Maldanado, J. Contreras-Ruiz, C. Cunniff, R. P. Erickson, D. Lev, M. Rogers, E. H. Zackai, and S. E. Plon. Association between osteosarcoma and deleterious mutations in the RECQL4 gene in RothmundThomson syndrome. J. Natl. Cancer Inst. 95:669–674, 2003. Ying, K. L., J. Oizumi, and C. J. R. Curry. Rothmund-Thomson syndrome associated with trisomy 8 mosaicism. J. Med. Genet. 27: 258–260, 1990.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Genetic Epidermal Syndromes with Café-au-lait Macules Sections Familial Multiple Café-au-lait Spots Nancy Burton Esterly Neurofibromatosis Nancy Burton Esterly, Eulalia Baselga, and Sheila S. Galbraith Neurofibromatosis 1 with Noonan Syndrome Nancy Burton Esterly McCune–Albright Syndrome Sheila S. Galbraith and Nancy Burton Esterly Segmental Neurofibromatosis Nancy Burton Esterly and Eulalia Baselga Silver–Russell Syndrome Nancy Burton Esterly, Eulalia Baselga, and Sheila S. Galbraith Watson Syndrome Nancy Burton Esterly

Familial Multiple Café-au-lait Spots Nancy Burton Esterly This syndrome is known as familial multiple café-au-lait spots or neurofibromatosis 6 and is inherited as an autosomal dominant trait (Riccardi, 1982). This entity has been described in several families (Arnsmeier et al., 1994; Brunner et al., 1993; Charrow et al., 1993) and is believed to be distinct from neurofibromatosis 1. The disorder is characterized by multiple café-au-lait spots that are indistinguishable from those of neurofibromatosis 1 in size, number, and configuration. Adults with this syndrome have six or more café-au-lait spots that are 1.5 cm in diameter or larger. Children with this disorder have six or more café-au-lait macules that are at least 0.5 cm in diameter. Other manifestations of neurofibromatosis 1, including the presence of neurofibromas, are absent. Although intertriginous freckling and Lisch nodules are not regarded as regular features of this syndrome, these findings have been documented in an occasional patient with multiple familial café-au-lait spots (Arnsmeier et al., 1994). Nevertheless, the usual absence of intertriginous freckling in patients with familial multiple café-au-lait spots has led some investigators to propose that the pathogenesis of the café-au-lait spots and freckling in these two disorders may differ (Charrow et al., 1993). Linkage analysis with probes proximal to, distal to, and within the neurofibromatosis 1 gene indicate that this condition is genetically distinct from neurofibromatosis 1 (Abeliovich et al., 1995; Brunner et al., 1993; Charrow et al., 1993). This information is significant prognostically because these patients are not at risk for the complications of neurofibromatosis 1. Unfortunately, the definitive diagnosis of familial multiple café-au-lait spots is contingent upon the finding of affected adult family members in two generations with caféau-lait spots only and no other stigmata of neurofibromatosis

1. Because café-au-lait spots may be the only manifestation of neurofibromatosis 1 in a young child, in the absence of affected adult relatives, a specific diagnosis cannot be made with certainty. The child should be thoroughly evaluated and followed closely to watch for the emergence of other manifestations of neurofibromatosis 1.

References Abeliovich, D., Z. Gelman-Kohan, S. Silverstein, I. Lerer, J. Chemke, S. Merin, and J. Zlotosora. Familial café au lait spots: a variant of neurofibromatosis type I. J. Med. Genet. 32:985–986, 1995. Arnsmeier, S. L., V. M. Riccardi, and A. S. Paller. Familial multiple cafe-au-lait spots. Arch. Dermatol. 130:1425–1426, 1994. Brunner, H. G., T. Hulsebos, P. M. Steijlen, D. J. der Kinderen, A. V. D. Steen, and B. C. Hamel. Exclusion of the neurofibromatosis 1 locus in a family with inherited cafe-au-lait spots. Am. J. Med. Genet. 46:472–474, 1993. Charrow, J., R. Listernick, and K. Ward. Autosomal dominant multiple cafe-au-lait spots and neurofibromatosis-1: evidence of non-linkage. Am. J. Med. Genet. 45:606–608, 1993. Riccardi, V. M. Neurofibromatosis: Clinical heterogeneity. Curr. Probl. Cancer 7:1–34, 1982.

Neurofibromatosis Nancy Burton Esterly, Eulalia Baselga, and Sheila S. Galbraith

Historical Background Neurofibromatosis is included in the group of disorders known as neurocutaneous diseases or phakomatoses. Although the first description of a patient with neurofibromatosis has been attributed to Mark Akenside in 1768, portrayals of this disorder might exist in thirteenth century manuscripts and in the works of Aldrovandi, a sixteenth century naturalist, physician, and philosopher from Bologna (Mulvihill, 1990; Ragge, 1994). During the nineteenth century several illustrated descriptions were published including those 809

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of Smith, Virchow, and Hitchcock. In 1882, Friedrich von Recklinghausen was the first to suggest that the characteristic tumors in affected individuals were of neural origin (Ragge, 1994). Thomson, in 1900, pointed out the genetic nature of the disorder and Prieser and Davenport in 1918 proposed an autosomal dominant pattern of inheritance. This suggestion was confirmed by Crowe, Schull, and Neel in their classic monograph on neurofibromatosis (Crowe et al., 1956; Dunn, 1987). The studies of Riccardi and others led to the recognition that neurofibromatosis consists of multiple heterogeneous disorders which share certain features. Neurofibromatosis 1 is the most frequent type accounting for 85–90% of all patients with any form of neurofibromatosis (Riccardi, 1982). In 1982, Riccardi proposed a classification system delineating six disorders to which Goltz added two more entities. However, the consensus statement from the National Institutes of Health (NIH) consensus meeting confirmed the existence of only types 1, 2, and 5 (segmental), stating that the remaining types were too poorly defined to merit identification as separate entities (Ruggieri and Huson, 1999).

Epidemiology and Genetics Neurofibromatosis 1 is one of the most common single gene disorders and occurs in approximately 1:3000–4000 live births. It is found in all ethnic groups and both sexes and is inherited as an autosomal dominant trait with nearly 100% penetrance but highly variable expressivity (Listernick and Charrow, 1990; National Institutes of Health Consensus Development Conference, 1988b; Riccardi, 1981). Roughly 50% of cases are spontaneous mutations reflecting a mutation rate 100-fold greater than the average rate for a single locus (Listernick and Charrow, 1990). Paternal germline mutations are responsible for 90% or more of the cases. There is possibly a paternal age effect as well (Lazaro et al., 1994; Ragge, 1994; Riccardi, 1993b).

Clinical Findings The clinical features of this syndrome are numerous but primarily involve the tissues derived from the neural crest, chiefly the skin, eyes, nervous system, and skeleton (Brownstein and Little, 1983; Crawford and Bagamery, 1986; Listernick and Charrow, 1990; Ragge, 1994; Riccardi, 1982) (Table 45.1). There is considerable variation in the manifestations even within a given family, complicating the diagnosis. Furthermore, some findings are age related making diagnosis difficult in some infants and small children. The cutaneous features include café-au-lait macules, intertriginous freckling and neurofibromas. Café-au-lait spots are hyperpigmented macules (Fig. 45.1) that may develop anywhere on the body surface and vary in color from tan to almost black depending on basic skin color (Figs 45.2 and 45.3). They are occasionally two-toned and vary in size from a few millimeters to several centimeters. Sometimes a particular café-au-lait spot encompasses half the torso. Typical caféau-lait spots tend to be ovoid in shape, smooth bordered and may have a hypopigmented halo when colliding with a 810

Table 45.1. Manifestations of neurofibromatosis type 1. Cutaneous Multiple café-au-lait macules Intertriginous macules or “freckling” (Crowe sign) Multiple neurofibromas Pruritus Plexiform neurofibromas Malignant peripheral nerve sheath tumors Other large congenital melanocytic nevi (increased risk of melanoma), xanthogranulomas (increased risk of juvenile leukemia), blue-red macules, pseudoatrophic macules Ocular Lisch nodules (asymptomatic melanocytic hamartomas of the iris) (~100% of adults) Optic gliomas (pilocytic astrocytomas) of the optic nerve and chiasm (15–19%) Neurofibromas of the eyelids; plexiform neurofibromas (± glaucoma, strabismus) Neurologic Neurofibromas and plexiform neurofibromas with peripheral nerve involvement; rarely, visceral neurofibromas (e.g., bowel, bladder, liver) Unidentified bright objects (focal areas of increased signal on T2weighted images) on cranial magnetic resonance imaging (?heterotopic gray matter) (29–60%) Learning disability (up to 40%) Speech abnormalities (e.g., hypernasality, reduced rate, imprecise consonants) (30–40%) Brain tumors (particularly optic gliomas) (≥18%) Idiopathic megalencephaly (12%) Spinal cord tumors (especially plexiform intraspinal or paraspinal neurofibromas) (6%) Dural ectasia (e.g., lateral thoracic meningoceles) Mental retardation (2–5%) Seizures (3–5% of patients without intracranial lesions or other known causes); nonspecific overt electroencephalogram abnormalities (14%) Other: headaches, poor coordination Skeletal Kyphoscoliosis, often progressive and severe with acute angulation (26–60%) Posterior (± anterior) scalloping of the vertebrae Notching, thinning of ribs (twisted “ribbon ribs”) (Anterolateral) bowing of the tibia, fibula, or both Subperiosteal hematoma; subperiosteal bone proliferation Dysplasia of the lesser wing of the sphenoid bone (~1%) (Congenital) pseudoarthroses (0.5–6%) Other Pulmonary disease (10–20%): fibrosing alveolitis, upper lobe bullous disease Short stature (12%) Constipation (10%) Wilms tumor Rhabdomyosarcoma (particularly the triton tumor) Myeloid leukemia and myeloproliferative syndrome in children Secondary hypertension (£1%): renal artery stenosis (children), pheochromocytoma (adults) From: Salmon and Frieden (1995).

GENETIC EPIDERMAL SYNDROMES WITH CAFÉ-AU-LAIT MACULES

Fig. 45.1. Multiple café-au-lait spots with neurofibromas, typical of neurofibromatosis 1 (see also Plate 45.1, pp. 494–495).

Fig. 45.3. Multiple café-au-lait spots and neurofibromas (see also Plate 45.3, pp. 494–495).

Fig. 45.2. Multiple café-au-lait spots. Six such spots are highly suggestive of neurofibromatosis (see also Plate 45.2, pp. 494–495).

Mongolian spot. These lesions may be present at birth or develop during the first several months after birth. Most are present by 2 years (Riccardi, 1992). In one study, 97% of children under 6 years of age with confirmed neurofibromatosis 1 had at least the requisite number of café-au-lait spots for diagnosis (Obringer et al., 1989). In contrast, intertriginous freckling is almost invariably of later onset although freckling was found in 81% of children with neurofibromatosis 1 under 6 years of age (Obringer et al., 1989). Unlike café-au-lait spots which are found in many diseases as well as normal skin, intertriginous freckling is almost pathognomonic for neurofibromatosis 1 (Fig. 45.4). Axillary and inguinal “freckles” are miniature café-au-lait macules. They occur in non–sun-exposed areas and have no relation to ephelides clinically or histologically. These small hyperpigmented macules may also develop at the base of the neck, in the inframammary area, or diffusely over the trunk (Riccardi, 1992). Iridial Lisch nodules (Fig. 45.5) are melanocytic hamartomas of variable size and color that do not alter vision and usually require a slit-lamp examination for detection (Perry

Fig. 45.4. Miniature café-au-lait spots in the axillae, called Crowe’s sign, in neurofibromatosis 1 (see also Plate 45.4, pp. 494–495).

and Font, 1982; Williamson et al., 1991). They are another identifying feature of neurofibromatosis 1 and can be found in approximately 75% of patients, 6 or more years of age (Lubs et al., 1991; Zehavi et al., 1986). Their prevalence increases with age, in one series occurring in only 40% of children under age 10 but in 90% of those patients over 10 and 100% of those over 21 years of age (Zehavi et al., 1986). Lubs et al. (1991) identified Lisch nodules in 5% of children under 3, 43% at 34 years, 55% at 56 years and 100% of affected adults over 21 years of age. 811

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Fig. 45.5. Typical Lisch nodule (see also Plate 45.5, pp. 494–495).

Neurofibromas are a cardinal feature of neurofibromatosis 1 but, except for the plexiform type (Figs 45.3 and 45.6) which may be congenital, they tend to develop later than the pigmented lesions. They may be asymptomatic, painful, or pruritic. Dermal neurofibromas are skin colored to reddish papules and nodules, at times pedunculated, that vary greatly in size from a few millimeters to several centimeters. They can be invaginated by an examining finger, a unique feature called “buttonholing.” An uncommon variant of this type of neurofibroma is the pseudoatrophic macule (Norris et al., 1985; Westerhof and Konrad, 1982). Subcutaneous neurofibromas are firm, mobile nodules that often cause localized pain or tenderness. Plexiform neurofibromas arise from the larger nerves and nerve roots and at times penetrate deeply into muscle, bone, and viscera. These tumors can greatly distort the involved tissues causing hypertrophy and large pendulous masses that on palpation resemble a “bag of worms.” Neurofibromas most commonly develop around puberty and during pregnancy and may be progressive throughout adulthood. Only 17% of children under 6 years have neurofibromas whereas most affected adults have at least a few (Obringer et al., 1989; Riccardi, 1981). Macular hyperpigmentation and hypertrichosis often signal an underlying plexiform neurofibroma. However, these pigmented lesions differ from the café-au-lait spot both in color and in the absence of a sharply demarcated border. They are often a red-brown or gray-brown color. A hair whorl localized to the posterior midline may be a marker for an underlying vertebral defect (Riccardi, 1993b). Patients with neurofibromatosis 1 are at risk for certain associated neural and non-neural malignancies that develop in an age-related pattern. In a Swedish cohort of 212 affected individuals and their relatives followed for 40 years, malignant neoplasms and central nervous system tumors occurred in 40% of the probands (Sorensen, 1986). Tumors associated with neurofibromatosis 1 include optic nerve gliomas (Lewis et al., 1984; Listernick et al., 1989; Lund and Skovby, 1991), other central nervous system gliomas, malignant schwannomas, neurofibrosarcomas (overall risk 5%), Wilms tumors, 812

Fig. 45.6. Plexiform neurofibroma (see also Plate 45.6, pp. 494–495).

pheochromocytomas, rhabdomyosarcomas particularly in the urogenital organs, and myelogenous leukemia (Bader and Miller, 1978; Matsui et al., 1993; Zvulunov et al., 1995). Juvenile xanthogranuloma has also been associated with myelogenous leukemia in the setting of neurofibromatosis 1 (Zvulunov et al., 1995). Xanthogranulomas are seen in 20% of children with neurofibromatosis 1 (Newell et al., 1973). Approximately 10% of patients with neurofibromatosis 1 may complain of pruritus (Riccardi, 1981). This may be related to the presence of mast cells within neurofibromas and appears to be proportional to the number and size of mast cells. No clinical predictive indicators were found in a study of 50 children with neurofibromatosis 1 and malignant tumors (Schneider et al., 1986). In addition to a predisposition to tumors, other abnormalities possibly associated with neurofibromatosis 1 include cognitive impairment, speech disorders, seizures, mental retardation, short stature and hypertension, and cardiovascular malformations (Lin et al., 2000, Listernick and Charrow, 1990; Mulvihill, 1990). Neurofibromatosis 2, bilateral acoustic neurofibromatosis or central neurofibromatosis, affects about one in 50 000 patients (Martuza and Roswell, 1977). It is also an autosomal

GENETIC EPIDERMAL SYNDROMES WITH CAFÉ-AU-LAIT MACULES

dominant disorder, although approximately two-thirds of the patients appear to represent new mutations. The gene for neurofibromatosis 2, nf2, is located on the long arm of chromosome 22 on band 11.2 and encodes the protein merlin (Rouleau et al., 1987). Cutaneous schwannomas, welldemarcated, rough-surfaced papules with overlying hypertrichosis, are the most common skin tumors present in neurofibromatosis 2 (Martuza et al., 1985). Neurofibromatosis 2 is characterized by bilateral acoustic neuromas (vestibular schwannomas) (Martuza and Roswell, 1977; Mulvihill, 1990). Other central nervous system tumors in neurofibromatosis 2 include intracranial and spinal meningiomas, ependymomas, spinal astrocytomas, paraspinal and cranial nerve schwannomas. Ocular findings in neurofibromatosis 2 include cataracts, retinal hamartomas, and astrocytomas (Kaye et al., 1992). The acoustic neuromas can result in hearing loss, tinnitus, and dizziness (Martuza and Roswell, 1977; Mulvihill, 1990). Seizures and headache may suggest neurologic involvement in neurofibromatosis 2. Neurofibromatosis 3 is the mixed type, characterized by features of neurofibromatosis 1 and neurofibromatosis 2 (National Institutes of Health Consensus Development Conference, 1988a; Riccardi, 1987). Neurofibromatosis 4 is characterized by diffuse café-au-lait macules (CALM) and neurofibromas in addition to central nervous system neoplasms. In neurofibromatosis 5 or segmental neurofibromatosis, cutaneous findings such as CALM, neurofibromas, intertriginous freckling, and/or Lisch nodules are localized to a segment (Miller and Sparkes, 1977). Rarely, it can be bilateral or involve more than one segment. There have been over 30 reported cases of neurofibromatosis 5 to date. Most cases have been sporadic. Neurofibromatosis 5 may result from spontaneous postzygotic somatic mutation of the nf1 gene (Jung, 1988). Familial CALM without neurofibromas is known as neurofibromatosis 6 (Arnsmeier et al., 1994). Its inheritance pattern is autosomal dominant. Genetic linkage analysis of neurofibromatosis 6 families does not reveal an allelic mutation in the nf1 gene (Charrow et al., 1993). The criteria for the CALM, that is based on their number and size, are the same as in neurofibromatosis 1. These patients may have Lisch nodules and intertriginous freckling, certain skeletal abnormalities and learning disorders, but they do not develop the complete clinical presentation of neurofibromatosis 1. Late-onset neurofibromatosis is known as neurofibromatosis 7, and it usually occurs in patients over 20 years of age (National Institutes of Health Consensus Development Conference, 1988a; Riccardi, 1987). Neurofibromatosis that cannot be classified into any of the above subtypes is called NFNOS (neurofibromatosis not otherwise specified).

Associated Disorders A possible variant of neurofibromatosis 1 includes Watson syndrome, an autosomal dominant disorder, associated with CALM, pulmonary stenosis, mental retardation, and short

stature (Allanson et al., 1991). These patients may also have intertriginous freckling, neurofibromas, Lisch nodules, and macrocephaly. Genetic linkage analysis shows allelic mutations similar to the nf1 gene. Certain neurofibromatosis 1 patients have clinical features that resemble Noonan syndrome (Mendez and Opitz, 1985; Wyre, 1978). It is an autosomal dominant disorder associated with a large deletion mutation that encompasses the nf1 gene locus. Noonan syndrome is characterized by a distinctive facies (hypertelorism, low set ears, downslanting palpebral fissures), webbed neck, congenital heart disease (particularly pulmonary stenosis), skeletal abnormalities (kyphoscoliosis, short stature, pectus deformity, cubitus valgus), and cryptorchidism. Poliosis and gray hair have been reported in an occasional patient (Kaplan and Shapiro, 1968). Preliminary data suggest that patients with familial sinal neurofibromatosis may have mutations in the nf1 gene on genetic analysis. Five members of a three-generation family that showed clinical homogeneity unusual for neurofibromatosis, with multiple spinal cord tumors and café-au-lait spots, showed segregation within the neurofibromatosis 1 locus (Ars et al., 1998; Ruggieri and Huson, 1999).

Histology Several investigators have attempted to characterize the caféau-lait spot of neurofibromatosis 1 histologically to distinguish it from café-au-lait spots in normal individuals or in other disorders. Evaluations of the density of the melanocyte population and the presence or absence of melanin macroglobules have yielded disparate findings (Benedict et al., 1968; Frenk and Marazzi, 1984; Johnson and Charneco, 1970; Martuza et al., 1985; Takahashi, 1976). In some studies, neurofibromatosis 1 patients were found to have an increased number of melanocytes in both normal and hyperpigmented skin (Benedict et al., 1968; Frenk and Marazzi, 1984). In other studies an increased number of melanocytes was documented only in their café-au-lait spots (Johnson and Charneco, 1970; Takahashi, 1976). Another study examined the keratinocyte:melanocyte ratio in skin from neurofibromatosis 1 and found lower ratios in both the café-au-lait spots and the normally pigmented skin compared with skin from unaffected individuals (Frenk and Marazzi, 1984). Studies of café-au-lait macules (Ishida and Jimbow, 1987) have considered not only population density but coloration and structure of the melanocytes. Ishida and Jimbow compared biopsy specimens from light-brown, brown, and darkbrown macules of patients with neurofibromatosis 1 and found that the population density of melanocytes was related to depth of coloration. Light-brown and brown macules had a normal number of melanocytes compared with the patient’s normal skin whereas dark-brown macules showed a significant increase in the melanocyte population. Furthermore, compared with the patient’s normally pigmented skin, there was a change in structure of the melanocytes with an increase in area and perimeter of the whole cell in light-brown and 813

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brown macules and a decrease in these parameters in darkbrown lesions (Ishida and Jimbow, 1987). Grossman et al. studied only light colored (tan) café-au-lait macules from both neurofibromatosis 1 and normal individuals and found two histologic subtypes: (1) normal numbers of melanocytes, and (2) increased numbers of melanocytes. Both subtypes were identified in macules from normal and affected individuals. Melanin macroglobules, originally called macromelanosomes, were initially thought to be pathognomonic for neurofibromatosis 1 (Johnson and Charneco, 1970). They have since been shown to be present in numerous other conditions (Slater et al., 1986). These structures can be detected in paraffin-embedded sections (Morris et al., 1982) as well as on split-dopa preparations (Martuza et al., 1985). It has subsequently been shown that melanin macroglobules are the result of fusion of melanosome complexes with phagolysosomes. Although they seem to occur in greater numbers in neurofibromatosis 1 than in other disorders, they are no longer considered diagnostically significant (Martuza et al., 1985). In tissue culture melanocytes from café-au-lait spots of a patient with neurofibromatosis-1 produced more melanin macroglobules than melanocytes from unaffected skin from the same patient and from normal skin. However on electron microscopy the melanin macroglobules of the cultured cells were found to be incompletely developed (Kaufmann et al., 1989). Histologic descriptions of the freckle-like café-au-lait spots are few in number but support the postulate that they represent small café-au-lait spots (Johnson and Charneco, 1970). No detailed histologic descriptions exist of the pigmented skin overlying plexiform neurofibromas.

Laboratory Investigations There are no requisite laboratory studies. Choice of studies should be predicated on the pertinent history and physical findings. However, some authorities believe it is appropriate to obtain a baseline magnetic resonance imaging study of the head for optic glioma which is often silent and may therefore be missed on physical examination (Listernick and Charrow, 1990).

Diagnosis The diagnostic criteria for neurofibromatosis 1 formulated at a National Institutes of Health Consensus Development Conference in 1987 have been widely accepted by physicians in all specialties (National Institutes of Health Consensus Development Conference, 1988b). Diagnosis of neurofibromatosis 1 is confirmed by the presence of two or more of the following components of the syndrome: (1) six or more café-au-lait macules larger than 5 mm in prepubertal children or greater than 15 mm in postpubertal individuals; (2) two or more neurofibromas of any type or one plexiform neurofibroma; (3) freckling in the axillary or inguinal regions; (4) optic glioma; (5) two or more Lisch nodules; (6) a distinctive osseous lesion (sphenoid dysplasia, thinning of long bone cortex, pseudarthrosis); or (7) a first-degree relative with neu814

rofibromatosis by the above criteria. The validity of these criteria has been substantiated by the appropriate classification of 94% of 160 children 6 years or under when these criteria were applied (Obringer et al., 1989). The presence of at least one of the following clinical manifestations supports the diagnosis of neurofibromatosis 2: (1) bilateral acoustic neuromas; (2) a parent, sibling, or offspring with neurofibromatosis 2 and either unilateral acoustic neuroma or two of the following: neurofibroma, meningioma, glioma, schwannoma, juvenile posterior subcapsular cataracts.

Differential Diagnosis The differential diagnosis is broad because of the extreme variability of expression of neurofibromatosis 1. Among the disorders that must be considered are neurofibromatosis 2, segmental neurofibromatosis, multiple familial café-au-lait spots (neurofibromatosis 6), Noonan syndrome, McCune– Albright syndrome, Silver–Russell syndrome, Watson syndrome, and Jaffe Campanacci syndrome (Salmon and Frieden, 1995).

Pathogenesis Neurofibromatosis 1 has been regarded as a neurocristopathy, a generalized disorder involving predominantly, but not exclusively, cells of neural crest origin. The nf1 gene is located in the pericentromeric region of the long arm of chromosome 17. It has been cloned and characterized and is a large gene (>300kb), a finding that may account for its high mutation rate. Several types of germline mutations have been documented including macrodeletions, microdeletions, translocations, insertions, and point mutations. However correlation of genotypes with specific phenotypes is still lacking (Goldberg and Collins, 1991; Gasparini et al., 1996; Ragge, 1994; Viskochil, 2002). The disorder is progressive with onset early in embryological development. A specific product of a small portion of the nf1 gene called neurofibromin has been identified in many tissues. This protein exhibits structural and functional homology to the GAP (guanidine triphosphatase activating protein) family of proteins. Neurofibromin appears to regulate the ras protooncogene that influences growth and differentiation of a variety of tissues. Neurofibromin functions as a tumor suppressor, downregulating ras in normal cells. Such downregulation does not occur in cells in which both copies of the nf1 gene are altered. It has been suggested that a two hit process similar to that which is responsible for retinoblastoma might explain the effects of mutations of the nf1 gene. One mutation is inherited and the “second hit” (somatic mutation) destroys the functional gene, allowing uncontrolled expression of ras. The mutated gene could result in the proliferation of benign tumors such as neurofibromas. Additional mutations, e.g., in the p53 tumor suppressor gene, may account for the development of malignant tumors (Gutmann and Collins, 1993; Origone et al., 2003; Ragge, 1994; Riccardi, 1993b; Salmon and Frieden, 1995).

GENETIC EPIDERMAL SYNDROMES WITH CAFÉ-AU-LAIT MACULES

Animal Models There are promising animal models of neurofibromatosis 1, but there are no animal models of neurofibromatosis 2. The bicolor damsel fish, Pomacentrus partitus, has pigmented skin tumors that resemble neurofibromas (Mulvihill, 1990; Riccardi, 1989). Another model uses transgenic mice which are produced by insertion of HTLV1 (human T lymphocyte virus) tat gene or mutant human nf1 gene into fertilized mouse eggs (Hinrichs et al., 1987; Riccardi, 1993a). Similar to human neurofibromas, the tumors that developed in the mice are of nerve sheath origin, may be formed by proliferation of fibroblasts, show an increased growth during pregnancy, and can develop along the path of cranial nerves (Hinrichs et al., 1987). Infiltration by mast cells is characteristic of human neurofibromas and transgenic tat tumors, respectively. Nude mouse xenografts have been constructed to study neurofibromatosis 2 tumors (Riccardi, 1993a). In addition, retroviruses are being developed to produce the wild-type gene in meningiomas and schwannomas that have a mutant nf2 gene.

Treatment Treatment is totally dependent on the clinical findings in a given individual. If possible, patients with neurofibromatosis 1 should be referred to multidisciplinary clinics where they can be seen by specialists in neurology, genetics, dermatology, ophthalmology, orthopedic surgery, and plastic surgery as well as a general pediatrician. Some patients also require the services of psychologists, speech pathologists, and occupational and physical therapists. Genetic counseling is mandatory. DNA-linkage testing for confirmation of diagnosis in individuals with minimal findings is a costly but accurate way to confirm or exclude the diagnosis of neurofibromatosis 1 in a familial setting. Prenatal diagnosis is also possible in families with established neurofibromatosis 1 (Hofman and Boehm, 1992; Origone, 2000). This technique is not applicable in sporadic cases of the disorder because of the tremendous variability in the phenotypic expression of the gene. The prognosis cannot be predicted early in life.

References Allanson, J. E., M. Upadhyaya, G. H. Watson, M. Partington, A. Mackenzie, D. Lahey, H. MacLeod, M. Sarfarazi, W. Broadhead, P. S. Harper, and S. M. Huson. Watson syndrome: is it a subtype of type 1 neurofibromatosis? J. Med. Genet. 28:752–756, 1991. Arnsmeier, S. L., V. M. Riccardi, and A. S. Paller. Familial multiple cafe-au-lait spots. Arch. Dermatol. 130:1425–1426, 1994. Ars, E., H. Kruger, A. Gaona, P. Casquero, J. Rosell, V. Volpini, E. Serra, C. Lazaro, and X. Estivill. A clinical variant of neurofibromatosis type 1: Familial spinal neurofibromatosis with a frameshift mutation in the NF1 gene. Am. J. Hum. Genet. 62:834–841, 1998. Bader, J. L., and R. W. Miller. Neurofibromatosis and childhood leukemia. J. Pediatr. 92:925–929, 1978. Benedict, P. H., G. Szabó, T. B. Fitzpatrick, and S. J. Sinesi. Melanotic macules in Albright’s syndrome and in neurofibromatosis. J. Am. Med. Assoc. 205:618–626, 1968.

Brownstein, S., and J. M. Little. Ocular neurofibromatosis. Ophthalmology 90:1595–1599, 1983. Charrow, J., R. Listernick, and K. Ward. Autosomal dominant multiple cafe-au-lait spots and neurofibromatosis-1: evidence of nonlinkage. Am. J. Med. Genet. 45:606–608, 1993. Crawford, A. H., Jr., and N. Bagamery. Osseous manifestations of neurofibromatosis in childhood. J. Pediatr. Orthoped. 6:72–88, 1986. Crowe, F. W., W. J. Schull, and J. V. Neel. A Clinical, Pathological and Genetic Study of Multiple Neurofibromatosis. Springfield, IL: Charles C. Thomas, 1956. Dunn, D. W. Neurofibromatosis in childhood. Curr. Probl. Pediatr. 17:449–497, 1987. Frenk, E., and A. Marazzi. Neurofibromatosis of von Recklinghausen: a quantitative study of the epidermal keratinocyte and melanocyte populations. J. Invest. Dermatol. 83:23–25, 1984. Gasparini, P., L. D’Agruma, P. G. Cillis, P. Balestrazzi, R. Mingarelli, and L. Zelante. Scanning the first part of the neurofibromatosis type 1 gene by RNA SCP identification of three novel mutations and of two new polymorphisms. J. Hum. Genet. 97:492–495, 1996. Goldberg, N. S., and F. S. Collins. The hunt for the neurofibromatosis gene. Arch. Dermatol. 117:1705–1707, 1991. Gutmann, D. H., and F. S. Collins. Neurofibromatosis type 1: Beyond positional cloning. Arch. Neurol. 50:1185–1193, 1993. Hinrichs, S. H., M. Nerenberg, R. K. Reynolds, G. Khoury, and G. Jay. A transgenic mouse model for human neurofibromatosis. Science 237:1340–1343, 1987. Hofman, K. J., and C. D. Boehm. Familial neurofibromatosis type 1: clinical experience with DNA testing. J. Pediatr. 120:394–398, 1992. Ishida, O., and K. Jimbow. A computed image analyzing system for quantitation of melanocyte morphology in cafe-au-lait macules of neurofibromatosis. J. Invest. Dermatol. 88:287–291, 1987. Johnson, B. L., and D. R. Charneco. Café au lait spot in neurofibromatosis and in normal individuals. Arch. Dermatol. 102:442–446, 1970. Jung, E. G. Segmental neurofibromatosis. Neurofibromatosis 1:306– 311, 1988. Kaplan, B. S., and L. Shapiro. Poliosis overlying a neurofibroma. Arch. Dermatol. 98:631–633, 1968. Kaufmann, D., W. Krone, R. Hochsattel, and R. Martin. A cell culture study on melanocytes from patients with neurofibromatosis-1. Arch. Dermatol. Res. 281:510–513, 1989. Kaye, L. D., A. D. Rothner, G. R. Beauchamp, S. M. Meyers, and M. L. Estes. Ocular findings associated with neurofibromatosis type II. Ophthalmology 99:1424–1429, 1992. Lazaro, C., A. Ravvella, and A. Gaona. Neurofibromatosis type 1 due to germ-line mosaicism in a clinically normal father. N. Engl. J. Med. 331:1403–1407, 1994. Lewis, R. A., P. Gerson, K. A. Axelson, V. M. Riccardi, and R. P. Whitford. von Recklinghausen neurofibromatosis II: Incidence of optic glioma. Ophthalmology 91:929–935, 1984. Lin, A. E., P. H. Birch, B. R. Korf, R. Tenconi, M. Niimura, M. Poyhonen, K. Armfield Uhas, M. Sigorini, R. Virdis, C. Romano, E. Bonioli, P. Wolkenstein, E. K. Pivnick, M. Lawrence, and J. M. Friedman. Cardiovascular malformations and other cardiovascular abnormalities. Am. J. Med. Genet. 95:108–117, 2000. Listernick, R., and J. Charrow. Nerurofibromatosis type 1 in childhood. J. Pediatr. 116:845–853, 1990. Listernick, R., J. Charrow, M. J. Greenwald, and N. B. Esterly. Optic gliomas in children with neurofibromatosis type 1. J. Pediatr. 114:788–792, 1989. Lubs, M.-L. E., M. S. Bauer, M. E. Formas, and B. Djokic. Lisch nodules in neurofibromatosis type 1. N. Engl. J. Med. 324:1264–1266, 1991. Lund, A. M., and F. Skovby. Optic gliomas in children with neurofibromatosis type 1. Eur. J. Pediatr. 150:835–838, 1991. Martuza, R. L., and Roswell E. Neurofibromatosis 2 (bilateral acoustic neurofibromatosis). N. Engl. J. Med. 318:684–838, 1977.

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CHAPTER 45 Martuza, R. L., I. Philippe, T. B. Fitzpatrick, J. Zwaan, Y. Seki, and J. Lederman. Melanin macroglobules as a cellular marker of neurofibromatosis: A quantitative study. J. Invest. Dermatol. 85:347–350, 1985. Matsui, M., T. Sawada, M. Tanimura, N. Nagahara, K. Kobayashi, and J. Akatsuka. Neurofibromatosis type 1 and childhood cancer. Cancer 72:2746–2754, 1993. Mendez, H. M. M., and J. M. Opitz. Noonan syndrome: a review. Am. J. Hum. Genet. 21:493–506, 1985. Miller, R. M., and R. S. Sparkes. Segmental neurofibromatosis. Arch. Dermatol. 133:837–838, 1977. Morris, T. J., W. G. Johnson, and D. N. Silvers. Giant pigment granules in biopsy specimens from café-au-lait spots in neurofibromatosis. Arch. Dermatol. 118:385–388, 1982. Mulvihill, J. J. Neurofibromatosis 1 (Recklinghausen disease) and neurofibromatosis 2 (bilateral acoustic neurofibromatosis). Ann. Intern. Med. 113:39–52, 1990. National Institutes of Health Consensus Development Conference. Neurofibromatosis. Neurofibromatosis 1:172–178, 1988a. National Institutes of Health Consensus Development Conference. Neurofibromatosis Conference Statement. Arch. Neurol. 45:575– 578, 1988b. Newell, G. B., O. J. Stone, and J. F. Mullins. Juvenile xanthogranuloma and neurofibromatosis. Arch. Dermatol. 107:262, 1973. Norris, J. F. B., A. G. Smith, P. J. H. Fletcher, T. L. Marshall, and M. J. Hand. Neurofibromatous dermal hypoplasia: a clinical, pharmacological and ultrastructural study. Br. J. Dermatol. 112:435–441, 1985. Obringer, A. C., A. T. Meadows, and E. H. Zackai. The diagnosis of neurofibromatosis-1 in the child under the age of 6 years. Am. J. Dis. Child. 143:717–719, 1989. Origone, P., E. Bonioli, E. Panucci, S. Costabel, F. Ajmar, and D. A. Coviello. The Genoa experience of prenatal dignosis in NF1. J. Prenat. Diag. 20:719–724, 2000. Origone, P., R. Defferrari, K. Mazzocco, C. Lo Consolo, B. De Bernardi, and C. P. Tonini. Homozygous activation of NF1 gene in a patient with familial NF1 and disseminated neuroblastoma. Am. J. Med. Genet. 118A:309–313, 2003. Perry, H. D., and R. L. Font. Iris nodules in von Recklinghausen’s neurofibromatosis: Electron microscopic confirmation of their melanocytic origin. Arch. Ophthalmol. 100:1635–1640, 1982. Ragge, N. Clinical and genetic patterns of neurofibromatosis 1 and 2. Br. J. Ophthalmol. 77:662–672, 1994. Riccardi, V. M. Von Recklinghausen neurofibromatosis. N. Engl. J. Med. 305:1617–1626, 1981. Riccardi, V. M. Neurofibromatosis: Clinical heterogeneity. Curr. Probl. Cancer 7:1–34, 1982. Riccardi, V. M. Neurofibromatosis. Neurol. Clin. 5:337–349, 1987. Riccardi, V. M. Genetic alterations and growth factors in the pathogenesis of von Recklinghausen neurofibromatosis. Neurofibromatosis 2:292–298, 1989. Riccardi, V. M. Neurofibromatosis: Phenotype, Natural History and Pathogenesis, 2nd edn. Baltimore, MD: Johns Hopkins University Press, 1992. Riccardi, V. M. A controlled multiphase trial of ketotifen to minimize neurofibroma-associated pain and itching. Arch. Dermatol. 129:577–581, 1993a. Riccardi, V. M. Molecular biology of the neurofibromatoses. Semin. Dermatol. 12:266–273, 1993b. Rouleau, G. A., W. Wertelecki, J. L. Haines, W. J. Hobbs, J. A. Trofatter, B. R. Seizinger, R. L. Martuza, D. W. Superneau, P. M. Conneally, and J. F. Gusella. Genetic linkage of bilateral acoustic neurofibromatosis to a DNA marker on chromosome 22. Nature 329:246–248, 1987. Ruggieri, M., and Huson, S. M. The neurofibromatoses. An overview. Ital. J. Neurol. Sci. 20:89–108, 1999.

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Salmon, J. K., and I. J. Frieden. Congenital and genetic disorders of hyperpigmentation. Curr. Probl. Dermatol. 7:145–196, 1995. Schneider, S., A. C. Obringer, E. Zackai, and A. T. Meadows. Childhood neurofibromatosis: Risk factors for malignant disease. Cancer Genet. Cytogenet. 21:347–354, 1986. Slater, C., M. Hayes, N. Saxe, C. Temple-Camp, and P. Beighton. Macromelanosomes in the early diagnosis of neurofibromatosis. Am. J. Dermatopathol. 8:284–289, 1986. Takahashi, M. Studies on café au lait spots in neurofibromatosis and pigmented macules of nevus spilus. Tohoku J. Exp. Med. 118:255–273, 1976. Viskochil, D. Genetics of neurofibromatosis 1 and the NF1 gene. J. Child Neurol. 17:562–570, 2002. Westerhof, W., and K. Konrad. Blue-red macules and pseudoatrophic macules. Arch. Dermatol. 118:577–581, 1982. Williamson, T. H., A. Garner, and A. T. Moore. Structure of Lisch nodules in neurofibromatosis type 1. Ophthalmic Paediatr. Genet. 12:11–17, 1991. Wyre, H. W., Jr. Cutaneous manifestations of Noonan’s syndrome. Arch. Dermatol. 114:929–930, 1978. Zehavi, C., A. Romano, and A. M. Goodman. Iris (Lisch) nodules in neurofibromatosis. Clin. Genet. 29:51–55, 1986. Zvulunov, A., Y. Barak, and A. Metzker. Juvenile xanthogranuloma, neurofibromatosis, and juvenile chronic myelogenous leukemia: world statistical analysis. Arch. Dermatol. 131:904–908, 1995.

Neurofibromatosis 1 with Noonan Syndrome Nancy Burton Esterly Noonan syndrome is an autosomal dominant disorder with an estimated incidence of 1 in 1000 to 2500 live births (Buehning and Curry, 1995). It is characterized by short stature, cardiac defects (mainly septal defects, patent ductus arteriosus, and pulmonary stenosis), dysmorphic facies (hypertelorism, micrognathia, downslanting palpebral fissures, and low set ears), a webbed neck, and cryptorchidism as well as skeletal anomalies. Transient congenital lymphedema, large numbers of melanocytic nevi, and abnormal hair are additional features (Mendez and Opitz, 1985; Wyre, 1978). Because of the extreme variability in expressivity, it is often difficult to categorize these patients with certainty, and some authors believe the disorder is genetically heterogeneous (Borochowitz et al., 1989). Coexistent features of neurofibromatosis 1 and Noonan syndrome have been described in isolated cases and in multiple members of a few families (Allanson et al., 1985; Buehning and Curry, 1995; Colley et al., 1989; Mendez, 1985). Several theories have been proposed to explain this overlap syndrome including: (1) coincidental occurrence of neurofibromatosis 1 and Noonan syndrome; (2) close gene loci subject to chromosomal aberrations; (3) a broad phenotype for Noonan syndrome; (4) a broad phenotype for neurofibromatosis 1; and (5) a genetically distinct entity represented by features of both disorders. This controversy is unlikely to be settled until many more patients are studied. Recent evidence suggests that more than one mechanism may be operative in the occurrence of this syndrome (Ahlbom et al., 1995; Baralle et al., 2003; Carey, 1998; Flintoff et al., 1993; Mendez, 1985; Sharland et al., 1992; Stern et al., 1992).

GENETIC EPIDERMAL SYNDROMES WITH CAFÉ-AU-LAIT MACULES

References Ahlbom, E., N. Dahl, P. Zetterquist, G. Anneren. Noonan syndrome with café-au-lait spots and multiple lentigines syndrome are not linked to the neurofibromatosis type I locus. Clin. Genet. 48:85–89, 1995. Allanson, J. E., J. G. Hall, and M. I. Van Allen. Noonan phenotype associated with neurofibromatosis. Am. J. Med. Genet. 21:457– 462, 1985. Baralle, D., C. Mattocks, K. Kalidas, F. Elmslie, J. Whittaker, M. Lees, N. Ragge, M. Patton, R. Winter, C. ffrench-Constant. Different mutations in the NF1 gene are associated with neurofibromatosisNoonan syndrome (NFNS). Am J. Med Genet. 119A:1–8, 2003. Borochowitz, Z., N. Berant, and H. Dar. The neurofibromatosisNoonan syndrome: genetic heterogeneity versus clinical variability. Neurofibromatosis 2:309–314, 1989. Buehning, L., and C. J. Curry. Neurofibromatosis — Noonan syndrome. Pediatr. Dermatol. 12:267–271, 1995. Carey, J. C. Neurofibromatosis-Noonan syndrome [editorial comment]. Am J. Med Genet. 75:263–264, 1998. Colley, A., J. Clayton-Smither, and D. Donnai. Noonan syndrome and neurofibromatosis type 1 segregating independently in a family. Am. J. Hum. Genet. 45:A43, 1989. Flintoff, W. F., M. Bahuau, and S. Lyonnet. No evidence for linkage to the type 1 or type 2 neurofibromatosis loci in Noonan syndrome families. Am. J. Med. Genet. 46:700–705, 1993. Mendez, H. M. M. The neurofibromatosis-Noonan syndrome. Am. J. Med. Genet. 21:471–476, 1985. Mendez, H. M. M., and J. M. Opitz. Noonan syndrome: a review. Am. J. Hum. Genet. 21:493–506, 1985. Sharland, M., R. Taylor, and M. A. Patton. Absence of linkage of the Noonan syndrome to the neurofibromatosis type 1 locus. J. Med. Genet. 29:188–190, 1992. Stern, H. J., H. M. Saal, and J. S. Lee. Clinical variability of type 1 neurofibromatosis: is there a neurofibromatosis-Noonan syndrome? J. Med. Genet. 29:184–287, 1992. Wyre, H. W., Jr. Cutaneous manifestations of Noonan`s syndrome. Arch. Dermatol. 114:929–930, 1978.

45.7

McCune–Albright Syndrome Sheila S. Galbraith and Nancy Burton Esterly

Historical Background During the early 1930s, Albright observed two women with precocious puberty, a bone disorder, and “pigmented nevi” (Albright et al., 1937). In 1936, several months before Albright’s report was published, McCune described a patient with similar clinical findings (McCune, 1936). Hence the entity is now known as McCune–Albright syndrome (OMIM #174800).

Synonym Albright’s syndrome.

Clinical Findings The syndrome in its complete form is characterized by the triad of bone lesions termed polyostotic fibrous dysplasia, a hyperfunctioning endocrine system, and cutaneous lesions of light-brown pigmentation. The pigmented macules of McCune–Albright syndrome are commonly called café-au-lait macules. The lesions may be present at birth, but most develop in infancy. Sites of predilec-

45.8 Figs 45.7 and 45.8. Classic large café-au-lait spots observed in patients with McCune–Albright syndrome (see also Plates 45.7 and 45.8, pp. 494–495).

tion include the head, trunk, sacral area, and buttocks. They may vary in size and number but are usually large with irregular borders. The borders are jagged and are said to mimic the contour of the “coast of Maine.” Many authors use this feature to distinguish these lesions from the smooth-bordered pigmented macules usually observed in neurofibromatosis. However, this criterion is not absolute. The macules are frequently in a segmental or linear distribution and tend to follow the lines of Blaschko (Rieger et al., 1994) (Figs 45.7 and 45.8). Polyostotic fibrous dysplasia, the skeletal abnormality observed in McCune–Albright syndrome, frequently affects the long bones and manifests as bone pain or pathologic fractures. This most commonly presents between the ages of 4 and 817

CHAPTER 45

8 years (de Sanctis et al., 1999). The classic radiographic finding consists of cystlike spaces in the cortex of the bone. The extent of involvement is variable. The bony lesions and the pigmented macules often occur in the same distribution. The association of multiple endocrinopathies is well established and is due to autonomous function of the affected glands (D’Armiento et al., 1983). Precocious puberty and hyperthyroidism are the most common abnormalities. de Sanctis et al. (1999) found a 50% probability of developing precocious puberty by 4 years of age in affected females. Other endocrinopathies include Cushing syndrome, hypersomatism, hyperprolactinemia, hyperparathyroidism, and acromegaly.

Histology Histologically, there is increased melanin in the basal layer of the epidermis without an increase in number of melanocytes.

Diagnosis The diagnosis of McCune–Albright syndrome is a clinical one and laboratory tests are generally not helpful in establishing the diagnosis. There may be an increase in serum alkaline phosphatase or hormonal abnormalities if an endocrinopathy is present. Mutation analysis can be performed to detect activating mutations of GNAS1 (guanine nucleotide-binding protein, a-stimulating activity polypeptide 1); however, the analysis has limitations as the mutation is present at low levels in lymphocytes and affected tissue which contains higher levels may not be readily available for analysis (Aldred and Trembath, 2000). Radiographic features of fibrous dysplasia are variable and include pseudocystic, sclerotic, and Paget types (Becelli et al., 2002).

Pathogenesis Most cases of McCune–Albright syndrome are sporadic, with only a few familial cases reported. Molecular genetic studies have supported the concept of a lethal autosomal dominant gene surviving only in a mosaic state by postzygotic somatic mutations. The segmental distribution of both the bone and skin lesions supports this explanation. Weinstein et al. have identified a missense mutation of the codon Arg 201 in the alpha subunit of the stimulatory G protein gene (GNAS1) present on chromosome 20q13.2. G proteins regulate the activity of adenylyl cyclase. Other inherited defects in this protein have been found to cause human disease as well. It is known that the defect in McCune–Albright syndrome is an “activating mutation” which results in markedly reduced GTPase activity and subsequent constitutive activation of adenylyl cyclase which catalyzes the formation of cyclic AMP (cAMP) (Weinstein et al., 1991). The accumulation of cAMP, in turn, activates protein kinase A which phosphorylates transcription factors and alters the expression of several genes whose promoters contain cAMP-responsive elements. This results in increased levels of c-fos, c-jun, interleukin (IL)-6, and IL-11 and affects the transcription of downstream genes, leading to excessive function of responsive cells such as melanocytes (due to increased levels of tyrosinase), skeletal 818

progenitor cells (producing abnormal osteoblasts), and endocrine cells (Marie, 2001). Happle (1987) has proposed that the distribution of pigmented lesions in this syndrome is due to the presence of two cell populations and represents mosaicism. This hypothesis is supported by the findings of Schwindinger et al. (1992). They have demonstrated in these patients mosaicism in the gene that encodes for the alpha subunit of the stimulatory G protein. Interestingly they have found higher levels of mutant alleles in skin biopsies from pigmented than in the normal skin of affected patients (Schwindinger et al., 1992).

Prognosis Patients afflicted with this disorder usually have a normal life span; however, the orthopedic deformities may cause significant morbidity in some patients. Massive overgrowth of the skull and jaw can lead to neurologic damage, blindness, deafness, vestibular dysfunction, and death.

Treatment Bisphosphonates can be used to reduce bone pain and fractures and may slow progression of bone disease (de Sanctis et al., 1999). Surgical therapy can be attempted for patients with involvement of the skull or craniofacial bones (Becelli et al., 2002). Medications such as ketoconazole and testolactone can slow the progression of precocious puberty (de Sanctis et al., 1999). Laser treatment of the pigmented lesion results in temporary lightening but most of the pigment returns within a year.

References Albright, F., A. M. Butler, A. O. Hampton, and P. Smith. Syndrome characterized by osteitis fibrosa disseminata, areas of pigmentation, and endocrine dysfunction, with precocious puberty in females. N. Engl. J. Med. 216:727–746, 1937. Aldred, M. A., and R. C. Trembath. Activating and inactivating mutations in the human GNAS1 gene. Hum. Mutat. 16:183–189, 2000. Becelli, R., M. Perugini, G. Cerulli, A. Carboni, and G. Renzi. Surgical treatment of fibrous dysplasia of the cranio-maxillo-facial area. Minerva Stomatol. 51:293–300, 2002. D’Armiento, M., G. Reda, A. Camagna, and L. Tardella. McCuneAlbright syndrome evidence for autonomous multiendocrine hyperfunction. J. Pediatr. 102:584–586, 1983. de Sanctis, C., R. Lala, A. Balsamo, R. Bergamaschi, M. Cappa, M. Cisternino, V. de Sanctis, M. Lucci, A. Franzese, L. Ghizzoni, A. M. Pasquino, M. Segni, F. Rigon, G. Sagesse, S. Bertelloni, F. Buzi. McCune–Albright syndrome: a longitudinal clinical study of 32 patients. Pediatr. Endocrinol. Metab. 12:817–826, 1999. Happle, R. Lethal genes surviving by mosaicism: a possible explanation for sporadic birth defects involving the skin. J. Am. Acad. Dermatol. 16:899–906, 1987. Marie, P. J. Cellular and molecular basis of fibrous dysplasia. Histol. Histopathol. 16:981–989, 2001. McCune, D. J. Osteitis fibrosa cystica: the case of a nine year old girl who also exhibits precocious puberty, multiple pigmentation of the skin and hyperthyroidism. Am. J. Dis. Child. 52:743–744, 1936. Rieger, E., R. Kofler, M. Borkenstein, J. Schwingshandl, H. P. Soyer, and H. Kerl. Melanotic macules following Blaschko’s lines in

GENETIC EPIDERMAL SYNDROMES WITH CAFÉ-AU-LAIT MACULES McCune-Albright syndrome. Br. J. Dermatol. 130:215–220, 1994. Schwindinger, W. F., C. A. Francomano, and M. A. Levine. Identification of a mutation in the gene encoding the alpha subunit of the stimulatory G protein of adenylyl cyclase in McCune-Albright syndrome. Proc. Natl. Acad. Sci. U. S. A. 89:5152–5156, 1992. Weinstein, L. S., A. Shenker, P. V. Gejman, M. J. Merino, E. Friedman, and A. M. Spiegel. Activating mutations of the stimulatory G protein in the McCune-Albright syndrome. N. Engl. J. Med. 325:1688–1695, 1991.

Segmental Neurofibromatosis Nancy Burton Esterly and Eulalia Baselga

Historical Background This atypical form of neurofibromatosis, first called sectorial neurofibromatosis, was originally described by Crowe et al. in their landmark monograph published in 1956. Several other classification systems were devised subsequently, but the term “segmental neurofibromatosis” became the most widely accepted. This disorder was also known as neurofibromatosis 5 in a classification system devised by Riccardi (1982, 1984); however, use of this term has fallen from favor (Schultz et al., 2002).

Epidemiology Segmental neurofibromatosis is a relatively rare condition in which the cutaneous features of café-au-lait spots, intertriginous freckling, and neurofibromas occur in a segmental pattern. According to Riccardi’s criteria this syndrome is not familial and is purely cutaneous in its manifestations (Riccardi, 1982). The true incidence is unknown but in a prospective series of infants and children with multiple caféau-lait spots (six spots greater than 5 mm in diameter) 15% were eventually assigned this diagnosis (Korf, 1992). Epidemiologic data reportedly show a higher incidence in whites and in females (2:1) with two frequency peaks between 10 to 30 years and 50 to 70 years of age (Conejo-Mir et al., 1989).

Clinical Findings The typical patient has café-au-lait spots, freckling, and/or cutaneous neurofibromas limited to one segment of the body (Fig. 45.9), usually in a unilateral distribution respecting the midline (Riccardi, 1982; Ruggieri, 2001). Rarely multiple segments may be involved or the changes may be bilateral (Cecchi et al., 1992; Crowe et al., 1956; Moss and Green, 1994; Trattner et al., 1990). Some patients have neurofibromas in the deeper tissues. Patients with pigmentary lesions only (Menni et al., 1994; Micali et al., 1993) and neurofibromas only (Cecchi et al., 1992; Conejo-Mir et al., 1989; Oranje et al., 1985; Roth et al., 1987; Trattner et al., 1990) have also been reported under this rubric. A few patients have also had Lisch nodules ipsilateral to the tumors and pigmented lesions (Moss and Green, 1994; Weleber and Zonana, 1983). Roth et al. pointed out that not all case reports have adhered to the classification criteria established by Riccardi for neu-

Fig. 45.9. Café-au-lait spots localized to one segment of the integument (see also Plate 45.9, pp. 494–495).

rofibromatosis 5 (Riccardi, 1982; Roth et al., 1987) and suggested division of these patients into four subsets: (1) unilateral segmental neurofibromas with or without café-au-lait spots and freckling, no systemic involvement, nonfamilial; (2) unilateral segmental cutaneous lesions, deep involvement, nonfamilial; (3) unilateral segmental, no deep involvement, familial (Rubenstein et al., 1983); and (4) bilateral segmental cutaneous lesions, no deep involvement, nonfamilial (Roth et al., 1987). The café-au-lait spots may have their onset at birth and usually increase in number and size over the next several years.

Histology There is very little information regarding biopsy findings in the pigmented lesions. Menni et al. took biopsies from a 10year-old girl with pigmented macules only. Histologic examination revealed a considerable amount of melanin pigment in the epidermal basal layer and normal numbers of melanocytes. On electron microscopy, numerous subepidermal nerves were observed below the dermoepidermal junction along with intraepidermal macromelanosomes (Menni et al., 1994). An increased number of nerves at the dermoepidermal junction has been described as a characteristic feature of the café-aulait macules in neurofibromatosis 1 (Mihara et al., 1992). Electron microscopy of a café-au-lait spot from a 16-year-old girl with neurofibromatosis 5 showed melanosome complexes in the basal keratinocytes. The melanocytes appeared normal with melanosomes in various stages of melanization (Micali et al., 1993).

Laboratory Investigations There are no diagnostic laboratory studies. 819

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Diagnosis The patient should be examined completely to exclude other disorders characterized by café-au-lait spots and freckling (Hager et al., 1997). Confusion may also arise with cases of unilateral or segmental lentiginosis or speckled lentiginous nevus, both distinguishable from café-au-lait spots by histologic evaluation (Moss and Green, 1994; Selvaag et al., 1994; Trattner and Hodak, 1994).

Pathogenesis Segmental neurofibromatosis is believed to be the result of a postzygotic somatic mutation in primitive neural crest cells (Riccardi, 1981, 1984). This would cause genomic mosaicism for the neurofibromatosis 1 gene mutation reflected by a limited expression of the 1 phenotype and resulting in a nontransmissible condition, as are most cases of neurofibromatosis 5. However, were somatic mosaicism to be accompanied by gonadal mosaicism, the offspring of such an individual would be at risk, albeit small, for the development of neurofibromatosis 1 (Boltshauser et al., 1989; Moss and Green, 1994; Riccardi and Lewis, 1988). Rarely, both parent and child have appeared to have neurofibromatosis 5 (Rubenstein et al., 1983; Sloan et al., 1990; Tinehert et al., 2000), possibly due to a heritable predisposition to somatic mutation at the neurofibromatosis 1 locus (Moss and Green, 1994).

Prognosis and Treatment Because the outcome is unclear for an individual with localized lesions, periodic follow-up is imperative in patients with segmental neurofibromatosis. Caution should also be exercised in administering genetic counseling, which should be deferred until after puberty when the absence of Lisch nodules and internal neurofibromas is of greater prognostic significance. The significance of unilateral Lisch nodules is unknown. It has been speculated that those patients are at the same risk for having offspring with localized or generalized neurofibromatosis as are the patients with segmental neurofibromatosis and no Lisch nodules (Sloan et al., 1990). The prognosis is generally excellent as these individuals are not at risk for the other complications of neurofibromatosis.

References Boltshauser, E., H. Stocker, and M. Machler. Neurofibromatosis 1 in a child of a parent with segmental neurofibromatosis (-5). Neurofibromatosis 2:244–245, 1989. Cecchi, R., A. Giomi, F. Tuci, L. Brunetti, and G. Seghieri. Bilateral segmental neurofibromatosis. Dermatology 185:59–61, 1992. Conejo-Mir, J. S., A. H. Saval, and F. C. Martinez. Segmental neurofibromatosis. J. Am. Acad. Dermatol. 20:681–682, 1989. Crowe, F. W., W. J. Schull, and J. V. Neel. A Clinical, Pathological and Genetic Study of Multiple Neurofibromatosis. Springfield, IL: Charles C. Thomas, 1956, pp. 3–81. Hager, C. M., P. R. Cohen, and J. A. Tschen. Segmental neurofibromatosis: case reports and review. J. Am. Acad. Dermatol. 37:864–869, 1997.

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Korf, B. R. Diagnostic outcome in children with multiple cafe au lait spots. Pediatrics 90:924–927, 1992. Menni, S., S. Cavicchini, A. Brezzi, and R. Piccinno. A case of segmental macular neurofibromatosis. Acta Dermatol. Venereol. 74:329, 1994. Micali, G., D. Lembo, S. Giustini, and S. Calvieri. Segmental neurofibromatosis with only macular lesions. Pediatr. Dermatol. 10:43–45, 1993. Mihara, M., H. Nakayama, T. Aki, T. Inoue, and S. Shimao. Cutaneous nerves in cafe au lait spots with halos in infants with neurofibromatosis. Arch. Dermatol. 128:957–961, 1992. Moss, C., and S. H. Green. What is segmental neurofibromatosis? Br. J. Dermatol. 130:106–110, 1994. Oranje, A. P., V. D. Vuzevski, T. J. Kalis, W. F. M. Arts, T. Van Joost, and E. Stolz. Segmental neurofibromatosis. Br. J. Dermatol. 112:107–112, 1985. Riccardi, V. M. Von Recklinghausen neurofibromatosis. N. Engl. J. Med. 305:1617–1626, 1981. Riccardi, V. M. Neurofibromatosis: clinical heterogeneity. Curr. Probl. Cancer 7:1–34, 1982. Riccardi, V. M. Neurofibromatosis heterogeneity. J. Am. Acad. Dermatol. 10:518–519, 1984. Riccardi, V. M., and R. A. Lewis. Penetrance of von Recklinghausen neurofibromatosis: a distinction between predecessors and descendants. Am. J. Hum. Genet. 42:284–289, 1988. Roth, M. R., R. Martinez, and W. D. James. Segmental neurofibromatosis. Arch. Dermatol. 123:917–920, 1987. Rubenstein, A. E., J. L. Bader, and A. A. Aron. Familial transmission of segmental neurofibromatosis. Neurology 33 (Suppl 2):76, 1983. Ruggieri, M. Mosaic (segmental) neurofibromatosis type I (NF1) and type 2 (NF2); No longer neurofibromatosis type 5 (NF5). Am. J. Med. Genet. 101:178–180, 2001. Schultz, E. S., D. Kaufman, S. Tinschert, H. Schell, P. von den Driesch, G. Schuler. Segmental neurofibromatosis. Dermatology 204:296–297, 2002. Selvaag, E., P. Thune, and T. E. Larsen. Segmental neurofibromatosis presenting as a giant naevus spilus. Acta Derm. Venereol. 74:327, 1994. Sloan, J. B., D. F. Fretzin, and D. A. Bovenmyer. Genetic counseling in segmental neurofibromatosis. J. Am. Acad. Dermatol. 22:461–467, 1990. Tinschert, S., I. Naumann, E. Stegmann, A. Buske, D. Kaufman, G. Thiel, and D. Jenne. Segmental neurofibromatosis is caused by somatic mutation of the neurofibromatosis type I (NF1) gene. Eur. J. Hum. Genet. 8:455–459, 2000. Trattner, A., and E. Hodak. Segmental neurofibromatosis clinically appearing as a nevus spilus. Int. J. Dermatol. 33:75, 1994. Trattner, A., M. David, E. Hodak, E. Ben-David, and M. Sandbank. Segmental neurofibromatosis. J. Am. Acad. Dermatol. 23:866–869, 1990. Weleber, R. G., and J. Zonana. Iris hamartomas (Lisch nodules) in a case of segmental neurofibromatosis. Am. J. Ophthalmol. 96:740–743, 1983.

Silver–Russell Syndrome Nancy Burton Esterly, Eulalia Baselga, and Sheila S. Galbraith

Historical Background In 1953, Silver et al. described two patients with body asymmetry, dwarfism, elevated urinary gonadotropins, and several other peculiar abnormalities (Silver et al., 1953). A year later, Russell (1954) described five children with short stature and similar facies, two of whom also had hemiatrophy. Subse-

GENETIC EPIDERMAL SYNDROMES WITH CAFÉ-AU-LAIT MACULES

quently, Black and Rimoin suggested that these represented the same clinical entity and it should be designated Silver–Russell syndrome (Black, 1961; Rimoin, 1969). Attempts to separate the Silver from the Russell syndrome (Gareis et al., 1971), depending on whether asymmetry is present or absent, generally have not been accepted (Robichaux et al., 1981; Tanner et al., 1975; Vestermark, 1970). In 1986, Partington described an X-linked variant of Silver–Russell syndrome with extensive pigmentary anomalies (Partington, 1986).

Epidemiology and Genetics Silver–Russell syndrome affects both sexes equally and all ethnic groups. Most cases are sporadic, although familial occurrences has been reported (Callahan, 1970; Duncan et al., 1990; Escobar et al., 1978; Fuleihan et al., 1971; Gareis et al., 1971; Gray and Evans, 1959; Partington, 1986; Rimoin, 1969; Robichaux et al., 1981; Silver, 1964; Silver and Gruskay, 1957; Taussig et al., 1973; Warkany et al., 1961). Autosomal dominant, autosomal recessive, and X-linked inheritance have been documented in different families; moreover, the disorder is extremely variable and is regarded as heterogeneous both clinically and genetically (Callahan, 1970; Duncan et al., 1990; Escobar et al., 1978; Fuleihan et al., 1971; Gray and Evans, 1959; Partington, 1986; Rimoin, 1969; Robichaux et al., 1981; Samm et al., 1990; Silver, 1964).

hypospadias, and small testes and penis (Angehrn et al., 1979; Weiss and Garnick, 1981). Café-au-lait macules are present from up to 27% (Escobar et al., 1978) to 45% of the cases (Silver, 1964). They usually number only one or two with a few patients having more than six (Silver, 1964). The size of the pigmented macules varies from less than 1 cm to 4 cm in diameter. The borders may be smooth or jagged. Occasionally, areas of darker pigmentation overlay the café-au-lait macules (Silver, 1964). In the kindred described in 1985 by Partington, the pigmentary abnormalities were so extensive and singular that the clinical picture he described is considered a variant of Silver–Russell syndrome (Partington, 1986). The affected members of this family developed after 1 year of age a large number of café-au-lait macules (more than 50) in a period of months as well as larger poorly demarcated areas of brown pigmentation and small achromic macules. The hyperpigmented areas extended progressively to cover most of the trunk and limbs but spared the face. The achromic macules varied in size from less than 1 cm to more than 2 cm. Sometimes they were seen superimposed on the areas of diffuse pigmentation.

Histology There are no reports describing the histopathologic findings in the café-au-lait macules, areas of diffuse pigmentation or in the achromic lesions.

Clinical Findings

Laboratory Investigations

There is considerable variability in the clinical expression of this syndrome (Angehrn et al., 1979; Donnai et al., 1989; Escobar et al., 1978; Patton, 1988; Silver, 1964; Vestermark, 1970). The two main features are low birthweight dwarfism and body asymmetry. Other features of the syndrome include a small triangular face, turned down corners of the mouth, short and/or incurved fifth fingers, syndactyly, and other abnormalities of the toes. Affected individuals have been noted to have café-au-lait macules, variation in the pattern of sexual development, precocious puberty, premature estrogenization of the urethral or vaginal mucosa, and retarded bone age in relation to the sexual development. Affected children are small for dates at birth. The length and weight of patients with this disorder are below the third percentile but the growth curve runs parallel to the normal curve (Tanner et al., 1975). The bone age is usually retarded but, until puberty, matches the retardation in growth (Tanner et al., 1975). The head circumference is proportionally less reduced than the height and, therefore, affected individuals have relative macrocephaly. The body asymmetry may not be noticed at birth and it may involve an entire side of the body (hemihypertrophy) or be limited to the extremities, face, or spine. Motor development is usually delayed but mental development is normal in most cases. Early puberty has been described and, although urinary gonadotropins were elevated in patients from the original report, no consistent abnormality has been found thereafter. Additional genital abnormalities include cryptorchidism,

Results from laboratory tests of individuals affected by the Silver–Russell syndrome are usually normal. In the original report, urinary gonadotropin levels were elevated but no consistent abnormality has been found thereafter (Curi et al., 1967; Sami, 1969; Tanner et al., 1975; Vestermark, 1970). Growth hormone levels are usually normal although there are a few reported cases of coexistent growth hormone deficiency (Eeckels et al., 1970; Hall, 1978; Nishi et al., 1982; O’Brien et al., 1978; Saal et al., 1985). Bone X-rays demonstrate retarded development of ossification centers. Hand radiographs may show, between the ages of 2 and 10 years, a constellation of findings suggestive of Silver–Russell syndrome which includes delayed maturation, clinodactyly, and fifth, middle, or distal phalangeal hypoplasia, ivory epiphysis, and a second metacarpal pseudoepiphysis (Herman et al., 1987; Moseley et al., 1966).

Pathogenesis The growth deficiency in Silver–Russell syndrome does not appear to be due to a deficiency of growth hormone or growth factors. In these individuals, abnormalities have been detected on chromosomes 7, 11, 15, 17, and 18 supporting the belief that the syndrome is heterogeneous (Preece, 2002). In addition, approximately 10% of Silver–Russell syndrome patients have been found to have uniparental disomy in which the involved chromosome is always of maternal derivation. This association, manifest by the presence of a maternally 821

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imprinted gene on chromosome 7, suggests that the syndrome may be due to a double dose of maternal genetic material rather than a deficiency of paternal expressed genes (Polychronakos and Kukuvitis, 2002; Wakeling et al., 1998).

Differential Diagnosis The differential diagnosis of Silver–Russell syndrome includes other cases of low birthweight dwarfism, particularly the autosomal recessive 3M syndrome, which also displays clinodactyly and a narrow facies but characteristically presents with tall vertebral bodies, prominent heels, and lacks other features of Silver–Russell syndrome. The phenotype of Silver–Russell has been reported in patients with mosaic trisomy 18, a diploid–triploid mosaicism and a 45,X/46,XY mosaicism (Patton, 1988). Neonatal progeroid syndrome of Wiedemann–Rautenstrauch shares with Silver–Russell syndrome the relatively large head and triangular facial shape, but natal teeth, large hands, and significant development delay are present in the neonatal progeroid syndrome (Donnai et al., 1989). Neurofibromatosis may be considered, although in Silver–Russell syndrome, the number of café-au-lait macules is usually limited, and the other associated typical findings are lacking. Children with McCune–Albright syndrome may have café-au-lait macules, sexual precocity, and some degree of asymmetry; however, they also have characteristic bony changes.

Treatment and Prognosis The prognosis is usually good. The body asymmetry and short stature tend to improve with age. In one study, a third of the patients achieved normal height (ranging from the 5th to the 40th percentile for age and sex). Treatment with growth hormone does not correct the growth pattern (Tanner et al., 1975; Vestermark, 1970).

References Angehrn, V., M. Zachmann, and A. Prader. Silver-Russell syndrome; observations in 20 patients. Helv. Paediatr. Acta 34:297–308, 1979. Black, J. Low birth weight dwarfism. Arch. Dis. Child. 36:633–644, 1961. Callahan, K. A. Asymmetrical dwarfism, or Silver’s syndrome, in two siblings. Med. J. Aust. 2:789–792, 1970. Curi, J. F., R. C. Vanucci, H. Grossmann, and M. New. Elevated serum gonadotropins in Silver’s syndrome. Am. J. Dis. Child. 114:658– 661, 1967. Donnai, D., E. Thompson, J. Allanson, and M. Baraitser. Severe SilverRussell syndrome. J. Med. Genet. 26:447–451, 1989. Duncan, P. A., J. G. Hall, L. R. Shapiro, and B. K. Vibert. Threegeneration dominant transmission of the Silver-Russell syndrome. Am. J. Med. Genet. 35:245–250, 1990. Eeckels, R., M. van der Schueren-Lodeweyckx, and R. Wolter. Plasma growth hormone determination in Silver-Russell syndrome. Helv. Paediatr. Acta 25:363–370, 1970. Escobar, V., S. Gleiser, and D. D. Weaver. Phenotypic and genetic analysis of the Silver-Russell syndrome. Clin. Genet. 13:278–288, 1978. Fuleihan, D. S., V. M. Der Kaloustian, and S. S. Najjar. The RussellSilver syndrome: report of three siblings. J. Pediatr. 78:654–657, 1971.

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Gareis, F. J., D. W. Smith, and R. L. Summit. The Russell-Silver syndrome without asymmetry. J. Pediatr. 79:775–781, 1971. Gray, O. P., and P. R. Evans. Dwarfism? Cockayne? Russell type. Proc. R. Soc. Med. 52:304–305, 1959. Hall, J. G. Microphallus, growth hormone deficiency and hypoglycemia in Russell-Silver syndrome. Am. J. Dis. Child. 132:1149, 1978. Herman, T. E., J. D. Crawford, R. H. Cleveland, and D. C. Kushner. Hand radiographs in Russell-Silver syndrome. Pediatrics 79:743–744, 1987. Moseley, J. E., R. E. Moloshok, and R. H. Freiberger. The Silver syndrome: congenital asymmetry, short stature and variations in sexual development: roentgen features. Am. J. Roentgenol. 97:74–81, 1966. Nishi, Y., Y. Nakanishi, S. Kawagushi, and T. Usui. Silver-Russell syndrome and growth hormone deficiency. Acta Paediatr. Scand. 71:1035–1036, 1982. O’Brien, J. E., A. Sadeghi-Nejad, and M. Feingold. Growth hormone deficiency in a patient with Silver-Russell syndrome. J. Pediatr. 93:152–153, 1978. Partington, M. W. X-linked short stature with skin pigmentation: evidence for heterogeneity of the Russell-Silver syndrome. Clin. Genet. 29:151–156, 1986. Patton, M. A. Russell-Silver syndrome. J. Med. Genet. 25:557–560, 1988. Preece, M. The genetics of the Silver-Russell syndrome. Rev. Endocrin. Metab. Dis. 3:369–379, 2002. Polychronakos, C. and Kukuvitis, A. Parental genomic imprinting in endocrinopathies. E. J. Endocrin. 147:561–569, 2002. Rimoin, D. C. The Silver syndrome in twins. Birth Defects Orig. Art. Ser. 5:183–186, 1969. Robichaux, V., A. Fraikor, B. Favara, and M. Richer. Silver-Russell syndrome: a family with symmetric and asymmetric siblings. Arch. Pathol. Lab. Med. 105:157–159, 1981. Russell, A. A syndrome of “intra-uterine” dwarfism recognizable at birth with cranio-facial dysostosis, disproportionately short arms, and other anomalies (5 examples). Proc. R. Soc. Med. 47:1040–1044, 1954. Saal, H. M., R. A. Pagon, and M. G. Pipin. Reevaluation of RussellSilver syndrome. J. Pediatr. 107:733–737, 1985. Sami, D. Two examples of Silver’s syndrome. Clin. Pediatr. (Phila.) 8:602–603, 1969. Samn, M., K. Lewis, and B. Blumberg. Monozygotic twins discordant for the Russell-Silver syndrome. Am. J. Med. Genet. 37:543–545, 1990. Silver, H. K. Asymmetry, short stature and variations in sexual development. Am. J. Dis. Child. 107:495–515, 1964. Silver, H. K., and F. L. Gruskay. Syndrome of congenital and hemihypertrophy and elevated urinary gonadotropins. Am. J. Dis. Child. 93:559–562, 1957. Silver, H. K., W. Kiyasu, J. George, and W. C. Deamer. Syndrome of congenital hemihypertrophy, shortness of stature and elevated urinary gonadotropins. Pediatrics 12:368–376, 1953. Tanner, J. M., H. Lejarraga, and N. Cameron. The natural history of the Silver-Russell syndrome: a longitudinal study of thirty-nine cases. Pediatr. Res. 9:611–623, 1975. Taussig, L. M., G. D. Braunstein, B. J. White, and R. L. Christiansen. Silver-Russell dwarfism and cystic fibrosis in a twin. Am. J. Dis. Child. 125:495–503, 1973. Vestermark, S. Silver’s syndrome. Acta Paediatr. Scand. 59:435–439, 1970. Wakeling, E. L., S. Abu-Amero, S. M. Price, P. Stanier, R. C. Trembath, G. E. Moore, and M. A. Preece. Genetics of SilverRussell syndrome. Hormone Research 2:32–36, 1998. Warkany, J., B. B. Munroe, and B. S. Sutherland. Intrauterine growth retardation. Am. J. Dis. Child. 102:249–279, 1961. Weiss, G. R., and M. B. Garnick. Testicular cancer in a Russell–Silver dwarf. J. Urol. 126:836–837, 1981.

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Watson Syndrome Nancy Burton Esterly First described in 1967 by Watson, this rare autosomal dominant syndrome consists of multiple café-au-lait spots, intertriginous freckling, mental deficiency, short stature, and pulmonary stenosis. Lisch nodules have also been documented in these patients (Riccardi, 1991). Thus, the clinical features overlap both neurofibromatosis 1 and Noonan syndrome (see above). Linkage analysis suggests that this disorder is an allelic variant of neurofibromatosis 1 (Tassabehji et al., 1993; Upadhyaya et al., 1990).

References Riccardi, V. M. Neurofibromatosis: Past, present, and future. N. Engl. J. Med. 324:1283–1284, 1991. Tassabehji, M., T. Strachan, M. Sharland, A. Colley, D. Donnai, R. Harris, and N. Thakker. Tandem duplication within a neurofibromatosis type 1 (NF1) gene exon in a family with features of Watson syndrome and Noonan syndrome. Am. J. Hum. Genet. 53:90–95, 1993. Upadhyaya, M., M. Sarfarazi, and S. Huson. Linkage of Watson’s syndrome to chromosome 17 markers. J. Med. Genet. 27:209, 1990. Watson, G. H. Pulmonary stenosis, cafe-au-lait spots, and dull intelligence. Arch. Dis. Child. 42:303–307, 1967.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Genetic Epidermal Pigmentation with Lentigines Sections Lentigo Simplex Mary K. Cullen Lentigo Senilis et Actinicus Mary K. Cullen Centrofacial Lentiginosis Mary K. Cullen LEOPARD Syndrome Mary K. Cullen Carney Complex Mary K. Cullen Other Lentiginoses Mary K. Cullen

Lentigo Simplex

lentigo senilis et actinicus, will be discussed immediately after the lentigo simplex section.

Mary K. Cullen

Historical Background Introduction The word lentigo (plural lentigines) was derived from the Latin lens to describe a lentil-shaped pigmented macule. Lentigines arise from melanocytic hyperpigmentation that is secondary to increased numbers of active basilar melanocytes, and from melanotic hyperpigmentation due to increased melanin production.

Lentiginoses Syndromes with multiple lentigines in a characteristic, nonrandom pattern are known as lentiginoses. In 1912, 1926, and 1929, Hammer, Decking, and Siemens, respectively, discussed heredity of the lentiginoses (Decking, 1926; Hammer, 1912; Siemens, 1929). Rarely, generalized lentigines may occur as an isolated phenomenon, without familial aggregation, appearing at birth, early infancy, or during adulthood (Kaufman and Ryan, 1976; Uhle and Norvell, 1988). In general each lentiginosis has a defined set of associated somatic abnormalities. Nitelea et al. noted that an almost constant feature of the lentiginoses is an association with developmental, and occasionally neuropsychiatric, anomalies. They further concluded that lentigines can be considered the cutaneous signature of polydysplastic, often hereditary syndromes (Nitelea and Dociu, 1974). Previous reviews by Cockayne (1933), Meirowsky (1933, 1942), and Brown (1943) contained no mention of any anomalies associated with the lentiginoses. The following lentiginoses are discussed in subsequent sections of this chapter: centrofacial neurodysraphic, LEOPARD syndrome, Carney complex/myxoma syndrome, and other less well-known lentiginoses. A particular induced type of lentigo, 824

The clinical distinction between lentigines and ephelides (freckles) can be difficult. Because of this, reports of lentigines without histologic support may or may not be describing lentigines. As early as 1924 Siemens described lentigines as different from ephelides by lack of influence of sunlight on their genesis (Siemens, 1924, 1926, 1929). The lentigo was at one time considered to be a nonelevated nevus (Darier, 1909; Kissmeyer, 1927; Scholtz, 1932). This grouping was challenged by Zeisler and Becker in 1936, and later Lund in 1960, who defined lentigo as an entity separate and distinct from nevomelanocytic nevus. In 1954, Ormsby and colleagues set out histologic criteria for distinguishing lentigines from freckles, the key finding being the melanocytic hyperplasia present in lentigines but not ephelides (Ormsby and Montgomery, 1954). In ephelides there is increased melanin but the number of melanocytes corresponds to that of the surrounding skin (Gorlin et al., 1969).

Epidemiology A large clinical survey by Alper and Holmes (1983) noted multiple lentigines present in 18.5% of 492 black newborns and 0.04% of 2682 white newborns. Pigmented lesions were found in 51% of 100 black newborns and 81% of those lesions examined microscopically were consistent with lentigo (Fox et al., 1979). Similar random studies in adults surveying palms, soles, and genitalia are available without histologic conformation of lentigo simplex (Allyn et al., 1963; Cullen, 1962). However, statistics are available from lesions excised for other purposes. Of 100 pigmented lesions excised in children ages 1–10 years, 14% were classified as lentigo (Stegmaier and Montgomery, 1953).

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

The most common histologic pattern of darkly pigmented lesions excised from acral sites of darkly pigmented races is lentigo simplex (Coleman et al., 1980b; Freinkel and Rippey, 1976). Freinkel studied 70 black adults in South Africa and noted 57% showed some pigmentation of the feet, but only 6% had circumscribed dense black spots. None of the spots examined histologically were nevi, but did largely fulfill the criteria for lentigines (Freinkel and Rippey, 1976). Darkskinned individuals are more likely to develop palmar and plantar (Coleman et al., 1980b), and nail bed (Goldberg, 1986) lentigines. Clinically, atypical plantar lentigo simplex has been reported (McCarthy, 1987), and 159 pigmented spots, consisting of 110 on the soles as well as 49 on the dorsal foot were studied clinically and histopathologically: (1) junctional nevi were most commonly observed at both sites and (2) lesions tended to be darker in color and bigger in size as they proceeded from the simple lentigo to the junctional and compound nevi (Hattori, 1990). Pigmented bands of the nail bed may have histologic features of lentigo simplex. They are rare in white people (Allyn et al., 1963), present in up to 20% of Japanese (Rhodes, 1993), and present in equal or higher frequencies in more darkly pigmented races (Higashi, 1968; Kato et al., 1989). (See also Chapter 55.) Aside from various lentiginoses or syndromes associated with lentigines, there is no known genetic predisposition for lentigo simplex (Rhodes, 1993).

Clinical Findings Lentigines can occur as isolated lesions of the skin, nails, and mucous membranes, or as multiple lesions associated with the lentiginoses. The perioral and periocular pigmented macules of Peutz–Touraine–Jeghers syndrome are lentigines (BanseKupin and Douglass, 1986; Jeghers et al., 1949; Utsunomiya et al., 1975; Yamada et al., 1981). They are generally present at birth, but may first appear early in life increasing in number until puberty (Zeisler and Becker, 1936). Freckles tend to first appear at 6–8 years of age, later than most lentigines (Gorlin et al., 1969). When a lentigo does appear during the first decade of life, it is known as the juvenile variety of lentigo simplex (Rhodes, 1993). Lentigo simplex is usually a sharply circumscribed, light brown to dark brown, variegated to uniformly colored, large or small macule (Rhodes, 1993) (Fig. 46.1). The juvenile form is usually less than 5 mm in diameter. Wood’s lamp examination is useful in defining margins of lentigo simplex. Lentigines often increase in number and darkness with Addison disease, pregnancy, and other conditions involving increased melanocyte stimulating hormone (Schweizer-Cagianut et al., 1982; Shenoy et al., 1984). Mucosal lentigines have been described on oral labia (Shapiro and Zegarelli, 1971), in the oral cavity (Buchner et al., 1991), and on the uterine cervix (Schneider et al., 1981). Isolated lentigo simplex on mucocutaneous sites may be influenced by genetic and/or racial factors (Rhodes, 1993). A particular type of mucosal lentigo demonstrating involvement of

Fig. 46.1. Sharply circumscribed dark brown macule typical of lentigo simplex (see also Plate 46.1, pp. 494–495).

the dermal–epidermal junction is known as a “jentigo” (Buchner et al., 1991; Marchesi et al., 1992). See also the section on Other lentiginoses later in this chapter. Genital lentigines usually occur as an isolated finding of uncertain significance (see Chapter 56, p. 1081). Vulvar and penile pigmented lesions may have histologic features of lentigo simplex (Leicht et al., 1988; Rock et al., 1990; Rudolph, 1990), and unique varieties of lentigo simplex on genitalia have been called genital lentiginosis (Barnhill et al., 1990). Barnhill et al. (1990) studied the clinical and histopathologic characteristics of melanotic macules of the penis and vulva. Clinically the lesions were regarded as atypical in appearance. There was no evidence of cytologic atypia and histologic criteria for lentigo were met. Vulvar lentigines have been reviewed by Hood and Lumadue (1992) and Kanj et al. (1992). Milde and colleagues (1992) described a single pigmented vulvar macule clinically consistent with a lentigo, however histologic examination revealed a lesion of Dowling–Degos disease. They suggested a differential diagnosis including vulvar melanosis, genital lentigo, malignant melanoma, acanthosis nigricans maligna and benigna, and syndromes occurring concomitantly with pigmentation of the mucosa. Penile lentigo was reviewed by Kopf and Bart (1982) in a discussion of balanopreputial pigmentation. Atypical penile lentigines (Bhawan and Cahn, 1984) and early malignant melanoma of glans and male urethra are rare and may be clinically indistinguishable from penile lentigo (Primus et al., 1990). See also the section on Other lentiginoses and Chapter 56. Melanonychia striata longitudinalis (see Chapter 55), pigmented nail streak, is due to a focal increase in number and/or activity of melanocytes in the nail matrix, resulting in the continuous production of large amounts of melanin which are transferred to the nail cells and grow out with the nail plate. Histologically, the streaks are usually lentigo simplex, benign melanocytic hyperplasia, or nevocellular nevus. However, atypical melanocytic hyperplasia and acral lentiginous melanoma do occur. These bands are common in darkly pigmented peoples but sufficiently uncommon in Caucasians as 825

CHAPTER 46

to be highly suggestive of a malignant melanoma (Baran and Haneke, 1984; Goldberg, 1986). In 1986 Goldberg reported a white patient with multiple pigmented streaks of the nail, one lesion was biopsied and was consistent with a lentigo (Goldberg, 1986). See also Chapter 55. Another variety of lentigo simplex may occur in scars following excision of melanoma (Jackson, 1979) and giant congenital nevomelanocytic nevi (Rhodes, unpublished observation).

Routine Histology and Histochemistry Lever and Shaumberg-Lever (1983) described the lentigo simplex as a proliferation of rete pegs and solitary nevus cells that are characteristically scattered within the stratum basale. While it is not clear whether the pigment cells are nevocytes or melanocytes, they will be referred to as melanocytes in these chapters, consistent with current usage. Microscopically, lentigines are characterized by both a greater number of melanocytes and a greater amount of melanin per unit skin area than is observed in nonlentiginous skin from the same subject. The basal layer melanocytic hyperplasia seen in lentigo simplex is without the nest formation typical of nevi (Ruiz-Maldonado et al., 1983). Epidermal keratinocyte hyperplasia consisting of elongated, club-shaped, and often fused rete ridges penetrating deeply into the dermis, is also a component of lentigo simplex (Rhodes, 1993). Rowe (1966) compared the volume of epidermis and dermal papillae in lentigo, psoriasis, and actinic keratoses. In all five cases they found the increase in epidermal volume paralleled the volume change in the dermal papillae. In childhood some flat pigmented lesions show transitions from lentigo to junctional nevus but do not appear to progress with time and are distinct from junctional and compound nevi (MacKie, 1992). Giant pigment granules (melanin macroglobules, macromelanosomes) may occur in lentigo simplex (Hirone and Eryu, 1978), as well as the lentigines of LEOPARD syndrome (Weiss and Zelickson, 1977), in solar lentigines (Rhodes et al., 1991), and in many other pigmented lesions such as the café-au-lait spots. Some lentigines on acral (palm, soles, nail bed) and mucosal sites may show melanocytic atypia (Bhawan and Cahn, 1984; Coleman et al., 1980b; Folberg et al., 1985; Kopf and Bart, 1982; Saida, 1989; Takagi et al., 1974). Ultrastructural studies of lentigo simplex have been discussed in depth by Fukushiro and Nagai (1967) and Hirone and Eryu (1978). An ultrastructural study of penile lentiginosis in five patients revealed more extensive alterations to melanocytes than previously reported and emphasized the fact that depigmentation is an essential element of the condition. In hyperpigmented areas, melanocytes were increased in number along the basal layer of the epithelium, were hyperactive, and in some cases contained bizarre melanosomes. In two cases there was suggestion of a defect in melanosome transfer to keratinocytes. Lymphocytes were closely opposed to melanocytes and, in hypopigmented areas, were clearly involved in their disintegration (Breathnach et al., 1992). Histologic examination of 826

the glans penis lentigo by Landthaler and colleagues (1989) revealed acanthosis, basal hyperpigmentation, and melanocytes without atypia. The most prominent feature by ultrastructural analysis was the presence of single melanosomes within the keratinocytes (Landthaler et al., 1989). In 1981 Hundeiker et al. described the “nevoid lentigo” lesion and discussed its histopathology.

Other Studies Marchesini and colleagues performed reflectance spectrophotometry on various cutaneous pigmented lesions including lentigo simplex. Their results suggested that detailed analysis of the reflectance spectrum may generate information that could ultimately be used as an aid in clinical diagnosis of cutaneous pigmented lesions (Marchesini et al., 1991). Stolz et al. described the distinctive pattern of benign lentigines when viewed with skin surface dermoscopy. Lentigines are sharply demarcated from adjacent skin and are characterized by a regular, symmetric pigment network, thickened webs, and occasional dilated holes. This pattern is due to the long and heavily pigmented rete ridges (Stolz et al., 1993). Asahina and colleagues compared the DNA ploidy patterns and histologic features of subungual lentigines and nevi. One subungual pigmented lesion that demonstrated a polyploid DNA pattern also contained abnormal melanocytes, possibly with the potential to undergo malignant transformation. They therefore concluded that DNA ploidy analysis can provide additional information that helps in the evaluation of subungual melanotic lesions (Asahina et al., 1993). Hastrup and Hou-Jensen (1993) compared the histologic features of 1000 melanocytic lesions, and found that slight nuclear atypia was usual in “common” nevi and lentigines. Giannini et al. morphometrically evaluated the nuclei of melanocytes in lentigines simplexes and junctional, compound, and intradermal acquired melanocytic nevi. They showed that as melanocytic lesions “evolved” from lentigo to dermal nevi the melanocytic nuclei become larger and rounder (Giannini et al., 1988). A histochemical study of a-D-mannosidase revealed that normal human melanocytes (resting state, activated, lentigo simplex) exhibit either no or just detectable activity, as do melanocytes in the initial phase of lentigo maligna. On the other hand, melanocytes in the initial stage of neoplastic transformation (dysplastic nevi, advanced stage of lentigo maligna) and also melanoma cells in disorders of low malignant potential (initial nevogenic melanoma, superficial spreading melanoma) displayed a high activity uniformly throughout the cell population (Elleder et al., 1986). Accumulation of Fe3+ and Mn2+ ions in melanins increases in the order: hair (black), retina and choroid (brown), malignant melanoma of eye and skin, and lentigo and nevus of skin (Okazaki et al., 1985).

Differential Diagnosis Lentigo simplex may appear clinically similar to pigmented actinic keratosis, lentigo senilis et actinicus (especially darkly pigmented varieties), junctional nevomelanocytic nevus, caféau-lait macule, pigmented seborrheic keratosis, freckles, and

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

lentigo maligna (Braun-Falco et al., 1986). The differential diagnosis of circumscribed palmar-plantar pigmented lesions includes lentigo simplex, nonspecific pigmentation, and nevomelanocytic nevus (Coleman et al., 1980a; Freinkel and Rippey, 1976). Patterned lentigines with associated somatic abnormalities are present in centrofacial lentiginosis, LEOPARD syndrome, myxoma syndrome (Carney complex, NAME, and LAMB), Peutz–Touraine–Jeghers syndrome, and cardiac pre-excitation syndrome. Melanonychia striata longitudinalis histologically, either benign melanocytic hyperplasia, lentigo simplex, nevocellular nevus, atypical melanocytic hyperplasia or acral lentiginous melanoma may be found (Baran and Haneke, 1984).

Pathogenesis The pathogenesis of these lesions is unknown. Melanin macroglobules in melanocytes and keratinocytes of lentigo simplex may be the result of aberrant melanogenesis and melanocyte dysfunction (Kaufman and Ryan, 1976). It is the belief of some authors that there may be a transition from lentigo simplex to nevomelanocytic nevus (Silverman et al., 1975; Stegmaier and Montgomery, 1953). However, the earliest acquired nevus biopsied in the first several years of life was histologically consistent with a nevomelanocytic nevus (Clemmensen and Kroon, 1988), suggesting that the nevus and lentigo simplex each have a separate histogenesis (Rhodes, 1993). Others have suggested that lentigo simplex precedes the lichen planus (LP)-like keratosis (Laur et al., 1981), an argument that is much stronger for the actinic lentigo. See lentigo senilis et actinicus section. The presence of lentigo simplex in association with the diverse somatic abnormalities occurring in Peutz–Touraine– Jeghers syndrome (Jeghers et al., 1949; Peutz, 1921; Touraine and Couder, 1945; Utsunomiya et al., 1975), LEOPARD syndrome (Capute et al., 1969; Gorlin et al., 1969; Nordlund et al., 1973), and Carney complex/myxoma syndrome (Carney et al., 1985; Rhodes et al., 1984) suggests a developmental neural crest disorder, significantly influenced by genetic factors (Rhodes, 1993). Information is insufficient at present to predict the natural history of genital lentiginosis or its relation to mucocutaneous melanoma (Barnhill et al., 1990). Autophagic degradation of melanosomes is the proposed mechanism for the formation of macromelanosomes in lentigo simplex and melanocytic nevi (Horikoshi et al., 1982).

Animal Models Nash and Paulsen described generalized lentigines on a silver cat with histologic confirmation of lentigo simplex. The lentigo lesions were similar to those described in orange, cream, red, and tricolored cats (Nash and Paulsen, 1990). These cats represent a possible animal model for lentigo simplex. Sherwood et al. used the skin of a miniature black pig to examine laser destruction of pigment cells (Sherwood et al., 1989).

Treatment In general no treatment is needed for a benign-appearing lentigo simplex. Darkly or irregularly pigmented varieties of lentigo simplex should be examined histologically for cellular atypia and varieties of lentigo simplex completely excised. Lukacs summarized the treatment and prognosis of lentigo in 1972. Using miniature black pig skin, Sherwood et al. (1989) found the most effective laser destruction of epidermal pigment cells occurred at a wavelength of 504 nm. Cosmetic improvement of lentigines by laser treatment has been most extensively examined for solar lentigo (see Chapter 64 and the section on lentigo senilis et actinicus). Wilder and Smith (1970) reported successful treatment of a benign facial lentigo with diamond abrasion. In 1993 Graham included lentigo in a review of advances in cryosurgical treatment during the previous decade. See also Chapters 59, 63 and 64. Penile lentigo was treated with azelaic acid in one patient, resulting in some improvement within four months (Landthaler et al., 1989). Treatment of streaked nail pigment was reviewed by Baran and Haneke (1984).

Course and Prognosis The natural history of a isolated lentigo simplex is not known. The hypothesis that lentigo simplex evolves into an ordinary nevomelanocytic nevus is without strong evidence for support. Conversely, atypical varieties of lentigo simplex may have a precursor role for some acral and mucosal melanomas (Coleman et al., 1980a; Collins, 1984; Folberg et al., 1985; Freinkel and Rippey, 1976; Lewis, 1967; Rhodes, 1982; Rippey and Rippey, 1977; Rudolph, 1990; Saida, 1989; Seiji and Takahashi, 1974; Takagi et al., 1974).

References Allyn, B., A. W. Kopf, M. Kahn, and V. H. S. Witten. Incidence of pigmented nevi. J. Am Med. Assoc. 186:890–893, 1963. Alper, J. C., and L. B. Holmes. The incidence and significance of birthmarks in a cohort of 4,641 newborns. Pediatr. Dermatol. 1:58–68, 1983. Asahina, A., H.-I. Chi, and F. Otsuka. Subungual pigmented nevus: evaluation of DNA ploidy in six cases. J. Dermatol. 20:466–472, 1993. Banse-Kupin, L. A., and M. C. Douglass. Localization of PeutzJeghers macules to psoriatic plaques. Arch. Dermatol. 122:679– 683, 1986. Baran, R., and E. Haneke. [Diagnosis and therapy of streaked nail pigmentation] [German] Diagnostik und Therapie der streifenformigen Nagelpigmentierung. Hautarzt 35:359–365, 1984. Barnhill, R. L., L. S. Albert, S. K. Shama, M. A. Goldenhersh, A. R. Rhodes, and A. J. Sober. Genital lentiginosis: a clinical and histopathologic study. J. Am. Acad. Dermatol. 22:453–460, 1990. Bhawan, J., and T. M. Cahn. Atypical penile lentigo. Case report. J. Dermatol. Surg. Oncol. 10:99–100, 1984. Braun-Falco, O., C. Schmoeckel, and C. Geyer. [Pigmented actinic keratoses] [German] Pigmentierte aktinische Keratosen. Hautarzt 37:676–678, 1986. Breathnach, A. S., L. Balus, and A. Amantea. Penile lentiginosis. An ultrastructural study. Pigment Cell Res. 5:404–413, 1992. Brown, E. E. Lentigenes: Their Possible Significance. Arch. Dermatol. Syphil. 47:804–815, 1943.

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lanosomes and autophagic giant melanosome complexes in nevocellular nevi, lentigo simplex and malignant melanoma. J. Cutan. Pathol. 9:329–339, 1982. Hundeiker, M. [Nevoid lentigo: Differential histological diagnosis] [German] Naevoide Lentigo: histologische differential diagnose. Pathologe 2:111–112, 1981. Jackson, R. Pigmented lesion following complete removal of melanoma. Arch. Dermatol. 115:1089–1090, 1979. Jeghers, H., V. A. McKusick, and K. A. Katz. Generalized intestinal polyposis and melanin spots of the oral mucosa, lips and digits: A syndrome of diagnostic significance. N. Engl. J. Med. 241:993– 1005,1031–1036, 1949. Kanj, L. F., N. G. Rubeiz, A. M. Mroueh, and A. G. Kibbi. Vulvar melanosis and lentiginosis: a case report. J. Am. Acad. Dermatol. 27(5 Pt 1):777–778, 1992. Kato, T., Y. Usuba, H. Takematsu, N. Kumasaka, Y. Tanita, K. Hashimoto, Y. Tomita, and H. Tagami. A rapidly growing pigmented nail streak resulting in diffuse melanosis of the nail: A possible sign of subungual melanoma in situ. Cancer 64:2191– 2197, 1989. Kaufman, M., and G. Ryan. Remission of the murine S91 melanoma, following treatment with tetracycline bound S91 melanoma tumor proteins. Cancer Cyto.:11–21, 1976. Kissmeyer, A. Etudes sur les nevi pigmentaires de la peau humaine. Paris: Legrand, éd., 1927. Kopf, A. W., and R. S. Bart. Tumor conference: 43:penile lentigo. J. Dermatol. Surg. Oncol. 8:637–639, 1982. Landthaler, M., W. Stolz, and O. Braun-Falco. [Lentigo of the glans penis] [German] Lentigo der Glans penis. Hautarzt 40:222–225, 1989. Laur, W. E., R. E. Posey, and J. D. Waller. Lichen planus-like keratosis: A clinicohistopathologic correlation. J. Am. Acad. Dermatol. 4:329–336, 1981. Leicht, S., G. Youngberg, and C. Diaz-Miranda. Atypical pigmented penile macules. Arch. Dermatol. 124:1267–1270, 1988. Lever, W. F., and G. Schaumburg-Lever. Histopathology of the Skin, 6th ed. Philadelphia: JB Lippincott Co., 1983. Lewis, M. G. Malignant melanoma in Uganda (The relationship between pigmentation and malignant melanoma on the soles of the feet). Br. J. Cancer 21:483–495, 1967. Lukacs, I. [Treatment and prognosis of lentigo] [German] Behandlung und prognose eines naevus naevocellularis. Munch. Med. Wochenschr. 114:409, 1972. Lund, H. Z. Pigmented Cell Neoplasms: Problems in the diagnosis of early lesions. Proceedings of the National Cancer Conference 4:619–622, 1960. MacKie, R. M. Melanocytic naevi and malignant melanoma. In: Rook/Wilkinson/Ebling Texbook of Dermatology, 5th ed., R. H. Champion, J. L. Burton, and F. J. G. Ebling (eds). Oxford: Blackwell Scientific Publications, 1992. Marchesi, L., L. Naldi, A. Di Landro, L. Cavalieri d’Oro, A. Brevi, and T. Cainelli. Segmental lentiginosis with jentigo histologic pattern. Am. J. Dermatopathol. 14:323–327, 1992. Marchesini, R., M. Brambilla, C. Clemente, M. Maniezzo, A. E. Sichirollo, A. Testori, D. R. Venturoli, and N. Cascinelli. In vivo spectrophotometric evaluation of neoplastic and non-neoplastic skin pigmented lesions. Reflectance measurements. Photochem. Photobiol. 53:77–84, 1991. McCarthy, D. J. Lentigo simplex. J. Am. Podiatr. Med. Assoc. 77:539–543, 1987. Meirowsky, E. Handbuch der Haut und Geschlechtskr. Jadassohn 4(2e parte):652–657, 1933. Meirowsky, E. Moles and malformations of the skin in relation to inheritance and phylogenesis. Br. J. Dermatol. 54:99–121,126–153, 1942. Milde, P., G. Goerz, and G. Plewig. [Dowling-Degos disease with exclusively genital manifestations]. Hautarzt 43:369–372, 1992.

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES Nash, S., and D. Paulsen. Generalized lentigines in a silver cat. J. Am. Vet. Med. Assoc. 196:1500–1501, 1990. Nitelea, O. G., and I. Dociu. Lentiginoses and anomalies of development. Case report of a family with lentiginosis associated with dysraphism and mental debility [Lentiginoses et anomalies de dévelopement, Observation familiale de lentiginose avec dysraphisme et débilité mentale, French]. Dermatologica 148:108–115, 1974. Nordlund, J. J., A. B. Lerner, I. M. Braverman, and J. McGuire. Multiple lentigines syndrome: A case study. Arch. Dermatol. 107:259–261, 1973. Okazaki, M., K. Kuwata, Y. Miki, S. Shiga, and T. Shiga. Electron spin relaxation of synthetic melanin and melanin-containing tissues studied by electron spin echo and electron spin resonance. Arch. Biochem. Biophys. 242:197–205, 1985. Ormsby, O. S., and K. Montgomery. Diseases of the Skin, 8th ed. Philadelphia: Lea and Febiger, 1954. Peutz, J. L. A. Very remarkable case of familial polyposis of mucous membrane of intestinal tract and nasopharynx accompanied by peculiar pigmentations of skin and mucous membrane. Nederl. Maatsch. N. F. 10:134–146, 1921. Primus, G., H. P. Soyer, J. Smolle, G. Mertl, K. Pummer, and H. Kerl. Early “invasive” malignant melanoma of the glans penis and the male urethra: Report of a case and review of the literature. Eur. Urol. 18:156–159, 1990. Rhodes, A. R. Acral lentiginous melanoma in situ: Discussion [review]. Plast. Reconstr. Surg. 70:617–619, 1982. Rhodes, A. R. Neoplasms: benign neoplasias, hyperplasias, and dysplasias of melanocytes. In: Dermatology in General Medicine, 4th ed., T. B. Fitzpatrick, A. Eisen, K. Wolff, I. M. Freedberg, and F. K. Austen (eds). New York: McGraw-Hill, Inc., 1993, pp. 996–1077. Rhodes, A. R., R. A. Silverman, T. J. Harrist, and A. R. Perez-Atayde. Mucocutaneous lentigines, cardiomucocutaneous myxomas, and multiple blue nevi: the “LAMB” syndrome. J. Am. Acad. Dermatol. 10:72–82, 1984. Rhodes, A. R., L. S. Albert, R. L. Barnhill, and M. A. Weinstock. Suninduced freckles in children and young adults. A correlation of clinical and histopathologic features. Cancer 67:1990–2001, 1991. Rippey, J. J., and E. Rippey. Malignant melanoma with adjacent Hutchinson’s melanotic freckle in Black Africans. Pathology 9:105–109, 1977. Rock, B., A. F. Hood, and J. A. Rock. Prospective study of vulvar nevi. J. Am. Acad. Dermatol. 22:104–106, 1990. Rowe, L. Volume of epidermis and dermal papillae in psoriasis, actinic keratoses, and lentigo. J. Invest. Dermatol. 46:374–377, 1966. Rudolph, R. I. Vulvar melanosis. J. Am. Acad. Dermatol. 23(5 Pt 2):982–984, 1990. Ruiz-Maldonado, R., L. Trevizo, L. Tamayo, M. F. de los Rios, M. Skurovich, J. Carrillo, D. Dominguez, and V. del Castillo. Progressive cardiomyopathic lentiginosis: report of six cases and one autopsy. Pediatr. Dermatol. 1:146–153, 1983. Saida, T. Malignant melanoma in situ on the sole of the foot: Its clinical and histopathologic characteristics. Am. J. Dermatopathol. 11:124–130, 1989. Schneider, V., S. T. Zimberg, and S. Kay. The pigmented portio: benign lentigo of the uterine cervix. Diagn. Gynecol. Obstet. 3:269–272, 1981. Scholtz, W. Handbuch der Haut und Geschlechtskr. Jadassohn 12(2e parte):543–549, 1932. Schweizer-Cagianut, M., F. Salomon, and C. E. Hedinger. Primary adrenocortical nodular dysplasia with Cushing’s syndrome and cardiac myxomas: A peculiar familial disease. Virchows Arch. A. Pathol. Anat. Histopathol. 397:183–192, 1982. Seiji, M., and M. Takahashi. Malignant melanoma with adjacent intraepidermal proliferation. Tohoku J. Exp. Med. 114:93–107, 1974.

Shapiro, L., and D. J. Zegarelli. The solitary labial lentigo: A clinicopathologic study of twenty cases. Oral Surg. Oral Med. Oral Pathol. 31:87–92, 1971. Shenoy, B. V., P. C. Carpenter, and J. A. Carney. Bilateral primary pigmented nodular adrenocortical disease. Rare cause of the Cushing’s syndrome. Am. J. Surg. Pathol. 8:335–344, 1984. Sherwood, K. A., S. Murray, A. K. Kurban, and O. T. Tan. Effect of wavelength on cutaneous pigment using pulsed irradiation. J. Invest. Dermatol. 92:717–720, 1989. Siemens, H. W. Zur kenntnis der epheliden mit bemerkungen uber haarbzeichung und haarfarbenbestimung. Arch. Dermatol. Syphil. 147:1–60(ephelides), 210–227(heredity of nevi), 1924. Siemens, H. W. Beiträge zur klinischen kenntris der lentigines. Arch. Dermatol. Syphil. 152:372–380, 1926. Siemens, H. W. Handbuch der Haut und Geschlechtskr. Jadassohn 3:139–141, 1929. Silverman, S., Jr., J. S. Greenspan, and T. M. Christie. Junctional nevus of the oral mucosa: Light and electron microscopic observations. Oral Surg. Oral Med. Oral Pathol. 39:259–267, 1975. Stegmaier, O. C., and H. Montgomery. Histopathologic studies of pigmented nevi in children. J. Invest. Dermatol. 20:51, 1953. Stolz, W., O. Braun-Falco, P. Bilek, M. Landthaler, and A. B. Cognetta. Lentigines. In: Color Atlas of Dermatoscopy. Oxford: Blackwell Scientific Publications, 1993. Takagi, M., G. Ishikawa, and W. Mori. Primary malignant melanoma of the oral cavity in Japan (with special reference to mucosal melanosis). Cancer 34:358–370, 1974. Touraine, A., and F. Couder. Periorificial lentiginosis and visceral polyposis [Lentiginose péri-orificielle et polypose viscerale, French]. Bull. Soc. Franc. Dermatol. Syphiligr. 5:313, 1945. Uhle, P., and S. S. Norvell Jr. Generalized lentiginosis. J. Am. Acad. Dermatol. 18(2 Pt 2):444–447, 1988. Utsunomiya, J., H. Gocho, T. Miyanaga, E. Hamaguchi, and A. Kashimure. Peutz-Jeghers syndrome: its natural course and management. Johns Hopkins Med. J. 136:71–82, 1975. Weiss, L. W., and A. S. Zelickson. Giant melanosomes in multiple lentigines syndrome. Arch. Dermatol. 113:491–494, 1977. Wilder, L. W., and B. Smith. Benign lentigo of the face. Treatment with a diamond abrader: a case report. J. Kansas Med. Soc. 71:196(passim), 1970. Yamada, K., A. Matsukawa, Y. Hori, and A. Kukita. Ultrastructural studies on pigmented macules of Peutz-Jeghers syndrome. J. Dermatol. 8:367–377, 1981. Zeisler, E. P., and S. W. Becker. Generalized lentigo: its relation to systemic nonelevated nevi. Arch. Dermatol. Syphil. 33:109–125, 1936.

Lentigo Senilis et Actinicus Mary K. Cullen

Historical Background Hutchison described pigmented macules that increased with age in his 1892 exposition regarding tissue dotage. In 1950, Cawley and Curtis defined the solar lentigo as a unique lesion that appeared mostly in older individuals and consisted of a localized proliferation of intraepidermal melanocytes in elongated epidermal rete ridges. The presence of melanocytic proliferation in solar lentigo was confirmed in 1963 by Hodgson. Montagna et al. later emphasized that the proliferation involved both melanocytes and keratinocytes (Montagna et al., 1980). 829

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Synonyms Actinic lentigo, liver spot, age spot, lentigo senilis, lentigo solaris, sun-induced lentigo, and senile lentigo. It is also a component of photoaging. Specific variants include: PUVA (psoralen and ultraviolet radiation) lentigo, sun bed or tanning bed lentigo, sunburn freckle, radiation lentigo, and reticulated black solar lentigo or “ink-spot” lentigo.

Epidemiology Solar lentigines are more prevalent than ephelides (freckles), increase in prevalence and number with age, and are most prevalent on the trunk. They occur more frequently in males than in females, unlike ephelides which tend to be higher in number in females (Bastiaens et al., 1999). They are present in 90% of Caucasians older than 60 years. The occurrence of solar lentigo was thought to be like that of ephelides, existing along a continuum with the highest prevalence among persons with red hair who sunburned easily and tanned poorly (Brues, 1950). However, recent studies by Bastiaens et al. (1999) have shown a direct association of freckles, but not solar lentigines, with hair color and skin type. Solar lentigo is rare among individuals with darkly pigmented skin (Rhodes et al., 1983c). A large case–control study of the Central Malignant Melanoma Registry of the German Dermatological Society noted actinic lentigines were found more frequently with increasing age and were significantly related to both a tendency to freckling in adolescence and two or more sunburns after the age of 20 (Garbe et al., 1994a). Amongst Central Italian adult males, those with phototype I or II skin and solar lentigines also had significantly higher numbers of nevi (Ballone et al., 1999). The most common pigmented lesions in sun-exposed skin are ephelides, actinic lentigines, pigmented solar keratoses, seborrheic keratoses, and lentigo maligna (Ortonne, 1990a, b). PUVA lentigines (Fig. 46.2) are solar lentigines induced by photochemotherapy with a Psoralen (oral unless stated otherwise) and UltraViolet A radiation (Kanerva and Lauharanta, 1985; Kanerva et al., 1984; Rhodes et al., 1983a, c). According to Rhodes, they tend to occur in 40–50% of patients an average of 5.7 years after starting therapy and their frequency and severity directly correlate with the total number of treatments (Rhodes et al., 1983c). Likewise, Cox et al. noted PUVA lentigines in 46% of treated patients, most frequently in those patients actively receiving PUVA and in those who had received high cumulative UVA doses (Cox et al., 1987). A similar incidence of PUVA lentigines was noted in Japanese patients, 42% of 214, in whom the psoralen was applied topically. As with systemic psoralen, lentigines increased correspondingly with the number of PUVA treatments and the cumulative UVA dose (Takashima et al., 1990). Some PUVA lentigines were noted to persist up to seven years after discontinuing therapy (Cox et al., 1987). Perhaps fading of PUVA lentigines with time (unlike solar lentigines) explains why on 10 year follow-up only 20% of 198 subjects having received long-term photochemotherapy were found by Abdullah and Keczkes to have PUVA lentigines (Abdullah and Keczkes, 830

A

B Fig. 46.2. Multiple PUVA-induced lentigines in a young woman (A). Close-up view of the lesions (B) (see also Plate 46.2, pp. 494–495).

1989). As with sun-induced lentigines, the lowest risk for PUVA-induced lentigines is in darkly pigmented skin (Rhodes et al., 1983c; Sigg and Pelloni, 1989). In some cases single phototoxic doses of PUVA have been followed by development of PUVA lentigines in six to eight months (Konrad et al., 1977). Sun-bed lentigines, secondary to the use of UVA tanning beds (Kadunce et al., 1990; Roth et al., 1989; Salisbury et al., 1989), are thought to require 50 or more exposures for development (Rhodes, 1993). Reticulated or “ink-spot” lentigines are present primarily in individuals with type I skin (Kaddu et al., 1997) and are thought to appear following a single severe sunburn. Additionally, Peter and colleagues (1997) have described the “radiation lentigo.” It has clinical and histologic similarity to the actinic lentigo, but is induced by a single large dose of ionizing radiation.

Clinical Description Lentigo senilis et actinicus, more commonly known as solar or actinic lentigo, is a circumscribed pigmented macule induced by natural or artificial sources of UV radiation and is frequently present in older Caucasians. The solar lentigo, a clinical marker of photodamage (Holzle, 1992), may persist

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

46.3

46.4 Figs. 46.3 and 46.4. Typical actinic lentigines on the forehead and neck of two patients (see also Plates 46.3 and 46.4, pp. 494–495).

indefinitely with minimal or no fading in the absence of sun exposure (Figs 46.3 and 46.4). This is in contrast to ephelides (freckles) that generally occur in Caucasian children and fade or disappear when sun exposure is discontinued. Because solar lentigines may also occur in children and young adults

(Rhodes et al., 1983a, b, 1991), it is not always possible clinically to differentiate solar lentigines from ephelides. The solar lentigo is dealt with in great depth in several excellent reviews (Altman, 1984; Bolognia, 1993, 1995; Braun-Falco and Schoefinius, 1971; Goldberg and Altman, 1984; Ortonne, 1990a, b). Solar lentigines are circumscribed pigmented macules occurring on sun-exposed, pigment-challenged skin. The diameter of lentigines ranges from less than 1 mm to a few centimeters, and lentigines can become confluent in areas of severe sun damage. They are usually light brown, occasionally black. When examined, using 10¥ hand lens magnification of the mineral oil-covered surface, the pigment pattern in solar lentigo is reticulated (Rhodes et al., 1983a). Solar lentigines not apparent in visible light may be seen with Wood’s lamp illumination in the dark. It must be kept in mind that lentigines in patients receiving chronic photochemotherapy may appear in sites traditionally considered to be sun protected (Kanerva et al., 1984; Rhodes et al., 1983a, c). The “ink-spot” lentigo, or reticulated black solar lentigo, looks like a spot of ink on the skin. Its jet black color, irregular border, limitation to sun-exposed skin, and low number of lesions may raise suspicions for melanoma. However, characteristic features are usually sufficient to allow the clinical diagnosis of benign lentigo. In patients with numerous solar lentigines, each averaged only one to two ink-spot lentigines (Bolognia, 1992). Stolz et al. (1993) described the distinctive pattern of “ink-spot” lentigines when viewed with skin surface dermoscopy, also known as epiluminescence light microscopy. Lesions are sharply demarcated from adjacent skin and are characterized by markedly thickened webs of pigment network. Although there are irregular and wide meshes, a monomorphous overall appearance is typical. Dilated holes, similar to those seen in lentigo simplex, are present but the hole size can vary markedly. Bizarre asymmetric patterns can sometimes be found (Stolz et al., 1993). Additionally, pigment spots homogeneous in color, shape, and size and regularly superimposed on and juxtaposed to the pigment network are seen with epiluminescence. Histologically these spots were determined to represent individual hyperpigmented keratinocytes (Haas, 2002). Sunburn freckles are a variant of solar lentigines with acute onset after intense UV radiation (Holzle, 1992) (Fig. 46.5). The oral labial melanotic macule has gross morphologic characteristics similar to solar lentigo, but its relation to a sunsensitive phenotype or excessive sun exposure has not been well documented (Sexton and Maize, 1987; Spann et al., 1987; Weathers et al., 1976). Xeroderma pigmentosum is associated with lentigines on sun-exposed skin within the first five years of life, which persist despite sun avoidance (Robbins and Moshell, 1979). Lentigines in xeroderma pigmentosum, like those secondary to photochemotherapy, are often darkly and irregularly pigmented (Gschnait et al., 1980; Konrad et al., 1977; Rhodes et al., 1983a; Robbins and Moshell, 1979) (see Chapter 48). Some pigmented macules believed to be solar lentigo may represent early lentigo maligna, often combined 831

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Fig. 46.5. Eruptive lentigines following severe sunburn (see also Plate 46.5, pp. 494–495).

with actinic keratoses in markedly sun-damaged skin (Sanchez et al., 1981).

Histology Solar lentigo shows elongated epidermal rete ridges with clubshaped or budlike extensions, frequent branching and fusing of rete ridges, and a thinned or atrophic epidermis between rete. This is combined with hypermelanosis and hyperplasia of strongly dopa-positive, dendritic, and functionally active epidermal melanocytes without the nesting pattern typical of melanocytic nevi. Compared with melanocytes in adjacent normal skin, solar lentigo melanocytes are usually normal in size and cellular detail (Braun-Falco and Schoefinius, 1971; Cawley and Curtis, 1950; Gschnait et al., 1980; Hodgson, 1963; Montagna et al., 1980; Nakagawa et al., 1984; Rhodes et al., 1983a, 1991). Mihm and Googe and others consider solar lentigines to be nevomelanocytic proliferation (Bentley et al., 1994; Estivariz and Iturriza, 1985; Mihm and Googe, 1990; Nordlund et al., 1993; Plott and Wagner, 1990; Suvanprakorn et al., 1985) although this remains to be substantiated. A scant to moderate perivascular mononuclear cell dermal infiltrate is usually associated with scattered melanin-

832

laden macrophages (Montagna et al., 1980; Rhodes et al., 1983a, 1991; Sanchez et al., 1981). To Montagna and colleagues, the histological changes of solar lentigo suggested concurrent proliferation of keratinocytes and melanocytes (Montagna et al., 1980). Despite a 15–20% reduction in melanocyte density in human skin (sun-exposed and nonexposed) for every ten years between the ages of 27 and 65 (Quevedo et al., 1969), dopa-reactive melanocytes consistently show a twofold greater density in human skin exposed to UVR versus nonexposed skin (see Chapter 27). Regardless of age, melanocytes in UVRexposed skin seemingly retain their ability to proliferate in response to experimental UVR (Gilchrest et al., 1979; Quevedo et al., 1969). In contrast ephelides consist of hypermelanosis with a reduced number of functionally active melanocytes (Breathnach, 1957; Breathnach and Wyllie, 1964; Wilson and Kligman, 1982). Electron microscopic studies of solar lentigines show abundant, larger than normal melanosome complexes in adjacent keratinocytes (Montagna et al., 1980). Compared with melanocytes in sun-protected skin, melanocytes in solar lentigo reveal increased melanogenic activity manifested by marked dopa reactivity, elongated dendrites, large numbers of normalappearing melanosomes, enlarged perikarya with welldeveloped rough endoplasmic reticula, numerous mitochondria, and hypertrophic Golgi complexes (Nakagawa et al., 1984). Melanin macroglobules have been noted in solar lentigines (Rhodes et al., 1991). Occasionally, cellular atypia of melanocytes, and even melanoma in situ, may be detected in lesions clinically diagnosed as solar lentigo (Rhodes et al., 1991; Sanchez et al., 1981). Solar lentigo appears to be a risk factor for malignant melanoma. Chromometric analysis of pigment changes in solar lentigines, before and two months after sunlight exposure, revealed significant increases in pigment without changes in adjacent healthy skin (Adhoute et al., 1994). PUVA lentigines have elongated epidermal rete ridges containing an increased number of melanocytes, frequently residing above the epidermal basal unit, with large cell bodies and occasional atypical cellular morphology. The long and numerous dendrites often extend high into the epidermis (Abel et al., 1985; Gschnait et al., 1980; Kanerva et al., 1984; Konrad et al., 1977; Rhodes et al., 1983a; Ros et al., 1983). PUVA lentigo melanocytes have sharply invaginated nuclear contours, nuclear pseudoinclusions, double nuclei, striking melanosomal alterations, melanin macroglobules, and occasional manifestations of autophagocytosis. Keratinocytes in PUVA lentigo contain melanosomes that are more likely to be large and single than their small, aggregated counterparts in solar lentigo (Kanerva et al., 1984; Nakagawa et al., 1984). Although PUVA lentigines typically persist at least one to two years after therapy is discontinued (Rhodes et al., 1983c), the epidermal melanocytic cellular atypia may persist for many years (Abel et al., 1985; Kanerva et al., 1984; Ros et al., 1983). Sunbed lentigines demonstrate melanocytic hyperplasia and occasional cellular atypia (Kadunce et al., 1990; Roth et al.,

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

1989; Salisbury et al., 1989). Histologically the lesions resemble PUVA lentigines except that epidermal hypertrophy is not observed (Meyskens et al., 1986). Similarly, xeroderma pigmentosum lentigines show hyperplasia of variably atypical melanocytes. The “ink-spot” lentigo (reticulated black solar lentigo) demonstrates lentiginous hyperplasia of the epidermis, minimally increased melanocyte number, and marked hyperpigmentation of the basal layer with “skip” areas at the rete ridges (Bolognia, 1992). This histologic description was expanded to include sharply circumscribed hyperpigmentation of the lower epidermis with accentuation at the tips of elongated and clubbed rete ridges. Also noted was an infiltrate of melanophages in the upper dermis (Kaddu et al., 1997). The “radiation lentigo” was noted to have increased melanin in both melanocytes and basal keratinocytes. There was slight melanocytic hyperplasia, but no cellular atypia. However, signs of ionizing radiation damage were also present in the form of epidermal atrophy, flattening of rete ridges, telangiectatic vessels, and hyalinization of connective tissue (Peter et al., 1997).

Differential Diagnosis Several different kinds of pigmented lesion are displayed in sun-damaged skin, the most common of which are senile lentigines (Holzle, 1992). Flat, reticular, and pigmented seborrheic keratoses, pigmented and pigmented-spreading actinic keratoses, large-cell acanthoma, lentigo simplex, and junctional nevomelanocytic nevus may clinically resemble an actinic lentigo (Braun-Falco et al., 1986; Holzle, 1992; Klinker and Jonsson, 1994; Rhodes, 1993; Subrt et al., 1983). Malignant melanoma and lentigo malignant melanoma must be excluded, especially in xeroderma pigmentosum lentigines and PUVA lentigines which are often darkly and/or irregularly pigmented (Gschnait et al., 1980; Holzle, 1992; Konrad et al., 1977; Rhodes et al., 1983a; Robbins and Moshell, 1979).

Pathogenesis Solar lentigo is a marker of intermittent high-intensity UVR, cumulative UVR, and/or a unique susceptibility to the proliferative, stimulatory, and/or mutagenic effects of UVR. Although repeated UVR exposure is known to increase epidermal melanocyte number, the acute hyperplastic response should decrease by two months after UVR is discontinued (Szabó, 1967). A direct causal relation between chronic UVB (UVR type B) irradiation and solar lentigo has been demonstrated in a study using RAG-1 mice grafted with human newborn foreskin (Atillasoy, 1998). However, UVR-induced melanocytic hyperplasia cannot totally account for the circumscribed and persistent nature of solar lentigines, nor their occurrence in some people but not in others (Rhodes et al., 1983c). Indications of somatic mutation in UVR induced lentigo include enhanced pigment production in response to UVR, relatively large cell size, marked dopa reactivity, qualitative melanosomal abnormalities, and persistent hyperplasia of

melanocytes (Abel et al., 1985; Jimbow and Uesugi, 1982; Kanerva et al., 1984; Nakagawa et al., 1984; Rhodes et al., 1983a, c; Ros et al., 1983; Wilson and Kligman, 1982). An initiating role for UVR in the pathogenesis of solar lentigo is supported by lesion distribution on sun-exposed skin, appearance during chronic photochemotherapy as well as with cosmetic UVA tanning bed use (Gschnait et al., 1980; Kanerva et al., 1984; Konrad et al., 1977; Rhodes, 1993; Rhodes et al., 1983a, c), and the association with sunburns received after the age of 20 (Garbe et al., 1994b). It is possible that melanocytic hyperplasia induced by early childhood UVR may be responsible for the sequential progression of melanocytic neoplasia (Farber and Cameron, 1980; Mitchell, 1963; Rhodes et al., 1989, 1991) or for some varieties of melanocytic dysplasia detected later in life such as lentigo maligna and junctional dysplastic melanocytic nevi (Rhodes, 1993). A similar function for melanocytic proliferation induced in adulthood has not been proposed. “Fixed” cellular atypia and hyperplasia of intraepidermal melanocytes, noted in some solar lentigines, may be one step of an UVR induced dysplastic and neoplastic process (Rhodes, 1993). Nikkels and colleagues (1991) compared PUVA- and sunlight-induced lentigines and concluded that UVR of different wavelengths may act differently on melanocytes and/or on the interrelation between melanocytes and keratinocytes, and that the pathophysiology of the two forms of lentigo is probably different. An etiologic relationship between solar lentigo and the solitary lichen planus-like keratosis (LPK) is suggested by the frequent coexistence of both in a single lesion. It is widely believed that LPK evolves from solar lentigo (Barranco, 1985; Goldenhersh et al., 1986; Mehregan, 1975; Roewert and Ackerman, 1992). However, Prieto and colleagues (1993) found solar lentigo adjacent to LP-like keratosis in only 7% of the 71 lesions they examined. Potential etiologic relationships also include the large cell acanthoma. Described in 1970, the kinship of large cell acanthoma to solar lentigo and LP-like keratosis was first suggested in 1978 (Rahbari and Pinkus, 1978; Roewert and Ackerman, 1992). This suggestion has been countered by those who believe that solar lentigo and large cell acanthoma are histopathologically and clinically distinct lesions (Ambrojo et al., 1990; Sanchez Yus et al., 1992). Stern et al. (1993) proposed solar lentigo as the most common precursor lesion for malignant melanoma in xeroderma pigmentosum. Histologically, they found solar lentigo lateral to and contiguous with melanoma in 88% of cases accompanied by areas of apparent transition. Significantly fewer solar lentigines, 22%, were contiguous with the basal cell carcinoma controls, and all lacked transitional areas (Stern et al., 1993). Kraemer and colleagues (1989) thought the atypical solar lentigo in xeroderma pigmentosum might reflect hypermutability of melanocytes related to impaired UVRinduced damage repair capacity.

Animal Models Yucatan swine skin with UV-stimulated hyperpigmentation is

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a model for solar lentigines (Nair et al., 1993). Sunlight was found to induce lentigines (and skin cancers) in Angora goats in a highly site-specific manner (Green et al., 1996). RAG-1 mice grafted with human newborn foreskin, and treated with UVB light or the combination of a carcinogen (7,12dimethyl(a)benzanthracene, DMBA) and UVB developed solar lentigines (Atillasoy, 1998). Pigmented hairless mice irradiated with UVB also develop pigmented spots clinically and histologically consistent with solar lentigines. The latent period until appearance and the extent of development of these spots were both dependent on the exposure period (Naganumaa, 2001).

Prevention It may be possible to prevent solar lentigo by sun avoidance, protective clothing, sunscreens, and blocking agents. Patients who have PUVA lentigines should be examined periodically for atypical evolution.

Treatment Bleaching creams containing hydroquinone are not usually effective (Rhodes, 1993). In 1975 Kligman found that actinic lentigines were resistant to topical treatment with a combination of tretinoin, hydroquinone, and dexamethasone in a hydrophilic ointment (Kligman and Willis, 1975). Griffiths, however, later noted improvement of several parameters of photoaging, including lightening of actinic lentigines, with topical tretinoin. Fading correlated with a reduction in epidermal melanin content (Griffiths et al., 1993). Additional topical methods employed since 1996 include: Cook body peel, trichloroacetic acid (TCA) with tretinoin, mequinol with TCA, high concentration retinoic acid, tazarotene, adapalene, pyruvic acid, and hydroquinone with cyclodextrin. See also Chapter 59. Slight application of liquid nitrogen may allow remission of lesions for as much as one year. Beacham (1990) used cryosurgery followed by topical tretinoin and topical hydroquinone. Karakashian applied liquid nitrogen with a handheld probe and the “Frigipoint” instrument to treat a wide variety of lesions including solar lentigo (Karakashian and Sweren, 1989). Likewise, Hosaka et al. (1995) found the “CRYOMINI” instrument, with a removable disk-shaped copper tip, extremely effective for cryosurgical treatment of senile lentigines. Stern compared cryosurgery with two laser modalities for the treatment of solar lentigo (Stern et al., 1994). Solar lentigo is being treated with an increasing variety of lasers including: low-fluence carbon dioxide (Dover et al., 1988), monoline argon laser (514 nm) with long 200 ms or 300 ms pulse lengths (Trelles et al., 1992), flashlamp-pulsed tunable dye laser (Day and Pardue, 1993), 510 nm pigmented lesion dye laser (Grekin et al., 1993), and Q-switched ruby laser (Hruza et al., 1991). Additional lasers in use for actinic lentigines since 1996 include: intense pulsed light, alexandrite, Q-switched alexandrite, HGM K1 krypton, 532 nm diodepumped vanadate and frequency-doubled neodymium:yttrium 834

aluminum garnet (Nd:YAG) with TCA. See also Chapters 63 and 64. Conscientious application of sunscreens and use of blocking agents is necessary after each of the above treatments to prevent new lesions (Rhodes, 1993). Multiple benign lichenoid keratoses arising from pre-existing senile lentigines will apparently disappear either spontaneously or with topical corticosteroid treatment (Barranco, 1985).

Prognosis The presence of sun-induced pigmented macules in adults is associated with an estimated two- to fourfold increase in nonmelanoma skin cancer risk and an estimated two- to sixfold increase in melanoma risk (Rhodes et al., 1991). Torres and colleagues (1994) reported a patient with a documented solar lentigo who later developed a malignant melanoma in situ at the same location. This potential progression, from solar lentigo to malignant melanoma, has been accepted and discussed by Stern and Haupt (1991). Moderate to large numbers of actinic lentigines are considered an independent risk factor for malignant melanoma (Garbe et al., 1989, 1994a; Green et al., 1988; MacKie et al., 1989). Using combined results of four large case–control studies over one decade, the frequency of actinic lentigines, number of common melanocytic nevi, and number of atypical melanocytic nevi were found by multivariate regression analysis to be the most important independent risk factors for melanoma development (Garbe, 1995). Solar lentigines may enlarge, darken, and become more irregular and “fixed” over time, similar to the progression of lentigo maligna (see Melanoma discussion), but longitudinal studies of solar lentigo are lacking. Solar lentigo may be a heterogeneous population, including lesions that fade and lesions that do not fade after UVR avoidance. It is possible that solar lentigo in some individuals evolves to varieties of intraepidermal melanocytic dysplasia similar to lentigo maligna (Rhodes, 1993). It is possible that lentigo maligna develops from solar lentigo, and this possibility cannot be discarded by studies reporting the absence of solar lentigo in histologic contiguity with lentigo maligna (Mehregan, 1975), because a small solar lentigo may be obscured or obliterated by an evolving melanocytic dysplasia (Rhodes, 1993). Unlike solar lentigines, ordinary PUVA lentigines have been noted to fade and resolve over time (Abdullah, 1989). The long-term course of the atypical PUVA lentigo is unknown (Rhodes et al., 1983a). Lever and Farr examined 54 patients who had received a cumulative UVA dose greater than 2000 J/cm2 each. In the 13% that lacked PUVA lentigines, actinic keratoses and squamous cell carcinomas were also lacking. Based on these data, it has been hypothesized that the absence of PUVA lentigines may be a useful indicator of a lower risk of any PUVA-induced skin malignancy (Lever and Farr, 1994). Patients on chronic hemodialysis were investigated for the presence of several cutaneous “aging” markers. Senile lentigo was present in 20% of the patients but, unlike two other solar damage lesions (actinic keratoses and Favre–Racouchot

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

disease), its frequency did not increase with the duration of chronic hemodialysis (Tercedor et al., 1995).

References Abdullah, A. N., and K. Keczkes. Cutaneous and ocular side-effects of PUVA photochemotherapy: a 10-year follow-up study [review]. Clin. Exp. Dermatol. 14:421–424, 1989. Abel, E. A., H. Reid, C. Wood, and C. H. Hu. PUVA-induced melanocytic atypia: is it confined to PUVA lentigines? J. Am. Acad. Dermatol. 13:761–768, 1985. Adhoute, H., R. Grossman, M. Cordier, and B. Soler. Chromametric quantification of pigmentary changes in the solar lentigo after sunlight exposure. Photodermatol. Photoimmunol. Photomed. 10:93–96, 1994. Altman, A. Benign skin changes associated with chronic sunlight exposure. Cutis 34:33–38,40, 1984. Ambrojo, P., A. Aguilar, L. Requena, and E. Sanchez Yus. Achromic verrucous large cell acanthoma. J. Cutan. Pathol. 17:182–184, 1990. Atillasoy, E. S., J. T. Seykora, P. W. Soballe, R. Elenitsas, M. Nesbit, D. E. Elder, K. T. Montone, E. Sauter, and M. Herlyn. UVB induces atypical melanocytic lesions and melanoma in human skin. Am. J. Pathol. 152:1179–1186, 1998. Ballone, E., M. Passamonti, G. Lappa, G. Di Blasio, and P. Fazii. Pigmentary traits, nevi and skin phototypes in a youth population of Central Italy. Eur. J. Epidemiol. 15:189–195, 1999. Barranco, V. P. Multiple benign lichenoid keratoses simulating photodermatoses: evolution from senile lentigines and their spontaneous regression. J. Am. Acad. Dermatol. 13:201–206, 1985. Bastiaens, M. T., R. G. Westendorp, Vermeer, B. J., and J. N. Bavinck. Ephelides are more related to pigmentary constitutional host factors than solar lentigines. Pigment Cell Res. 12:316–322, 1999. Beacham, B. E. Solar-induced epidermal tumors in the elderly [review]. Am. Fam. Physician 42:153–160, 1990. Bentley, N. J., T. Eisen, and C. R. Goding. Melanocyte-specific expression of the human tyrosinase promoter: Activation by the microphthalmia gene product and role of the initiator. Mol. Cell. Biol. 14:7996–8006, 1994. Bolognia, J. L. Reticulated black solar lentigo (“ink spot” lentigo). Arch. Dermatol. 128:934–940, 1992. Bolognia, J. L. Dermatologic and cosmetic concerns of the older woman [review]. Clin. Geriatr. Med. 9:209–229, 1993. Bolognia, J. L. Aging skin [review]. Am. J. Med. 98:99S-103S, 1995. Braun-Falco, O., and H. H. Schoefinius. [Lentigo senilis: Review and studies] [German] Lentigo senilis: Ubersicht und eigene Untersuchungen. Hautarzt 22:277–283, 1971. Braun-Falco, O., C. Schmoeckel, and C. Geyer. [Pigmented actinic keratoses] [German] Pigmentierte aktinische Keratosen. Hautarzt 37:676–678, 1986. Breathnach, A. S. Melanocytic distribution in forearm epidermis of freckled human subjects. J. Invest. Dermatol. 29:253–261, 1957. Breathnach, A. S., and L. M. Wyllie. Electron microscopy of melanocytes and melanosomes in freckled human epidermis. J. Invest. Dermatol. 42:389–394, 1964. Brues, A. M. Linkage of bodybuild with sex, eye colour, and freckling. Am. J. Hum. Genet. 2:215, 1950. Cawley, E. P., and A. C. Curtis. Lentigo senilis. Arch. Dermatol. Syphil. 62:635, 1950. Cox, N. H., S. K. Jones, D. J. Downey, E. J. Tuyp, J. L. Jay, H. Moseley, and R. M. Mackie. Cutaneous and ocular side-effects of oral photochemotherapy: results of an 8-year follow-up study. Br. J. Dermatol. 116:145–152, 1987. Day, T. W., and C. C. Pardue. Preliminary experience with a flashlamp-pulsed tunable dye laser for treatment of benign pigmented lesions. Cutis 51:188–190, 1993.

Dover, J. S., B. R. Smoller, R. S. Stern, S. Rosen, and K. A. Arndt. Low-fluence carbon dioxide laser irradiation of lentigines. Arch. Dermatol. 124:1219–1224, 1988. Estivariz, F. E., and F. C. Iturriza. An investigation on proopiomelanocortin and processed peptides from the teleost fish Prochilodus platensis. Peptides 6:817–824, 1985. Farber, E., and R. Cameron. The sequential analysis of cancer development [review]. Adv. Cancer Res. 31:125–226, 1980. Garbe, C. [Risk factors for the development of malignant melanoma and identification of risk groups in German-speaking regions] [review] [German] Risikofaktoren fur die Entwicklung maligner Melanome und Identifikation von Risikopersonen im deutschsprachigen Raum. Hautarzt 46:309–314, 1995. Garbe, C., S. Kruger, R. Stadler, I. Guggenmoos-Holzmann, and C. E. Orfanos. Markers and relative risk in a German population for developing malignant melanoma. Int. J. Dermatol. 28:517–523, 1989. Garbe, C., P. Buttner, J. Weiss, H. P. Soyer, U. Stocker, S. Kruger, M. Roser, J. Weckbecker, R. Panizzon, F. Bahmer, W. Tilgen, I. Guggenmoos-Holzmann, and C. E. Orfanos. Risk factors for developing cutaneous melanoma and criteria for identifying persons at risk: multicenter case-control study of the Central Malignant Melanoma Registry of the German Dermatological Society. J. Invest. Dermatol. 102:695–699, 1994a. Garbe, C., P. Buttner, J. Weiss, H. P. Soyer, U. Stocker, S. Kruger, M. Roser, J. Weckbecker, R. Panizzon, F. Bahmer, W. Tilgen, I. Guggenmoos-Holzmann, and C. E. Orfanos. Associated factors in the prevalence of more than 50 common melanocytic nevi, atypical melanocytic nevi, and actinic lentigines: multicenter case-control study of the Central Malignant Melanoma Registry of the German Dermatological Society. J. Invest. Dermatol. 102:700–705, 1994b. Gilchrest, B. A., F. B. Blog, and G. Szabo. Effects of aging and chronic sun exposure on melanocytes in human skin. J. Invest. Dermatol. 73:141–143, 1979. Goldberg, L. H., and A. Altman. Benign skin changes associated with chronic sunlight exposure. Cutis 34:33–38, 1984. Goldenhersh, M. A., R. L. Barnhill, H. M. Rosenbaum, and K. S. Stenn. Documented evolution of a solar lentigo into a solitary lichen planus-like keratosis. J. Cutan. Pathol. 13:308–311, 1986. Green, A., G. Beardmore, V. Hart, D. Leslie, R. Marks, and D. Staines. Skin cancer in a Queensland population. J. Am. Acad. Dermatol. 19:1045–1052, 1988. Green, A., R. Neale, R. Kelly, I. Smith, E. Ablett, Meyers, B., and P. Parsons. An animal model for human melanoma. Photochem. Photobiol. 64:577–580, 1996. Grekin, R. C., R. M. Shelton, J. K. Geisse, and I. Frieden. 510-nm pigmented lesion dye laser. Its characteristics and clinical uses. J. Dermatol. Surg. Oncol. 19:380–387, 1993. Griffiths, C. E., L. J. Finkel, C. M. Ditre, T. A. Hamilton, C. N. Ellis, and J. J. Voorhees. Topical tretinoin (retinoic acid) improves melasma. A vehicle-controlled, clinical trial. Br. J. Dermatol. 129:415–421, 1993. Gschnait, F., K. Wolff, H. Honigsmann, G. Stingl, W. Brenner, E. Jaschke, and K. Konrad. Long-term photochemotherapy: histopathological and immunofluorescence observations in 243 patients. Br. J. Dermatol. 103:11–22, 1980. Haas, N., B. Hermes, and B. M. Henz. Detection of a novel pigment network feature in reticulated black solar lentigo by high-resolution epiluminescence microscopy. Am J Dermatopathol. 24:213–217, 2002. Hodgson, C. Senile lentigo. Arch. Dermatol. 87:197, 1963. Holzle, E. Pigmented lesions as a sign of photodamage. Br. J. Dermatol. 127 Suppl 41:48–50, 1992. Hosaka, Y., T. Onizuka, M. Ichinose, S. Yoshimoto, F. Okubo, S. Hori, and A. Keyama. Treatment of nevus of Ota by liquid nitrogen cryotherapy. Plast. Reconstr. Surg. 95:703–711, 1995.

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CHAPTER 46 Hruza, G. J., J. S. Dover, T. J. Flotte, M. Goetschkes, S. Watanabe, and R. R. Anderson. Q-switched ruby laser irradiation of normal human skin. Histologic and ultrastructural findings. Arch. Dermatol. 127:1799–1805, 1991. Hutchison, J. Tissue Dotage. Arch. Surg. 3:315, 1892. Jimbow, K., and T. Uesugi. New melanogenesis and photobiological processes in activation and proliferation of precursor melanocytes after UV-exposure: ultrastructural differentiation of precursor melanocytes from Langerhans cells. J. Invest. Dermatol. 78:108–115, 1982. Kaddu, S., H. P. Soyer, I. H. Wolf, E. Rieger, and H. Kerl. Reticular lentigo. Hautarzt. 48:181–5, 1997. Kadunce, D. P., M. W. Piepkorn, and J. J. Zone. Persistent melanocytic lesions associated with cosmetic tanning bed use: “sunbed lentigines.” J. Am. Acad. Dermatol. 23:1029–1031, 1990. Kanerva, L., and J. Lauharanta. PUVA lentigo. Photodermatology 2:49–51, 1985. Kanerva, L., K. M. Niemi, and J. Lauharanta. A semiquantitative light and electron microscopic analysis of histopathologic changes in phototherapy-induced freckles. Arch. Dermatol. Res. 276:2–11, 1984. Karakashian, G. V., and R. J. Sweren. Frigipoint: a new cryosurgical instrument. J. Dermatol. Surg. Oncol. 15:514–517, 1989. Kligman, A. M., and I. Willis. A new formula for depigmenting human skin. Arch. Dermatol. 111:40–48, 1975. Klinker, M., and N. Jonsson. Simulation of solar lentigo by spreading pigmented actinic keratosis [letter]. Acta Derm. Venereol. 74:406–407, 1994. Konrad, K., F. Gschnait, and K. Wolff. Ultrastructure of poikiloderma-like pigmentary changes after repeated experimental PUVAoverdosage. J. Cutan. Pathol. 4:219, 1977. Kraemer, K. H., M. Herlyn, S. H. Yuspa, W. H. Clark, K. Townsend, G. Neises, and V. J. Hearing. Reduced DNA repair in cultured melanocytes and nevus cells from a patient with xeroderma pigmentosum. Arch. Dermatol. 125:263–268, 1989. Lever, L. R., and P. M. Farr. Skin cancers or premalignant lesions occur in half of high-dose PUVA patients. Br. J. Dermatol. 131:215–219, 1994. Lever, W. F., and G. Schaumburg-Lever. Histopathology of the Skin, 7th ed. Philadelphia: JB Lippincott, 1990, p. 756. MacKie, R. M., T. Freudenberger, and T. C. Aitchison. Personal riskfactor chart for cutaneous melanoma. Lancet 2:487–490, 1989. Mehregan, A. H. Lentigo senilis and its evolutions. J. Invest. Dermatol. 65:429–433, 1975. Meyskens, F. L., Jr., L. Edwards, and N. S. Levine. Role of topical tretinoin in melanoma and dysplastic nevi. J. Am. Acad. Dermatol. 15:822–825, 1986. Mihm, M. C., and P. B. Googe. Problematic Pigmented Lesions: A Case Method Approach. Philadelphia: Lea and Febiger, 1990, p. 3. Mitchell, R. E. The effect of prolonged solar radiation on melanocytes of the human epidermis. J. Invest. Dermatol. 41:199, 1963. Montagna, W., F. Hu, and K. Carlisle. Reinvestigation of solar lentigines. Arch. Dermatol. 116:1151–1154, 1980. Naganumaa, M., E. Yagi, and M. Fukuda. Delayed induction of pigmented spots on UVB-irradiated hairless mice. J. Dermatol. Sci. 25:29–35, 2001. Nair, X., P. Parab, L. Suhr, and K. M. Tramposch. Combination of 4-hydroxyanisole and all-trans retinoic acid produces synergistic skin depigmentation in swine. J. Invest. Dermatol. 101:145–149, 1993. Nakagawa, H., A. R. Rhodes, T. K. Momtaz, and T. B. Fitzpatrick. Morphologic alterations of epidermal melanocytes and melanosomes in PUVA lentigines: a comparative ultrastructural investigation of lentigines induced by PUVA and sunlight. J. Invest. Dermatol. 82:101–107, 1984. Nikkels, A., T. Ben Mosbah, C. Pierard-Franchimont, M. de la Brassinne, and G. E. Pierard. Comparative morphometric study of

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eruptive PUVA-induced and chronic sun-induced lentigines of the skin. Anal. Quant. Cytol. Histol. 13:23–26, 1991. Nordlund, J. J., R. M. Halder, and P. Grimes. Management of vitiligo. Dermatol. Clin. 11:27–33, 1993. Ortonne, J. P. The effects of ultraviolet exposure on skin melanin pigmentation [review]. J. Int. Med. Res. 18(Suppl. 3):8C-17C, 1990a. Ortonne, J. P. Pigmentary changes of the ageing skin. Br. J. Dermatol. 122(Suppl 35):21–28, 1990b. Peter, R. U., P. Gottlober, N. Nadeshina, G. Krahn, Plewig, G., and P. Kind. Radiation lentigo. A distinct cutaneous lesion after accidental radiation exposure. Arch Dermatol. 133:209–211, 1997. Plott, R. T., and R. F. Wagner. Modern treatment approaches to vitiligo. Cutis 45:311–316, 1990. Prieto, V. G., M. Casal, and N. S. McNutt. Lichen planus-like keratosis: A clinical and histological reexamination. Am. J. Surg. Pathol. 17:259–263, 1993. Quevedo, W. C., Jr., G. Szabó, and J. Virks. Influence of age and UV on the population of DOPA-positive melanocytes in human skin. J. Invest. Dermatol. 52:287–290, 1969. Rahbari, H., and H. Pinkus. Large cell acanthoma: One of the actinic keratoses. Arch. Dermatol. 114:49–52, 1978. Rhodes, A. R. Neoplasms: benign neoplasias, hyperplasias, and dysplasias of melanocytes. In: Dermatology in General Medicine, 4th ed., T. B. Fitzpatrick, A. Eisen, K. Wolff, I. M. Freedberg, and F. K. Austen (eds). New York: McGraw-Hill, Inc., 1993, pp. 996–1077. Rhodes, A. R., T. J. Harrist, and T. K. Momtaz. The PUVA-induced pigmented macule: a lentiginous proliferation of large, sometimes cytologically atypical, melanocytes. J. Am. Acad. Dermatol. 9:47–58, 1983a. Rhodes, A. R., J. W. Melski, A. J. Sober, T. J. Harrist, M. C. Mihm Jr., and T. B. Fitzpatrick. Increased intraepidermal melanocyte frequency and size in dysplastic melanocytic nevi and cutaneous melanoma: A comparative quantitative study of dysplastic melanocytic nevi, superficial spreading melanoma, nevocellular nevi, and solar lentignes. J. Invest. Dermatol. 80:452–459, 1983b. Rhodes, A. R., R. S. Stern, and J. W. Melski. The PUVA lentigo: an analysis of predisposing factors. J. Invest. Dermatol. 81:459–463, 1983c. Rhodes, A. R., M. C. Mihm Jr., and M. A. Weinstock. Dysplastic melanocytic nevi: a reproducible histologic definition emphasizing cellular morphology. Modern Pathology 2:306–319, 1989. Rhodes, A. R., L. S. Albert, R. L. Barnhill, and M. A. Weinstock. Suninduced freckles in children and young adults. A correlation of clinical and histopathologic features. Cancer 67:1990–2001, 1991. Robbins, J. H., and A. N. Moshell. DNA repair processes protect human beings from premature solar skin damage: evidence from studies on xeroderma pigmentosum. J. Invest. Dermatol. 73:102– 107, 1979. Roewert, H. J., and A. B. Ackerman. Large-cell acanthoma is a solar lentigo. Am. J. Dermatopathol. 14:122–132, 1992. Ros, A. M., G. Wennersten, and B. Lagerholm. Long-term photochemotherapy for psoriasis: a histopathological and clinical follow-up study with special emphasis on tumour incidence and behavior of pigmented lesions. Acta Derm. Venereol. 63:215–221, 1983. Roth, D. E., S. J. Hodge, and J. P. Callen. Possible ultraviolet A-induced lentigines: a side effect of chronic tanning salon usage. J. Am. Acad. Dermatol. 20:950–954, 1989. Salisbury, J. R., H. Williams, and A. W. du Vivier. Tanning-bed lentigines: ultrastructural and histopathologic features. J. Am. Acad. Dermatol. 21:689–693, 1989. Sanchez, J. L., F. Ramos-Caro, and A. B. Ackerman. Histopathologic findings in supposed solar lentigines of Puerto Ricans. In: Pathology of Malignant Melanoma, A. B. Ackerman (ed.). New York: Masson Publishers, 1981, p. 107. Sanchez Yus, E., E. del Rio, and L. Requena. Large-cell acanthoma is a distinctive condition. Am. J. Dermatopathol. 14:140–147, 1992.

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES Sexton, F. M., and J. C. Maize. Melanotic macules and melanoacanthomas of the lip. Am. J. Dermatopathol. 9:438–444, 1987. Sigg, C., and F. Pelloni. Frequency of acquired melanonevocytic nevi and their relationship to skin complexion in 939 schoolchildren. Dermatologica 179:123–128, 1989. Spann, C. R., L. G. Owen, and S. J. Hodge. The labial melanotic macule. Arch. Dermatol. 123:1029–1031, 1987. Stern, J. B., and H. M. Haupt. Solar lentigo joins the usual suspects as a possible malignant melanoma precursor [Abstract]. J. Cutan. Pathol. 18:392, 1991. Stern, J. B., G. L. Peck, H. M. Haupt, H. C. Hollingsworth, and T. Beckerman. Malignant melanoma in xeroderma pigmentosum: search for a precursor lesion. J. Am. Acad. Dermatol. 28:591–594, 1993. Stern, R. S., J. S. Dover, J. A. Levin, and K. A. Arndt. Laser therapy versus cryotherapy of lentigines: a comparative trial. J. Am. Acad. Dermatol. 30:985–987, 1994. Stolz, W., O. Braun-Falco, P. Bilek, M. Landthaler, and A. B. Cognetta. Lentigines. In: Color Atlas of Dermatoscopy. Blackwell Scientific Publications, 1993. Subrt, P., J. L. Jorizzo, P. Apisarnthanarax, E. S. Head, and E. B. Smith. Spreading pigmented actinic keratosis. J. Am. Acad. Dermatol. 8:63–67, 1983. Suvanprakorn, P., S. Dee-Ananlap, C. Pongsomboon, and S. N. Klaus. Melanocyte autologous grafting for the treatment of leukoderma. J. Am. Acad. Dermatol. 13:968–974, 1985. Szabó, G. Photobiology of melanogenesis: Cytological aspects with special reference to differences in racial coloration. In: Advances in Biology of Skin–The Pigmentary System, vol. 8, W. Montagna, and F. Hu (eds). London: Pergamon Press, 1967, pp. 379–396. Takashima, A., E. Matsunami, K. Yamamoto, S. Kitajima, and N. Mizuno. Cutaneous carcinoma and 8-methoxypsoralen and ultraviolet A (PUVA) lentigines in Japanese patients with psoriasis treated with topical PUVA: a follow-up study of 214 patients. Photodermatol. Photoimmunol. Photomed. 7:218–221, 1990. Tercedor, J., B. Lopez-Hernandez, J. M. Rodenas, M. DelgadoRodriguez, S. Cerezo, and S. Serrano-Ortega. Multivariate analysis of cutaneous markers of aging in chronic hemodialyzed patients. Int. J. Dermatol. 34:546–550, 1995. Torres, J. E., S. M. Torres, and J. L. Sanchez. Melanoma in situ on facial skin damaged by sunlight. Am. J. Dermatopathol. 16:171– 174, 1994. Trelles, M. A., W. Verkruysse, J. W. Pickering, M. Velez, J. Sanchez, and P. Sala. Monoline argon laser (514 nm) treatment of benign pigmented lesions with long pulse lengths. J. Photochem. Photobiol. B 16:357–365, 1992. Weathers, D. R., R. L. Corio, B. E. Crawford, J. S. Giansanti, and L. R. Page. The labial melanotic macule. Oral Surg. Oral Med. Oral Pathol. 42:196–205, 1976. Wilson, P. D., and A. M. Kligman. Experimental induction of freckles by ultraviolet-B. Br. J. Dermatol. 106:401–406, 1982.

Centrofacial Lentiginosis Mary K. Cullen

Historical Aspects Centrofacial lentiginosis with associated dysplasias was first described in 1941 by Touraine. An overview of his findings and hypotheses (Touraine, 1941a) was followed by detailed descriptions of 30 cases (Touraine, 1941c). This “newly described, congenital, neuroectodermal disease” was a triad of lentigines limited to the medial face, neuropsychiatric problems, and dysraphic malformations. Pathogenesis was postu-

lated to be a syphilis-induced genetic mutation that, once present, was transmitted in an autosomal dominant fashion (Touraine, 1941a, c, 1958). Touraine’s third account of the syndrome, in 1942, came to the attention of Delay and Pichot (1946) who subsequently described a patient doubly affected with centrofacial lentiginosis and tuberous sclerosis. Both diseases were present in their full syndromic forms including profound mental retardation (oligophrenia). All of the patient’s three sisters had typical centrofacial lentigines, two had dysraphia and none demonstrated altered intelligence. The authors concluded that mental retardation was a variably penetrant aspect of centrofacial lentiginosis. In 1947 Musumeci presented a family from Sicily with centrofacial lentiginosis. The mother, whose sole manifestation was lentigines, had six children. Four displayed Touraine’s full triad and two lacked only the lentigines. Musumeci questioned whether the correlation of centrofacial lentigines with dysraphia and neuropsychiatric disorders was more than coincidence (Musumeci, 1947). Not until 1953 was another group of unrelated patients presented (Mercadal-Peyri, 1953). Twenty-five subjects from Barcelona, Spain, with this syndrome had been identified among 2000 school children. All had lentigines and dysraphic anomalies. Mental retardation occurred in 88%. Familial involvement was not investigated. Degos (1953) and Lutz (1957) described centrofacial lentiginosis in dermatology texts but apparently did not agree with its designation as a neuroectodermal disease (Touraine, 1958). Touraine’s fourth and final publication regarding centrofacial lentiginosis included two new cases and appeared in 1958. He described an association between adiposogenital dystrophy (Fröhlich syndrome) and radiological changes of the sella turcica. His previously expounded conclusions were reemphasized, regarding not only the triad of components, but also the proposed pathogenic role of syphilis. Centrofacial lentiginosis had received minimal attention in the 17 years succeeding his initial descriptions and he used the abstract to express his perplexity. “I first described this previously unpublished syndrome in 1941. Subsequently, my three publications have apparently been unnoticed. Further, to my knowledge, studies confirming the existence and accuracy of this polydysplasia are rare. And finally, most of the recent treatises in dermatology have ignored centrofacial lentiginosis as an entity” (Touraine, 1958). Touraine pleaded with readers to further examine his hypotheses, and noted that evaluation by other investigators was necessary to clarify the syndromic elements and to substantiate or invalidate his conclusions. The following year Montagnani and Mariotti (1959) presented a family with centrofacial lentiginosis. Electroencephalography (EEG) in four cases exhibited slight abnormalities. Pituitary injury was the hypothesized cause of the syndrome. No further reports appeared until 1971, when Dociu et al. described seven apparently unrelated persons, all of whom had neuropsychiatric problems, dysraphia, and a high degree of bony dysmorphism (especially of the spine and 837

CHAPTER 46

skull). Lentigines, numbering 30 to 100, were exhibited on the noses, cheeks, and foreheads of all seven subjects. The importance and broad range of osseous and neurophysical problems was emphasized. Congenital ocular anomalies were also reported. Frequent reference in the centrofacial lentiginosis literature is made to a 1974 article by Nitelea and Dociu. A family with lentiginosis, dysraphism, and mental debility was reported. However, the distribution of lentigines was not consistent with centrofacial lentiginosis and the family was not designated as such by the authors. A comprehensive survey of the skeletal anomalies occurring in centrofacial lentiginosis was provided by Murgu et al. in 1975. Of their 39 cases, 11 had come to medical attention because of illness, and the remaining 28 were elicited by inquiry “within some collectives.” An increased tendency for precocious arthritis was noted and joined the list of syndromic associations. Murgu et al. commented that the frequent dysmorphic anomalies, growth disturbances, and arthritic tendencies in patients with centrofacial lentiginosis “awaken the interest of the radiologist.” Radiologists were encouraged to pay attention to these skeletal manifestations, as they were potential indicators of learning and vocational training disabilities. In 1976 Dociu et al. discussed 40 cases of centrofacial lentiginosis. All patients had strict centrofacial localization of the lentigines with none on the rest of the skin. The subjects were grouped according to the method by which they were identified. Eleven subjects (identified by profound psychiatric difficulties) demonstrated frequent and marked bone dysmorphism that appeared to correlate with the severity of their psychiatric disease. Twenty-three students (found by surveying 1500 vocational school students), when compared with a control group of 23 unaffected students, evidenced an increased number of bone abnormalities, dysraphic anomalies, endocrine dysfunction and neurological disease. Six previously unidentified factory workers were also investigated. No investigation was made of any of the subjects’ families.

A Comment on the Scientific Literature Accounts of centrofacial lentiginosis in the scientific and medical literature are seemingly limited to 14 in number. Of these, seven were multiply authored by two or three of the following: Dociu, Galaction-Nitelea, and Murgu (Dociu et al., 1971, 1972, 1974a, b, 1976; Murgu et al., 1975; Nitelea and Dociu, 1974) and four come from Touraine himself (Touraine, 1941a, c, 1942, 1958). Consequently, these accounts emanate from only five discrete authors or author groups. These reports are confined to the 35 years extending from 1941 to 1976, and they are published in five different languages, French (Delay, 1946; Dociu, 1971; Mercadal-Peyri, 1953; Touraine, 1941a, c, 1942, 1958), German (Dociu, 1972), Italian (Montagnani, 1959; Murgu, 1975; Musumeci, 1947), Rumanian (Dociu, 1974a, b), and English (Dociu, 1976). At the time of this writing Dociu et al.’s. 1976 exposition is the sole English 838

language report and also the last mention of centrofacial lentiginosis in the literature.

Synonyms La lentiginose centrofaciale et dysplasias associeés (Touraine, 1941a, c), la lentiginose centrofaciale neurodysraphique (Touraine, 1958), lentiginose dysraphique médiofaciale (Delay and Pichot, 1946), centrofacial neurodysraphic lentiginosis, la lentiginose centrofaciale de Touraine, la lentiginosi centrofaciale neurodisrafica di Touraine.

Clinical Description Centrofacial lentiginosis is a neurocutaneous syndrome characterized by centrofacial lentigines, dysraphic malformations, and neuropsychiatric problems (Touraine, 1941a, c, 1958). Touraine concluded that these three “independent traits” demonstrated such a high frequency of association as to exclude the possibility of coincidence (Touraine, 1941a, c, 1958). Musumeci (1947), on the other hand, questioned whether the frequency of association was truly greater than that of chance, and Delay and Pichot (1946) considered mental retardation a variably penetrant aspect of the centrofacial lentiginosis syndrome.

Lentigines Numerous lentigines are observed on the nose and cheeks in a “butterflylike” pattern. The spots generally are restricted to a horizontal band across the central face. Occasionally there are also lentigines on the forehead, eyelids, and upper lip. The number of lentigines and their extent is variable. On any one individual’s face, as few as 30 and as many as 100 lentigines have been reported (Dociu et al., 1971). As a rule they are not present on the body, limbs, or mucous membranes. Other types of pigmented lesions such as the nevus spilus, café-aulait macules, and nevus verrucous (junctional nevus?) are almost always absent. The number of lentigines is not known to correlate with the extent of associated anomalies. Lentigines are described as appearing in the first year of life, accumulating until 8–10 years, then regressing (Dociu et al., 1976). The extent and frequency to which lentigines fade after puberty, as they do in many other lentiginoses, has not been addressed. Touraine’s oldest patient exhibiting lentigines was 40 years of age (Touraine, 1958), whereas Dociu et al. (1976) were cognizant of individuals older than 54 years who still had visible lentigines.

Status Dysraphicus, the Dysraphic State, Dysraphia Dysraphia is defined as a collection of developmental anomalies thought to be secondary to incomplete fusion of the developing neural tube. This group of anomalies and associated psychiatric problems were well described before the introduction of centrofacial lentiginosis syndrome and frequently were noted in cases of congenital syphilis (Curtius and Lorenz, 1933; Touraine, 1937b). Most centrofacial lentiginosis anomalies are consistent with those previously described for the “dysraphic state” (Touraine, 1937b). They include

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

broad forehead (“olympian”), pectus carinatum (“chicken chest”), absent xiphoid process, umbilical hernia, cervicodorsal kyphosis, spina bifida, sacral dimple (foveola coccygea), lumbar hypertrichosis, coalescence of the eyebrows, high arched palate, absence or separation of upper medial incisors, poorly attached teeth, protrusion of the sternal angle of Louis, scoliosis, widened lumbar vertebral saddles, short and wide body habitus, bilateral clubfoot, and palmoplantar hyperhidrosis with bodily hypohidrosis (Touraine, 1941c). One or more of these anomalies were present in 50% of Touraine’s initial cases (Touraine, 1958) and in 100% of Mercadal-Peyri’s (1953) cases.

Endocrine Disturbances Delayed puberty (Dociu et al., 1976; Musumeci, 1947) and adiposogenital syndrome (Dociu et al., 1972, 1976; Touraine, 1941c) have been reported. Adiposogenital syndrome, known as Fröhlich syndrome when caused by an anterior pituitary tumor, is characterized primarily by obesity, genital hypoplasia, and altered radiographic appearance of the sella turcica. Touraine considered it to be present, in partial or complete form, in 20% of his original subjects. Conversely, in 1958 he described its frequency as mirroring that occurring in dysraphia, or 50–67% of cases (Touraine, 1958).

Metabolic Dysfunction Neuropsychiatric Disorders Neurologic manifestations in Touraine’s patients included infantile hemiplegia, infantile seizures, and epileptic seizures (Touraine, 1941c). Frequent mental retardation and epilepsy in most patients with centrofacial lentiginoses was noted by Dociu in 1976. Psychiatric impairment has been the subject of disagreement. The reported association of psychiatric disorders with centrofacial lentiginosis ranges from none (Dociu et al., 1976) to greater than 90% (Touraine, 1958). Dociu et al. did not find an increased incidence of psychiatric problems in patients identified by school survey. However, they did note that the vocational school students had been relatively preselected by entrance tests, schooling, and medical examinations. Conversely, 29 of Touraine’s 32 cases (91%) and 22 of MercadalPeyri’s 25 cases (88%) were reported to have psychiatric problems (Mercadal-Peyri, 1953; Touraine, 1958). Indeed, re-evaluation of Mercadal-Peyri’s cases suggested to Dociu et al. (1976) that 100% of them exhibited psychiatric disturbances. Psychiatric problems noted were poor school performance, significantly decreased intelligence, persistent nocturnal bed wetting beyond childhood, continued bowel and bladder incontinence into adulthood, introversion with apathy, profound memory impairment, attention difficulties, emotional instability, and physical and mental agitation (Touraine, 1941a, c, 1958). A small group of the patients that Dociu et al. presented in 1976 (psychiatric referral patients) demonstrated a strong correlation between the extent of neuropsychiatric difficulties and the degree of dysraphic involvement.

Associated Disorders Small Sella Turcica Alterations of the sella turcica have been reported by Murgu et al. (1975), Dociu et al. (1976), and others. As measured radiographically, small sella turcica was present in 54% of Dociu’s psychiatric referral subjects, 33% of six factory workers, 21% of 23 students, and, interestingly, 26% of 23 control students.

Vertigo with nausea, excessive sweating, a labile pulse and blood pressure, or damp and cyanotic extremities (Dociu et al., 1976) have been noted in some individuals with centrofacial lentiginosis.

Congenital Ocular Anomalies Malformation of the pupillary membrane, nevus of the iris, and poor refraction, were described in 1971 by Dociu et al.

Epidemiology Inheritance Familial involvement was demonstrated in 63% of Touraine’s 30 cases (19 subjects were part of eight families), but only 12% of Mercadal-Peyri’s 25 cases. Inheritance was direct in four of Touraine’s eight families and skipped a generation in the other four. Within sibships the ratio of subjects with lentigines to those without was 15:13, strongly suggesting dominant mendelian inheritance (Touraine, 1941c). Three families meeting the criteria for centrofacial lentiginosis syndrome have been reported in the literature, i.e., a sibship of four with no mention of the parents (Delay and Pichot, 1946), a mother with six children (Musumeci, 1947), and a third family (Montagnani and Mariotti, 1959). Family histories for the other published cases were either not investigated, not reported, no longer available, or in German, Romanian, or Italian.

Prevalence Touraine reasoned that the syndrome was not rare because 25 of his initial 30 cases came to attention in the year preceding his description (Touraine, 1941c). However, the prevalence was not extensively studied. When two large groups were surveyed, centrofacial lentiginosis syndrome was found in 1.25% of 2000 school children (Mercadal-Peyri, 1953), and 1.54% of 1500 vocational school adolescents (Dociu et al., 1976).

Sexual Distribution A male to female ratio of 2:1 was noted in the affected vocational students, although the ratio of males to females for the entire student body was not available for comparison (Dociu et al., 1976). A 2.7: 1 ratio was noted in Dociu’s group of 11 affected individuals with profound psychiatric disturbances (Dociu et al., 1976). 839

CHAPTER 46

Unknown. The described cases apparently occurred in European Caucasians from France, Spain, Rumania, and Sicily.

turbances or somatic malformations, and lentigines are also present on palms, soles, and buttocks. (See Other Lentiginoses section, this chapter.)

Age

Pathogenesis

Manifestations of this congenital disorder tend to be noted early in life. Skeletal and other dysraphic anomalies are generally discovered at birth. Lentigines appear in the first year of life, accumulate until 8–10 years, then tend to regress (Dociu et al., 1976). Decreased mental intelligence is often discovered in childhood, though psychiatric disturbances may not be apparent until later.

Not known. Speculations include the following.

Ethnic Distribution

Histology When histologic examination was performed, pigmented spots were consistent with lentigo simplex.

Laboratory Findings and Investigations Dociu et al. found abnormal urinary 17 corticosteroids and simple goiters in several of their affected patients. In addition, hypocalcemia and hyperphosphaturia were often associated with metabolic dysfunction in these patients (see above) (Dociu et al., 1976). Abnormal electroencephalographic studies have been reported (Dociu et al., 1972, 1974a, 1976; Montagnani and Mariotti, 1959). Tests for syphilis in affected individuals and their families were performed in 1941, 1946, and 1953 (Delay and Pichot, 1946; Mercadal-Peyri, 1953; Touraine, 1941c) as noted in the section on pathogenesis below.

Criteria for Diagnosis Butterfly pattern of facial lentigines suggests the diagnosis. As a rule there are no lentigines or pigmentary changes elsewhere on the skin (Touraine, 1941a, c, 1958).

Differential Diagnosis Carney complex and myxoma syndrome patients display a similar mediofacial distribution of lentigines, but other lentigines and pigmented lesions are present as well as mucocutaneous myxoid tumors. Cardiac myxomas, an important component of Carney complex/myxoma syndrome, have not been described in the centrofacial lentiginosis syndrome. (See Carney complex/myxoma syndrome below.) LEOPARD syndrome includes lentigines over the entire body, highly concentrated over the neck and upper trunk and sparse on the face. Although skeletal anomalies and endocrine involvement (delay of growth and puberty) overlap slightly with similar aspects of centrofacial lentiginosis, deafness, cardiac conduction defects, and pulmonary stenosis do not. Lentigines and other pigmented macules observed in the Peutz–Jeghers syndrome occur in large numbers and are prominent in perioral and perianal locations as well as on mucous membranes. There is an associated gastrointestinal polyposis with tendency to malignant degeneration. Patterned lentiginosis in black people (O’Neill and James, 1989) demonstrates a similar centrofacial distribution of lentigines. However, the subjects do not manifest neuropsychiatric dis840

Syphilis-induced Mutation Hypothesis Results of laboratory testing for syphilis in families with centrofacial lentiginosis led Touraine to conclude that Treponema pallidum was a potential causative agent responsible for a genetic mutation that was then inherited in an autosomal dominant fashion (Touraine, 1941a, c). He stated that “the role of congenital syphilis seems to be large because it was found in 8 of 12 families tested. An interesting fact is that syphilis has not been found in families where lentigines existed in the parents but was uniformly found in families that did not demonstrate parental lentigines. The role of syphilis seems small or nonexistent when centrofacial lentiginosis exists in the parental generation. It seems to be of greater importance in families without preexisting disease. The role of Treponema pallidum in causing the mutation which leads to mendelian transmission of the lentiginosis and its associated dysplasias was proposed by Touraine but not substantiated. This is an example of Lamarckian genetics that certain infectious agents acting as a mutational factor lead to anomalies that are transmitted from generation to generation by classical genetics. This theory was stated to be proved in the Castor Rex rabbit and also for the dysraphic state (Touraine, 1941a, c). This theory of pathogenesis was again espoused in his 1958 paper, although without further evidence than that offered in 1941 (Touraine, 1958). In a commentary accompanying Touraine’s initial description, Milian wrote: “I have observed for a long time this lentigo (in a butterfly distribution on the nose and cheeks as in lupus erythematosus) exclusively in little children with congenital syphilis. My conclusion was that this was the stigmata of congenital syphilis. Therefore it is not surprising that in the children with these lentigines we see enuresis, behavior problems, stealing, diverse dystrophy, and all the usual associations with congenital syphilis” (Milian, 1941). No one in Delay and Pichot’s sibship of four, reported in 1946, was infected with syphilis. The parents were not tested for syphilis, nor was their clinical presentation described. Parental syphilis was present in 16% (4) of the school children Mercadal-Peyri reported in 1953, but it is unclear how many (if any) of these families demonstrated stigmata of centrofacial lentiginosis, or what the prevailing rate of syphilis was in that community. The issue of syphilis is not addressed further. However, it is interesting to note that penicillin, the first cure for syphilis, became widely available in the mid to late 1940s.

Other Hypotheses Mercadal-Peyri pointed out the relative frequency of alcoholism among parents of affected children and raised the

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

question of the mutational action of intoxicants such as alcohol (Mercadal-Peyri, 1953). Montagnani and Mariotti (1959) proposed hypothalamic injury as the cause.

Mechanistic Suggestions Touraine thought skull-base dysraphia explained the mechanism by which centrofacial lentiginosis syndrome was created. “In this particular disease dysraphia may extend to both the brain thus accounting for neuropsychiatric problems, and the hypophysis explaining the pigmentation and adiposogenital syndrome” (Touraine, 1941a, c, 1958). Bremer wrote about both the etiology and the pathology of status dysraphicus, in 1926 and 1927, respectively. The high rate of association between dysraphia and adipsogenital syndrome, according to Bremer, could be explained by the gliomatosis which frequently accompanied dysraphia.

Animal Models No animals are known to resemble the centrofacial lentiginosis syndrome. However, Touraine refers to anomalies caused by “certain infectious agents” which are then transmitted from generation to generation by mendelian genetics. He makes the non-referenced statement that “this fact has been shown in the castorrex rabbit, and also for the dysraphic state” (Touraine, 1941a).

Treatment Lentigines are treated for cosmetic purposes only (see Chapters 59–64). Supportive medical, psychiatric, and educational care are important for other anomalies.

Prognosis There are no reports of altered mortality in association with the centrofacial lentiginosis syndrome.

Additional Background Information Prior to 1941, “dysraphic” disorders (problems secondary to failure of complete neural tube fusion) had been of particular interest to Touraine. This included publication of at least eight treatises between 1933 and 1938 delineating the spectrum of malformations and psychiatric difficulties associated with the dysraphic state (Touraine, 1941c). His interests also extended to pigment involvement in neuroectodermal diseases including neurofibromatosis, cutaneous melanoblastosis (characterized by the presence of melanoblasts in cutaneous growths), and the periorificial lentiginosis and visceral polyposis of Peutz (Peutz, 1921; Touraine, 1937a, 1941b, 1949; Touraine and Couder, 1945). In fact, because Touraine’s own work with Peutz syndrome (Touraine and Couder, 1945) predated that of Jeghers (Jeghers et al., 1949) by four years, he was offended by the appellation “Peutz–Jeghers syndrome” (Touraine, 1958).

References Bremer,

F.

W.

Klinishe

Untersuchungen

zur

Ätiologie

der

Syringomyelie, der “status dysraphicus.” Dtsch Z. Nervenheilk. 95:1–103, 1926. Bremer, F. W. Die pathologische anatomische Bearüngdes status dysraphicus. Dtsch Z. Nervenheilk. 99:104–123, 1927. Curtius, F., and I. Lorenz. Uber den Status dysraphicus. Klinisch–erbbiologische und rassehygienische Untersuchungen an 35 Fallen von Status dysraphicus und 17 Fallen von Syringomyelie. Zeitschr. f. d. Ges. Neurol. u. Psychiatrie 149:1–49, 1933. Degos, R. Dermatologie. In: Collection médico-chirurgical à revision annuelle, Publiée sous la direction de Pasteur Vallerg-Radot et Jean Hamburger (eds). Paris: Flammarion. éd., 1953, p. 756 (as cited by Touraine, 1958). Delay, J., and P. Pichot. Sclérose tubéreuse de Bourneville et lentiginose dysraphique médio-faciale. Rev. Neurol. (Paris) 78:233–234, 1946. Dociu, I., O. Galaction-Nitelea, N. Sirjita, and V. Murgu. Touraine’s centrofacial lentiginosis (CFL) (7 cases) [La lentiginose centrófaciale de Touraine (LCF) (à propos de 7 cas). French]. Presse Med. 79:1111–1112, 1971. Dociu, I., O. Galaction-Nitelea, N. Sirjita, and V. Murgu. Centrofacial lentiginosis, observations on 9 patients [Die centrofaciale lentiginose, beobachtungen an 9 patienten, German]. Hautarzt 23:389– 392, 1972. Dociu, I., O. Galaction-Nitelea, V. Murgu, and D. Gheorghiu. Centrofacial lentiginosis (CFL); study of 23 cases [Lentiginoza centrofaciala (LCF); studiu pe 23 de cazuri, Rumanian]. Revista de Pediatrie, Obstetrica Si Ginecologie-Pediatria 23:407–411, 1974a. Dociu, I., O. Galaction-Nitelea, N. Sirjita, and V. Murgu. Touraine’s centrofacial lentiginosis (CFL) [Lentiginoza centrofaciala Touraine (LCF), Rumanian]. Neurologia, Psihiatria, Neurochirurgia 19:21– 26, 1974b. Dociu, I., O. Galaction-Nitelea, N. Sirjita, and V. Murgu. Centrofacial lentiginosis: A survey of 40 cases. Br. J. Dermatol. 94:39–43, 1976. Jeghers, H., V. A. McKusick, and K. A. Katz. Generalized intestinal polyposis and melanin spots of the oral mucosa, lips and digits: A syndrome of diagnostic significance. N. Engl. J. Med. 241:993– 1005,1031–1036, 1949. Lutz, W. Lehrbuch d. Haut u. Geschl. Krankheiten, 2e édit: 536, 1957. (As cited by Touraine, A. La Presse Médicale 66:1611, 1958). Mercadal-Peyri, J. VIIIe Congrès des dermatologistes et syphilographes de langue française (Nancy-Vittel, 29–31 Mai 1953). Communications et discussions: 172–176, 1953. As cited in Touraine, 1958. Milian, M. Commentary. Appended to: Touraine, A. Bull. Soc. Franç. Dermatol. Syphiligr. 48:518–521, 1941. Montagnani, A., and F. Mariotti. The neurodysraphic centro-facial lentiginosis of Touraine (A case report). [La lentiginosi centrofaciale neuro-disrafica di Touraine (A proposito di un caso clinico)]. Minerva Dermatol. 34:588, 1959. Murgu, V., I. Dociu, N. Sirjita, and O. Galaction-Nitelea. Osseous anomalies of centrofacial lentiginoses (39 cases), [Anomalies osseuses dans la lentiginose centro-faciale, A propos de 39 cas, Italian]. J. Radiol. Electrol. Med. Nucl. 56:75–81, 1975. Musumeci, V. [Studio clinico e critico sulla lentigginosi centro-faciale. Italian]. G. Ital. Dermatol. Sifilol. 88:502–511, 1947. Nitelea, O. G., and I. Dociu. Lentiginoses and anomalies of development. Case report of a family with lentiginosis associated with dysraphism and mental debility [Lentiginoses et anomalies de dévelopement, Observation familiale de lentiginose avec dysraphisme et débilité mentale, French]. Dermatologica 148:108–115, 1974. O’Neill, J. F., and W. D. James. Inherited patterned lentiginosis in blacks [review]. Arch. Dermatol. 125:1231–1235, 1989. Peutz, J. L. A. Very remarkable case of familial polyposis of mucous membrane of intestinal tract and nasopharynx accompanied by

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CHAPTER 46 peculiar pigmentations of skin and mucous membrane. Nederl. Maatsch. N. F. 10:134–146, 1921. Touraine, A. Bull. Soc. Franç. Dermatol. Syphiligr. 14 January: 81. 1937a. Touraine, A. The dysraphic state [L’etat dysraphique, French]. Le progres medical 65:361, 1937b. Touraine, A. Centro-facial lentiginosis and associated dysplasias [Lentiginose centro-faciale et dysplasies associées, French]. Bull. Soc. Franc. Dermatol. Syphiligr. 48:518–521, 1941a. Touraine, A. A new congenital neuro-ectodermatosis: Neurocutaneous Melanoblastosis [Une nouvelle neuro-ectodermose congénitale: La Mélanoblastose Neuro-cutanée, French]. Presse Med. 49:1087–1088, 1941b. Touraine, A. A novel congenital neuro-ectodermatosis: Centrofacial lentiginosis and associated dysplasias [Une nouvelle neuroectodermose congénitale: La lentiginose centrofaciale et les dysplasies associée, French]. Ann. Dermatol. Syphiligr. (Paris) 8:453–473, 1941c. Touraine, A. A unique congenital neuroectodermatosis: Centro-facial neuro-dysraphic lentiginosis and its associated dysplasias [Une neuroectodermose congenitale inédite: La lentiginose neurodysraphique centro-faciale et ses dysplasies associées, French]. Sem. Hopital Paris 18:53–59, 1942. Touraine, A. Neuro-cutaneous melanosis [Les mélanoses neurocutanées, French]. Ann. Dermatol. Syphiligr. (Paris) 9:489–524, 1949. Touraine, A. Centro-facial neuro-dysraphic lentiginosis [La Lentiginose centro-faciale neuro-dysraphique French]. Presse Med. 66:1611, 1958. Touraine, A., and F. Couder. Periorificial lentiginosis and visceral polyposis [Lentiginose péri-orificielle et polypose viscerale, French]. Bull. Soc. Franc. Dermatol. Syphiligr. 5:313, 1945.

LEOPARD Syndrome Mary K. Cullen

Historical Background In 1902 Darier presented a patient with clinical entity known as “lentiginosis profusa.” In 1921 Jordan and in 1927 Almqvist discussed the clinical features of the entity known as profuse lentigines. In 1926 Weber presented a patient who had developed full manifestations of von Recklinghausen neurofibromatosis 21 years after lentigines were noted. In 1936 Zeisler and Becker presented a large pedigree of “generalized lentigo.” Touraine, who had earlier described centrofacial lentiginosis and Peutz–Touraine–Jeghers syndrome, in 1941 described a person with “lentigo profusa.” Zeisler also presented a 24-year-old female with multiple lentigines and osseus abnormalities. Lentigines spared the face and showed a marked increase in number up to puberty (Zeisler and Becker, 1936). Pipkin and Pipkin (1950) subsequently presented a large pedigree with eight affected members, three generations of whom had lentigines, features suggesting an autosomal dominant mode of inheritance. In 1957 a few individuals with pulmonary stenosis, nevi, and deafness were noted within a large study of congenital heart disease (Lamy et al., 1957). Lewis et al. (1958), and later Koroxenidis et al. (1966), described an African American family in whom deafness, cardiac defects, mild facial and skeletal defects, and retarded growth were all present. There was no mention of unusual pigmentation. 842

Moynahan (1962) is credited with the initial description of generalized lentigo as a syndrome. He reported three unrelated patients with reduced stature and normal intelligence. The two girls had delayed puberty, one with only a single hypoplastic ovary. Both girls were thought to have congenital mitral stenosis, termed endomyocardial fibroelastosis. The boy had hypospadias and an undescended right testicle with a normal clinical cardiologic examination. Biopsy of a pigmented spot showed a junctional nevus. Other reports followed. In 1966 Walther et al. described echocardiographic abnormalities in a family with generalized lentigo for which recessive inheritance was suggested (Walther et al., 1966). In 1968 Walther and colleagues reported another family with the LEOPARD syndrome with an autosomal pattern of inheritance. Features present in individuals within the family included multiple lentigines, cardiac abnormalities, short stature, delayed puberty, hypospadias, and strabismus (Walther, 1968). The same year Matthews reported a family with multiple generalized lentigines in three generations, electrocardiographic (EKG) abnormalities in two generations, and cardiac murmurs but no clinically significant cardiac disease. No abnormal growth, hearing loss, genital abnormalities, or mental retardation were associated (Matthews, 1968). Prominent spotty pigmentation of the periorbital skin with cardiac defects (subaortic and subpulmonic stenosis, probable biventricular hypertrophy) and hyperflexibility of the metacarpophalangeal joints were found. A relation between this cardiocutaneous syndrome and familial obstructive cardiomyopathy was suggested (Kraunz and Blackmon, 1968). Moynahan (1970) reported the first autopsy findings of a patient with this syndrome. Gorlin et al. (1969) postulated that multiple lentigines represented one facet of a more generalized hereditary syndrome and proposed the acronym LEOPARD (Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness, sensorineural type). LEOPARD syndrome was to encompass those disorders previous known as generalized lentigo, cardiocutaneous syndrome (Kraunz and Blackmon, 1968; Walther, 1968; Walther et al., 1966), familial pulmonary stenosis (Campbell, 1962; Carleton et al., 1958; Coblentz and Mathivat, 1952; Koretsky, 1969), pulmonary stenosis with dwarfism and undescended testicles (Campbell, 1962), and dysplastic pulmonary valve syndrome (Koretsky, 1969). Somerville and Bonham-Carter (1972) were the first to carefully document and define the range of cardiologic findings present in the multiple lentigines syndrome. Likewise, Polani and Moynahan (1972) emphasized the importance of cardiomyopathy in the syndrome. Summit in 1969, and then Selmanowitz et al. in 1970, noted that the LEOPARD syndrome shared a constellation of manifestations with other genodermatoses, notably von Recklinghausen neurofibromatosis (NF) and Noonan syndrome. A direct relation between NF and LEOPARD syndrome was proposed by Selmanowitz in 1971. Using linkage analysis, Ahlbom and colleagues (1995) were apparently able to demonstrate that LEOPARD syndrome was not linked to the

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

NF1 gene. However, in 2002 Digilio et al. showed that LEOPARD syndrome and Noonan syndrome are allelic disorders on the NF1 (PTPN11) gene of chromosome 12q24. Legius et al. (2002) then described a point mutation resulting in a tyrosine-to-cystine substitution at amino acid 279. Threonine-to-methionine changes (amino acid 468) were also implicated in LEOPARD syndrome. In 2003 Conti et al. described a third change, that of glutamine-to-proline at amino acid 506. This finding was surprising in light of the fact that changes at amino acids 501–504 cause Noonan syndrome. (See also Chapter 57 for a discussion of the genetics.)

Synonyms Lentiginosis profusa (Darier, 1902), lentiginosis profusa syndrome (Walther, 1968), generalized lentigines/generalized lentigo (Zeisler and Becker, 1936), cardiocutaneous syndrome (Kraunz and Blackmon, 1968; Walther, 1968; Walther et al., 1966), familial obstructive cardiomyopathy (Kraunz and Blackmon, 1968), diffuse lentigo (Kraunz and Blackmon, 1968), multiple lentigines/multiple lentigos syndrome (Gorlin et al., 1969), little leopard (Benthem et al., 1977; Pickering et al., 1971), progressive cardiomyopathic lentiginosis syndrome (Polani and Moynahan, 1972), lentiginocardiomyopathic syndrome (Jurko et al., 1978), Moynahan’s syndrome (Goudie et al., 1985), spotting heart disease (Goldstein and Dunn, 1987), and congenital lentiginosis (Jurko et al., 1991). Current usage of the term lentigines profusa may refer to generalized lentigines without associated anomalies (Ber Rahman, 1996). Although the acronym LEOPARD is the most commonly used term, it is not uniformly accepted. Selmanowitz and colleagues (1971) argued that it does not specify the nature of the cutaneous lentigines in addition to questioning the tastefulness of the term. “Multiple lentigines syndrome” was preferred over “lentiginosis profusa” by Selmanowitz because the number of lentigines present was sometimes small and “multiple means more than one while profuse means abundance” (Selmanowitz et al., 1971). Colomb and colleagues noted that LEOPARD used as a mnemonic identified only some of the possible associated features (Colomb and Morel, 1984). They also emphasized that manifestation of all LEOPARD features in one individual was extremely rare. “Lentiginocardiomyopathic syndrome” was suggested by Jurko et al. in 1978 for a similar reason. They noted that the major syndrome features got lost among the many possible features present in the mnemonic LEOPARD.

colleagues features with the strongest penetrance are also those most constantly found and include EKG abnormalities, pulmonary stenosis, growth retardation, and sensorineural deafness (Butenschon et al., 1979). However, Bleehen (1992) lists deafness as one of the inconstant features.

Clinical Description The features most commonly associated as proposed by Gorlin et al. in 1969 are: ∑ lentigines ∑ EKG abnormalities ∑ ocular hypertelorism ∑ pulmonary stenosis ∑ abnormalities of genitalia ∑ retardation of growth ∑ deafness, sensorineural.

Lentigines Lentigines are considered the cutaneous hallmark of LEOPARD syndrome (Ruiz-Maldonado et al., 1983). Most are 5 mm or less in diameter, round to oval in shape, and dark brown to black in color (Rhodes, 1993) (Figs 46.6–46.9). Usually profuse in number, lentigines are distributed over the entire body surface, except mucous membranes. They appear on both sun-exposed and protected areas of the skin and lips

46.6

Epidemiology The inheritance of LEOPARD syndrome is autosomal dominant with high penetrance and variable expression (Gorlin and Sedano, 1969). This was well illustrated in the family presented by Seuanez et al. in 1976 in which five of six family members exhibited only lentigines, while the sixth had lentigines, cardiac defects, ocular hypertelorism, genital abnormalities, and growth retardation. According to Butenschon and

46.7 Figs 46.6 and 46.7. Note the extensive skin involvement by innumerable lentigines (see also Plates 46.6 and 46.7, pp. 494–495).

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Selmanowitz et al., 1971), or as an isolated finding (Nitelea and Dociu, 1974; Walther et al., 1966).

Other Cutaneous Findings

Fig. 46.8. Close-up view of the lentigines of this syndrome (see also Plate 46.8, pp. 494–495).

A few additional cutaneous lesions have been described including abnormal palmar dermatoglyphics (Capute, 1969; Capute et al., 1969; Hornstein and Weidner, 1971; Paver and Coleman, 1971; Selmanowitz, 1971; Selmanowitz et al., 1971), interdigital webs (Selmanowitz, 1971; Selmanowitz et al., 1971; Watson, 1967), onychodystrophy (Selmanowitz, 1971; Selmanowitz et al., 1971), alopecia areata (Kristensen and Thestrup-Pedersen, 1989), hyperelasticity (Voron et al., 1976), multiple granular cell “myoblastomas” (Capute et al., 1969; Selmanowitz, 1971; Selmanowitz et al., 1971), and morphea with acro-osteolysis (Dacey et al., 1998).

Associated Disorders Ocular Hypertelorism, Facial and Skeletal Malformations

Fig. 46.9. Multiple lentigines in a patient with LEOPARD syndrome (see also Plate 46.9, pp. 494–495).

with the highest concentration on the neck and upper trunk (Rhodes, 1993; Selmanowitz et al., 1971). Lentigines appear at or shortly after birth, increase in number with age until puberty (Gorlin et al., 1969; Zeisler and Becker, 1936) or adulthood, then remain unchanged or fade in midlife. One individual has been reported with perioral predominance of the lentigines (Kraunz and Blackmon, 1968), while another developed her first lentigines at the age of 35 years (Kuhn et al., 1977).

MacEwen and Zaharko (1973) described and discussed the range of bony abnormalities seen in individuals with the LEOPARD syndrome. Later, Colomb and Morel (1984) held the primordial syndromic signs to be osseous. Ocular hypertelorism (Zeisler and Becker, 1936) and mild mandibular prognathism (Pipkin and Pipkin, 1950) are the most frequent of a variety of skeletal abnormalities. Others include: pectus carinatum or excavatum (David, 1973; Lassonde et al., 1970; Matthews, 1968; Nordlund et al., 1973; Pipkin and Pipkin, 1950; Poznanski et al., 1971; Voron et al., 1976; Watson, 1967), dorsal kyphosis (Koroxenidis et al., 1966), lateral flattening of the chest (Matthews, 1968), winging of the scapulae (Gorlin et al., 1969), and cervical spine fusion. Bony malformation of the chest has even been cited as causing death by chronic respiratory insufficiency in one case (Peter and Kemp, 1990). Hyposmia (Swanson et al., 1971) and Chiari malformation (cerebellar herniation) have also been described (Agha and Hashimoto, 1995). Ho, O’Donnell, and Rodrigo described supernumerary teeth and discussed the dental care risks of LEOPARD syndrome (Ho et al., 1989). Multiple dental anomalies, basilar impression, and platybasia were present in a patient reported by Peixoto et al. (1981). Uthoff (1987) reviewed dental defects and their relevance. Rodrigo et al. (1990) covered the use of dental anesthesia in affected individuals. Additional dental anomalies were present in a patient presented by Yam et al. (2001).

Other Pigmentation The prevalence of café-au-lait spots is increased in those with LEOPARD syndrome (Ortonne et al., 1980; Watson, 1967), and a few large café noir spots have been noted on the trunk (Gorlin et al., 1969). Large patch lentigines are frequently present, often several per individual (Capute et al., 1969; Lynch, 1970). Crowe’s sign (axillary freckling) has also been noted in LEOPARD syndrome (David, 1973; Selmanowitz, 1971; Selmanowitz et al., 1971; Voron et al., 1976). Localized hypopigmentation has rarely been noted at the sites of previous lentigines (Selmanowitz, 1971; 844

Deafness, Sensorineural Deafness, when present, is apparent by 9 months after birth and is of the neurosensory type (Capute et al., 1969; Lamy et al., 1957; Lewis et al., 1958). Moatti et al. (1990) included one patient with LEOPARD syndrome in their review of congenital sensorineural deafness.

EKG and Structural Cardiac Anomalies Cardiac abnormalities are frequent and are primarily conduction defects (Smith et al., 1970); EKG abnormalities include

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the S1, S2, and S3 patterns. Pulmonary valvular dysplasia is common (Koroxenidis et al., 1966; Kraunz and Blackmon, 1968; Lewis et al., 1958; Matthews, 1968; Walther et al., 1966; Watson, 1967) and is characterized by a pulmonary systolic ejection murmur without an ejection click. Some patients have had subaortic or subpulmonic defects, primary pulmonary hypertension (Blieden et al., 1981), atrial septal defect (Gorlin et al., 1969), subaortic stenosis (Kraunz and Blackmon, 1968), and congenital mitral valve stenosis “fibroelastosis” (Moynahan, 1962). Several patients with this syndrome died at an early age from an obstructive cardiomyopathy (Polani and Moynahan, 1972). Alday and Moreyra (1984) presented one patient in whom hypertrophic cardiomyopathy in childhood preceded other manifestations of the syndrome. Many excellent reviews cover various aspects of the cardiac involvement in LEOPARD syndrome: cardiovascular changes and malformations (Bochkova et al., 1982; Einhorn et al., 1986; Janus et al., 1977; Smith et al., 1970), hereditary cardiocutaneous syndromes (Schmidt, 1980), cardiac testing (Gorlin et al., 1969, 1971a, b; Miric et al., 1986; Munoz et al., 1995; Sakamoto et al., 1983; Schmidt, 1975), and cardiocutaneous diseases (Chan, 1990).

Abnormalities of Genitalia Moynahan (1962) initially described gonadal hypoplasia (absent ovary, hypoplastic ovary, hypospadias). Subsequent reports have added hypogonadotropism (Swanson et al., 1971), unilateral undescended testicle (Koroxenidis et al., 1966; Moynahan, 1962; Watson, 1967), and clitoral hypertrophy (Peixoto et al., 1981).

Growth Retardation and Endocrine Abnormalities Delayed puberty, reduced stature and growth retardation (Koroxenidis et al., 1966; Moynahan, 1962; Walther, 1968; Walther et al., 1966) are frequently present in LEOPARD syndrome. Isolated cases of pituitary insufficiency (Malpuech et al., 1979), and Graves disease (Yamamura et al., 1978) have occurred. Balducci et al. (1982) described the full endocrinologic evaluation of a child with LEOPARD syndrome.

and anal ectopy (Peixoto et al., 1981) have been described in individuals with this syndrome. LEOPARD syndrome has coexisted with various disease states including: Gerstmann tetrad syndrome (Garty et al., 1989), Noonan syndrome (Blieden et al., 1983), NF/Noonan syndrome (Tullu et al., 2000), Werner syndrome (Lazarov et al., 1995), Marfan syndrome (Torok et al., 1989, 1990), elastic dystrophy (Claudy et al., 1985), pigmented villonodular synovitis (Wendt et al., 1986), depression (Kuhn et al., 1977), and familial psychosis (Loyd and Tsuang, 1980; Loyd et al., 1982).

Other Commentaries, Case Reports and Literature Reviews English Literature Capute et al. (1969), Char (1971), Cleland et al. (1983), Colomb and Morel (1984), Cooles (1986), Cronje and Feinberg (1973), Gorlin and Sedano (1969), Gorlin et al. (1971a, b), Herzberg and Naevi (1965), Konigsmark (1969a, b), Lassonde et al. (1970), Laude et al. (1977), Macmillan and Vickers (1969), Malathi et al. (1984), Moynahan and Polani (1967), Nordlund et al. (1973), Passarge (1975), Rosen (1942), Seuanez et al. (1976), Sommer et al. (1971), Song and Toppet (1974), Ting and Ng (1983), Voron et al. (1976). Other Literature ∑ French: Battin et al. (1972), Ben Charnia et al. (1985),

Deschamps and Didier (1972), Laugier et al. (1972), Pernot et al. (1971, 1972). ∑ German: Butenschon et al. (1979), Dorittke (1992), Senn et al. (1984). ∑ Portuguese: Goncalves et al. (1987), Hornstein and Weidner (1971). ∑ Spanish: Olivan del Cacho et al. (1982), Roman (1987). ∑ Italian: Isaia et al. (1982), Morace et al. (1971), Pennelli et al. (1988). ∑ Polish: Pasyk (1977). ∑ Japanese: Takatsu et al. (1977), Tsukahara et al. (1996). ∑ Dutch: Benthem et al. (1977). ∑ Slovak: Jurko et al. (1978, 1991). ∑ Hebrew: Garty et al. (1983). ∑ Norwegian: Ogreid et al. (1986). ∑ Russian: Mikhailova (1971), Morova et al. (1991).

Other Associations Defective ocular motility (Pipkin and Pipkin, 1950), nystagmus (Zeisler and Becker, 1936), strabismus (Walther, 1968), premature cataracts (Howard, 1979), coloboma of the iris, retina and choroid (Rudolph et al., 2001), choristoma (Choi et al., 2003), hypoplastic fifth digit (Koroxenidis et al., 1966), hyperflexibility of the metacarpophalangeal joints (Kraunz and Blackmon, 1968), mental retardation (Watson, 1967), partial agenesis of the corpus callosum (Bonioli et al., 1999), cerebral arteriovenous malformations (Cabanas et al., 1985), other blood vessel alterations (Torok et al., 1989, 1990), peripheral polyaneurysms (Yagubyan et al., 2004), renal anomalies (Jurecka et al., 1983; Swanson et al., 1971), lymphedema (Wendt et al., 1986), and macroglossia, megacolon,

Routine Histology and Histochemistry The histology of lentigines in LEOPARD syndrome was reviewed by Selmanowitz (1971). Pigmented macules are histologically consistent with lentigines, characterized by melanocytic hyperplasia, increased melanin in melanocytes and basal keratinocytes, and elongation of rete ridges (Mosher et al., 1993). However, at the ultrastructural level there are several modifications. Melanocytes are not only glutted with melanosomes (Nordlund et al., 1973), but also contain packets of melanosomes known as either giant melanosomes (Bhawan et al., 1976; Selmanowitz, 1975; Weiss and Zelickson, 1977) or autophagosomes (Nordlund et al., 1973). Bhawan et al. (1976) demonstrated that these spherical and 845

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giant melanosomes are lysosome-like structures. Both Selmanowitz (1971, 1975) and Bhawan et al. (1976) considered the giant melanosome similar to those previously described in café-au-lait spots of NF. In 1984, Hull and Epinette noted that giant melanosomes may occasionally be found in several common pigmented lesions including benign lentigines and typical nevi. Nevertheless, their ultrastructural comparison of giant melanosomes present in various disease states revealed two major groups: (1) Those of LEOPARD syndrome, ocular albinism, and some lesions of dysplastic nevus syndrome composed of microvesicles without the usual limiting membrane or filamentous internal structure of normal melanosomes; and (2) those found in café-au-lait spots of NF, nevus spilus, and vitiliginous achromia (Hull and Epinette, 1984). Ordinarily melanosomes within Caucasian keratinocytes are arranged in groups which are thought to be determined by the melanosome size and racial background. Although melanosomes in Caucasian LEOPARD syndrome keratinocytes are of normal size and shape, they have been found to be distributed as singlets (Bhawan et al., 1976). Also, in contrast with the prevailing concept that only mature melanosomes are transferred to keratinocytes, immature melanosomes have been detected in keratinocytes of LEOPARD syndrome lentigines (Bhawan et al., 1976). Fryer and Pope (1992) found large accumulations of melanosomes in Langerhans cells, while Ono and colleagues (1984) reported unusual collections of dermal Merkel cells in LEOPARD syndrome lentigines.

Laboratory Findings and Investigations EKG Abnormal orientation of the QRS, possibly with widening of the QRS and bundle branch block, abnormal P waves and prolongation of the PR interval, and ST and T wave changes.

Other Hematologic evaluation did not reveal hematopoietic defects in the patient studied (Van Voolen and Selmanowitz, 1976).

Criteria for Diagnosis Identification of precise diagnostic criteria for the LEOPARD syndrome has not been straightforward. The reasons were best stated by Gorlin, Anderson, and Blaw in 1969: “Until a specific test permits exact identification of affected individuals, it will not be possible to define the limits of the syndrome or to determine the etiologic relationship of the increasing number of individuals and pedigrees currently being viewed as examples of this syndrome. It may well be that several distinct subtypes will gradually be identified, of different etiology and with somewhat different constellations of clinical signs.” Seven years later Voron et al. (1976) again commented on the marked variability in expression of the syndrome and acknowledged both the lack of a single pathognomonic finding, and the existence of few patients with all the major

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features. An excellent, extensive, review of 81 cases from the literature and one new case led to the creation of 12 detailed diagnostic categories and a proposal of minimal diagnostic criteria for the multiple lentigines syndrome. The categories were: Lentigines, Other cutaneous, Cardiac structural, Cardiac electrocardiographic, Cardiac symptoms, Genitourinary, Endocrine, Neurologic, Cephalofacial dysmorphism, Short Stature, Skeletal, and Family History consistent with autosomal dominant inheritance. Minimal diagnostic criteria rely on these categories as follows: (1) multiple lentigines and features from at least two other categories; OR (2) if no lentigines are present, features from at least three other categories AND a first-degree relative with multiple lentigines syndrome as defined in (1). Seventy-four of the reviewed cases met criteria for the first group, six fit the second group, and only two cases were excluded (Voron et al., 1976).

Differential Diagnosis Patterned lentigines with associated somatic abnormalities are also present in centrofacial lentiginosis, Carney complex/ myxoma syndrome, Peutz–Touraine–Jeghers syndrome and cardiac pre-excitation syndrome. Complete medical examination and family history, particularly cardiac, are necessary to distinguish between these syndromes. Centrofacial lentiginosis exhibits lentigines restricted to a medial band across the face, with mental retardation and epilepsy as frequent characteristics. The skeletal features overlap minimally with those of the LEOPARD syndrome. EKG changes, cardiac anomalies, other cutaneous lesions, sensorineural deafness, ocular hypertelorism, and genitourinary disturbances have not been described. Carney complex/ myxoma syndrome patients often have multiple cardiac and cutaneous myxomatous lesions. Endocrine overactivity, caused by adrenal hyperplasia, testicular tumors, and ovarian tumors, is frequent. Those patients without myxomatous lesions have all had endocrine overactivity or a first-degree relative with myxoma syndrome. Peutz–Touraine–Jeghers syndrome patients almost always have lentigines on the oral mucosa. Additionally the syndrome is characterized by mucocutaneous pigmentation, intestinal polyposis, and hamartomatous and malignant neoplasms in extraintestinal sites (Jeghers et al., 1949; Peutz, 1921; Rhodes, 1993; Touraine and Couder, 1945). Cardiac, auditory, cephalofacial, and genitourinary anomalies are not part of the syndrome. Noonan syndrome patients with several features of the LEOPARD syndrome, but who lack lentigines and a positive family history have been described (Summit, 1969; Voron et al., 1976).

Pathogenesis An unknown alteration of the neural crest cells during development is widely believed to be the basis of this disorder. Two groups have focused on different syndrome manifestations to support this hypothesis: Selmanowitz and colleagues (1971) suggested that an underlying neurogenic disorder, possibly mutant stem cells in the embryonic neural crest, might account

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

for the melanocytic alterations, granular cell neoplasms, oculomotor defects, hearing loss, and cardiac conduction defects. Later Peixoto et al. (1981) reasoned that the presence of dental anomalies and megacolon may represent involvement of the dental papillae and myenteric plexus, favoring the view that the syndrome results from a derangement of the neural crest element.

Animal Models Hudson and Ruben (1962) described hereditary deafness in the Dalmatian dog with abnormal spotting. Voron et al. (1976) have suggested that this may represent a partial canine analog to the LEOPARD syndrome observed in humans.

Treatment of Lentigines As with other lentigines, lesions are considered benign and no treatment is required. However, because the long-term course and malignant potential are not known, periodic examinations to detect early atypical changes in lentigines are recommended. A single case of malignant melanoma arising in one of the lentigines has been reported (Jurecka et al., 1983). There are now several methods for esthetic treatment of lentigines. An overview of these options is presented in the Lentigo Simplex and Lentigo Senilis et Actinicus sections in this chapter. A few case reports have specifically dealt with the treatment of LEOPARD syndrome lentigines. Dermabrasion eradicated many of the facial lentigines in a patient of Selmanowitz et al. with LEOPARD syndrome. The authors suggested waiting until the lentigines have stabilized post puberty before attempting abrasion (Selmanowitz et al., 1971). Van Voolen and Selmanowitz (1976) reported considerable cosmetic improvement following facial dermabrasion and light electrodesiccation. Shelley described a disturbed young woman in whom lentigines were eradicated after years of repetitive autodermabrasion (factitial erosive lesions), which was surprisingly nonscarring. Her relatively inaccessible back was the only site of residual multiple lentigines (Shelley, 1982). Fitzpatrick successfully used topical monobenzylether of hydroquinone, however partial eyebrow leukotrichia also occurred (Rhodes, 1993). Intense pulsed light has also been reported to be effective in a patient with LEOPARD syndrome (Kontoes et al., 2003). (See also Chapters 59, 63, and 64.)

Prognosis These individuals probably have a normal life span, altered by the presence, extent, and early diagnosis of heart disease.

References Agha, A., and K. Hashimoto. Multiple lentigines (Leopard) syndrome with Chiara I malformation. J. Dermatol. 22:520–523, 1995. Ahlbom, B. E., N. Dahl, P. Zetterqvist, and G. Anneren. Noonan syndrome with cafe-au-lait spots and multiple lentigines syndrome

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CHAPTER 46 peculiar pigmentations of skin and mucous membrane. Nederl. Maatsch. N. F. 10:134–146, 1921. Pickering, D., B. Laski, D. C. Macmillan, and V. Rose. “Little leopard” syndrome. Description of 3 cases and review of 24. Arch. Dis. Child. 46:85–90, 1971. Pipkin, A. E., and S. W. Pipkin. A pedigree of generalized lentigo. J. Hered. 41:79–82, 1950. Polani, P. E., and E. J. Moynahan. Progressive cardiomyopathic lentiginosis. Q. J. Med. 41:205–225, 1972. Poznanski, A. K., A. M. Stern, and J. C. Gall Jr. Skeletal anomalies in genetically determined congenital heart disease. Radiol. Clin. No. Am. 9:435–458, 1971. Rhodes, A. R. Neoplasms: benign neoplasias, hyperplasias, and dysplasias of melanocytes. In: Dermatology in General Medicine, 4th ed., T. B. Fitzpatrick, A. Eisen, K. Wolff, I. M. Freedberg, and F. K. Austen (eds). New York: McGraw-Hill, Inc., 1993, pp. 996– 1077. Rodrigo, M. R., C. H. Cheng, Y. T. Tai, and D. O’Donnell. “Leopard” syndrome. Anaesthesia 45:30–33, 1990. Roman, P. Sindrome Leopard. Piel 2:22–25, 1987. Rosen, I. Lentigenes. Arch. Dermatol. Syphil. 45:979–980, 1942. Rudolph, G., C. Haritoglou, P. Kalpadakis, K. P. Boergen, and T. Meitinger. [LEOPARD syndrome with iris-retinachoroid coloboma. Discordant findings in monozygotic twins (MIM # 151 100)]. Ophthalmologe 98:1101–1103, 2001. Ruiz-Maldonado, R., L. Trevizo, L. Tamayo, M. F. de los Rios, M. Skurovich, J. Carrillo, D. Dominguez, and V. del Castillo. Progressive cardiomyopathic lentiginosis: report of six cases and one autopsy. Pediatr. Dermatol. 1:146–153, 1983. Sakamoto, T., Y. Hada, S. Sugiura, T. Serizawa, K. Amano, T. Yamaguchi, K. Takenaka, R. Takikawa, H. Takahashi, I. Hasegawa, and K. Suga. [Generalized lentigo in a case of hypertrophic cardiomyopathy with right ventricular outflow tract obstruction] [Japanese]. J. Cardiogr. 13:1029–1040, 1983. Schmidt, B. [Cardiac arrhythmias in vector cardiograms in the “Leopard” syndrome (author’s transl.)] [German Die kardialen Reizleitungsstorungen im Vektrocardiogramm beim Leopard Syndrom. Klin. Padiatr. 187:367–369, 1975. Schmidt, H. [Hereditary syndromes with congenital cardiac defects and abnormalities of the skin especially in the LEOPARD syndrome (author’s transl)] [German] Hereditare Syndrome mit Herzfehlern und Anomalien der Haut unter besonderer Berucksichtigung des LEOPARD-Syndroms. Dermatol. Monatsschr. 166:622–628, 1980. Selmanowitz, V. J. Lentiginosis profusa syndrome (multiple lentigines syndrome). II. Histological findings, modified Crowe’s sign, and possible relationship to von Recklinghausen’s disease. Acta Derm. Venereol. 51:387–393, 1971. Selmanowitz, V. J. Lentiginosis profusa syndrome. IV. Giant pigment granules (light microscopy). Acta Derm. Venereol. 55:481–484, 1975. Selmanowitz, V. J., and N. Orentreich. Lentiginosis profusa in daughter and mother: multiple granular cell “myoblastomas” in the former. Arch. Dermatol. 101:615–616, 1970. Selmanowitz, V. J., N. Orentreich, and J. M. Felsenstein. Lentiginosis profusa syndrome (multiple lentigines syndrome). Arch. Dermatol. 104:393–401, 1971. Senn, M., O. M. Hess, and H. P. Krayenbuhl. [Hypertrophic cardiomyopathy and lentiginosis] [German]. Schweiz. Med. Wochenschr. 114:838–841, 1984. Seuanez, H., F. Mane-Garzon, and R. Kolski. Cardio-cutaneous syndrome (the “LEOPARD” syndrome): Review of the literature and a new family. Clin. Genet. 9:266–276, 1976. Shelley, W. B. Factitial dermatitis as the presenting sign of multiple

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lentigines syndrome: Therapeutic effect of autodermabrasion. Arch. Dermatol. 118:260–262, 1982. Smith, R. F., L. U. Pulicicchio, and A. V. Holmes. Generalized lentigo: electrocardiographic abnormalities, conduction disorders and arrhythmias in three cases. Am. J. Cardiol. 25:501–506, 1970. Somerville, J., and R. E. Bonham-Carter. The heart in lentiginosis. Br. Heart J. 34:58–66, 1972. Sommer, A., S. B. Contras, J. M. Craenen, and D. M. Hosier. A family study of the leopard syndrome. Am. J. Dis. Child. 121:520–523, 1971. Song, M., and M. Toppet. [Leopard syndrome] [French] Syndrome leopard. Arch. Belges Dermatol. 30:95–97, 1974. Summit, R. L. The Noonan syndrome. In: The Clinical Delineation of Birth Defects. Part V, Phenotypic Aspects of Chromosomal Aberrations, D. Bergsma (ed.). New York: The National Foundation March of Dimes, 1969, p. 39. Swanson, S. L., R. J. Santen, and D. W. Smith. Multiple lentigines syndrome: new findings of hypogonadotrophism, hyposmia, and unilateral renal agenesis. J. Pediatr. 78:1037–1039, 1971. Takatsu, T., T. Tanaka, H. Morita, N. Sontani, and K. Imamura. [Lentiginosis profusa syndrome and the heart: Leopard syndrome and hypertrophic obstructive cardiomyopathy] [Japanese]. Nippon Rinsho 35:1724–1737, 1977. Ting, H. C., and S. C. Ng. The leopard (multiple lentigines) syndrome: A case report. Med. J. Malaysia 38:98–101, 1983. Torok, L., L. Szentendrei, M. Szili, and S. Budai. [Progressive cardiomyopathic lentiginosis (Leopard syndrome, Moynahan syndrome)] [Hungarian]. Orv. Hetil. 130:1531–1532,1535–6, 1989. Torok, L., L. Szentendrei, M. Szili, and S. Budai. [Progressive cardiomyopathic lentiginosis (LEOPARD syndrome) in 3 patients, combined with Marfan syndrome] [German]. Z. Hautkr. 65:197–201, 1990. Touraine, A. La melanoblastose neurocutanée. Bull. Soc. Franc. Dermatol. Syphiligr. 51:421–431, 1941. Touraine, A., and F. Couder. Periorificial lentiginosis and visceral polyposis [Lentiginose péri-orificielle et polypose viscerale, French]. Bull. Soc. Franc. Dermatol. syphiligr. 5:313, 1945. Tullu, M. S., M. N. Muranjan, V. C. Kantharia, R. C. Parmar, D. R. Sahu, S. B. Bavdekar, and B. A. Bharucha. NeurofibromatosisNoonan syndrome or LEOPARD syndrome? A clinical dilemma. J. Postgrad. Med. 46:98–100, 2000. Uthoff, D. [Leopard syndrome — dental relevance] [German] Das Leopardsyndrom — zahnarztliche Belange. Zahnarzt. Prax. 38:414–416, 1987. Van Voolen, G. A., and V. J. Selmanowitz. Lentiginosis profusa. III. Aesthetic and sanguineous aspects. Cutis 17:325–329, 1976. Voron, D. A., H. H. Hatfield, and R. K. Kalkhoff. Multiple lentigines syndrome: case report and review of the literature. Am. J. Med. 60:447–456, 1976. Walther, R. J. Cardiocutaneous syndrome. N. Engl. J. Med. 278:1127, 1968. Walther, R. J., B. J. Polansky, and I. A. Grotis. Electrocardiographic abnormalities in a family with generalized lentigo. N. Engl. J. Med. 275:1220–1225, 1966. Watson, G. H. Pulmonary stenosis, cafe-au-lait spots, and dull intelligence. Arch. Dis. Child. 42:303–307, 1967. Weber, F. P. A child recovering from aphasia and right-sided hemiplegia: Attacks of Jacksonian epilepsy on the right side. Proc. R. Soc. Med. 20:2, 1926. Weiss, L. W., and A. S. Zelickson. Giant melanosomes in multiple lentigines syndrome. Arch. Dermatol. 113:491–494, 1977. Wendt, R. G., F. Wolfe, D. McQueen, P. Murphy, H. Solomon, and M. Housholder. Polyarticular pigmented villonodular synovitis in children: evidence for a genetic contribution. J. Rheumatol. 13:921– 926, 1986.

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES Table 46.1. Cardiac myxomas with cutaneous pigmentation and mucinous tumors.* Sp (spotty pigmentation)

1957 1960 1960 1962 1965 1965 1973 1974 1976 1977 1980 1980 1982 1984 1985

M (myxoma)

EO (endocrine overactivity)

s (skin)†

h (heart)

Ms

Mh Mh Mh Mh

Sp Ms Sp Sp Sp Sp Sp Sp Sp Sp Sp

Ms Ms

Ms Ms Ms Ms Ms Ms

Mh Mh Mh Mh Mh Mh Mh Mh

Familial

EO ad EO ad EO ad

EO EO EO EO EO EO

ad, tsc tsc, lc tsc ad ad, tsc, pacr ad

EO ad, tsc

+ +

Author(s)

Lund Frankenfeld Kracht and Tam Honey and Axelrad de Moor et al. Roper et al. Rees et al. Ruder et al. Fligiel et al. Chandraratna et al. Atherton et al. (“NAME syndrome”) Proppe and Scully 1980 Schweizer-Cagianut (“Swiss syndrome”) Rhodes (“LAMB syndrome”) Carney (“Carney complex”)

*Modified from Koopman and Happle (1991, Table 1). †Includes breast lesions. ad, adrenal; t, testicular; sc, Sertoli cell (large cell calcifying type); lc, Leydig cell; p, pituitary; acr, acromegaly.

Yagubyan, M., J. M. Panneton, N. M. Lindor, E. Conti, A. Sarkozy, and A. Pizzuti. LEOPARD syndrome: a new polyaneurysm association and an update on the molecular genetics of the disease. J. Vasc. Surg. 39:897–900, 2004. Yam, A. A., M. Faye, A. Kane, F. Diop, D. Coulybaly-Ba, A. TambaBa, N. G. Mbaye, and I. Ba. Oro-dental and craniofacial anomalies in LEOPARD syndrome. Oral. Dis. 7:200–202, 2001. Yamamura, M., T. Inoue, T. Kojima, M. Miyamoto, Y. Ban, and S. Iino. [Multiple lentigines syndrome complicated with Graves’ disease (author’s transl)] [Japanese]. Nippon Naika Gakkai Zasshi 67:623–629, 1978. Zeisler, E. P., and S. W. Becker. Generalized lentigo: its relation to systemic nonelevated nevi. Arch. Dermatol. Syphil. 33:109–125, 1936.

Carney Complex Mary K. Cullen

Historical Background The myxoma syndrome (Rhodes, 1993) is characterized by cutaneous and mucocutaneous lentigines, multiple blue nevi, and cutaneous, mucocutaneous and cardiac myxomata. The syndrome includes NAME and LAMB syndromes and the Carney complex. Carney definitively included endocrine overactivity in the syndrome in the report published in 1985 (Carney et al., 1985). This broadly inclusive syndrome was categorized as a complex of spotty pigmentation, cardiac and cutaneous myxomas, and endocrine overactivity by J. A. Carney in 1985. Individuals with LAMB syndrome or NAME syndrome lentiginoses have the same clinical characteristics that are now

called Carney complex. “Myxoma syndrome” has been proposed as a replacement for the labels LAMB and NAME syndromes and Carney complex (Rhodes, 1993). A combined term, Carney complex/myxoma syndrome, will be used here. Reports of the various features of the syndrome appeared long before this constellation of findings was considered to be a unique syndrome. The first report for each particular combination of characteristics in the myxoma syndrome is noted in Table 46.1. Rees et al. (1973) described a patient who presented with hemiparesis secondary to an embolized atrial myxoma fragment. Histology of one of the patient’s pigmented spots was interpreted as that of a simple lentigo. Several years later, Atherton et al. (1980) reviewed the slides and considered the lesions to be ephelides, not lentigines. This example epitomizes the problems associated with retrospectively determining whether cases in the literature are consistent with Carney complex/myxoma syndrome. In many early cases before the syndrome was well characterized the association between myxomatous tumors and skin pigmentation was not widely known. Consequently the presence or absence of pigmented lesions was not mentioned. The pigmented spots associated with this syndrome have been variously designated as ephelides (Atherton et al., 1980), lentigines (Rees et al., 1973), blue nevi (Atherton et al., 1980), and “Peutz–Jegherslike” pigmentation (Schweizer-Cagianut et al., 1982). The syndrome is now considered one of the lentiginoses because the lentigo is usually the predominant lesion with several other types of pigmented lesions possible. Atherton et al. in 1980 were the first to report a single

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person in whom all four characteristics of the myxoid syndrome were present. Multiple subcutaneous tumors from this individual, along with a palatal tumor from the patient of Rees, were histologically interpreted as myxoid fibromas or were strongly suggestive of neurofibromas with myxoid degeneration. Atherton et al. considered it important that the association of “these distinctive cutaneous manifestations” with cardiac myxomas be known so that physicians would detect as quickly as possible the myxoma that can cause severe neurological sequelae or even death. With these concerns in mind the acronym “NAME” syndrome was proposed from the following features: Nevi, Atrial myxomas, Myxoid neurofibromas, and Ephelides. In 1980, Proppe and Scully described a family with four involved members and confirmed the genetic basis for the syndrome. Two of the reported patients demonstrated all but one of the characteristics of the complex, i.e., they did not have cardiac myxomas. The manifestations were limited to endocrine overactivity in the other two. All four individuals demonstrated two separate types of endocrine overactivity. All had testicular Sertoli cell tumors, three had adrenal involvement, and a fourth had acromegaly. In 1982 SchweizerCagianut reported the first familial combination of Cushing syndrome secondary to adrenocortical nodular hyperplasia, cardiac myxomas, cutaneous myxoid masses, and pigmented macules. Also in 1982, Rosenzweig et al. described adrenocorticotropin independent hypercortisolemia and testicular tumors in a patient with a pituitary tumor and gigantism. A review of all literature on atrial myxomas published during a period of 50 years was reported in 1979 (Bulkley and Hutchins, 1979). Other case reports and discussions of cardiac myxomas and a clinicopathologic study of cardiac myxomas by Beohr and colleagues were published in the ensuing few years (Beohr et al., 1983; Dashkoff et al., 1978; Pavie et al., 1981). In 1984 Vidaillet et al. compared clinical features of 16 patients with NAME syndrome with 63 consecutive Mayo Clinic patients with sporadic cardiac myxomas. Patients with NAME syndrome tended to be younger (mean age 24) than patients with sporadic myxomas (mean age 56). No sex predominance was evident, a finding that contrasts with the observation that there is a fourfold higher incidence of females with the sporadic cardiac myxoma. Twenty-five percent of the sporadic tumors exhibited unusual cardiac attachments compared with 75% of those from NAME syndrome individuals. In the sporadic group only three individuals demonstrated multiple or extracardiac myxomas. Recurrent cardiac myxomas, familial myxomas, endocrine tumors, and pigmented spots were not recorded in those with sporadic myxomas. A larger study and a review of cardiac tumors associated with hereditary syndromes appeared in the cardiology literature in 1987 and 1988, respectively (Vidaillet, 1988; Vidaillet et al., 1987). A female subject with pigmented lesions on the skin and genital mucosa, skin and tongue nodules, and an atrial myxoma was presented by Rhodes et al. in 1984. Pigmented lesions from the face and genitalia were histologically consis852

tent with lentigines. Cutaneous tumors were myxomas and myxoid fibromas. The authors offered “LAMB” syndrome as an alternative to the NAME syndrome because lentigines were the predominant pigmented lesions, not ephelides (E). LAMB is the acronym derived from Lentigines; Atrial myxomas; Mucocutaneous myxomas; and Blue nevi. The following year Carney et al. (1985) published a review of published cases and suggested that the syndrome complex could be divided into three general categories using the following features: spotty hyperpigmentation, myxomatous tumors, or endocrine overactivity. Associated features included those previous described with the NAME and LAMB syndromes, myxomas of the breast and/or heart (not limited to atria), mucous membrane pigmentation, and neoplasia of the adrenal, testicular, or pituitary glands. Subsequently, patients or families with both cardiac myxomas and lentigines often carry the diagnosis of Carney complex. However, Koopman and Happle (1991) considered Carney complex an extension of the NAME syndrome. Reed and colleagues (1986) reviewed the literature of lentiginosis with atrial myxomas. One of the accomplishments of the work of Carney and colleagues (1985) was the definitive inclusion of endocrine overactivity as part of these three syndromes. Various forms of excessive hormone production associated with cardiac myxomas had been reported since 1960 (Kracht and Tamm, 1960). However, when endocrine overactivity was present, it was often considered as an additional clinical finding. Consequently, the presence of endocrine hyperactivity in family members was not sought in many cases. Cushing syndrome secondary to primary pigmented adrenocortical nodular dysplasia has been extensively reviewed (Young et al., 1989), therefore only a brief summary will be included here. A familial form of Cushing syndrome, with adrenal hyperplasia, was reported by Arce et al. in 1978. Review of the adrenal histology was consistent with that of primary nodular adrenocortical dysplasia (SchweizerCagianut et al., 1980). In 1980 Schweizer-Cagianut et al. reported a sibship with “a new form of Cushing’s syndrome due to an inborn error of the adrenals.” Adrenal pathology of primary cortical nodular dysplasia was present in the absence of hypothalamic-pituitary dysfunction. Family members also demonstrated cutaneous fibromas, bilateral breast calcifications, intracranial hemorrhage of unknown etiology at a young age, death of a 4-year-old child secondary to an atrial myxoma, delayed puberty, and short stature. No mention was made of pigmented spots but, interestingly, the expected postadrenalectomy hyperpigmentation (Nelson syndrome) did not develop in these patients (Schweizer-Cagianut et al., 1980). Two more reports of familial adrenocortical nodular dysplasia appeared in 1980 (Atherton et al., 1980; Proppe and Scully, 1980). Proppe and Scully’s description included bilateral large cell calcifying Sertoli cell tumors of the testis as well as cardiac myxomas in the same family. In the family described by Atherton et al. two forms of endocrine overactivity were present in each of four family members. Furthermore, two of

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

the individuals had spotty pigmentation and cardiac myxomas. In 1982, Schweizer-Cagianut et al. expanded the clinical descriptions of the family reported in 1980 (SchweizerCagianut et al., 1980) by including cardiac myxoma, cutaneous myxomatous tumors, and “Peutz–Jeghers-like” hyperpigmentation. They noted that the association of familial adrenocortical nodular dysplasia, Cushing syndrome, and cardiac myxomas had not been previously reported (Schweizer-Cagianut et al., 1982). Inspired by the 1980 Schweizer-Cagianut et al. report, Carney and colleagues searched the Mayo Clinic records and world literature for patients exhibiting both Cushing syndrome and cardiac myxomas. Therefore, Carney noted, a second report by Schweizer-Cagianut et al. (1982) “confirmed what we had already discovered” (Carney, 1990a). The resultant triad of spotty pigmentation, myxomas, and endocrine overactivity was published by Carney et al. in 1985. Multiple aspects of Cushing syndrome secondary to primary pigmented nodular disease are explored in a review by Grant et al. (1986). Myxoma syndrome has also been significantly discussed in the nonEnglish literature by Ortonne (1986), André et al. (1988) [French], and Koyano et al. (1990). An association with psammomatous melanotic schwannoma was added in 1990 (Carney, 1990b). Carney complex was also reviewed in the context of endocrine–skin interactions (Feingold and Elias, 1988).

“Myxoma syndrome” (Rhodes, 1993) has recently come into use and is inclusive of Carney complex, NAME syndrome, and LAMB syndrome. “Carney complex” (OMIM #160980), coined by Bain (1986), is currently the most frequently used name. Although “Swiss syndrome” was proposed as an alternative in 1987 and again in 1990 (Hedinger, 1987; Salomon et al., 1990), it has not come into general use. Swiss syndrome would credit both the country and first name of the reporter initially presenting patients with all of the syndrome characteristics in a familial pattern (Schweizer-Cagianut et al., 1982). Familial Cushing syndrome (Rees et al., 1973), NAME syndrome (Atherton et al., 1980), LAMB syndrome (Rhodes et al., 1984), and syndrome myxoma (Vidaillet et al., 1987) have also been used. Two variations of above include cardiac myxoma syndrome, and familial myxoma syndrome.

tance pattern. Reviews note that 50% of the cases of Carney complex demonstrate autosomal dominant transmission with variable penetrance and incomplete expressivity (Arce et al., 1978; Carney, 1995a; Carney et al., 1986b; Danoff et al., 1987). The remainder of the patients appear to have sporadic syndromes (Carney, 1985b, 1995a). The index case of LAMB syndrome was apparently sporadic because grandparents, parents, two maternal aunts, four paternal uncles, and five siblings manifested no features of the syndrome (Rhodes et al., 1984). In 1996 Carney complex/myxoma syndrome was linked to the short arm of chromosome 2, 2p16 (Stratakis, 1996). In 1998, Stratakis et al. further described two kindreds with Carney complex/myxoma syndrome that did not show linkage to 2p16. Other reports followed confirming the genetic heterogeneity. Ovarian tumors from patients with Carney complex/myxoma syndrome often have mutations at the 2p16 locus (Stratakis, 2000). In 1998, another locus was described on 17q2, the long arm of chromosome 17 (Casey et al., 1998). This 17q2 site was confirmed in 1999 (Goldstein, 1999). In 2000 Kirschner et al. noted that mutations in the 17q22–24 site were within the gene for PRKAR1A (protein kinase A type 1-a regulatory subunit), a gene thought to act as a tumor suppressor gene (Kirschner, 2000a). At the same time, Casey et al. (2000) noted that both mutant and wild-type PRKAR1A alleles were present in the same tissues from patients with Carney complex/myxoma syndrome. All Carney complex mutations identified on chromosome 17 at that time were functionally null mutations of PRKAR1A. Carney complex is the first human disease recognized to be caused by the PKA holoenzyme — a critical component of cellular signaling (Kirschner, 2000b). Stratakis et al. (2000) then determined that 40% of the kindreds had mutations at 17q22–24. A majority of patients worldwide have been Caucasian and only a small minority have been of African descent (Carney, 1995a, b). No apparent sex predominance exists (Vidaillet et al., 1984). More than 150 people worldwide have been diagnosed with Carney complex since 1985 (Carney, 1995a, b). The diagnosis is typically made in young persons and is usually evident by the age of 20. The majority of patients have come to medical attention because of noncutaneous tumorous lesions (Carney, 1995a).

Epidemiology

General Clinical Findings and Criteria for Diagnosis

Laboux et al. (1978) and Gaillard et al. (1988) contributed reports of monozygotic twins with cardiac myxomas to the literature of “familial atrial myxoma.” The inheritance of the myxoma syndrome is described as being either autosomal dominant (AD) or X-linked (Rhodes, 1993). NAME syndrome was proposed to be autosomal dominant but X-linked transmission was not ruled out until Koopman and Happle (1991) reported a family with two of four children affected, one each male and female. Familial Carney complex cases were thought to be either dominant or X-linked (Carney et al., 1985). In 1986 Carney and colleagues confirmed the dominant inheri-

NAME Syndrome

Synonyms

∑ Nevi (blue) ∑ Atrial myxomas not limited to the atrial chambers ∑ Myxoid neurofibromas (Atherton et al., 1980)

OR Mucinosis, cutaneous (Koopman and Happle, 1991) ∑ Ephelides (Atherton et al., 1980) OR Endocrine overactivity (Koopman and Happle, 1991). Ephelides Atherton described a profusion of pigmented lesions, the majority small (up to 1 cm in diameter) and pale brown with 853

CHAPTER 46

Fig. 46.10. Small pigmented macules of the fingertip (see also Plate 46.10, pp. 494–495).

Fig. 46.11. Heart myxoma (see also Plate 46.11, pp. 494–495).

a tendency to confluence in areas of high density. Interspersed among these were darker brown macules of a similar size. The pale brown macules were always more prominent in summer months, especially those on the face, neck, upper trunk, and extensor aspects of the arms. The patient and patient’s family often had red or light brown hair and blue eyes (Atherton et al., 1980).

site of the presenting lesion in at least one case. The most serious component of Carney complex is the cardiac myxoma (Atherton et al., 1980; Champagnac et al., 1990; Salomon et al., 1990). The most common presentation is that of lentigines with cardiac myxoma. Early appreciation of the cutaneous lesions and their significance allows affected patients and their primary relatives to be screened. This is especially important for cardiac myxomas. Family history may be suggestive if it includes deaths or embolic events in children or young adults.

“Ink-spot” macules These are heavily pigmented, unusual macules “resembling small spots of spattered India ink” (Koopman and Happle, 1991). Endocrine overactivity Cushing syndrome (Kracht and Tamm, 1960) is secondary to primary, pigmented, nodular adrenocortical hyperplasia (Carney, 1990b). No postadrenalectomy hyperpigmentation (Nelson syndrome) has been reported. Testicular tumors are either the large cell calcifying Sertoli cell type (Ruder et al., 1974) or the Leydig cell type (Fligiel et al., 1976). Acromegaly or gigantism are usually secondary to growth hormone secreting pituitary tumors (Proppe and Scully, 1980).

LAMB Syndrome ∑ Lentigines: brown and black macules (Fig. 46.10) ∑ Atrial myxomas: breast or cardiac myxomas (Fig. 46.11)

that are not limited to the atrial chambers myxomas: skin (subcutaneous) and mucosal sites including the oral mucosa (Atherton et al., 1980; Rhodes et al., 1984), palate (Atherton et al., 1980), ocular conjunctiva (Atherton et al., 1980), sclera (Koopman and Happle, 1991), and genital mucosa (Rhodes et al., 1984) ∑ Blue nevi (Atherton et al., 1980; Rhodes et al., 1984).

Spotty Skin Pigmentation This can be cutaneous (Figs 46.1–46.14) and/or mucocutaneous. Lentigines and blue nevi usually predominate with both present in 10% of cases. Ephelides, if present, may be profuse in number (Atherton et al., 1980). Junctional nevi and compound nevi may also be present (Carney, 1995b). Pigmented spots are commonly overlooked. However, it is important to note that the spots are apparent prior to detection of noncutaneous aspects or clinical involvement in more than 50% of the cases (Carney, 1995b). Occasionally they are noted at birth. Myxomas These tend to be multiple in each involved organ, affect the heart, skin, and breast, and commonly recur post excision (Carney, 1995b).

∑ Mucocutaneous

Carney Complex Considered a multisystem tumorous disorder (Carney, 1995b), this syndrome exhibits as its earliest manifestations cutaneous pigmented spots and myxoid masses. However, each of the organs or systems affected, except the pituitary, has been the 854

Cardiac More than 65% of patients with the complex have cardiac myxoma(s); half occur singly, the other half are multiple (Carney, 1985a; Carney et al., 1985). Considered the most important component of the complex, “silent” tumors can suddenly cause serious embolic devastation in 20% of patients, and death in another 20% (Carney, 1995a). Fatalities have occurred in children as young as 4 years of age (Schweizer-Cagianut et al., 1980). Initial detection of cardiac myxomas has ranged from the age of 6 to 57 years with a mean age of detection at 24 years (Carney, 1995a). Cardiac myxomas can be the presenting sign of Carney complex.

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

Fig. 46.14. Periorificial pigmented macules in a patient with heart and cutaneous myxomas (see also Plate 46.14, pp. 494–495).

Breast (Myxoid Mammary Fibroadenomas) These occur in 25% of female patients and are usually bilateral (Carney, 1995b).

Fig. 46.12. Lentigines of the mouth (see also Plate 46.12, pp. 494–495).

Endocrine Overactivity Cushing Syndrome This is present in 20% of patients. It is an otherwise rare, pituitary-independent form, due to primary pigmented nodular adrenocortical disease (Carney et al., 1985; Findlay et al., 1993; Shenoy et al., 1984; Travis et al., 1989). An outstanding feature in some cases is osteoporosis (Doppman et al., 1989). Onset of adrenal signs or symptoms commonly occurs from 8 to 19 years of age. Mean age for discovery by surgical exploration is 19 years, by autopsy 23 years (Carney, 1995a). Acromegaly Growth hormone secreting pituitary adenomas cause gigantism or acromegaly in 10% of the patients with Carney complex. Age at onset of symptoms ranged from 11 to 27 years (Carney et al., 1985).

Fig. 46.13. Orbital lentigines (see also Plate 46.13, pp. 494–495).

Testicular Tumors These generally occur in 30–50% of affected males and 75% of the time they are multicentric and bilateral (Carney et al., 1985; Proppe and Scully, 1980). Detected in patients aged 5–33 years, the mean age of discovery is 17 years. The tumors are predominantly of the rare large cell calcifying Sertoli cell type (Carney, 1995a; Ruder et al., 1974), however, Leydig cell (Fligiel et al., 1976) and adrenocortical rest cell (Carney, 1995b) tumors have also been noted.

Cutaneous Skin myxomas are present in more than 33% of patients and are multiple in 75%. They appear from infancy to 38 years and are rarely present at birth. More than 50% of those individuals with cutaneous myxomas have undergone excision of a lesion by the age of 20 (Carney, 1995b). This may be the primary presenting lesion of the syndrome in some cases (Radin and Kempf, 1995; Senff et al., 1988). One should always suspect an occult cardiac myxoma in the presence of multiple cutaneous myxomas (Reinecke et al., 1987).

Schwannomas Schwannomas are peripheral nerve tumors. These tend to be the rare psammomatous melanotic type. Roughly 50% of reported cases worldwide have occurred in patients with Carney complex, even as the initial presentation (Martin-Reay et al., 1991; Radin and Kempf, 1995). In a series of 121 persons with Carney complex psammomatus melanotic schwannomas were present in 14% of them. Most are benign but approximately 10% are malignant, extensively metastatic, and fatal (Carney, 1995a, b). These statistics 855

CHAPTER 46

suggest that approximately 2% of Carney complex patients may develop malignant schwannomas.

Clinical Description and Presentation Pigmented Lesions Pigmented spots are consistent with normal lentigines, ephelides, or blue nevi. Lentigines These are widespread, small (0.2–2 mm), brown to dark brown or black spots, round, irregularly shaped or jagged, and generally nonelevated or only slightly elevated (Carney, 1995b). Numbering few to extensive, they arise in the first years of life or occasionally are congenital. Areas of blotchy brown patches in the heavily pigmented patients show scattered foci of deeper pigmentation within. In order of decreasing frequency they are found in the following locations: face especially centrofacial, but also periorbital, nasal bridge, perioral (Fig. 46.14); face mucocutaneous labial vermilion borders (50%) (Cook et al., 1987), buccal mucosa (5% of patients), eyelids, conjunctiva especially lacrimal caruncle and conjunctival semilunar fold (Kennedy et al., 1987), sclera; ears; neck; trunk; limbs; dorsal hands; and vulva, especially labia minora. Areas of infrequent involvement are palms and soles (Ohara et al., 1993), the anal verge, scalp, fingertips, and glans penis (Carney, 1995a, b). Blue Nevi The blue to black, large (up to 8 mm), domed lesions are typical blue nevi and average one lesion per patient, but may be multiple. Usually located on the face, trunk, or limbs, they are rarely seen on hands or feet (Carney et al., 1985). Cutaneous Myxomas Frequently small (1 cm or less), smooth, nonulcerated sessile papules or subcutaneous nodules, they can also be very large (greater than 5 cm) (Fig. 46.15). Occasionally lesions are papillomatous, rough, pedunculated or cystic (Carney et al., 1986a). Most are white, skin-colored, opalescent, or dusky pink

(Carney, 1995b). Grouped lesions are not common but have been noted on the arm, vulva, and nipples (Carney, 1995b). Favored locations for cutaneous myxomas are eyelid (Grossniklaus et al., 1991; Kennedy et al., 1991), external ear canal (Ferreiro and Carney, 1994; Grossniklaus et al., 1991), and nipples (Carney, 1995b). Sites, in decreasing frequency, are: head and neck (38%); trunk (23%); perineum (genitalia, buttocks, and groin) (17%); and arms (13%) (Carney, 1995b); less commonly shoulders and axillae (13%) (Handley et al., 1992); and legs (10%). Oral mucosa (Carson et al., 1988; Cook et al., 1987; Handley et al., 1992; Quintal et al., 1994) and forehead lesions have also been noted. Hands and feet are spared.

Associated Disorders Myxomas Cardiac Cardiac myxoma, cardiac infarction (Balk et al., 1979), congestive heart failure (Peterson and Serrill, 1984), death (Schuiki and Hedinger, 1987), or devastating central nervous system embolic events (Neau et al., 1993) may be the presenting signs of Carney complex/myxoma syndrome, particularly in individuals lacking a large number of facial lentigines (Carney, 1995a). Transient ischemic attacks have also been the initial presentation (Fressinaud et al., 1993). In contrast to the classical location of myxomas in the right atrial fossa ovalis for those with the sporadic form of cardiac tumors (Milner et al., 1990), cardiac myxomas in Carney complex patients may occur in any of the four heart chambers. The locations in decreasing order of frequency are the left atrium, right atrium, right ventricle, and left ventricle. Most frequently there is a single myxoma in one cardiac chamber. Multiple tumors in one chamber as well as individual tumors in multiple chambers have been reported, including ten patients with two chambers involved, four with four or more distinct tumors one of whom had myxomas in all four chambers (Carney, 1995a, b). Other commentaries and case reports appearing in the cardiology literature: Bennett et al., 1990; Chaudron et al., 1992; Sommariva et al., 1993; Vatterott et al., 1987; Yen et al., 1992; Zabala Arguelles et al., 1994. Mammary As a rule, these myxoid fibroadenomas occur as one or more discrete, bilateral, asymptomatic masses in the breast. However, several exceptions have occurred. One woman developed more than 20 lesions. In a few cases pain and tenderness has been noted, and one woman presented with unilateral breast enlargement (Carney and Toorkey, 1991b). These lesions are not restricted to females (Leedman et al., 1986). The term myxoid fibroadenomatosis has been used in cases of multiple lesions.

Fig. 46.15. Cutaneous myxomas in a patient with spotty cutaneous pigmentation and cardiac myxoma (see also Plate 46.15, pp. 494–495). (Courtesy of Dr. Mary K. Cullen.)

856

Mammary Ductal Adenomas Ductal adenoma of the breast was found in four women with Carney complex coincident with the typical myxoid mesenchymal lesions. The lesion was bilateral in two women. Only two tumors were symptomatic (bloody nipple dis-

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

charge). Five of six tumors formed palpable masses close to the areola. Mammograms, as well as microscopic examination, suggested carcinoma. None recurred or metastasized. It was postulated that the mammary ductal adenoma is a component of the Carney complex (Carney and Toorkey, 1991a).

Primary Pigmented Nodular Adrenal Disease This adrenal neoplasm causes hypercortisolism in a pituitaryindependent, adrenal-dependent manner. The excessive steroid production typically manifests itself clinically during adrenarche (the beginning of puberty) as Cushing syndrome (Bohm et al., 1983). The clinical presentation of Cushing syndrome in this group of patients may be atypical and includes findings such as severe osteoporosis or short stature (Doppman et al., 1989).

Testicular Tumor(s) These are asymptomatic or can cause sexual precocity or gynecomastia. The testicular masses are clinically firm or rock hard and nontender (Carney, 1995b). They are usually only found on physical examination, either when Carney complex is suspected or as part of a work-up for precocious sexual development.

Pituitary Tumors Enlarged sella turcica and elevated plasma growth hormone levels were noted in all patients with pituitary symptoms (Carney, 1995a). Adrenocorticotropic hormone (ACTH) (Carson et al., 1988) and prolactin (Handley et al., 1992) secreting adenomas have also been reported.

Psammomatous Melanotic Schwannoma This particular histological type of schwannoma generally affects the upper gastrointestinal tract (esophagus and stomach) and paravertebral sympathetic nerve chains. Cutaneous forms are rare but have been reported (Handley et al., 1992; Thornton et al., 1992; Utiger and Headington, 1993).

Other Follicular tumors of the thyroid may have an association with Carney complex (Schuiki and Hedinger, 1987). Danoff et al. found the rare visceral tumor fibrolamellar hepatoma in an affected family of three generations. Myxoid leiomyoma (Haught et al., 1991) and myxoid tumor of the uterus (Barlow et al., 1983) have also been described. Ovarian tumors may regularly occur in a small percentage of Carney complex/ myxoma patients (Stratakis, 2000a). Additional commentaries and case reports have appeared in the general medical literature (Carney, 1987; Konstantinov et al., 1994; Mansell et al., 1991; Rowe et al., 1988; Shapiro et al., 1982).

Histology and Histochemistry Pigmented Lesions Lentigo hyperpigmentation of the basal epidermal layer and hyperplasia of melanocytes with or without elongation of the rete pegs (Carney, 1995b). Numbers of melanosomes per

melanocyte, particularly in the dendritic areas may be increased (Koopman and Happle, 1991). Blue nevus localized upper dermal accumulation of spindle shaped melanocytes with elongated, dendritic processes. Other pigmented lesions included one or more of the following lesions: ephelides — hyperpigmentation of the basal epidermis; junctional nevus — nodular accumulation of melanocytes at the dermal–epidermal junction; compound nevus — junctional nevus with melanocytic aggregates in the upper dermis; atypical blue nevus — compound nevus with large amounts of melanin, epitheloid, and spindly epitheliod melanocytes with large atypical nuclei, a single large nucleolus, and often dermal melanophages, prominent and commonly perivascular (Carney, 1995b).

Cutaneous Myxomas The usual anatomic location within the skin in decreasing frequency is the dermis, subcutis, or both (Carney et al., 1986a). Almost 50% of the dermal tumors contact the epidermis and a few are follicular. The majority are solid but on occasion have microcystic areas. About 30% are combined solid and cystic and the remainder are cystic. Most of the cystic areas contain only mucinous matrix; a few contain solid, spherical tissue nodules that can be attached to the cyst wall. The sharply circumscribed cysts, with the appearance of mucin-filled sacs, do not have a distinct capsule but may be delimited by fine fibrous membranes. Five subcuticular encapsulated tumors have been reported (Carney et al., 1986a). Abundant myxoid ground substance/ mucinous matrix is usually basophilic. Contained within are mesenchymal, mast, and inflammatory cells; fibers (collagen and reticulin); and capillaries. Copious thin-walled channels, lacking red blood cells, are regularly present. These vessels, probably lymphatic, with their large and sometimes widely dilated lumens form an impressive component of the tumors. A cuff of spindle cells, usually single layered, or a “tenuous” mantle of collagen surrounds these delicate appearing vessels. Histochemical analyses revealed a negative periodic acid-Schiff reaction, positive Alcian blue staining at pH 2.5 but negative at pH 1.0. Positive colloidal iron staining was nearly or completely removed by testicular hyaluronidase predigestion, indicating that proteoglycans are plentiful in the mucinous matrix (Carney, 1995b). Twenty-five percent of the myxomas examined by Carney and colleagues had an epithelial component in one of the following forms: keratinous cysts demonstrating an epidermoid mode of keratinization (infundibular or epidermoid cysts); keratinous cysts with proliferating cells extending outward from the cyst wall and foci suggesting poorly induced trichofolliculomas (with or without focal sebaceous differentiation); or a unique anastomosing pattern of thin epithelial strands (Carney, 1995a). The tumor cells, with a shape reminiscent of fibroblasts, are plump and polygonal or stellate and spindly. Stellate cells have elongated multipolar or bipolar dendritic extensions. Polygonal cells predominate in the smallest lesions and also at the periphery of larger ones; spindle cells prevail toward the center of the lesion. Both cell types appear multinucleated. 857

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Commonly round, the pale nuclei are difficult to see. Many are lobulated, irregular, or cleaved. Large or bizarre nuclei and mitotic figures are very rare. Intranuclear vacuole(s) are characteristic. Cytoplasm is poorly delimited, mildly acidophilic, and negative for S-100 protein. Nucleoli are small and inconspicuous, seldom prominent (Carney, 1995b). A pathologic study of superficial angiomyxomas led Allen and colleagues (1988) to surmise that superficial angiomyxoma, cutaneous focal mucinosis, trichogenic myxoma, trichogenic adnexal tumors, trichodiscoma, myxoid perifollicular fibromas, trichofolliculomas, fibrofolliculomas and the cutaneous myxomas, Carney complex, NAME and LAMB syndromes are all closely related. Furthermore, they concluded that the solitary superficial angiomyxoma with no epithelial elements is the most common manifestation of these myxoid tumors.

documented support, as cited by Schweizer-Cagianut et al. (1980), quickly followed (Arce et al., 1978; De Gennes et al., 1970; Donaldson et al., 1981; Flattet and Hedinger, 1980; Klevit et al., 1966; Kracht and Tamm, 1960; Levin, 1966; Meador et al., 1967; Mosier et al., 1960; Rose et al., 1952; Ruder et al., 1974). Shimizu reviewed Cushing disease in 1993.

Psammomatous Melanotic Schwannoma Black, brown, or blue encapsulated masses. Large and small, polygonal, epithelial-like, and spindle cells have cytoplasmic melanin and are present in an organoid pattern along with laminated calcification, fat, and areas suggestive of schwannoma and nevus. S-100 protein immunostaining is positive (Carney, 1995a, b).

Testicular Tumors Cardiac Myxomas There are histologically indistinguishable from sporadic cardiac myxomas (Allen et al., 1988). An unusual cardiac myxoma was described in which histological examination revealed numerous well-formed glandular structures at the base of the lesion. Mucin stains were positive and immunoperoxidase studies showed epithelial-like areas with positive staining for eosin 5-maleimide (EMA), carcinoembryonic antigen (CEA), and CAM 5.2 (Thornton et al., 1993), raising concerns that the presence of these glandlike structures may lead to the erroneous diagnosis of secondary adenocarcinoma (Thornton and Walsh, 1993). Right atrial myxomas have been reviewed by Loire and Delaye (1992). McCarthy et al. (1989) examined cardiac myxomas by DNA flow cytometry and found that five of five from Carney complex patients had a tetraploid pattern while only 20% of the sporadic type were tetraploid. Conversely, Rocco et al. (1991) examined 16 cardiac myxomas from Carney complex patients and found that 15 were diploid and only one was tetraploid.

Breast Myxomas Well-circumscribed tumors with or without encapsulation. Histologically they are myxoid fibroadenomas, except the stroma is more delicate, less cellular, and more myxoid than the stroma of the usual fibroadenoma (Carney, 1995b). Clinically normal mammary tissue contains irregular collections of stromal myxoid ground substance, suggestive of a multifocal myxomatous disorder (Carney, 1995a).

Primary Pigmented Nodular Adrenal Disease Adrenal weight varies. External and cut adrenal surfaces are studded with small (less than 4 mm) black, brown, dark green, red, or yellow nodules. Microscopically, nodules contain enlarged, globular cortical cells with granular acidophilic cytoplasm that often has lipofuscin. Cortices are atrophic and zonally disorganized between nodules. Also known as cortical microadenomatosis or primary cortical nodular dysplasia (Schweizer-Cagianut et al., 1980), primary pigmented nodular adrenal disease was first described by Chute et al. (1949) as a pathologic finding in pediatric Cushing syndrome. Well858

Well-demarcated tumors are either yellow and calcified (largecell calcifying Sertoli cell tumor), or brown and soft (steroidtype Leydig cell tumor or adrenocortical rest tumor). Other commentaries and case reports appearing in the surgical and pathology literature: Cheung and Thompson, 1989; Herrmann et al., 1990; McCarthy et al., 1986; Oemus and Rath, 1990; Tatebe et al., 1994; van Gelder et al., 1992.

Laboratory Investigations General Autosomal dominant mode of inheritance indicates importance of examining first- and second-degree relatives, particularly for cardiac myxomas (Carney, 1995a).

Cardiac Myxoma Lifelong echocardiographic screening is recommended, scheduled yearly from the age of 5 years, because cardiac myxomas tend to be recurrent in these patients. The same frequency can be used for relatives with one or more diagnostic criteria of the complex even if asymptomatic (Bier et al., 1989). Ohshima et al. (1990) performed echocardiographic follow-up at sixmonth intervals for at least five years. Screening is usually transthoracic, however in one instance cardiac myxomas were only detected with transesophageal echocardiography (Agostini et al., 1991).

Primary Pigmented Nodular Adrenal Disease For patients or relatives presenting with Cushing syndrome and at least one other criterion for Carney complex, 24-hour urinary cortisol and low-dose dexamethasone suppression are good screening tests. Multiple, small adrenocortical nodules detected by computed tomography (CT) and magnetic resonance imaging (MRI) may be diagnostic, especially with atypical presentations of Cushing syndrome (Doppman et al., 1989; Ohara et al., 1993).

Pituitary Tumors CT scan of the head is important since a large tumor may be present without a clinically detectable visual field defect (Handley et al., 1992). When patients present with acromegaly

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

or gigantism, plasma somatomedin C level is the best screening test for a growth hormone producing pituitary adenoma.

Testicular Tumors Physical examination for palpable nodules. Testicular ultrasonography may detect occult calcified masses. If sexual precocity is present check pituitary gonadotropins. Low levels suggest suppression of pituitary function by an endogenous source of sex hormones, probably the testis.

Ovarian Tumors Ovarian ultrasonography is recommended at diagnosis and at regular follow-up intervals (Stratakis, 2000).

cortical maldevelopment (rather than true neoplasia) leading to autonomous adrenocortical hyperfunction. Iseli and Hedinger (1985) extended the idea by suggesting a malformative process of the adrenal gland zona reticularis. Teding van Berkhout et al. consider this an inherited disease of immunological origin because serum immunoglobulin preparations from two affected sisters and their apparently unaffected mother stimulated both adrenocortical cell growth and cortisol production in a cytochemical bioassay system. Circulating growth factors are also suspected to be involved in the pathogenesis (Teding van Berkhout et al., 1986, 1989).

Neoplasms in General Psammomatous Melanotic Schwannoma There are no good screening tests for these tumors.

Differential Diagnosis

Elevated circulating levels of insulinlike growth factors raise the possibility of abnormal stimulation of somatic growth and may be linked to development of soft-tissue neoplasms in these patients (Wilsher et al., 1986).

Multiple Endocrine Neoplasia (MEN) Similarities include involvement of multiple endocrine glands, multicentric distribution, bilateral occurrence in paired organs, and autosomal dominant inheritance. However, pigmentation, myxomas, and testicular tumors are not a part of MEN.

Animal Models None.

Treatment Lentigines

Peutz–Jeghers Syndrome Mucocutaneous pigmentation is similar and both syndromes include the rare large-cell calcifying Sertoli cell tumor of the testis. Peutz–Jeghers patients often have ovarian tumors and exhibit numerous blue nevi but lack cardiac and cutaneous myxomas. No ovarian tumors have been described in Carney complex and blue nevi are commonly singular. (See discussion of Peutz–Jeghers syndrome earlier in the chapter.)

LEOPARD Syndrome Mucocutaneous pigmentation and cardiac involvement (hypertrophic cardiomyopathy) occur in LEOPARD syndrome. However, six of the seven criteria for the syndrome do not occur in Carney complex (see section on LEOPARD syndrome earlier in this chapter).

Centrofacial Lentiginosis Only facial distribution of lentigines is similar (See section describing Centrofacial lentiginosis).

Neurofibromatosis 1 Pigmented spots include axillary freckling, café-au-lait, and hypopigmented macules. Cardiac myxomas do not occur and subcutaneous tumors are neurofibromas, not myxomas.

Pathogenesis Carney complex is a disorder of mesenchymal, melanocytic, adrenocortical, and pituitary tissues of unknown etiology.

Cushing Syndrome Due to Pigmented Multinodular Adrenocortical Dysplasia Bohm and colleagues (1983) postulated an inherited adreno-

See Chapters 59–64.

Cutaneous Myxomas Simple excision is frequently followed by one or several recurrences.

Mammary Myxoid Fibroadenoma Simple excision for uncomfortable or painful tumors. Occasional patients have required simple mastectomy because of pain (Carney and Toorkey, 1991b).

Cardiac Myxomas Excision, removing as much associated endocardium as practical (Loire and Termet, 1991; Ohshima et al., 1990), with concomitant thorough search for multiple heterotopic tumors (Ohshima et al., 1990). Multiple operations are common due to recurrence and development of new tumor(s). Sellke et al. (1990) review their experience in treating cardiac myxomas, several of them from two patients with Carney complex. Baillot et al. (1992) reported a similar series including one patient with Carney complex.

Cushing Syndrome Bilateral total adrenalectomy is curative. Unilateral adrenalectomy has resulted in persistent abnormal adrenocortical function. Nelson syndrome following adrenalectomy has not occurred in any patients to date (Carney, 1995b). Markers of bone turnover (BGP, ICTP) and of collagen turnover (PIIINP) may be a useful prognostic index after treatment of Cushing syndrome (Ambrosi et al., 1995). Replacement therapy with glucocorticoids and mineralocorticoids is essential. 859

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Testicular Tumors Conservative treatment is recommended. Some very small tumors, detected only by ultrasonography, are currently being carefully followed (Carney, 1995b). Tumor enucleation has been performed in several instances. Larger tumors can be excised. Carney warns that bilateral orchiectomy should be done only if careful evaluation of the clinical findings deems it necessary (Carney, 1995a). There has been only one report of a metastasizing testicular tumor in patients with a myxoma syndrome; it was a fatal Leydig cell carcinoma (Koopman and Happle, 1991).

Pituitary Tumors Transsphenoidal hypophysectomy.

Psammomatous Melanotic Schwannoma Majority are benign and cured by complete local excision. The 10% that are malignant metastasize widely with a fatal outcome (Carney, 1995a; Radin and Kempf, 1995).

Prognosis Normal life span if cardiac myxomas are recognized and removed in a timely manner. Approximately 2% of patients will develop a fatal malignant psammomatous melanotic schwannoma (Carney, 1995a; Radin and Kempf, 1995).

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myxoid neurofibromata, cutaneous pigmented lesions, and endocrine abnormalities. Aust. N. Z. J. Med. 16:393–396, 1986. Yen, R. S., B. Allen, R. Ott, and M. Brodsky. The syndrome of right atrial myxoma, spotty skin pigmentation, and acromegaly. Am. Heart J. 123:243–244, 1992. Young, W. F., Jr., J. A. Carney, B. U. Musa, N. M. Wulffraat, J. W. Lens, and H. A. Drexhage. Familial Cushing’s syndrome due to primary pigmented nodular adrenocortical disease: Reinvestigation 50 years later [see comments]. N. Engl. J. Med. 321:1659–1664, 1989. Zabala Arguelles, J. I., E. Maroto Alvaro, C. Maroto Monedero, E. J. Garcia Fernandez, R. Minguez Garcia, P. Valles Serrano, C. Moreno Belzue, and R. Arcas Meca. [The myxoma syndrome: cardiac myxoma, cutaneous pigmented lesions and peripheral and endocrine neoplasms. Apropos 2 cases]. Rev. Esp. Cardiol. 47:113–115, 1994.

Other Lentiginoses Mary K. Cullen

Introduction The major lentiginosis syndromes are discussed in individual sections of this chapter. “Other lentiginoses” includes reported cases of lentigines for which either the criteria for, or the range of, associated disease(s) are still evolving.

Agminated Lentigines Agminated lentiginosis (AL) is characterized by numerous lentigines arranged in a group and often confined to a body segment with a sharp midline demarcation (Micali et al., 1994). Many introductions to reports about AL state that most cases of AL have no known associated disorders. However, Trattner and Metzker presented the largest group of patients with AL, in which four of the nine patients had other health problems: café-au-lait macules (CALM), vitiligo, acanthosis nigricans, cutis marmorata, celiac disease, and asthma (Trattner and Metzker, 1993). Additionally, almost 70% of the 46 cases in the literature (for whom this was addressed) had associated conditions. These associations are discussed below.

Synonyms Unilateral lentigines (McKelway, 1904), partial unilateral lentiginosis (PUL), multifocal PUL (Micali et al., 1994), segmental lentiginosis, lentiginous mosaicism, macular nevus unilateralis, and zosteriform speckled lentiginosis (Fig. 46.16) In the past, the term “zosteriform lentiginous nevus” (Port et al., 1978; Ruth et al., 1980), has been used in reference to agminated lentigines. As currently used, the term zosteriform lentiginous nevus refers to a lesion that contains both lentigines and true nevi (see further description at the end of this chapter).

Background The term “agminated lentigines” was recently proposed (Rhodes, 1993) and will be used here because it is the most straightforward and inclusive. The first published description of pigmented macules in a segmental distribution is attributed to McKelway (1904). His report, as well as several others, preceded the more recent practice of distinguishing between 863

CHAPTER 46

Fig. 46.16. Zosteriform speckled lentiginosis (see also Plate 46.16, pp. 494–495).

lentigines and ephelides (freckles) on clinical, histological, and etiological grounds. In fact, McKelway thought he was describing unusual ephelides: “The appearance of lentigo, or freckles, is so well known that no elaborate description need be attempted in this article. [They are] usually found in individuals of a light complexion and having light, especially red, hair. The exciting cause is generally exposure to the rays of the sun . . .”. Because freckles and lentigines have at times been used interchangeably, all reports of macular pigmented lesions in a grouped pattern will be included in this discussion. In some instances the histologic “jentigo” pattern (nests of grouped melanocytes within a lentigo) has been found (Marchesi et al., 1992; Matsudo et al., 1973; Ruth et al., 1980). These cases are included unless clinical descriptions were consistent with nevus spilus (Matsudo et al., 1973). McKelway’s patient had pigmented spots present on the entire left side of the body. “From the roots of the hair to the knee was more or less thickly covered with freckles, varying in size, the areas of pigmentation were largest and most numerous on her left breast and chest, although there were many on her left cheek, arm, forearm, and the left half of her abdomen. There were a number on her left thigh, especially on the inner surface” (McKelway, 1904). McKelway stated that the distribution of pigmented macules is generally bilateral, but referred to a statement by Hyde (no reference) that “rare instances are recorded in which the symptoms exist only upon one side of the body” (McKelway, 1904). In the 1916 first edition of his Dermatology text Sutton mentions that Crocker (no reference) had previously described localized unilateral pigmented macules (Sutton, 1916). Two ensuing cases, included contralateral findings of supernumerary nipple and capillary nevus, respectively (Adamson, 1911; MacLeod, 1913). These three cases, and one other (Gron, 1928), were summarized as follows by Sutton and Sutton in 1939: “Lentiginous pigmentation of a localized or unilateral distribution has been noted in a few instances, and it probably represents macular nevus unis lateralis or ichthyosis hystrix.” Shumate (1941) was the first to report bilateral involvement with grouped lentigines accompanying a more extensive unilat864

eral distribution. Cappon described the first ipsilateral associated finding (skin on the involved side was somewhat uneven and coarser than the other side) in 1948. He noted that the lentigines tended to crowd towards the midline at which they abruptly stopped (Cappon, 1948). Almost 25 years later Davis and Shaw (1964) published the next case in the literature. Their patient, whom they termed “a human mosaic for skin pigmentation,” demonstrated segmental lentigines in a checkerboard distribution (alternating segments from side to side of the body). After an additional hiatus of nine years, in 1973 Pickering described a patient with left truncal lentigines and ipsilateral facial vascular nevi and intracranial vascular abnormalities. The latter caused focal seizures (Pickering, 1973). For many years thereafter, discussions of segmental or unilateral lentigines included Jacksonian seizures as a possible associated finding. In 1978, Port and colleagues suggested that segmental lentiginosis be established as an entity separate from unilateral and partial unilateral lentiginosis. They described lentigines in a zosteriform distribution which is now simply considered one of several possible presentations for agminated lentigines. In 1980 Ruth and colleagues presented a case of “zosteriform lentiginous nevus” in which histological examination revealed nests of melanocytes at the dermal–epidermal junction (so-called jentigo pattern) within the clinically apparent lentigines (Ruth et al., 1980). However, the lentigines in their case, unlike those described by Matsudo et al. in 1973, did not exhibit the lightly pigmented background consistent with nevus spilus. In 1992 Marchesi and colleagues described a patient with lentigines mostly limited to the skin area served by the trigeminal nerve. Histologic analysis of a pigmented macule matched that of Ruth et al. (1980). Marchesi et al. noted that “similar cases have been described in the literature under the term ‘zosteriform lentiginous nevus,’ which in our opinion makes for confusion since the same term has also been used to describe cases that fit the diagnostic criteria for speckled lentiginous nevus (nevus spilus).” See discussion on nevus spilus below for further details. In 1980 Thompson and Diehl presented a patient who had partial unilateral lentiginosis and one CALM greater than 15 mm in diameter, raising the possibility that this was a form of segmental neurofibromatosis (NF) (Thompson and Diehl, 1980). Gerhards and Hamm added three more cases in 1992 all with café-au-lait macules as follows: six CALM <13 mm, five CALM ~15 mm, and two CALM 49 mm (Gerhards and Hamm, 1992). In 1987 Roth et al. discussed a patient with segmental NF and contralateral lentiginosis in the corresponding dermatome (Roth et al., 1987). Allegue et al. offered a similar case in 1989 (Allegue et al., 1989). Theiler et al. (1991), and later Moss and Green (1994) described cases of partial unilateral lentiginosis in which one or more correlation with NF was present (CALM, Lisch nodules, family history of NF). Also in 1989 another disease entity, the myxoma syndrome (Carney complex/LAMB/NAME lentiginoses), was suggested by Goskel and Kural’s patient in whom both segmental lentigines and an intracardiac myxoma were present (Goksel and Kural, 1989). In 1994 Holder and colleagues

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

described a PUL (partial unilateral lentiginosis) patient with blue nevi (reminiscent of the LAMB/myxoma lentiginosis) and a nerve palsy in the same distribution as the lentigines (Holder et al., 1994). Right bundle branch block (reminiscent of the LEOPARD lentiginosis) was also present. In 1994 Micali et al. reviewed the PUL literature in conjunction with a new case (Micali et al., 1994). The unusual pattern of agminated lentigines in their patient was dubbed “multifocal PUL.” A “skip” area was present (not previously reported) in which a band from the breast to the upper abdomen, and the corresponding area posteriorly, was without lentigines. Bilateral involvement, similar to that of Shumate’s patient (1941), was also present. Interestingly, all patients with bilateral or alternating segmental lentigines (Davis and Shaw, 1964; Gron, 1928; Micali et al., 1994; Parslew and Verbov, 1995; Shumate, 1941) have had involvement of the right face and neck. The chin was involved in three of the five cases. A total of 48 cases of AL have been reported in the literature and 65% are female. The number of males and females with right-sided involvement is almost equivalent. However, 70% of those with left-sided involvement are female, as are four of the five individuals with complex bilateral involvement. Additional case reports not specifically mentioned above include: Bronson (1895), Brocq (1921), Schubert (1924), Hughes et al. (1983), Rosenblum (1985), and Aguilar et al. (1992). The most recent review of AL, at the time of this writing, was that of Piqué et al. in 1995. In Table 46.3 all cases found in the literature are listed by side of the body involved, then further subdivided by sex. Male to female ratio, (M:F) for each side of the body is indicated in bold. The presence or absence of associated findings is indicated by + or (–), respectively. Cases with an asterisk(*) have involvement of both sides of the body and are also listed with the combination cases.

ciations. Port et al. (1978) noted that central nervous system abnormalities are uncommon with the limited dermatomal type of unilateral lentigines, but may be associated with the nondermatomal distribution. Cutaneous findings include coarse and uneven skin in the same distribution as the lentigines (Cappon, 1948), diffuse xerosis with hyperkeratotic area(s) (Micali et al., 1994; Port et al., 1978), supernumerary nipple (Adamson, 1911), vascular nevi (MacLeod, 1913; Pickering, 1973), cutis marmorata (Trattner and Metzker, 1993), blue nevi (Holder et al., 1994), acanthosis nigricans (Trattner and Metzker, 1993), and vitiligo (Piqué et al., 1995; Trattner and Metzker, 1993). Extracutaneous associations include cerebrovascular anomalies (Hughes et al., 1983; Pickering, 1973) seizures (Pickering, 1973), mental deficiency (Cappon, 1948; McKelway, 1904; Port et al., 1978), neuromuscular anomalies (Holder et al., 1994; Port et al., 1978), cardiac involvement (Goksel and Kural, 1989; Holder et al., 1994), skeletal anomalies (Gerhards and Hamm, 1992), celiac disease (Trattner and Metzker, 1993), asthma (Trattner and Metzker, 1993), endocrine disturbance (Thompson and Diehl, 1980), iron deficiency anemia (Piqué et al., 1995), and sickle cell anemia (Davis and Shaw, 1964; Hughes et al., 1983).

Epidemiology

∑ Segmental neurofibromatosis: Size, number and location of

There is no known inheritance pattern (Trattner and Metzker, 1993), sex or racial predilection. Considered rare, partial unilateral lentiginosis may simply be infrequently reported, as suggested by the accumulation of nine cases in eight years by Trattner et al. (1993), and six in two years by Piqué et al. (1995).

café-au-lait macules is important in the distinction (Goldberg, 1995; Micali et al., 1994). ∑ Lentiginosis syndromes: Complete medical examination and family history, particularly cardiac, are necessary to rule out this possibility.

Clinical Description

Treatment

AL first become manifest at birth or early childhood as small, circumscribed, light brown macules 2–10 mm in diameter, confined to a localized area of the body. They occur in one of several patterns: segmental, dermatomal, curvilinear, or swirled. Several authors have noted café-au-lait macules in association with AL (Gerhards and Hamm, 1992; Hughes et al., 1983; Micali et al., 1994; Moss and Green, 1994; Piqué et al., 1995; Roth et al., 1987; Theiler et al., 1991; Thompson and Diehl, 1980; Trattner and Metzker, 1993), and segmental lentigines have been described contralateral to coexisting segmental neurofibromatosis (Allegue et al., 1989; Roth et al., 1987; Theiler et al., 1991). Individual case reports have included a variety of other asso-

As with other lentigines, lesions are considered benign and no treatment is required. Because the long-term course and malignant potential are not known, periodic examinations to detect early atypical changes in lentigines are recommended. For esthetic reasons, there are now several methods for treating lentigines. Rosenblum (1985) described successful cryosurgical eradication of lentigines distributed in a unilateral segmental pattern. See also Section 8, Treatment of Pigmentary Disorders.

Histopathology The microscopic appearance is that of lentigo simplex: slight to moderate elongation of the rete ridges, increased numbers of melanocytes, and increased melanin in both melanocytes and basal cells. Intraepidermal melanocytic dysplasia may occur in varieties of agminated lentigines (Rhodes, 1993). Holder and colleagues noted some melanocytes with nuclear atypia (Holder et al., 1994).

Differential Diagnosis ∑ Nevus spilus: Wood’s lamp examination may be needed to

demonstrate background pigmentation.

Pathogenesis Multiple hypotheses exist with, as yet, no clear-cut answer. Chromosomal mosaicism confined to neural crest melanoblasts has been implicated in the pathogenesis (Pickering, 865

Table 46.3 All cases of agminated lentiginosis, reviewed by Piqué et al. (1995). RIGHT: M:F 9:11 F Marchesi F Rosenblum F Rhodes F Gerhards patient #1 F Goskel F Trattner patient #8 F Holder F Thompson F Schubert F Brocq F Roth *F Parslew

Face and neck, mostly within trigeminal nerve distribution Face nose, cheek, periorbital Neck and supraclavicular area Axilla and upper thorax Trunk, shoulder, and upper arm Chest, upper back Leg midcalf to ankle Midback and abdomen to ankle, ulnar surface of arm C4 to T5 distribution Neck, shoulder, chest Shoulder and back Lower back Face patch Neck, shoulder, upper arm Forehead, cheek, lips, chin, anterior neck; B (bilateral) nose Upper third of face, chin, neck to mandible, arm, hand, chest, shoulder to scapula Midface, lower back and trunk/abdomen to ankle Cheek Cheek, neck, shoulder, axilla, arm Face, neck, shoulder, arm, chest Face and neck Axilla, arm, chest, upper abdomen Chest, axilla, upper arm Body clavicle/occipital hairline to ankle Side of body Glans penis and penile shaft Chest, back, hip, arm, hand

(–) (–) (–) + + (–) + + ? ? +

M M M M M M M M M *M

R R R R R R R R R R R R L R R L R R R R R R R R R L

Micali

*F

Davis, checkerboard distribution

L

Face, neck, arm, upper back, chest Cheek, neck, shoulder Face Upper back, neck, shoulder, axilla, chest Breast and arm Buttock and thigh Neck and shoulder Trunk above clavicle to umbilicus Inferior neck to groin, anterior and posterior Side of the body, from the roots of the hair to the knee Nipple to inguinal fold anterior and iliac crest posteriorly Trunk axilla to buttock Face Upper thorax, areolar, and axillary Axilla and upper thorax Chest, axilla, upper arm Neck, shoulder and upper arm Face patch Breast and axilla, and upper abdomen to umbilicus; B neck, chin and cheeks in distribution of mandibular branch of trigeminal nerve Middle third of face (below eye to upper lip), lower back and trunk/abdomen to ankle Upper third of face (with inferior aspect just below eye), chin and neck to mandible, arm, hand, chest and shoulder to scapula Hemithorax, leg Abdomen and lumbar area Shoulder and arm Cranial nerve C4T5 distribution with abrupt midline ending Temple, neck, arm, upper back, chest Anterior chest, scapula, shoulder Posterior neck to waist: posterior, lateral and anterior trunk Chest, back, hip, arm, hand; B postauricular {L>R}, cheeks and posterior neck {R>L}

(–) + + (–) + + + + + + + + + + + +

*F

L L L L L L L L L L L L L L L L L R L

*F *F

Shumate Davis, checkerboard distribution

Rhodes Trattner patient #1 Trattner patient #7 Piqué patient #2 Trattner patient #3 Theiler Cappon Gron patient #2 Rhodes Gron patient #1 B, cheeks and posterior neck {R>L}, postauricular {L>R}; LEFT: M:F 7:16 F Trattner patient #5 F Trattner patient #6 F Trattner patient #9 F Trattner patient #2 F Piqué patient #5 F Piqué patient #4 F Piqué patient #7 F Pickering F Allegue F McKelway F Adamson F MacLeod F Bronson F Theiler F Gerhards patient #2 F Moss *F Parslew

R M Gerhards M Aguilar M Piqué patient #3 M Hughes M Trattner patient #4 M Ruth M Port *M Gron patient #1 COMBINATION: M:F 1:4 *F Shumate, 1941; *F Parslew

L L L L L L L L

*F

Micali

R L R L

*F

Davis, checkerboard distribution

L R

*M

866

Gron patient #1

L

Forehead, cheek, lips, chin, anterior neck; B nose Neck, shoulder, upper arm Face patch Axilla, upper abdomen; B neck, chin and cheeks in mandibular branch trigeminal nerve distribution Middle third of face (below eye to upper lip), lower back and trunk/abdomen to ankle Upper third of face (with inferior aspect just below eye), chin and neck to mandible, arm, hand, chest and shoulder to scapula Chest, back, hip, arm, hand; B postauricular (L>R), cheeks and posterior neck (R>L)

(–) (–) + (–) + (–) (–) + + + (–) (–) +

(–) + +

+ + (–) + + (–) + + (–) (–) + +

+

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

1973), and a clone of abnormal cells which has arisen early in development and occupied a considerable area of the body surface has also been suggested (Goudie et al., 1985). The not uncommon presence of café-au-lait macules suggests a possible association with segmental neurofibromatosis (Gerhards and Hamm, 1992; Hughes et al., 1983; Moss and Green, 1994; Piqué et al., 1995; Theiler et al., 1991; Thompson and Diehl, 1980; Trattner and Metzker, 1993). Reports of segmental lentigines coexisting with segmental neurofibromatosis (Allegue et al., 1989; Roth et al., 1987), or Lisch nodules and CALM (Moss and Green, 1994; Theiler et al., 1991) further suggest a common etiology. Segmental lentiginosis is thought by some to be a forme fruste of neurofibromatosis (Cappon, 1948; Thompson and Diehl, 1980). Some reports of “new” lentiginoses may actually represent incomplete expression of one of the well-described lentiginosis syndromes. For example, right bundle branch block (Holder et al., 1994) may be LEOPARD syndrome while reports of cardiac myxoma (Goksel and Kural, 1989) and blue nevi (Holder et al., 1994) may be Carney complex.

Animal models Chimeric and mosaic coat color mice have been used for genetic and developmental studies (Goudie et al., 1985).

Bannayan–Riley–Ruvalcaba Syndrome (BRR) (OMIM#153480) Synonyms Ruvalcaba–Myhre–Smith, Bannayan–Zonana, Zonana–Smith, and Riley–Smith.

Bannayan–

Background In 1980 Ruvalcaba, Myhre, and Smith first reported two adult patients with macrocephaly, pigmented penile macules, and hamartomatous intestinal polyps (Ruvalcaba et al., 1980). By 1988, when Gretzula and colleagues reviewed the literature, added two cases, and proposed a syndromic definition (Gretzula et al., 1988), ten other cases had been reported (DiLiberti et al., 1983, 1984). Minimal criteria for this autosomal dominantly inherited syndrome included (1) macrocephaly, (2) a normal CT scan of the brain, and (3) at least two of the following: the syndrome in a first-degree relative, pigmented penile macules, hamartomatous intestinal polyps, subcutaneous lipomata, lipid storage myopathy, or prominent Schwalbe lines and corneal nerves. Other possible abnormalities included angiolipomas, motor or developmental delay, unusual craniofacial appearance, ocular and skeletal abnormalities. Fargnoli et al. (1996) reported two kindreds with lentigines of the penis and vulva, cutaneous vascular malformations, acanthosis nigricans, central nervous system vascular malformations, and thyroid tumors. Additionally, they reported verrucous skin changes similar to those seen in Cowden syndrome/disease (CS), suggesting a common genetic pathway for CS (OMIM#158350) and BRR. They further proposed the syndrome be named Bannayan– Riley–Ruvalcaba syndrome to reflect overlap of three

autosomal dominant syndromes: Ruvalcaba–Myhre–Smith, Bannayan–Zonana, and Riley–Smith (Fargnoli et al., 1996). CS is another rare, autosomal dominantly inherited neoplastic disorder characterized by multiple hamartomas (including gastrointestinal like BRR). CS patients have an increased risk of malignant disease of the breast, thyroid, and endometrium. The first molecular mapping of BRR came when Arch et al. (1997) reported a BRR patient with a chromosomal deletion at 10q23.2-q24.1. This gene region includes the PTEN gene, 10q23.3, mutations of which had previously been described as a susceptibility locus in CS. PTEN encodes a dualspecificity phosphatase which performs a major function in cellular growth, death, adhesion, and migration. PTEN (also called MMAC1 or TEP1), a tumor suppressor gene, is somatically mutated in several types of cancer. Because of phenotypic overlap between CS and BRR, and their similar cytogenetic and molecular findings, Arch et al. suggested that the syndromes are allelic. Longy et al. (1998) reported three additional mutations in PTEN in five patients with BRR from three unrelated families. Their results supported previous findings of PTEN mutations as causal in sporadic and familial BRR. In 1997 the 10q23 of BRR localization was confirmed (Zigman et al., 1997). Analysis of the PTEN gene in a mother with CS and her son with BRR demonstrated the same heterozygous mutation in both. This led to the suggestion that CS and BRR are one entity (Zori et al., 1998). When DNA from 37 CS families and seven BRR families was screened, germline PTEN mutations were identified in 81% of CS families and 57% of BRR families (Marsh et al., 1998). Similarly, DNA from 43 BRR individuals showed a 60% mutation rate (Marsh et al., 1999). Further, genotype–phenotype analysis in the CS families revealed an association between the presence of a PTEN mutation and malignant breast disease (Marsh et al., 1998). Additional analysis suggested an association of PTEN mutation and cancer or breast fibroadenoma in any given CS, BRR or BRR/CS overlap family (Marsh et al., 1999). The presence of lipomas was also correlated with PTEN mutation in BRR patients (Marsh et al., 1999). Thus, PTEN–mutation-positive CS and BRR may be different presentations of a single syndrome, suggesting both should receive equal attention with respect to cancer surveillance (Marsh, 1999). This was reiterated and extended by Hendriks et al. in 2003. They recommended BRR cases with a mutation in PTEN undergo the same surveillance protocol for malignant tumors as is currently recommended for CS. Additionally they proposed a yearly hemoglobin test from early infancy for the early detection of intestinal hamartomas (Hendriks et al., 2003). Because of multiple cancers associated with CS, and benign neoplasias in BRR, De Vivo and colleagues (2000) looked at the general population to see if germline PTEN mutations signaled a genetic predisposition to multiple primary tumors. The PTEN genes from 103 women with more than one primary tumor at different anatomical sites were screened. Two novel germline heterozygous mutations were found in five of the 867

CHAPTER 46

cases. Neither mutation was observed in 115 controls free of diagnosed cancer. Their data suggest that germline mutations in PTEN may be a more frequent predisposing factor for cancers in women than had been previously thought (De Vivo et al., 2000). Because no BRCA1 or BRCA2 gene mutations are detected in up to 40% of familial breast cancer cases, Figer et al. (2002) looked for PTEN mutations in familial breast cancer. A large group of Israeli women with familial breast cancer, but lacking a BRCA1 or BRCA2 gene mutation, was screened for PTEN mutations. Their results suggested that PTEN does not play a major role in predisposing to hereditary breast cancer in Israeli women (Figer et al., 2002). Zhou et al. (2002) then reported a novel germline alteration in the PTEN gene (IVS7–15Æ53del39) of an African American individual with features of CS. Further investigation revealed that 12 of 42 (28.6%) African American controls, but not individuals of Caucasian or Japanese origin, also carried this heterozygous 39-bp deletion in PTEN (Zhou et al., 2002). Future investigations may reveal more population-based polymorphisms in the PTEN gene that appear to be independent of the increased cancer risk found in BRR and CS.

Animal Models Murine Gimm et al. (2000) looked at the pattern of PTEN expression during development using RNA expression data in mouse embryos and monoclonal antibody localization in human tissue. They compared the relationship of the temporal and spatial PTEN expression pattern to: the clinical manifestations of CS and BRR, the somatic genetic data in sporadic cancers, and the murine knockout models. They observed mainly highlevel PTEN expression in tissues (e.g., skin, thyroid, and central nervous system) known to be involved in CS and BRR. Also observed was high PTEN expression in tissues (e.g., peripheral nervous system, autonomic nervous system, and upper gastrointestinal tract) not known to be commonly involved in CS, BRR, or sporadic tumorigenesis (Gimm et al., 2000).

this entity have hamartomas of both melanocytic and vascular proliferations. PPV is divided into multiple types, which are extensively discussed elsewhere in this text. However, lentigines are only one of several possible pigmented lesions comprising the melanocytic component of PPV and as such are not included in the formal PPV definitions. This is interesting because phakomatosis comes from the Greek phakos, defined as a: lentil, lentil-shaped object, or a spot on the body. We have included here several reports of uncommon lentiginoses that fulfill the general criteria for PPV set out by Ota et al. In 1960 Bandler described a lentiginosis presumed to be either an incomplete form of Peutz–Jeghers syndrome or an entirely separate entity (Bandler, 1960). The patient had multiple hemangiomas of the small intestine, which caused repeated episodes of severe gastrointestinal bleeding. Lentigines were present in the Peutz–Jeghers distribution. This entity has been called mucocutaneous lentigines with hemangiomas. In 1988 Zahorcsek et al. presented the case of a young woman with multiple areas of a progressive circumscribed lentiginosis, a nevus spilus, and nevi flammei on the face, neck, and trunk. The authors called it progressive circumscript lentiginosis — phakomatosis IIIa because the association of lentigines, nevus spilus, and nevus flammeus suggested a phakomatosis (Zahorcsek, 1988). PPV type III includes nevus flammeus and nevus spilus. Joshi et al. (1999) described a patient with multiple lentigines on the face and trunk in addition to a nevus flammeus, Becker nevus, and a café-au-lait macule. They considered this new combination consistent with PPV type Ia (Joshi et al., 1999). Type I PPV includes nevus flammeus, nevus pigmentosus, and verrucous epidermal nevus.

Lentiginosis with Arterial Dissections

Caenorhabditis elegans daf-18 is the C. elegans homolog of PTEN. Rouault et al. (1999) demonstrated that mutations of daf-18 contribute to the development of neoplasia through an aberrant activation of the PI 3-kinase signaling cascade.

A previously unreported association between lentigines and arterial dissections was described in 1995 by Schievink et al. Two families were described in which multiple members had one or both of the features above with or without evidence of cystic medial necrosis of the aorta. The histologically proved lentigines were scattered in a generalized distribution, but predominated on the extremities including palms, soles, fingers, and toes. The lentigines usually appeared in childhood, increased in number through early adulthood, then faded or disappeared. A 33-year-old male had fading of his lentigines over the ensuing 12 years, likewise, a 30-year-old female noted fading over the next 12 years (Schievink et al., 1995).

Tay Syndrome

Cardiac Pre-excitation Lentiginosis

An autosomal recessive disorder was described by Tay et al. (1974) in which two Malaysian sisters were found to have multiple lentigines, vitiligo, café-au-lait macules, premature canities (early graying of the hair), growth and mental retardation, hepatic cirrhosis, aminoaciduria, hypersplenism, and multiple skeletal defects.

A familial syndrome of cardiac pre-excitation (causing arrhythmias and sudden death) and generalized cutaneous lentigines distinct from LEOPARD syndrome was reported by Vidaillet and colleagues (1987). Cardiac pre-excitation is also known as accessory atrioventricular pathways.

Phakomatosis Pigmentovascularis (PPV)

Isolated Mucocutaneous Melanotic Pigmentation (IMMP)

First described in 1947 by Ota and colleagues, individuals with

Newly described in 2000 by Boardman and colleagues, indi-

868

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES

viduals with this lentiginosis had Peutz–Jeghers syndrome (PJS)-like pigmentation but lacked polyposis. Patient records covering 51 years were reviewed to determine if those with IMMP had an elevated cancer risk as is true with PJS. Surprisingly, it was found that females (but not males) had an increased relative risk for cancer of 3.2. Even more significantly, the relative risk for breast and gynecologic cancers was 7.8 in affected females (Boardman et al., 2000). The importance of recognizing this entity cannot be understated. While the lentigines themselves are benign, the associated increased cancer risk is significant. Recognition will allow early and increased oncologic screening in females.

Lentiginoses without Systemic Abnormalities Isolated Generalized Lentigines Nonfamilial, generalized lentigines may occur in the absence of systemic abnormalities. Like the syndromic lentigines they are usually present at birth or develop in early infancy. However, they may not appear until adulthood (Kaufmann et al., 1976; Uhle and Norvell, 1988). Some reports of generalized lentigines as an isolated finding have been included in the LEOPARD syndrome discussion. It is important to rule out IMMP (above) before reassuring patients (especially female) that it is benign.

Inherited Patterned Lentiginosis in Black People Generalized lentiginosis has been described in ten adult black patients, ages 25–52 years, with onset during infancy or early childhood, familial clustering in an autosomal pattern in seven patients, and without somatic abnormalities. Face, lips, extremities, buttocks, and palmoplantar, but not mucosal, surfaces were involved. O’Neill (1989) commented that this process may not be rare. Microscopic features of the two biopsies performed included basilar hyperpigmentation, and slight rete elongation in one of two biopsies. No melanocytic hyperplasia was demonstrated. The described pigmentation is clinically most consistent with lentigines, however this is not fully supported histologically.

Eruptive Lentiginosis Widespread occurrence of several hundred lentigines over a few months to years is typical of eruptive lentiginosis. This is usually found in adolescents and young adults who have no evidence of systemic abnormalities. Histologic examination shows the typical picture of a simple lentigo, with or without melanin macroglobules. In 1956 Degos and Carteaud described telangiectatic papules that darkened and became slightly raised keratotic or depressed scaly lentigines (Degos and Carteaud, 1956). These are different from eruptive nevi (Sanderson, 1960), and the two entities can be distinguished histologically. Eruptive lentiginosis was discussed by Bologa and colleagues in 1965. Later Eady and colleagues (1977) reported a patient in whom “a great number of lentigines” developed in the course of two years. Light and electron microscopy confirmed the diagnosis of lentigo and demonstrated giant melanosomes within the lesions (Eady et al., 1977).

Fig. 46.17. Nevus spilus (see also Plate 46.17, pp. 494–495).

Laugier–Hunziker Syndrome In 1970 Laugier and Hunziker described lentigines that appeared in early to mid-life in otherwise healthy persons. The pigmented lesions were primarily on the lips and buccal mucosa. Approximately 50% of the patients demonstrate nail pigmentation, usually longitudinal melanonychia (Lenane et al., 2001). Palms, soles, and other cutaneous surfaces are also frequently involved. This syndrome is synonymously known as: essential cutaneo-mucous hyperpigmentation (Seoane Leston, 1998), idiopathic lenticular mucocutaneous pigmentation (Gerbig, 1996), Laugier disease, and acquired idiopathic benign lentiginosis.

White Lentiginosis In 1994 Grosshans and colleagues described an adult female patient with congenital guttate hyper- and hypomelanotic macules. The numerous guttate lesions were flat and pigmented on the light-exposed areas of her limbs, and flat or papulokeratotic and depigmented on her trunk. They were histologically characterized by lentiginous hyperplasia of the epidermis with elongated club-shaped rete ridges, loss of pigmentation, and no disturbance of keratinization. They 869

CHAPTER 46

proposed the name “white lentiginosis” for their newly described entity (Grosshans et al., 1994).

Nevus Spilus A circumscribed pigmented (usually lightly) macule or patch containing scattered more darkly pigmented macules (Fig. 46.17; see also Chapter 57). Histopathology is consistent with lentigo for the background macule, and junctional and compound nevi in the darker speckled lesions (Lever and Schaumburg-Lever, 1990). Some cases reported as zosteriform lentiginous nevus have been clinically consistent with nevus spilus (Port et al., 1978).

Speckled Lentiginous Nevus Thought to be distinct from nevus spilus and Becker nevus (nevus spilus tardus) by some, the lesional background pigmentation is histologically consistent with lentigo simplex (Stewart et al., 1978). Agminated blue nevus combined with lentigo is a peculiar variant of speckled lentiginous nevus (Marchesi et al., 1993).

Isolated Reports of Lentigines Associated with Cutaneous Disorders Psoriasis with acquired lentigines confined to resolving plaques in two patients who had not received PUVA (psoralen and UVA) therapy (Burrows et al., 1994) and Dowling–Degos disease with profuse lentigines (Korstanje et al., 1989) have been described.

References Adamson, D. A case of unilateral pigmentary naevus. Br. J. Dermatol. 23:77, 1911. Aguilar, A., M. A. Gallego, E. Del Río, and E. Martínez. Partial unilateral lentiginosis. Actas. Dermo-Sifilogr. 83:646–648, 1992. Allegue, F., A. España, J. M. Fernández-García, and A. Ledo. Segmental neurofibromatosis with contralateral lentiginosis. Clin. Exp. Dermatol. 14:448–450, 1989. Arch, E. M., B. K. Goodman, R. A. Van Wesep, D. Liaw, K. Clarke, Parsons R, V. A. McKusick, and M. T. Geraghty. Deletion of PTEN in a patient with Bannayan-Riley-Ruvalcaba syndrome suggests allelism with Cowden disease. Am. J. Med. Genet. 71:489–493, 1997. Bandler, M. Haemangiomas of the small intestine associated with mucocutaneous pigmentation. Gastroenterology 38:641–645, 1960. Bologa, E. I., M. Bene, and P. Pasztor. [Considerations on the eruptive lentiginosis of the face] [French] Considerations sur la lentiginose eruptive de la face. Ann. Dermatol. Syphiligr. (Paris) 92:277–286, 1965. Boardman, L. A., M. R. Pittelkow, F. J. Couch, D. J. Schaid, S. K. McDonnell, L. J. Burgart, D. A. Ahlquist, J. A. Carney, D. I. Schwartz, S. N. Thibodeau, and L. C. Hartmann. Association of Peutz-Jeghers-like mucocutaneous pigmentation with breast and gynecologic carcinomas in women. Medicine (Baltimore) 79:293–298, 2000. Brocq, L. Précis Atlas de Pratique Dermatologique. Paris, 1921. Bronson, E. B. A case of symmetrical cutaneous atrophy of the extremities. J. Cutan. Genito-Urin. Dis. 13:1–10, 1895. Burrows, N. P., S. Handfield-Jones, B. E. Monk, R. A. Sabroe, J. M. Geraghty, and P. G. Norris. Multiple lentigines confined to psoriatic plaques. Clin. Exp. Dermatol. 19:380–382, 1994.

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Cappon, D. Case of unilateral lentigines with mental deficiency. Br. J. Dermatol. 60:371–374, 1948. Davis, D. G., and M. W. Shaw. An unusual human mosaic for skin pigmentation. N. Engl. J. Med. 270:1384–1389, 1964. Degos, R., and A. Carteaud. Lentiginose profuse keratosique. Ann. Dermatol. Venereol. 83:125, 1956. De Vivo, I., D. M. Gertig, S. Nagase, S. E. Hankinson, R. O’Brien, F. E. Speizer, R. Parsons, and D. J. Hunter. Novel germline mutations in the PTEN tumour suppressor gene found in women with multiple cancers. J. Med. Genet. 37:336–341, 2000. DiLiberti, J. H., R. G. Weleber, and S. Budden. RuvalcabaMyhre-Smith syndrome: a case with probable autosomal-dominant inheritance and additional manifestations. Am. J. Med. Genet. 15:491–495, 1983. DiLiberti, J. H., A. N. D’Agostino, R. H. Ruvalcaba, and J. R. Schimschock. A new lipid storage myopathy observed in individuals with the Ruvalcaba-Myhre-Smith syndrome. Am. J. Med. Genet. 18:163–167, 1984. Eady, R. A., J. J. Gilkes, and E. W. Jones. Eruptive naevi: Report of two cases, with enzyme histochemical, light and electron microscopical findings. Br. J. Dermatol. 97:267–278, 1977. Fargnoli, M. C., S. J. Orlow, J. Semel-Concepcion, and J. L. Bolognia. Clinicopathologic findings in the Bannayan-Riley-Ruvalcaba syndrome. Arch. Dermatol. 132:1214–1218, 1996. Figer, A., A. Kaplan, M. Frydman, D. Lev, J. Paswell, M. Z. Papa, B. Goldman, E. Friedman. Germline mutations in the PTEN gene in Israeli patients with Bannayan-Riley-Ruvalcaba syndrome and women with familial breast cancer. Clin. Genet. 62:298–302, 2002. Gerbig, A. W., and Hunziker T. Idiopathic lenticular mucocutaneous pigmentation or Laugier-Hunziker syndrome with atypical features. Arch. Dermatol. 132:844–845, 1996. Gerhards, G., and H. Hamm. [Unilateral lentiginosis — a segmental neurofibromatosis without neurofibromas]. Hautarzt 43:491–495, 1992. Gimm, O, T., J. Attie-Bitach, A. Lees, M. Vekemans, and C. Eng. Expression of the PTEN tumour suppressor protein during human development. Hum. Mol. Genet. 9:1633–1639, 2000. Goksel, S., and T. Kural. Lentiginosis and right atrial myxoma. Eur. Heart J. 10:769–771, 1989. Goldberg, N. S. Partial unilateral lentiginosis [letter]. J. Am. Acad. Dermatol. 32:144–146, 1995. Goudie, R. B., A. S. Jack, and B. M. Goudie. Genetic and developmental aspects of pathological pigmentation patterns. Chapter 7: Syndromes associated with lentigines. Curr. Top. Pathol. 74:115– 120, 1985. Gretzula, J. C., O. Hevia, L. S. Schachner, J. H. DiLiberti, R. H. Ruvalcaba, J. R. Schimschock, R. G. Weleber, F. Halal, M. H. Lipson, B. Blumberg, and P. J. Weber. Ruvalcaba-Myhre-Smith syndrome. Pediatr. Dermatol. 5:28–32, 1988. Gron, K. Lentiginosis profusa unilateralis. Acta Derm. Venereol. 8:466–473, 1928. Grosshans, E., D. Sengel, and E. Heid. [White lentiginosis]. Ann. Dermatol. Venereol. 121:7–10, 1994. Hendriks, Y. M., J. T. Verhallen, J. J. van der Smagt, S. G. Kant, Y. Hilhorst, L. Hoefsloot, K. B. Hansson, P. J. van der Straaten, H. Boutkan, M. H. Breuning, H. F. Vasen, and A. H. Brocker-Vriends. Bannayan-Riley-Ruvalcaba syndrome: further delineation of the phenotype and management of PTEN mutation-positive cases. Fam. Cancer. 2:79–85, 2003. Holder, J. E., R. A. Graham-Brown, and R. D. Camp. Partial unilateral lentiginosis associated with blue naevi. Br. J. Dermatol. 130:390–393, 1994. Hughes, G. S., Jr., H. K. Park, and B. E. Jones. Partial unilateral lentiginosis in a black patient with sickle cell anemia [letter]. J. Am. Acad. Dermatol. 8:563–565, 1983. Joshi A., V. K. Garg, S. Agrawal, A. Agarwalla, and A. Thakur. Portwine-stain (nevus flammeus), congenital Becker’s nevus, cafe-au-

GENETIC EPIDERMAL PIGMENTATION WITH LENTIGINES lait-macule and lentigines: phakomatosis pigmentovascularis type Ia — a new combination. J. Dermatol. 26:834–836, 1999. Kaufmann, J. et al. 1976. Lentiginosa profusa. Dermatologica 153:116. Quoted in Rhodes, A. R. Neoplasms: benign neoplasias, hyperplasias, and dysplasias of melanocytes. In: Dermatology in General Medicine, 4th ed., T. B. Fitzpatrick, A. Eisen, K. Wolff, I. M. Freedberg, and F. K. Austen (eds). New York: McGraw-Hill, Inc., 1993, pp. 996–1077. Korstanje, M. J., R. F. Hulsmans, F. J. Hulsmans, and A. M. J. van der Kley. Dowling-Degos disease associated with profuse lentigines: Case report from 244th Netherlands Society for Dermatology and Venerology. Br. J. Dermatol. 121:529–530, 1989. Laugier, P., and N. Huziker. Pigmentation melaniques lentigulaire, essentielle de la muqueuse jugale et des levres. Arch. Belges Dermatol. Syphil. 26:391, 1970. Lenane P., D. O. Sullivan, C. O. Keane, and S. O. Loughlint. The Laugier-Hunziker syndrome. J. Eur. Acad. Dermatol. Venereol. 15:574–577, 2001. Lever, W. F., and G. Schaumburg-Lever. Histopathology of the Skin, 7th ed. Philadelphia: J. B. Lippincott, 1990, p. 772. Longy, M., V. Coulon, B. Duboue, A. David, M. Larregue, C. Eng, P. Amati, J. L. Kraimps, A. Bottani, D. Lacombe, and D. Bonneau. Mutations of PTEN in patients with Bannayan-Riley-Ruvalcaba phenotype. J. Med. Genet. 35:886–889, 1998. MacLeod, J. M. H. A case of pigmented naevi-like freckles. Br. J. Dermatol. 25:191, 1913. Marchesi, L., L. Naldi, A. Di Landro, L. Cavalieri d’Oro, A. Brevi, and T. Cainelli. Segmental lentiginosis with jentigo histologic pattern. Am. J. Dermatopathol. 14:323–327, 1992. Marchesi, L., L. Naldi, A. Parma, F. Locati, and T. Cainelli. Agminate blue nevus combined with lentigo. A variant of speckled lentiginous nevus? Am. J. Dermatopathol. 15:162–165, 1993. Marsh, D. J., V. Coulon, K. L. Lunetta, P. Rocca-Serra, P. L. Dahia, Z. Zheng, D. Liaw, S. Caron, B. Duboue, A. Y. Lin, A. L. Richardson, J. M. Bonnetblanc, J. M. Bressieux, A. Cabarrot-Moreau, A. Chompret, L. Demange, R. A. Eeles, A. M. Yahanda, E. R. Fearon, J. P. Fricker, R. J. Gorlin, S. V. Hodgson, S. Huson, D. Lacombe, and C. Eng. Mutation spectrum and genotype–phenotype analyses in Cowden disease and Bannayan–Zonana syndrome, two hamartoma syndromes with germline PTEN mutation. Hum. Mol. Genet. 7:507–515, 1998. Marsh, D. J., J. B. Kum, K. L. Lunetta, M. J. Bennett, R. J. Gorlin, S. F. Ahmed, J. Bodurtha, C. Crowe, M. A. Curtis, M. Dasouki, T. Dunn, H. Feit, M. T. Geraghty, J. M. Graham, Jr, S. V. Hodgson, A. Hunter, B. R. Korf, D. Manchester, S. Miesfeldt, V. A. Murday, K. L. Nathanson, M. Parisi, B. Pober, C. Romano, and C. Eng. PTEN mutation spectrum and genotype-phenotype correlations in Bannayan-Riley-Ruvalcaba syndrome suggest a single entity with Cowden syndrome. Hum. Mol. Genet. 8:1461–1472, 1999. Matsudo, H., W. B. Reed, D. Homme, W. Bartok, and R. Horowitz. Zosteriform lentiginous nevus. Arch. Dermatol. 107:902–905, 1973. McKelway, J. I. Lentigo: unilateral distribution. Report of a case. N. Y. Med. J. 80:197–198, 1904. Micali, G., M. R. Nasca, D. Innocenzi, and D. Lembo. Agminated lentiginosis: case report and review of the literature [review]. Pediatric. Dermatol. 11:241–245, 1994. Moss, C., and S. H. Green. What is segmental neurofibromatosis? Br. J. Dermatol. 130:106–110, 1994. Ota, M., T. Kawamura, and N. Ito. Phacomatosis pigmentovascularis. Jpn. J. Dermatol. 52:1–3, 1947. O’Neill, J. F., and W. D. James. Inherited patterned lentiginosis in blacks [review]. Arch. Dermatol. 125:1231–1235, 1989. Parslew, R., and J. L. Verbov. Partial lentiginosis. Clin. Exp. Dermatol. 20:141–142, 1995. Pickering, J. G. Partial unilateral lentiginosis with associated developmental abnormalities. Guys Hospital Rep. 122:361–370, 1973. Piqué, E., A. Aguilar, M. C. Farina, M. A. Gallego, P. Escalonilla, and

L. Requena. Partial unilateral lentiginosis: report of seven cases and review of the literature [review]. Clin. Exp. Dermatol. 20:319–322, 1995. Port, M., J. Courniotes, and M. Podwal. Zosteriform lentiginous naevus with ipsilateral rigid cavus foot. Br. J. Dermatol. 98:693–698, 1978. Rhodes, A. R. Neoplasms: benign neoplasias, hyperplasias, and dysplasias of melanocytes. In: Dermatology in General Medicine, 4th ed., T. B. Fitzpatrick, A. Eisen, K. Wolff, I. M. Freedberg, and F. K. Austen (eds). New York: McGraw-Hill, Inc., 1993, pp. 996–1077. Rosenblum, G. A. Cryotherapy of lentiginous mosaicism. Cutis 35:543–544, 1985. Roth, M. R., R. Martinez, and W. D. James. Segmental neurofibromatosis. Arch. Dermatol. 123:917–920, 1987. Rouault, J. P., P. E. Kuwabara, O. M. Sinilnikova, L. Duret, D. Thierry-Mieg, and M. Billaud. Regulation of dauer larva development in Caenorhabditis elegans by daf-18, a homologue of the tumour suppressor PTEN. Curr Biol. 9:329–332, 1999. Ruth, W. K., J. D. Shelbourne, and B. V. Jegasothy. Zosteriform lentiginous nevus. Arch. Dermatol. 116:476, 1980. Ruvalcaba, R. H., S. Myhre, and D. W. Smith. Sotos syndrome with intestinal polyposis and pigmentary changes of the genitalia. Clin. Genet. 18:413–416, 1980. Sanderson, K. V. Eruptive telangiectatic cellular naevi. Br. J. Dermatol. 27:302–311, 1960. Schievink, W. I., V. V. Michels, B. Mokri, D. G. Piepgras, and H. O. Perry. Brief report: a familial syndrome of arterial dissections with lentiginosis. N. Engl. J. Med. 332:576–579, 1995. Schubert. 1924. Zentralballt f. Haut. u. Geschlechtskrankheiten 12:128. Quoted in Gron, K. Lentiginosis profusa unilateralis. Acta Derm. Venereol. 8:466–473, 1928. Seoane Leston, J. M., J. Vazquez Garcia, A. M. Cazenave Jimenez, A. de la Cruz Mera, and A. Aguado Santos. Laugier-Hunziker syndrome. A clinical and anatomopathologic study. Presentation of 13 cases. Rev. Stomatol. Chir. Maxillofac. 99:44–48, 1998. Shumate, C. A. Pigmented unilateral naevus-like lentigo. Arch. Dermatol. 43:410, 1941. Stewart, D. M., J. Altman, and A. H. Mehregan. Speckled lentiginous nevus. Arch. Dermatol. 114:895–896, 1978. Sutton, R. L. Diseases of the Skin, 1st ed. St. Louis: Mosby, 1916. Sutton, R. L., and R. L. Sutton Jr. Diseases of the Skin, 10th ed. St. Louis: Mosby, 1939. Tay, C. H., E. Rajagopalan, E. McEvoy-Bowe, E. P. C. Tock, and J. L. Da Costa. A recessive disorder with growth and mental retardation, peculiar facies, abnormal pigmentation, hepatic cirrhosis and aminoaciduria. Acta Paediatr. Scand. 63:777–782, 1974. Theiler, R., H. Stocker, and E. Boltshauser. [The classification of atypical forms of neurofibromatosis] [German]. Schweiz. Med. Wochenschr. 121:446–455, 1991. Thompson, G. W., and A. K. Diehl. Partial unilateral lentiginosis. Arch. Dermatol. 116:356, 1980. Trattner, A., and A. Metzker. Partial unilateral lentiginosis. J. Am. Acad. Dermatol. 29(5 Pt 1):693–695, 1993. Uhle, P., and S. S. Norvell Jr. Generalized lentiginosis. J. Am. Acad. Dermatol. 18(2 Pt 2):444–447, 1988. Vidaillet, H. J., Jr., C. S. Burton 3rd., N. M. Ramirez, M. R. Gilbert, V. S. Behar, D. B. Hackel, and L. D. German. An unusual variant of familial preexcitation. Am. J. Cardiol. 59:371–373, 1987. Zahorcsek, Z., A. Schmelas, and I. Schneider. [Progressive circumscript lentiginosis — phakomatosis pigmentovascularis III/A] [German] Progrediente zirkumskripte Lentiginose — Phakomatosis pigmentovascularis III/A. Hautarzt 39:519–523, 1988. Zhou, X. P., H. Hampel, J. Roggenbuck, N. Saba, T. W. Prior, and C. Eng. A 39-bp deletion polymorphism in PTEN in African American individuals: implications for molecular diagnostic testing. J. Mol. Diagn. 4:114–117, 2002.

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CHAPTER 46 Zigman, A. F., J. E. Lavine, M. C. Jones, C. R. Boland, and J. M. Carethers. Localization of the Bannayan-Riley-Ruvalcaba syndrome gene to chromosome 10q23. Gastroenterology 113:1433–1437, 1997.

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Zori, R. T., D. J. Marsh, G. E. Graham, E. B. Marliss, and C. Eng. Germline PTEN mutation in a family with Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome. Am. J. Med. Genet. 80:399–402, 1998.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

47

Genetic Epidermal Syndromes: Localized Hyperpigmentation Sections Anonychia with Flexural Pigmentation Nancy Burton Esterly, Eulalia Baselga, and Beth A. Drolet Incontinentia Pigmenti Sheila S. Galbraith and Nancy Burton Esterly Periorbital Hyperpigmentation Nancy Burton Esterly, Eulalia Baselga, and Beth A. Drolet Pigmentary Demarcation Lines Sheila S. Galbraith and Nancy Burton Esterly Dowling–Degos Disease Sheila S. Galbraith and Nancy Burton Esterly

Anonychia with Flexural Pigmentation Nancy Burton Esterly, Eulalia Baselga, and Beth A. Drolet In 1975 Verbov described four members of an English family with absent or rudimentary nails, macular pigmentation of the flexures, dry, sparse hair, and abnormal dermatoglyphics (Verbov, 1975). Brown macules and hypopigmented patches are seen in the axillae and groin. One patient had hyperpigmentation on the lateral border of the areola. Generalized hypohidrosis, diminished dermatoglyphics, and loss of epidermal ridges over the palms and soles are also reported (Table 47.1). The hair is coarse and slow growing. The histologic features are not documented. The entity is inherited as an autosomal dominant trait and there are no systemic manifestations. There is no known treatment.

Reference Verbov, J. Anonychia with bizarre flexural pigmentation — an autosomal dominant dermatosis. Br. J. Dermatol. 92:469–474, 1975.

Incontinentia Pigmenti Sheila S. Galbraith and Nancy Burton Esterly

Historical Background Incontinentia pigmenti is an uncommon genetic disorder characterized by distinctive skin manifestations as well as ocular, dental, skeletal, and central nervous system abnormalities. Two early reports of probable incontinentia pigmenti were those of Garrod (1906) and Adamson (1908), each of whom described a female patient with peculiar skin pigmentation and multiple other anomalies. In 1925, three other affected individuals were identified. Bardach reported on monozygotic twin girls (Bardach, 1925). Bloch presented a young girl with bizarre pigmentation of the skin and retinal detachment to the Swiss Dermatological Society (Bloch, 1926). Two years later

Sulzberger provided a more detailed account of that same patient (Sulzberger, 1927). It was Bloch who proposed the name incontinentia pigmenti. The disorder is sometimes referred to by the eponym Bloch–Sulzberger disease.

Synonyms Bloch–Sulzberger syndrome, Asboe–Hansen disease, Bloch– Siemens syndrome, Siemens–Bloch pigmented dermatosis, melanoblastosis cutis linearis, and nevus pigmentosus systematicus.

Epidemiology Incontinentia pigmenti occurs worldwide in all racial groups and almost exclusively in females. About 55% of affected girls have a positive family history for the disorder.

Genetics The inheritance pattern is that of an X-linked dominant trait which is lethal for hemizygous affected males. Studies of multigenerational pedigrees have shown that affected females have approximately twice as many daughters as sons and have an increased incidence of spontaneous abortions of male fetuses (Ashley and Burgdorf, 1987; Gorski and Burright, 1993). The few documented living affected males have had either a 47 XXY chromosomal genotype (Prendiville et al., 1989) or have been presumed to have a halfchromatid mutation (Lenz, 1975). These boys are mosaic for two cell lines, one containing a normal X chromosome and the other a mutated X chromosome. Additional mechanisms to explain survival of affected boys include an unstable premutation (Traupe and Vehring, 1994) and genetic heterogeneity.

Clinical Findings Incontinentia pigmenti was originally thought to have a sporadic form (termed IP1) which was mapped to chromosome Xp11 and a familial form (termed IP2) mapped to chromosome Xq28. A review of the original reports of IP1, however, revealed that most of these cases represented pigmentary 873

CHAPTER 47 Table 47.1. Genetic diseases with reticulated epidermal hyperpigmentation. Disease

Inheritance* Distribution

Special features

Associated findings

Acropigmentation of Kitamura Acropigmentation of Dohi Dermatopathia pigmentosa reticularis

AD AD AD

Acral Acral Generalized

Lesions are atrophic Hypopigmented macules Hypopigmented macules

Anonychia with flexural pigmentation Mendes da Costa disease

AD

Flexural

Hypopigmented macules

XLR

Generalized, face and limbs

Dowling–Degos disease

AD

Naegeli–Franceschetti– Jadassohn syndrome Epidermolysis bullosa with mottled pigmentation

AD

Flexural, then generalized Generalized

Blistering early; hypopigmented macules Onset delayed

Palmar pits; breakage of epidermal ridges Predominant in males Sweating disorders; decreased dermatoglyphics; alopecia; onychodystrophy; palmar/plantar keratoses Sparse hair; hypohidrosis; decreased dermatoglyphics; absent/rudimentary nails Microcephaly; mental retardation; atrichia; short conical fingers

AD

Trunk, proximal limbs

Blistering disorder

Perioral pits; keratoses; epidermal cysts Keratoderma; enamel hypoplasia; hyperhidrosis; nail dystrophy Keratoderma; palmar/plantar; carious teeth; photosensitivity

* AD, autosomal dominant; XLR, X-linked recessive.

mosaicism and were not true cases of incontinenia pigmenti (Sybert, 1994, 1998). Since then, there have been no further reports linking the Xp11 locus with definitive cases of incontinentia pigmenti, and it has been accepted that Xq28 is the only true locus for incontinentia pigmenti. The terms IP1 and IP2 have been discarded (Berlin et al., 2002). The clinical expression of the disorder has been attributed to random inactivation of the X chromosome which can cause extreme variation in the constellation of findings from one patient to another. Migeon et al. have postulated that selection against cells expressing the mutated gene may help to ameliorate the deleterious phenotype in females (Curtis et al., 1994; Migeon et al., 1989). The characteristic skin findings, which occur in more than 96% of affected children, consist of four distinct types of lesions that usually appear sequentially but that may overlap or be skipped in a given individual (Carney, 1976; Landy and Donnai, 1993). In approximately 90% of patients, the cutaneous lesions are present at birth. In the remainder onset is during the first year or, rarely, even later (Carney, 1976). Stage 1 lesions are inflammatory macules, papules, vesicles, and pustules, most often in linear array and located primarily on the limbs (Fig. 47.1) and trunk following the lines of Blaschko. For approximately the first four months of life these lesions occur in crops, persist for days to weeks and then involute spontaneously. Occasionally they recur periodically in older children, especially at times of acute febrile illnesses. Stage 2 lesions are hyperkeratotic and verrucous and may be present at birth but more often develop during early infancy, waning by 6 months of age. Although often relatively inconspicuous they tend to be linear in configuration, follow the Blaschko lines, and may persist in some patients until adulthood (Bessems et al., 1988; Dahl et al., 1975). 874

Fig. 47.1. Early stage: vesicular lesions following the lines of Blaschko (see also Plate 47.1, pp. 494–495).

The stage 3 lesions which consist of streaks and whorls of gray-brown pigment (Fig. 47.2) that end at the midline and follow the Blaschko lines. The trunk is more often involved in this phase, particularly the axillae and groin. It is not always appreciated that the pigmented lesions do not occur specifically at sites of phase 1 and 2 lesions and, therefore, do not represent postinflammatory pigmentary change. These lesions may be visible at birth but more often arise during infancy or early childhood, fading totally or partially during adolescence or young adulthood (Landy and Donnai, 1993; Salmon and Frieden, 1995). The stage 4 lesions are a late manifestation of the disease and frequently go unrecognized and are much more subtle than their predecessors. They consist of atrophic, hypo- or

GENETIC EPIDERMAL SYNDROMES: LOCALIZED HYPERPIGMENTATION

Fig. 47.3. Stage 4 lesions: note the prominent depigmentation (see also Plate 47.3, pp. 494–495).

Fig. 47.2. Stage 3 lesions with pigmentary abnormalities found in this disorder (see also Plate 47.2, pp. 494–495).

depigmented bands or streaks that are hairless and anhidrotic, fail to tan with sun exposure, and arise on normal skin (Fig. 47.3). They are most prominent on the flexor aspects of the legs where their reticulated arrangement has been referred to as a “Chinese letter pattern” (Ashley and Burgdorf, 1987; Moss and Ince, 1987; Nazzaro et al., 1990; PascualCastroviejo et al., 1994). Hypopigmented streaks may be the only cutaneous manifestation of the disease in affected adults and are seen only occasionally in children. Additional cutaneous findings include alopecia and nail dystrophy. Approximately 35–40% of patients have mild partial or patchy alopecia most commonly involving the vertex. Textural changes resembling woolly hair have also been described (Landy, 1993). The nail dystrophy, which occurs in less than 10% of patients, is relatively innocuous and consists mainly of ridging and pitting of the nail plates. A rare manifestation, painful subungual tumors, associated with underlying lytic bony deformities are always of late onset (adolescence or adulthood) and occur more frequently on the fingers. These tumors have the same histologic pattern as the phase 2 verrucous skin lesions (Mascaro et al., 1985; Simmons et al., 1986).

Approximately 80% of patients have extracutaneous abnormalities which may not be evident during infancy (Carney, 1976). Dental defects occur in 80% of cases over 1 year of age and because they persist indefinitely are of considerable diagnostic importance. Ocular abnormalities are also quite common occurring in 35–40% of patients (Catalano, 1990; Francois, 1984; Spallone, 1987). Although a figure of 30% is often cited for central nervous system involvement (from Carney’s review of 653 cases in the world’s literature), a more recent review citing experience with over 100 cases suggests that less than 10% of children with incontinentia pigmenti have mental or motor retardation (Landy and Donnai, 1993). Additional systemic features include breast anomalies and hypoplasia, skeletal defects and body asymmetry, and cardiovascular anomalies (Carney, 1976; Landy and Donnai, 1993; Miteva and Nikolova, 2001). The more common extracutaneous anomalies are listed in Table 47.2.

Histology The histologic findings of the skin lesions in incontinentia pigmenti varies with the stage but certain histologic features are shared. However, there is no unanimity of opinion as to the exact findings in any stage. The first stage is characterized by marked spongiosis as well as intraepidermal vesicles filled with eosinophils and surrounded by dyskeratotic cells singly or in clusters. Ultrastructural studies demonstrate some intracellular alterations of the melanocytes and keratinocytes, an intact basement membrane, normal distribution of pigment throughout the basal epidermal layer and absence of pigment in the dermis. A moderate mixed dermal infiltrate of melanophages and Schwann cells containing melanosomes has also been described (Caputo et al., 1975; Schaumberg-Lever and Lever, 1973). One study of a transitional lesion (stage 1–2) notes breaches in the basement membrane and a second population of melanocytes with abnormal dendrites and containing large vacuoles and myelin figures high in the epidermis. Numerous melanophages containing ingested melanosomes and lysosome-like structures also appeared to be lying free in the upper 875

CHAPTER 47 Table 47.2. Noncutaneous features of incontinentia pigmenti. Dental Delayed eruption of teeth Hypo- or anodontia Abnormally shaped teeth (pegged) Ocular Strabismus (most common) Cataracts Microphthalmos Optic atrophy Proliferative retinopathy Fibrovascular retinal membrane Retinal detachment Central nervous system Microcephaly; hydrocephaly Seizures Mental and motor retardation Hypotonia Spastic paralysis Ataxia Ischemic cerebrovascular accidents Hydrocephalus

dermis (Guerrier and Wong, 1974). These latter findings have not generally been confirmed by others. Second stage lesions have a hyperkeratotic, papillomatous and acanthotic epidermis composed of dyskeratotic cells with abnormally clumped tonofilaments and lacking in desmosomes. In the lower epidermis and upper dermis large macrophages can be seen phagocytizing both melanosomes and dyskeratotic keratinocytes. Clusters of intra- and extracellular pigment granules are found in the dermal papillae. Mast cells and Schwann cells are present in the dermis as well. Basal layer melanocytes are normal but numerous melanocyte dendrites extend into the spinous layer. Dyskeratotic cells are once again present (Caputo et al., 1975; Schaumberg-Lever and Lever, 1973). Third stage lesions have given the disease its name. Large numbers of melanin-laden macrophages are present in the dermis. The epidermis is remarkable for dyskeratotic cells and increased melanin in the basal cell layer. However, the segments of the basal layer overlying the groups of melanophages have a marked diminution of pigment. Collagen in the lower dermis is thickened. The basement membrane is normal (Schaumberg-Lever and Lever, 1973; Zillikens et al., 1991). Schwann cells containing varying numbers of melanosomes populate the upper dermis along with the melanophages (Schaumberg-Lever and Lever, 1973) and have been seen in the epidermis as well (Worret et al., 1988). In the fourth or hypopigmented stage, the epidermis is slightly thinned, the dermis is thickened and homogenized, and dermal appendageal structures are reduced or absent. Melanocytes and keratinocytes are normal in size, number, and shape on ultrastructural examination. Rare melanophages may be present in the dermis (Ashley and Burgdorf, 1987; 876

Moss and Ince, 1987; Nazzaro et al., 1990). Zillikins et al. (1991) found a reduced number of melanocytes in the basal cell layer and round eosinophilic bodies of an amorphous substance thought to resemble degenerated keratinocytes in the upper dermis. They also, unexpectedly, found the same amount of melanin granules in the basal layer as in the stage 3 lesions but less melanin in the upper epidermal layers.

Laboratory Findings Abnormal laboratory studies include peripheral eosinophilia ranging from 5% to 65%, most pronounced during the first four months of life concurrent with stage 1 vesiculopustular lesions (Carney, 1976). Defective neutrophil chemotaxis and impaired lymphocyte transformation have been documented in a few affected individuals (Dahl et al., 1975; Jessen et al., 1978; Menni et al., 1990). Neuroradiologic studies have been performed in a few patients with neurologic manifestations and have demonstrated hypoplasia of the corpus callosum and focal atrophy of the cerebrum and cerebellum (Mangano and Barbagallo, 1993; Pascual-Castroviejo et al., 1994) as well as radiologic findings indicative of hemorrhagic necrosis (Chatkupt et al., 1993). Periventricular white matter damage and porencephalic cysts have also been found on magnetic resonance images (Mirowski and Caldemeyer, 2000).

Criteria for Diagnosis The diagnosis is almost always made on the basis of the distinctive clinical skin lesions and confirmed by examination of a skin biopsy. Peripheral eosinophilia during the first stage as well as the presence of characteristic abnormalities in other organs provide additional evidence for the diagnosis. Landy and Donnai (1993) have proposed diagnostic criteria for incontinentia pigmenti (Table 47.3); however the utility of these criteria has not yet been established.

Differential Diagnosis Differential diagnosis includes several neonatal vesiculopustular eruptions during the first stage, most notably herpes simplex infection, bacterial infections, mastocytosis, epidermolysis bullosa, Langerhans cell histiocytosis, candidiasis, and varicella-zoster infections. The verrucous second stage can be confused with verruca vulgaris, X-linked chondrodysplasia punctata, and linear epidermal nevi. The hyperpigmented lesions resemble those of linear and whorled hypermelanosis, Naegeli–Franceschetti–Jadassohn syndrome, dermatopathia pigmentosa reticularis, focal dermal hypoplasia, chromosomal mosaicism, and chimerism. The lesions of the hypopigmented stage can resemble hypomelanosis of Ito, but they do not appear until adolescence or adulthood. None of these entities have the sequential cutaneous changes seen in incontinentia pigmenti. A skin biopsy confirming the diagnosis is helpful during the neonatal period.

Pathogenesis Incontinentia pigmenti has recently been mapped to chromosome Xq28. The 23kb gene encodes nuclear factor kB essen-

GENETIC EPIDERMAL SYNDROMES: LOCALIZED HYPERPIGMENTATION Table 47.3. Diagnostic criteria for incontinentia pigmenti. Family history

Major criteria

Minor criteria

No evidence of IP in a first degree female relative*

Typical neonatal rash Erythema, vesicles, eosinophilia Typical hyperpigmentation mainly on trunk, following lines of Blaschko, fading by adolescence Linear, atrophic, hairless lesions Suggestive history or evidence of typical rash, hyperpigmentation, atrophic hairless lesions Vertex alopecia Dental anomalies Retinal disease Multiple male miscarriages

Dental anomalies Alopecia Abnormal nails

Evidence of IP in a first degree female relative†

Retinal disease

*At lease one major criterion is necessary for diagnosis in cases with no family history. Minor criteria support the diagnosis; complete absence should induce a degree of uncertainty. †Presence of any one or more of the major criteria strongly suggests a diagnosis of IP in cases with definitive family history.

tial modulator (NEMO), and consists of ten exons. A single mutation resulting in the deletion of exons 4–10 accounts for more than 80% of cases of incontinentia pigmenti (Aradhya et al., 2001). NEMO is otherwise known as the g-subunit of the inhibitor kB kinase (IKKg) and controls nuclear factor kB (NF-kB). (NF-kB) is a transcription factor that regulates the expression of many genes involved in immune and inflammatory responses and protects against apoptosis induced by tumor necrosis factor. In the majority of cells, NF-kB is inactivated by the inhibitor kB (IkB) protein. When TNF receptors are activated, IkB is phosphorylated by the inhibitor kB kinase (IKK), causing degradation of IkB. NF-kB is then released and allowed to control transcription. The IKK complex is made up of IKKa, IKKb, and IKKg (NEMO). NEMO is therefore essential in the activation of NF-kB. In incontinentia pigmenti, there is a population of NEMO deficient cells with disruption of this signaling pathway which results in absence of NF-kB activity, leading to apoptosis. The degree of cutaneous expression in incontinentia pigmenti reflects the percentage of cells that express the mutated X chromosome (Berlin et al., 2002). Eotaxin, a eosinophilselective chemokine, has been found to be involved in the first stage of incontinentia pigmenti (Berlin et al., 2002). Eotaxin expression is increased by inflammatory cytokines such as interleukin (IL)-1a and tumor necrosis factor (TNF) a and causes epidermal eosinophil infiltration. After degranulation, eosinophil proteases cause degradation of tonofilaments and desmosomes, resulting in blister formation (Jean-Baptiste et al., 2002). Once the number of NEMO deficient cells decrease secondary to apoptosis, the inflammation subsides and the vesicular stage ends. The second stage of incontinentia pigmenti is in part due to the inflammation associated with the first stage. The hyperproliferative cytokeratins 6 and 17 have been found to progressively increase during the inflammatory stage. The resultant hyperproliferation is likely due to the proliferation

of normal IKKg (NEMO) keratinocytes (Berlin et al., 2002). The exact pathogenesis of the third stage of incontinentia pigmenti is unclear. Neither inflammation nor death of defective IKKg (NEMO) cells provide an adequate explanation for the hyperpigmentation.The fourth stage of incontinentia pigmenti likely represents postinflammatory dermal scarring (Berlin et al., 2002). Mutations involving other alleles of the NEMO gene have also been identified which result in phenotypes different from incontinentia pigmenti (Berlin, 2002; Smahi, 2002). Anhidrotic ectodermal dysplasia and immunodeficiency (EDA-ID) is an X-linked recessive disorder characterized by features of ectodermal dysplasia with recurrent infections caused by encapsulated bacterial pathogens. It is caused by milder missense mutations or small deletions in the NEMO gene rather than a severe truncation of NEMO as in incontinentia pigmenti; thus, there is not complete loss of NF-kB activation. The disorder affects only males who may have a family history of incontinentia pigmenti. Some authors believe that EDA-ID and incontinentia pigmenti form a continuum, while others believe that they are distinct entities (Happle, 2003). Mutations in the stop codon of the NEMO gene result in a combination of EDA-ID, osteopetrosis, and lymphedema (OL-EDA-ID).

Treatment Management will depend on the manifestations in a particular child. Skin lesions can be treated symptomatically and parents can be reassured that the lesions will run their course and heal without scarring. An ophthalmologic examination in infancy is mandatory and a dental evaluation should be performed by 1 year of age. If neurologic abnormalities are apparent or suspected, neurological consultation and magnetic resonance imaging are appropriate. A careful physical examination and frequent follow-up examinations will detect associated abnormalities that might not be immediately apparent 877

CHAPTER 47

in the neonatal period. A thorough family history and, if possible, examination of other family members may yield helpful information. Genetic counseling should be offered to all families of an affected child.

Prognosis The prognosis will vary with the particular constellation of defects (O’Brien and Feingold, 1985). Most patients do well unless neurologically impaired or afflicted with a severe ophthalmologic defect. The pigmentary lesions usually fade partially or completely by adolescence or early adulthood.

References Adamson, H. G. Congenital pigmentation with atrophic scarring associated with other congenital abnormalities. Proc. R. Soc. Med. 1 (Part 1):9–10, 1908. Aradhya, S., H. Woffendin, T. Jakins, T. Bardaro, T. Esposito, A. Smahi, C. Shaw, M. Levy, A. Munni, M. D’Urso, R. A. Lewis, S. Kenwrick, and D. L. Nelson. A recurrent deletion in the ubiquitously expressed NEMO (IKK-gamma) gene accounts for the vast majority of incontinentia pigmenti mutations. Hum. Mol. Genet. I:2171–2179, 2001. Ashley, J. R., and W. H. C. Burgdorf. Incontinentia pigmenti: Pigmentary changes independent of incontinence. J. Cutan. Pathol. 14:248–250, 1987. Bardach, M. Systematisierte naevusbildungen bei einem eineiigen Zwillingspaar. Z. Kinderheilkd. 39:542–550, 1925. Berlin, A. L., A. S. Paller, and L. S. Chan. Incontinentia pigmenti: a review and update on the molecular basis of pathophysiology. J. Am. Acad. Dermatol. 47:169–187, 2002. Bessems, P. J., B. A. Jagtman, W. J. van de Staak, R. F. Hulsmanss, and K. J. Croughs. Progressive, persistent, hyperkeratotic lesions in incontinentia pigmenti. Arch. Dermatol. 124:29–30, 1988. Bloch, B. Eigentumliche, bisher nicht beschriebene pigmentaffektion (incontinentia pigmenti). Schweiz. Med. Wochenschr. 56:404–405, 1926. Caputo, R., F. Gianotti, and M. Innocenti. Ultrastructural findings in incontinentia pigmenti. Int. J. Dermatol. 14:46–55, 1975. Carney, R. G., Jr. Incontinentia pigmenti. A world statistical analysis. Arch. Dermatol. 112:535–542, 1976. Catalano, R. A. Incontinentia pigmenti. Am. J. Ophthalmol. 110:696–700, 1990. Chatkupt, S., A. O. Gozo, L. J. Wolansky, and S. Sun. Characteristic MR findings in a neonate with incontinentia pigmenti. Am. J. Roentgenol. 160:372–374, 1993. Curtis, A. R., S. Lindsay, E. Boye, A. J. Clarke, S. J. Landy, and S. S. Bhattacharya. A study of X chromosome activity in two incontinentia pigmenti families with probable linkage to Xq28. Eur. J. Hum. Genet. 2:51–58, 1994. Dahl, M. V., G. Matula, R. Leonards, and D. L. Tuffanelli. Incontinentia pigmenti and defective neutrophil chemotaxis. Arch. Dermatol. 111:1603–1605, 1975. Francois, J. Incontinentia pigmenti (Bloch-Sulzberger syndrome) and retinal changes. Br. J. Ophthalmol. 68:19–25, 1984. Garrod, A. E. Peculiar pigmentation of the skin in an infant. Trans. Clin. Soc. Lond. 39:216, 1906. Gorski, J. L., and E. N. Burright. The molecular genetics of incontinentia pigmenti. Semin. Dermatol. 12:255–265, 1993. Guerrier, C. J. W., and C. K. Wong. Ultrastructural evolution of the skin in incontinentia pigmenti (Bloch-Sulzberger). Study of 6 cases. Dermatologica 149:10–22, 1974. Happle, R. A fresh look at incontinentia pigmenti (comment). Arch. Dermatol. 139:1206–1208, 2003. Jessen, R. T., D. E. Van Epps, J. S. Goodwin, and J. Bowerman. Incon-

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tinentia pigmenti. Evidence for both neutrophil and lymphocyte dysfunction. Arch. Dermatol. 114:1182–1186, 1978. Jean-Baptiste, S., E. A. O’Toole, M. Chen, J. Guitart, A. Paller, and L. S. Chan. Expression of eotaxin, an eosinophil-selective chemokine, parallels eopsinophil accumulation in the vesiculobullous stage of incontinentia pigmenti. Clin. Exp. Immunol. 127:470–478, 2002. Landy, S. J., and D. Donnai. Incontinentia pigmenti (Bloch-Sulzberger syndrome). J. Med. Genet. 30:53–59, 1993. Lenz, W. Half chromatid mutations may explain incontinentia pigmenti in males. Am. J. Hum. Genet. 27:690–691, 1975. Mangano, S., and A. Barbagallo. Incontinentia pigmenti: clinical and neuroradiologic features. Brain Dev. 15:362–366, 1993. Mascaro, J. M., J. Palou, and P. Vives. Painful subungual keratotic tumors in incontinentia pigmenti. J. Am. Acad. Dermatol. 13:913–918, 1985. Menni, S., R. Piccinno, A. Biolchini, and A. Plebani. Immunologic investigations in eight patients with incontinentia pigmenti. Pediatr. Dermatol. 7:275–277, 1990. Migeon, B. R., J. Axelman, S. J. de Beur, D. Valle, G. A. Mitchell, and K. M. Rosenbaum. Selection against lethal alleles in females heterozygous for incontinentia pigmenti. Am. J. Hum. Genet. 44:100–106, 1989. Mirwoski, G. W., and K. S. Caldemeyer. Incontinentia pigmenti. J. Am. Acad. Dermatol. 43:517–518, 2000. Miteva, L., and A. Nikolova. Incontinentia pigmenti: a case associated with cardiovascular anomalies. Pediatr. Dermatol. 18:54–56, 2001. Moss, C., and P. Ince. Anhidrotic and achromians lesions in incontinentia pigmenti. Br. J. Dermatol. 116:839–849, 1987. Nazzaro, V., A. Brusasco, C. Gelmetti, E. Ermacora, and R. Caputo. Hypochromic reticulated streaks in incontinentia pigmenti: An immunohistochemical and ultrastructural study. Pediatr. Dermatol. 7:174–178, 1990. O’Brien, J. E., and M. Feingold. Incontinentia pigmenti. A longitudinal study. Am. J. Dis. Child. 139:711–712, 1985. Pascual-Castroviejo, I., M. C. Roche, V. Martinez Fernandez, M. Perez-Romero, R. M. Escudero, J. J. Garcia-Penas, and M. Sanchez. Incontinentia pigmenti: MR demonstration of brain changes. Am. J. Neuroradiol. 15:1521–1527, 1994. Prendiville, J. S., J. L. Gorski, C. K. Stein, and N. B. Esterly. Incontinentia pigmenti in a male infant with Klinefelter syndrome. J. Am. Acad. Dermatol. 20:937–940, 1989. Salmon, J. K., and I. J. Frieden. Congenital and genetic disorders of hyperpigmentation. Curr. Probl. Dermatol. 7:145–196, 1995. Schaumberg-Lever, G., and W. F. Lever. Electron microscopy of incontinentia pigmenti. J. Invest. Dermatol. 61:151–158, 1973. Simmons, D. A., M. F. Kegel, R. K. Scher, and Y. C. Hines. Subungual tumors in incontinentia pigmenti. Arch. Dermatol. 122:1431– 1434, 1986. Smahi, A., G. Courtois, S. H. Rabia, R. Doffinger, C. Bodemer, A. Munnich, J. L. Casanova, and A. Israel. The NF-kappaB signalling pathway in human diseases: from incontinentia pigmenti to ectodermal dysplasias and immune-deficiency syndromes. Hum. Mol. Genet. 11:2371–2375, 2002. Spallone, A. Incontinentia pigmenti (Bloch-Sulzberger syndrome): seven case reports from one family. Br. J. Ophthalmol. 71:629–634, 1987. Sulzberger, M. B. Uber eine bisher nicht beschriebene congenitale pigmentanomalie (incontinentia pigmenti). Arch. Dermatol. Syphil. 154:19–32, 1927. Sybert, V. P. Incontinentia pigmenti nomenclature [letter]. Am. J. Hum. Genet. 55:209–211, 1994. Sybert, V. P. A case revisited: recent presentation of incontinentia pigmenti in association with a previously reported X; autosome translocation. Am. J. Med. Genet. 75:334, 1998.

GENETIC EPIDERMAL SYNDROMES: LOCALIZED HYPERPIGMENTATION Traupe, H., and K. H. Vehring. Unstable pre-mutation may explain mosaic disease expression of incontinentia pigmenti in males. Am. J. Med. Genet. 49:397–398, 1994. Worret, W. I., R. E. Nordquist, and W. H. C. Burgdorf. Abnormal cutaneous nerves in incontinentia pigmenti. Ultrastruct. Pathol. 12:449–454, 1988. Zillikens, D., A. Mehringer, W. Lechner, and G. Burg. Hypo- and hyperpigmented areas in incontinentia pigmenti. Light and electron microscopic studies. Am. J. Dermatopathol. 13:57–62, 1991.

Periorbital Hyperpigmentation Nancy Burton Esterly, Eulalia Baselga, and Beth A. Drolet

Clinical Findings Periorbital hyperpigmentation or dark circles around the eyes is a common finding in otherwise healthy people. Folk opinion holds that they reflect fatigue and lack of sleep. In the older literature, French physicians connected the disorder with biliary diseases and called it masque biliaire or lunettes biliaires (Aguilera-Maruri and Aguilera-Diaz, 1969). Adlercreutz analyzed sixty women with dark circles around the eyes and found a common association with infections of the urinary tract, unspecified gynecological diseases, and biliary dyspepsia and dyskinesia (Adlercreutz, 1949). These associations, however, have never been confirmed in subsequent reports (Goodman and Belcher, 1969). Peters et al. in 1918 described this form of hyperpigmentation as an autosomal dominant disorder affecting 20 members in five generations of a single kindred. The recognition of this pigmentary disorder in additional large pedigrees (Franceschetti et al., 1953; Fulk, 1984; Goodman and Belcher, 1969; Hunzinger, 1962) confirms its hereditable nature. The incidence of this abnormality is difficult to assess since it is probably overlooked and underreported. Both males and females are similarly affected and it is seen more frequently in individuals with dark complexions (Fulk, 1984; Gellin, 1981) (Figs 47.4 and 47.5). Clinical expression is variable in different members of the family. The hyperpigmentation usually starts during childhood in the lower eyelids and progresses with age to involve the entire periorbital area. There is marked variation in expression of this trait. The most severely affected individuals may have a raccoonlike appearance. The degree of darkness fluctuates, becoming more intense with fatigue, worry, anxiety, and illness. The youngest patient reported was 9 years old. However, some parents state that the hyperpigmentation starts during infancy. There is no erythema or dermatitis preceding the hyperpigmentation. A family history of atopy is usually absent in the affected patients.

Fig. 47.4. Mild periorbital hyperpigmentation (see also Plate 47.4, pp. 494–495).

Fig. 47.5. Idiopathic periorbital hyperpigmentation (see also Plate 47.5, pp. 494–495).

Differential Diagnosis Differential diagnosis includes other causes of periorbital pigmentation. Malignant melanoma and periorbital ecchymosis have to be considered. Certain drugs including antipsychotic medications, gold, and chemotherapeutic agents may result in periorbital pigmentation. Erythema dyschromicum perstans may also affect the lids (Ing et al., 1992). However, in this latter disorder the pigmentation is more patchy, there is no family history and the histopathologic features are those of a lichenoid dermatitis. Dark circles around the eyes (allergic shiners) have been described as a stigma of atopy. Allergic shiners may cause lower lid discoloration and edema and are attributed to venous stasis resulting from mucosal edema of the nose and paranasal sinuses (Carlson, 1981).

Histology

Treatment

Histopathologically there is orthokeratosis with an increased number of melanin granules in the basal cells and lower Malpighian layers as well as melanin-containing macrophages in the upper dermis (Fulk, 1984; Goodman and Belcher, 1969). The pathogenesis is unknown.

Treatment for this condition is probably unnecessary because the condition is often overlooked or considered a familial trait.

References Adlercreutz, E. Periocular hyperpigmentation (“masque biliaire”) and its relation to biliary and genito-urinary tract diseases. Acta Med. Scand. 135:63–67, 1949.

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CHAPTER 47 Aguilera-Maruri, C., and L. Aguilera-Diaz. Dark circles around the eyes. Cutia 5:979–982, 1969. Carlson, R. E. Allergic shiners. J. Am. Med. Assoc. 246:835, 1981. Franceschetti, A., W. Jadassohn, and R. Paillard. Démonstration d’un syndrome de Sjögren associé a una hyperpigmentation famililale des paupières. Dermatologica 106:277, 1953. Fulk, C. S. Primary disorders of hyperpigmentation. J. Am. Acad. Dermatol. 10:1–16, 1984. Gellin, G. A. Dark circles around the eyes. J. Am. Med. Assoc. 245:1165, 1981. Goodman, M., and W. Belcher. Periorbital hyperpigmentation. Arch. Dermatol. 100:169–174, 1969. Hunzinger, N. A. A propos de l’hyperpigmentation familiale des paupières. Journal de Genetique Humaine 11:16, 1962. Ing, E. B., J. R. Buncic, B. A. Weiser, J. de Nanassy, and L. Boxall. Periorbital hyperpigmentation and erythema dyschromicum perstans. Can. J. Ophthalmol. 27:353–355, 1992. Peters, R. Auffallende Dunkelfarbung der unteren Lider als erbliche Anomalie. Zbl. Prakt. Augenheilk. 42:8–11, 1918.

Table 47.4. Classification of pigmentary demarcation lines (modified from James and Carter, 1987). Type

Location

A

Lateral aspect of upper anterior portion of the arms across pectoral area Posteromedial portion of the lower limbs Vertical hypopigmented line in the pre- and parasternal areas Posteromedial area of the spine Bilateral aspect of the chest, marking from the mid third of the clavicle to the periareolar skin Lines on the face

B C D E F

Pigmentary Demarcation Lines Sheila S. Galbraith and Nancy Burton Esterly

Historical Background In 1913, Matzumoto first described pigmentary demarcation lines on the upper and lower extremities of Japanese patients. In 1938, Futcher described demarcation lines in the black population (Futcher, 1938; Selmanowitz and Krivo, 1973). Since first described, pigmentary demarcation lines have been classified into five types designated by the letters A–E (James and Carter, 1987; Miura, 1951; Selmanowitz and Krivo, 1975). Demarcation lines occurring in association with pregnancy were first described by Fulk (1984).

Fig. 47.6. Typical pigmentary demarcation lines on the flexural aspects of the arm (type 1) (see also Plate 47.6, pp. 494–495).

Synonyms Futcher or Voight lines.

Epidemiology These lines, also referred to as Voigt lines or Futcher lines, have been noted to occur in other locations and in all ethnic groups, although they are more distinct and common in black people. Familial occurrences of these lines have been reported (Ito, 1965; Kisch and Nasuhoglu, 1953; Selmanowitz and Krivo, 1973; Vazquez et al., 1986).

Clinical Findings Pigmentary demarcation lines have been classified in five types (A–E) based on their location (Table 47.4). More recently, Malakar and Dhar (2000) described pigmentary lines on the face which they labeled as type F pigmentary demarcation lines. Types A and C are the best characterized and more common types, with a reported prevalence of 19.5–77% in black people (Futcher, 1938; James and Carter, 1987; Kisch and Nasuhoglu, 1953; Vollum, 1971), 4.3% in Japanese (Ito, 1965) and 14% in white people (James and Carter, 1987). Ito (1965) reported a ratio of affected women to men of 10:1. However, in all other series, the incidence has been similar in both sexes (Futcher, 1938; James and Carter, 1987; Kisch and 880

Fig. 47.7. Close-up view of Fig. 47.6 (see also Plate 47.7, pp. 494–495).

Nasuhoglu, 1953; Vollum, 1971). Within a given race, the prevalence does not depend on the depth of pigmentation of the skin. Pigmentary demarcation lines are usually bilateral and symmetrical (Figs 47.6 and 47.7) although unilateral variants have been described in up to 16% of the cases (James and Carter, 1987). More than one type of line may coexist in a single patient (James et al., 1984). Pigmentary demarcation lines usually become visible during the first 6 months of life but they may be apparent at birth (James and Carter, 1987). In most individuals they become more noticeable with age

GENETIC EPIDERMAL SYNDROMES: LOCALIZED HYPERPIGMENTATION

(Selmanowitz and Krivo, 1975; Vollum, 1971). Onset during pregnancy has been described for type B lines. Type A demarcation lines (see Plate 27.10, pp. 494–495, and Figs 47.6 and 47.7), the most common type, extend from the humoral head down the anterolateral aspect of the arm to the elbow. They may extend proximally to the pectoral area and distally to the base of the first metacarpal bone. A dorsal counterpart on the posterior aspect of the upper arm has been described (James and Carter, 1987; Miura, 1951; Vollum, 1971). Type B demarcation lines (see Fig. 27.10) extend from the perineal region along the posteromedial aspect of the leg to the lateral or inner malleolus. Type B lines have been reported to develop both during early and late pregnancy (Fulk, 1984; James and Carter, 1987; James et al., 1984; Ozawa et al., 1993; Vazquez et al., 1986). They tend to disappear in the postpartum period (Ozawa et al., 1993; Vazquez et al., 1986) but persistence after delivery has been described (James et al., 1984). They usually appear in women that show other pigmentary changes of pregnancy such as pigmentation of the linea alba, genitalia, and areola (James et al., 1984; Vazquez et al., 1986). Type C pigmentary demarcation line is a mediosternal hypopigmented fine line (see Plate 27.11, pp. 494–495) or band that often arches from the xiphoid process to the midsternum superiorly (Kisch and Nasuhoglu, 1953; Matzumoto, 1913; Selmanowitz and Krivo, 1975). It may extend to the abdomen overlying the linea alba. Type D, or the dorsal spinal line, is rare and has only been reported in Japanese and black patients (James and Carter, 1987). Type E lines are not usually true lines but hypopigmented lanceolate macules, bands or streaks on the chest of black individuals. They are often symmetrical, oriented obliquely, and distributed along an imaginary line traced from the middle third of the clavicle to the periareolar skin (James and Carter, 1987; Selmanowitz and Krivo, 1975). Type F lines were recently described as straight or curved convex lines sharply demarcating a relatively darker zone from a lighter area over the face (Malakar and Dhar, 2000). The lines are almost always bilateral and correspond to the distribution of the ophthalmic, maxillary, and mandibular divisions of the trigeminal nerve.

Histology Histopathologic examination of pigmentary demarcation lines has demonstrated both normal and increased epidermal pigmentation. Futcher (1938) could not find any microscopic differences in the amount of pigmentation between both halves of a biopsy taken across a type A line. Other authors, however, when comparing biopsies from the darker and the lighter skin have noted increased melanin in the basal cell layer with normal numbers of melanocytes on the darker side (James et al., 1984; Matzumoto, 1913; Miura, 1951; Vazquez et al., 1986).

Diagnosis and Differential Diagnosis Diagnosis depends on the recognition of the particular distribution of these lines. A skin biopsy is not necessary and, when performed, the darker skin has to be compared to the paler skin. Type E lines may be confused with the ash leaf macules

of tuberous sclerosis. Ash leaf macules tend to be more numerous and widespread, not bilaterally symmetrical, and are associated with other features of tuberous sclerosis.

Pathogenesis The pathophysiologic mechanisms responsible for pigmentary demarcation lines are unknown but various explanations have been proposed. Some authors postulate that they may be of phylogenetic importance since, in animals, the dorsal skin is usually more pigmented to provide better protection against ultraviolet light and for thermoregulation (Matzumoto, 1913). Others believe that the pigmentary demarcation lines coincide with the marginal lines of cutaneous innervation which separate dermatomes arising from nonconsecutive dorsal roots (Dupre et al., 1977; Miura, 1951; Vollum, 1971). Ozawa et al. (1993) postulated that the appearance of type B lines during pregnancy may be secondary to compression of S1 and S2 nerves by the enlarged uterus. A hormonal influence has also been suggested for pregnancy associated lines (Vazquez et al., 1986). Finally, the possibility of a dominantly inherited condition has been raised since familial cases have been described (Ito, 1965; James et al., 1984; Selmanowitz and Krivo, 1973).

Treatment Treatment is not necessary since these lines are viewed as normal variants and of no cosmetic concern.

References Dupre, A., J. Maleville, J. Lassere, and O. Aillos. Les dermatoses axiales. Ann. Dermatol. Venereol. 104:304–308, 1977. Fulk, C. S. Primary disorders of hyperpigmentation. J. Am. Acad. Dermatol. 10:1–16, 1984. Futcher, P. H. A peculiarity of pigmentation of the upper arm of Negroes. Science 88:570–571, 1938. Ito, K. The peculiar demarcation of pigmentation along the so-callled Voigt’s line among the Japanese. Dermatol. Int. 4:45–47, 1965. James, W. J., and J. M. Carter. Pigmentary demarcation lines: A population survey. J. Am. Acad. Dermatol. 16:584–590, 1987. James, W. J., M. A. Guill, T. G. Berger, and O. G. Rodman. Pigmentary demarcation lines associated with pregnancy. J. Am. Acad. Dermatol. 11:438–440, 1984. Kisch, B., and A. Nasuhoglu. A mediosternal depigmentation line in Negroes. Exp. Med. Surg. 11:265–267, 1953. Malakar, S., and S. Dhar. Pigmentary demarcation lines over the face. Dermatology 200:85–86, 2000. Matzumoto, S. H. Uber eine eigentumliche Pigmentverteilung an den Voigtschen Linien. Arch. Dermatol. Syphil. 118:157–164, 1913. Miura, O. On the demarcation lines of pigmentation observed among the Japanese, on inner sides of their extremities and on anterior and posterior sides of their medial regions. Tohoku J. Exp. Med. 54:135–139, 1951. Ozawa, H., M. Rokugo, and H. Aoyama. Pigmentary demarcation lines of pregnancy with erythema. Dermatology 187:134–136, 1993. Selmanowitz, V. J., and J. M. Krivo. Hypopigmented markings in Negroes. Int. J. Dermatol. 12:229–235, 1973. Selmanowitz, V. J., and J. M. Krivo. Pigmentary demarcation lines: Comparison of Negroes with Japanese. Br. J. Dermatol. 93:371–377, 1975. Vazquez, M., M. Ibañez, and J. L. Sánchez. Pigmentary demarcation lines during pregnancy. Cutis 38:263–266, 1986.

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CHAPTER 47

Fig. 47.8. Reticulated pigmentation of the flexures (see also Plate 47.8, pp. 494–495). Vollum, D. I. Skin markings in Negro children from the West Indies. Br. J. Dermatol. 85:260–263, 1971.

Dowling–Degos Disease Sheila S. Galbraith and Nancy Burton Esterly

Historical Background Dowling–Degos disease is a rare entity first described as a form of acanthosis nigricans by Dowling and Freudenthal in 1938. Degos and Ossipowski recognized it as a distinct entity in 1954, and Wilson-Jones and Grice characterized it as “reticulate pigmented anomaly of the flexures” in 1978.

Fig. 47.9. Acneiform perioral pits (see also Plate 47.9, pp. 494–495).

macules are commonly seen in the central flexural areas such as the vault of the axilla and in the inguinal creases. Some of the lesions may be palpable but never are velvety or papillomatous. Once established, the pigmentation is thought to be permanent. The mucous membranes are spared, although genital lesions have been described (O’Goshi, 2001). Other cutaneous findings include pitted acneiform scars in the perioral region. In addition there are comedolike, hyperkeratotic follicular papules on the neck and axillae and epidermal cysts. The hyperkeratotic papules are thought to arise in areas of friction. Other reported associated findings include hidradenitis suppurativa, keratoacanthoma, and squamous cell carcinoma (Bedlow and Mortimer, 1996; Fenske et al., 1991; Weber, 1990).

Synonyms Reticulated pigmented anomaly of the flexures, dark dot disease.

Epidemiology and Genetics The inheritance is autosomal dominant with variable penetrance and expressivity (Brown, 1982). There is a slight female predominance (Kim et al., 1999).

Clinical Findings Dowling–Degos disease is characterized by progressive, reticulate pigmentation of the flexures (Fig. 47.8) and acneiform perioral pits (Fig. 47.9). The pigmentary changes are gradual and the onset is delayed beginning in early adulthood (30–40 years). The distribution of the pigmented lesions is symmetric. There are numerous, asymptomatic, discrete, round or oval, 1–3 mm, brown macules that resemble ephelides. The macules gradually become confluent in a “lacelike” or reticulated pattern. They are usually brown-gray in color although some authors report “deeply pigmented” or black macules (Dowling and Freudenthal, 1938). Initially the groin and axilla are affected. As the disorder progresses the neck, inframammary creases, trunk, proximal arms, and antecubital fossae become involved. Pigmentation may rarely be found on the wrists, dorsa of the hands, fingers and toes. The confluent 882

Histology The histologic findings of Dowling–Degos disease are relatively unique. The epidermis is slightly atrophic with long, narrow, branched rete ridges that intertwine at their bases. This pattern has been called “antler-like” (Smith et al., 1971). The pigmentation is a result of increased melanin at the tips of the rete ridges. There is no pigmentary incontinence or increase in the number of melanocytes. Hair follicles may be involved as well.

Differential Diagnosis This entity has been confused with many other autosomal dominant disorders of pigmentation (Table 47.5). The pigmentation of Kitamura’s reticulate acropigmentation is typically distributed in acral areas. However, features of both Dowling–Degos disease and Kitamura’s disorder have been described in patients of the same kindred (Crovato and Rebora, 1986). Many authors believe these disorders represent one entity (Crovato and Rebora, 1986; Rebora and Crovato, 1984). Haber’s syndrome is a disorder characterized by pigmented keratotic papules on the axilla, neck, and torso as well as pitted scars on the face and persistent facial erythema (Kikuchi, 1988). This disorder is now considered to be a variant of Dowling–Degos disease, however. The differential

GENETIC EPIDERMAL SYNDROMES: LOCALIZED HYPERPIGMENTATION Table 47.5. Genetic diseases with reticulated epidermal hyperpigmentation. Disease

Inheritance* Distribution

Special features

Associated findings

Acropigmentation of Kitamura Acropigmentation of Dohi Dermatopathia pigmentosa reticularis

AD AD AD

Acral Acral Generalized

Lesions are atrophic Hypopigmented macules Hypopigmented macules

Anonychia with flexural pigmentation Mendes da Costa disease

AD

Flexural

Hypopigmented macules

XLR

Generalized, face and limbs

Dowling–Degos disease

AD

Naegeli–Franceschetti– Jadassohn syndrome Epidermolysis bullosa with mottled pigmentation

AD

Flexural, then generalized Generalized

Blistering early; hypopigmented macules Onset delayed

Palmar pits; breakage of epidermal ridges Predominant in males Sweating disorders; decreased dermatoglyphics; alopecia; onychodystrophy; palmar/plantar keratoses Sparse hair; hypohidrosis; decreased dermatoglyphics; absent/rudimentary nails Microcephaly; mental retardation; atrichia; short conical fingers

AD

Trunk, proximal limbs

Blistering disorder

Perioral pits; keratoses; epidermal cysts Keratoderma; enamel hypoplasia; hyperhidrosis; nail dystrophy Keratoderma; palmar/plantar; carious teeth; photosensitivity

* AD, autosomal dominant; XLR, X-linked recessive.

diagnosis also includes the Crowe sign of neurofibromatosis, confluent and reticulated papillomatosis of Gougerot and Carteaud, and acanthosis nigricans. The pigmentation seen in confluent and reticulated papillomatosis of Gougerot and Carteaud usually involves the back and chest. Individuals with acanthosis nigricans have associated endocrinopathies and the onset is earlier than observed in Dowling–Degos disease. The uniform, velvety texture and the histologic findings of acanthosis nigricans distinguish this disorder.

Pathogenesis and Prognosis The pathogenesis is unknown. There are no systemic manifestations and patients have a normal life span.

Treatment Many topical medications including azelaic acid, retinoic acid, hydroquinone, and corticosteroids, as well as systemic retinoids, have been tried with minimal improvement (Kim et al., 1999). Wenzel et al. (2003) reported successful long-term treatment of Dowling–Degos disease with the erbium:YAG laser.

References Bedlow, A. J., and P. S. Mortimer. Dowling-Degos disease associated with hidradentitis suppurativa. Clin. Exp. Dermatol. 21:305–306, 1996. Brown, W. G. Reticulate pigmentary anomaly of the flexures: Case reports and genetic investigation. Arch. Dermatol. 118:490–493, 1982. Crovato, F., and A. Rebora. Reticulate pigmented anomaly of the flex-

ures associating reticulate acropigmentation: one single entity. J. Am. Acad. Dermatol. 14:359–361, 1986. Degos, R., and B. Ossipowski. Dermatose pigmentaire reticulee des plis. Ann. Dermatol. Syphiligr. 81:147–151, 1954. Dowling, G. B., and W. Freudenthal. Acanthosis nigricans. Br. J. Dermatol. 50:467–471, 1938. Fenske, N. A., C. E. Groover, C. W. Lober, and C. G. Espinoza. Dowling-Degos disease, hidradenitis suppurativa, and multiple keratoacanthomas: a disorder that may be caused by a single underlying defect in pilosebaceous epithelial proliferation. J. Am. Acad. Dermatol. 24:888–892, 1991. Kikuchi, I., F. Crovato, and A. Rebora. Haber’s syndrome and Dowling-Degos disease. Int. J. Dermatol. 27:96–97, 1988. Kim, Y. C., M. D Davis, C. F. Schanbacher, and W. P. Su. DowlingDegos disease (reticulate pigmented anomaly of the flexures): a clinical and histopathologic study of 6 cases. J. Am. Acad. Dermatol. 40:462–467, 1999. O’Goshi, K., T. Terui, and H. Tagami. Dowling-Degos disease affecting the vulva. Acta Derm. Venereol. 81:148, 2001. Rebora, A., and F. Crovato. The spectrum of Dowling-Degos disease. Br. J. Dermatol. 110:627–630, 1984. Smith, E. L., G. B. Dowling, and E. Wilson-Jones. Acquired axillary and inguinal pigmentation: An epidermal naevoid abnormality not to be confused with acanthosis nigricans. Br. J. Dermatol. 85:295–296, 1971. Weber, L. A., G. R. Kantor, and W. F. Bergfeld. Reticulate pigmented anomaly of the flexures (Dowling-Degos disease): a case report associated with hidradentitis suppurativa and squamous cell carcinoma. Cutis 45:446–450, 1990. Wenzel, G., W. Petrow, K. Tappe, R. Gerdsen, W. P. Uerlich, and T. Bieber. Treatment of Dowling-Degos disease with Er:YAG-laser: results after 2.5 years. Dermatol. Surg. 29:1161–1162, 2003. Wilson-Jones, E., and K. Grice. Reticulate pigmented anomaly of the flexures. Arch Dermatol. 114:1150–1157, 1978.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

48

Genetic epidermal syndromes: disorders of aging Sections Acrogeria Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, and Beth A. Drolet Metageria Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, and Beth A. Drolet Progeria Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, and Beth A. Drolet Xeroderma Pigmentosum Anita P. Sheth, Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, and Beth A. Drolet Werner Syndrome Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, Beth A. Drolet, and Cindy L. Lamerson

Acrogeria Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, and Beth A. Drolet

Historical Background In 1940, Gottron reported a brother and a sister whose hands and feet appeared old since infancy. At least 32 other cases have been reported since then (Beauregard and Gilchrest, 1987; de Groot et al., 1980; Matsumura et al., 1993; Meurer et al., 1993).

Synonym

Gilkes et al., 1974; Meurer et al., 1993). The hair is usually normal but there is recession of the hair line. Various nail abnormalities have been noted including onychogryphosis, koilonychia and atrophy (Beauregard and Gilchrest, 1987; de Groot et al., 1980). Teeth are normal. The skeletal system is the only other affected system. The most frequent abnormalities are short stature, micrognathia, and small hands and feet. Other abnormalities include acro-osteolysis, spina bifida, osteoporosis, congenital dislocation of hips, and pes planus (Beauregard and Gilchrest, 1987; Ho et al., 1987). Several systemic signs associated with other premature aging syndromes such as cataracts, arteriosclerosis, and diabetes mellitus are lacking. Life expectancy is therefore normal.

Gottron’s syndrome.

Associated Disorders Epidemiology Most cases of acrogeria are sporadic, although familial occurrence has been reported in a few instances (Blaszczyk et al., 2000; de Groot et al., 1980; Gottron, 1940; Huttova et al., 1967; Kaufman et al., 1985). Both autosomal recessive and autosomal dominant inheritance have been suggested. Acrogeria affects females three times more frequently than males (de Groot et al., 1980; Meurer et al., 1993).

Clinical Findings The cutaneous changes are usually evident at birth or develop during early infancy. There is atrophy, fine wrinkling and loss of subcutaneous tissue of the distal extremities. Mottled hyperpigmentation at acral sites is found in a third of the patients (Meurer et al., 1993). The pigmentary changes may also affect the axillae and anogenital folds (Bruckner-Tuderman et al., 1987; de Groot et al., 1980). The skin of the trunk may be affected to a lesser extent and an abnormal visibility of the venous pattern is typical (Venencie et al., 1984). The face is usually pinched with a small pointed nose and wrinkled skin. Easy bruisability and impaired wound healing may occur (Beauregard and Gilchrest, 1987; de Groot et al., 1980; 884

Acrogeria has been associated with elastosis perforans serpiginosa in several cases (de Groot et al., 1980; Gottron, 1940; Laugier et al., 1959; Tafelkruyer and Mees, 1978; Venencie et al., 1984). A single patient with coexistent psoriasis and sarcoidosis has been reported (Meurer et al., 1993).

Histology Histologic examination of the skin shows loss of subcutaneous tissue and atrophy of the dermis. The collagen fibers may have a swollen appearance and stain poorly. The elastic fibers often appear fragmented. Epidermal changes are minimal and include both atrophy and acanthosis, hyperkeratosis and increased pigmentation in the basal layer. Ultrastructurally the collagen fibers appear hypoplastic and immature. The elastic fibers appear as amorphous masses with frayed edges. There is abundant granular material in the extracellular space of unknown significance (de Groot et al., 1980; Laurent et al., 1975).

Diagnosis and Differential Diagnosis Diagnosis relies on the recognition of atrophic skin in acral locations. Age of onset, lack of other systemic findings, and

GENETIC EPIDERMAL SYNDROMES: DISORDERS OF AGING Table 48.1. Progeroid syndromes with hyperpigmentation. Werner

Progeria

Acrogeria

Metageria

Mandibuloacral dysplasia

Age of onset Inheritance Facies

Adolescence AR Beaked nose

Atrophy; calluses; ulcers; diffuse or mottled hyperpigmentation

Childhood AR “Andy Gump” like; loss of teeth Acral atrophy; mottled hyperpigmentation

Eyes Hair

Cataracts Premature graying

Prominent eyes Alopecia

Stature Sexual development Systemic findings

Small Absent Atherosclerosis; diabetes Reduced

Small Absent Atherosclerosis

Birth Sporadic, AD?, AR? Pinched face, beaked nose, micrognathia, wrinkled Acral atrophy, wrinkling, mottled hyperpigmentation; prominent veins on trunk Normal Normal; recession of hair line Normal Normal No

Birth ?Possibly AR Pinched face

Skin

Infancy ?AR and AD reported Plucked bird; prominent scalp veins; midfacial cyanosis Sclerodermalike; mottled hyperpigmentation

Reduced

Normal

Life span

Acral atrophy; mottled hyperpigmentation and telangiectasia on trunk; ulcers Prominent eyes Fine and thin Tall Normal Diabetes; atherosclerosis Reduced

Prominent eyes Alopecia Small Normal No; insulin resistance? Normal

AD, autosomal dominant; AR, autosomal recessive.

normal life expectancy differentiate acrogeria from other progeroid syndromes (Table 48.1). Differentiation from metageria may be more difficult and simultaneous occurrence of metageria and acrogeria in the same family has been reported (Kaufman et al., 1985). Some authors have coined the term acrometageria to emphasize that they may represent a spectrum of the same disease (Greally et al., 1992). Acrogeria has several features in common with Ehlers Danlos type IV such as readily visible veins, easy bruisability, and thin facies. However, in acrogeria the skin findings predominate and the hemorrhagic episodes from internal organs and arterial ruptures do not occur.

Laboratory Investigations Laboratory and chromosomal studies are normal including urinary levels of hyaluronic acid and glycosaminoglycans (Konohana et al., 1986).

Pathogenesis The pathogenesis and underlying genetic defect in acrogeria are not fully understood. Mutations in the COL3A1 gene, particularly with glycine substations, have been identified as a potential source for the variable clinical phenotypes found in acrogeria and vascular rupture (Jansen et al., 2000; Pope et al., 1996). The specific DNA mutation appears to be unique to each family which may interest the clinician in further molecular diagnostic tests. Pope et al. (1980) and Bouillie et al. (1986) found a tissue deficiency of collagen type III and proposed that acrogeria represents a form of Ehlers Danlos type IV. These findings have not been confirmed by others (Bruckner-Tuderman et al., 1987; Greally et al., 1992).

References Beauregard, S., and B. Gilchrest. Syndromes of premature aging. Dermatol. Clin. 5:109–121, 1987. Blaszczyk, M., A. Depaepe, L. Nuytinck, M. Glinska-Ferenz, and S. Jablonska. Acrogeria of the Grottron type in a mother and son. Eur. J. Dermatol. 10: 36–40, 2000. Bouillie, M., P. Venencie, E. Thomine, H. Ogier, A. Puissant, and P. Lauret. Syndrome d’Ehlers-Danlos type IV: A type d’acrogeria. Ann. Dermatol. Venereol. 113:1077–1085, 1986. Bruckner-Tuderman, L., A. Vogel, and U. W. Schnyder. Fibroblasts of an acrogeria patient produce normal amounts of type I and III collagen. Dermatologica 174:157–165, 1987. de Groot, W. P., J. Tafelkruyer, and M. J. Woerdeman. Familial acrogeria. Br. J. Dermatol. 103:213–223, 1980. Gilkes, J. J. H., D. E. Sharvill, and R. S. Wells. The premature aging syndromes: Report of eight cases and description of a new entity named metageria. Br. J. Dermatol. 91:243–262, 1974. Gottron, H. Familiaere acrogeria. Arch. Dermatol. Syphil. 181:571– 583, 1940. Greally, J. M., L. Y. Boone, S. G. Lenkey, S. L. Wenger, and M. W. Steele. Acrometageria: a spectrum of “premature aging” syndromes. Am. J. Med. Genet. 44:34–339, 1992. Ho, A., S. J. White, and J. E. Rasmussen. Skeletal abnormalities of acrogeria, a progeroid syndrome. Skeletal Radiol. 16:463–468, 1987. Huttova, M., M. Rusnak, and G. Lysa. Akrogeria. Cesk. Pediatr. 22:233–237, 1967. Jansen, T., A. de Paepe, N. Luytinck, and G. Plewig. COL3A1 mutation leading to acrogeria (Gottron type). Br. J. Dermatol. 142:178–180, 2000. Kaufman, I., B. Thiele, and G. Mahrle. Simultaneous occurrence of metageria and Gottron’s acrogeria in one family. Z. Hautkrankh. 60:975–984, 1985. Konohana, A., S. Miyakawa, S. Tajima, and T. Nishikawa. Decreased collagen and hyaluronic acid content in lesional skin of acrogeria. Dermatologica 172:241–244, 1986.

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CHAPTER 48 Laugier, P., C. Gomet, and F. Woringer. Acrogeria (Gottron) et keratose folliculare sepigineuse (Lutz). Bull. Dermatol. 66:80–92, 1959. Laurent, R., A. Oppermann, and P. Agache. Acrogeria: Clinical, histopathologic and ultrastructural study. Ann. Anat. Pathol. 20: 43–56, 1975. Matsumura, Y., H. Hamanaka, and T. Okuwa. A case of acrogeria. J. Dermatol. 20:572–576, 1993. Meurer, A., G. Lohmoler, and C. Keller. Gottron’s acrogeria and sarcoidosis. Clin. Invest. 71:387–391, 1993. Pope, F. M., P. Narcisi, A. C. Nicholls, D. Germaine, G. Pals, and A. J. Richards. COL3A1 mutations cause variable clinical phenotypes including acrogeria and vascular rupture. Br. J. Dermatol. 135:163–181, 1996. Pope, F. M., A. C. Nicholls, P. M. Jones, R. S. Wells, and D. Lawrence. EDS IV (acrogeria): new autosomal dominant and recessive types. J. R. Soc. Med. 73:180–186, 1980. Tafelkruyer, J., and R. Mees. Acrogeria and elastosis perforans serpiginosa. Dermatologica 156:309–312, 1978. Venencie, P. Y., F. C. Powell, and R. K. Winkelmann. Acrogeria with perforating elastoma and bony abnormalities. Acta. Derm. Venereol. 64:348–351, 1984.

Metageria Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, and Beth A. Drolet

Historical Background Metageria was first described by Gilkes et al. (1974) in two prematurely aged patients who could not be classified as having a previously known syndrome. Since then there have been only two other reports of metageria. One patient was from a family in which two other members were diagnosed as having acrogeria (Kaufman et al., 1985). Greally et al. (1992) described an additional patient with clinical features of both acrogeria and metageria and proposed the term acrometageria.

Clinical Findings Patients with metageria are noted to be abnormal at birth or during early infancy. They exhibit a generalized absence of subcutaneous fat, a tall thin habitus, and birdlike facies with a beaked nose and prominent eyes. The skin of the hands and feet appears atrophic. Around puberty, telangiectasia and mottled hyperpigmentation develop on the upper chest, neck, and extremities. The scalp hair is fine. One of the reported patients developed peripheral vasospasm resulting in leg ulcers and gangrene of her feet. Systemic manifestations include diabetes mellitus and atherosclerosis. Life span is probably reduced from complications of the systemic manifestations. The mode of inheritance is difficult to ascertain with only three reported cases.

Histology Histopathologic findings are not provided in the reported cases.

886

Diagnosis and Differential Diagnosis Diagnosis relies on the recognition of the clinical features. Differential diagnosis includes other progeroid syndromes (Table 48.2). Differentiation from acrogeria may be very difficult since there is some overlap between these two entities.

Pathogenesis An in vitro study of fibroblasts from a patient suggests that the defect resides in the type I collagen transcription because the fibroblasts were able to express mRNA and protein at a near normal level following incubation with a transforming growth factor-b (Hunzelmann et al., 1997). Nevertheless, the genetic defect remains unknown.

References Gilkes, J. J. H., D. E. Sharvill, and R. S. Wells. The premature aging syndromes: Report of eight cases and description of a new entity named metageria. Br. J. Dermatol. 91:243–262, 1974. Greally, J. M., L. Y. Boone, S. G. Lenkey, S. L. Wenger, and M. W. Steele. Acrometageria: a spectrum of “premature aging” syndromes. Am. J. Med. Genet. 44:34–339, 1992. Hunzelmann, N., U. Ueberham, B. Eckes, K. Herrmann, and T. Krieg. Transforming growth factor-beta reverses expression of type (I) collagen in cultured fibroblasts of a patient with metageria. Biochim. Biophys. Acta. 1360:65–70, 1997. Kaufman, I., B. Thiele, and G. Mahrle. Simultaneous occurrence of metageria and Gottron’s acrogeria in one family. Z. Hautkrankh. 60:975–984, 1985.

Progeria Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, and Beth A. Drolet

Historical Background This syndrome was first described by Hutchinson in 1886, in a report of a 3-year-old boy with an overall aged appearance. The term progeria was coined by Gilford (1904) who further described Hutchinson’s patient and reported an additional patient. In 1972, DeBusk reviewed the clinical characteristics of 60 patients with this syndrome.

Synonym Hutchinson–Gilford syndrome.

Epidemiology The incidence of progeria has been estimated to be approximately 1 per 4–8 million births with a male to female ratio of 2:1 (Beauregard and Gilchrest, 1987). Most of the reported patients are white (DeBusk, 1972).

Clinical Findings Affected patients appear normal at birth and the clinical manifestations of the syndrome develop during the first year of life. Neonatal progeroid syndrome (Wiedemann–Rautenstrauch syndrome) occurs at birth and is clinically distinct from progeria which includes a normal lamin A genome (Cao and

GENETIC EPIDERMAL SYNDROMES: DISORDERS OF AGING

of the hair has been noted. Hyperplastic scars and keloidlike lesions on the extremities have been described in a single case report (Jimbow et al., 1988). Nails may be dystrophic, small, short, thin, and discolored. Dentition is frequently delayed and incomplete. Progeria arises from de novo (not inherited) point mutations in lamin A (Eriksson et al., 2003). A recessive inheritance has been suggested by some authors on the basis of the reports of sibships with more than one affected member (Gabr et al., 1960; Khalifa, 1989; Macial, 1988; Parkash et al., 1990). Others have hypothesized that progeria is a sporadic dominant condition because there is a paternal age effect and the frequency of parental consanguinity is low (Brown et al., 1985; DeBusk, 1972).

Histology Histopathologically, the skin from sclerotic areas shows a normal or atrophic epidermis, increased melanin in the basal epidermal layers, thickening and hyalinization of collagen bundles, and loss of epidermal appendages (Badame, 1989; DeBusk, 1972; Fleischmajer and Nedwich, 1973; Gabr et al., 1960; Ishii, 1976). Histopathologic description of the hyperpigmented areas has not been reported. Light microscopy and electron microscopy of hair from two patients showed twisting of the shafts, longitudinal grooves, irregular diameters, and minor cuticular defects (Fleischmajer and Nedwich, 1973; Gillar et al., 1991).

Laboratory Investigations Fig. 48.1. Typical aged appearance of a patient with progeria (see also Plate 48.1, pp. 494–495).

Hegele, 2003) and the absence of hyperpigmentation. The classic clinical features that give the patients a prematurely aged and wasted appearance include generalized absence of subcutaneous fat; dwarfism; alopecia of the scalp, eyebrows, and lashes; sparse body hair; prominent scalp veins; small face with marked craniofacial disproportion; micrognathia; beaked nose; circumoral cyanosis; and protruding eyes and ears (Fig. 48.1). These facial peculiarities give these patients a strikingly similar appearance and a plucked bird facies. Several skeletal abnormalities are also typical such as shortened clavicles, coxa vara, progressive joint contractures that result in a “horse riding” stance, and accelerated osteoporosis that leads to frequent fractures. Sexual development is absent but intelligence is normal. Systemic manifestations are those derived from accelerated atherosclerosis. Dermatologic manifestations include sclerodermatous skin changes on the abdomen and upper thighs; thin, wrinkled, and loose skin on the hands and feet; and mottled hyperpigmentation. The pigmentary changes are more prominent over sunexposed areas and gradually increase with age (Brown et al., 1985; DeBusk, 1972; Gillar et al., 1991). Premature graying

Molecular diagnostics for de novo mutations of lamin A in exon 11 may prove to be useful in diagnosis and in prenatal detection (Eriksson et al., 2003). There are no consistent metabolic, endocrine, or serum lipid abnormalities. Increased urinary excretion of hyaluronic acid has been reported (Brown et al., 1985; Sweeney and Weiss, 1992) but has been challenged by a cohort study of 19 patients in whom neither serum nor urinary hyaluronan were elevated (Gordon et al., 2003). Characteristic radiographic findings are hypoplasia of the facial bones, patent fontanelles, wormian bones, resorption of the distal clavicles, narrowing of the ribs, thinned cortices of the long bones, coxa vara, distal resorption of phalanges, fishmouth vertebral bodies, and diffuse osteoporosis (DeBusk, 1972).

Diagnosis and Differential Diagnosis The diagnosis of progeria rests on recognition of the clinical features and can usually be made during the first or second year of life. Differential diagnosis includes other progeroid syndromes such as Werner syndrome, acrogeria, metageria, familial mandibuloacral dysplasia, the neonatal progeroid syndrome, poikiloderma congenitale of Rothmund–Thomson and Cockayne syndrome (Table 48.1).

Pathogenesis The pathogenesis of progeria stems from de novo point muta-

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tions of C to T nucleotide substitution in lamin A (Eriksson et al., 2003). The gene encodes proteins that form the nuclear lamina which supports the nuclear membrane. The mutation leads to an activation of a cryptic splice site, resulting in a truncated protein. Three patients without this mutation are believed to arise from a somatic rescue event. Mice homozygous for a point mutation in lamin A (a different substitution from Eriksson) have been shown to develop features of progeria (Mounkes et al., 2003). Altered metabolism of hyaluronic acid has been implicated because of its importance as extracellular matrix. However, elevation of urinary hyaluronic acid is not a reliable clinical marker of this disease. And it may not be the primary defect (Sweeney and Weiss, 1992). In vitro studies of skin fibroblasts from patients with progeria have shown growth deficiency (Danes, 1971; Goldstein, 1969); a decreased DNA repair capacity (Epstein et al., 1973); C-myc overexpression (Nakamura et al., 1989); increased levels of elastin and collagen IV mRNA (Colige et al., 1991; Sephel et al., 1988); and altered glycoprotein binding patterns (Clark and Weiss, 1995). These findings have not been confirmed. Brown et al. (1990) suggested that the gene for progeria may be located on chromosome 1, which has been confirmed (Eriksson et al., 2003). Progeria is considered one of the growing list of laminopathies—diseases that can affect striated muscle, adipose tissue, and bone.

Prognosis Patients with progeria usually die at an average age of 13 years from myocardial infarction or congestive heart failure (Badame, 1989). No effective therapy is available. Administration of growth hormone to a single patient accelerated growth (Brown et al., 1990). Physicians should be aware of the psychosocial challenges in a young, terminal patient (Livneh et al., 1995). Patients should be entered into the progeria registry by writing to: Progeria Research Foundation, PO Box 3453, Peabody, MA 01961.

References Badame, A. J. Progeria. Arch. Dermatol. 125:540–544, 1989. Beauregard, S., and B. Gilchrest. Syndromes of premature aging. Dermatol. Clin. 5:109–121, 1987. Brown, W. T., F. J. Kieras, G. E. Houck, R. Dutkowski, and E. C. Jenkins. A comparison of adult and childhood progerias: Werner syndrome and Hutchinson-Gilford progeria syndrome. Adv. Exp. Med. Biol. 190:229–244, 1985. Brown, W. T., J. Abdenur, P. Goonewardena, R. Alemzadeh, M. Smith, S. Friedman, C. Cervantes, S. Bandyopadhyay, A. Zaslav, S. Kunaporn, A. Serotkin, and F. Lifshitz. Hutchinson-Gilford progeria syndrome: clinical, chromosomal and metabolic abnormalities. Am. J. Hum. Genet. 47 Suppl:A50, 1990. Cao, H., and R. A. Hegele. LMNA is mutated in Hutchinson-Gilford progeria but not in Wiedemann-Rautenstrauch progeroid syndrome. J. Hum. Genet. 48:271–274, 2003. Clark, M. A., and A. S. Weiss. Hutchinson-Gilford progeria types defined by differential binding of lectin DSA. Biochim. Biophys. Acta 1270:142–148, 1995.

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Colige, A., J. C. Roujeau, F. De la Rocque, B. Nusgens, and C. M. Lapiere. Abnormal gene expression in skin fibroblasts from a Hutchinson-Gilford patient. Lab. Invest. 64:799–806, 1991. Danes, B. S. Progeria: a cell culture study on aging. J. Clin. Invest. 50:2000–2009, 1971. DeBusk, F. L. The Hutchinson-Gilford progeria syndrome. Report of four cases and review of the literature. J. Pediatr. 80:697–724, 1972. Epstein, J., J. R. Williams, and J. B. Little. Deficient DNA repair in human progeroid cells. Proc. Natl. Acad. Sci. U. S. A. 70:977, 1973. Eriksson, M., W. T. Brown, L. B. Gordon, M. W. Glynn, J. Singer, L. Scott, M. R. Erdos, C. M. Robbins, T. Y. Moses, P. Berglund, A. Dutra, E. Pak, S. Durkin, A. B. Csoka, M. Boehnke, T. W. Glover, and F. S. Collins. Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome. Nature 423:293– 298, 2003. Fleischmajer, R., and A. Nedwich. Progeria (Hutchinson-Gilford). Arch. Dermatol. 107:253–258, 1973. Gabr, M., N. Hashem, M. Hashem, A. Fahmi, and M. Safouh. Progeria, a pathologic study. J. Pediatr. 57:70–77, 1960. Gilford, H. Progeria: a form of senilism. Practitioner 73:188–217, 1904. Gillar, P. J., C. I. Kaye, and J. W. McCourt. Progressive early dermatologic changes in Hutchinson-Gilford progeria syndrome. Pediatr. Dermatol. 8:199–206, 1991. Goldstein, S. Lifespan of cultured cells in progeria. Lancet 1:424, 1969. Gordon, L. B., I. A. Harten, A. Calabro, G. Sugumaran, A. B. Csoka, W. T. Brown, V. Hascall, and B. P. Toole. Hyaluronan is not elevated in urine or serum in Hutchinson-Gilford Progeria Syndrome. Hum. Genet. 113:178–187, 2003. Hutchinson, J. Congenital abscence of hair and mammary glands with atrophic condition of the skin and its appendages in a boy whose mother had been almost totally bald from alopecia areata from the age of six. Med. Chirurg. Trans. 69:473–477, 1886. Ishii, T. Progeria: autopsy report of one case, with a review of pathologic findings reported in the literature. J. Am. Geriatr. Soc. 24:193–202, 1976. Jimbow, K., H. Kobayashi, M. Ishii, A. Oyanagi, and A. Ooshima. Scar and keloid-like lesions in progeria: an electron-microscopic and immunohistochemical study. Arch. Dermatol. 124:1261–1266, 1988. Khalifa, M. M. Hutchinson-Gilford progeria syndrome: report of a Libyan family and evidence of autosomal recessive inheritance. Clin. Genet. 35:125–132, 1989. Livneh, H., R. F. Antonak, and S. Maron. Progeria: medical aspects, psychosocial perspectives, and intervention guidelines. Death Studies 19:433–452, 1995. Macial, A. T. Evidence for autosomal recessive inheritance of progeria (Hutchinson-Gilford). Am. J. Med. Genet. 31:483–487, 1988. Mounkes, L. C., S. Kozlov, L. Hernandez, T. Sullivan, and C. L. Stewart. A progeroid syndrome in mice is caused by defects in Atype lamins. Nature. 423:298–301, 2003. Nakamura, K. D., A. Turturro, and R. W. Hart. Elevated c-myc expression in progeria fibroblasts. Biochem. Biophys. Res. Commun. 155:996–1000, 1989. Parkash, H., S. S. Sidhu, R. Raghavan, and R. N. Deshmukh. Hutchinson-Gilford progeria: familal occurrence. Am. J. Med. Genet. 36:431–433, 1990. Sephel, G. C., A. Sturrock, M. G. Giro, and J. M. Davidson. Increased elastin production by progeria fibroblasts is controlled by the steady-state levels of elastin m-RNA. J. Invest. Dermatol. 90:643– 647, 1988. Sweeney, K. J., and A. S. Weiss. Hyaluronic acid in progeria and the aged phenotype? Gerontology 38:139–152, 1992.

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Xeroderma Pigmentosum Anita P. Sheth, Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, and Beth A. Drolet

Historical Background Xeroderma pigmentosum is a heterogeneous group of genetic diseases that are caused by faulty DNA repair mechanisms. Xeroderma pigmentosum was recognized in the late 1800s by Moritz Kaposi who described a patient with classic findings of photosensitivity and skin cancers (Hebra and Kaposi, 1874). Kaposi coined the term xeroderma pigmentosum which means “dry, pigmented skin.” The hallmark of the disease is congenital photosensitivity and, ultimately, the sequelae of ultraviolet (UV)–light-induced damage.

Epidemiology

Fig. 48.2. Two sisters with xeroderma pigmentosum showing the typical mottled pigmentation and severe photophobia (courtesy of Dr. Viziam Chetty) (see also Plate 48.2, pp. 494–495).

Xeroderma pigmentosum has been reported from all countries of the world. The overall prevalence worldwide is estimated to be about 1–4 per million. A predominance of cases have been reported from the Middle East, Europe, and Japan where the incidence in some isolated groups may be as high as 10–25 per million population. The gene frequency is calculated to be 1:200 individuals in the latter populations. The estimated frequency in the United States is 1 per 250 000 to 1 per million and it has been seen in the Native American population. The high incidence in some countries is attributed to consanguinity of the parents (Kraemer et al., 1987; Robbins et al., 1974).

General Clinical Findings Xeroderma pigmentosum is an autosomal recessive disease characterized by abnormal DNA repair in cells exposed to ultraviolet light. The defect causes marked photosensitivity, premature aging of the skin, and cutaneous neoplasms. The exact genetic defects are still not fully understood in all forms of xeroderma pigmentosum but defective excision repair of UV-induced DNA damage is found in most individuals. Heterozygotes do not have sun sensitivity but do seem to have an increase in skin cancers (Swift and Chase, 1979). Prenatal diagnosis and carrier detection are now available (Parshad et al., 1990; Ramsay et al., 1974). The skin is normal at birth but cutaneous manifestations appear early in life and are initially manifest as photosensitivity. In severe forms photosensitivity and photophobia (Fig. 48.2) may be apparent after the first exposure to sun. In 75% of the cases the symptoms begin in the first years of life. Patients experience prolonged erythema and blistering after minimal exposure to ultraviolet light with wavelengths in the 280–340 nm range (UVB). Xeroderma pigmentosum represents a heterogeneous group of disorders. The various subtypes are designated by the letters A–G and a variant group. These subtypes are distinguished by different DNA repair deficiencies (Lambert et al., 1995; Robbins et al., 1974). Those in a given complementation group are thought to have the same or similar genetic defect. There are some features common to all the subtypes. In the

Fig. 48.3. Note the photodistribution of the skin involvement.

first few years of life (Fig. 48.3), all patients develop xerosis, extensive freckling, lentigines (Fig. 48.4), mottling of skin, telangiectases, and atrophy and scarring in skin chronically exposed to sun. With increasing age and cumulative sun 889

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Fig. 48.4. Close-up view of the facial lesions showing lentigines and freckles.

Fig. 48.5. A basal cell epithelioma under the right eye of a 4-yearold boy with xeroderma pigmentosum (see also Plate 48.3, pp. 494–495).

exposure all patients develop actinic keratoses, keratoacanthomas and cutaneous malignancies. Squamous and basal cell carcinomas (Fig. 48.5) and melanomas are the most common cancers affecting these individuals, but sarcomas may develop. Patients with xeroderma pigmentosum have approximately a 1000-fold increased risk of developing one of the epidermal malignancies compared to the general population. The median age of onset of nonmelanoma skin cancers is 8 years of age. The number of skin cancers is closely related to the amount of sun exposure. The majority of the tumors occur in sunexposed skin (Kraemer et al., 1987, 1994). As the exposure to sunlight increases the skin becomes markedly atrophic with fine telangiectases. Traumatic erosions and ulcerations may leave disfiguring scars giving the skin a taut appearance. The atrophy and xerosis can cause microstomia, ectropion and mutilation of the ears and finger tips. All of these findings are usually limited to the sun-exposed areas and are accompanied by severe dryness, scaling, and wrinkling. Many of the changes mimic solar damage observed in elderly patients. However, the dermal dystrophy seen his890

tologically in the skin from the latter individuals is absent in patients with xeroderma pigmentosum. Periocular and ocular complications can cause severe morbidity in patients with xeroderma pigmentosum. Photophobia and lacrimation are early signs. The eye changes in patients with xeroderma pigmentosum are primarily observed in the anterior portion of the eye, i.e., conjunctiva, cornea, and lids. The abnormalities are thought to be due to exposure of these structures to ultraviolet light. On examination they exhibit conjunctivitis, keratitis, and ectropion. The eyelids also show characteristic skin changes with freckles, atrophy, and loss of the eyelashes (Goyal et al., 1994; Kraemer et al., 1987). Corneal opacities and ulceration, as well as tumors of the eyelid, may cause loss of vision. Ocular neoplasms do occur, in particular malignant melanoma (Johnson et al., 1989). Jacyk noted an early development of ocular neoplasms in black South Africans, four of whom developed neoplasms by the age of 3 years (Jacyk, 1999). Patients may also develop neoplasms on the oral mucosa, especially on the anterior portion of the tongue. Leukemia and other internal malignancies have been reported with a higher than expected incidence (Kraemer et al., 1987). Neurologic deficits are detected in 20–30% of patients with xeroderma pigmentosum. Severe, progressive degenerative disease of the central nervous system occurs primarily in patients in complementation group A. Some patients have microcephaly, delayed motor development, intellectual impairment, sensorineural deafness, peripheral neuropathy, and dementia. Many have signs of dysfunction of the basal ganglia, cerebellum, and corticospinal tracts. Very few patients have the full constellation of signs. The combination of xeroderma pigmentosum, short stature, immature sexual development, and neurologic defects is defined as the De Sanctis–Cacchione syndrome named after De Sanctis and Cacchione who first described a patient with xeroderma pigmentosum and neurologic deficits. Many patients with this syndrome have mild neurologic manifestations such as hyporeflexia or sensorineural deafness for high frequency often detectable only by testing (Lambert et al., 1995; Mimaki et al., 1986; Robbins et al., 1974). There is very significant phenotypic variation within each complementation group. Complementation group A has patients with the most severe neurologic deficits. Other patients in this group have minimal or no neurologic abnormalities. Complementation group B patients are rare. There are reports of at least three patients in this group with features of both xeroderma pigmentosum and Cockayne syndrome (Brumback et al., 1978; Harper, 1994; Lambert et al., 1995). Patients with features of both xeroderma pigmentosum and Cockayne syndrome are also found in groups D and G. Patients in group C do not have neurologic abnormalities whereas patients in group D have either a delayed onset of neurologic abnormalities (second decade) or no neurologic abnormalities (Kraemer, 1980). A patient with a trichothiodystrophy and photosensitivity (without xeroderma pigmentosum) has been assigned to group D (Stefanini et al.,

GENETIC EPIDERMAL SYNDROMES: DISORDERS OF AGING

1986). Patients in complementation group E and group F have mild cutaneous abnormalities with no neurologic abnormalities. Patients in complementation group G have neurologic abnormalities without skin cancer. Those individuals in the variant group rarely have neurologic abnormalities and the cutaneous and ocular findings are variable (Ichihashi and Fujiwara, 1981; Lambert et al., 1995).

Pigmentary Features The pigmentary features of xeroderma pigmentosum are freckles, lentigines, hypopigmentation and hyperpigmentation, and malignant melanoma. The pigmentary changes also begin in early childhood and are initially limited to sunexposed areas. Multiple brown to black macules appear on the face, most prominently around the eyes and nose. These lesions are often described as freckles. They may be pinpoint to a centimeter in size and as they enlarge become irregular and jagged in shape. The face often takes on a “dappled” appearance as the macules coalesce and are intermingled with areas of round or angular hypopigmentation. Hyperpigmented macules may also be seen on the palms, soles, lips, tongue, conjunctivae, and genitals. All of the pigmentary abnormalities seem to be related to the amount of sun exposure. Usually the initial lesions are an excessive number of freckles or lentigines out of proportion to the child’s age. The median age of freckling is 1.5 years. Lentigines also occur at an early age (Ichihashi and Fujiwara, 1981; Lambert et al., 1995). Hypopigmented macules can be seen in areas not exposed to sunlight such as the buttocks (Cesarini et al., 1975). As the child ages, UVB-induced neoplasms begin to appear. The median age of onset of neoplasia is 8 years, and the frequency of basal cell carcinoma and squamous cell carcinoma is increased 2000-fold by age of 20 years. Keratoses, keratoacanthomas, basal cell carcinomas, squamous cell carcinomas, and melanomas have all been described. The frequency of melanoma varies greatly depending on the series (3–50%), and probably reflects the population studied. Malignant melanoma was diagnosed in 42 of 830 patients (5%) at a median age of 19. This frequency is 2000 times that for the general population of the United States. Two-thirds of the melanomas occurred on the face, head, or neck. This distribution differs significantly from melanoma in the population at large in whom only one fifth of the cancers develop in these areas. Superficial spreading, lentigo maligna melanoma, nodular melanoma, and isolated metastatic melanoma have all been reported (Takebe et al., 1989). Many authors suggest that the solar lentigines observed in xeroderma pigmentosum are precursors for malignant melanoma. Stern et al. (1993) found solar lentigines lateral to or contiguous with 14 of the 16 melanomas histologically examined. Not all patients with xeroderma pigmentosum have an increased risk of malignant melanoma. Patients in complementation group A rarely develop malignant melanoma. Those in groups F and G tend not to develop any type of skin cancer (Kraemer, 1980, 1989; Lambert et al., 1995; Yamamura et al., 1989).

Histology The histopathology of skin with characteristic cutaneous changes of xeroderma pigmentosum includes epidermal atrophy resembling that of aged skin. The epidermis is thin and the rete ridges flattened. The dermal collagen shows basophilic degeneration. Irregular proliferation of rete pegs, heavily laden with pigment, is a distinctive feature (Harper, 1994). The histologic features of the neoplasms are similar to lesions in normal individuals. It has been hypothesized that the solar lentigo is the precursor lesion of malignant melanoma in patients with xeroderma pigmentosum (Stern et al., 1993). Electron microscopic studies have shown abnormalities of keratinocytes, melanocytes, desmosomes, endoplasmic reticulum, and the mitochondria. The melanocytes are abnormal with polymorphic melanosomes. There often are giant pigment granules in melanocytes or keratinocytes (Guerrier et al., 1973). Melanosomes have been identified within vacuoles of fibroblasts (Plotnick and Lupulescu, 1983). In hypopigmented macules the number of melanocytes is decreased (Cesarini et al., 1975).

Laboratory Findings Cells, usually fibroblasts, exposed to ultraviolet radiation exhibit low levels of unscheduled DNA synthesis. This abnormality is used to confirm a diagnosis of xeroderma pigmentosum. The degree of the defect assists in classifying patients into different complementation groups. Prenatal diagnosis has been done by measuring UV-induced unscheduled DNA synthesis in cultured cells from amniotic fluid (Ramsay et al., 1974; Regan et al., 1971, Cleaver et al., 1994).

Criteria for Diagnosis The diagnosis of xeroderma pigmentosum is based on clinical examination and can be confirmed by studies on unscheduled DNA synthesis in fibroblasts exposed to ultraviolet light. The typical freckles, lentigines, hyper- and hypopigmented macules, the photophobia, and extreme photosensitivity usually make the diagnosis straightforward.

Differential Diagnosis The diagnosis of xeroderma pigmentosum during infancy or early childhood may be difficult because of minimal cutaneous changes. The diagnosis is often missed or the patient is incorrectly diagnosed as having a different disorder of photosensitivity. Cutaneous disorders characterized by photosensitivity such as the porphyrias, Cockayne syndrome, trichothiodystrophy, and the basal cell nevus syndrome must be considered when making the diagnosis of xeroderma pigmentosum. Other forms of photosensitivity and premature aging syndromes such as progeria, acrogeria, Rothmund–Thomson syndrome, Bloom syndrome, and Hartnup syndrome must also be differentiated. However, these other entities usually are easily differentiated from xeroderma pigmentosum when the characteristic clinical and laboratory findings are present. 891

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Pathogenesis

Animal Models

The cells of patients with xeroderma pigmentosum have an impaired capacity to repair damage to DNA following exposure to ultraviolet light or to chemicals that damage DNA. Cleaver demonstrated that these cells in culture show significantly decreased rates of unscheduled DNA synthesis after exposure to ultraviolet light (Cleaver, 1968). Setlow et al. (1969) demonstrated that cells from these individuals are defective in removing the pyrimidine dimer (thymine dimer) induced by UV light. Repair of the 6–4 photoproduct has been found to be defective (Lambert et al., 1995). Attempts to understand the DNA repair defects in xeroderma pigmentosum have resulted in the discovery of eight different subtypes of this disorder, designated complement groups A–G and XP-variant. A complement group designation is made on the basis of the ability of fused cells from two unrelated patients with xeroderma pigmentosum to correct each other’s DNA repair defect. This implies that the two fused cell strains have different but complementary DNA repair defects, in that each supplies what the other is lacking. Research has shown a complex, sequential interaction of XP proteins with other molecules in the nucleotide excision repair system, but the essential function of each XP protein can be characterized. XP-A, XP-C, and XP-E protein recognize DNA damage. Then XP-B and XP-D unwind the DNA helix. XP-G and XP-F incise DNA and remove the mutated strand. XPvariant is the polymerase eta that restores complementary nucleotides. All except XP-D and XP-F are located in different chromosomes (Norgauer et al., 2003). The various complement groups exhibit interesting clinical and epidemiological differences. The DNA repair capability, expressed as unscheduled DNA synthesis, shows a clear correlation with the onset and severity of clinical symptoms. Most of the well-documented cases of xeroderma pigmentosum correspond to the A, C, D, and variant groups. Groups A and D usually demonstrate the greatest photosensitivity. The relative frequency of certain tumor types also varies depending on the complement group. The XP-A group develops multiple squamous cell carcinomas whereas the milder variants, XP-E and the variant group, have a predominance of basal cell carcinomas. Lentigo maligna melanomas are much more prominent in the XP-D group. In the XP-variant form patients display severe skin disease but have normal excision repair (Burk et al., 1971). These patients are thought to have defective post-replication repair mechanisms (Boyer et al., 1990; Lambert et al., 1995; Robbins et al., 1975). The defective genes for some of the types of xeroderma have been cloned and located on various chromosomes. Their specific functions are being studied (Lambert et al., 1995; Nishigori et al., 1994). There also is an abnormality in defense against oxidative damage. Low levels of natural killer cell activity and depressed induction of interferon g by leukocytes is commonly observed in these patients (Gaspari et al., 1993; Mariani et al., 1992; Norris et al., 1988, 1990).

Two nonhuman systems have been developed to study the repair defects seen in xeroderma pigmentosum. They are rodent cell lines which have been heavily mutagenized for XPA or XP-C and mutants of two species of yeast (Lambert et al., 1995). Immortal cell lines of XP-E and XP-F are also available (Itoh et al., 2000). XP-C keratinocytes from patients can be genetically corrected with a retroviral vector and restored in vitro with DNA repair capacity (Arnaudea-Begard et al., 2003). This opens the door to further investigations in gene therapy where conventional treatments have been noncurative.

892

Therapy The prognosis of these patients is generally poor. Untreated patients die from metastatic cutaneous carcinoma at an early age. Survival may be prolonged with early and diligent sun protection (Lynch et al., 1977). Oral retinoids have been used prophylactically to reduce the incidence of skin cancers (Kraemer et al., 1988). Imiquimod 5% cream has decreased the development of facial basal cell carcinomas in a brother and sister with XP with variable tolerability (Weisberg and Varghese, 2002). A case report suggests that topical pseudocatalase has reduced number of cryotherapy sessions needed in a XP-C patient by curtailing the formation of free oxygen radicals (Schallreuter, 1999). In a randomized, double blinded study of 30 patients, topical T4 endonuclease V, a bacterial DNA repair enzyme, delivered in a liposomal vehicle reduced the incidence of actinic keratoses and basal cell carcinomas in treated patients with good tolerability (Yarosh et al., 2001). Prevention of malignancies and other oral, ocular, and cutaneous lesions by strict avoidance of ultraviolet light is most important. Regular use of sunscreens is not sufficient. Patients must wear protective clothing and eye wear as well as adopt a lifestyle which minimizes sun exposure if they are to succeed in decreasing the number of cutaneous malignancies (BechThomsen et al., 1991; Johnson, 1994; Kondoh et al., 1994). Normal vitamin D levels can be maintained even with rigorous photoprotection documented by results from a six-year study of eight patients (Sollitto et al., 1997). Cutaneous malignancies are generally treated with electrodesiccation and curettage or by excision including Mohs micrographic surgery. Dermabrasion, chemical peels, and oral retinoid therapy have all been used as prophylactic measures against the development of skin cancers with variable success (Konig et al., 1998, Kraemer et al., 1988, 1992; Nelson et al., 1995; Wee and Ahn, 1999). Squamous cell carcinomas have been successfully treated with etretinate in two case reports for xeroderma pigmentosum variant (Berth-Jones and Graham-Brown, 1990; Finkelstein et al., 1996). Others suggest that facial resurfacing with monobloc full-thickness skin can increase a period free of neoplasm for patients with large number of invasive tumors; however, new lesions may develop under the graft because of remnant tissue (Atabay et al., 1991; Sonmez Ergun et al., 2002). Patients should

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be examined at frequent intervals and premalignant and malignant lesions should be treated appropriately. Patients should be entered into the xeroderma pigmentosum registry by writing to: Xeroderma Pigmentosum Registry, Room C520 Medical Science Building, UMDNJ New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey, 07103–2714, USA.

References Arnaudea-Begard, C., F. Brellier, O. Chevallier-Lagente, J. Joeijmakers, F. Bernard, A. Sarasin, and T. Magnaldo. Genetic correction of DNA repair-deficient/cancer-prone xeroderma pigmentosum group C keratinocytes. Hum. Gene Ther. 14:983–986, 2003. Atabay, K., C. Celebi, S. Cenetoglus, N. K. Baran, and Z. Kiymaz. Facial resurfacing in xeroderma pigmentosum with monoblock full thickness skin graft. Plast. Reconstr. Surg. 87:1121, 1991. Bech-Thomsen, N., H. C. Wulf, and S. Ullman. Xeroderma pigmentosum lesions related to ultraviolet transmittance by clothes. J. Am. Acad. Dermatol. 24(2 Pt 2):365–368, 1991. Berth-Jones, J., and R. A. C. Graham-Brown. Xeroderma pigmentosum variant: response to etretinate. Br. J. Dermatol. 122:559–561, 1990. Boyer, J. C., W. K. Kaufmann, B. P. Brylawski, and M. Cordeiro-Stone. Defective postreplication repair in xeroderma pigmentosum variant fibroblasts. Cancer Res. 50:2593–2598, 1990. Brumback, R. A., F. W. Yoder, A. D. Andrews, G. L. Peck, and J. H. Robbins. Normal pressure hydrocephalus: recognition and relationship to neurological abnormalities in Cockayne’s syndrome. Arch. Neurol. 35:337–345, 1978. Burk, P. G., M. A. Lutzner, and D. D. Clarke. Ultraviolet stimulated thymidine incorporation in xeroderma pigmentosum lymphocytes. J. Lab. Clin. Med. 77:759–767, 1971. Cesarini, J. P., G. Bioulac, G. Moreno, and M. Prunieras. Hypopigmented macules of sun-exposed skin in xeroderma pigmentosum. An electron microscopic study. J. Cutan. Pathol. 2:128–139, 1975. Cleaver, J. E. Defective repair replication of DNA in xeroderma pigmentosum. Nature 218:652–656, 1968. Cleaver, J. E., J. P. Volpe, W. C. Charles, and G. H. Thomas. Prenatal diagnosis of xeroderma pigmentosum and Cockayne syndrome. Prenat. Diagn. 14:921–928, 1994. Finkelstein, E., A. Lazarov, and S. Halevy. Treatment of xeroderma pigmentosum variant with low-dose etretinate. Br. J. Dermatol. 134:815–816, 1996. Gaspari, A. A., T. A. Fleisher, and K. H. Kraemer. Impaired interferon production and natural killer cell activation in patients with the skin cancer-prone disorder, xeroderma pigmentosum. J. Clin. Invest. 92:1135–1142, 1993. Goyal, J. L., V. A. Rao, R. Srinivasan, and K. Agrawal. Oculocutaneous manifestations in xeroderma pigmentosa. Br. J. Ophthalmol. 78:295–297, 1994. Guerrier, C. J., M. A. Lutzner, V. Devico, and M. Prunieras. An electron microscopical study of the skin in 18 cases of xeroderma pigmentosum. Dermatologica 146:211–222, 1973. Harper, J. Genetics and genodermatoses. In: Textbook of Dermatology, R. H. Champion, J. L. Burton, and F. J. G. Ebling (eds). Oxford: Blackwell Scientific Publications, 1994, pp. 305–372. Hebra, F., and M. Kaposi. On Diseases of the Skin Including the Exanthemata, 3rd ed. London: The New Sydenham Society, 1874, pp. 252–258. Ichihashi, M., and Y. Fujiwara. Clinical and photobiological characteristics of Japanese xeroderma pigmentosum variant. Br. J. Dermatol. 105:1–12, 1981. Itoh, T., S. Linn, T. Ono, and M. Yamaizumi. Reinvestigation of the

classification of five cell strains of xeroderma pigmentosum group E with reclassification of three of them. J. Invest. Dermatol. 114: 1022–1029, 2000. Jacyk, W. K. Xeroderma pigmentosum in black South Africans. Inter. J. Dermatol. 38:511–514, 1999. Johnson, J. A. Prevention of skin cancer in xeroderma pigmentosum [letter]. J. Am. Acad. Dermatol. 31:1078, 1994. Johnson, M. W., G. L. Skuta, M. C. Kincaid, G. C. Nelson, and J. R. Wolter. Malignant melanoma of the iris in xeroderma pigmentosum. Arch. Ophthalmol. 107:402–407, 1989. Kondoh, M., M. Ueda, K. Nakagawa, and M. Ichihashi. Siblings with xeroderma pigmentosum complementation group A with different skin cancer development: importance of sun protection at an early age. J. Am. Acad. Dermatol. 31:993–996, 1994. Konig, A., H. C. Friederich, R. Hoffmann, and R. Happle. Dermabrasion for the treatment of xeroderma pigmentosum. Arch. Dermatol. 134:241–242, 1998. Kraemer, K. H. Xeroderma pigmentosum. A prototype disease of environmental-genetic interaction. Arch. Dermatol. 116:541–542, 1980. Kraemer, K. H. Heritable diseases with increased sensitivity to cellular injury. In: Dermatology in General Medicine, 4th ed., T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M. Freedberg, and K. F. Austen (eds). New York: McGraw-Hill, Inc., 1989, pp. 1974– 1991. Kraemer, K. H., M. M. Lee, and J. Scotto. Xeroderma pigmentosum: cutaneous, ocular and neurologic abnormalities in 830 published cases. Arch. Dermatol. 123:241–250, 1987. Kraemer, K. H., J. J. DiGiovanna, A. N. Moshell, R. E. Tarone, and G. L. Peck. Prevention of skin cancer in xeroderma pigmentosum with use of oral isotretinoin. N. Engl. J. Med. 318:1633–1637, 1988. Kraemer, K. H., J. J. DiGiovanna, and G. L. Peck. Chemoprevention of skin cancer in xeroderma pigmentosum. J. Dermatol. 19:715– 718, 1992. Kraemer, K. H., M.-M. Lee, A. D. Andrews, and W. C. Lambert. The role of sunlight and DNA repair in melanoma and nonmelanoma skin cancer. The xeroderma pigmentosum paradigm. Arch. Dermatol. 130:1018–1021, 1994. Lambert, W. C., H. R. Kuo, and M. W. Lambert. Xeroderma pigmentosum. [Review]. Dermatol. Clin. 13:169–209, 1995. Lynch, H. T., B. C. Frichot III, and J. F. Lynch. Cancer control in xeroderma pigmentosum. Arch. Dermatol. 13:193–195, 1977. Mariani, E., A. Facchini, M. C. Honorati, E. Lalli, E. Berardesca, P. Ghetti, S. Marinoni, F. Nuzzo, G. C. Astaldi Ricotti, and M. Stefanini. Immune defects in families and patients with xeroderma pigmentosum and trichothiodystrophy. Clin. Exp. Immunol. 88: 376–382, 1992. Mimaki, T., N. Itoh, J. Abe, T. Tagawa, K. Sato, H. Yabuuchi, and H. Takebe. Neurological manifestations in xeroderma pigmentosum. Ann. Neurol. 20:70–75, 1986. Nelson, B. R., D. J. Fader, M. Gillard, S. R. Baker, and T. M. Johnson. The role of dermabrasion and chemical peels in the treatment of patients with xeroderma pigmentosum. J. Am. Acad. Dermatol. 32:623–626, 1995. Nishigori, C., S. Moriwaki, H. Takebe, T. Tanaka, and S. Imamura. Gene alterations and clinical characteristics of xeroderma pigmentosum group A patients in Japan. Arch. Dermatol. 130:191–197, 1994. Norgauer, J., M. Idzko, E. Panther, O. Hellstern, and Y. Herouy. Xeroderma pigmentosum. Eur. J. Dermatol. 13:4–9, 2003. Norris, P. G., G. A. Limb, A. S. Hamblis, and J. L. M. Hawk. Impairment of natural-killer cell activity in xeroderma pigmentosum [letter]. N. Engl. J. Med. 319:1668–1669, 1988. Norris, P. G., G. A. Limb, A. S. Hamblin, A. R. Lehmann, C. F. Arlett,

893

CHAPTER 48 J. Cole, A. P. W. Waugh, and J. L. M. Hawk. Immune function, mutant frequency, and cancer risk in the DNA repair defective genodermatoses xeroderma pigmentosum, Cockayne’s syndrome and trichothiodystrophy. J. Invest. Dermatol. 94:94–100, 1990. Parshad, R., K. K. Saord, K. H. Kraemer, G. M. Jones, and R. E. Tarone. Carrier detection in xeroderma pigmentosum. J. Clin. Invest. 85:135–138, 1990. Plotnick, H., and A. Lupulescu. Ultrastructural studies of xeroderma pigmentosum. J. Am. Acad. Dermatol. 9:876–882, 1983. Ramsay, C. A., T. M. Coltart, S. Blunt, S. A. Pawsey, and F. Giannelli. Prenatal diagnosis of xeroderma pigmentosum: Report of the first successful case. Lancet 2:1109–1112, 1974. Regan, J. D., R. B. Setlow, M. M. Kaback, R. R. Howell, E. Klein, and G. Burgess. Xeroderma pigmentosum: a rapid sensitive method for prenatal diagnosis. Science 174:147–150, 1971. Robbins, J. H., K. H. Kraemer, M. A. Lutzner, B. W. Festoff, and H. G. Coon. Xeroderma pigmentosum: an inherited disease with sun sensitivity, multiple cutaneous neoplasms and abnormal DNA repair. Ann. Intern. Med. 80:221–248, 1974. Robbins, J. H., K. H. Kraemer, and B. A. Flaxman. DNA repair in tumor cells from the variant form of xeroderma pigmentosum. J. Invest. Dermatol. 64:150–155, 1975. Schallreuter, K. U. Pseudocatalase treatment in xeroderma pigmentosum: a case report. Br. J. Dermatol. 140:1190–1191, 1999. Setlow, R. B., J. D. Regan, J. German, and W. L. Carrier. Evidence that xeroderma pigmentosum cells do not perform the first step in the repair of ultraviolet damage to their DNA. Proc. Natl. Acad. Sci. U. S. A. 64:1035–1041, 1969. Sollitto, R. B., K. H. Kraemer, and J. J. DiGiovanna. Normal vitamin D levels can be maintained despite rigorous photoprotection: six years’ experience with xeroderma pigmentosum. J. Am. Acad. Dermatol. 37:942–947, 1997. Sonmez Ergun, S., D. Iscen Cek, and C. Demirkesen. Is facial resurfacing with monobloc full-thickness skin graft a remedy in xeroderma pigmentosum? Plast. Reconstr. Surg. 110:1290–1293, 2002. Stefanini, M., P. Lagomarsini, C. F. Arlett, S. Marinoni, C. Borrone, F. Crovato, G. Trevisan, G. Cordone, and F. Nuzzo. Xeroderma pigmentosum (complementation group D) mutation is present in patients affected by trichothiodystrophy with photosensitivity. Hum. Genet. 74:107–112, 1986. Stern, J. B., G. L. Peck, H. M. Haupt, H. C. Hollingsworth, and T. Beckerman. Malignant melanoma in xeroderma pigmentosum: search for a precursor lesion. J. Am. Acad. Dermatol. 28:591–594, 1993. Swift, M., and C. Chase. Cancer in families with xeroderma pigmentosum. J. Natl. Cancer Inst. 62:1415–1421, 1979. Takebe, H., C. Nishigori, and K. Tasumi. Melanoma and other skin cancer in xeroderma pigmentosum patients and mutations in their cells. J. Invest. Dermatol. 92:236S–238S, 1989. Wee, S. Y., and D. Ahn. Facial resurfacing in xeroderma pigmentosum with chemical peeling. Plast. Reconst. Surg. 103:1464–1467, 1999. Weisberg, N. K., and M. Varghese. Therapeutic response of a brother and sister with xeroderma pigmentosum to imiquimod 5% cream. Dermatol. Surg. 28:518–523, 2002. Yarosh, D., J. Klein, A. O’Connor, J. Hawk, E. Rafal, and P. Wolf. Effect of topically applied T4 endonuclease V in liposomes on skin cancer in xeroderma pigmentosum: a randomized study. Lancet. 357:926–929, 2001. Yamamura, K., M. Ichihashi, T. Hiramoto, M. Ogosifi, K. Nishioka, and Y. Fusiwara. Clinical and photobiological characteristics of xeroderma pigmentosum complementation group F: A review of cases from Japan. Br. J. Dermatol. 121:471–480, 1989.

894

Werner Syndrome Nancy Burton Esterly, Eulalia Baselga, Peter M. H. Chan, Beth A. Drolet, and Cindy L. Lamerson

Historical Background Werner syndrome was first described in 1904 by Otto Werner in his doctoral thesis, “On Cataract in Conjunction with Scleroderma.” He reported four siblings with a senile appearance, shortness of stature, cataracts, and sclerodermatous skin changes involving the feet and hands. Oppenheimer and Kugel in 1934 clearly delineated Werner syndrome from Rothmund syndrome and suggested its eponym. Thannhauser, in a comprehensive review in 1945, recognized the recessive inheritance of the syndrome and listed 12 principal characteristics. Epstein et al. (1966) described three new patients and reviewed 122 reported cases in a landmark article on Werner syndrome.

Synonyms Pangeria; adult progeria.

Epidemiology and Genetics The incidence of Werner syndrome is low. The estimated prevalence is 1 to 2.2 cases per million population (Epstein et al., 1966). In Japan, where the incidence of Werner syndrome seems high, it is 1/500 000 (Goto et al., 1985). Werner syndrome affects both sexes equally and has been reported in all races (Goto et al., 1978; Jaiyesimi et al., 1991; Murata and Nakashim, 1982). No consistent manifestations have been identified in individuals heterozygous for this disorder.

Clinical Findings The 12 principal clinical characteristics listed by Thannhauser include: (1) shortness of stature; (2) premature graying of the hair; (3) premature baldness; (4) scleropoikiloderma; (5) trophic ulcers of the legs; (6) juvenile cataracts; (7) hypogonadism; (8) tendency to diabetes; (9) calcification of blood vessels; (10) osteoporosis; (11) metastatic calcifications; and (12) tendency to occur in brothers and sisters (Thannhauser, 1945). Other clinical features that subsequently have been described include an aged “birdlike” facies, high-pitched voice, irregular dental development, muscle wasting in the lower limbs with calcified deposits in ligaments and tendons, and high incidence of neoplasms (Epstein et al., 1966; Murata and Nakashim, 1982; Salk, 1982; Tsuchiya et al., 1991; Walton et al., 2000; Zucker-Franklin et al., 1968). Patients are usually in good health until near puberty when cessation of growth is the earliest manifestation. The other features of the syndrome develop during the third decade of life but the diagnosis is usually delayed until the next decade of life (Epstein et al., 1966). Death ensues at an average age of 47 years, the principal causes being myocardial infarction, cerebrovascular accidents, and neoplasia. Dermatologic findings include premature graying and baldness; sclerodermalike skin changes with taut, fine, shiny,

GENETIC EPIDERMAL SYNDROMES: DISORDERS OF AGING

48.6

48.7 Figs 48.6 and 48.7. Sclerodermalike changes of the hands (Fig. 48.6) and feet (Fig. 48.7).

bound-down skin involving the extremities (Figs 48.6 and 48.7) and face; torpid ulcerations on the extremities; hyperkeratotic calluses over bony prominences; ulcerations on the soles; and subcutaneous calcifications and disorders of pigmentation. The pigmentary changes (Fig. 48.8) that have been described include diffuse hyperpigmentation, localized areas of hyperpigmentation intermingled with depigmentation on the extremities, and freckled or lentiginous pigmentation (Epstein et al., 1966). Telangiectases, although usually present, are not a prominent feature and the skin appears more sclerotic than poikilodermatous.

Associated Disorders Werner syndrome has been reported in association with lupus erythematosus (Kogure et al., 1995), myelodysplastic syndrome (Cottoni et al., 1994), basal cell epithelioma (Morita and Miwa, 1995), and LEOPARD syndrome in single patient (Lazarov et al., 1995). Predisposition for atypical osteosarcomas is noted (Ishikawa et al., 2000).

Histology Histopathologic features of the sclerodermalike areas include

Fig. 48.8. Large hyperpigmented macules on the legs (see also Plate 48.4, pp. 494–495).

epidermal atrophy, mild hyperkeratosis, increased melanization of basal keratinocytes, variable dermal fibrosis, microangiopathy similar to that seen in diabetes mellitus, and subcutaneous atrophy (Epstein et al., 1966; Fleischmajer and Nedwich, 1973; Ishii and Hosoda, 1975; Zucker-Franklin et al., 1968). Biochemical analyses of the sclerodermalike skin have demonstrated increased levels of hexosamines, hydroxyproline, and dermatan sulfate in the dermis (Fleischmajer and Nedwich, 1973; Salk, 1982). Some authors have not confirmed the presence of abnormalities in dermal collagen (Ishii and Hosoda, 1975). Histopathologic findings of a lentiginous macule from a single description consist of epidermal hyperplasia with elongation of the rete ridges and an increased number of single melanocytes in the basal cell layer with melanophages in the upper dermis (Lazarov et al., 1995). Electron microscopy from the same lesion showed increased numbers of melanocytes containing individual melanosomes of normal size and the presence of numerous bundles of microfilaments in the perinuclear zone of the melanocytes (Lazarov et al., 1995).

Laboratory Investigations Immunoblot analysis is used to make a diagnosis without determining the mutated sequence. Monoclonal antibodies directed to RecQ type DNA helicase reveal that the patient’s leukocytes lacked this molecule (Shimizu et al., 2002). Laboratory abnormalities in Werner syndrome include high urinary levels of hyaluronidase (Goto et al., 1985; Tokunaga et al., 1975), an abnormal glucose tolerance test with relative insulin resistance, hyperlipidemia and hypercoagulable state (Goto and Kato, 1995), and variable gonadotropin levels. Muscle enzymes, plasma lipids, cholesterol, and growth hormone levels are usually normal. X-ray findings include osteoporosis, arterial and soft tissue calcifications, osteomyelitic or osteomyelitis-like lesions of the extremities, and foot deformities (Zucker-Franklin et al., 1968). 895

CHAPTER 48

Diagnosis Diagnosis rests on the recognition of signs of premature aging and other features of the syndrome.

breakages but, unlike other syndromes with chromosome instability, they do not show increased susceptibility to clastogens and the frequency of sister chromatid exchange is normal (Gebhart et al., 1988).

Differential Diagnosis Differential diagnosis includes Rothmund–Thomson syndrome, systemic sclerosis, hereditary sclerosing poikiloderma, and other premature aging syndromes such as progeria of children (Hutchinson–Gilford syndrome) and acrogeria (Table 48.1). In Rothmund–Thomson syndrome onset of skin changes and cataracts occurs in early infancy and the skin is poikilodermatous but not taut. In systemic sclerosis and hereditary sclerosing poikiloderma, cataracts and other features of premature aging are lacking. Furthermore, systemic features of scleroderma are not present in Werner syndrome. Progeria and acrogeria usually are manifest shortly after birth and are not associated with cataracts although the skin changes can be very similar.

Pathogenesis The genetic defect causing Werner syndrome has been identified. The gene RECQL2 is located on 8p12. At least 25 mutations have been described among 34 exons of the gene resulting in a truncated protein that renders the nuclear localization signal defective (Lebel, 2001). As a result, the protein is not transported into the nucleus. The gene product WRN is about 1432 amino acids in length and encodes an enzyme that possesses helicase, ATPase, and exonuclease activities (Goto et al., 1992; Thomas et al., 1993; Ye et al., 1995; Yu et al., 1996). WRN’s primarly function is DNA repair but it has been implicated in DNA replication, recombination, telomere metabolism, and apoptosis (Opresko et al., 2003). At least 14 WRN interacting proteins are known. For instance, WRN in collaboration with ATR/ATM kinases help prevent genomic instability after DNA damage during the S phase of DNA replication (Pichierri et al., 2003). The gene product is also shown to limit cellular senescence in fibroblasts that overexpress the MYC oncoprotein (Grandori et al., 2003). It is hypothesized that Werner patients have decreased susceptibility to tumors associated with myc overexpression, such as Burkitt or large B-cell lymphoma. Significant research has revealed the intricate and complex role that WRN has in DNA metabolism, yet much remains to be discovered about its cellular localization and interactions with other proteins. Growth of skin fibroblasts in vitro from patients with Werner syndrome is more difficult than that of normal skin fibroblast. In addition fibroblasts from an individual with Werner syndrome have a reduced life span (Epstein et al., 1966; Salk, 1982; Salk et al., 1981b). Cytogenetic studies have demonstrated that skin fibroblast cell lines and cultured lymphocytes from these patients are usually composed of several clones, each marked by a distinctive, apparently balanced translocation, a phenomenon that has been referred to as “variegated translocation mosaicism” (Salk et al., 1981a; Scappaticci et al., 1982, 1990). Cells from an affected individual have a higher frequency of spontaneous chromosomal 896

Treatment Therapy of patients with Werner syndrome is mainly supportive. Skin ulcers present a very difficult problem and are frequently complicated by secondary infection and the development of gangrene (Zalla, 1980). Split thickness skin grafts have been used with some success (Bingham and Anderson, 1970). Diabetes can generally be controlled by diet alone or with an oral hypoglycemic agent. Cataract surgery is frequently complicated by wound dehiscence and degenerative corneal changes (Jonas et al., 1987; Petrohelos, 1963). Osteoporosis has been treated in a single patient with subcutaneous administration of insulinlike growth factor with resultant increase in bone mass (Rubin et al., 1994). Patients should be entered into the Werner syndrome registry by writing to: Werner’s Syndrome Registry, Department of Pathology University of Washington, School of Medicine, Box 357470, Seattle, Washington 98195.

References Bingham, H. L., and P. C. Anderson. Coverage of cutaneous ulcers in Werner’s syndrome. Acta Derm. Venereol. 50:237–239, 1970. Cottoni, F., S. Scapaticci, R. Faedda, E. Capra, and A. Murgia. Werner’s syndrome associated with myelofibrosis. J. Am. Acad. Dermatol. 30:1034–1036, 1994. Epstein, C. J., G. M. Martin, A. L. Schultz, and A. G. Motulsky. Werner’s syndrome: A review of its symptomatology, natural history, pathologic features, genetics and relationship to the natural aging process. Medicine (Baltimore) 45:177–221, 1966. Fleischmajer, R., and A. Nedwich. Werner’s syndrome. Am. J. Med. 54:111–118, 1973. Gebhart, E., R. Bauer, U. Raub, M. Schinzel, K. W. Ruprecht, and J. B. Jonah. Spontaneous and induced chromosomal instability in Werner syndrome. Hum. Genet. 80:135–139, 1988. Goto, M., Y. Horiuchi, K. Tanimoto, T. Ishi, and H. Nakashima. Werner’s syndrome: analysis of 15 cases with a review of the Japanese literature. J. Am. Geriatr. Soc. 26:341–347, 1978. Goto, M., and Y. Kato. Hypercoagulable state indicates an additional risk factor for atherosclerosis in Werner’s syndrome. Thromb. Haemost. 73:576–578, 1995. Goto, M., F. Takeuchi, K. Tanimoto, and T. Miyamoto. Clinical, demographic and genetic aspects of the Werner syndrome in Japan. Adv. Exp. Med. Biol. 190:245–261, 1985. Goto, M., M. Rubenstein, J. Weber, K. Woods, and D. Drayna. Genetic linkage of Werner’s syndrome to five markers on chromosome 8. Nature 355:735–738, 1992. Grandori, C., W. Kou-Juey, P. Fernandez, C. Ngouenet, J. Grim, B. E. Clurman, M. J. Moser, J. Oshima, D. W. Russell, K. Swisshelm, S. Frank, B. Amati, R. Dalla-Favera, and R. J. Monnat, Jr. Werner syndrome protein limits MYC-induced cellular senescence. Genes Dev. 17:1569–1574, 2003. Ishii, T., and Y. Hosoda. Autopsy report of one case, with a review of the pathologic findings reported in the literature. J. Am. Geriatr. Soc. 23:145–154, 1975. Ishikawa, Y., R. W. Miller, R. Machinami, H. Sugano, and M. Goto. Aytpical osteosarcomas in Werner Syndrome. Jpn. J. Cancer Res. 91:1345–1349, 2000.

GENETIC EPIDERMAL SYNDROMES: DISORDERS OF AGING Jaiyesimi, A. E., A. I. Sada, and O. Jimoh. Werner’s syndrome in a Nigerian: case report. Cent. Afr. J. Med. 37:63–67, 1991. Jonas, J. B., K. W. Rupecht, P. Schmitz-Valckenberg, D. Brambring, D. Platt, E. Gebhart, D. O. Schachtschabel, and G. O. Naumann. Ophthalmic surgical complications in Werner’s syndrome: report on 18 eyes of nine patients. Ophthalmic Surg. 18:760–764, 1987. Kogure, A., Y. Oshima, N. Watanabe, T. Ohba, M. Miyata, M. Ohara, T. Nishimaki, and R. Kasukawa. A case of Werner’s syndrome associated with systemic lupus erythematosus. Clin. Rheumatol. 14: 199–203, 1995. Lazarov, A., E. Finkelstein, I. Avinoach, L. Kachko, and S. Halevy. Diffuse lentiginosis in a patient with Werner’s syndrome — a possible association with incomplete leopard syndrome. Clin. Exp. Dermatol. 20:46–50, 1995. Lebel, M. Werner syndrome: a generic and molecular basis of a premature aging disorder. Cell Mol. Life Sci. 58:857–867, 2001. Morita, K., and M. Miwa. Werner’s associated with basal cell epithelioma. J. Dermatol. 22:693–695, 1995. Murata, K., and H. Nakashim. Werner’s syndrome: Twenty-four cases with a review of the Japanese medical literature. J. Am. Geriatr. Soc. 30:303–308, 1982. Oppenheimer, B. S., and V. H. Kugel. Werner’s syndrome — a heredofamilial disorder with scleroderma, bilateral juvenile cataracts, precocious graying of hair and endocrine stigmatization. Trans. Assoc. Am. Physician 49:358–370, 1934. Opresko, P., W. Cheng, C. V. Kobbe, J. A. Harrigan, and V. A. Bohr. Werner syndrome and the function of the Werner protein; what they can teach us about the molecular aging process. Carcinogenesis 24: 791–802, 2003. Petrohelos, M. A. Werner’s syndrome: A survey of three cases with a review of the literature. Am. J. Ophthalmol. 56:941, 1963. Pichierri, P., F. Rosselli, and A. Franchitto. Werner’s syndrome protein is phosphorylated in ATR/ATM-dependent manner following replication arrest and DNA damage induced during the S phase of the cell cycle. Oncogene 22:1491–1500, 2003. Rubin, C. D., B. Reed, K. Sakhaee, and C. V. Pak. Treating a patient with the Werner syndrome and osteoporosis using recombinant human insulin-like growth factor. Ann. Intern. Med. 121:665–668, 1994. Salk, D. Werner’s syndrome: A review of recent research with an analysis of connective tissue metabolism, growth control of cultured cells, and chromosomal aberrations. Hum. Genet. 62:1–15, 1982. Salk, D., K. Au, H. Hoehn, and G. M. Martin. Cytogenetics of Werner syndrome cultured skin fibroblasts: variegated translocation mosaicism. Cytogenet. Cell Genet. 30:92–107, 1981a. Salk, D., E. Bryant, K. Au, H. Hoehn, and G. M. Martin. Systematic

growth studies, cocultivation, and cell hybridization studies of Werner syndrome cultured skin fibroblast. Hum. Genet. 58:310– 316, 1981b. Scappaticci, S., D. Cerimele, and M. Fraccaro. Clonal structural chromosomal rearrangements in primary fibroblast cultures and in lymphocytes of patients with Werner’s syndrome. Hum. Genet. 62:16–24, 1982. Scappaticci, S., A. Forabosco, G. Borroni, G. Orecchia, and M. Fraccaro. Clonal structural chromosomal rearrangements in lymphocytes of four patients with Werner’s syndrome. Ann. Genet. 33:5–8, 1990. Shimizu, T., Y. Tateishi, M. Sugimoto, T. Kawabe, T. Matsumoto, and H. Shimizu. Diagnosis of Werner syndrome by immunoblot analysis. Clin. Exp. Dermatol. 27:157–159, 2002. Thannhauser, S. J. Werner’s syndrome (progeria of the adult) and Rothmund syndrome: two types of closely related heredofamilial atrophic dermatosis with juvenile cataracts and endocrine features. A critical study of five new cases. Ann. Intern. Med. 23:559–626, 1945. Thomas, W., M. Rubenstein, M. Goto, and D. Drayna. A genetic analysis of the Werner syndrome region on human chromosome 8p. Genomics 16:685–690, 1993. Tokunaga, M., T. Futami, E. Wakamatsu, M. Endo, and Z. Yosizawa. Werner’s syndrome as “hyaluronuria.” Clin. Chim. Acta. 62:89–96, 1975. Tsuchiya, H., K. Tomita, M. Ohno, M. Inaoki, and A. Kawashima. Werner’s syndrome combined with quintuplicate malignant tumors: A case report and review of literature data. Jpn. J. Clin. Oncol. 21:135–142, 1991. Walton, N. P., T. J. Brammar, and N. P. Colemann. The musculoskeletal manifestations of Werner’s syndrome. J. Bone Joint Surg. (Br.) 82:885–888, 2000. Werner, O. Uber katarakt in verbinding mit sclerodermie. Ph.D. Dissertation. Kiel University, Schmidt and Klaunig, Kiel, 1904. Ye, L., J. Nakura, N. Mitsuda, Y. Fujioka, K. Kamino, T. Ohta, Y. Jinno, N. Niikawa, T. Miki, and T. Ogihara. Genetic association between chromosome 8 microsatellite (MS8–134) and Werner syndrome (WRN): chromosome microdissection and homozygosity mapping. Genomics 28:566–569, 1995. Yu, C. E., J. Shima, U. H. Fu, E. M. Wijsman, F. Hisama, R. Allsch, S. Matthews, J. Nakura, T. Miki, S. Ouais, G. M. Martin, J. Mulligan, and G. D. Schellenberg. Positional cloning of the Werner’s syndrome gene. Science 272:258–262, 1996. Zalla, J. A. Werner’s syndrome. Cutis 25:275–278, 1980. Zucker-Franklin, D., H. Rifkin, and H. G. Jacobson. Werner’s syndrome: An analysis of ten cases. Geriatrics 23:123–135, 1968.

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Congenital Epidermal Hypermelanoses Sections Dyskeratosis Congenita Susan Bayliss Mallory, Peggy L. Chern, and Sharon A. Foley Ectodermal Dysplasias Susan Bayliss Mallory, Peggy L. Chern, and Sharon A. Foley Transient Neonatal Pustular Melanosis Susan Bayliss Mallory, Peggy L. Chern, and Sharon A. Foley Universal Acquired Melanosis Susan Bayliss Mallory, Peggy L. Chern, and Sharon A. Foley

Dyskeratosis Congenita Susan Bayliss Mallory, Peggy L. Chern, and Sharon A. Foley

Clinical Findings Dyskeratosis congenita is a rare genodermatosis with three major cutaneous manifestations, reticulated hyperpigmentation, dystrophy of the nails, and leukoplakia of the mucous membranes (Aguilar et al., 1974; Cheesbrough and Kinmont, 1978; Cohen and Callen, 1992; Cole et al., 1929; Connor and Teague, 1981; Davidson and Connor, 1988; Drachtman and Alter, 1992, 1995; Duprey and Steger, 1988; Esterly, 1986; Gutman et al., 1978; Kint et al., 1987; Loh et al., 1987; Lucky, 1988; Ogden et al., 1988; Sirinavin and Trowbridge, 1975; Wang and Wong, 1975; Zeisler, 1926). The classic triad usually appears between the ages of 4 and 10 years. Reticulated pigmentation on the neck, face, trunk, and upper thighs begins initially and is often mistaken for dirty skin (Mallory, 1991) (Figs 49.1 and 49.2). Poikiloderma arises later in life manifested by telangiectases and atrophy. Nails become dystrophic with longitudinal ridges and grooves or show pterygium formation (Aguilar et al., 1974; Dodd et al., 1985; Reichel et al., 1992). Loss of dermatoglyphics is characteristic, with fingertips appearing shiny. Leukoplakia of the mucous membranes can be seen on the tongue, buccal mucosa, vagina, rectum, gastrointestinal tract, or genitourinary tract (Cole et al., 1929; Wald and Diner, 1974). Other cutaneous features include palmoplantar hyperkeratosis with erythema, hyperhidrosis of the palms and soles, bullae of the palms and soles induced by trauma, and generalized hyperpigmentation. These changes are progressive and become more obvious with time. One case report describes a child with pronounced hyperpigmentation along the lines of Blaschko. The reticulated telangiectatic hyperpigmentation was superimposed over the typical diffuse cutaneous manifestations in an inverted V-shape midline with semiarcs in subcostal areas (Baselga et al., 1998).

Associated Findings Dyskeratosis congenita is usually recognized by its cutaneous 898

signs and symptoms, however, every organ system in the body may be involved. Most significantly, the bone marrow may be involved with 50% of affected children having hematologic findings (De Boeck et al., 1981; Trowbridge et al., 1977). Pancytopenia of the Fanconi type is the most common abnormality showing neutropenia, anemia, thrombocytopenia, and bone marrow dysplasia. Bone marrow changes are slowly progressive, leading to severe pancytopenia (Dodd et al., 1985; Friedland et al., 1985; Jacobs et al., 1984; Ling et al., 1985; Oehler et al., 1994; Phillips et al., 1992). In addition to the hematologic complications, immune abnormalities have been reported (Giannetti and Seidenari, 1980; Juneja et al., 1987; Wiedemann et al., 1984). Decreased cellular immunity with decreased T cell response to mitogens as well as negative delayed hypersensitivity (anergy) has been demonstrated (Wiedemann et al., 1984). Immunoglobulin levels may be low or elevated. Because of the leukopenia and immunologic dysfunction, an increased susceptibility to infection can be seen, although documentation of immune dysfunction has not always been made in some patients with numerous infections (Lee et al., 1992). Skeletal manifestations include increased bone fragility (Kelly and Stelling, 1982), osteoporosis, intracranial calcifications (Duprey and Steger, 1988; Mills et al., 1979), and aseptic necrosis of the hip (Kalb et al., 1986). Radiographs may show radiolucencies in the diaphyses as well as coarse trabecular patterns in the metaphyses (Kelly and Stelling, 1982). Enamel dystrophy with numerous carious teeth and severe periodontitis may be seen. Gastrointestinal complaints are usually linked to areas of leukoplakia. Dysphasia may be caused by esophageal involvement (Sawant et al., 1994). Rectal plaques may cause irritation. Leukoplakia has been noted in the genitourinary tract, involving the urethra, vagina, and cervix (Olsen et al., 1981). Small testes and scrotum and increased numbers of urinary tract infections have been reported. Eye findings include blepharitis, conjunctivitis, and lacrimal duct obstruction with epiphora, ectropion, or congenital cataracts (Womer et al., 1983). Although most children have normal mentation, 42% are reported to have neurologic abnormalities, including mild

CONGENITAL EPIDERMAL HYPERMELANOSES

Fig. 49.1. Reticulated pigmentation of the trunk (see also Plate 49.1, pp. 494–495).

to moderate mental retardation (Sirinavin and Trowbridge, 1975; Womer et al., 1983).

Histology Histologically, areas of poikiloderma demonstrate focal epidermal atrophy, mononuclear cell infiltrates, and telangiectasias (Cheesbrough and Kinmont, 1978). Deposits of amyloid have been described in the upper dermis intermixed with melanophages (Llistosella et al., 1984).

Fig. 49.2. Close-up view of the pigmentary changes (see also Plate 49.2, pp. 494–495).

telomerase function; pathology of DKC results in compromised telomerase function which may limit the proliferative capacity of human somatic cells in epithelia and blood (Heiss et al., 1998; Mitchell et al., 1999). Autosomal dominant dyskeratosis congenita is caused by mutations in TERC, assigned to chromosome 3q21-q28, a telomerase RNA gene known to interact with dyskerin (Vulliamy et al., 2001).

Treatment Pathogenesis Most reports of dyskeratosis congenita have suggested Xlinked recessive form of inheritance, with the majority of cases occurring in males. However, families with an autosomal dominant pattern have been reported (Tchou and Kohn, 1982) as well as autosomal recessive. There have been 18 reported cases of affected females (Joshi et al., 1994). Several mothers of male patients have had leukoplakia of the oral cavity, but no other manifestations consistent with dyskeratosis congenita. The gene for X-linked dyskeratosis congenita has been assigned to the Xq28 chromosome by linkage analysis (Connor et al., 1986). X-linked dyskeratosis congenita is caused by mutation in the gene encoding dyskerin (DKC1) which is involved in

Allogeneic bone marrow transplantation is the treatment of choice in patients with complete bone marrow failure (Ivker et al., 1993). Etretinate plus topical retinoid application may provide partial remission of leukoplakia oris.

Prognosis The prognosis for children with dyskeratosis congenita is guarded. Patients rarely survive past 50 years of age. No specific treatment exists. Most of the reported patients have died from infections, hemorrhage, or malignancies early in life. Malignancies of the skin and mucous membranes usually occur between 20 and 50 years of age (Kawaguchi et al., 1990). Leukoplakia leading to squamous cell carcinoma may 899

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involve the mouth, tongue, pharynx, esophagus, anus, or rectum (Anil et al., 1994; Ogden et al., 1993).

References Aguilar, A. R., F. Gomez, R. T. Sierra, and P. Larripa. Dyskeratosis congenita Zinsser-Cole-Engmann form with abnormal karyotype. Dermatologica 148:98–103, 1974. Anil, S., V. T. Beena, M. A. Raji, P. Remani, R. Ankathil, and T. Vijayakumar. Oral squamous cell carcinoma in a case of dyskeratosis congenita. Ann. Dent. 53:15–18, 1994. Baselga, E., B. A. Drolet, P. van Tuinen, N. B. Esterly, and R. Happle. Dyskeratosis congenita with linear areas of severe cutaneous involvement. Am. J. Med. Genet. 75: 492–296, 1998. Cheesbrough, M. J., and P. D. C. Kinmont. Dyskeratosis congenita. Br. J. Dermatol. 99:29–30, 1978. Cohen, L. M., and J. P. Callen. Dyskeratosis congenita: a classic case. Clin. Cases Dermatol. 4:2–6, 1992. Cole, H. N., J. E. Rauschkolb, and J. Toomey. Dyskeratosis congenita with pigmentation, dystrophia unguis and leukokeratosis oris. Arch. Dermatol. Syphil. 21:71–95, 1929. Connor, J. M., and R. H. Teague. Dyskeratosis congenita: Report of a large kindred. Br. J. Dermatol. 105:321–325, 1981. Connor, J. M., D. Gatherer, F. C. Gray, L. A. Pirrit, and N. A. Affara. Assignment of the gene for dyskeratosis congenita to Xq28. Hum. Genet. 72:348–351, 1986. Davidson, H. R., and J. M. Connor. Dyskeratosis congenita. J. Med. Genet. 25:843–846, 1988. De Boeck, K., H. Degreef, R. Verwilghen, L. Corbeel, and M. Casteels-Van Daele. Thrombocytopenia: first symptom in a patient with dyskeratosis congenita. Pediatrics 67:898–903, 1981. Dodd, H. J., S. Devereux, and I. Sarkany. Dyskeratosis congenita with pancytopenia. Clin. Exp. Dermatol. 10:73–78, 1985. Drachtman, R. A., and B. P. Alter. Dyskeratosis congenita: clinical and genetic heterogeneity. Report of a new case and review of the literature. Am. J. Pediatr. Hematol. Oncol. 14:297–304, 1992. Drachtman, R. A., and B. P. Alter. Dyskeratosis congenita. Dermatol. Clin. 13:33–39, 1995. Duprey, P. A., and J. W. Steger. An unusual case of dyskeratosis congenita with intracranial calcifications. J. Am. Acad. Dermatol. 19:760–762, 1988. Esterly, N. B. Nail dystrophy in dyskeratosis congenita and chronic graft-versus-host disease. Arch. Dermatol. 122:506–507, 1986. Friedland, M., J. D. Lutton, R. Spitzer, and R. D. Levere. Dyskeratosis congenita with hypoplastic anemia: a stem cell defect. Am. J. Hematol. 20:85–87, 1985. Giannetti, A., and S. Seidenari. Deficit of cell-mediated immunity, chromosomal alterations and defective DNA repair in a case of dyskeratosis congenita. Dermatologica 160:113–117, 1980. Gutman, A., A. Frumkin, A. Adam, N. Bloch-Shtacher, and L. A. Rozenszajn. X-linked dyskeratosis congenita with pancytopenia. Arch. Dermatol. 114:1667–1671, 1978. Heiss, N. S., S. W. Knight, Vulliamy, T. J., S. M. Klauck, S. Wiemann, P. J. Mason, A. Poustka, and I. Dokal. X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions. Nature Genet. 19:32–38, 1998. Ivker, R. A., J. Woosley, and S. D. Resnick. Dyskeratosis congenita or chronic graft-versus-host disease? A diagnostic dilemma in a child eight years after bone marrow transplantation for aplastic anemia. Pediatr. Dermatol. 10:362–365, 1993. Jacobs, P., N. Saxe, W. Gordon, and M. Nelson. Dyskeratosis congenita: Haematologic, cytogenetic, and dermatologic studies. Scand. J. Haematol. 32:461–468, 1984. Joshi, R. K., D. N. Atukorala, A. Abanmi, and A. Kudwah. Dyskeratosis congenita in a female. Br. J. Dermatol. 130:520–522, 1994. Juneja, H. S., F. F. B. Elder, and F. H. Gardner. Abnormality of platelet size and T-lymphocyte proliferation in an autosomal recessive

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form of dyskeratosis congenita. Eur. J. Haematol. 39:306–310, 1987. Kalb, R. E., M. E. Grossman, and C. Hutt. Avascular necrosis of bone in dyskeratosis congenita. Am. J. Med. 80:511–513, 1986. Kawaguchi, K., H. Sakamaki, Y. Onozawa, and M. Koike. Dyskeratosis congenita (Zinsser-Cole-Engman syndrome): An autopsy case presenting with rectal carcinoma, non-cirrrhotic portal hypertension, and Pneumocystis carinii pneumonia. Virchows Arch. A. Pathol. Anat. Histopathol. 417:247–253, 1990. Kelly, T. E., and C. B. Stelling. Dyskeratosis congenita: radiologic features. Pediatr. Radiol. 12:31–36, 1982. Kint, A., C. Oomen, M. L. Geerts, and F. Breuillard. Melanose diffuse congenitale. Ann. Dermatologie Venereologie 114:11–16, 1987. Lee, B. W., H. K. Yap, T. C. Quah, A. Chong, and C. C. Seah. T cell immunodeficiency in dyskeratosis congenita. Arch. Dis. Child. 67:524–526, 1992. Ling, N. S., N. A. Fenske, R. L. Julius, C. G. Espinoza, and L. A. Drake. Dyskeratosis congenita in a girl simulating chronic graftversus-host disease. Arch. Dermatol. 121:1424–1428, 1985. Llistosella, E., A. Moreno, and J. M. deMoragas. Dyskeratosis congenita with macular cutaneous amyloid deposits. Arch. Dermatol. 120:1381–1382, 1984. Loh, H. S., M. L. Koh, and Y. C. Giam. Dyskeratosis congenita in two male cousins. Br. J. Oral Maxillofac. Surg. 25:492–499, 1987. Lucky, A. W. Pigmentary abnormalities in genetic disorders. Dermatol. Clin. 6:193–203, 1988. Mitchell, J. R., E. Wood, and K. Collins. A telomerase component is defective in the human disease dyskeratosis congenita. Nature 402: 551–555, 1999. Mallory, S. B. What syndrome is this characteristic of? Dyskeratosis congenita. Pediatr. Dermatol. 8:81–83, 1991. Mills, S. E., P. H. Cooper, B. E. Beacham, and K. E. Greer. Intracranial calcifications and dyskeratosis congenita. Arch. Dermatol. 115:1437–1439, 1979. Oehler, L., E. Reiter, J. Friedl, E. Kabrna, O. A. Haas, A. Rosenkranz, K. Lechner, and K. Geissler. Effective stimulation of neutropoiesis with rh G-CSF in dyskeratosis congenita: a case report. Ann. Hematol. 69:325–327, 1994. Ogden, G. R., E. Connor, and D. M. Chisholm. Dyskeratosis congenita: report of a case and review of the literature. Oral Surg. Oral Med. Oral Pathol. 65:586–591, 1988. Ogden, G. R., D. P. Lane, and D. M. Chisholm. p53 expression in dyskeratosis congenita: a marker for oral premalignancy? J. Clin. Pathol. 46:169–170, 1993. Olsen, T. G., G. L. Peck, and R. H. Lovegrove. Acute urinary tract obstruction in dyskeratosis congenita. J. Am. Acad. Dermatol. 4:556–560, 1981. Phillips, R. J., M. Judge, D. Webb, and J. I. Harper. Dyskeratosis congenita: delay in diagnosis and successful treatment of pancytopenia by bone marrow transplantation. Br. J. Dermatol. 127:278–280, 1992. Reichel, M., A. C. Grix, and R. R. Isseroff. Dyskeratosis congenita associated with elevated fetal hemoglobin, X-linked ocular albinism, and juvenile-onset diabetes mellitus. Pediatr. Dermatol. 9:103–106, 1992. Sawant, P., N. M. Chopda, D. C. Desai, U. R. Dave, R. P. Satarkar, and S. A. Nanivadekar. Dyskeratosis congenita with esophageal stricture and dermatological manifestations. Endoscopy 26:711–712, 1994. Sirinavin, C., and A. A. Trowbridge. Dyskeratosis congenita: clinical features and genetic aspects. Report of a family and review of the literature. J. Med. Genet. 12:339–354, 1975. Tchou, P.-K., and T. Kohn. Dyskeratosis congenita: an autosomal dominant disorder. J. Am. Acad. Dermatol. 6:1034–1039, 1982. Trowbridge, A. A., C. Sirinavin, and J. W. Linman. Dyskeratosis congenita: hematologic evaluation of a sibship and review of the literature. Am. J. Hematol. 3:143–152, 1977.

CONGENITAL EPIDERMAL HYPERMELANOSES Vulliamy, T., A. Marrone, Goldman, F., A. Dearlove, Bessler, M., P. J. Mason, and I. Dokal. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature 413: 432–435, 2001. Wald, C., and H. Diner. Dyskeratosis congenita with associated periodontal disease. Oral Surg. 37:736–744, 1974. Wang, S. R., and C. K. Wong. Dyskeratosis congenita. Dermatologica 150:305–310, 1975. Wiedemann, H. P., J. McGuire, J. M. Dwyer, J. Sabetta, J. B. L. Gee, G. J. W. Smith, and J. Loke. Progressive immune failure in dyskeratosis congenita. Arch. Intern. Med. 144:397–399, 1984. Womer, R., J. E. Clark, P. Wood, H. Sabio, and T. E. Kelly. Dyskeratosis congenita: two examples of this multisystem disorder. Pediatrics 71:603–609, 1983. Zeisler, E. P. A unique case of reticular pigmentation of the skin with atrophy. Arch. Dermatol. 13:685–687, 1926.

1999; McGrath et al., 2001). The p63-expressing cells are thought to represent a stem cell population responsible for regeneration of overlying tissues. These cells are found in normal tissues in the basal layer of the skin, cervix, tongue, esophagus, mammary glands, prostate, and uroepithelium (Brunner et al., 2002). The p63 syndrome family also includes two disorders that lack skin and hair manifestations and will not be further discussed: nonsyndromic split hand/foot malformation (SHFM) and limb-mammary syndrome (LMS). Mutations in the p63 gene have highly pleiotropic effects with a clear genotype–phenotype correlation for each syndrome. In the following section, we will discuss only the ectodermal dysplasias which have cutaneous pigmentary manifestations. These anomalies often present as patchy, mottled, or reticulated hyperpigmentation.

Ectodermal Dysplasias Susan Bayliss Mallory, Peggy L. Chern, and Sharon A. Foley

Introduction Ectodermal dysplasias are disorders in which the embryologically affected structures are derived primarily from ectoderm such as hair, skin, nails, teeth, sweat glands, and skeletal tissue. The number of distinctive syndromes in which there is a defect in one or more epidermal appendages is so large (more than 100) that Freire-Maia and Pinheiro originally proposed a classification system based on which ectodermal derivatives are affected. According to their classification, numbers are used to indicate involvement (hair =1, teeth =2, nails =3 and sweat glands =4) and the disorders are grouped into two groups: in group A are conditions with defects in at least two “classic” structures and in group B are conditions with defects in only one of the four structures along with at least one other ectodermal defect (Freire-Maia and Pinheiro 1984, 1988; Pinheiro and Freire-Maia 1994). For example, Christ–Siemens– Touraine syndrome, one of the most common of the ectodermal dysplasias, would be classified into subgroup 1–2–3–4 because there are defects in all four ectodermal derivatives. In the light of recent information on the molecular basis and biologic functions in ectodermal dysplasias, a new clinical–genetic classification has been proposed that separates ectodermal dysplasias into two groups: the first group includes disorders with a defect in developmental regulation and epithelial–mesenchymal interaction, the second group includes disorders in which there is a structural protein defect (Priolo and Laguna, 2001). Another proposed classification groups these disorders by causative gene into four functional groups: cell–cell communication and signaling, adhesion, transcription regulation, and development (Lamartine, 2003). Mutation studies have provided a molecular basis for classification of several of the ectodermal dysplasias into a syndromic family. Three of the ectodermal dysplasias — EEC syndrome, AEC syndrome, and ADULT syndrome — have been found to be caused by mutations in the p63 gene, on chromosome 3q27, a member of the p53 family which is expressed in embryonic ectoderm and in the basal regenerative layers of epithelial tissue in the adult (Amiel et al., 2001; Celli et al.,

Hypohidrotic Ectodermal Dysplasia (X-linked Anhidrotic Ectodermal Dysplasia, Christ–Siemens–Touraine Syndrome; OMIM #305100) The Christ–Siemens–Touraine syndrome has become accepted as a general term for the varied conditions of hypohidrotic ectodermal dysplasia. Affected individuals have sparse to absent scalp and vellus body hair, hypodontia and adontia, paucity of sweat glands, and decreased mucus production in the respiratory tree (Bartstra et al., 1994). The nails are usually normal but may be thin. Associated clinical features include dry skin with a tendency to develop eczema, frequent upper respiratory infections, difficulties in lacrimation, absence or hypoplasia of the areolae and/or breasts, and a characteristic facies with a saddle nose and mid-facial hypoplasia (Fig. 49.3). Cutaneous features consist of a pigmented wrinkled periorbital skin reminiscent of atopics (Elejalde and de Elejalde, 1983; Lowry et al., 1966). Periorbital hyperpigmentation is often present at birth. The disease is X-linked recessive and has been mapped to chromosome Xq12-q13.1 (Bartstra et al., 1994; Champlin and Mallory, 1989; Rook et al., 1992; Zonana et al., 1988, 1989). It is caused by mutations in the EDA gene which is expressed in keratinocytes, hair follicles, sweat glands, and other adult and fetal tissues (Kere et al., 1996). This gene codes for a novel transmembrane protein, ectodysplasin, which has a role in the epithelial–mesenchymal interactions that regulate ectodermal appendage formation (Ezer et al., 1999).

Ectodermal Dysplasia, Ectrodactyly, Cleft Lip and Palate [EEC Syndrome; OMIM *12900 (EEC1), OMIM *602077 (EEC2), OMIM #604292 (EEC3)] Rudiger and coworkers (1970) first described the association of ectodermal dysplasia with ectrodactyly (lobsterclaw deformity of the hand) and cleft lip and palate. Other abnormalities associated with this disorder include partial anodontia, carious teeth, abnormal nails, and hypoplastic nipples. Blue irides and blepharitis have also been described. The cutaneous changes are characterized by fair, scaly, diffusely hypopigmented skin. The hair tends to be sparse, wiry, and hypopigmented. 901

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hyperpigmentation of the forearms and axillae has been reported with AEC (Fosko et al., 1992). Drut et al. (2002) described a 2-year-old girl with familial AEC associated with familial reticulate pigmentation and bilateral Wilms tumor. Patchy hyperpigmented skin lesions were seen in the child on the hands and feet; the mother and maternal grandmother had similar progressive pigmented skin lesions involving the extremities and the face. Bowen and Armstrong (1976) described three sisters with ankyloblepharon, syndactyly, and mental retardation in addition to ectodermal dysplasia and cleft palate. Mottled hyperpigmentation of the axillae and inguinal areas was seen in all three of these patients. This syndrome appears to represent the same pathologic entity as AEC with a slightly different clinical phenotype (Zenteno, 1999). AEC syndrome is caused by missense mutations in the SAM domain — important in protein–protein interactions — of the p63 gene which maps to chromosome 3q27 (McGrath et al., 2001).

ADULT Syndrome (Acro-Dermato-Ungual-LacrimalTooth Syndrome; OMIM #103285)

Fig. 49.3. Typical clinical facies of a patient with anhidrotic ectodermal dysplasia.

Numerous melanocytic nevi have been reported (Fosko et al., 1992; Pashayan et al., 1974; Solomon and Keuer, 1980). EEC syndrome (EEC1) has been mapped to chromosome 7q11.2-q21.3 (Qumsiyeh, 1992). A variant found in a Dutch kindred described by Maas et al. has been dubbed EEC2 and is mapped to chromosome 19 (Maas et al., 1996; O’Quinn et al., 1997). A third form, EEC3, has been shown to be caused by heterozygous missense mutations in the DNA binding domain of the p63 gene on chromosome 3q27 in nine unrelated families (Celli et al., 1999).

AEC Syndrome (Ankyloblepharon, Ectodermal Dysplasia, Cleft Lip and Palate); Hay–Wells Syndrome (OMIM #106260) This disorder was first described in 1976 by Hay and Wells in seven patients from four families. It is an autosomal dominant disorder characterized by ectodermal dysplasia, ankyloblepharon, lacrimal duct abnormalities, and cleft lip and palate. Limb anomalies are infrequent but syndactyly is the most common finding. In four of the original seven patients, palmoplantar keratoderma was also seen. A cutaneous abnormality characterized by a reticulated 902

Propping and colleagues described a family with at least seven living persons who were affected by an autosomal dominant syndrome with variable expression which bore a resemblance to EEC syndrome but lacked facial clefting. It is characterized by electrodactyly, nail dysplasia, and hypoplastic nipples. Excessive freckling is a characteristic sign of the syndrome, and loss of permanent teeth usually occurs before age 25 (Propping and Zerres, 1993). ADULT syndrome has been reported to be caused by mutations in the p63 gene which maps to chromosome 3q27; this mutation is different than the gene mutation in EEC or AEC and causes an amino acid substitution outside the DNA-binding region (Amiel et al., 2001).

Anonychia with Bizarre Flexural Pigmentation (OMIM #106750) Verbov reported a 26-year-old nulliparous woman with a constellation of features. These included congenitally absent or rudimentary nails, thin, dry and peeling palmar and plantar skin, coarse sparse hair, and diminished sweating. A diagnostic feature is the presence of mottled hyper- and hypopigmentation in the axillae, groin, and gluteal cleft. There was also hypopigmentation of the nipples and areolae. Two affected relatives had similar findings. It is thought to be transmitted in an autosomal dominant fashion (Verbov, 1975).

Hidrotic Ectodermal Dysplasia (Clouston Syndrome; OMIM #129500) This syndrome applies to a group of individuals initially described in 1929 with a phenotype similar to that of the Christ–Siemens–Touraine syndrome except that patients sweat normally (Solomon et al., 1987). This autosomal dominant disease is characterized by dystrophic nails, scant or absent hair, and dental anomalies. Less common findings include hyperkeratosis of the palms (Fig. 49.4) and soles, epilepsy,

CONGENITAL EPIDERMAL HYPERMELANOSES

mixed hearing defect. It is thought to be autosomal recessive. Multiple pigmented nevi and multiple ephelides may be prominent on the face and back (Pinheiro et al., 1983).

Ectodermal Dysplasia, Tricho-odonto-onychial Type (OMIM #129510)

Fig. 49.4. Palmar hyperkeratosis in a patient with Clouston syndrome (see also Plate 49.3, pp. 494–495).

deafness, mental retardation, polydactyly, syndactyly, and sensorineural hearing loss. Cutaneous changes are characterized by a patchy hyperpigmentation which tends to be localized over the bony prominences as well as the eyelids, axillae, pubic area, and areolae (Elejalde and de Elejalde, 1983; Hazen et al., 1980; Pinheiro et al., 1981). Fulk and colleagues reported a case of ectodermal dysplasia with features of slow-growing scalp hair, sparse facial and body hair, palmoplantar keratoderma, and oligospermia with infertility (Fulk, 1982). The author reported that this patient had features most consistent with hidrotic ectodermal dysplasia. Cutaneous changes showed reticulated pigmentation. At age 5 years, small punctate hyperpigmented macules appeared on the dorsal surfaces of the hands and feet. Through the years, the macules extended proximally to the shoulders and hips and became reticulated. The macules remained most prominent on the hands and feet. Histopathologic examination of a pigmented macule revealed increased basal melanization. This syndrome is caused by mutations in the GJB6 gene, on chromosome 13q12, which encodes connexin-30 (Lamartine et al., 2000).

Tricho-rhino-phalangeal Syndrome (OMIM #190350) This autosomal dominant disorder is characterized by a pearshaped nose, sparse fine hair, and digital epiphyseal coning resulting in short crooked fingers. Affected patients are of normal intelligence. Patients can have thin hypopigmented hair (Solomon and Keuer, 1980). This syndrome is caused by mutations in the gene for TRPS1, on chromosome 8q24.12, which codes for a zincfinger protein that is a putative transcription factor (Momeni et al., 2000).

Tricho-odonto-onychodysplasia Pinheiro et al. (1983) described four women who had severe hypotrichosis, enamel hypoplasia affecting deciduous and permanent teeth, dystrophic nails, supernumerary nipples, and a

Tsakalokos et al. (1986) reported a mother and daughter from a large autosomal dominant pedigree with a seemingly “new” form of ectodermal dysplasia. The main findings were hypotrichosis (with trichorrhexis nodosa), hypodontia, focal linear dermal hypoplasia of the nose, absent breasts, ridged fingernails and dysplastic toenails, and mild sensorineural deafness. Both patients had hyperpigmentation of the skin. The daughter had light-brown pigmentation of the face which was more prominent around the eyes and chin. Similar pigment was present on the trunk, limbs, axillae, and anterior chest. On the back the pigment was irregular with a tendency to become retiform. The mother had light-brown pigmentation over the back of the axillae, eyes, and mouth, and also had numerous lentigines, ephelides, and guttate hypomelanosis. Skin biopsies from hyperpigmented areas revealed normal epidermis with possible focal atrophy and increased melanophages in the upper dermis. Melanin pigment was observed in the basal cells and in the more superficial layers of the epidermis. The number of melanocytes was normal, and there was no incontinence of pigment.

Dento-oculo-cutaneous Syndrome Ackerman and colleagues first presented this unique constellation of dental, ocular, and cutaneous abnormalities involving 20 members of a family of English-German ancestry (Ackerman et al., 1973). The most prominent feature was the presence of dental abnormalities including taurodontism and pyramidal (single conical root) and fused molar roots. Fingernails are dystrophic with longitudinal ridges and distal splitting. The philtrum is thick and wide with ectropion of the lower lids. Affected individuals have hyperpigmented and indurated areas over the interphalangeal joints of the fingers, simulating knuckle pads (Ackerman et al., 1973; Pinheiro et al., 1981).

Johanson–Blizzard Syndrome (OMIM #243800) These patients demonstrate aplastic nasal alae, aplasia cutis congenita of the scalp, absence of permanent teeth buds, hypotonia, microcephaly, and malabsorption due to pancreatic deficiency. There is frequent occurrence of hypopigmented, thin, sparse scalp hair (Johanson and Blizzard, 1971; Solomon and Keuer, 1980).

Tricho-oculo-dermo-vertebral Syndrome (OMIM #601701) Freire-Maia first described this syndrome which is an autosomal recessive ectodermal dysplasia of hypotrichosis, onychodysplasia, hyperkeratosis, and kyphoscoliosis. The skin is dry and scaly with hyperchromic (ichthyosiformlike) spots and hyperkeratosis (Alves et al., 1981). 903

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Naegeli–Franceschetti–Jadassohn Syndrome (OMIM #161000) This syndrome, also discussed in Chapter 44, is characterized by reticulate pigmentation beginning in early childhood, diminished sweating, palmoplantar keratosis and defective dentition (Franceschetti and Jadassohn, 1954; Itin et al., 1993; Kudo et al., 1995). The reticulate pigmentation can partially decrease with age (Papini, 1994). The gene for this syndrome maps to chromosome 17q11.2-q21 (Sprecher et al., 2002).

Tricho-odonto-onycho-dermal (TOOD) Syndrome Pinheiro et al. (1981) described this disorder in the youngest son in an outbred sibship of four males. It is characterized by hypotrichosis, aplasia cutis congenita of the scalp, dental abnormalities, palmar keratosis, dermatoglyphic changes, and onychodysplasia. Xerotic skin with hypochromic atrophic poikilodermalike spots also occur. Histopathologic examination revealed focal areas of hyperkeratosis and an increased amount of pigment in the epidermis.

Focal Facial Dermal Dysplasia (OMIM #136500) This disorder was described in three large kindreds with autosomal dominant inheritance. Characterized by focal wrinkling and puckering of the skin at the temples, these areas are often hypohidrotic, hyperpigmented, and look like marks made by forceps. There may be associated multiple vertical linear depressions on the forehead and chin. Nails, hair, teeth, and psychomotor development are normal (McGeoch and Reed, 1971; Solomon et al., 1987).

Ectodermal Dysplasia with Abnormal Thumbs Lucky and colleagues (1980) first described this autosomal dominant disorder characterized by hypoplastic digitalized thumbs, alopecia, and endocrine abnormalities. Patients have a diffuse deep hyperpigmentation with superimposed raindrop-shaped hypopigmented macules. The hypopigmented macules are most pronounced over the genital area, inner thighs, axillae, neck, palms, and soles. Skin changes were present at birth. Histopathology from a reticulated area revealed only scattered inflammatory cells in the upper dermis with an otherwise unremarkable epidermis.

Thumb Deformity and Alopecia (OMIM #188150) Winter et al. (1988) described a family with a mother and three sons with mild short stature, sparse scalp hair, hypoplastic thumbs, single upper central incisors, and transient urticarialike reactions on the hands and arms. The patients also had increased pigmentation in the groin, lower abdomen, and neck within which were small dots of depigmentation giving a raindrop appearance. Inheritance is most likely autosomal dominant.

Tricho-onycho-hypohidrotic Ectodermal Dysplasia Freire-Maia et al. (1975) reported a 2-year-old girl with hypohidrotic ectodermal dysplasia with normal teeth, peculiar facies, psychomotor and growth retardation, bilateral nuclear cataracts, and pigmentary disturbances. Skin changes consisted 904

of hyper- and hypochromic spots on the feet, knees, arms, neck, and forehead. This disorder represents one specific variant of the many syndromes classified in the tricho–onycho– hypohidrotic subgroup of ectodermal dysplasias.

References Ackerman, J. L., A. L. Ackerman, and A. B. Ackerman. A new dental, ocular and cutaneous syndrome. Int. J. Dermatol. 12:285–289, 1973. Alves, A. F., P. A. dos Santos, E. Castelo-Branco-Neto, and N. Freire-Maia. An autosomal recessive ectoderma dysplasia syndrome of hypotrichosis, onycho-dysplasia, hyperkeratosis and kyphoscoliosis, cataract and other manifestations. Am. J. Med. Genet. 10:213–218, 1981. Amiel, J., G. Bougeard, C. Francannet, V. Raclin, A. Munnich, S. Lyonnet, and T. Frebourg. TP63 gene mutation in ADULT syndrome. Eur. J. Hum. Genet. 9: 642–645, 2001. Bartstra, H. L. J., R. F. H. J. Hulsmans, P. M. Steijlen, M. Ruige, C. E. M. Die-Smulders, and J. J. Cassiman. Mosaic expression of hypohidrotic ectodermal dysplasia in an isolated affected female child. Arch. Dermatol. 130:1421–1423, 1994. Bowen, P., and H. B. Armstrong. Ectodermal dysplasia, mental retardation, cleft lip/palate and other anomalies in three sibs. Clin. Genet. 9:35–42, 1976. Brunner, H. G., B. C. J. Hamel, and H. van Bokhoven. P63 gene mutations and human developmental syndromes. Am. J. Med. Genet. 112:284–290, 2002. Celli, J., P. Duijf, B. C. J. Hamel, M. Bamshad, B. Kramer, A. P. T. Smits, R. Newbury-Ecob, R. C. M. Hennekam, G. Van Buggenhout, A. van Haeringen, C. G. Woods, A. J. van Essen, R. de Waal, G. Vriend, D. A. Haber, A. Yang, F. McKeon, H. G. Brunner, and H. van Bokhoven. Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell 99: 143–153, 1999. Champlin, T. L., and S. B. Mallory. Hypohidrotic ectodermal dysplasia: A review. J. Arkansas Med. Soc. 86:115–117, 1989. Drut, R., D. Pollono, and R. M. Drut. Bilateral nephroblastoma in familial Hay-Wells syndrome associated with familial reticulate pigmentation of the skin. Am. J. Med. Genet. 110: 164–169, 2002. Elejalde, B. R., and M. M. de Elejalde. Pigmentary characteristics of the ectodermal dysplasias. J. Clin. Dysmorphol. 1:2–8, 1983. Ezer, S., M. Bayes, O. Elomaa, D. Schlessinger, J. Kere. Ectodysplasin is a collagenous trimeric type II membrane protein with a tumor necrosis factor-like domain and co-localizes with cytoskeletal structures at lateral and apical surfaces of cells. Hum. Mol. Genet. 8: 2079–2086, 1999. Fosko, S. W., K. S. Stenn, and J. L. Bolognia. Ectodermal dysplasias associated with clefting: Significance of scalp dermatitis. J. Am. Acad. Dermatol. 27:249–256, 1992. Franceschetti, A., and W. Jadassohn. A propos de l’incontinentia pigmenti delimitation de deux syndromes differents figurant sous le meme terme. Dermatologica 108:1–28, 1954. Freire-Maia, N., and M. Pinheiro. Ectodermal Dysplasias: A Clinical Genetic Study. New York: Alan R. Liss, 1984. Freire-Maia, N., and M. Pinheiro. Ectodermal dysplasias — some recollections and a classification. In: C. F. Salinas, J. M. Opitz, and N. W. Paul (eds). Recent Advances is Ectodermal Dysplasias. New York: Alan R. Liss, 1988, pp. 3–14. Freire-Maia, N., V. A. Fortes, L. C. Pereira, J. M. Opitz, F. A. Marcallo, and I. J. Cavalli. A syndrome of hypohidrotic ectodermal dysplasia with normal teeth, peculiar facies, pigmentary disturbances, psychomotor and growth retardation, bilateral nuclear cataract and other signs. J. Med. Genet. 12:308–310, 1975. Fulk, C. S. Hidrotic ectodermal dysplasia. J. Am. Acad. Dermatol. 6:476–480, 1982. Hazen, P. G., I. Zamora, W. E. Bruner, and W. A. Muir. Premature

CONGENITAL EPIDERMAL HYPERMELANOSES cataracts in a family with hidrotic ectodermal dysplasia. Arch. Dermatol. 116:1385–1387, 1980. Itin, P. H., S. Lautenschlager, R. Meyer, B. Mevorah, and T. Rufli. Natural history of the Naegeli-Franceschetti-Jadassohn syndrome and further delineation of its clinical manifestations. J. Am. Acad. Dermatol. 28:942–950, 1993. Johanson, A., and R. Blizzard. A syndrome of congenital aplasia of the alae nasi, deafness, hypothyroidism, dwarfism, absent permanent teeth, and malabsorption. J. Pediatr. 79:982–987, 1971. Kere, J., A. K. Srivastava, O. Montonen, J. Zonana, N. Thomas, B. Ferguson, F. Munoz, D. Morgan, A. Clarke, P. Baybayan, E. Y. Chen, S. Ezer, U. Saarialho-Kere, A. de la Chapelle, D. Schlessinger. X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein. Nat. Genet. 13:409–416, 1996. Kudo, Y., S. Fujiwara, S. Takayasu, H. Ooki, and A. Ogawa. Reticulate pigmentary dermatosis associated with hypohydrosis and short stature: a variant of Naegeli-Franceschetti-Jadassohn syndrome? Int. J. Dermatol. 34:30–31, 1995. Lamartine, J. Towards a new classification of ectodermal dysplasias. Clin. Exp. Dermatol. 28: 351–355, 2003. Lamartine, J., G. M. Essenfelder, Z. Kibar, I. Lanneluc, E. Callouet, D. Laoudj, G. Lemaitre, C. Hand, S. J. Haylick, J. Zonana, S. Antonarakis, U. Radhakrishna, D. P. Kelsell, A. L. Christianson, A. Pitaval, V. Der Kaloustian, C. Fraser, C. Blanchet-Bardon, G. A. Rouleau, and G. Waksman. Mutations in GJB6 cause hidrotic ectodermal dysplasia. Nat. Genet. 26: 142–144, 2000. Lowry, R. B., G. F. Robinson, and J. R. Miller. Hereditary ectodermal dysplasia. Clin. Pediatr. (Phila.) 5:395–402, 1966. Lucky, A. W., N. B. Esterly, and W. W. Tunnessen. Ectodermal dysplasia and abnormal thumbs. J. Am. Acad. Dermatol. 2:379–384, 1980. Maas, S. M., T. P. V. M. de Jong, P. Buss, and R. C. M. Hennekam. EEC syndrome and genitourinary anomalies: an update. Am. J. Med. Genet. 63: 472–478, 1996. McGeoch, A. H., and W. B. Reed. Familial focal facial dermal dysplasia. Birth Defects Orig. Art. Ser. 7:96–99, 1971. McGrath, J. A., P. H. G. Duijf, V. Doetsch, A. D. Irvine, R. de Waal, K. R. J. Vanmolkot, V. Wessagowit, A. Kelly, D. J. Atherton, W. A. D. Griffiths, S. J. Orlow, A. van Haeringen, M. G. E. M. Ausems, A. Yang, F. McKeon, M. A. Bamshad, H. G. Brunner, B. C. J. Hamel, and H. van Bokhoven. Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63. Hum. Mol. Genet. 10: 221–229, 2001. Momeni, P., G. Glockner, O. Schmidt, D. von Holtum, B. Albrecht, G. Gillessen-Kaesbach, R. Hennekam, P. Meinecke, B. Zabel, A. Rosenthal, B. Horsthemke, and H.-J. Ludecke. Mutations in a new gene, encoding a zinc-finger protein, cause tricho-rhino-phalangeal syndrome type I. Nat. Genet. 24: 71–74, 2000. O’Quinn, J. R., R. C. M. Hennekam, L. B. Jorde, and M. Bamshad. Syndromic ectrodactyly with severe limb, ectodermal, urogenital, and palatal defects maps to chromosome 19 [abstract]. Am. J. Hum. Genet. 61 (suppl.): A289 only, 1997. Papini, M. Natural history of the Naegeli-Franceschetti-Jadassohn syndrome [letter]. J. Am. Acad. Dermatol. 31(5 Pt 1):830, 1994. Pashayan, H. M., S. Pruzansky, and L. Solomon. The EEC syndrome: Report of six patients. Birth Defects Orig. Art. Ser. 10:105–127, 1974. Pinheiro, M., and N. Freire-Maia. Ectodermal dysplasias: a clinical classification and a causal review. Am. J. Med. Genet. 53:153–162, 1994. Pinheiro, M., L. C. Pereira, and N. Freire-Maia. A previously undescribed condition: tricho-odonto-onycho-dermal syndrome. A review of the tricho-odonto-onychial subgroup of ectodermal dysplasias. Br. J. Dermatol. 105:371–382, 1981. Pinheiro, M., F. M. Newton, and A. J. Roth. Trichoodontoonychial dysplasia — a new meso-ectodermal dysplasia. Am. J. Med. Genet. 15:67–70, 1983.

Priolo, M., and C. Laguna. Ectodermal dysplasias: a new clinicalgenetic classification. J. Med. Genet. 38: 579–585, 2001. Propping, P., and K. Zerres. ADULT-syndrome: an autosomal-dominant disorder with pigment anomalies, ectrodactyly, nail dysplasia, and hypodontia. Am. J. Med. Genet. 45:642–648, 1993. Qumsiyeh, M. B. EEC syndrome (ectrodactyly, ectodermal dysplasia and cleft lip/palate) is on 7p11.2-q21.3 [letter]. Clin. Genet. 42: 101 only, 1992. Rook, A., D. S. Wilkinson, and F. J. G. Ebling. Textbook of Dermatology, 5th ed. London: Blackwell Scientific Publications, 1992, pp. 334–348. Rudiger, R. A, W. Haase, and E. Passarge. Association of ectrodactyly, ectodermal dysplasia, and cleft lip/palate. Am. J. Dis. Child. 120:160–163, 1970. Solomon, L. M., and E. J. Keuer. The ectodermal dysplasias. Problems of classification and some newer syndromes. Arch. Dermatol. 116:1295–1299, 1980. Solomon, L. M., B. Cook, and W. Klipfel. The ectodermal dysplasias. Dermatol. Clin. 5:231–237, 1987. Sprecher, E., P. Itin, N. V. Whittock, J. A. McGrath, R. Meyer, J. J. DiGiovanna, S. J. Bale, J. Uitto, and G. Richard. Refined mapping of Naegeli-Franceschetti-Jadassohn syndrome to a 6 cM interval on chromosome 17q11.2-q21 and investigation of candidate genes. J. Invest. Derm. 119: 692–698, 2002. Tsakalokos, N., F. H. Jordann, J. J. F. Taljaard, and S. F. Hough. A previously undescribed ectodermal dysplasia of the trich-odontoonychial subgroup in a family. Arch. Dermatol. 122:1047–1053, 1986. Verbov, J. Anonychia with bizarre flexural pigmentation–an autosomal dominant dermatosis. Br. J. Dermatol. 92:469–474, 1975. Winter, R. M., K. D. MacDermot, and F. J. Hill. Sparse hair, short stature, hypoplastic thumbs, single upper central incisor and abnormal skin pigmentation: a possible new form of ectodermal dysplasia. Am. J. Med. Genet. 29:209–216, 1988. Zenteno, J. C., C. Venegas, and S. Kofman-Alfero. Evidence that AEC syndrome and Bowen–Armstrong syndrome are variable expressions of the same disease. Pediatr Dermatol. 16:103–107, 1999. Zonana, J., A. Clarke, M. Sarfarazi, N. S. T. Thomas, K. Roberts, K. Marymee, and P. S. Harper. X-linked hypohidrotic ectodermal dysplasia: localization within the region Xq11–21.1 by linkage analysis and implications for carrier detection and prenatal diagnosis. Am. J. Hum. Genet. 43: 75–85, 1988. Zonana, J., M. Sarfarazi, N. S. T. Thomas, A. Clarke, K. Marymee, and P. S. Harper. Improved definition of carrier status in X-linked hypohidrotic ectodermal dysplasia by use of restriction fragment length polymorphism-based linkage analysis. J. Pediatr. 114:392– 399, 1989.

Transient Neonatal Pustular Melanosis Susan Bayliss Mallory, Peggy L. Chern, and Sharon A. Foley

Historical Background In 1976, Ramamurthy and colleagues described a dermatosis characterized by the presence of pustules and hyperpigmented macules seen in full-term newborns, more commonly in black infants. This had previous been described by Marino (1965) as toxic erythema presenting at birth.

Clinical Findings Lesions are usually present at birth and progress from pustules to scaly lesions (Auster, 1978). The vesicopustules last 24–48 hours (Laude, 1995). When the pustules rupture, a fine white collarette of scale is left that is replaced by a hyperpigmented 905

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Fig. 49.5. Hyperpigmented macules in a black patient with transient neonatal pustular melanosis.

Fig. 49.6. Close-up view of the pustules.

macule. The hyperpigmented macules are more commonly seen in black newborns (Merlob et al., 1982) (Figs 49.5 and 49.6). These macules have been confused with erythema toxicum neonatorum (Levy and Cothran, 1962; Marino, 1965) or lentigines in the past (Perrin et al., 1961) but unlike lentigines these lesions fade within weeks to months. The most common areas affected are the trunk, chin, neck, buttocks, abdomen, and thighs but any area may be involved (Wyre and Murphy, 1979). Unlike erythema toxicum neonatorum, lesions are not usually surrounded by erythema (Auster, 1980). The cause of this disorder is unknown. A Wright stain or a Giemsa stain of the pustule shows polymorphonuclear leukocytes with occasional eosinophils. Gram stain is negative for bacteria and pustules are sterile (Gupta and Rasmussen, 1988).

Ferrandiz, C., W. Coroleu, M. Ribera, J. C. Lorenzo, and A. Natal. Sterile transient neonatal pustulosis is a precocious form of erythema toxicum neonatorum. Dermatology 185:18–22, 1992. Fretzin, D. F. Biopsy in vesiculobullous disorders. Cutis 20:639–645, 1977. Gupta, A. K., and J. E. Rasmussen. What’s new in pediatric dermatology? J. Am. Acad. Dermatol. 18:239–259, 1988. Laude, T. A. Approach to dermatologic disorders in black children. Semin. Dermatol. 14:15–20, 1995. Levy, H. L., and F. Cothran. Erythema toxicum neonatorum present at birth. Am. J. Dis. Child. 103:125–127, 1962. Marino, L. J. Toxic erythema present at birth. Arch. Dermatol. 92:402–403, 1965. Merlob, P., A. Metzker, and S. H. Reisner. Transient neonatal pustular melanosis. Am. J. Dis. Child. 136:521–522, 1982. Perrin, E., J. Sutherland, and S. Baltazar. Inquiry into the nature of lentigines neonatorum: Demonstration of a statistical relationship with squamous metaplasia of the amnion. Am. J. Dis. Child. 102:260–261, 1961. Ramamurthy, R. S., M. Reveri, N. B. Esterly, D. F. Fretzin, and R. S. Pildes. Transient neonatal pustular melanosis. J. Pediatr. 88:831– 835, 1976. Wyre, H. W., and M. O. Murphy. Transient neonatal pustular melanosis. Arch. Dermatol. 115:458, 1979.

Histology Histologically, there are intracorneal or subcorneal collections of neutrophils and some eosinophils in the early stages (Barr et al., 1979; Ferrandiz et al., 1992; Fretzin, 1977; Gupta and Rasmussen, 1988). The hyperpigmented macules demonstrate an increase in basalar melanocytes.

Laboratory Findings Bacterial infection should be ruled out by Gram stain and culture. Herpes simplex infection as well as candidiasis should be excluded in a sick neonate. Although neonates may acquire scabies, this is uncommon and a scraping to demonstrate a mite may be appropriate.

Therapy This is a benign disorder with no systemic manifestations and it requires no treatment.

References Auster, B. Transient neonatal pustular melanosis. Cutis 22:327–328, 1978. Auster, B. Pustular eruptions of the neonate. Curr. Concepts Skin. Dis. 1:2–8, 1980. Barr, R. J., L. M. Globerman, and F. A. Werber. Transient neonatal pustular melanosis. Int. J. Dermatol. 18:636–638, 1979.

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Universal Acquired Melanosis Susan Bayliss Mallory, Peggy L. Chern, and Sharon A. Foley Universal acquired melanosis (carbon baby) was described by Ruiz-Maldonado et al. (1978) in a Mexican baby boy who developed increased pigmentation on the face and extremities beginning at age 15 days. Eventually the skin and oral mucosa became completely black. However, the palms and soles were spared and his hair was fine, sparse and black. Histologic examination showed increased melanization and an occasional melanophage. There were a normal number of basal melanocytes with increased type 3 and 4 melanosomes.

References Ruiz-Maldonado, R., L. Tamayo, and J. Fernandez-Diez. Universal acquired melanosis. Arch. Dermatol. 114:775–778, 1978.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

50

Acquired Epidermal Hypermelanoses Sections Acanthosis Nigricans Norman Levine and Cynthia Burk Acromelanosis Progressiva Norman Levine and Cynthia Burk Becker Nevus Norman Levine and Cynthia Burk

Café-au-lait Spots Norman Levine and Cynthia Burk Carcinoid Syndrome Norman Levine and Cynthia Burk Confluent and Reticulated Papillomatosis Norman Levine and Cynthia Burk Cutaneous Amyloidosis Norman Levine and Cynthia Burk Dermatosis Papulosa Nigra Norman Levine and Cynthia Burk Ephelides (Freckles) Norman Levine and Cynthia Burk Erythema ab Igne Norman Levine and Cynthia Burk Erythema Dyschromicum Perstans Norman Levine and Cynthia Burk Erythromelanosis Follicularis Faciei et Colli Norman Levine and Cynthia Burk Erythrose Péribuccale Pigmentaire of Brocq Norman Levine and Cynthia Burk Extracutaneous Neuroendocrine Melanoderma Norman Levine and Cynthia Burk Felty Syndrome and Rheumatoid Arthritis Norman Levine and Cynthia Burk Hyperpigmentation Associated with Human Immunodeficiency Virus (HIV) Infection Philippe Bahadoran Melanoacanthoma Norman Levine and Cynthia Burk Phytophotodermatitis Norman Levine and Cynthia Burk Polyneuropathy, Organomegaly, Endocrinopathy, M Protein, and Skin Changes: POEMS Syndrome James J. Nordlund Urticaria Pigmentosum and Mastocytosis James J. Nordlund Poikiloderma of Civatte Vlada Groysman and Norman Levine Riehl’s Melanosis Scott Bangert and Norman Levine Atrophoderma of Pasini et Pierini James J. Nordlund, Norman Levine, Charles S. Fulk, and Randi Rubenzik Hyperpigmentation Associated with Scleromyxedema and Gammopathy Kazunori Urabe, Juichiro Nakayama, and Yoshiaki Hori Ichthyosis Nigricans, Keratoses, and Epidermal Hyperplasia James J. Nordlund Morphea and Scleroderma James J. Nordlund Pigmentary Changes Associated with Addison Disease Cindy L. Lamerson and James J. Nordlund Pigmentary Changes Associated with Cutaneous Lymphomas Debra L. Breneman

Acanthosis Nigricans Norman Levine and Cynthia Burk

Historical Background Although Addison may have seen a case of acanthosis nigricans before 1855, possibly misdiagnosed as Addison disease (Schwartz, 1994), the first documented case of acanthosis nigricans was noted by Unna in 1889. His student, Sigmund Pollitzer, reported this finding in 1890. In the same year Janovsky published an independent description of acanthosis nigricans. In 1893, Darier reported two patients with acanthosis nigricans, or what he called “dystrophie papillaire et

pigmentaire,” one of whom died of an abdominal carcinoma. Curth (1943) advanced the concept of malignant acanthosis nigricans as a cutaneous marker for aggressive internal malignancy. She later categorized acanthosis nigricans into four types: malignant, benign, syndromic, and pseudo-acanthosis nigricans (Curth, 1968). Since then, others have reclassified acanthosis nigricans in various ways; however, each makes the basic distinction between malignant and nonmalignant types. In 1976 Kahn et al. linked nonmalignant acanthosis nigricans with states of insulin resistance. This concept has evolved to the point where it appears that all nonmalignant acanthosis nigricans is the product of an insulin-resistant state (Cruz and Hud, 1992). Acanthosis nigricans is currently viewed as 907

CHAPTER 50

a common cutaneous manifestation of endocrinopathy, and an occasional concomitant finding in some malignancies.

Synonyms Dystrophie anomaly.

papillaire

et

pigmentaire,

acral

acanthotic

Epidemiology The incidence of acanthosis nigricans in the adult population is unknown. In an unselected population of 1412 school-aged children, physical evidence of acanthosis nigricans was present in 7.1%. Overall, the incidence in girls and boys is nearly equal. Acanthosis nigricans is common among black people, Hispanics, and Native Americans (Stuart et al., 1989). but less prevalent among white people (Stoddart et al., 2002). Acanthosis nigricans is most commonly associated with obesity, insulin resistance, hyperinsulinemia, and type 2 diabetes mellitus (Stoddart et al., 2002). One study found acanthosis nigricans in 74% of 34 adult obese patients (Hud et al., 1992). Acanthosis nigricans and obesity are independently and positively associated with hyperinsulinemia in adolescents (Mukhtar et al., 2001), and acanthosis nigricans was found in more than half of adolescents who weighed at least 200% of their ideal body weight (Schwartz 1994; Stuart et al., 1994). Acanthosis nigricans is now more common in young people due to the increased incidence of noninsulin-dependent diabetes (Mukhtar et al., 2001). Some Native American tribes have higher rates of inherited insulin resistance, resulting in noninsulin-dependent diabetes mellitus, obesity, and acanthosis nigricans. In a study examining a random sample of 2205 Cherokee Nation members aged 5–40 years, the prevalence of acanthosis nigricans was 34.2%, while the prevalence of hyperinsulinemia was 47.2% (Stoddart et al., 2002). Acanthosis nigricans is also common in patients with polycystic ovarian disease, independent of body weight (Dunaif, 1991). Acanthosis nigricans has been reported during pregnancy in association with gestational diabetes as well as glucose intolerance secondary to reversible insulin resistance. In the majority of these cases, skin lesions resolved post-partum (Kroumpouzos et al., 2002). Malignant acanthosis nigricans is relatively rarely seen in patients with cancer. At one time, malignant acanthosis nigricans was said to occur in approximately 20% of all acanthosis nigricans cases, but it is likely to be much less common (Brown and Winkelmann, 1968). With the increasing identification of acanthosis nigricans in benign conditions, the percentage of reported malignant cases decreased significantly (Brown and Winkelmann, 1968). Malignant acanthosis nigricans affects men and women equally, without racial or geographic predilection (Schwartz, 1994). Acanthosis nigricans that is associated with malignancy is a disease of adults, with the youngest reported case being a 17-year-old female (Braverman, 2002). Malignant acanthosis nigricans may present either before or after clinical tumor presentation (Curth 1976; Gross et al., 1984; Matsuoka et al., 1993). Tumors associated with acanthosis nigricans are often 908

Fig. 50.1. Hyperpigmented lesions in the axillae (see also Plate 50.1, pp. 494–495).

Fig. 50.2. Velvety hyperpigmented plaques of acanthosis nigricans in the groin.

advanced (Yeh et al., 2000). Eighty to ninety percent of cases of malignant acanthosis nigricans are associated with abdominal malignancies, approximately 60% of which are gastric adenocarcinomas (Braverman, 2002). Other sites include pituitary, colon, esophagus, gall bladder, kidney, liver, lung, ovary, pancreas, prostate, thyroid, rectum, and uterus. Rarely, other tumors are associated with acanthosis nigricans, including choriocarcinoma, lymphosarcoma, anaplastic carcinoma of the lung or stomach, squamous cell carcinoma of the cervix, lung, or bladder, transitional cell carcinoma of the kidney, reticulum cell sarcoma, Hodgkin lymphoma, carcinoid tumor, osteosarcoma, mycosis fungoides, and pheochromocytoma (Matsuoka et al., 1993; Braverman, 2002).

Clinical Findings A typical patient presents with a symmetric eruption, most commonly involving the axillae (Fig. 50.1), the groin (Fig. 50.2), and/or the nape of the neck (Fig. 50.3). Frequently, acanthosis nigricans presents initially only in one site. Intertriginous areas such as the popliteal and antecubital fossae may also be affected. The disease presents with tan-to-brown velvety plaques with poorly demarcated margins. This has

ACQUIRED EPIDERMAL HYPERMELANOSES 100

Percentage with AN

80

60

40

20

0 Fig. 50.3. Acanthosis nigricans on the folds of the neck.

been described as the “appearance of an unwashed neck” (Bonnekoh et al., 1993). Acrochordons (skin tags) sometimes are within the plaques and may appear on the eyelids, neck, and axilla (Hidalgo, 2002). Acanthosis nigricans and acrochordons develop in obese individuals, and this correlates with the severity of the obesity (Hidalgo, 2002). Occasionally, hyperpigmentation alone can be the first clinical sign. Though the patient is usually asymptomatic, lesions are occasionally pruritic, particularly in the variety associated with malignancy. Atypical manifestations include lesions in inframammary folds, the periumbilical area, the vulva, the scrotum, and the perianal region. Patients may also have verrucous plaques over the interphalangeal joints (Braverman, 2002; Matsuoka et al., 1993; Schwartz, 1994). In a severe form, referred to as “tripe palms,” the hands may develop excessively deep sulci, producing a yellow rugated surface resembling a honeycomb. This keratoderma of either the palmar or plantar surfaces of the hands and feet are features typical of malignant acanthosis nigricans (Yeh et al., 2000; Skiljevic et al., 2001). Rarely, nail changes, progressive hair loss of the scalp, axillae, limbs, and eyebrows, hyperkeratosis of the nipple and areola, and mucous membrane involvement may occur (Azizi et al., 1980; de Graciansky et al., 1951; Ive, 1963; Matsuoka et al., 1993; Schwartz, 1994). Lesions may also occur on the lips, tongue, buccal mucosa, and vulva (Braverman, 2002; Brown and Winkelmann, 1968; Cobenour and Gamble, 1971; Matsuoka et al., 1993). Acanthosis nigricans lesions have developed at sites of subcutaneous insulin injections in patients with diabetes mellitus (Fleming and Simon, 1986; Schwartz, 1994; Stone, 1993). Malignant acanthosis nigricans is clinically indistinguishable from its benign counterpart (Rogers, 1991). Although there are many exceptions, the clinical presentations which should raise the question of malignancy include symptomatic acanthosis nigricans, atypical locations, and extensive lesions (Rogers, 1991; Schwartz, 1994). All of the classifications of acanthosis nigricans describe the same lesion. The benign types can be divided into the following groups: familial; obesity- or endocrine-related; syndromic; and medication induced. Syndromic acanthosis nigricans can

120-150

151-200

201-250

>250

Ideal body weight, % Fig. 50.4. The incidence of acanthosis nigricans increases as body weight increases. [Reprinted with permission from: Hud, J. A., Jr, J. B. Cohen, J. M. Wagner, and P. D. Cruz, Jr. Prevalence and significance of acanthosis nigricans in an adult obese poulation. Arch. Dermatol. 128:941–944, 1992. Copyright 1992, American Medical Association.]

be further subdivided into type A, type B, and other genetic syndromes. Familial acanthosis nigricans is an autosomal dominant condition that develops before puberty. Lesions usually stabilize after several years, although, in some cases, they may regress. Acral acanthotic anomaly is a type of benign acanthosis nigricans seen occasionally in black or Hispanic people, in whom lesions are limited to the dorsae of the hands and feet (Krishnaram, 1991; Schwartz, 1994). Endocrinopathy-related acanthosis nigricans occurs in insulin-resistant or hyperinsulinemic states, especially obesity, polycystic ovarian syndrome, and noninsulin-dependent diabetes mellitus, as well as Cushing syndrome, Addison disease, streak gonads, male hypogonadism, and hyper- and hypothyroidism. Obesity-related acanthosis nigricans frequently resolves after significant weight loss (Fig. 50.4). The clinical course of acanthosis nigricans follows that of the patient’s insulin resistance with onset of insulin resistance correlating directly with the appearance of acanthosis nigricans (Dunaif et al., 1991; Hardaway and Gibbs, 2002; Matsuoka et al., 1993). Overt diabetes mellitus, however, is not common in this type of acanthosis nigricans (Cruz and Hud 1992; Hud et al., 1992). Two constellations of signs are defined as type A and type B syndromic acanthosis nigricans. Type A syndrome, also known as HAIR-AN syndrome (hyperandrogenemia, insulin resistance, and acanthosis nigricans), appears in early childhood, and generalizes rapidly at the onset of puberty. Virilization, enlarged kidneys and adrenal glands, and accelerated somatic growth are frequently seen. Female patients often are hirsute and have polycystic ovaries. It may be more common in black people (Schwartz, 1994). Type B acanthosis nigricans is characterized by autoimmu909

CHAPTER 50 Table 50.1. Syndromes associated with acanthosis nigricans. Type A syndrome (HAIR-AN syndrome) Type B syndrome Hirschowitz syndrome Ataxia telangiectasia Rabson syndrome Leprechaunism Wilson disease Chondrodystrophy with dwarfism Pyramidal tract degeneration Alström syndrome Crouzon syndrome Capozucca syndrome Streak gonads Rud syndrome Bloom syndrome Pituitary neoplasms Gigantism Acromegaly Familial pineal body hypertrophy

Rabson–Mendenhall syndrome Benign encephalopathy Prader–Willi syndrome Pituitary hypogonadism Stein–Leventhal syndrome Lipoatrophic diabetes mellitus Lawrence–Seip syndrome Bartter syndrome Werner syndrome Lupoid hepatitis Lupus erythematosus Dermatomyositis Scleroderma Hashimoto thyroiditis Phenylketonuria Beare–Stevenson syndrome Costello syndrome Pseudoacromegaly Lawrence–Moon–Bardet–Biedl syndrome

acanthosis nigricans have elevated levels of thyroid stimulating hormone (TSH), growth hormone (GH), melanocytestimulating hormone (MSH), and androgens (Givens et al., 1974; Millard and Gould, 1976; Moller, 1978; Nordlund and Lerner, 1975; Weiss et al., 1995). Type A syndrome generally produces high plasma testosterone levels. Patients with type B syndrome may have circulating antibodies to the insulin receptor, leukopenia, or high anti-DNA antibody titers (Kahn et al., 1976; Moller and Flier, 1991; Schwartz, 1994). Hyperlipidemia is a feature of lipodystrophy in Lawrence–Seip syndrome, frequently the only sign in heterozygotes (Mork et al., 1986; Schwartz, 1994; Seip and Trygstad, 1963). Transforming growth factor-a (TGF-a) may be elevated in both the serum and in the urine, rarely in patients with malignant acanthosis nigricans (Yeh et al., 2000).

Criterion for Diagnosis The criterion for diagnosis of acanthosis nigricans is clinical presentation of pigmented, velvety plaques. The sites affected most commonly are the axilla, nape of the neck, and the groin.

Differential Diagnosis nity, including anti-insulin receptor antibodies. The clinical course of acanthosis nigricans parallels the insulin resistance, which may relapse and remit along with the immunologic disease (Cruz and Hud, 1992). Diabetes mellitus is a typical feature. Premenopausal women may also have hyperandrogenism. Putative autoimmune diseases seen in type B syndrome include systemic lupus erythematosus, Sjögren syndrome, vitiligo, Hashimoto thyroiditis, and scleroderma (Matsuoka et al., 1993; Moller and Flier, 1991; Rendon et al., 1989; Schwartz, 1994). Miscellaneous other rare, benign hereditary conditions with acanthosis nigricans include Lawrence–Seip syndrome (lipodystrophic diabetes), leprechaunism, Hirschowitz syndrome, Rabson–Mendenhall syndrome, and pseudoacromegaly syndrome (Table 50.1). Additionally, a high prevalence of acanthosis nigricans in patients with trisomy 21 has recently been described (Hirschler et al., 2002). Certain medications may induce acanthosis nigricans. These include nicotinic acid, diethylstilbestrol, insulin, glucocorticoids, pituitary extracts, oral contraceptives, methyltestosterone, topical fusidic acid, and triazinate. The cutaneous process may normalize following withdrawal of the offending agent, at least in the case of nicotinic acid (Brown and Winkelmann, 1968; Darmstadt et al., 1991; Mellor-Pita et al., 2002; Teknetzis et al., 1993).

Laboratory Findings and Investigations Laboratory findings vary according to the type of acanthosis nigricans. Patients with benign acanthosis nigricans exhibit elevated fasting plasma insulin levels that are significantly higher than those of nonacanthotic patients (Hidalgo, 2002). Hyperglycemia may occur, particularly in obese patients with noninsulin-dependent diabetes mellitus. Some patients with 910

The main diseases in the differential diagnosis of acanthosis nigricans are Dowling–Degos disease, confluent and reticulated papillomatosis (CRP), Becker nevus, and tinea versicolor. Acanthosis nigricans may occasionally have a similar appearance to hyperkeratotic seborrheic keratoses, melanocytic nevi, linear epidermal nevi, and ichthyosis hystrix. It may also resemble the hyperpigmentation of pellagra, Addison disease, and pemphigus vegetans. Early plaques may have some similarities to atopic dermatitis. Dowling–Degos disease (reticulate pigmented anomaly of the flexures) has a distribution similar to acanthosis nigricans, usually affecting the axilla, groin, and other intertriginous sites with reticulated brown plaques. However, unlike acanthosis nigricans, the lesions have little or no substance. Both demonstrate a normal number of melanocytes histologically; Dowling–Degos disease differs from acanthosis nigricans in its lack of papillomatosis, its follicular involvement, and its elongated rete ridges with melanin deposition at the lower tips. The lesions of CRP may appear on the neck, back, and chest as red papules or brown verrucous plaques, mimicking acanthosis nigricans. However, they are not found on mucous membranes, and rarely appear in the axillae or groin. As noted earlier, Becker nevus, like acanthosis nigricans, is usually a brown plaque with skin thickening. However, it may have coarse hypertrichosis on its surface. Histologically, it is identified by smooth muscle hyperplasia and increased amount of melanin, which are not present in acanthosis nigricans. Tinea versicolor can masquerade as acanthosis nigricans with its brown coloration, especially when it appears in flexural areas, on the sides of the chest, or on the neck. However, unlike acanthosis nigricans, tinea versicolor lesions are barely palpable, and have a reticulated pattern with fine scale. Seborrheic keratoses may be confused with acanthosis nigricans because of their brown, black, or yellow color and

ACQUIRED EPIDERMAL HYPERMELANOSES

hyperkeratotic rough surface; however, they are usually well demarcated, round to oval in shape, and are smaller than acanthosis nigricans lesions. Unlike acanthosis nigricans, their number increases with age. Melanocytic nevi are pigmented, sometimes verrucous lesions which may simulate acanthosis nigricans. They differ from acanthosis nigricans in that these nevi are asymmetric, rarely grow larger than 0.5 to 1.0 cm, and have no predilection for intertriginous areas. Histologically, they are composed of an abnormal number of melanocytes in the dermis, unlike acanthosis nigricans, which usually has a normal number of pigment-producing cells, limited to the epidermis. Epidermal nevi, including linear and verrucous types, can resemble acanthosis nigricans. They both may be flesh-colored, brown, or gray-brown. The nevi appear as verrucous papules which may coalesce into plaques. Other types, such as ichthyosis hystrix, can have a bilateral distribution, giving the appearance of acanthosis nigricans-like symmetry. Unlike acanthosis nigricans, they tend to become inflamed, pruritic, and scaly. They rarely appear after puberty. The thickened, hyperpigmented plaques of pellagra may mimic acanthosis nigricans. Sites such as dorsal hands and neck are common to both. Unlike acanthosis nigricans, lesions may be fissured and may worsen after sun exposure. Pellagra affects areas over bony prominences and scalp, which are uncommon sites of involvement in acanthosis nigricans. Addison disease can cause generalized, diffuse brown hyperpigmentation. In contradistinction to acanthosis nigricans, the lesions are not palpable. Pemphigus vegetans may present with verrucous, hyperpigmented plaques involving both cutaneous and mucous membrane surfaces. Unlike acanthosis nigricans, the lesions are often eroded as well as verrucous. On histologic examination pemphigus is characterized by intraepidermal vesiculation and acantholysis, whereas acanthosis nigricans displays none of these features. Atopic dermatitis can present with poorly-demarcated plaques which may be hyperpigmented. Atopic dermatitis and acanthosis nigricans may affect comparable skin sites, such as the nuchal region and intertriginous areas such as the popliteal and antecubital fossae. However, epithelial disruption distinguishes this from acanthosis nigricans. Unlike acanthosis nigricans, pruritus is a prominent symptom, although acanthosis nigricans lesions may occasionally itch.

Pathology Light Microscopy Acanthosis nigricans lesions have a histologic picture which consists of marked hyperkeratosis, prominent finger-like papillomatosis, varying but mild acanthosis, and minimal melanocytic hyperplasia. The interpapillary crypts contain keratotic material. Slight hyperpigmentation of the basal layer is occasionally present, but the gross color is due to hyperkeratosis, not melanin deposition. Usually, there is no inflammation, but a sparse dermal mononuclear infiltrate may be present (Brown and Winkelmann, 1968; Darmstadt et al., 1991; Krishnaram, 1991). There is no histologic difference

between the benign and malignant types of acanthosis nigricans (Brown and Winkelmann, 1968; Farmer and Hood, 1990).

Immunohistochemistry Glycosaminoglycan deposition in the epidermis and papillary dermis may be present (Rogers, 1991). Hyaluronic acid deposits have been associated with polycystic ovary syndrome, other endocrinopathies, and one case of malignant pheochromocytoma (Matsuoka et al., 1993). Tumors associated with acanthosis nigricans have been shown to express TGF-a and increased numbers of epidermal growth factor (EGF) receptors. TGF-a and EGF share a common receptor, the EGF receptor, which may be present in abnormally high numbers in skin affected with acanthosis nigricans (Ellis et al., 1987; Wilgenbus et al., 1992).

Pathogenesis Growth factors which stimulate keratinocytes and dermal fibroblasts at the level of cell receptors may be the proximate cause of acanthosis nigricans. Insulin and TGF-a are the best studied. In normal concentrations, insulin binds preferentially to its classical receptor, initiating its metabolic and proliferative effects. In high concentrations, as in patients with insulinresistant states, insulin has an increased affinity for insulinlike growth factor-1 (IGF-1) (somatomedin C) receptors (“specificity spillover effect”). IGF-1 receptors are present on keratinocytes; thus insulin is capable of activating IGF-1 receptors, stimulating DNA synthesis and epidermal growth (Uyttendaele et al., 2003). The genetic syndromes associated with acanthosis nigricans, such as lipoatrophic diabetes, leprechaunism, type A syndrome, and Rabson–Mendenhall syndrome, have identifiable heterogeneous mutations in the insulin receptor gene (Accili et al., 1992; Flier et al., 1985; Grasinger et al., 1993; Matsuoka et al., 1993; Schwartz, 1994). These abnormalities are associated with acanthosis nigricans, but do not correlate directly with the individual clinical syndromes (Accili et al., 1992). Obesity, hirsutism, and hyperandrogenism may have additive effects which aggravate insulin resistance (Flier et al., 1985). Unlike benign acanthosis nigricans, which is related to insulin action, malignant acanthosis nigricans probably reflects the effects of ectopic growth factors. Tumor products, such as IGF-1 and TGF-a, stimulate epidermal proliferation by binding to skin IGF-1 and EGF receptors (Ellis et al., 1987; Matsuoka et al., 1987; Wilgenbus et al., 1992). Increased plasma levels of growth factors may be operative in other dermatologic paraneoplastic syndromes, such as the sign of Leser–Trelat, florid cutaneous papillomatosis, tylosis, paraneoplastic pemphigus, acquired hypertrichosis lanuginosa, and pachydermoperiostosis, as well as in acanthosis nigricans (Ive, 1963; Matsuoka et al., 1993; Schwartz, 1994; Schwartz and Burgess, 1978). Medications which cause acanthosis nigricans act via a mechanism which has not yet been elucidated (Rogers, 1991; Schwartz, 1994; Teknetzis et al., 1993). 911

CHAPTER 50

Animal Models Acanthosis nigricans occurs in dogs, most notably dachshunds, but also in German shepherds and Lhasa apsos. The majority suffer from an underlying endocrinopathy, as in humans, although associated malignancy has been found. Gartner induced cutaneous lesions resembling acanthosis nigricans in dogs by removing their adrenal glands and gonads (Bornfors, 1958; Brown and Winkelmann, 1968; Curth and Slanetz, 1939; Schwartz, 1994).

Treatment The primary goal of treatment is to correct the underlying disease which leads to the development of acanthosis nigricans. Regression of acanthosis nigricans can occur with improvement of underlying endocrinopathies. Physical activity, calorie restriction, and weight reduction are all protective against hyperinsulinemia and subsequent acanthosis nigricans (Mukhtar et al., 2001). Insulin therapy is usually not indicated even in patients with insulin resistance, since most patients are not diabetic (Cruz and Hud 1992; Schwartz, 1994). Treatment with octreotide, a synthetic analog of somatostatin, reduces insulin secretion and therefore should theoretically reduce the insulin binding to IGF-1 receptors (Hidalgo, 2002). Because acanthosis nigricans is a cutaneous manifestation of hyperinsulinemia and insulin resistance, its detection could be important as a noninvasive and cost-effective tool in type 2 diabetes mellitus screening (Mukhtar et al., 2001). Specific hormone therapy, such as thyroid replacement, estrogen, oral contraceptives, oral hypoglycemic agents, and recombinant IGF-1, have shown benefit in some patients (Dix et al., 1986; Hardaway and Gibbs, 2002; Matsuoka et al., 1993; Ober 1985; Schwartz 1994). Cyclophosphamide or other immune modulators have been tried in type B acanthosis nigricans (Matsuoka et al., 1993). In patients with malignant acanthosis nigricans, resolution of acanthosis nigricans may occur with chemotherapy. Specifically, chemotherapy is beneficial because it decreases epithelial proliferation (Anderson et al., 1999). Treatment directed at the acanthosis nigricans lesions themselves include topical corticosteroids, oral retinoids, topical tretinoin gel, podophyllin, and emollient creams (Anderson et al., 1999; Darmstadt et al., 1991; Katz 1980; Skiljevic et al., 2001). The vitamin D3 analog, calcipitriol, has been shown to be effective in treating acanthosis nigricans by inhibiting the proliferation of keratinocytes and reducing the hyperkeratosis and papillomatosis (Hidalgo, 2002). The acrochordons associated with acanthosis nigricans can be removed via snipping with curved scissors, cryotherapy, or electrodesiccation (Hidalgo, 2002). In drug-induced acanthosis nigricans, discontinuation of the offending drug may result in acanthosis nigricans regression (Coates et al., 1992). Less conventional treatments have been used as well. One patient achieved palliation of malignant acanthosis nigricans after radiation therapy with two courses of electron beam therapy (Weiss et al., 1995). Oral cyproheptadine has been used to inhibit tumor product release and diminish paraneoplastic acanthosis nigricans (Greenwood and Tring, 1982). A 912

few patients with lipodystrophic diabetes have had improvement of acanthosis nigricans while taking dietary fish oil supplements (Sherertz, 1988). Ketoconazole therapy and dermabrasion have also been suggested (Curth and Gibbs, 1967; Schwartz, 1994; Tercedor et al., 1992).

Prognosis Acanthosis nigricans is a chronic condition. The patient’s prognosis depends upon the underlying pathology which is responsible for the acanthosis nigricans. In malignant acanthosis nigricans, diminution of lesions frequently occurs with excision of related cancers; lesions may reappear with recurrence or metastasis (Schwartz, 1994). The longest time between acanthosis nigricans diagnosis and tumor detection is reported to be 16 years; the interval is usually much shorter (Curth et al., 1962). The malignancies in question are often extremely aggressive; the average survival after detection is two years (Curth et al., 1962; Matsuoka et al., 1993) and the average survival being 12 months (Braverman, 2002). However, many patients who receive early cancer therapy survive, one even as long as 14 years after tumor resection (Brown and Winkelmann, 1968).

References Accili, D., F. Barbetti, A. Cama, H. Kadowaki, T. Kadowaki, E. Imano, R. Levy-Toledano, and S. I. Taylor. Mutations in the insulin receptor gene in patients with genetic syndromes of insulin resistance and acanthosis nigricans. J. Invest. Dermatol. 98(6 Suppl):77S-81S, 1992. Anderson, S. H., M. Hudson-Peacock, A. F. Muller. Malignant acanthosis nigricans: potential role of chemotherapy. Br. J. Dermatol. 141:714–716, 1999. Andreev, V. C. Malignant acanthosis nigricans. Semin. Dermatol. 3:265–272, 1984. Azizi, E., H. Trau, M. Schewach-Millet, V. Rosenberg, S. Schneebaum, and R. Michalevicz. Generalized malignant acanthosis nigricans [letter]. Arch. Dermatol. 116:381, 1980. Bonnekoh, B., A. Wevers, H. Spangenberger, G. Mahrle, and T. Krieg. Keratin pattern of acanthosis nigricans in syndromelike association with polythelia, polycystic kidneys, and syndactyly. Arch. Dermatol. 129:1177–1182, 1993. Bornfors, S. Acanthosis nigricans in dogs: A study of aetiology and medical treatment with special attention to hypophyseal-thyroid function. Acta Endocrinol. Suppl. 37:1, 1958. Braverman, I. M. Skin manifestations of internal malignancy. Clin. Geriatr. Med. 18:1–19, 2002. Brown, J., and R. K. Winkelmann. Acanthosis nigricans: A study of 90 cases. Medicine 47:33–51, 1968. Coates, P., D. Shuttleworth, and A. Rees. Resolution of nicotinic acidinduced acanthosis nigricans by substitution of an analogue (acipimox) in a patient with type V hyperlipidaemia. Br. J. Dermatol. 126:412–414, 1992. Cobenour, W., and J. W. Gamble. Acanthosis nigricans: A review of literature and report of case. J. Oral Surg. 29:48–51, 1971. Cruz, P. D., Jr., and J. A. Hud Jr. Excess insulin binding to insulinlike growth factor receptors: proposed mechanism for acanthosis nigricans. J. Invest. Dermatol. 98(6 Suppl):82S–85S, 1992. Curth, H. O. Cancer associated with acanthosis nigricans: review of literature and report of a case of acanthosis nigricans with cancer of the breast. Arch. Surg. 47:517–552, 1943. Curth, H. O. Significance of acanthosis nigricans. Arch. Dermatol. 66:80, 1952.

ACQUIRED EPIDERMAL HYPERMELANOSES Curth, H. O. The Necessity of Distinguishing Four Types of Acanthosis Nigricans. Berlin: Springer-Verlag, 1968. Curth, H. O. Classification of acanthosis nigricans. Int. J. Dermatol. 15:592–593, 1976. Curth, H. O., and R. G. Gibbs. Benign acanthosis nigricans. Arch. Dermatol. 96:345–346, 1967. Curth, H. O., and C. A. Slanetz. Acanthosis nigricans and cancer of the liver in a dog. Am. J. Cancer 37:216–223, 1939. Curth, H. O., A. W. Hilberg, and G. F. Machacek. The site and histology of the cancer associated with malignant acanthosis nigricans. Cancer 15:364–382, 1962. Darier, J. Dystrophie papillaire et pigmentaire. Ann. Dermatol. Syphiligr. (Paris) 4:865–875, 1893. Darmstadt, G. L., B. K. Yokel, and T. D. Horn. Treatment of acanthosis nigricans with tretinoin. Arch. Dermatol. 127:1139–1140, 1991. de Graciansky, P., C. Grupper, and P. Lefort. Acanthosis nigricans atypique. Bull. Soc. Franc. Dermatol. Syphiligr.:483–484, 1951. Dix, J. H., W. J. Levy, and C. Fuenning. Remission of acanthosis nigricans, hypertrichosis, and Hashimoto’s thyroiditis with thyroxine replacement. Pediatr. Dermatol. 3:323–326, 1986. Dunaif, A., G. Green, R. G. Phelps, M. Lebwohl, W. Futterweit, and L. Lewy. Acanthosis nigricans, insulin action, and hyperandrogenism: clinical, histological, and biochemical findings. J. Clin. Endocrinol. Metab. 73:590–595, 1991. Ellis, D. L., S. P. Kafka, J. C. Chow, L. B. Nanney, W. H. Inmann, M. E. McCadden, and L. E. King. Melanoma growth factors, acanthosis nigricans, the sign of Leser-Trelet, and multiple acrochordons: A possible role for alpha-transforming growth factor in cutaneous paraneoplastic syndromes. N. Engl. J. Med. 317:1582–1587, 1987. Farmer, E. R., and A. F. Hood. Pathology of the Skin. Norwalk, CT: Appleton and Lange, 1990. Fleming, M. G., and S. I. Simon. Cutaneous insulin reaction resembling acanthosis nigricans. Arch. Dermatol. 122:1054–1056, 1986. Flier, J. S., R. C. Eastman, K. L. Minaker, D. Matteson, and J. W. Rowe. Acanthosis nigricans in obese women with hyperandrogenism: Characterization of an insulin-resistant state distinct from the type A and B syndromes. Diabetes 34:101–107, 1985. Givens, J. R., I. J. Kerber, W. L. Wiser, R. N. Andersen, S. A. Coleman, and S. A. Fish. Remission of acanthosis nigricans associated with polycystic ovarian disease and a stromal luteoma. J. Clin. Endocrinol. Metab. 38:437–455, 1974. Grasinger, C. C., R. A. Wild, and I. J. Parker. Vulvar acanthosis nigricans: a marker for insulin resistance in hirsute women. Fertil. Steril. 59:583–586, 1993. Greenwood, R., and F. C. Tring. Treatment of malignant acanthosis nigricans with cyproheptadine. Br. J. Dermatol. 106:705–710, 1982. Gross, G., H. Pfister, B. Hellenthal, and M. Hagedorn. Acanthosis nigricans maligna: Clinical and virological investigations. Dermatologica 168:265–272, 1984. Hardaway, C. A., and N. F. Gibbs. What syndrome is this? RabsonMendenhall syndrome. Pediatr. Dermatol. 19:267–270, 2002. Hidalgo, G. Dermatological complications of obesity. Am. J. Clin. Dermatol. 2:497–506, 2002. Hirschler, V., C. Aranda, A. Oneto, C. Gonzalez, M. Jadzinsky. Is acanthosis nigricans a marker of insulin resistance in obese children? Diabetes Care 25:2353, 2002. Hud, J. A., Jr., J. B. Cohen, J. M. Wagner, and P. D. Cruz Jr. Prevalence and significance of acanthosis nigricans in an adult obese population. Arch. Dermatol. 128:941–944, 1992. Ive, F. A. Metastatic carcinoma of cervix with acanthosis nigricans, bullous pemphigoid and hypertrophic pulmonary osteoarthropathy. Proc. R. Soc. Med. 56:910, 1963. Kahn, C. R., J. S. Flier, R. S. Bar, J. A. Archer, P. Gorden, M. M. Martin, and J. Roth. The syndromes of insulin resistance and acan-

thosis nigricans: Insulin-receptor disorders in man. N. Engl. J. Med. 294:739–745, 1976. Katz, R. A. Treatment of acanthosis nigricans with oral isotretinoin. Arch. Dermatol. 116:110–111, 1980. Kierland, R. R. Acanthosis nigricans: an analysis of data in twentytwo cases and a study of its frequency in necropsy material. J. Invest. Dermatol. 9:299–305, 1947. Krishnaram, A. S. Unilateral nevoid acanthosis nigricans [letter]. Int. J. Dermatol. 30:452–453, 1991. Kroumpouzos, G., G. Avgerinou, and S. R. Granter. Acanthosis nigricans without diabetes during pregnancy. Br. J. Dermatol. 146:925–928, 2002. Kuzuya, H., N. Matsuura, M. Sakamoto, H. Makino, Y. Sakamoto, T. Kadowaki, Y. Suzuki, M. Kobayashi, Y. Akazawa, M. Nomura, Y. Yoshimasa, M. Kasuga, K. Goji, S. Nagataki, H. Oyasu, and H. Imura. Trial of insulinlike growth factor I therapy for patients with extreme insulin resistance syndromes. Diabetes 42:696–705, 1993. Matsuoka, L. Y., J. Goldman, J. Wortsman, D. Kleinsmith, and C. E. Kupchella. Antibodies against the insulin receptor in paraneoplastic acanthosis nigricans. Am. J. Med. 82:1253–1256, 1987. Matsuoka, L. Y., J. Wortsman, and J. Goldman. Acanthosis nigricans. Clin. Dermatol. 11:21–25, 1993. Mellor-Pita, S., M. Yebra-Bango, J. Alfaro-Martinez, and E. Suarez. Acanthosis nigricans: a new manifestation of insulin resistance in patients receiving treatment with protease inhibitors. Clin. Infect. Dis. 34:716–717, 2002. Millard, L. G., and D. J. Gould. Hyperkeratosis of the palms and soles associated with internal malignancy and elevated levels of immunoreactive human growth hormone. Clin. Exp. Dermatol. 1:363–368, 1976. Moller, D. E., and J. S. Flier. Insulin resistance: mechanisms, syndromes, and implications. N. Engl. J. Med. 325:938–948, 1991. Moller, H. Phototoxicity of Dictamnus alba. Contact Derm. 4:264–269, 1978. Mork, N. J., G. Rajka, and J. Halse. Treatment of acanthosis nigricans with etretinate (Tigason) in a patient with LawrenceSeip syndrome (generalized lipodystrophy). Acta Derm. Venereol. 66:173–174, 1986. Mukhtar, Q., G. Cleverley, R. E. Voorhees, and J. W. McGrath. Prevalence of acanthosis nigricans and its association with hyperinsulinemia in New Mexico adolescents. J. Adolesc. Health 28:372–376, 2001. Nordlund, J. J., and A. B. Lerner. On the cause of acanthosis nigricans. N. Engl. J. Med. 293:200, 1975. Ober, K. P. Acanthosis nigricans and insulin resistance associated with hypothyroidism. Arch. Dermatol. 121:229–231, 1985. Pollitzer, S. Acanthosis Nigricans. London: HK Lewis and Co., 1890. Rendon, M. I., P. D. Cruz Jr., R. D. Sontheimer, and P. R. Bergstresser. Acanthosis nigricans: a cutaneous marker of tissue resistance to insulin. J. Am. Acad. Dermatol. 21:461–469, 1989. Robbins, S. L., R. S. Cotran, and V. Kimar. Robbins’ Pathologic Basis of Disease, 4th ed. Philadelphia: Saunders, 1989. Rogers, D. L. Acanthosis nigricans. Semin. Dermatol. 10:160–163, 1991. Schwartz, R. A. Acanthosis nigricans. J. Am. Acad. Dermatol. 31:1–19, 1994. Schwartz, R. A., and G. H. Burgess. Florid cutaneous papillomatosis. Arch. Dermatol. 114:1803–1806, 1978. Seip, M., and O. Trygstad. Generalized lipodystrophy. Arch. Dis. Child. 38:447–453, 1963. Sherertz, E. F. Improved acanthosis nigricans with lipodystrophic diabetes during dietary fish oil supplementation. Arch. Dermatol. 124:1094–1096, 1988. Skiljevic, D. S., M. M. Nikolic, A. Jakovljevic, and D. D. Dobrosavljevic. Generalized acanthosis nigricans in early childhood. Pediatr. Dermatol. 18:213–216, 2001.

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CHAPTER 50 Stone, O. J. Acanthosis nigricans — decreased extracellular matrix viscosity: cancer, obesity, diabetes, corticosteroids, somatotrophin. Medical Hypotheses 40:154–157, 1993. Stoddart, M. L., K. S. Blevins, E. T. Lee, W. Wang, P. R. Blackett; Cherokee Diabetes Study. Association of acanthosis nigricans with hyperinsulinemia compared with other selected risk factors for type 2 diabetes in Cherokee Indians: the Cherokee Diabetes Study. Diabetes Care 25:1009–1014, 2002. Stuart, C. A., C. J. Pate, and E. J. Peters. Prevalence of acanthosis nigricans in an unselected population. Am. J. Med. 87:269–272, 1989. Stuart, C. A., M. M. Smith, C. R. Gilkison, S. Shaheb, and R. M. Stahn. Acanthosis nigricans among native Americans: an indicator of high diabetes risk. Am. J. Publ. Health 84:1839–1842, 1994. Teknetzis, A., I. Lefaki, D. Joannides, and A. Minas. Acanthosis nigricans-like lesions after local application of fusidic acid. J. Am. Acad. Dermatol. 28:501–502, 1993. Tercedor, J., J. M. Rodenas, M. T. Herranz, A. Vidal, and M. MunozTorres. Effect of ketoconazole in the hyperandrogenism, insulin resistance and acanthosis nigricans (HAIR-AN) syndrome [letter; comment]. J. Am. Acad. Dermatol. 27(5 Pt 1):786, 1992. Uyttendaele, H., T. Koss, et al. Generalized acanthosis nigricans in an otherwise healthy young child. Pediatr. Dermatol. 20:254–256, 2003. Weiss, E., H. Schmidberger, R. Jany, C. F. Hess, and M. Bamberg. Palliative radiotherapy of mucocutaneous lesions in malignant acanthosis nigricans. Acta Oncol. 34:265–267, 1995. Wilgenbus, K., A. Lentner, R. Kuckelkorn, S. Handt, and C. Mittermayer. Further evidence that acanthosis nigricans maligna is linked to enhanced secretion by the tumour of transforming growth factor alpha. Arch. Dermatol. Res. 284:266–270, 1992. Yeh, J. S., S. E. Munn, T. A. Plunkett, P. G. Harper, D. J. Hopster, and A. W. du Vivier. Coexistence of acanthosis nigricans and the sign of Leser-Trelat in a patient with gastric adenocarcinoma: a case report and literature review. J. Am. Acad. Dermatol. 42(2 Pt 2): 357–362, 2000.

Acromelanosis Progressiva

Fig. 50.5. Typical blue-black lesion on the fingers of a child with acromelanosis progressiva.

(Furuya and Mishima, 1962). The mucous membranes and nails are spared (Gonzalez and Vazquez Botet, 1980). One of the children had associated seizures (Furuya and Mishima, 1962) and the other was otherwise normal (Gonzalez and Vazquez Botet, 1980).

Pathology The main pathologic finding is a proliferation of normal melanocytes in the basal epidermis. In some areas, the melanocytes are clustered, but no true nesting is present. Melanin is noted mainly along the basal cell layer of the epidermis with some upward migration of this pigment. The dermis is uninvolved, except for a sparse perivascular mononuclear infiltrate (Gonzalez and Vazquez Botet, 1980).

Norman Levine and Cynthia Burk

Differential Diagnosis Historical background Thomas (1923) first described hyperpigmentation on the dorsal digits in infants, which he called “spitzenpigment.” Bloom made a similar observation in 1950 in a patient with associated hypertrichosis and café-au-lait spots. Weidman (1969) described a 10-year-old child with pigmentation of the fingers and toes, which slowly progressed over several years. Furuya and Mishima (1962) were the first to report a patient whose initial acral pigmentation became more widespread. One additional case, now termed acromelanosis progressiva, has been described (Gonzalez and Vazquez Botet, 1980).

Synonyms Spitzenpigment, acropigmentation, acromelanosis.

Clinical Findings The lesions first appear in early childhood as symmetrical blue-black lesions over the distal digits (Fig. 50.5), toes, and the perianal area. The pigmentation subsequently spreads proximally to the thighs, buttocks, genitals, and abdomen 914

Reticulate acropigmentation of Kitamura bears some resemblance to acromelanosis progressiva. However, it is far more common. It is transmitted via an autosomal dominant mode of inheritance and presents in the first and second decades of life. Like acromelanosis, it presents with acral pigmentation, but it is characterized by a reticulate pattern of hyperpigmented macules. There are no areas of hypopigmentation (Griffiths, 1976). The classic histopathology involves the presence of digitated and filiform elongated rete ridges, with clumps of heavy melanin pigmentation at their tips (Al Hawsawi, 2002). There is also epidermal atrophy and an increase in the number of basal melanocytes (Alfadley, 2000). Acropigmentation of Dohi (Komaya, 1924) presents in infancy and/or early childhood with reticulate hyperpigmented and hypopigmented macules of the dorsa of hands and feet. Acropigmentation of Dohi is most often inherited in an autosomal dominant pattern; however, a few cases have been inherited as an autosomal recessive trait. Histopathologically, there is an increase in melanin pigments at the basal layer and through the epidermis within the areas of hyperpigmentation. In the areas of hypopigmentation, there is a decrease or an

ACQUIRED EPIDERMAL HYPERMELANOSES

absence of melanin pigments. Overall, melanocyte number is unaffected (Alfadley, 2000). Universal acquired melanosis has been described in a neonate with progressive brown pigmentation which eventuated in generalized jet-black skin by age 3 (Ruiz-Maldonado et al., 1978). Electron microscopy showed the pigment to be distributed in a Negroid pattern in spite of the fact that the patient was white.

Pathogenesis The origin of this peculiar pigmentary abnormality is not known. The histologic findings are identical to those of a lentigo, with increased numbers of epidermal melanocytes. Thus, this may be an epidermal hamartoma of melanocytes; the dermal counterpart might be the nevus of Ota, the Mongolian spot, and the blue nevus (Furuya and Mishima, 1962; Gonzalez and Vazquez Botet, 1980).

Treatment and Prognosis No therapies have been described for acromelanosis progressiva. Although long-term follow-up on these few cases has not been reported, there is no mention of spontaneous resolution once the pigment is established.

References Alfadley, A., A. Al Ajlan, B. Hainau, K. T. Pedersen, and I. Al Hoqail. Reticulate acropigmentation of Dohi: a case report of autosomal recessive inheritance. J. Am. Acad. Dermatol. 43:113–117, 2002. Al Hawsawi, K., K. Al Aboud, A. Alfadley, and D. Al Aboud. Kitamura-Dowling Degos disease overlap: a case report. Int. J. Dermatol. 41:518–520, 2002. Bloom, D. Acropigmentation in a child. Arch. Dermatol. 62:475, 1950. Furuya, T., and Y. Mishima. Progressive pigmentary disorder in Japanese child. Arch. Dermatol. 86:412–418, 1962. Gonzalez, J. R., and M. Vazquez Botet. Acromelanosis: A case report. J. Am. Acad. Dermatol. 2:128–131, 1980. Griffiths, W. A. D. Reticulate acropigmentation of Kitamura. Br. J. Dermatol. 95:437–443, 1976. Komaya, G. Pigmentanomalie der extremitaten. Arch. Dermatol. Syphil. 147:389–393, 1924. Ruiz-Maldonado, R., L. Tamayo, and J. Fernandez-Diez. Universal acquired melanosis. Arch. Dermatol. 114:775–778, 1978. Thomas, E. Uber das Spitzen pigment des Kleinekindes. Munch. Med. Wochenschr. 70:1102, 1923. Weidman, A. I. Acropigmentation (acromelanosis): Report of a case. Cutis 5:1119–11120, 1969.

Becker Nevus Norman Levine and Cynthia Burk

Historical Background In 1923, Stokes described a patient with a pigmented hairy plaque, which when biopsied, displayed dermal smooth muscle hyperplasia. Twenty-six years later, Becker (1949) reported almost identical findings. Since 1965, the condition has been named after Becker (Copeman and Wilson-Jones, 1965).

Fig. 50.6. Becker nevus of the trunk (see also Plate 50.2 pp. 494–495).

Synonyms Becker melanosis, pigmented hairy epidermal nevus, nevus pilaris, melanosis naeviformis Becker, nevus tardif de Becker.

Epidemiology Becker nevus occurs in all races; it is four to six times more common in males than in females (Tymen et al., 1981). It occurs in approximately 0.5% of the population (Hsu et al., 2001).

Clinical Findings Becker nevus is characterized by an irregular macule or patch, often with hypertrichosis (Terheyden et al., 1998). The hair associated with Becker nevus usually develops several years after the hyperpigmentation and is often coarse and dark. Becker nevus is most commonly located unilaterally on the shoulder, upper arm, anterior chest, or scapula (Fig. 50.6). However, there have been reports of Becker nevi occurring on the face, neck, and lower extremities. It is often minimally substantive (Copeman and Wilson-Jones, 1965). The lesions typically present during the adolescent years; however, both congenital and familial cases have been reported. Familial cases demonstrate autosomal dominance with incomplete penetrance and variable expressivity (Book et al., 1997). There appears to be a spectrum of disease which includes lesions present at birth with prominent hamartomatous changes of dermal smooth muscle and less skin darkening. These have been called smooth muscle hamartomas or congenital Becker nevus (Berger and Levin 1984; Johnson and Jacobs, 1989; Karo and Gange 1981). The clinical appearance of these lesions closely resembles those of the acquired plaques in adolescent boys. Most Becker nevi occur as isolated defects. However, ipsilateral bony abnormalities, acneiform eruptions (Burgeen and Ackerman, 1978), and breast hypoplasia have been described (Glinick et al., 1983; Lucky et al., 1981) in patients with Becker nevi. In addition, cystic lymphangioma, ipsilateral foot enlargement, pectus carinatum, neurofibroma, segmental odontomaxillary dysplasia, and connective tissue nevus have been 915

CHAPTER 50

described in patients with Becker nevus (Oyler et al., 1997). Skeletal abnormalities such as hemivertebrae, spina bifida occulta, scoliosis, cervical ribs, and minor sternal anomalies have also been associated with Becker nevus. Other associated abnormalities include accessory scrotum, congenital adrenal hyperplasia, supernumerary nipples, aplasia of the ipsilateral pectoralis major muscle, and localized lipoatrophy (SantosJuanes et al., 2002). Lichen planus has also been associated within Becker nevus (Terheyden et al., 1998). The acneiform eruptions are thought to be caused by an increased number of androgen receptors within the affected skin. The onset around puberty, the male predominance, and the hypertrichosis all suggest a critical role of local androgen sensitivity.

Pathology Microscopic examination of a Becker nevus reveals regular acanthosis, papillomatosis, variable hyperkeratosis, increased basal layer pigmentation, dermal thickening, and an absence of nevus cells in the dermis. Hyperplasia of hair follicles and sebaceous glands can also occur as well as lengthening and clubbing of the rete ridges. The number of melanocytes is probably normal (Frenk and Delacre, 1970), but one report suggests that it is increased (Tate et al., 1980). Melanocytes surrounding keratinocytes contain an increased density of pigment granules (Oyler et al., 1997). In the dermis there are hyperplastic smooth muscle fibers which are randomly arranged without relationship to hair follicles (Haneke, 1979). These are almost identical to the changes seen in what has been called smooth muscle hamartoma, many of which have some increased epidermal pigmentation (Berger and Levin 1984; Johnson and Jacobs, 1989). Variable hyperplasia of different cell types may be found in these lesions, such as keratinocytes, fibroblasts, melanocytes, sebaceous and pilar epithelium, nerve cells, and smooth muscle cells (Hsu et al., 2001). There are no nevus cell nests such as one would see in melanocytic nevi.

Criteria for Diagnosis The diagnosis of a Becker nevus is usually made on the following grounds: ∑ sharply demarcated hyperpigmented plaque with coarse hair; ∑ the lesion usually presents or enlarges at the time of puberty; and ∑ pathological examination reveals basilar hyperpigmentation without nests of nevus cells. There are also increased smooth muscle fibers in the dermis.

Differential Diagnosis The differential diagnosis includes those conditions with large hyperpigmented patches and plaques. Congenital hairy nevi can have a similar appearance to the congenital variant of Becker nevus. However, these often lack the sharp margination and have a pebbly surface, which is lacking in Becker nevi. Nevus of Ito presents as a brown-gray patch over the posterior trunk. It differs from a Becker nevus in that it lacks hair 916

and has a poorly demarcated border. The differential diagnosis also includes lichen simplex chronicus, postinflammatory hyperpigmentation, and café-au-lait patch.

Pathogenesis The exact cause of this anomaly is unknown. The basic change in the pigmentary system in these lesions appears to be one of an inability of the melanocytes to be downregulated once stimulatory factors activate them (Urbanek and Johnson, 1978). One such stimulatory influence could be endogenous androgens. Becker nevi often appear at about the time of puberty and share features of other androgen-mediated tissues such as coarse terminal hair growth, smooth muscle hyperplasia, and dermal thickening (Person and Longcope, 1984). Lesional tissue has been shown to contain increased numbers of androgen receptors which may be the cause or the result of increased androgen effects at this localized site (Person and Longcope, 1984).

Treatment A Becker nevus has no medical import; thus treatment is for cosmetic purposes only. If the lesion is small, surgical excision is possible. However, many lesions are too large to permit primary closure. Serial partial excisions are possible in these cases. Laser technology has been introduced in the management of pigmented lesions (Geronemus, 1992; Goldberg, 1993; Nelson and Applebaum, 1992). A single Becker nevus has been treated in this way; the results were difficult to assess from the discussion, but this may be a worthwhile avenue for further investigation (Tse et al., 1994). A Q-switched ruby laser can reduce the hyperpigmentation (Adams, 1997).

Prognosis A Becker nevus is benign and has no potential to undergo malignant degeneration. Once established, it remains for the rest of one’s life. However, there have been reports of the hyperpigmentation fading over a number of years (Hsu et al., 2001).

References Adams, S. P. Dermacase. Becker’s nevus. Can. Fam. Physician. 43:855, 863, 1997. Becker, S. W. Concurrent melanosis and hypertrichosis in distribution of nevus unius lateris. Arch. Dermatol. Syphil. 60:155–160, 1949. Berger, T. G., and M. W. Levin. Congenital smooth muscle hamartoma. J. Am. Acad. Dermatol. 11:709–712, 1984. Book, S. E., A. T. Glass, et al. Congenital Becker’s nevus with a familial association. Pediatr. Dermatol. 14:373–375, 1997. Burgeen, B. L., and A. B. Ackerman. Acneform lesions in Becker’s nevus. Cutis 21:617–619, 1978. Copeman, P. W. M., and E. Wilson-Jones. Pigmented hairy epidermal nevus (Becker). Arch. Dermatol. 92:249–252, 1965. Frenk, E., and T. J. Delacre. Zür ultrastruktur der Beckareschen melanose. Hautarzt 9:397–400, 1970. Geronemus, R. G. Q-switched ruby laser therapy of nevus of Ota. Arch. Dermatol. 128:1618–1622, 1992. Glinick, S. E., J. C. Alper, H. Boggars, and J. A. Brown. Becker’s melanosis: Associated abnormalities. J. Am. Acad. Dermatol. 9:509–511, 1983.

ACQUIRED EPIDERMAL HYPERMELANOSES Goldberg, D. J. Benign pigmented lesions of the skin. Treatment with the Q-switched ruby laser. J. Dermatol. Surg. Oncol. 19:376–379, 1993. Haneke, E. The dermal component of melanosis naeviformis Becker. J. Cutan. Pathol. 6:53–58, 1979. Hsu, S., J. Y. Chen, and P. Subrt. Becker’s melanosis in a woman. J. Am. Acad. Dermatol. 45(6 Suppl): S195–196, 2001. Johnson, M. D., and A. H. Jacobs. Congenital smooth muscle hamartoma: A report of six cases and a review of the literature. Arch. Dermatol. 125:820–822, 1989. Karo, K. R., and R. W. Gange. Smooth-muscle hamartoma: Possible congenital Becker’s nevus. Arch. Dermatol. 117:678–679, 1981. Lucky, A. W., M. Saruk, and A. B. Lerner. Becker’s nevus associated with limb asymmetry. Arch. Dermatol. 117:243, 1981. Nelson, J. S., and J. Applebaum. Treatment of superficial cutaneous pigmented lesions by melanin-specific selective photothermolysis using the Q-switched ruby laser. Ann. Plast. Surg. 29:231–237, 1992. Oyler, R. M., D. A. Davis, and J. T. Woosley. Lymphangioma associated with Becker’s nevus: a report of coincident hamartomas in a child. Pediatr. Dermatol. 14:376–379, 1997. Person, J. R., and C. Longcope. Becker’s nevus: an androgenmediated hyperplasia with increased androgen receptors. J. Am. Acad. Dermatol. 10:235–238, 1984. Santos-Juanes, J., C. Galache, J. R. Curto, M. P. Carrasco, A. Ribas, and J. Sanchez del Rio. Acneiform lesions in Becker’s nevus and breast hypoplasia. Int. J. Dermatol. 41:699–700, 2002. Stokes, J. H. Nevus pilaris with hyperplasia of nonstriated muscle. Arch. Dermatol. Syphil. 7:479–481, 1923. Tate, P. R., S. J. Hodge, and L. G. Owen. A quantitative study of melanocytes in Becker’s nevus. J. Cutan. Pathol. 7:404–409, 1980. Terheyden, P., B. Hornschuh, S. Karl, J. C. Becker, and E. B. Brocker. Lichen planus associated with Becker’s nevus. J. Am. Acad. Dermatol. 38(5 Pt 1): 770–772, 1998. Tse, Y., V. J. Levine, S. A. McClain, and R. Ashinoff. The removal of cutaneous pigmented lesions with the Q-switched ruby laser and the Q-switched neodymium: yttrium-aluminum-garnet laser. A comparative study. J. Dermatol. Surg. Oncol. 20:795–800, 1994. Tymen, R., J. F. Forestier, B. Boutet, and B. Colomb. Nevus tardif de Becker. Ann. Dermatol. Venereol. 108:41–46, 1981. Urbanek, R. W., and W. C. Johnson. Smooth muscle hamartoma associated with Becker’s nevus. Arch. Dermatol. 114:104–106, 1978.

Fig. 50.7. A large isolated café-au-lait spot in a baby.

Fig. 50.8. Small café-au-lait spot on the scrotum.

Café-au-lait macule, circumscribed melanotic macule, hypermelanotic macule, circumscribed hypermelanosis.

included more than 4000 newborns (Tekin et al., 2001). The vast majority of infants with café-au-lait spots have no associated abnormalities. Solitary café-au-lait spots are common in the general population affecting up to a third of normal children; however, multiple lesions are unusual and raise the suspicion of a multiple-system disorder (Landau and Krafchik, 1999). There are several multiorgan disorders which include them, in addition to neurofibromatosis, including McCune–Albright syndrome, tuberous sclerosis, Bloom syndrome, Watson syndrome, and ring chromosome syndrome. The association with café-au-lait spots and ataxia-telangectasia, Silver–Russell syndrome, juvenile xanthogranuloma, and LEOPARD syndrome is less clear but there may be an association.

Epidemiology

Clinical Findings

Café-au-lait spots are a common finding at birth (Alper et al., 1979; Whitehouse, 1966) but the incidence rate depends on the race of the neonate. Café-au-lait spots were noted in 0.3% of Caucasians and 18% of African-Americans in a study that

Café-au-lait spots present as uniformly pigmented tan-brown, oval or round macules or patches. The pigmented border may be either smooth or jagged (Figs 50.7–50.9). Those café-aulait spots associated with McCune–Albright syndrome often

Café-au-lait Spots Norman Levine and Cynthia Burk

Historical Background Although the first written description of café-au-lait spots is lost to history, it first became an important cutaneous sign in the late 1800s when von Recklinghausen first described the neural nature of neurofibromas and others noted café-au-lait macules as a part of the syndrome (Marie and Bernard, 1896).

Synonyms

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Differential Diagnosis Café-au-lait spots must be distinguished from other conditions which produce pigmented macules. A congenital melanocytic nevus is present at birth and is often uniformly brown. Most of these lesions acquire some substance and/or hair early in life, while café-au-lait spots remain macular throughout life. Becker nevus may present at or near birth and have an almost identical appearance to café-au-lait spots. As the child ages, the lesions acquire coarse hair and may become palpable. The increased dermal smooth muscle seen on skin biopsy clinches the diagnosis of Becker nevus. Nevus spilus is characterized by a light-brown patch, but differs from a café-au-lait spot in that it is stippled with dark-brown macules (Levine, 1995). Ephelides, congenital nevus, lentigo, nevocellular nevus, and segmental pigmented disorder are all other skin lesions to consider in the differential diagnosis (Landau and Krafchik, 1999). There are several related congenital pigmentary abnormalities, collectively called dermal melanosis; these include nevus of Ota, nevus of Ito, Mongolian spot, and perhaps, blue nevus. All of these conditions impart a blue or gray hue to the skin because of the deep dermal melanin deposition, which differentiates them from café-au-lait spots. In darkly pigmented children, it may be very difficult to distinguish blue from brown colors. The most common of these conditions, Mongolian spots almost always resolve in the first few years of life; café-au-lait spots are stable.

Pathogenesis Fig. 50.9. Café-au-lait spot showing a nevoid distribution.

have characteristic jagged edges; those spots associated with neurofibromatosis 1 usually have smooth margins (Cohen et al., 2000). Generally, more lesions are found in areas like the trunk, buttocks, and lower limbs and tend to spare the head, neck, and upper extremities (Cohen et al., 2000). In newborns, lesions are mainly concentrated to the buttock area and range in size from 0.2 cm to 4 cm, whereas in older children lesions are mainly centered on the trunk and can be 1.5–30 cm in size (Landau and Krafchik, 1999).

Pathology Light microscopic examination reveals increased epidermal melanin with normal numbers of melanocytes. Ultrastructural examination shows increased pigment without giant pigment granules (macromelanosomes), which are a feature of the caféau-lait spots that occur in neurofibromatosis.

Criteria for Diagnosis ∑ Lightly pigmented macules or patches. ∑ Fewer than six lesions with a diameter of 1.5 cm or larger.

If present, this is almost pathognomonic of neurofibromatosis (Korf, 1992). 918

The etiology of these congenital lesions is not known. It appears that there are a normal number of melanocytes, which are more active at synthesizing melanin.

Treatment There are no medical indications for treating café-au-lait spots. They have no malignant potential and are usually in locations of little esthetic significance. The Q-switched ruby laser, the Q-switched Nd-YAG laser, and the pulsed dye laser have all recently been used with variable responses (Shimboshi and Kamide, 1997).

Prognosis These innocuous macules and patches increase in size only in proportion to the growth of the child. They may lighten slightly as one ages, but usually remain unchanged throughout adult life.

References Alper, J., L. B. Holmes, and M. C. Mihm Jr. Birthmarks with serious medical significance: nevocellular nevi, sebaceous nevi, and multiple cafe au lait spots. J. Pediatr. 95:696–700, 1979. Carpo, B. G., J. M. Gravelink, and S. V. Gravelink. Laser treatment of pigmented lesions in children. Semin. Cutan. Med. Surg. 18:233– 243, 1999. Cohen, J. B., C. K. Janniger, and R. A. Schwartz. Café-au-lait spots. Cutis 66:22–24, 2000.

ACQUIRED EPIDERMAL HYPERMELANOSES Korf, B. R. Diagnostic outcome in children with multiple cafe au lait spots. Pediatrics 90:924–927, 1992. Landau, M., and B. R. Krafchik. The diagnostic value of café-au-lait macules. J. Am. Acad. Dermatol. 40(6 Pt 1): 877–890, 1999. Levine, N. Pigmentary abnormalities. In: Pediatric Dermatology, 2nd ed., L. A. Schachner, and R. C. Hansen (eds). New York: Churchill Livingstone, 1995, pp. 541–542. Marie, P., and A. Bernard. Presentation d’un malade atteint de neuro-fibromaose generalisée. Bull. Mem. Soc. Med. Hop. Paris 13:200–203, 1896. Shimboshi, T., and R. Kamide. Long term follow-up in treatment of solar lentigo and café-au-lait macules with Q-switched ruby laser. Aesth. Plast. Surg. 21:445–448, 1997. Tekin, M., J. N. Bodurtha, and V. M. Riccardi. Café-au-lait spots: the pediatrician’s perspective. Pediatr. Rev. 22:82–90, 2001. von Recklinghausen, F. D. Ueber die multiplen Fibrome der Haut und ihre Beziehung zu den multiplen Neuromen. Berlin: Hirschwald, 1882. Whitehouse, D. Diagnostic value of café-au-lait spots in children. Arch. Dis. Child. 41:316–319, 1966.

Carcinoid Syndrome Norman Levine and Cynthia Burk

Historical Background The carcinoid syndrome is an uncommon complication of malignant carcinoid tumors, occurring in about 4% of all patients with carcinoid tumors (Wilson, 1970). Since its initial description by Thorsen et al. (1954), over 200 cases have been reported, undoubtedly a low estimate of the total number that have occurred. Recent reports indicate that the estimated annual incidence is between 1 and 8.4/100 000 (Rohaizak and Farndon, 2002).

stenosis/insufficiency, endocardial fibrosis, pulmonary valve stenosis, and rarely, sexual dysfunction, vasospastic angina, retroperitoneal fibrosis, rheumatoid arthritis, and mental status changes. These clinical signs associated with carcinoid tumors are dependent on the monoamine elaborated; these include catecholamines, histamine, bradykinins, adrenocorticotropin, vasopressin, and prostaglandins (Feldman, 1987). The location of the primary tumor dictates the types of symptoms and signs of the syndrome. For example midgut carcinoids are more likely to produce carcinoid syndrome than are foregut tumors, but if carcinoid syndrome does occur with a foregut neoplasm, there is often very profound flushing. Midgut carcinoids are associated with a more transient flush which is more cyanotic in appearance, telangiectasias, cardiac lesions, and pulmonary fibrosis (Kaplan, 1991). Diarrhea occurs with tumors from all locations. Although much is known about these various mediators, it is not entirely clear exactly which one is associated with the various signs and symptoms. The cardinal cutaneous sign of carcinoid syndrome is cutaneous flushing. The color can vary from bright red or violaceous to pink-orange. It is usually limited to the upper torso, neck, and face (Fig. 50.10). Most paroxysms last for only a few minutes, although severe attacks can last much longer and be associated with such intense and widespread flushing that periorbital edema and a decrease in blood pressure can ensue. Most episodes occur randomly, but some patients note that emotional stress, physical exertion, ethanol ingestion, or

Synonyms Carcinoidosis (Weichert, 1970).

Epidemiology Carcinoid syndrome occurs mostly in those patients with malignant carcinoid tumors of the intestinal tract, particularly the small intestine. Rarely, the syndrome may arise from bronchial, stomach, or appendix neoplasms. There is no race or sex predilection for either the primary tumor or for the fullblown syndrome.

Clinical Findings Enterochromaffin cells are embryologically related to thyroid C cells, adrenal medullary cells, and melanocytes, all derived from the neural crest. These cells concentrate amino acid precursors of aromatic amines and produce bioamines; the acronym for these cells is APUD and the tumors are sometimes called “apudomas.” Other such tumors include thyroid adenomas, pituitary adenomas, parathyroid adenomas, and adrenal cortical adenomas. Up to 18% of patients with malignant carcinoid tumors present with the carcinoid syndrome, which may include signs and symptoms of facial flushing with telangiectasia, lacrimation, diarrhea/nausea, bronchospasm/wheezing, tricuspid

Fig. 50.10. Red violaceous flush of carcinoid syndrome (see also Plate 50.3, pp. 494–495).

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defecation can precipitate an attack (Feingold and Elias, 1988). After years of chronic flushing episodes, many people develop fixed telangiectasias of the face and neck. Cutaneous hyperpigmentation occasionally occurs with carcinoid syndrome. Three different circumstances lead to a change in skin pigment. Carcinoid tumors secrete large amounts of serotonin; the precursor for this amine is tryptophan. When excess tryptophan is diverted for this synthesis, a deficiency of another tryptophan product, niacin, may result which may lead to the clinical signs of pellagra. The eruption of this vitamin deficiency consists of gray-black, keratotic plaques in a photodistribution, localized to the dorsal forearms, legs, and trunk (Castiello and Lynch, 1972). Carcinoid tumors rarely can secrete excess adrenocorticotropic hormone (ACTH). This can result in generalized hyperpigmentation, similar to what is seen in Addison disease (Rodriguez Vaca et al., 1987). The third type of skin pigment change occurs over the forehead, wrists, thighs, and trunk; it consists of yellow-brown, slightly atrophic plaques (Stampien et al., 1993). Many other cutaneous findings have been reported in the carcinoid syndrome. These include pruritus, scleroderma, cutaneous metastases, pyoderma gangrenosum, and erythema annulare centrifugum (Donaldson, 2000; Wilkin and Demis, 1995). A few primary carcinoid tumors of the skin have also been described (Bart et al., 1990; Van Dijk and ten Seldam, 1975).

Criteria for Diagnosis

Pathology

Pathogenesis

Microscopically, carcinoid tumors consist of small round cells in monotonous sheets with uniform nuclei and cytoplasm. They stain positive with argyrophil, argentaffin, chromogranin, and nonspecific enolase. It is difficult to distinguish benign versus malignant carcinoid tumors via pathological appearance alone.

The mediators secreted by the tumor cells are almost certainly responsible for the signs and symptoms of carcinoid syndrome. However, it has been very difficult to correlate specific clinical findings with the effects of a given mediator. One exception to this may be the generalized hyperpigmentation, which seems to be a direct result of excess ACTH secretion (Rodriguez Vaca et al., 1987). Another well-described association is the pellagralike findings in those patients with high circulating levels of serotonin. As noted above, tryptophan is diverted from niacin synthesis to serotonin synthesis, leaving the affected patient with a niacin deficiency state which leads to pellagra. The cutaneous sclerosis associated with the carcinoid syndrome may also be related to an impairment of tryptophan metabolism (Ratnavel et al., 1994). There are several candidates for mediators of the flushing and diarrhea that are so common in these patients. These include histamine, 5-hydroxytryptamine (5-HT), prostaglandins, vasoactive intestinal peptide, glucagon, gastrin, calcitonin, kallikrein, substance P, and substance K (Kaplan, 1991). It is possible that combinations of these mediators are responsible for the clinical findings.

Diagnostic Evaluation Light microscopic examination of flushed skin is nondiagnostic. The hyperpigmented skin reveals increased epidermal melanin. The evaluation of carcinoid syndrome is based on the observation that most of these tumors secrete large amounts of serotonin. This amine is metabolized in the blood to 5hydroxyindoleacetic acid (5-HIAA) which is then excreted through the kidneys. Elevated levels of this metabolite in the urine are highly suggestive of the presence of a carcinoid tumor. Since many foods can affect 5-HIAA excretion, an elimination diet is necessary before this determination is undertaken (Wilkin and Demis, 1995). Selective abdominal vein catheterization with blood sampling for serotonin can localize the site of the tumor and can identify the rare cases where urine 5-HIAA levels are normal (Davis and Rosenberg, 1961; Kuwada, 2000). When clinical findings are equivocal, one can attempt to provoke a flushing episode. Ethanol, pentagastrin, or minute doses of epinephrine may be used for this purpose. 920

∑ Metastatic carcinoid tumor. ∑ Symptom complex of paroxysmal flushing and diarrhea. ∑ Urinary 5-HIAA levels above 15 mg per day (in most

instances).

Differential Diagnosis The differential diagnosis of carcinoid syndrome includes other conditions associated with flushing (Wilkin, 1983). Systemic mastocytosis can produce flushing and diarrhea. Elevated urinary histamine and its metabolites and a normal urinary 5-HIAA clearly separate it from carcinoid syndrome. Menopausal flushing can simulate that seen in those with carcinoid tumors. Often, the same triggers are operative for both processes. The drenching sweats and nighttime episodes are more common in those with menopausal symptoms. If there is any doubt, a urinary 5-HIAA measurement will separate these two entities. Pheochromocytoma is a tumor of the adrenal gland which secretes a catecholamine which can cause occasional episodes of paroxysmal flushing. However, the flushed sensation appears only after an attack of headache, tachycardia, chest pain, and pallor (Wilkin, 1983). Rosacea occurs in individuals who are prone to flush. Facial telangiectasias identical to those noted in carcinoid syndrome are an essential part of the disease. The other clinical manifestations of carcinoid syndrome are absent and urinary 5-HIAA is normal.

Treatment If the carcinoid tumor can be surgically excised, the signs and symptoms of the syndrome can disappear (Rodriguez Vaca et al., 1987). However, great care by the anesthesiologist is needed to prevent a carcinoid crisis with hypotension and

ACQUIRED EPIDERMAL HYPERMELANOSES

cardiac arrest from occurring (RuDusky, 1999). Hepatic resection as well as hepatic artery embolization have been used with a good performance status for removing metastatic disease (Filosso et al., 2002). Although the radiosensitivity of these tumors has not been reported, chemotherapy regimens including the combination of 5-fluorouracil and streptozocin have been tried, with moderate success (Filosso et al., 2002). Thus, it should be used as a late treatment for patients with carcinoid tumors/syndrome. Somatostatin and its longer-acting analogs octreotide and lanreotide are the drugs of choice for managing malignant carcinoid syndrome as they have been proved to improve symptoms and decrease the tumor progression (Ganim and Norton 2000; O’Toole et al., 2000; Filosso et al., 2002; Leong and Pasieka, 2002). Treatment with recombinant interferon-a may control the symptoms of the disease and in some cases increase mean survival time (Filosso et al., 2002). Serotonin antagonists such as methysergide or ondansetron, a 5-HT3 antagonist, can be used to slow gastric emptying and improve both diarrhea and nausea (Wymenga et al., 1998). However, these agents do little for the flushing. Cyproheptadine has antiserotonin and antihistamine effects and is sometimes useful in the treatment of the flush. Clonidine is another agent of value in histamine-mediated flushing. Treatment for the wheezing and bronchospasm should include systemic corticosteroids, but not epinephrine, which could initiate a vasomotor attack in these patients. For the patient who is at risk for the pellagralike reaction, niacin supplementation is essential. Many foods containing tyramine can precipitate a flushing paroxysm; these include tomatoes, eggplant, and avocados. Alcohol and hot, spicy foods can also evoke an attack. The patient should be given a list of the foods to eat in moderation or to avoid altogether.

Prognosis The prognosis depends on the site and the stage of the disease at diagnosis. Rectal and appendageal tumors do not affect survival; lesions in other intestinal sites have a five-year survival of 65% with lymph node involvement and only an 18% survival if there are hepatic metastases, a situation common in the carcinoid syndrome. Once flushing ensues, the median survival is less than three years. Patients with greatly elevated urinary 5-HIAA (greater than 150 mg/day) can expect to live only about 13 months (Kaplan, 1991). Nearly half of all patients with carcinoid tumors who die from the disease succumb to heart failure (Ganim, 2000).

References Bart, R. S., H. Kamino, J. Waisman, A. Lindner, and S. Colen. Carcinoid tumor of skin: report of a possible primary case. J. Am. Acad. Dermatol. 22:366–370, 1990. Castiello, R. J., and P. J. Lynch. Pellagra and the carcinoid syndrome. Arch. Dermatol. 105:574–577, 1972. Davis, R. B., and J. C. Rosenberg. Carcinoid syndrome associated with hyperserotoninemia and normal 5-hydroxyindoleacetic acid excretion. Am. J. Med. 30:167–174, 1961. Donaldson, D. Carcinoid tumours — the carcinoid syndrome and

serotonin (5-HT): a brief review. J. Royal Soc. Health 120:78, 2000. Feingold, K. R., and P. M. Elias. Endocrine–skin interactions. Cutaneous manifestations of adrenal disease, pheochromocytomas, carcinoid syndrome, sex hormone excess and deficiency, polyglandular autoimmune syndromes, multiple endocrine neoplasia syndromes, and other miscellaneous disorders [review]. J. Am. Acad. Dermatol. 19:1–20, 1988. Feldman, J. M. Carcinoid tumors and syndrome. Semin. Oncol. 14:237–260, 1987. Filosso, P. L., E. Ruffini, A. Oliaro, E. Papalia, G. Donati, and O. Rena. Long-term survival of atypical bronchial carcinoids with liver metastases, treated with octreotide. Eur. J. Cardiothorac. Surg. 21:913–917, 2002. Ganim, R. B., and J. A. Norton. Recent advances in carcinoid pathogenesis, diagnosis and management. Surg. Oncol. 9:173–179, 2000. Kaplan, L. M. Endocrine tumors of the gastrointestinal tract and pancreas. In: Harrison’s Principles of Internal Medicine, J. D. Wilson et al. (eds). New York: McGraw-Hill, 1991, pp. 1386–1393. Kuwada, S. K. Carcinoid tumors. Semin. Gastrointest. Dis. 11:157– 161, 2000. Leong, W. L., and J. L. Pasieka. Regression of metastatic carcinoid tumors with octreotide therapy: two case reports and a review of the literature. J. Surg. Oncol. 79:180–187, 2002. O’Toole, D., M. Ducreux, G. Bommelaer, J. L. Wemeau, O. Bouche, Catus F, J. Blumberg, and P. Ruszniewski. Treatment of carcinoid syndrome: a prospective crossover evaluation of lanreotide versus octreotide in terms of efficacy, patient acceptability, and tolerance. Cancer 88:770–776, 2000. Rohaizak, M., and J. R. Farndon. Use of octreotide and lanreotide in the treatment of symptomatic non-resectable carcinoid tumours. Aust. N. Z. J. Surg. 72:635–638, 2002. RuDusky, B. M. Carcinoid — a diagnostic and therapeutic dilemma [comment]. Chest 116:1142–1143, 1999. Ratnavel, R. C., N. P. Burrows, and R. J. Pye. Scleroderma and the carcinoid syndrome. Clin. Exp. Dermatol. 19:83–85, 1994. Rodriguez Vaca, M. D., M. Angel, I. Halperin, J. Freixenet, M. Marti, M. J. Martinez Osaba, J. Sanchez Lloret, A. Palacin, and E. Vilardell. Diagnosis of lung carcinoid with cutaneous hyperpigmentation eight years after bilateral adrenalectomy. J. Endocrinol. Invest. 10:537–540, 1987. Stampien, T. M., I. Thomas, and R. A. Schwartz. Cutaneous pigmentation as a sign of systemic disease. In: Pigmentation and Pigmentary Disorders, N. Levine (ed.). Boca Raton, FL: CRC Press, 1993, pp. 376–377. Thorson, A., G. Biorck, and G. Bjorkman. Malignant carcinoid of the small intestine with metastases to the liver, valvular disease of the right side of the heart (pulmonary stenosis and tricuspid regurgitation without septal defects), peripheral vasomotor symptoms, bronchoconstriction, and an unusual type of cyanosis: A clinical and pathologic syndrome. Am. Heart Assoc. J. 47:795–817, 1954. Van Dijk, C., and L. E. J. ten Seldam. A possible primary cutaneous carcinoid. Cancer 36:1016–1020, 1975. Weichert, R. F. The neural ectodermal origin of the peptide secreting endocrine glands: A unifying concept for the etiology of multiple endocrine adenomatosis and the inappropriate secretion of peptide hormones by non-endocrine tumors. Am. J. Med. 49:232–241, 1970. Wilkin, J. K. Flushing reactions. Rec. Adv. Dermatol. 6:157–187, 1983. Wilkin, J. K., and D. J. Demis. Carcinoidosis (carcinoid syndrome). In: Clinical Dermatology, J. D. Demis (eds). Philadelphia: Lippincott-Raven Publishers, 1995, pp. 1–7. Wilson, H. Carcinoid syndrome. Curr. Probl. Surg. 11:36–41, 1970. Wymenga, A. N., E. G. de Vrieds, M. K. Leijsma, I. P. Kema, and J. H. Kleibeuker. Effects of ondansetron on gastrointestinal symptoms in carcinoid syndrome. Eur. J. Cancer 34:1293–1294, 1998.

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Confluent and Reticulated Papillomatosis Norman Levine and Cynthia Burk

Historical Background Two French dermatologists, Gougerot and Carteaud (1927, 1932), were the first to report a series of cases of an unusual papillomatous disease. They described three slightly different variants, confluent and reticulated papillomatosis (CRP), punctate, pigmented, verrucous papillomatosis, and nummular and confluent papillomatosis. Only the first variety is seen with any frequency today. Subsequently, this condition was reported in the United States (Wise and Sachs, 1937). Confluent and reticulated papillomatosis has now been seen in all parts of the world. Approximately 50 cases have been reported since 1978 (Lee et al., 1994).

Fig. 50.11. Localized pigmented confluent and reticulated papillomatosis (see also Plate 50.4, pp. 494–495).

Synonyms Cutaneous papillomatosis, CRP of Gougerot and Carteaud, nummular and confluent papillomatosis, papillomatose pigmentee innominee, papillomatose pigmentee confluente et reticulee innominee, atrophie brilliante, parakeratose brilliante (Waisman, 1995).

Epidemiology Early studies reported that CRP was more common in women and in black people (Hamilton et al., 1980). A review of many published studies led the investigators to refute this claim (Lee et al., 1994). They found no difference in the incidence between the sexes or between any racial group. The typical onset is around age 20. Most of the reported cases have been sporadic, although there have been a few familial occurrences (Baden, 1965). Confluent and reticulated papillomatosis is 2.5 times more common in women and two times more common in black people (Hamilton et al., 1980; Montemarano et al., 1996; Solomon and Laude, 1996).

Fig. 50.12. Close-up view of the patient in Figure 50.11.

Clinical Findings A typical patient with CRP presents with 1–2 mm red, verrucous, minimally scaly papules in the inframammary, interscapular, and epigastric region; these enlarge and eventually coalesce into brown plaques (Figs 50.11–50.13). The lesions lead to an accentuation of the natural skin folds on the neck and in the axillae. As the lesions spread peripherally, the advancing borders take on a reticulated appearance (Hamilton et al., 1980; Lee et al., 1994). The mucous membranes, palms, and soles are spared.

Pathology

Fig. 50.13. Extensive confluent and reticulated papillomatosis of Gougerot and Carteaud (see also Plate 50.5, pp. 494–495).

Light microscopic examination reveals variable hyperkeratosis, and a normal basal layer. Epidermal atrophy over the dermal papillary tips may alternate with mild to moderate acanthosis. Papillomatosis is always present. In the dermis, there is a mild perivascular lymphocytic infiltrate, vascular dilatation, and occasional upper dermal edema (Hamilton et al., 1980).

Electron microscopic examination shows an alteration of cornified cell structures showing snake coil-like, or trianglelike stacks, an increase in the Odland bodies in the stratum granulosum, and an increased number of melanosomes in the stratum corneum (Montemarano et al., 1996). There are also an increased number of transitional cells between the stratum

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granulosum and the stratum corneum (Inaloz et al., 2002), an increase in the number of lamellar granules in the granular cell layer, and an increased number of melanosomes in the upper epidermal layers. The basal layer melanocytes appear normal (Chang et al., 1996; Jimbow et al., 1992).

Criteria for Diagnosis The diagnostic features that should make one consider the diagnosis of CRP are as follows (Lee et al., 1994): ∑ Hyperpigmented papules and plaques involving the chest and/or back with a reticulated peripheral margin. ∑ No hyphae suggestive of tinea versicolor present on potassium hydroxide preparation or skin biopsy. ∑ Histologic changes including hyperkeratosis, papillomatosis, acanthosis alternating with mild atrophy, and evidence of mild dermal inflammation and vasodilatation.

Differential Diagnosis The skin diseases to be considered include tinea versicolor, acanthosis nigricans, cutaneous amyloidosis, keratosis follicularis, certain forms of ichthyosis, erythrokeratoderma variabilis, and epidermodysplasia verruciformis. The main condition that must be differentiated from CRP is tinea versicolor. In fact, the two skin problems are so similar clinically, and even mycologically on occasion (Thomsen, 1979), some believe that CRP is, indeed, a variety of tinea versicolor (Roberts and Lachapelle, 1969). The arguments against this notion are discussed in the section on pathogenesis. Although the distribution and morphology of the full-blown lesions is similar, the histologic changes are distinct enough to diagnose these as distinct entities. Acanthosis nigricans can sometimes be confused with CRP. Histopathologically, they are indistinguishable (Ginarte et al., 2002). The lesions of acanthosis nigricans are usually velvety rather than scaly. Although the neck is commonly involved in both diseases, acanthosis nigricans appears prominently in the axillae and groin area while CRP presents on the central back and chest. Mucous membrane lesions can be found in acanthosis nigricans while this does not occur in CRP (Hamilton et al., 1980).

Pathogenesis The etiology of CRP is not known; however, several theories have been proposed. Because of its clinical similarity to tinea versicolor, some consider CRP to be of fungal origin. The response to antifungal agents such as selenium sulfide has also led to this conclusion (Nordby and Mitchell, 1986). However, this agent probably is working in CRP as an inhibitor of keratinization rather than as an antimicrobial drug (Sheth, 1983). Meischer was the first to suggest that CRP was a disorder of keratinization (Meischer, 1954). Many others have voiced this same view since that time (Bruynzeel-Koomen and de Witt, 1984; Jimbow et al., 1992; Lee et al., 1991). Electron microscopic studies have provided strong evidence that this is the case (Jimbow et al., 1992; Lee et al., 1991). Early investigators believed that CRP was associated with

endocrine dysfunction because of its clinical similarity to acanthosis nigricans (Waisman, 1953). There have been occasional cases of CRP appearing in patients with endocrine dysfunction such as Cushing disease, hypothyroidism (Waisman, 1995), diabetes mellitus, obesity, and menstrual irregularities (Chang et al., 1996; Montemarano et al., 1996). The vast majority of cases have no associated endocrine abnormalities, however; thus, this theory is no longer seriously considered. Other theories of causation with little corroborative evidence include photosensitivity (Vassileva et al., 1989), cutaneous amyloidosis (Groh and Schnyder, 1983), genetic factors (Henning and de Wit, 1981), and atopy (Chang et al., 1996).

Treatment Many different therapeutic modalities have been tried in CRP, but most have fallen by the wayside. These include keratolytics, superficial radiation, thyroid replacement, phototherapy, anticholinergic agents, hydroquinone, selenium sulfide, weight reduction, and crude liver extract, presumably for its vitamin A content (Hamilton et al., 1980). The treatment of choice for CRP is minocycline, 100–200 mg per day for weeks to months (Baalbaki, 1993; Chang et al., 1996; Petit et al., 1989; Puig, 1995; Sassolas, 1992). Still, other antibiotics may also be efficacious, including oral fusidic acid (1000 mg daily), clarithromycin (500 mg daily for five weeks), erythromycin (1000 mg daily), and azithromycin (500 mg daily). Complete clearing after treatment with these various antibiotics raises the possibility that reticulated papillomatosis is caused by a bacterial infection (Jang et al., 2001). Retinoids have been used in several patients with CRP; the results are very encouraging. Isotretinoin (Hodge and Ray, 1991; Lee et al., 1994) and etretinate (Baalbaki et al., 1993; Bruynzeel-Koomen and de Witt, 1984; Hirokawa et al., 1994) have both worked well in this disease. Long-term treatment is necessary since relapse occurs promptly after discontinuation of either of these drugs. In addition, topical tacalcitol and calcipotriol (both vitamin D analogs which regulate cell differentiation and inhibit the proliferation of keratinocytes) have been effective (Ginarte et al., 2002).

Prognosis CRP is a chronic disease with a slow, irregular progression. With few exceptions (Thomsen, 1979) there is little tendency for spontaneous resolution.

References Baalbaki, S. A., J. A. Malak, and M. A. al-Khars. Confluent and reticulated papillomatosis. Treatment with etretinate. Arch. Dermatol. 129:961–963, 1993. Baden, H. P. Familial cutaneous papillomatosis. Arch. Dermatol. 92:394–395, 1965. Bruynzeel-Koomen, C., and R. F. E. de Witt. Confluent and reticulated papillomatosis successfully treated with the aromatic etretinate. Arch. Dermatol. 120:1236–1237, 1984. Chang, S. N., S. C. Kim, S. H. Lee, and W. S. Lee. Minocycline treatment for CRP. Cutis 57:454–457, 1996.

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CHAPTER 50 Ginarte, M., J. M. Fabeiro, and J. Toribio. Confluent and reticulated papillomatosis (Gougerot-Carteaud) successfully treated with tacalcitol [comment]. J. Dermatol. Treat. 13:27–30, 2002. Gougerot, H., and A. Carteaud. Papillomatose pigmentee innominee. Bull. Soc. Franc. Dermatol. Syphiligr. 34:719–721, 1927. Gougerot, H., and A. Carteaud. Neue formen der papillomatose. Arch. Dermatol. Syphil. 165:232–267, 1932. Groh, V., and U. Schnyder. Nosologie der Papillomatose papuleuse confluente et réticulée (Gougerot-Carteaud). Hautarzt 34:81–86, 1983. Hamilton, D., V. Tavafoghi, J. C. Shafer, and G. W. Hambrick Jr. Confluent and reticulated papillomatosis of Gougerot and Carteaud: Its relation to other papillomatoses. J. Am. Acad. Dermatol. 2:401– 410, 1980. Henning, J. P., and R. F. de Wit. Familial occurrence of CRP. Arch. Dermatol. 117:809–810, 1981. Hirokawa, M., M. Matsumoto, and H. Iizuka. Confluent and reticulated papillomatosis: a case with concurrent acanthosis nigricans associated with obesity and insulin resistance. Dermatology 188:148–151, 1994. Hodge, J. A., and M. C. Ray. Confluent and reticulated papillomatosis: response to isotretinoin. J. Am. Acad. Dermatol. 24:654, 1991. Inaloz, H. S., G. K. Patel, and A. G. Knight. Familial CRP. Arch. Dermatol. 138:276–277, 2002. Jang, H. S., C. K. Oh, J. H. Cha, S. H. Cho, K. S. Kwon. Six cases of CRP alleviated by various antibiotics. J. Am. Acad. Dermatol. 44:652–655, 2001. Jimbow, M., O. Talpash, and K. Jimbow. Confluent and reticulated papillomatosis: clinical, light and electron microscopic studies. Int. J. Dermatol. 31:480–483, 1992. Lee, M. P., M. J. Stiller, S. A. McClain, J. L. Shupack, and D. E. Cohen. Confluent and reticulated papillomatosis: Response to high-dose oral isotretinoin therapy and reassessment of epidemiologic data. J. Am. Acad. Dermatol. 31:327–331, 1994. Lee, S. H., E. H. Choi, and W. S. Lee. Confluent and reticulated papillomatosis: a clinical, histopathological, and electron microscopic study. J. Dermatol. 18:725–730, 1991. Meischer, G. Erythrokeratodermia papillaris et reticularis. Dermatologica 108:303–314, 1954. Montemarano, A. D., M. Hengge, P. Sau, and M. Welch. Confluent and reticulated papillomatosis: response to minocycline. J. Am. Acad. Dermatol. 34(2 Pt 1): 253–256, 1996. Nordby, C., and A. Mitchell. Confluent and reticulated papillomatosis responsive to selenium sulfide. Int. J. Dermatol. 25:194–199, 1986. Petit, A., P. Evenou, and J. Civatte. Papillomatose confluente et reticulée de Gougerot et Carteaud: traitment par mynocicline. Ann. Dermatologie Venereologie 116:29–30, 1989. Puig, L., and J. M. de Moragas. Confluent and reticulated papillomatosis of Gougerot and Carteaud: minocycline deserves trial before etretinate [letter]. Arch. Dermatol. 131:109–110, 1995. Roberts, S., and J. Lachapelle. Confluent and reticulate papillomatosis (Gougerot-Carteaud) and Pityrosporum orbiculare. Br. J. Dermatol. 81:841–845, 1969. Sassolas, B., P. Plantin, and G. Guillet. Confluent and reticulated papillomatosis: treatment with minocycline. J. Am. Acad. Dermatol. 26:501–502, 1992. Sheth, R. A. A comparison of miconazole nitrate and selenium disulfide as anti-dandruff agents. Int. J. Dermatol. 22:123–125, 1983. Solomon, B. A., and T. A. Laude. Two patients with CRP: response to oral isotretinoin and 10% lactic acid lotion. J. Am. Acad. Dermatol. 35:645–646, 1996. Thomsen, K. Confluent and reticulated papillomatosis (GougerotCarteaud). Acta Derm. Venereol. Suppl. (Stockh.) 59:185–187, 1979. Vassileva, S., K. Pramatarov, and L. Popova. Ultraviolet light-induced CRP. J. Am. Acad. Dermatol. 21:413–414, 1989.

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Waisman, M. Cutaneous papillomatosis, pseudoacanthosis nigricans, and benign acanthosis nigricans. South. Med. J. 46:162–169, 1953. Waisman, M. Cutaneous papillomatosis (Gougerot-Carteaud). In: Clinical Dermatology, J. D. Demis (eds). Philadelphia: LippincottRaven Publishers, 1995, pp. 41–45. Wise, F., and W. Sachs. Cutaneous papillomatosis. Arch. Dermatol. Syphil. 36:475–485, 1937.

Cutaneous Amyloidosis Norman Levine and Cynthia Burk

Historical Background In the mid-1800s, Virchow first coined the term “amyloidosis” to describe starchlike substances in the skin and in other organs (Virchow, 1971). He was actually describing the ground substance that surrounded the amyloid fibrils. Since that time, efforts have been made to apply a uniform definition to these fibrils, which have some similarities in spite of greatly differing chemical compositions (Glenner, 1980). It is now known that all amyloids have two common characteristics: (1) they have straight, nonbranching filaments composed of polypeptide chains and a special three-dimensional structure, b-pleating, and (2) they all stain positively with Congo red (Eanes and Glenner, 1968).

Synonym b-fibrilloses.

Epidemiology Cutaneous amyloidosis is seen throughout the world, but is more common among Asians and Latin Americans. People of all ages may be affected but it is far more prevalent during adult life.

Clinical Findings There are several different clinical patterns associated with localized cutaneous amyloid deposition. The most common types are macular and lichen amyloidosis. These are probably variations on the same theme, since there are cases of a mixed picture in the same patient and macular amyloidosis becoming lichenoid over time (Iwasaki et al., 1991; Kibbi et al., 1992). In the purely macular variety, asymptomatic, poorly defined brown to gray-brown macules are found, most commonly in the interscapular area, over the shoulders (Fig. 50.14), on the face, or on the extremities (Black and WilsonJones, 1971; Brownstein and Hashimoto, 1972). The pigmentation may appear in parallel bands; this rippled hyperpigmentation occurs in no other pigmentary disorder other than macular amyloidosis. Lichen amyloidosis presents as pruritic, flesh-colored papules which coalesce into flat-topped (lichenoid), mildly keratotic plaques. The anterior legs (Figs 50.15 and 50.16) and upper back are frequent sites of lichen amyloidosis (Hashimoto and Yoong Onn, 1971). Mixed, or biphasic cutaneous amyloidosis consists of both the macular (Fig. 50.17) and lichenoid variety in the same patient in different lesions

ACQUIRED EPIDERMAL HYPERMELANOSES

Fig. 50.16. Typical hyperpigmented lesions on the thigh.

Fig. 50.14. Interscapular maculopapular and pigmented amyloidosis (see also Plate 50.6, pp. 494–495).

Fig. 50.17. Heavily pigmented macular amyloidosis (see also Plate 50.7, pp. 494–495).

Fig. 50.15. Pigmented cutaneous amyloidosis of the leg.

or in different parts of the same lesion (Brownstein et al., 1973). Another variation of lichen/macular amyloidosis is friction amyloidosis. This has been described after prolonged or vigorous use of nylon brushes or towels (Hashimoto et al., 1987; Macsween and Saihan, 1997; Tanigaki et al., 1985; Wong and Lin, 1988). Chronic rubbing and scratching for whatever reason probably causes the same changes in predisposed individuals. Nodular (tumefactive) amyloidosis usually presents in middle-aged women as one or a few firm, flesh-colored or yellowish papules or nodules on the face, scalp, or extremities.

Minor trauma may result in the lesions becoming hemorrhagic. Although nodular amyloidosis is almost always an isolated finding, it has been noted in a few patients with concomitant Sjögren syndrome (Haneda and Hamamatsu, 1979). Nodular amyloidosis may represent a localized plasma cell dyscrasia that can be associated with a monoclonal gammopathy, multiple myeloma, or lymphoplasmacytoid lymphoma (Badell et al., 1996; Hamzavi and Lui, 1999). Localized macular or biphasic amyloid deposition may be associated with depigmentation surrounded by hyperpigmentation. This is known as vitiliginous amyloidosis (Ishigaki et al., 1977). A similar poikilodermatous variety has also been described in patients with Riehl melanosis (Nagao and Iijima, 1974). Children may develop amyloid deposits in the skin after a severe sunburn. These lesions resemble colloid milia on the face. Adults may have a similar problem on the concha of the ears (Hicks et al., 1988). Both of these processes appear to be examples of actinically induced epidermal damage with 925

CHAPTER 50

amyloid synthesis and deposition. A number of Japanese patients have been described with asymptomatic amyloid deposition of the perianal area. The lesions present as hyperkeratotic linear plaques radiating from the anal verge (Vasily et al., 1978). A rare familial form of generalized macular or lichenoid amyloidosis has been described as an autosomal dominant disease. These patients manifest generalized hyperpigmentation with irregular hypopigmented macules (Yanagihara, 1981).

Electron microscopy of the amyloid deposits reveals filaments that are identical to those seen in AL amyloid, the type noted in systemic amyloidosis. Amyloid deposits preferentially around elastic fibrils. Melanosomes are also found in the amyloid deposits, suggesting that either keratinocytes containing melanosomes or melanocytes themselves undergo degeneration to produce these deposits (Hashimoto, 1995).

Criteria for Diagnosis ∑ Localized skin lesions containing amyloid fibrils ∑ No systemic manifestations of amyloidosis

Associated Disorders Multiple endocrine neoplasia (MEN) type 2a syndrome (Sipple syndrome) is an autosomal dominant phakomatosis characterized by hyperplasia and/or carcinoma of the thyroid gland, hyperplasia of the parathyroid gland and adrenal medulla, and/or pheochromocytoma (Gagel et al., 1988, 1989; Sipple, 1961; Szinnai et al., 2003). A specific mutation has been discovered for this phenotype (Barone et al., 1994; Kousseff, 1995). Many patients with this syndrome also have localized cutaneous amyloidosis (Chabre et al., 1992; Donovan et al., 1989; Gagel et al., 1988; Kousseff et al., 1991; Pacini et al., 1993; Robinson et al., 1992). Pleiotropy appears to be the most likely explanation for this association (Kousseff et al., 1991). Two other associations have been noted. Three patients with notalgia paresthetica, which is a localized peripheral neuropathy of the upper back, have also had amyloid deposition in the pruritic area (Goulden et al., 1994). This probably occurs in the same way as in friction amyloidosis, secondary to chronic rubbing and scratching. Two kindreds have been described with pachyonychia congenita and localized amyloid. The authors hypothesize that both conditions share abnormal keratin dynamics (Tidman et al., 1987).

Differential Diagnosis The differential diagnosis of macular amyloidosis includes conditions which produce macular hyperpigmentation. These include postinflammatory hyperpigmentation, melasma, nevus of Ito, and ashy dermatosis. Both postinflammatory hyperpigmentation and melasma can produce poorly demarcated brown patches. Amyloid is not noted on histologic examination. Although nevus of Ito and ashy dermatosis occur on the upper back, they both have a violaceous hue rather than a brown color. Amyloid is not deposited in these conditions. Lichen amyloidosis must be differentiated from other pruritic processes that produce papules and plaques; these include prurigo nodularis, lichen simplex chronicus, lichen planus, and pretibial myxedema. Although all of these conditions produce lichenoid plaques, only lichen amyloidosis has histologic evidence of dermal amyloid deposition on light microscopy. Nodular amyloidosis has a similar clinical appearance to many localized infiltrative processes such as lymphoma, colloid milium, sarcoidosis, mastocytoma, rheumatoid nodules, gouty tophi, and calcinosis cutis. Histologic examination is usually needed to sort out these possibilities.

Pathogenesis Pathology Light microscopic examination reveals similar lichenoid changes whether the lesions are macules or lichenoid papules. There is hyperkeratosis and variable degrees of keratinocyte degeneration and basal layer destruction. Amorphous eosinophilic material (amyloid) is deposited in a widened papillary dermis. Dermal melanophages and a sparse superficial perivascular infiltrate are also present (Kibbi et al., 1992). The nodular form of cutaneous amyloid differs in that the amyloid deposits extend into the deeper dermis and even the subcutis. Histochemical analysis shows that the amyloids present in the localized forms of amyloidosis contain keratins, presumably derived from epidermal keratinocytes (Ortiz-Romero et al., 1994). This is designated as amyloid K or AK amyloid (Hashimoto, 1984). Thus, most of these amyloid proteins will stain positive with antikeratin antibodies (Hamzavi and Lui, 1999). Nodular amyloid does not contain amyloid K, although other amyloid stains are positive. It is derived from immunoglobulin light chains (Ito et al., 1989), similar to that found in systemic amyloid. 926

Localized cutaneous amyloid fibrils are produced by a filamentous degeneration of keratin-type intermediate filaments (Hashimoto et al., 1987; Kamakiri et al., 1983). These may be apoptotic keratinocytes which drop into the upper dermis (Kamakiri et al., 1983). Specific antikeratin antibodies that react with cutaneous amyloid give further credence to the notion that these deposits originate in the suprabasal layer of the epidermis (Ortiz-Romero et al., 1994). This certainly explains why one might develop localized amyloid deposits after prolonged scratching or rubbing with nylon brushes or with sharp fingernails. Asymptomatic lesions of macular amyloid are more difficult to explain with this logic. Similarly, the cases of localized amyloid that appears in association with MEN 2a syndrome could be ascribed to cutaneous trauma. It seems that there is a genetic component in many cases, which may make one more prone to this disposition of apoptotic keratinocytes.

Treatment Most cases of localized cutaneous amyloidosis respond poorly to therapy. There are a few encouraging reports which tout systemic retinoids as being beneficial for lichen amyloidosis

ACQUIRED EPIDERMAL HYPERMELANOSES

(Helander and Hopsu-Havu, 1986; Marschalko et al., 1988). Low-dose cyclophosphamide has been reported as beneficial in reducing the symptoms of lichen amyloidosis (Paricha et al., 1987). One must weigh the risks of aggressive therapy such as this with the benefits in this relatively innocuous condition. Dermabrasion may successfully debulk cutaneous amyloid deposits and improve pruritus (Wong et al., 1982). Hypopigmenting agents such as hydroquinone are not useful in improving the appearance of the lesions of macular amyloidosis. Topical steroids with or without occlusion have been used for macular amyloidosis with limited success (Macsween and Saihan, 1997). Carbon dioxide laser vaporization has been successful in treating nodular amyloidosis. However, tissue friability and poor hemostasis may be of some concern with these patients (Hamzavi and Lui, 1999). A recent case report documents complete clearing in a patient with lichen amyloidosis and atopic dermatitis after ciclosporin therapy (Behr et al., 2001).

Prognosis The lesions of localized cutaneous amyloidosis seldom regress spontaneously. The course is variable; some people continue to develop new lesions or have old ones expand for years whereas others have only a single, stable patch or plaque.

References Badell, AO, Servitje, J. Graells, Curco N, J. Notario, and J. Peyri. Salivary gland lymphoplasmacytoid lymphoma with nodular cutaneous amyloid deposition and lambda chain paraproteinaemia. Br. J. Dermatol. 135:327–329, 1996. Behr, F. D., N. Levine, and J. Bangert. Lichen amyloidosis associated with atopic dermatitis: clinical resolution with cyclosporine. Arch. Dermatol. 137:553–555, 2001. Barone, V., F. Pacini, E. Martino, A. Loviselli, A. Pinchera, and G. Romeo. Identification of the Cys634 to Tyr mutation of the RET proto-oncogene in a pedigree with multiple endocrine neoplasia type 2A and localized cutaneous lichen amyloidosis. J. Endocrinol. Invest. 17:201–204, 1994. Black, M. M., and E. Wilson-Jones. Macular amyloidosis. Br. J. Dermatol. 84:199–209, 1971. Brownstein, M., and K. Hashimoto. Macular amyloidosis. Arch. Dermatol. 106:50–57, 1972. Brownstein, M. H., K. Hashimoto, and G. Greenwald. Biphasic amyloidosis: link between macular and lichenoid forms. Br. J. Dermatol. 88:25–29, 1973. Chabre, O., F. Labat, N. Pinel, F. Berthod, V. Tarel, and I. Bachelot. Cutaneous lesion associated with multiple endocrine neoplasia type 2A: lichen amyloidosis or notalgia paresthetica? Henry Ford Hosp. Med. J. 40:245–248, 1992. Chang, Y. T., S. F. Tsai, W. J. Wang, C. J. Hong, C. Y. Huang, and C. K. Wong. A study of apolipoproteins E and A-I in cutaneous amyloids. Br. J. Dermatol. 145:422–427, 2001. Donovan, D. T., M. L. Levy, E. J. Furst, B. R. Alford, T. Wheeler, J. A. Tschen, and R. F. Gagel. Familial cutaneous lichen amyloidosis in association with multiple endocrine neoplasia type 2A: a new variant. Henry Ford Hosp. Med. J. 37:147–150, 1989. Eanes, E. D., and G. G. Glenner. X-ray diffraction studies on amyloid filaments. J. Histochem. Cytochem. 16:673–677, 1968. Gagel, R. F., A. H. Tashjian Jr., T. Cummings, N. Papathanasopoulos, M. M. Kaplan, R. A. DeLellis, H. J. Wolfe, and S. Reichlin. The clinical outcome of prospective screening for multiple endocrine neoplasia type 2a: An 18 year experience. N. Engl. J. Med. 318:478–484, 1988.

Gagel, R. F., M. L. Levy, D. T. Donovan, B. R. Alford, T. Wheeler, and J. A. Tschen. Multiple endocrine neoplasia type 2a associated with cutaneous lichen amyloidosis [see comments]. Ann. Intern. Med. 111:802–806, 1989. Glenner, G. G. A retrospective and prospective overview of the investigations on amyloid and amyloidosis: The beta-fibrilloses. Amsterdam: Exerpta Medica, 1980. Goulden, V., A. S. Highet, and H. K. Shamy. Notalgia paraesthetica — report of an association with macular amyloidosis. Clin. Exp. Dermatol. 19:346–349, 1994. Hamzavi, I., and H. Lui. Excess tissue friability during CO2 laser vaporization of nodular amyloidosis. Dermatol. Surg. 25:726–728, 1999. Haneda, S., and T. Hamamatsu. Cutaneous amyloidosis associated with Sjögren’s syndrome. Hifu. Rinsho. 21:81–86, 1979. Hashimoto, K. Progress on cutaneous amyloid. J. Invest. Dermatol. 82:1, 1984. Hashimoto, K. Cutaneous amyloidoses. In: Clinical Dermatology. Hagerstown, Maryland: Williams and Wilkins, 1995, pp. 11–13. Hashimoto, K., and L. L. Yoong Onn. Lichen amyloidosus: Electron microscopic study of a typical case and a review. Arch. Dermatol. 104:648–667, 1971. Hashimoto, K., K. Ito, M. Kumakiri, and J. Headington. Nylon brush macular amyloidosis. Arch. Dermatol. 123:633–637, 1987. Helander, I., and V. K. Hopsu-Havu. Treatment of lichen amyloidosis by etretinate. Clin. Exp. Dermatol. 11:574–577, 1986. Hicks, B. C., P. J. Weber, K. Hashimoto, K. Ito, and D. M. Koreman. Primary cutaneous amyloidosis of the auricular concha. J. Am. Acad. Dermatol. 18:19–25, 1988. Ishigaki, M., T. Fugii, and M. Ohashi. Cutaneous amyloidosis with vitiligo. Jpn. J. Clin. Dermatol. (Tokyo) 31:513, 1977. Ito, K., K. Hashimoto, N. Kambe, and S. Van. Roles of immunoglobulins in amyloidogenesis in cutaneous nodular amyloidosis. J. Invest. Dermatol. 89:415–418, 1989. Iwasaki, K., M. Mihara, S. Nishiura, and S. Shimao. Biphasic amyloidosis arising from friction melanosis. J. Dermatol. 18:86–91, 1991. Kamakiri, M., K. Hashimoto, I. Tsukinaga, T. Kimura, and Y. Miura. Presence of basal lamina-like substance with anchoring fibrils within the amyloid deposits of primary localized cutaneous amyloidosis. J. Invest. Dermatol. 81:153–157, 1983. Kibbi, A. G., N. G. Rubeiz, S. T. Zaynoun, and A. K. Kurban. Primary localized cutaneous amyloidosis. Int. J. Dermatol. 31:95–98, 1992. Kousseff, B. G. Multiple endocrine neoplasia 2 (MEN 2)/MEN 2A (Sipple syndrome). Dermatol. Clin. 13:91–97, 1995. Kousseff, B. G., C. Espinoza, and G. A. Zamore. Sipple syndrome with lichen amyloidosis as a paracrinopathy: pleiotropy, heterogeneity, or a contiguous gene? J. Am. Acad. Dermatol. 25:651–657, 1991. Macsween, R. M., and E. M. Saihan. Nylon cloth macular amyloidosis. Clin. Exp. Dermatol. 22:28–29, 1997. Marschalko, M., J. Daroczy, and G. Soos. Etretinate for the treatment of lichen amyloidosis. Arch. Dermatol. 124:657–659, 1988. Nagao, S., and S. Iijima. Light and electron microscopic study of Riehl’s melanosis: Possible mode of its pigmentary incontinence. J. Cutan. Pathol. 1:165, 1974. Ortiz-Romero, P. L., C. Ballestin-Carcavilla, J. L. Lopez-Estebaranz, and L. Iglesias-Diez. Clinicopathologic and immunohistochemical studies on lichen amyloidosis and macular amyloidosis [letter]. Arch. Dermatol. 130:1559–1560, 1994. Pacini, F., L. Fugazzola, and G. Bevilacqua. Multiple endocrine neoplasia type 2A and cutaneous lichen amyloidosis: Description of a new family. Endocrine. Invest. 16:295–296, 1993. Paricha, J. S. et al. Low dose cyclophosphamide therapy in lichen amyloidosis. Indian J. Dermatol. Venereol. Leprol. 53:273–274, 1987.

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CHAPTER 50 Robinson, M. F., E. J. Furst, V. Nunziata, M. L. Brandi, J. P. Ferrer, M. J. Martins Bugalho, G. di Giovanni, R. J. Smith, D. T. Donovan, B. R. Alford, J. F. Hejtmancik, V. Colantuoni, L. Quadro, E. Limbert, I. Halperin, E. Vilardell, and R. F. Gagel. Characterization of the clinical features of five families with hereditary primary cutaneous lichen amyloidosis and multiple endocrine neoplasia type 2. Henry Ford Hosp. Med. J. 40:249–252, 1992. Sipple, J. H. The association of pheochromocytoma with carcinoma of thyroid gland. Am. J. Med. 31:163–166, 1961. Szinnai, G., C. Meier, P. Komminoth, U. W. Zumsteg. Review of multiple endocrine neoplasia type 2A in children: therapeutic results of early thyroidectomy and prognostic value of codon analysis. Pediatrics 111:E132–139, 2003. Tanigaki, T., S. Hata, and Y. Kitano. Epidemiological survey of “nylon clothes friction dermatosis” [Japanese]. Nippon Hifuka Gakkai Zasshi 95:1159–1164, 1985. Tidman, M. J., R. S. Wells, and D. M. MacDonald. Pachyonychia congenita with cutaneous amyloidosis and hyperpigmentation — a distinct variant. J. Am. Acad. Dermatol. 16:935–940, 1987. Vasily, D. B., S. G. Bhatia, and S. R. Uhlin. Familial primary cutaneous amyloidosis: Clinical, genetic and immunofluorescent studies. Arch. Dermatol. 114:1173–1176, 1978. Virchow, R. Cellular Pathology. New York: Dover Publications, 1971. Wong, C. K., and C. S. Lin. Friction amyloidosis. Int. J. Dermatol. 27:302–307, 1988. Wong, C.-K., D. Phil, and W.-M. Li. Dermabrasion for lichen amyloidosis. Arch. Dermatol. 118:302–304, 1982. Yanagihara, M. Ano-sacral cutaneous amyloidosis. Jpn. J. Dermatol. 91:463–468, 1981.

Dermatosis Papulosa Nigra Norman Levine and Cynthia Burk

Historical Background While visiting Jamaica and Central America in 1925, Aldo Castellani observed a rather common condition among the adult, indigenous population. He named this condition dermatosis papulosa nigra. A collaborative report with Duval (Castellani and Duval, 1929) established the basic clinical and pathologic findings.

Epidemiology Dermatosis papulosa nigra is seen almost exclusively in the Negroid race (Costa, 1944; Mascaro, 1963; Michael and Seale, 1930, 1933). Occasional cases among Mexicans, Filipinos, Vietnamese, and Europeans have been described (Grimes, 1983).

Clinical Findings The characteristic presentation is one of light-brown to black, well-circumscribed, smooth and rounded (85%), pedunculated (15%), or filiform (rare) papules measuring 0.1–0.5 cm. These papules are most densely located on the central and lateral cheeks with gradual fanning toward the forehead and neck (Fig. 50.18). Rarely, the upper torso is involved. Prepubertal onset is rare, with only one case reported in a 7-yearold (Babapour et al., 1993). There is a gradual increase in size and number of papules, peaking during the sixth decade. Light-skinned black people have a lower prevalence of involvement than those with darker skin. There is a female 928

Fig. 50.18. Small pigmented lesions on the temporal areas.

predominance approaching 2 to 1. Females also tend to have greater numbers of papules. The incidence among black people ranges from 35% (Hairston, 1964) up to 77% (Grimes, 1983). The papules are generally asymptomatic unless traumatized, and itching is seen in only 5% of cases. There appears to be a hereditary basis for this condition; approximately one half of affected individuals report at least one family member with this problem. There are no associated medical conditions.

Pathology Histopathologic examination of the early lesion shows acanthosis with broadening and downward projection of the rete pegs. There are increased numbers of mitotic figures and more pigment in the basal cell. There may be mild spongiosis. As a papule matures, the acanthotic center becomes more pronounced with fusing of the rete pegs. Intraepidermal horn cysts are common (Michael and Seale, 1929). Melanophages are present in the upper dermis. Mild papillary dermal edema and lymphohistiocytic infiltrate may be present, along with immature pilosebaceous structures in the involved and adjacent dermis. The histologic picture is much like that of an acanthotic seborrheic keratosis (Lever and Schaumburg-Lever, 1983).

Differential Diagnosis The differential diagnosis includes other pigmented papules. Pigmented nevi are less numerous, are smoother, and have a distinctive histopathology. Multiple minute digitate hyperkeratoses are not seen on the face (Balus et al., 1988). Adenoma sebaceum in the black may mimic dermatosis papulosa nigra clinically; only by pathologic examination can they be absolutely identified. Verrucae planae are usually less pigmented and often show signs of the isomorphic (Koebner) phenomenon. Histopathology is distinctive. Basal cell nevus syndrome may present with small pigmented papules but the histopathology is diagnostic. Fibroepitheliomas (skin tags) are common on the eyelids and periorbital areas but tend to be more pedunculated, and have much less acanthosis and hyalinization on histologic examination. The differential diagnosis also includes melanocytic nevi, follicular hamartomas, and

ACQUIRED EPIDERMAL HYPERMELANOSES

seborrheic keratoses. Typically, follicular hamartomas have a central umbilication that is not present in dermatosis papulosa nigra (Babapour et al., 1993).

Pathogenesis Although the pathogenesis of dermatosis papulosa nigra is unproved, there appears to be a genetic basis. The appearance at puberty with slow progression would suggest a hormonal effect on the pilosebaceous apparatus.

Treatment The lesions of dermatosis papulosa nigra are benign and generally do not psychologically or socially affect involved individuals. Some consider the papules to be attractive and a form of beauty mark. Various destructive modalities of removal have been reported to be effective and safe (Kenney, 1988). This would include “light abrasive curettage” where the papule is gently abraded with a curette using no anesthesia, allowing the remaining portion of the papule to involute spontaneously (Kauh, 1983). Light electrodesiccation, cryosurgery, shave excision, and chemical cautery can also be used. The carbon dioxide laser in the ultrapulsed mode may also be used to remove these lesions.

References Babapour, R., J. Leach, and H. Levy. Dermatosis papulosa nigra in a young child. Pediatr. Dermatol. 10:356–358, 1993. Balus, L., P. Donati, A. Amantea, and A. S. Breathnach. Multiple minute digitate hyperkeratosis. J. Am. Acad. Dermatol. 18:431– 436, 1988. Castellani, A. Observations on some diseases of Central America. J. Trop. Med. Hygiene 28:149–150, 1925. Castellani, A., and C. W. Duval. Dermatosis papulosa nigra. J. Trop. Med. 32:149–150, 1929. Costa, O. G. Dermatosis papulosa nigra. An. Brasil Dermatol. Sifil. 19:217–219, 1944. Grimes, P. E. Dermatosis papulosa nigra. Cutis 32:385–386, 1983. Hairston, M. A. Dermatosis papulosa nigra. Arch. Dermatol. 89:655– 658, 1964. Kauh, Y. C. A surgical approach for dermatosis papulosa nigra. Int. J. Dermatol. 22:590–592, 1983. Kenney, J. A., Jr. Dermatosis papulosa nigra and seborrheic keratosis. Ala. J. Med. Soc. 17:49–50, 1988. Lever, W. F., and G. Schaumburg-Lever. Histopathology of the Skin, 6th ed. Philadelphia: JB Lippincott Co., 1983. Mascaro, J. M. Castellani’s dermatosis papulosa nigra; historical note. Bull. Soc. Franc. Dermatol. Syphiligr. 70:535–537, 1963. Michael, J. C., and E. R. Seale. Dermatosis papulosa nigra. Arch. Dermatol. Syphil. 20:629–640, 1929. Michael, J. C., and E. R. Seale. Dermatosis papulosa nigra. J. Trop. Med. 33:12–15, 1930. Michael, J. C., and E. R. Seale. Dermatosis papulosa nigra. Arch. Dermatol. Syphil. 20:629–640, 1933.

Ephelides (Freckles) Norman Levine and Cynthia Burk

Introduction Ephelides (freckles) are small tan to dark-brown macules, seen mainly in children, on sun-exposed skin. Because the

Fig. 50.19. Ephelides on sun-exposed areas.

term freckle is sometimes used interchangeably with the term lentigo, there is often considerable confusion within the medical literature about what is actually being described (Jansson, 1958).

Clinical Findings Freckles are genetic in origin, following an autosomal dominant pattern (Jansson, 1958); they are especially common among persons of Celtic (Scottish–Irish–Welsh) ancestry. Freckles are seen only in sun-exposed areas, being most heavily concentrated on the face, upper back, and dorsal forearms (Frederich, 1959; Goldberg and Altman, 1984) (Fig. 50.19). Onset may be at any time but is often early in childhood when outdoor swimming begins. A study of 1456 New South Wales school children revealed an increasing number of freckles with increasing age, suggesting that continued sun exposure increases the degree of freckling (Nicholls, 1968). Boys and girls are equally affected. Redheads are most likely to have freckles (Brues, 1950). Most freckles are small macules, measuring 2–4 mm, but may be larger. Experimental evidence reveals the MFD (minimal freckle dose) to be 6 MED (minimal erythema dose) (Wilson and Kligman, 1982) and may appear after a single sunburn. Freckles help to protect the skin from further ultraviolet (UV) damage (Ito, 1950), and may be considered a sign of photoaging (Holzle, 1992). Freckles are often dynamic lesions. Most sunburn freckles appear after the skin has peeled, normally, within two to four weeks after the sunburn. Photochemotherapy in fair-skinned individuals may lead to frecklelike lesions but they are much more like lentigines, based on histopathology (Kanerva et al., 1984). Melanocytes exposed to radioactive thorium-X solution may lose their capacity to freckle (Breathnach, 1982). Excessive plasma and tissue levels of chloroquine due to renal failure may lead to lightening of the hair and to freckles (Dupre et al., 1985). Ephelides may appear in stretched scars (Breathnach, 1958). Freckles are of clinical significance because of their association with both melanoma (Bataille et al., 2000; Elwood et 929

CHAPTER 50

al., 1985; Evans et al., 1988; Marks, 1989; Scotto and Fears 1987) and nonmelanoma skin cancer (Bastiaens et al., 2001; Hogan et al., 1989, 1990). Recent studies indicate that having freckles is a moderate risk factor for developing cutaneous melanoma (Bakos et al., 2002; Grulich et al., 1996). Freckling is also correlated with increased numbers of nevocellular nevi (Bataille et al., 2000; Coombs et al., 1992; Fritschi et al., 1994; Sigg and Pelloni 1989). Ephelides, malignant melanoma, and nonmelanoma skin cancer are all strongly associated with the MC1R gene which plays an important role in skin pigmentation (Bastiaens et al., 2001). Freckles are seen in individuals with xeroderma pigmentosum. Axillary and intertriginous “frecklelike” macules are important markers in neurofibromatosis, but are not true freckles since they are not induced by ultraviolet light and do not fade. Freckles may be present in Gardner syndrome (Bazex and Bazex, 1978) or the NAME syndrome (Nevi, Atrial myxoma, Myxoid neurofibroma, and Ephelides) (Rolle et al., 1995). Patients with tyrosinase-positive albinism may have freckling due to accumulation of pigment over time (Findlay, 1962). Freckles and abnormalities of the eyebrows may serve as indicators for genetic abnormalities in the development of the teeth and jaws (Ulrich, 1990). Freckles may be related to a change that can occur in pheomelanin exposed to ultraviolet light. This may explain the predominance of freckling among redheads and blondes.

Pathology Light microscopy reveals a normal stratum corneum and Malpighian layer. The basal layer shows increased pigmentation without elongation of the rete pegs. The dermis is normal (Lever and Schaumburg-Lever, 1983). Large numbers of mature melanosomes and dendritic melanocytes as seen in dark-skinned individuals are characteristic (Breathnach, 1957). There seem to be an increased number of melanocytes in freckles compared to adjacent nonpigmented skin (Rhodes et al., 1991). Ultrastructural studies reveal elongated, fully melanized melanosomes with 90 Å (9 nm) striations (Breathnach and Wyllie, 1964).

Differential Diagnosis The clinical diagnosis of freckles is routine when occurring in a fair-skinned, red-headed child and limited to sun-exposed skin. Solar lentigines are the main point of confusion. These lesions tend to occur in older individuals, have a greater spectrum of shape, size, and color, and histologically show elongation of the rete pegs, increased numbers of melanocytes with some degree of cellular atypia, and dermal melanophages. Solar lentigines are slow to fade without therapy.

Treatment Treatment of freckles is for cosmetic reasons only. Application of hydroquinone 2–4% or combination glycolic acid/kojic acid lotion along with maximum UVA blocking sunscreen in the morning and retinoic acid (Goldfarb et al., 1990) in the 930

evening will significantly lighten freckles. Sun avoidance and the use of protective clothing are helpful in speeding the response to treatment. Phenol peeling removes freckles but runs the risk of toxicity and permanent hypopigmentation (Schuhmachers-Brendler, 1955; Zhao, 1992). Skillful use of liquid nitrogen (Kleine-Natrop, 1964) and dermabrasion is effective. Pigment lasers such as the Q-switched alexandrite laser, Q-switched 532 nm neodymium:yttrium aluminum garnet (Nd:YAG), Q-switched ruby (Nelson and Applebaum, 1992) offer an aggressive and effective way to remove freckles (Jang et al., 2000; Suh et al., 2001). Combination therapy of pigmented lasers and chemical peels may be useful in removing freckles (Lee et al., 2002). Intense pulsed light is another possible modality for the treatment of facial freckles (Huang et al., 2002; Kawada et al., 2002).

References Bakos, L. M., Wagner, R. M. Bakos, C. S. Leite, C. L. Sperhacke, K. S. Dzekaniak, and A. L. Gleisner. Sunburn, sunscreens, and phenotypes: some risk factors for cutaneous melanoma in southern Brazil. Int. J. Dermatol. 41:557–562, 2002. Bastiaens, M., J. ter Huurne, N. Gruis, Bergman W, R. Westendorp, B. J. Vermeer, and J. N. Bouwes Bavinck. The melanocortin-1-receptor gene is the major freckle gene. Hum. Mol. Genet. 10:1701– 1708, 2001. Bataille, V., H. Snieder, A. J. MacGregor, P. Sasieni, and T. D. Spector. Genetics of risk factors for melanoma: an adult twin study of nevi and freckles. J. Natl. Cancer Inst. 92:457–463, 2000. Bazex, A., and J. Bazex. Dermatology and polyposis of the gastrointestinal tract. Ann. Dermatol. Venereol. 105:851–858, 1978. Breathnach, A. S. Melanocytic distribution in forearm epidermis of freckled human subjects. J. Invest. Dermatol. 29:253–261, 1957. Breathnach, A. S. Melanocytic pattern of an area of freckled epidermis covering a stretched scar. J. Invest. Dermatol. 31:237–241, 1958. Breathnach, A. S. A long-term hypopigmentation effect of thorium-X on freckled skin. Br. J. Dermatol. 106:19–25, 1982. Breathnach, A. S., and L. M. Wyllie. Electron microscopy of melanocytes and melanosomes in freckled human epidermis. J. Invest. Dermatol. 42:389–394, 1964. Brues, A. M. Linkage of bodybuild with sex, eye colour, and freckling. Am. J. Hum. Genet. 2:215, 1950. Coombs, B. D., K. J. Sharples, K. R. Cooke, D. C. Skegg, and J. M. Elwood. Variation and covariates of the number of benign nevi in adolescents. Am. J. Epidemiol. 136:344–355, 1992. Crowe, F. W. Axillary freckling as a diagnostic aid in neurofibromatosis. Ann. Intern. Med. 61:1142–1143, 1964. Dupre, A., J. P. Ortonne, P. Viraben, and F. Arfeux. Chloroquineinduced hyperpigmentation of hair and freckles, associated with congenital renal failure. Arch. Dermatol. 121:1164–1166, 1985. Elwood, J. M., R. P. Gallagher, and G. P. Hill. Pigmentation and skin reaction to sun as risk factor for cutaneous melanoma: Western Canada Study. Br. Med. J. 288:99–102, 1984. Elwood, J. M., R. P. Gallagher, J. Davidson, and G. B. Hill. Sunburn, suntan and the risk of cutaneous malignant melanoma. Br. J. Cancer 51:543–549, 1985. Evans, R. D., A. W. Kopf, R. A. Lew, D. S. Rigel, R. S. Bart, R. J. Friedman, and J. K. Rivers. Risk factors for the development of malignant melanoma: Review of case-control studies. J. Dermatol. Surg. Oncol. 14:393–408, 1988. Findlay, G. H. On giant dendritic freckles and melanocytic reactions in the skin of the albino Bantu. S. Afr. J. Lab. Clin. Med. 8:68–72, 1962.

ACQUIRED EPIDERMAL HYPERMELANOSES Frederich, H. Beitrag zur Behandlung von Epheliden und Virginum. Medizinische 16:800–801, 1959. Fritschi, L. P., A. McHenry, R. M. Green, L. Green, and V. Siskind. Nevi in schoolchildren in Scotland and Australia. Br. J. Dermatol. 130:599–603, 1994. Goldberg, L. H., and A. Altman. Benign skin changes associated with chronic sunlight exposure. Cutis 34:33–38, 1984. Goldfarb, M. T., C. N. Ellis, and J. J. Voorhees. Topical tretinoin — its use in daily practice to reverse photoaging. Br. J. Dermatol. 122(Suppl. 35):87–91, 1990. Grulich, A. E., V. Bataille, A. J. Swerdlow, J. A. Newton-Bishop, J. Cuzick, P. Hersey, and W. H. McCarthy. Naevi and pigmentary characteristics as risk factors for melanoma in a high-risk population: a case-control study in New South Wales, Australia. Int. J. Cancer 67:485–491, 1996. Hogan, D. J., P. R. Lane, L. Gran, and L. Wong. Risk factors for squamous cell carcinoma of the skin in Saskatchewan, Canada. J. Dermatol. Sci. 1:97–101, 1990. Hogan, O. J., T. To, L. Gran, D. Wong, and P. R. Lane. Risk factors for basal carcinoma. Int. J. Dermatol. 28:591–594, 1989. Holzle, E. Pigmented lesions as a sign of photodamage. Br. J. Dermatol. 127 Suppl 41:48–50, 1992. Huang, Y. L., Y. L. Liao, S. H. Lee, and H. S. Hong. Intense pulsed light for the treatment of facial freckles in Asian skin. Dermatol. Surg. 28:1007–12; discussion p. 1012, 2002. Ito, M. Genetic studies on skin diseases: ephelides, dyschromatosis symmetrica hereditaria and xeroderma pigmentosum. Tohoku J. Exp. Med. 53:69–72, 1950. Jang, K. A., E. C. Chung, et al. Successful removal of freckles in Asian skin with a Q-switched alexandrite laser. Dermatol. Surg. 26:231–234, 2000. Jansson, H. Problems of differentiation of ephelides and lentigines based on analysis of frequency. Hautarzt 7:311–313, 1958. Kanerva, L., K. M. Niemi, and J. Lauharanta. A semiquantitative light and electron microscopic analysis of histopathologic changes in phototherapy-induced freckles. Arch. Dermatol. Res. 276:2–11, 1984. Kawada, A., H. Shiraishi, M. Asai, H. Kameyama, Y. Sangen, Y. Aragane, and T. Tezuka. Clinical improvement of solar lentigines and ephelides with an intense pulsed light source. Dermatol. Surg. 28:504–508, 2002. Kleine-Natrop, H. E. Nitrogen peeling in freckles and skin infection. Hautarzt 15:557–559, 1964. Lee, G. Y., H. J. Kim, and K. K. Wang. The effect of combination treatment of the recalcitrant pigmentary disorders with pigmented laser and chemical peeling. Dermatol. Surg. 28:1120–1123; discussion 1123, 2002. Lever, W. F., and G. Schaumburg-Lever. Histopathology of the Skin, 6th ed. Philadelphia: JB Lippincott Co., 1983. Marks, J. Freckles, moles, melanoma and the ozone layer: a tale of the relationship between humans and their environment. Med. J. Aust. 151:611–613, 1989. Marks, R. Freckles, moles, and melanoma. Aust. Fam. Physician 17:1025, 1988. Nelson, J. S., and J. Applebaum. Treatment of superficial cutaneous pigmented lesions by melanin-specific selective photothermolysis using the Q-switched ruby laser. Ann. Plast. Surg. 29:231–237, 1992. Nicholls, E. M. Genetic susceptibility and somatic mutation in the production of freckles, birthmarks and moles. Lancet 1:71–73, 1968. Rhodes, A. R., L. S. Albert, R. L. Barnhill, and M. A. Weinstock. Suninduced freckles in children and young adults. A correlation of clinical and histopathologic features. Cancer 67:1990–2001, 1991. Rolle, F., E. Cornu, Y. Ghossein, J. M. Bonnetblanc, J. Bensaid, and C. Christides. Cutaneous lentigines, freckles, and atrial myxomas [letter]. Ann. Thorac. Surg. 59:267–268, 1995.

Schuhmachers-Brendler, R. Corrective dermatology: therapy of freckles with phenol ether. Hautarzt 6:499–501, 1955. Scotto, J., and T. R. Fears. The association of solar ultraviolet and skin melanoma incidence among caucasians in the United States. Cancer Invest. 5:275–283, 1987. Sigg, C., and F. Pelloni. Frequency of acquired melanonevocytic nevi and their relationship to skin complexion in 939 schoolchildren. Dermatologica 179:123–128, 1989. Suh, D. H., K. H. Han, and J. H. Chung. The use of Q-switched Nd:YAG laser in the treatment of superficial pigmented lesions in Koreans. J. Dermatol. Treat. 12:91–96, 2001. Ulrich, K. Freckles and dysplasias of the eyebrows as indicators for genetic abnormalities of the development of the teeth and jaws. Stomatol. DDR 40:64–66, 1990. Wilson, P. D., and A. M. Kligman. Experimental induction of freckles by ultraviolet-B. Br. J. Dermatol. 106:401–406, 1982. Zhao, Q. M. [Clinical observation of face peeling treatment for ephelides and phenol excretion in urine]. Chung Hua Cheng Hsing Shao Shang Wai Ko Tsa Chih 8:179–181,247, 1992.

Erythema ab Igne Norman Levine and Cynthia Burk

Synonyms Erythema a colore (Bazex et al., 1963), ephelis ignealis, ephelis ab igne (Abraham, 1902), erythema caloricum, livedo reticularis e calore, Buscke heat melanosis, and toasted skin syndrome.

Clinical Findings Erythema ab igne is a reticulated dermatosis, which is a reaction to chronic, nonburning thermal exposure in the range of 43–47 ∞C. Skin lesions may not appear for three weeks after the heat exposure (Lin et al., 2002). The typical case of erythema ab igne occurs in a person who has repeatedly warmed bare legs in front of an open fireplace, radiator, or heater. Another common setting is the young girl with menstrual cramps who uses a heating pad on the abdomen. Other cases occur in cooks, stokers, blacksmiths, silversmiths, and foundrymen. Anyone with chronic pain, such as low back pain, that uses a heating device may develop erythema ab igne (Dvoretzky and Silverman, 1991). Cases of erythema ab igne have also been reported in individuals who take frequent hot baths, who use heating blankets or hot water bottles, who are in close contact with car heaters, and who use furniture/therapeutic chairs with built-in heating devices (Helm et al., 1997; Lin et al., 2002; Meffert and Davis 1996; Milligan and Graham-Brown 1989). Erythema ab igne may be linked to cancer-related pain secondary to the application of heat for pain relief (MacHale et al., 2000). In each case the skin and subdermal vascular plexus reacts with vasodilatation and hemostasis, forming a reticulated pattern that begins with erythema but in time becomes hyperpigmented (Fig. 50.20). In very chronic cases, the skin develops poikiloderma with epidermal atrophy, telangiectasia, and hyperkeratosis. The pattern on the legs can resemble that of excessive solar damage. There may be some itching or burning; however, most cases are asymptomatic. 931

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Fig. 50.20. Extensive involvement of the legs.

Fig. 50.21. Typical reticulated pattern of hyperpigmentation induced by erythema ab igne (see also Plate 50.8, pp. 494–495).

Associated Disorders Erythema ab igne is associated with a slight increase in malignancy when the etiology is due to a hydrocarbon heat source (Lin et al., 2002). Actinic keratoses and squamous cell carcinomas have been reported in a number of cases after a long latent period (Arrington and Lockman 1979; Ashby 1985; Helm et al., 1997). These include cases from the chronic heat of a sunken hearth (irori), and underfloor braziers covered with a quilt (kotatsu). Other cases include those from heated brick beds in northern China (kang cancers) (Laycock, 1948), coal-burning baskets in Kashmir in India (Kangri cancers) (Neve, 1923; Suryanarayan, 1973), peat fire cancers in Ireland (Cross, 1967), and benzene-burning pots in Japan (kairo cancers) (Kligman and Kligman, 1984). There is evidence of a destructive synergism between ultraviolet and infrared radiation (Kligman, 1982). A few cases of coexisting squamous cell carcinoma and Merkel cell carcinoma in an area of erythema ab igne have been reported (Jones et al., 1988; Iacocca et al., 1998). Abdominal erythema ab igne is increasingly being recognized as an important sign in chronic abdominal pain, especially from pancreatic disease (Ashby et al., 1985; Halliday et al., 1986; Mok and Blumgart 1984; Mucklow and Freeman, 1990; Raffle 1984). Erythema ab igne on the flank, back, or the upper abdomen (Fig. 50.21) may be the only cutaneous manifestation of splenomegaly, pancreatitis, peptic ulcer disease, pancreatic pseudocyst or pancreatic cancer (Butler, 1977; Tan and Bertucci, 2000). Other unusual sites such as the thigh, pubic area, and upper back suggest underlying malignancy such as gastric and renal carcinoma as well as bony metastases (Butler, 1977). A few cases of bullous lichen planus arising in an area of erythema ab igne have been reported (Horio and Imamura 1986; Flanagan et al., 1996). In addition, lymphedema has been associated with a hypertrophic or keloidal variant of erythema ab igne (Cox et al., 1996).

Pathology Light microscopic examination of early erythema ab igne shows atrophy of the Malpighian layer, increased epidermal and upper dermal melanin with dermal vasodilatation. 932

Advanced cases show epidermal vacuolation (Wilkinson, 1972), focal hyperkeratosis, and dyskeratosis. There is increased elastosis with fragmented collagen fibers (Johnson and Butterworth, 1971). Epidermal dysplasia within abnormal elastic tissue may also be seen (Cox et al., 1996). There is minimal basophilia and homogenization of elastic fibers which is prominent in solar elastosis (Shahrad and Marks, 1977). Melanophages and hemosiderin are seen in the dermis (Findlayson et al., 1966; Hartzell, 1912). Varying degrees of dermal lymphohistiocytic infiltration are present. Extravasation of red blood cells is inconstant.

Differential Diagnosis Livedo reticularis has a reticulated pattern with erythema but shows no hyperpigmentation. Poikiloderma atrophicans vasculare is more atrophic and telangiectatic and does not show the distinct vascular pattern of erythema ab igne. Majocchi disease tends to be more annular and superficial.

Treatment In early cases there is complete resolution by removal of the offending heating device. More advanced cases may respond somewhat to tretinoin and/or 5-fluorouracil cream (Helm et al., 1997; Sahl and Taira 1992). Associated cutaneous malignancies require surgical removal.

Prognosis Prognosis is good except those cases associated with internal disease or metastatic malignancy.

References Abraham, P. S. Ephelis ab igne. Br. Med. J. 14:100, 1902. Akasaka, T., and S. Kon. Two cases of squamous cell carcinoma arising from erythema ab igne. Nippon. Hifuka. Gakkai. Zasshi. Jpn. J. Dermatol. 99:735–742, 1989. Arrington, J. H., III, and D. S. Lockman. Thermal keratosis and squamous cell carcinoma in situ associated with erythema ab igne. Arch. Dermatol. 115:1226–1228, 1979. Ashby, M. Erythema ab igne in cancer patients. J. R. Soc. Med. 78:925–927, 1985.

ACQUIRED EPIDERMAL HYPERMELANOSES Ashby, M. A., P. Carmochan, and D. M. Tait. Erythema ab igne: a model of hyperthermic skin damage and carcinogenesis in humans? Int. J. Hypertherm. 1:391–393, 1985. Bazex, A. R., R. Salvador, A. Dupre, M. Parant, and B. Christol. Erytheme a calore. Bull. Soc. Franc. Dermatol. Syphiligr. 70:296, 1963. Cox, N. H., W. D. Paterson, and A. W. Popple. A reticulate vascular abnormality in patients with lymphoedema: observations in eight patients. Br. J. Dermatol. 135:92–97, 1996. Cross, F. On a turf (peat) fire cancer: malignant change superimposed on erythema ab igne. Proc. R. Soc. Med. 60:1307–1308, 1967. Dvoretzky, I., and N. R. Silverman. Reticular erythema of the lower back. Erythema ab igne. Arch. Dermatol. 127:405–406,408–409, 1991. Findlayson, G. R., W. M. Sams Jr., and J. G. Smith. Erythema ab igne — a histopathological study. J. Invest. Dermatol. 46:104–107, 1966. Flanagan, N., R. Watson, E. Sweeney, and L. Barnes. Bullous erythema ab igne. Br. J. Dermatol. 134:1159–1160, 1996. Halliday, C. E., A. K. Goka, and M. J. Farthing. Erythema ab igne: a sign of organic disease [letter]. J. R. Soc. Med. 79:249–250, 1986. Hartzell, M. B. Erythema ab igne. J. Cutan. Dis. 30:461, 1912. Helm, T. N., G. T. Spigel, and K. F. Helm. Erythema ab igne caused by a car heater. Cutis 59:81–82, 1997. Horio, T., and S. Imamura. Bullous lichen planus developed on erythema ab igne. J. Dermatol. 13:203–207, 1986. Iacocca, M. V., J. L. Abernethy, C. M. Stefanato, A. E. Allan, J. Bhawan. Mixed Merkel cell carcinoma and squamous cell carcinoma of the skin. J. Am. Acad. Dermatol. 39(5 Pt 2): 882–887, 1998. Johnson, W. C., and T. Butterworth. Erythema ab igne elastosis. Arch. Dermatol. 104:128–131, 1971. Jones, C. S., S. K. Tyring, P. C. Lee, and J. D. Fine. Development of neuroendocrine (Merkel cell) carcinoma mixed with squamous cell carcinoma in erythema ab igne. Arch. Dermatol. 124:110–113, 1988. Kligman, L. H. Intensification of ultraviolet-induced dermal damage by infrared radiation. Arch. Dermatol. Res. 272:229–238, 1982. Kligman, L. H., and A. M. Kligman. Reflections on heat. Br. J. Dermatol. 110:369–375, 1984. Lankisch, P. G., and W. Creutzfeldt. Erythema ab igne (livido reticularis e calorie): a skin manifestation of chronic pancreatic disease. Z. Gastroenterol. 24:119–120, 1986. Laycock, H. T. Kang Cancer. Br. Med. J. 1:982, 1948. Lin, S. J., C. J. Hsu, and H. C. Chiu. Erythema ab igne caused by frequent hot bathing. Acta Derm.Venereol. 82:478–479, 2002. MacHale, J., F. Chambers, and P. R. O’Connell. Erythema ab igne: an unusual manifestation of cancer-related pain. Pain 87:107–108, 2000. Meffert, J. J., and B. M. Davis. Furniture-induced erythema ab igne. J. Am. Acad. Dermatol. 34:516–517, 1996. Milligan, A., and R. A. Graham-Brown. Erythema ab igne affecting the palms. Clin. Exp. Dermatol. 14:168–169, 1989. Mok, D. W. H., and L. H. Blumgart. Erythema ab igne in chronic pancreatic pain: a diagnostic sign. J. R. Soc. Med. 77:299–301, 1984. Mucklow, E. S., and N. V. Freeman. Pancreatic ascites in childhood. Br. J. Clin. Pract. 44:248–251, 1990. Neve, E. F. Kangri-burn cancer. Br. Med. J. 2:1255–1256, 1923. Peterkin, G. A. G. Malignant change in erythema ab igne. Br. Med. J. 2:1599–1602, 1955. Raffle, E. J. Erythema ab igne in chronic pancreatic pain. J. R. Soc. Med. 77:706, 1984. Roth, D., and M. London. Acridine probe study into synergistic DNA denaturing action of heat and ultraviolet light in squamous cells. J. Invest. Dermatol. 69:368–372, 1977. Sahl, W. J., Jr., and J. W. Taira. Erythema ab igne: treatment with 5fluorouracil cream. J. Am. Acad. Dermatol. 27:109–110, 1992.

Shahrad, P., and R. Marks. The wages of warmth: changes in erythema ab igne. Br. J. Dermatol. 97:163–172, 1977. Suryanarayan, C. R. Kangri cancer in Kashmir Valley. J. Surg. Oncol. 5:327–333, 1973. Tan, S., and V. Bertucci. Erythema ab igne: an old condition new again. Can. Med. Assoc. J. 162:77–78, 2000. Wilkinson, D. S. Cutaneous reactions to mechanical and thermal injury. In: Textbook of Dermatology, 2nd ed., A. Rook, D. S. Wilkinson, and F. J. G. Ebling (eds). Oxford: Blackwell Scientific Publications, 1972, pp. 435–436.

Erythema Dyschromicum Perstans Norman Levine and Cynthia Burk

Historical Background In 1957 Ramirez described a group of patients which he called Los Cenicientos (ashen ones). He called the disease dermatosis cenicienta, a reference to Cinderella and her ashen smudges (Knox et al., 1968). Convit et al. suggested the name erythema chromicum figuratum melanodermicum, but in 1961 adopted the term erythema dyschromicum perstans (Convit et al., 1961a, b). Other cases which could possibly be erythema dyschromicum perstans include lichen atypiques on invisibles pigmentogenes (Gougerot, 1935a, b), roseola pigmentosa, pigmentation maculosa multiplex (Ito, 1955), and lichen pigmentosum in Japan, la melanodermite lichenoidea in Ethiopia (Greppi and Soustek, 1966), and lichen planus pigmentosus in India (Bhutani et al., 1974). The term ashy dermatosis of Ramirez is used in English-speaking countries.

Epidemiology Erythema dyschromicum perstans is most commonly seen in persons of intermediate skin color. Most reports come from Central America (Ramirez, 1966, 1967) but cases have been reported worldwide (Koves de Amini and Briceno-Maaz, 1967) and represent all skin types, even fair-skinned Caucasians (Byrne and Berger, 1974; Holst and Mobachen, 1974; Peachy, 1976). There is no gender preference; although most cases are seen in young adults, children as young as 5 years old have been reported (Urano-Suehissa et al., 1984).

Clinical Findings One sees variably sized ashy hyperpigmented patches primarily located on the torso, extremities, and/or face. There is no involvement of the palms, soles, scalp, nails, or mucosa. New cases may have a leading, erythematous edge, but this is normally absent soon afterwards (Convit et al., 1961b). Lesions may vary in color from slate-gray to blue-brown. Peripheral hypopigmentation is often seen in older lesions, which tends to accentuate the inner hyperpigmentation (Fig. 50.22). Wood’s light examination shows no accentuation of the hyperpigmentation. This is to be expected since the pigment is primarily dermal in origin. Individual lesions may be annular or polycyclic with overlapping borders. New lesions may appear in areas which have previously resolved. Oval lesions may follow skin tension lines. Up to 90% of the body may be involved in exceptional cases. There may be mild pruritus but 933

CHAPTER 50

Differential Diagnosis

Fig. 50.22. Blue-brown hyperpigmented macules at the late stage of erythema dyschromicum perstans (see also Plate 50.9, pp. 494–495).

typically the condition is asymptomatic. A unilateral case in a child with positive rheumatoid factor has been reported (Urano-Suehissa et al., 1984). Erythema dyschromicum perstans may occur simultaneously in patients with vitiligo (Osswald et al., 2001). Onset may be abrupt but there is slow progression with less than a 10% chance of spontaneous remission (Palatsi, 1977). General health is not affected and routine laboratory studies are normal.

Pathology Routine histology shows both early and late changes. Early, active lesions show a normal stratum corneum, slightly atrophic epidermis, varying degrees of spongiosis and lymphocytic exocytosis, basal vacuolar degeneration (Ackerman, 1978), and dermal lymphohistiocytic perivascular and/or lichenoid infiltrate. Colloid bodies, which represent degenerated extruded keratinocytes, are frequently seen in the dermis. Dermal hemosiderin may be present. Both increased epidermal melanin and dermal melanophages (melaninosis) (Pathak et al., 1983) are seen. Older lesions lack basal degeneration and a dense dermal infiltrate while dermal pigmentation increases. Split-dopa staining reveals increased numbers of epidermal melanocytes with an arborizing, dendritic proliferation and epidermal pigmentation. Contrarily, S-100 staining reveals the dermal pigmentation to be within melanophages. This dermal pigmentation produces the characteristic blue or ashy-gray color because of scattering and re-emission of blue light. Electron microscopy reveals cytoplasmic vacuoles and melanosome complexes within basal keratinocytes. Desmosomes are retracted. There is no evidence of basement membrane reduplication (Soter et al., 1969). Immunofluorescence studies show variable staining of dermal colloid bodies with IgG, IgM, IgA, C3, and C4 (Kark and Litt, 1980; Novick and Phelps, 1985; Tschen et al., 1980). There may be occasional IgG immunoglobulin staining of the basal and prickle cell layer. All immunofluorescence studies may be negative. 934

The differential diagnosis includes fixed-drug reactions, which may mimic erythema dyschromicum perstans quite convincingly. Histology may be similar but a careful history is diagnostic. The pigmentation of melasma is frequently enhanced by Wood’s light examination. Poikiloderma of Civatte shows significant telangiectasia. A number of clinically similar cases in New Guinea (Bereston, 1946; Wilson, 1946) proved to be due to Triquin, which is a combination of quinacrine, chloroquine, and hydroxychloroquine. Postinflammatory hyperpigmentation from pityriasis rosea has a different clinical course and histology, and resolves in a few weeks. Urticaria pigmentosa has a positive Darier sign. Late pinta has a positive darkfield examination, antitreponemal serology, and responds to penicillin therapy. Maculae cerulae is associated with pediculosis. Macular amyloid can be differentiated by histology. Argyria is more diffuse and histology is diagnostic. Riehl melanosis is associated with irritant and sensitizing topical agents, and lichen planus subtropicus has a distinct photoaccentuated pattern. There have been several cases of lichen planus in conjunction with erythema dyschromicum perstans; there are those that contend that erythema dyschromicum perstans is a subset of lichen planus rather than a form of fixed erythema. Several authors have debated this issue in detail (Berger et al., 1989; Kark and Litt 1980; Naidorf and Cohen 1982; Person and Rogers 1981). There has also been an association with erythema dyschromicum perstans and lichen planopilaris (Metin et al., 2001).

Pathogenesis The etiology of erythema dyschromicum perstans is unknown. However, there is evidence that the immune system plays a crucial role in the development of this condition. The first case from the United States seemed to resolve after treatment of the whipworm Trichuris trichiura with dithiazanine (Stevenson and Miura, 1966). Contact with optical whitener in washing powders has been suspected, but never substantiated (Pinol, 1971). Some have suggested that paraphenylenediamine hair dyes might trigger the condition (Bhutani, 1986). There has been one case with an association with licking ammonium nitrate fertilizer (Jablonska, 1975). Erythema dyschromicum perstans has also been associated with endocrinopathies, Xray contrast studies, and been linked to the hepatitis C virus (Goihman, 1998). Cell adhesion and activation molecules may be involved in the pathogenesis of erythema dyschromicum perstans (Baranda et al., 1997).

Treatment There is no single established therapy for erythema dyschromicum perstans. Various therapies have been tried with limited success such as sun protection, chemical peels, antibiotics, topical and systemic corticosteroids, estrogens, keratolytics, vitamins, isoniazid, griseofulvin, and chloroquine (Goihman 1998; Osswald et al., 2001). Clofazimine may be successful by decreasing the inflammatory component

ACQUIRED EPIDERMAL HYPERMELANOSES

in erythema dyschromicum perstans and by exerting its immunomodulating effects (Baranda et al., 1997). Dapsone is another treatment that may work by regulating the immune responses involved in the pathogenesis of erythema dyschromicum perstans (Goihman, 1998). The Q-switched ruby laser may prove to be useful as it is in nevus of Ota, which also has excessive dermal melanin.

References Ackerman, A. B. Histologic Diagnosis of Inflammatory Skin Diseases. Philadelphia: Lea and Febiger, 1978. Baranda, L. B., Torres-Alvarez, R. Cortes-Franco, B. Moncada, D. P. Portales-Perez, and R. Gonzalez-Amaro. Involvement of cell adhesion and activation molecules in the pathogenesis of erythema dyschromicum perstans (ashy dermatitis). The effect of clofazimine therapy. Arch. Dermatol. 133:325–329, 1997. Bereston, E. S. Lichenoid dermatitis: observation of 200 cases from the dermatology section, Medical Branch, Dewitt General Hospital, Auburn, California. J. Invest. Dermatol. 7:69, 1946. Berger, R. S., T. J. Hayes, and S. L. Dixon. Erythema dyschromicum perstans and lichen planus: Are they related? J. Am. Acad. Dermatol. 21:438–442, 1989. Bhutani, L. K., T. R. Bedi, A. K. Pandhi, and N. C. Nayak. Lichen planus pigmentosus. Dermatologica 149:43–50, 1974. Byrne, D. A., and R. S. Berger. Erythema dyschromicum perstans. Acta Derm. Venereol. 54:65–68, 1974. Convit, J., F. Kerdel-Vegas, and G. Rodriguez. Eritema discromico perstans. Dermatol. Venez. 2:118–164, 1961a. Convit, J., F. Kerdel-Vegas, and G. Rodriguez. Erythema dyschromicum perstans: A hitherto undescribed skin disease. J. Invest. Dermatol. 36:457–462, 1961b. Goihman, Y. Erythema dyschromicum perstans: response to dapsone therapy. Int. J. Dermatol. 37:796, 1998. Gougerot, M. H. Lichen atypiques on invisibles pigmentogenes reveles par des pigmentation. Bull. Soc. Franc. Dermatol. Syphiligr. 42:792–794, 1935a. Gougerot, M. H. Lichen atypiques ou invisibles pigmentogenes. Bull. Soc. Franc. Dermatol. Syphiligr. 42:894–898, 1935b. Greppi, C., and Z. Soustek. Laa melanodermite lichenoide in soggetti etiopici. Minerva Dermatol. 41:153, 1966. Henderson, C. D., J. A. Tschen, and D. G. Schaefer. Simultaneously active lesions of vitiligo and erythema dyschromicum perstans. Arch. Dermatol. 124:1258–1260, 1988. Holst, R., and H. Mobachen. Erythema dyschromicum perstans. Acta Derm. Venereol. 54:69–72, 1974. Ito, M. Pigmentation maculosa multiplex. Tokyo: Kanahara Shuppan, 1955. Jablonska, S. Ingestion of ammonium nitrate as a possible cause of erythema dyschromicum perstans (ashy dermatosis). Dermatologica 150:287–291, 1975. Kark, E. C., and R. L. Litt. Ashy dermatosis: A variant of lichen planus? Cutis 25:631–633, 1980. Knox, J. M., B. G. Dodge, and R. G. Freeman. Erythema dyschromicum perstans. Arch. Dermatol. 97:262–270, 1968. Koves de Amini, E., and T. Briceno-Maaz. Neuvos casos de eritema discrómico perstans: Dermaosis. Cenicienta. Med. Cutánea. 1:353–360, 1967. Metin, A., O. Calka, and S. Ugras. Lichen planopilaris coexisting with erythema dyschromicum perstans. Br. J. Dermatol. 145:522–523, 2001. Naidorf, K. F., and S. R. Cohen. Erythema dyschromicum perstans and lichen planus. Arch. Dermatol. 118:683–685, 1982. Novick, N. L., and R. Phelps. Erythema dyschromicum perstans. Int. J. Dermatol. 10:630–633, 1985. Osswald, S. S., L. H. Proffer, and C. R. Sartori. Erythema

dyschromicum perstans: a case report and review. Cutis 68:25–28, 2001. Palatsi, R. Erythema dyschromicum perstans: A follow-up study from Northern Finland. Dermatologica 155:40–44, 1977. Pathak, M. A., N. P. Sanchez, and T. B. Fitzpatrick. Erythema dyschromicum perstans. In: Biology and Diseases of Dermal Pigmentation, T. B. Fitzpatrick et al. (eds). Tokyo: University of Tokyo Press, 1983, pp. 191–208. Peachy, R. D. E. Ashy dermatosis. Br. J. Dermatol. 94:227–228, 1976. Person, J. R., and R. L. Rogers. Ashy dermatosis: An apoptotic disease? Arch. Dermatol. 117:701–704, 1981. Piñol, A. J. Dermatitis por blaqucadones opticos: Estudio de 103 obervaciones personales. Med. Cutanea Ibero-Lat-Am. 5:249–267, 1971. Piquero-Martin, J., R. Perez-Alfonzo, and V. Abrusci. Clinical trial with clofazimine for treating erythema dyschromicum perstans. Int. J. Dermatol. 28:198–200, 1989. Ramirez, C. O. Los cenicientos, problemo clinico. In: Memoria del Primer Congreso Centroamericano de Dermatologica. San Salvador, 1957; pp. 122–130. Ramirez, C. O. Dermatosis cenicienta: Estudio epidermiológico de 139 casos. Dermatologica (Mexico) 10:133–142, 1966. Ramirez, C. O. The ashy dermatosis (erythema dyschromicum perstans). Cutis 3:244–247, 1967. Soter, N. A., C. Wand, and R. G. Freeman. Ultrastructural pathology of erythema dyschromicum perstans. J. Invest. Dermatol. 52:155, 1969. Stevenson, J. R., and M. Miura. Erythema dyschromicum perstans (ashy dermatosis). Arch. Dermatol. 94:196, 1966. Tschen, J. A., E. A. Tschen, and M. H. McGavran. Erythema dyschromicum perstans. J. Am. Acad. Dermatol. 2:295–302, 1980. Urano-Suehissa, S., H. Tagami, and K. Iwatsuki. Unilateral ashy dermatosis occurring in a child. Arch. Dermatol. 120:1491–1493, 1984. Wilson, D. J. Eczematous and pigmentary lichenoid dermatitis: Atypical lichen planus: Preliminary report. Arch. Dermatol. 54:377–396, 1946.

Erythromelanosis Follicularis Faciei et Colli Norman Levine and Cynthia Burk

Historical Background In 1960 Kitamura et al. described six young Japanese men with the triad of preauricular erythema, hyperpigmentation, and fine follicular papules. Since then, about 33 cases have been described (Kim et al., 2001). Most cases are sporadic (Mishima and Rudner, 1966), but a brother and sister have been reported (Yanez et al., 1993), which suggests an autosomal recessive inheritance (Acay 1993; Perez-Bernal et al., 2000).

Clinical Findings Erythromelanosis follicularis faciei et colli usually begins around the time of puberty, mainly affecting males (Sodaify et al., 1994). Approximately 15 cases have been reported in women (Alcalay et al., 1986; Hodak et al., 1996; Ingber et al., 1986). It is usually bilateral but unilateral cases have been noted (Borkovic et al., 1984). It usually begins in the preauricular area and slowly extends outwardly towards the cheek, neck, and occasionally involves the auricle and eyelid (Seiko 935

CHAPTER 50

Fig. 50.23. Typical lesion of the face and neck. (Courtesy of Dr. Yoon Kee Park and Dr. Sungbin Im.)

Fig. 50.24. Same patient as in Figure 50.23 (see also Plate 50.10, pp. 494–495).

et al., 1991) (Figs 50.23 and 50.24). One sees pinpoint, pale follicular papules interlaced with a ruddy hyperpigmentation (Fig. 50.25) (Whittaker and Griffiths, 1987). Posteriorly, the condition is fairly well circumscribed but anteriorly and inferiorly there is a gradual feathering (Watt and Kaiser, 1981). On diascopy there is blanching of the erythema but not the hyperpigmentation. Wood’s light examination shows accentuation of the hyperpigmentation. Terminal hairs may be enlarged and vellus hairs are lost. Local hyperhidrosis has been described leading to bothersome rusting of eyeglasses (Andersen, 1980). There is often an associated keratosis pilaris of the arms or back. One case had associated calcinosis cutis (Lee and Yang, 1987). Photosensitivity is absent.

melanosomes (Janniger, 1993). A single immunofluorescence study of five patients showed IgM in a granular pattern at the dermoepidermal junction (Alcalay et al., 1986).

Pathology Routine histopathologic examination shows a mounding and perifollicular parakeratosis. Enlarged hair follicles and hair shafts are noted. The hair shaft medulla may show retained cellular debris. There is also basal hyperpigmentation. The dermis contains a mild to moderate superficial perivascular lymphohistiocytic infiltrate with vascular ectasia. Electron microscopic studies reveal homogeneous clumping and enlargement of both melanocytic and keratinocytic 936

Differential Diagnosis The differential diagnosis includes ulerythema oophryogenes which mainly involves the eyebrows, forehead, and cheeks and leads to atrophic scarring with no hyperpigmentation. Keratosis pilaris blanches on diascopy and does not have increased basal pigmentation. Erythrose péribuccale pigmentaire lacks follicular papules and is scaly. Becker melanosis is usually seen on the back or torso and has normal hair structures. Postinflammatory hyperpigmentation, melasma, and berloque dermatitis do not have follicular papules or telangiectasia. Poikiloderma of Civatte involves the lateral neck and upper chest, and shows distinctive sparing under the chin. Follicular papules are not prominent.

Treatment With the exception of topical retinoic acid, which is of minimal value, there have been few proved therapeutic options described. One group recommends 12% ammonium lactate and metronidazole 0.75% gel (Warren and Davis, 1995).

ACQUIRED EPIDERMAL HYPERMELANOSES Seiko, T., S. Takahashi, and M. Morohashi. A case of erythromelanosis follicularis faciei with a unique distribution. J. Dermatol. 18:167–170, 1991. Sodaify, M., S. Baghestani, F. Handjani, and M. Sotoodeh. Erythromelanosis follicularis faciei et colli. Int. J. Dermatol. 33:643–644, 1994. Warren, F. M., and L. S. Davis. Erythromelanosis follicularis faciei in women. J. Am. Acad. Dermatol. 32(5 Pt 2):863–866, 1995. Watt, T. L., and J. S. Kaiser. Erythromelanosis follicularis faciei et colli: a case report. J. Am. Acad. Dermatol. 5:533–534, 1981. Whittaker, S. J., and W. A. Griffiths. Erythromelanosis follicularis faciei et colli. Clin. Exp. Dermatol. 12:33–35, 1987. Yanez, S., J. A. Velasco, and M. P. Gonzalez. Familial erythromelanosis follicularis faciei et colli — an autosomal recessive mode of inheritance. Clin. Exp. Dermatol. 18:283–285, 1993.

Erythrose Péribuccale Pigmentaire of Brocq Norman Levine and Cynthia Burk

Historical Background In 1923, Brocq reported on a case of unusual perioral hyperpigmentation. The next 50 years produced few of a similar condition.

Synonyms

Fig. 50.25. Prominent hyperpigmentation associated with erythema.

References Acay, M. C. Erythromelanosis follicularis faciei et colli: a genetic disorder? Int. J. Dermatol. 32:542, 1993. Alcalay, J., A. Ingber, S. Halevi, M. David, and M. Sandbank. Erythromelanosis follicularis faciei in women. Br. J. Dermatol. 114:267, 1986. Andersen, B. L. Erythromelanosis follicularis faciei et colli. Case reports. Br. J. Dermatol. 102:323–325, 1980. Borkovic, S. P., R. A. Schwartz, and R. S. McNutt. Unilateral erythromelanosis follicularis faciei et colli. Cutis 33:163–168, 1984. Hodak, E., A. Ingber, J. Alcaley, and M. David. Erythromelanosis follicularis faciei in women [comment]. J. Am. Acad. Dermatol. 34:714, 1996. Ingber, A., E. Hodak, and E. Sandbank. A recent case of erythromelanosis follicularis faciei et colli in a female. Z. Hautkrankh. 61:1409–1410, 1986. Janniger, C. K. Erythromelanosis follicularis faciei et colli. Cutis 51:91–92, 1993. Kitamura, K., H. Kato, Y. Mishima, and S. Sonoda. Erythromelanosis follicularis faciei. Hautarzt 11:391–393, 1960. Lee, C. W., and I. S. Yang. Cutaneous calcinosis in erythromelanosis follicularis faciei et colli. Clin. Exp. Dermatol. 12:31–32, 1987. Mishima, Y., and E. Rudner. Erythromelanosis follicularis faciei et colli. Dermatologica 132:269, 1966. Perez-Bernal, A., M. A. Munoz-Perez, and E. Camacho. Management of facial hyperpigmentation. Am. J. Clin. Dermatol. 1:261–268, 2000.

L’erthrose pigmentée péribuccale, melanosis perioralis et peribuccalis (Braun-Falco et al., 1991), erythrosis pigmenta faciei (Korting and Denk, 1976), erythrosis pigmentosa peribuccalis (Cohen, 1948), erythrose péribuccale pigmentaire (Kaminsky and Asrilant, 1959; Tritsch and Greither, 1955), and pigmented peribuccal erythema of Brocq (Everett and Burgdorf, 1992).

Clinical Findings Erythrose péribuccale pigmentaire is most commonly seen in adult women but may rarely be seen in men. The condition affects the central face, especially, the perioral area and usually has a thin, unaffected grenz zone at the vermilion border. The hyperpigmentation may extend down as far as the nasolabialis sulcus (Perez-Bernal et al., 2000). Occasionally, the disorder extends over the jaws and to the temples (Tritsch and Greither, 1955). After recurrent episodes of erythema and a reddishbrown pigmentary response, a gray-brown hyperpigmentation ensues, usually with some degree of telangiectasia (Fig. 50.26). Although there may be considerable variation of erythema and discoloration, the process is fairly stable. There may be loss of vellus hairs. Should significant scaling develop, the condition is called “parakeratose pigmentogene peribuccale.” Although there are no strict diagnostic criteria, the combination of centro-facial, perioral, red-brown or gray-brown hyperpigmentation with a history or clinical evidence of vascular instability and telangiectasia should be sufficient for the diagnosis. Topical corticosteroids and photodynamic substances in some cosmetics may be factors in the development of erythrose péribuccale pigmentaire of Brocq (Perez-Bernal et al., 2000). 937

CHAPTER 50 of facial hyperpigmentation. Am. J. Clin. Dermatol. 1:261–268, 2000. Tritsch, H., and A. Greither. Erythrosis pigmenta of the face. Arch. Dermatol. Syphil. (Berlin) 199:221–227, 1955.

Extracutaneous Neuroendocrine Melanoderma Norman Levine and Cynthia Burk

Historical Background

Fig. 50.26. Typical péribuccale pattern of involvement.

Although there had been over 40 well-documented cases of hyperadrenocorticism occurring in association with neoplasia between 1928 and 1961, the relationship of an increased plasma and tumor tissue ACTH-like substance was not proved until 1961 (Christy, 1961). In 1963 the term “ectopic ACTH syndrome” was first used and the syndrome was clearly defined (Liddle et al., 1963).

Histopathology Light microscopy reveals basal epidermal hyperpigmentation. The dermis shows vascular dilatation, mild superficial perivascular lymphohistiocytic infiltration and melanophages (Obermayer and Becker, 1932).

Clinical Findings

The differential diagnosis includes perioral dermatitis, rosacea, seborrheic dermatitis, and estrogen-driven melasma, with or without complicating factors such as the abuse of topical, fluorinated corticosteroids, petrolatum, or photosensitizing cosmetic agents.

Signs and symptoms may be mild and insidious, delaying early diagnosis and treatment. Approximately 25% of patients with Cushing syndrome have the “ectopic ACTH syndrome.” Hypokalemic alkalosis, weakness, and edema are common; obesity, ecchymosis and striae may also be present. Associated internal disorders include impaired glucose tolerance and oligomenorrhea. Hirsutism, acne, and seborrhea may develop. The hypermelanosis is accentuated in sun-exposed areas (Fig. 50.27), creases, pressure points, oral mucosa, and the genitalia.

Treatment

Pathology

The hyperpigmentation may respond to various combinations of broad-spectrum sunscreen, hydroquinone, kojic acid, azelaic acid (Perez-Bernal et al., 2000), glycolic acid, retinoic acid, chemical peeling, dermabrasion, and laser resurfacing. Wood’s light examination may help guide therapy. Avoidance of sun and ultraviolet lamps is important. Pigmentation usually persists even after the cause has been removed (PerezBernal et al., 2000).

Routine histologic examination shows increased epidermal melanin without increased numbers of melanocytes. Melanin is most concentrated in the basal layer but is also prominent in the stratum malpighii. Dermal melanophages may be present (Lerner, 1955). The histologic picture is similar to that of a naturally darkly pigmented person.

Differential Diagnosis

References Braun-Falco, O. G., H. H. Plewig, K. Wolf, and R. K. Winkelman. Melanosis perioralis et peribuccalis. In: Dermatology, O. BraunFalco et al. (eds). Berlin: Springer-Verlag, 1991, p. 692. Brocq, J. L. L’erthrose pigmentée péri-buccale. Presse Med. 31:720– 728, 1923. Cohen, E. L. Erythrosis pigmentosa peribuccalis. Br. J. Dermatol. 60:203–211, 1948. Everett, M. A., and W. H. C. Burgdorf. Pigmented peribuccal erythema of Brocq. In: Clinical Dermatology, J. Demis (ed.). Philadelphia: J. B. Lippincott, 1992, pp. 11–12. Kaminsky, A., and M. Asrilant. Erythrose peribuccale pigmentaire. Rev. Assoc. Med. Argent. 73:427–429, 1959. Korting, G. W., and R. Denk. Circumscribed hyperpigmentation. In: Dermatology. Philadelphia: JB Lippincott, 1976, p. 381. Obermayer, M. E., and S. W. Becker. Erythrose peribuccale pigmentaire of Brocq: case with capillary and histologic study. Arch. Dermatol. Syphil. 26:444–450, 1932. Perez-Bernal, A., M. A. Munoz-Perez, and E. Camacho. Management

938

Laboratory Findings Laboratory studies show hypokalemia and elevated plasma and urinary 17-OH corticosteroids. Elevated plasma ACTH can arise from a pituitary tumor or from the adrenal gland (Bower and Gordon, 1965). These sources must be ruled out by magnetic resonance imaging (MRI) studies and dexamethasone suppression tests. There is evidence that certain tumors produce peptides which, much like corticotropinreleasing factor, act on the pituitary gland (Al Rustom et al., 1986). The ACTH produced by neoplasms is identical to pituitary ACTH (Liddle et al., 1969). There can also be an MSH action which is distinct from the ACTH action.

Pathogenesis Hyperpigmentation is caused by both ACTH and MSH action on the melanocytes. The most common tumor to cause the “ectopic ACTH syndrome” is bronchogenic oat cell carcinoma (Friedman et al., 1965; Liddle et al., 1969), accounting

ACQUIRED EPIDERMAL HYPERMELANOSES Bower, B. F., and G. S. Gordon. Hormonal effects of nonendocrine tumors. Annu. Rev. Med. 16:83–118, 1965. Christy, N. P. Adrenocorticotropic activity in the plasma of patients with Cushing’s syndrome associated with pulmonary neoplasm. Cancer Res. 1:85–86, 1961. Friedman, M., P. Marshall-Jones, and E. J. Ross. Cushing’s syndrome: Adrenocortical hyperactivity secondary to neoplasms arising outside the pituitary-adrenal system. Q. J. Med. 35:193–214, 1965. Gartner, L. A., and M. L. Voorhess. Adrenocorticotropic hormone-producing thymic carcinoid in a teenager. Cancer 71:106–111, 1993. Lerner, A. B. Melanin pigmentation. Am. J. Med. 19:902–924, 1955. Liddle, G. W., D. P. Island, R. L. Ney, W. E. Nicholson, and N. Shimizu. Nonpituitary neoplasm and Cushing’s syndrome. Arch. Intern. Med. 111:471–475, 1963. Liddle, G. W., W. E. Nicholson, D. P. Island, D. N. Orth, K. Abe, and S. C. Lowder. Clinical and laboratory studies of ectopic hormonal syndromes. Rec. Prog. Hormone Res. 25:283–305, 1969. Lohrenz, F. N., and G. S. Custer. ACTH-producing metastasis from carcinoma of the esophagus. Ann. Intern. Med. 62:1017–1022, 1965. Pearse, A. G. E. The neuroendocrine (APUD) cells of the skin. Am. J. Dermatopathol. 2:121–128, 1980. Upton, G. V., and T. T. Amaturda. Evidence for the presence of tumor peptides with corticotropin-releasing-factor-like activity in the ectopic ACTH syndrome. N. Engl. J. Med. 285:419–424, 1971. Waldrum, H. L., P. G. Burhol, J. A. Johnson, and A. G. Smith. MSHproducing gastric tumor. Acta Hepatogastroenterol. 24:386–388, 1977.

Felty Syndrome and Rheumatoid Arthritis Norman Levine and Cynthia Burk

Historical Background Fig. 50.27. Hyperpigmentation of the face.

for over half of cases. Other tumors include pancreatic carcinoma, esophageal carcinoma (Lohrenz and Custer, 1965), pheochromocytoma, thymoma (Gartner and Voorhess, 1993), ovarian carcinoma, gastric cancer (Waldrum et al., 1977), and various APUD tumors (Azzopardi et al., 1968; Pearse, 1980). The wide variety of causative tumors suggests that there may be a selective loss of repressors allowing ACTH and other active peptides to be produced (Upton and Amaturda, 1971). Additionally, exogenous sources of corticosteroids given for chronic diseases may produce these symptoms.

Treatment Unfortunately, by the time of diagnosis, there is often metastasis and, thus, a poor prognosis. Early diagnosis and treatment allows for the only chance of cure.

References Al Rustom, K., J. Gerard, and G. E. Pierard. Extrapituitary neuroendocrine melanoderma, unique association of extensive melanoderma with macromelanosomes and extrapituitary secretion of a high molecular weight neuropeptide related to pro-opiomelanocortin. Dermatologica 173:157–162, 1986. Azzopardi, J. G., M. C. Path, and E. D. Williams. Pathology of “nonendocrine” tumors associated with Cushing’s Syndrome. Cancer 22:274–286, 1968.

In 1924, while still a resident physician, Augustus Felty reported on his index case and four others from hospital records. The syndrome had been reported in Europe under a variety of names as early as 1896 (Still, 1896; Von Jaksch, 1896). The term Felty syndrome was first used in 1932 (Hanrahan and Miller, 1932), and is characterized by the triad of rheumatoid arthritis, splenomegaly, and leukopenia (neutropenia). Middle-aged individuals with chronic arthritis are most prone to this syndrome, but an occasional child has been reported. Most cases are sporadic but familial cases have been reported (Blendis et al., 1976; Runge et al., 1986). As in rheumatoid arthritis, there are numerous extra-articular manifestations. This syndrome has been extensively reviewed (Bowman, 2002; DeGruchy and Langley, 1961; Sienknecht et al., 1977; Spivak, 1977).

Clinical Findings Felty’s first five cases had hyperpigmentation as a clinical feature — most reports since have not emphasized this. Approximately 20% of Felty’s cases showed some degree of hyperpigmentation. Some may also have a yellow-brown pigmentation of sun-exposed areas (Ruderman, 1968) (Fig. 50.28). When hyperpigmentation is present, the source is generally of two types; vasculitis, especially, of the distal digits can lead to small, pigmented macules. There is general skin fragility in rheumatoid arthritis that leads to easy bruising and 939

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Pathology Histologic changes are those of a naturally dark or tanned person. There is increased epidermal melanization. Dermal hemosiderin may be present.

Treatment Treatment of hyperpigmentation in Felty syndrome and rheumatoid arthritis involves control of the primary disease process, sunscreens, and sun avoidance.

References

Fig. 50.28. Hyperpigmentation on sun-exposed areas in a patient with rheumatoid arthritis (see also Plate 50.11, pp. 494–495).

ecchymosis which can result in dermal hemosiderin deposition and iron-induced stimulation of melanocytes (Morgan and Chow, 1993). Rare cases associated with symptomatic porphyria have been described (Eales et al., 1972; Rimington et al., 1972). Medications used to treat arthritic symptoms may lead to hyperpigmentation. Gold therapy can produce a diffuse cutaneous pigmentation. Gold is preferentially taken up by the dermis in ultraviolet-exposed skin. This dermal gold stimulates melanin production leading to photoaccentuated hyperpigmentation (Leonard et al., 1986; Pelachyk et al., 1984). Antimalarial preparations (quinacrine, chloroquine, hydroxychloroquine) may bind to melanin with subsequent cutaneous and mucosal hyperpigmentation (Granstein and Sober, 1981). Methotrexate when taken weekly may induce photosensitivity and result in hyperpigmentation (Toussirot and Wendling, 1999). Busulfan and cyclophosphamide may both induce diffuse hyperpigmentation that is enhanced in sun-exposed areas, especially in patients with dark complexions (Toussirot and Wendling, 1999). Minocycline is used for its immunomodulatory properties in treating rheumatoid arthritis. This drug has also been associated with hyperpigmentation (Langevitz et al., 2000; Ozog et al., 2000). 940

Bennion, S. D. Hyperpigmentation in a patient with Felty’s syndrome and hemochromatosis. J. Assoc. Mil. Dermatol. 9:14–17, 1983. Blendis, L. M., K. L. Jones, E. B. Hamilton, and R. Williams. Familial Felty’s syndrome. Ann. Rheum. Dis. 35:279–281, 1976. Bowman, S. J. Hematological manifestations of rheumatoid arthritis. Scand. J. Rheum. 31:251–259, 2002. DeGruchy, G. C., and G. R. Langley. Felty’s syndrome. Australas. Ann. Med. 10:292–303, 1961. Eales, L., W. G. Sears, K. B. King, M. B. Levey, and C. Rimington. Symptomatic porphyria in a case of Felty’s syndrome, I, Clinical and routine biochemical studies. Clin. Chem. 18:459–461, 1972. Fam, A., and T. Paton. Nail pigmentation after parenteral gold therapy for rheumatoid arthritis. Arthritis Rheum. 27:119–120, 1984. Felty, A. R. Chronic asthma in the adult, associated with splenomegaly and leukopenia: a report of 5 cases of an unusual clinical syndrome. Bull. Johns Hopkins Hosp. 35:16–20, 1924. Granstein, R., and A. Sober. Drug- and heavy metal-induced hyperpigmentation. J. Am. Acad. Dermatol. 5:1–18, 1981. Hanrahan, E. M., Jr., and S. R. Miller. Effect of phlebectomy in Felty’s syndrome. J. Am. Med. Assoc. 99:1247–1249, 1932. Leonard, P., F. Moatamed, J. Ward, M. Piepkorn, E. Adams, and W. Knibbe. Chrysiasis: the role of sun exposure in dermal hyperpigmentation secondary to gold therapy. J. Rheumatol. 13:58–64, 1986. Langevitz, P., A. Livneh, I. Bank, and M. Pras. Benefits and risks of minocycline in rheumatoid arthritis. Drug Saf. 22:405–414, 2000. Lutterloh, C., and P. Shallenberger. Unusual pigmentation developing after prolonged suppressive therapy with quinacrine hydrochloride. Arch. Dermatol. Syphil. 53:349–354, 1946. Morgan, G. J., Jr., and W. S. Chow. Clinical features, diagnosis, and prognosis in rheumatoid arthritis. Curr. Opin. Rheumatol. 5:184– 190, 1993. Ozog, D. M., D. S. Gogstetter, G. Scott, and A. A. Gaspari. Minocycline-induced hyperpigmentation in patients with pemphigus and pemphigoid. Arch. Dermatol. 136:1133–1138, 2000. Pelachyk, J. M., W. F. Bergfeld, and J. T. McMahon. Chrysiasis following gold therapy for rheumatoid arthritis: ultrastructural analysis with x-ray energy spectroscopy. J. Cutan. Pathol. 11:491–494, 1984. Pinals, R. S. Felty’s syndrome. In: Textbook of Rheumatology, 4th ed., W. N. Kelly, C. B. Sledge, E. D. Harris, and S. Ruddy (eds). Philadelphia: WB Saunders, 1993, pp. 924–930. Rimington, C., W. G. Smears, and L. Eales. Symptomatic porphyria in a case of Felty’s syndrome. II. Biochemical investigations. Clin. Chem. 18:462–470, 1972. Ruderman, M., L. M. Miller, and R. S. Pinals. Clinical and serologic observations on 27 patients with Felty’s syndrome. Arthritis Rheum. 11:377–384, 1968. Runge, L. A., F. R. Davey, J. Goldberg, and P. R. Boyd. The inheritance of Felty’s syndrome in a family with several affected members. J. Rheumatol. 13:39–42, 1986. Sams, W., and J. Epstein. The affinity of melanin for chloroquine. J. Invest. Dermatol. 45:482–488, 1965. Sienknecht, C. W., M. B. Urowitz, and W. Pruzanski. Felty’s syn-

ACQUIRED EPIDERMAL HYPERMELANOSES

Fig. 50.29. Melasma-like hyperpigmentation.

Fig. 50.30. Diffuse meloderma.

drome: Clinical and serological analysis of 34 cases. Ann. Rheum. Dis. 36:500, 1977. Spivak, J. L. Felty’s Syndrome: An analytical review. Johns Hopkins Med. J. 141:156–162, 1977. Still, G. F. On a form of chronic joint disease in children. Med. Chirurg. Trans. 80:47–53, 1896. Toussirot, E. and D. Wendling. Methotrexate-induced hyperpigmentation in a rheumatoid arthritis patient. Clin. Exp. Rheumatol. 17: 751, 1999. Von Jaksch, A. Arthritis urica, Megalosplenie and Leukopenie. Dtsch. Med. Wochenschr. 36:634–638, 1896.

Hyperpigmentation Associated with Human Immunodeficiency Virus (HIV) Infection Philippe Bahadoran

Fig. 50.31. Small hyperpigmented macules of the fingertips.

Hyperpigmentation Types Probably Related to HIV Infection and/or its Therapeutics

described and are summarized in Table 50.2. Except for melasmalike pigmentation, that seems to occur alone, the other types of pigmentation are frequently associated with each other (Bendick et al., 1989; Gallais et al., 1992; GrauMassanes et al., 1990; Greenberg and Berger, 1990; Lacour et al., 1991; Langford et al., 1989; Merenich et al., 1989; PoizotMartin et al., 1991; Tadini et al., 1991).

Clinical and Histologic Features Hyperpigmentation occurs frequently in patients with HIV infections, especially in the late stage of the disease. Different patterns of cutaneous (Figs 50.29–50.31), mucosal (Fig. 50.32), and nail (Fig. 50.33) pigmentation have been

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Histologic and ultrastructural features indicative of melanotic pigmentation are found in most cases, except for clofazimine-induced pigmentation. Melanin is increased in the epidermis, especially in the keratinocytes of the basal layer (Esposito, 1987; Ficarra et al., 1990; Gaddoni et al., 1995; Gallais et al., 1992; Greenberg and Berger, 1990; Gregory and DeLeo, 1994; Hermanns-Lê et al., 1993; Langford et al., 1989). A moderate degree of dermal pigment incontinence is often noted in addition to epidermal pigmentation (Ficarra et al., 1990; Gallais et al., 1992; Greenberg and Berger, 1990; Gregory and DeLeo, 1994; Hermanns-Lê et al., 1993; Langford et al., 1989). There is no melanocyte proliferation, but ultrastructural studies indicate that these cells are activated (Ficarra et al., 1990; Hermanns-Lê et al., 1993; Zhang et al.,

Fig. 50.32. Hyperpigmented lesions of the tongue.

Fig. 50.33. Hyperpigmentation of the nail bed (see also Plate 50.12, pp. 494–495).

Table 50.2. Hyperpigmentation patterns probably related to HIV infection and/or its therapeutics. Hyperpigmentation

Clinical features

References

Facial and photodistributed hyperpigmentation

Light brown macules over the temporal and frontal regions Sometimes more spread, mimicking melasma May even extend to other photoexposed areas Occurs mostly at the late stage of HIV infection Pigmented macules on palms and soles, on dorsal and ventral aspects of fingers, and on finger pads Reported in severely immunocompromised patients Heterogeneous light to dark brown pigmentation Confers a “dirty” aspect Observed in patients with advanced HIV infection Oral pigmentation is the most frequent and is more often macular than diffuse Oral melanotic macules in HIV infection have a rapid progression rate Genital pigmentation was rarely reported Longitudinal melanonychia is the most frequent aspect Horizontal or diffuse nail discoloration is also possible Zidovudine (AZT) is clearly the dominant cause, pigmentation is dose dependent and reversible once AZT is stopped Nail pigmentation also occurs in HIV-positive patients without AZT

Lacour et al., 1993; Gaddoni et al., 1995; Gregory and DeLeo, 1994; Pascual et al., 1996

Acral hyperpigmentation

Diffuse hyperpigmentation

Mucosal hyperpigmentation

Nail hyperpigmentation

942

Bendick et al., 1989; Doutre et al., 1990; Gallais et al., 1992; Merenich et al., 1989 Esposito, 1987; Jing et al., 2000; HermannsLê et al., 1993; Lacour et al., 1991; Poizot-Martin et al., 1991 Cohen and Callen, 1992; Ficarra et al., 1990; Gallais et al., 1992; Greenberg and Berger, 1990; Grau-Massanes et al., 1990; Langford et al., 1989; Tadini et al., 1991 Azon-Masoliver and Mallolas, 1988; Bendick et al., 1989; Chandrasekar, 1989; Don et al., 1990; Furth and Kazakis, 1987; Greenberg and Berger, 1990; Grau-Massanes et al., 1990; Lacour et al., 1988; Panwalker, 1987; Tosti et al., 1990

ACQUIRED EPIDERMAL HYPERMELANOSES

1989). Similar findings, e.g., increase of melanin throughout the nail plate, and dendritic melanocytes, are observed in melanonychia (Greenberg and Berger, 1990; Tosti et al., 1990).

Etiology Hyperpigmentation during the course of HIV infection is rarely secondary to adrenal insufficiency (Dore et al., 1998; Esposito, 1987). Although opportunistic infections can cause adrenal lesions in HIV infection (Bricaire et al., 1998), adrenal function is nonetheless often normal in these patients (Verges et al., 1990). Folate or vitamin B12 deficiency can result in acquired cutaneous or mucosal pigmentation (Fitzpatrick and Ortonne, 1995), and low folate or vitamin B12 plasma levels occur frequently in acquired immune deficiency syndrome (AIDS) (Boudes et al., 1990), but a direct link between hyperpigmentation and vitamin deficiency has not been established in HIV infection. Hyperpigmentation of photoexposed areas in porphyria cutanea tarda was observed in association with AIDS (Pascual et al., 1996), but porphyria cutanea tarda might rather be linked to hepatitis C infection (Castanet et al., 1994). Zidovudine (AZT) was the first nucleoside analog to be approved as antiretroviral medication. AZT is undoubtedly a frequent cause of pigmented nails (Anders and Abele, 1989; Azon-Masoliver and Mallolas, 1988; Chandrasekar, 1989; Don et al., 1990; Furth and Kazakis, 1987; Grau-Massanes et al., 1990; Greenberg and Berger, 1990; Lacour et al., 1988; Panwalker, 1987; Tosti et al., 1990). Nail involvement ranges from toenail only involvement to complete fingernail and toenail involvement, including scattered nail involvement of toes and fingers. Nail pigmentation patterns associated with AZT include entire nail pigmentation, transverse banding, and multiple longitudinal bands. Pigmentation varies from faint blue, dark blue-gray, and brownish discolorations (Ward et al., 2002). AZT may sometimes induce oral pigmentation (Greenberg and Berger, 1990), acral pigmented macules (Bendick et al., 1989; Doutre et al., 1990), and diffuse melanoderma (Hermanns-Lê et al., 1993), but these types of pigmentation are also frequently reported without relation to this drug during the course of HIV infection (Ficarra et al., 1990; Gallais et al., 1992; Granel et al., 1997; Lacour et al., 1991; Langford et al., 1989; Poizot-Martin et al., 1991). As with other drugs, AZT-induced pigmentation is reversible upon discontinuation (Ward et al., 2002). The nail and mucocutaneous pigmentation induced by AZT is due to the deposition of melanin (Greenberg and Berger, 1990). Mice fed AZT exhibit a hyperpigmentation on their tails because of the presence of large quantities of melanin in the epidermis; they are therefore regarded as a possible animal model of AZT-induced hyperpigmentation (Roth et al., 1991). In this model the increased pigmentation is due to increased numbers of melanosomes within epidermal keratinocytes (Obuch et al., 1992). Emtricitabine (FTC) is a recent nucleoside analog used for antiretroviral therapy. Hyperpigmentation, usually affecting either the palms of the hands or the soles

of the feet, was reported on individuals with FTC, and is almost exclusive to patients of African origin (Nelson et al., 2004). Insulin resistance is a side effect in patients with HIV infection who are receiving treatment with protease inhibitors. A case of acanthosis nigricans, in the context of insulinresistance induced by protease inhibitors, was reported recently (Mellor-Pita et al., 2002). Hydroxyurea, a cytotoxic agent used in myeloproliferative disorders, has recently been introduced as an adjunct to antiretroviral therapy. Melanonychia and mucocutaneous hyperpigmentation due to hydroxyurea use in HIV-infected patients have been reported (Joyner et al., 1999; Laughon et al., 2000). High-dose rifabutin (600 mg daily) for mycobacterial infection can induce dark brown skin pigmentation, but not the lower dosage (300 mg daily) used for prophylaxis (Figueras et al., 1998; Smith and Flanigan, 1995). Clofazimine, which is also used for mycobacterial infection, induces skin hyperpigmentation, due to the accumulation in the skin of the drug itself and later of a ceroidlike substance (Granstein and Sober, 1981). Tetracosactide (ACTH), which is used for the treatment of elevated cranial pressure due to cerebral toxoplasmosis, induces a diffuse, Addisonlike hyperpigmentation (Granstein and Sober, 1981). Several drugs used for Kaposi sarcoma treatment — bleomycin, vinblastine, and cyclophosphamide — can cause pigmentary changes (Caumes et al., 1991; Granstein and Sober, 1981). Ketoconazole was suspected of causing cutaneous and oral pigmentation in HIVinfected patients (Langford et al., 1989; Poizot-Martin et al., 1991). Increased plasmatic levels of a-MSH were noted in a patient with severe melanoderma (Gallais et al., 1992). However, when a-MSH was measured in nine patients with AIDS and diffuse melanoderma, only one had an elevated value, which suggests that excessive production of a-MSH is a rare etiology of hyperpigmentation associated with AIDS (Lacour and Ortonne, 1996). In a study that aimed to assess the cutaneous response to UV irradiation in HIV patients each subject underwent an exposure of 6 MED. No significant differences between HIVinfected patients and controls were found for delayed pigmentation (Aubin et al., 1999).

Other Pigmented Dermatoses Occurring in HIV-Infected Patients The following entities are considered separately because erythema dyschromicum perstans and Riehl melanosis may represent the occurrence of otherwise well-defined pigmented dermatoses in the context of HIV infection, whereas in pigmented erythroderma, pigmentation is only a secondary event.

Erythema Dyschromicum Perstans This rare dermatitis was reported twice during the course of HIV infection. The histologic findings of basal cell vacuolization with dermal pigment incontinence confirmed the diagnosis in both cases (Nelson et al., 1992; Venencie et al., 1988). 943

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Riehl Melanosis One case of Riehl–melanosis-like facial pigmentation, with a large number of melanophages in the dermis, was reported in a Japanese patient with advanced HIV infection (Hanada et al., 1994).

Pigmented Erythroderma This severe skin disease occurs during the late stage of AIDS, and it is characterized by a pruriginous squamous erythroderma, followed in most patients by intense cutaneous pigmentation (Friedler et al., 1999; Janier et al., 1989; Kaplan et al., 1991; Picard-Dahan et al., 1996). Despite some clinical similarities with Sézary syndrome, histologic and molecular biology studies do not demonstrate features of cutaneous Tcell lymphomas. The cause of this peculiar HIV-associated erythroderma remains unknown. The mechanism of hyperpigmentation is also uncertain, but given the context in which it occurs, a postinflammatory pigmentation seems most likely.

Hypopigmentation HIV–infection-associated vitiligo has rarely been reported (de la Fuente et al., 1991; Duvic et al., 1987; Ivker et al., 1994). In addition, a case of vitiligo in association with HIV where a rising CD4 lymphocyte count due to highly active antiretroviral therapy (HAART) closely correlated with changes in the skin was reported recently (Anthony and Marsden, 2003). On the other hand, infectious causes of hypopigmentation, such as tinea versicolor achromians, post-zoster hypopigmented macules, secondary syphilis, or even leprae, may occur more frequently during the course of HIV infection.

Dysplastic Nevi and Melanoma There have been several case reports of melanoma (McGregor et al., 1992; Rivers et al., 1989; Tyndall et al., 1989; Van Ginkel et al., 1991) and dysplastic nevi (Betlloch et al., 1991; Duvic et al., 1989) among HIV-infected patients. In a comparative study, it was found that the total number of nevi was higher in a group of HIV-infected patients than in controls (Grob et al., 1996). However, patients and controls were not significantly different according to the number of large (equal or larger than 5 mm) or atypical nevi. Additional epidemiological data are warranted to confirm that the frequency or the gravity of melanocytic tumors is increased with HIV infection. Regarding the prognosis, it was reported recently that melanoma patients who were HIV-positive had a significantly shorter disease-free and overall survival compared with HIVnegative melanoma patients. There was an inverse relationship between CD4 cell counts and time to first melanoma recurrence (Rodrigues et al., 2002).

Conclusion Hypermelanosis significantly increases in individuals with HIV infection. Apart from nail pigmentation that is clearly linked with AZT, a defined etiology can rarely be determined for cutaneous and mucosal hyperpigmentation. However, the 944

search for treatable causes and for drug-induced hyperpigmentation must be done carefully in every case of hyperpigmentation occurring in an HIV-infected patient.

References Antony, F. C., and R. A. Marsden. Vitiligo in association with human immunodeficiency virus infection. J. Eur. Acad. Dermatol. Venereol. 17:456–458, 2003. Aubin, F, N. Parriaux, C. Robert, D. Blanc, C. Drobacheff, R. Laurent, and P. Humbert. Cutaneous reaction to ultraviolet irradiation in human-immunodeficiency-virus-infected patients. A casecontrol study. Dermatology 198:256–260, 1999. Azon-Masoliver, A., and J. Mallolas. Zidovudine-induced nail pigmentation. Arch. Dermatol. 124:1570–1571, 1988. Bendick, C., H. Rasokat, and G. Steigleder. Azidothymidine-induced hyperpigmentation of skin and nails. Arch. Dermatol. 125:1285– 1286, 1989. Betlloch, I., C. Amador, E. Chiner, F. Pasquau, J. L. Calpe, and A. Vilar. Eruptive melanocytic nevi in hurnan immunodeficiency virus infection. Int. J. Dermatol. 30:303, 1991. Boudes, P., J. Zittoun, and A. Sobel. Folate, vitamin B 12 and HIV infection. Lancet 335:1401–1402, 1990. Bricaire, F., C. Marche, and D. Zoubi. Adrenocortical lesions and AIDS. Lancet i:881, 1988. Castanet, J., J. P. Lacour, J. Bodokh, S. Bekri, and J. P. Ortonne. Porphyria cutanea tarda in association with human immunodeficiency virus infection: is it related to hepatitis C virus infection? Arch. Dermatol. 130:664–665,1994. Caumes, E., C. Katlama, G. Guermonprez, I. Bournerias, M. Danis, and M. Gentilini. Cutaneous side-effects of bleomycin in AIDS patients with Kaposi’s sarcoma. Lancet 336:1593, 1991. Chandrasekar, P. H. Nail discoloration and human immunodeficiency virus infection. Am. J. Med. 86:506–507, 1989. Cohen, L. M., and J. P. Callen. Oral and labial melanotic macules in a patient infected with human immunodeficiency virus. J. Am. Acad. Dermatol. 26:653–654, 1992. de la Fuente, J., E. Suarez, J. A. Arzuaga, P. Tebas, and J. Lopez de Letona. Vitiligo et SIDA. Nouv. Dermatol. 10:397–398, 1991. Don, P. C., F. Fusco, P. Fried, A. Batterman, F. P. Duncanson, T. H. Lenox, and N. C. Klein. Nail dyschromia associated with zidovudine. Ann. Intern. Med. 112:145–146, 1990. Dore, M. X., A. de La Blanchardiere, P. Lesprit, F. David, J. P. Beressi, J. Fiet, D. Sicard, and J. M. Decazes. Peripheral adrenal insufficiency in AIDS. Rev. Med. Intern. 19:23–28, 1998. Doutre, M.-S., C. Beylot, and J. Beylot. Macules pigmentées des extrémités au cours d’un traitement par zidovudine. Rev. Med. Interne 11:486, 1990. Duvic, M., L. Lowe, R. P. Rapini, S. Rodriguez, and M. L. Levy. Eruptive dysplastic nevi associated with human immunodeficiency infection. Arch. Dermatol. 125:397–401, 1989. Duvic, M., R. Rapimi, W. K. Hoots, and P. W. Mansell. Human immunodeficiency virus-associated vitiligo: expression of autoimmunity with immunodeficiency? J. Am. Acad. Dermatol. 17:656– 662, 1987. Esposito, R. Hyperpigmentation of skin in patients with AIDS. Br. Med. J. 294:840, 1987. Ficarra, G., E. J. Shillitoe, K. Adler-Storthz, D. Gaglioti, M. Di Pietro, R. Riccardi, and G. Forti. Oral melanotic macules in patients infected with human immunodeficiency virus. Oral Surg. Oral Med. Oral Pathol. 70:748–755, 1990. Fitzpatrick, T. B., and J. P. Ortonne. Abnormalities of pigmentation. In: Dermatology in General Medicine, 3rd ed., T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M. Freedberg, and K. F. Austen (eds). New York: McGraw Hill, 1995, p. 962. Figueras, C., L. Garcia, and J. M. Bertran. Hyperpigmentation in a

ACQUIRED EPIDERMAL HYPERMELANOSES patient with AIDS, receiving rifabutin for disseminated Mycobacterium genavense infection. Eur. J. Pediatr.157:612, 1998. Friedler, S., M. T. Parisi, E. Waldo, R. Wieczorek, G. Sidhu, and M. J. Rico. Atypical cutaneous lymphoproliferative disorder in patients with HIV infection. Int. J. Dermatol. 38:111–118, 1999. Furth, P., and A. Kazakis. Nail pigmentation changes associated with azidothymidine (zidovudine). Ann. Intern. Med. 107:350, 1987. Gaddoni, G., L. Baldassari, F. Albertini, and C. Misciali. Melasma and acquired immunodeficiency syndrome (AIDS). J. Eur. Acad. Dermatol. Venereol. 4:44–47, 1995. Gallais, V., J. P. Lacour, C. Perrin, G. Ghanem, J. Bodokh, and J. P. Ortonne. Acral hyperpigmented macules and longitudinal melanonychia in AIDS patients. Br. J. Dermatol. 126:387–391, 1992. Granel, F., F. Truchetet, and M. Grandidier. Longitudinal colored bands, oral and cutaneous pigmentation without taking zidovudine. Ann. Dermatol. Venereol. 124:460–462, 1997. Granstein, R., and A. Sober. Drug- and heavy metal-induced hyperpigmentation. J. Am. Acad. Dermatol. 5:1–18, 1981. Grau-Massanes, F., F. Millan, M. I. Febrer, C. Pujol, V. A. Alegre, M. Salavert, V. Navarro, and A. Aliaga. Pigmented nails and mucocutaneous pigmentation in HIV-positive patients treated with zidovudine. J. Am. Acad. Dermatol. 22:687, 1990. Greenberg, R., and T. Berger. Nail and mucocutaneous hyperpigmentation with azidothymidine therapy. J. Am. Acad. Dermatol. 22: 327–330, 1990. Gregory, N., and V. A. DeLeo. Clinical manifestations of photosensitivity in patients with human immunodeficiency virus infection [editorial]. Arch. Dermatol. 130:630–633, 1994. Hanada, K., I. Hashimoto, T. Baba, T. Tamura, and S. Kishibe. Melanosis Riehl-like facial pigmentation in a Japanese case of AIDS. J. Dermatol. 21:363–366, 1994. Grob, J. J., S. Bastuji-Garin, L. Vaillant, J. C. Roujeau, P. Bernard, B. Sassolas, and J. C. Guillaume. Excess of nevi related to immunodeficiency: a study in HIV-infected patients and renal transplant recipients. J Invest. Dermatol. 107:694–697, 1996. Hermanns-Lê, T., F. Gerardy-Goffin, M. Giet-Lesuisse, and G. E. Pierard. Ultrastructural study of azidothymidine-induced melanoderma in an AIDS patient [in French]. Ann. Pathol. 13:328–331, 1993. lvker, R., M. Goldaber, and M. R. Buchness. Blue vitiligo. J. Am. Acad. Dermatol. 30(5 Pt 2):829–831, 1994. Janier, M., C. Katlama, B. Flageul, F. Vaiensi, 1. Moulonguet, F. Sigaux, D. Dompmartin, and J. Civatte. The pseudo-Sezary syndrome with CD8 phenotype in a patient with the acquired immunodeficiency syndrome. Ann. Intern. Med. 110:738–740, 1989. Jing, W. A retrospective survey of mucocutaneous manifestations of HIV infection in Malaysia: analysis of 182 cases. J. Dermatol. 27:225–232, 2000. Joyner, S., D. Lee, P. Hay, and R. Lau. Hydroxyurea-induced nail pigmentation in HIV patients. HIV Med. 1:40–42, 1999. Kaplan, M. H., W. Hall, M. Susin, S. Pahwa, S. Z. Salahuddin, C. Heilman, J. Fetten, M. Coronesi, and B. F. Farber. Syndrome of severe skin disease, eosinophilia, and dermatopathic lymphadenopathy in patients with HTLV-II complicating human immunodeficiency virus infection. Am. J. Med. 91:300–307, 1991. Lacour, J. P., D. Dubois, and D. Ortonne. Mélanonychies en bandes au cours du traitement par la zidovudine. Ann. Dermatol. Venereol. 155:1091, 1988. Lacour, J. P., V. Gallais, J. Bodokh, G. Ghanem, and J. P. Ortonne. Cutaneous and mucous hyperpigmentation in AIDS [letter, in French]. Presse Med. 20:1686–1690, 1991. Lacour, J. P., G. Ghanem, and J. P. Ortonne. AIDS and pigmentation. In: Proceedings of the 18th World Congress of Dermatology, W. H. C. Burgdorf, and S. I. Katz (eds). New York: Parthenon Publishing Group, 1993, pp. 513–515. Lacour, J. P., and J. P. Ortonne. AIDS, hypermelanosis, melasma and alphaMSH. J. Eur. Acad. Dermatol. Venerol. 6:96–97, 1996.

Langford, A., H. D. Pohle, and H. Gelderblom. Oral hyperpigmentation in HIV infected patients. Oral Surg. Oral Med. Oral Pathol. 67:301–307, 1989. Laughon, S. K., L. L. Shinn, and J. R. Nunley. Melanonychia and mucocutaneous hyperpigmentation due to hydroxyurea use in an HIV-infected patient. Int. J. Dermatol. 39:928–931, 2000. McGregor, J. M., M. Newell, J. Ross, N. Kirkham, D. H. McGibbon, and C. Darley. Cutaneous malignant melanoma and human immunodeficiency virus (HIV) infection: a report of three cases. Br J. Dermatol. 126:516–519, 1992. Mellor-Pita, S., M. Yebra-Bango, J. Alfaro-Martinez, and E. Suarez. Acanthosis nigricans: a new manifestation of insulin resistance in patients receiving treatment with protease inhibitors. Clin. Infect. Dis. 34:716–717, 2002. Merenich, J. A., R. N. Hannon, R. H. Gentry, and S. M. Harrison. Azidothymidine-induced hyperpigmentation mimicking adrenal insufficiency. Am. J. Med. 86:469–470, 1989. Nelson, M. R., A. G. Lawrence, R. C. Staughton, and B. G. Gazzard. Erythema dyschromicum perstans in an HIV antibody-positive man [letter]. Br. J. Dermatol. 127:658–659, 1992. Nelson, M., and M. Schiavone. Emtricitabine (FTC) for the treatment of HIV infection. Int. J. Clin. Pract. 58:504–510, 2004. Obuch, M. L., G. Baker, R. I. Roth, T. S. Yen, J. Levin, and T. G. Berger. Selective cutaneous hyperpigmentation in mice following zidovudine administration. Arch. Dermatol. 28:508–513, 1992. Panwalker, A. P. Nail pigmentation in the acquired immunodeficiency syndrome. Ann. Intern. Med. 107:943–944, 1987. Pascual, C., V. Garcia-Patos, R. Bartralot, R. Pedragosa, M. Capdevila, J. Barbera, and A. Castells. Pigmentation cutanée, seule manifestation d’une porphyrie cutanée tardive chez un malade séropositif pour le VIH 1. Ann. Dermatol. Venereol. 123:262–264, 1996. Picard-Dahan, C., T. Le Guyadec, M. Grossin, N. Féton, M. Raphaël, A. M. Simonpoli, B. Crickx, and S. Belaich. Erythrodermies pigmentées au cours du SIDA: cinq cas. Ann. Dermatol. Venereol. 123:307–313, 1996. Poizot-Martin, I., A. Lafeuillade, C. Dhiver, L. Xeri, R. Bouabdallah, T. Gamby, and J. A. Gastaut. Cutaneo-mucosal hyperpigmentation in AIDS. 4 cases [in French]. Presse Med. 20:632–636, 1991. Rivers, J. K., A. W. Kopf, and A. H. Postel. Malignant melanoma in a man séropositive for the human immunodeficiency virus. J. Am. Acad. Dermatol. 20:1127–1128, 1989. Rodrigues, L. K., B. J. Klencke, K. Vin-Christian, T. G. Berger, R. I. Crawford, J. R. Miller 3rd, C. M. Ferreira, M. Nosrati, and M. Kashani-Sabet. Altered clinical course of malignant melanoma in HIV-positive patients. Arch. Dermatol. 138:765–770, 2002. Roth, R. I., G. Baker, and J. Levin. An animal model for the study of azidothymidine-induced hyperpigmentation. Lab. Invest. 64:437– 439, 1991. Smith, J. F., and T. P. Flanigan. Unusual pigmentation in patients with AIDS who are receiving rifabutin for bacteremia due to Mycobacterium avium/Mycobacterium intracellulare complexe. Clin. Infect. Dis. 21:1515, 1995. Tadini, G., M. D’Orso, M. Cusini, and E. Alessi. Oral mucosa pigmentation: a new side effect of azidothymidine therapy in patients with acquired immunodeficiency syndrome [letter]. Arch. Dermatol. 127:267–268, 1991. Tosti, A., G. Gaddoni, P. A. Fanti, A. D’Antuono, and F. Albertini. Longitudinal melanonychia induced by 3¢azidodeoxythymidine. Dermatologica 180:217–220, 1990. Tyndall, B., R. Finlayson, K. Mutimer, F. A. Billson, V. F. Munro, and D. A. Cooper. Malignant melanoma associated with human immunodeficiency virus infection in three homosexual men. J. Am. Acad. Dermatol. 20:587–591, 1989. Van Ginkel, C. J. W., R. Tjon Lim Sang, J. L. G. Blaauwgeers, J. K. M. Eeftinck Schattenkerk, W. J. Mooi, and H. J. Hulsebosch. Multiple primary malignant melanomas in an HIV-positive man. J. Am. Acad. Dermatol. 24:284–285, 1991.

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CHAPTER 50

Fig. 50.34. Hyperpigmented melanoacanthoma (see also Plate 50.13, pp. 494–495).

Fig. 50.35. Melanoacanthoma of the oral mucosa.

Venencie, P. Y., Y. Laurian, F. Lemarchand-Venencie, D. Lemay, and F. Verroust. Erythema dyschromicum perstans following human immunodeficiency virus seroconversion in an haemophiliac child. Arch. Dermatol. 124:1013–1014, 1988. Ward, H. A., G. G. Russo, and J. Shrum. Cutaneous manifestations of antiretroviral therapy. J. Am. Acad. Dermatol. 46:284–293, 2002.

Melanoacanthoma Norman Levine and Cynthia Burk

Historical Background In 1927, Bloch (1927) reported a case of an unusual pigmented lesion which he called “melanoepithelioma.” Mishima and Pinkus (1960) coined the term “melanoacanthoma” to describe a benign tumor characterized by proliferating dendritic melanocytes and keratinocytes. They included Bloch’s case but felt that he had failed to clearly identify this entity as separate from a pigmented seborrheic keratosis.

Clinical Findings There are two clinical forms of melanoacanthoma. The first (cutaneous) type is seen mainly in middle-aged to elderly Caucasians of either sex (Delacretaz, 1975; Reaves, 1972); it consists of a solitary, brown or black papule or small to giant-sized plaque (Fig. 50.34) which may be seen in the scalp, neck, back, abdomen, penis, eyelids, or extremities (Delacretaz 1975; Fatahzadeh and Sirois, 2002; Spott et al., 1972; Vion and Merot 1989). There is no malignant potential. These develop slowly and usually have a roughened surface. The second (mucosal) type occurs primarily in young or middle-aged black females (Tomich and Zunt, 1990). Approximately 37 cases of oral melanoacanthoma have been reported between 1978 and 2002 (Fatahzadeh and Sirois, 2002). It occurs on the lips, oropharynx, or oral mucosa (Fig. 50.35), including the hard palate, labial mocosa, alveolar mucosa, and fixed gingival mucosa (Fornatora et al., 2003; Matsuoka et al., 1982; Sexton and Maize 1987). It may be slightly verrucous but, generally, is flat and asymptomatic (Eisen and 946

Fig. 50.36. The clear cells in the mid epidermis are melanocytes.

Voorhees, 1991; Frey et al., 1984; Wright 1988). Pruritus, burning, or pain is infrequent. The lesions are solitary (90%), dark brown, black, or blue-black in color, and are 0.5–6.0 cm in size (Prince et al., 1984). They arise within a few weeks, and usually resolve after biopsy or removal of irritating stimuli. Cheek-biting, smoking, and ill-fitting dentures are the most common irritants. There are no associated medical conditions. Oral melanoacanthoma is unrelated to seborrheic keratosis (Fornatora et al., 2003). The lesion has no malignant potential. Clinical findings are nondiagnostic, thus biopsy is suggested (Fatahzadeh and Sirois, 2002).

Pathology Histopathologic examination of the cutaneous melanoacanthoma reveals a hyperplastic epidermis with papillomatosis, acanthosis, and pseudo-horn cyst formation. The distinguishing characteristic is dendritic melanocytes throughout the basal and all spinous layers (Figs 50.36 and 50.37). Most of the melanin is contained within the melanocytes. Ultrastructural study shows a partial blockage of melanin transfer to the

ACQUIRED EPIDERMAL HYPERMELANOSES

suggest that oral melanoacanthoma is a misnomer and have proposed the term, mucosal melanotic macule, reactive type (Horlick et al., 1988). The fact that these mucosal lesions arise and remit quickly following removal of offending stimuli would support this concept.

Treatment

Fig. 50.37. Close-up view of a dendritic melanocyte (FontanaMasson stain).

keratinocytes (Schlappner et al., 1978). Cells in mitosis may be seen but there is no atypia. Dermal melanophages are present. In the mucosal type, spongiotic intraepithelial vesicles (Zemtsov and Bergfeld, 1989), lymphohistiocytic dermal infiltrate admixed with eosinophils and increased vascularity may be seen (Fornatora et al., 2003). Strong HMB-45 reactivity is present in most cases of oral melanoacanthoma, making the diagnosis of melanoma difficult to exclude (Fornatora et al., 2003).

Differential Diagnosis The pigmented seborrheic keratosis does not have the prominent dendritic melanocytes with heavy pigmentation and melanin transfer blockage; although up to 38% of seborrheic keratosis demonstrate some degree of dendritic melanocytes (Simon et al., 1991). Some seborrheic keratoses show dendritic melanocytes within the islands of basaloid cells. Physiologic pigmentation shows basal hyperpigmentation. Nevocellular nevus (Buchner and Hansen, 1979), blue nevus, melanoma (Lambert et al., 1985, 1987), and pigmented basal cell carcinoma have distinctive pathologic findings. Dermatofibroma is a dermal tumor. The labial melanotic macule shows only increased numbers of melanocytes along the basal cell layer (Weathers et al., 1976). Amalgam tattoos show dermal granules. Kaposi sarcoma in HIV patients is a vascular lesion. Fixed drug eruption may affect the tongue (Tagami, 1973). Smoker’s melanosis is more diffuse than melanoacanthoma (Hedin, 1977).

Pathogenesis The pathogenesis of melanoacanthoma is unknown but it is suggested that the cutaneous melanoacanthoma is an irritated, pigmented seborrheic keratosis (Mevorah and Mishima, 1965; Simon et al., 1991). The mucosal type is a reactive lesion seen in black people due to their physiologic oral pigmentation in association with pigment lability following trauma or irritation. The term “melanoacanthosis” might better fit the clinicopathologic presentation (Tomich and Zunt, 1990). Others

Cutaneous melanoacanthomas may easily be removed surgically. A specimen for pathologic study should be obtained to rule out melanoma (Whitt et al., 1988). Mucosal lesions should also be biopsied since melanoma may be clinically identical to melanoacanthoma. Several oral melanoacanthomas have been reported to regress spontaneously after biopsy (Fatahzadeh and Sirois 2002; Fornatora et al., 2003). Removal of offending irritants generally leads to remission of oral melanoacanthoma.

References Bloch, B. Uber benigne nicht naevoide Melanoepitheliome der Haut nebst Bemerkungen uber das Wesen und die Genese der Dendritenzallon. Arch. Dermatol. Syphil. (Berlin) 153:20–40, 1927. Buchner, A., and L. S. Hansen. Pigmented nevi of the oral mucosa: A clinicopathologic study of 32 new cases and review of 75 cases from the literature. Part I. A clinicopathologic study of 32 new cases. Oral Surg. 48:131–142, 1979. Delacretaz, J. Melana Acanthome. Dermatologica 151:236–240, 1975. Eisen, D., and J. J. Voorhees. Oral melanoma and other pigmented lesions of the oral cavity. J. Am. Acad. Dermatol. 24:527–537, 1991. Fatahzadeh, M., and D. A. Sirois. Multiple intraoral melanoacanthomas: a case report with unusual findings. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodont. 94:54–56, 2002. Fornatora, M. L., R. F. Reich, S. Haber, F. Solomon, and P. D. Freedman. Oral melanoacanthoma: a report of 10 cases, review of the literature, and immunohistochemical analysis for HMB-45 reactivity. Am. J. Dermatopathol. 25:12–15, 2003. Frey, V. M., W. C. Lambert, R. D. Seldin, L. C. Schneider, and M. L. Mesa. Intraoral melanoacanthoma. J. Surg. Oncol. 27:93–96, 1984. Hedin, C. A. Smoker’s melanosis. Arch. Dermatol. 113:1533–1538, 1977. Horlick, H. P., R. R. Walther, D. J. Zagarelli, D. N. Silvers, and Y. D. Eliezri. Mucosal melanotic macule, reactive type: a simulation of melanoma. J. Am. Acad. Dermatol. 19:786–791, 1988. Lambert, M. W., W. C. Lambert, R. A. Schwartz, M. L. Mesa, R. H. Brodkin, A. H. Abbey, G. K. Potter, and W. P. Little Jr. Colonization of nonmelanocytic cutaneous lesions by dendritic melanocytic cells: a stimulant of acral-lentiginous (palmar-plantar-subungualmucosal) melanoma. J. Surg. Oncol. 28:12–18, 1985. Lambert, W. C., M. W. Lambert, M. L. Mesa, L. C. Scheider, C. G. Fischman, A. H. Abbey, R. H. Brodkin, M. D. Elam, W. A. Anderson, and G. K. Potter. Melanoacanthoma and related disorders: Simulants of acral-lentiginous (PPSM) melanoma. Int. J. Dermatol. 26:508–510, 1987. Matsuoka, L. Y., S. Glasser, and S. Barsky. Melanoacanthoma of the lip. Arch. Dermatol. 115:1116, 1979. Matsuoka, L. Y., S. Barsky, S. Barsky, and S. Glasser. Melanoacanthoma of the lip. Arch. Dermatol. 118:290, 1982. Mevorah, B., and Y. Mishima. Cellular response of seborrheic keratosis following croton oil irritation and surgical trauma with special reference to melanoacanthoma. Dermatologica 131:452–464, 1965.

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CHAPTER 50 Mishima, Y., and H. Pinkus. Benign mixed tumor of melanocytic and malphigian cells. Melanoacanthoma: Its relationship to Bloch’s benign non-nevoid melanoepithelioma. Arch. Dermatol. 81:539– 550, 1960. Prince, C., A. H. Mehregan, K. Hashimoto, and H. Plotnick. Large melanoacanthomas: A report of 5 cases. J. Cutan. Pathol. 11:309– 317, 1984. Reaves, C. E. Melanoacanthoma: A case report and review of the literature. Bull. Assoc. Mil. Dermatol. 22:55–58, 1972. Schlappner, O. L., G. Rowden, T. M. Philips, and Z. Raham. Melanoacanthoma: Ultrastructural and immunological studies. J. Cutan. Pathol. 5:127–141, 1978. Schneider, L. C., M. L. Mesa, and S. M. Haber. Melanoacanthoma of the oral cavity. Oral Surg. 52:284–287, 1981a. Schneider, L. C., M. L. Mesa, and L. Moore. Melanoacanthoma: A ten-year prospective study. J. Oral Pathol. 10:368, 1981b. Sexton, F. M., and J. C. Maize. Melanotic macules and melanoacanthomas of the lip. Am. J. Dermatopathol. 9:438–444, 1987. Simon, P., L. Requena, and Y. E. Sanchez. How rare is melanoacanthoma? Arch. Dermatol. 127:583–584, 1991. Spott, D. A., M. G. Wood, and C. L. Heaton. Melanoacanthoma of the eyelid. Arch. Dermatol. 115:898–899, 1972. Tagami, H. Pigmented macules of the tongue following fixed drug eruption. Dermatologica 147:157, 1973. Tomich, C. E., and J. L. Dorey. Melanoacanthoma of the oral mucosa. Annual Meeting of the American Academy of Oral Pathologists. 1979. Tomich, C. E., and S. L. Zunt. Melanoacanthosis (melanoacanthoma) of the oral mucosa. J. Dermatol. Surg. Oncol. 16:231–236, 1990. Vion, B., and Y. Merot. Melanoacanthoma of the penis shaft: Report of a case. Dermatologica 179:87–89, 1989. Weathers, D. R., R. L. Corio, B. E. Crawford, J. S. Giansanti, and L. R. Page. The labial melanotic macule. Oral Surg. Oral Med. Oral Pathol. 42:196–205, 1976. Whitt, J. C., D. R. Jennings, D. M. Arendt, and J. R. Vinton. Rapidly expanding pigmented lesion of the buccal mucosa. J. Am. Dent. Assoc. 117:620–622, 1988. Wright, J. M. Intraoral melanoacanthomas: a reactive melanocytic hyperplasia: Case report. J. Periodontol. 59:53–55, 1988. Wright, J. M., W. H. Bonnie, D. L. Byrd, and A. R. Dunsworth. Intraoral melanoacanthoma. J. Periodontol. 54:107–111, 1983. Zemtsov, A., and W. F. Bergfeld. Oral melanoacanthoma with prominent spongiotic intraepithelial vesicles. J. Cutan. Pathol. 16:365– 369, 1989.

biologic systems. In 1942, Klaber coined the term “phytophotodermatitis” to specify the skin’s reaction to sunlight after contact with certain plants (Heskel et al., 1983).

Synonyms Berloque dermatitis, “Club Med dermatitis”, phototoxic dermatitis, strimmer rash (“string trimmers’ dermatitis”), “celery burn,” dermatitis bullosa striata pretensis.

Epidemiology Phytophotodermatitis is an inflammatory cutaneous response to plant phototoxic chemicals, known as furocoumarins or psoralens. This in conjunction with ultraviolet light results in hyperpigmentation. The incidence varies geographically with the type of local flora and the likelihood of human–plant contact. The exact incidence of this problem is unknown. Individuals having physical contact with plants containing furocoumarins with subsequent sun exposure are at risk for developing phytophotodermatitis. Field workers, gardeners, chefs, and grocers are most commonly affected. In certain regions of the world, psoralen-containing plant species are endemic or are even used for esthetic foliage (Camm et al., 1976). Unsuspecting nature lovers have had severe consequences after rubbing phototoxic leaves on their skin as an insect repellent (Gawkrodger and Savin, 1983).

Clinical Findings People must be exposed to both psoralen-containing plants and other sources such as perfumes, and ultraviolet A (UVA) light to develop phytophotodermatitis. Psoralen exposure occurs after contact with crushed surfaces or the sap of the culprit plant. For example, sliced fruit permits exposure to psoralens in the peel (Egan and Sterling, 1993). In a typical patient, the first physical finding may be sharply demarcated, hyperpigmented macules or patches within days of exposure (Figs 50.38 and 50.39). The color of the lesions is usually tan or brown (Tunget et al., 1994). They appear at sites of plant

Phytophotodermatitis Norman Levine and Cynthia Burk

Historical Background For 3000 years, the sap of certain roots and flowers has been known to cause skin blistering (Levine, 1993; Moller, 1978). Some oils from these plants produced skin pigmentation, and were applied topically as therapy for vitiligo (Egan and Sterling, 1993). In 1916, Freund described berloque dermatitis, which occurred as a result of skin exposure to oil of bergamot in cologne (Egan and Sterling, 1993). Oppenheim speculated in 1932 that “itching blisters” occurring in meadow sunbathers were the result of photosensitization caused by either an insect in the field or perhaps the chlorophyll in the grass. He named the phenomenon “dermatitis bullosa striata pretensis.” Blum was one of the first to demonstrate that light of specific wavelengths has direct effects on 948

Fig. 50.38. Typical figurate pattern of hyperpigmentation caused by phytophotodermatitis.

ACQUIRED EPIDERMAL HYPERMELANOSES

contact and sun exposure. Hyperpigmentation may be the only clinical sequela if erythema-provoking doses of UVA light are not absorbed. If present, the acute dermatitis occurs within 24–48 hours of exposure (Egan and Sterling, 1993; Heskel et al., 1983), although in one case a 16-day interval between plant contact and sun exposure was reported (Tunget et al., 1994). Involvement is limited to light-exposed areas of skin, most commonly the face, neck, and dorsal aspects of the extremities. Patients may develop linear or bizarre-shaped, erythematous plaques and vesicles at the sites of plant contact. Vesicles may coalesce into bullae coexistent with the erythematous streaks. A mild sunburn-like erythema may be the only acute change. A burning sensation occasionally accompanies the lesions. Pruritus is not a characteristic symptom. Within 3–10 days, denudation of blisters and desquamation occurs (Egan and Sterling, 1993). The patient often has residual hyperpigmentation which may persist up to six months after exposure. Spontaneous resolution of the dermatitis usually occurs; however, one case severe enough to require five days of inpatient treatment has been reported (Gawkrodger and Savin, 1983; Webb and Brooke, 1995). Phototoxicity is a dose-related phenomenon, and should theoretically occur in 100% of individuals receiving the sufficient doses of psoralen and UVA radiation (Harber and Baer, 1972). Failure to elicit such a reaction happens because of variable psoralen content in plants and individual patient factors influencing absorption of phototoxins. Furocoumarin content in plants can vary seasonally and regionally in the same species (Finkelstein et al., 1994; Rook et al., 1992). As phytophotodermatitis is not an allergic or immune-mediated process, it can occur with the first exposure. Compared to the frequency of substrate exposures, dermatitis due to phytophototoxins is quite unusual.

Criteria for Diagnosis ∑ History of contact with psoralen-containing plant with sub-

sequent UVA light exposure. ∑ Physical examination shows hyperpigmented patches, which

may be preceded by linear erythema and vesicles or bullae on sun-exposed areas of skin.

Differential Diagnosis The differential diagnosis of berloque dermatitis includes other causes of hyperpigmentation, eruptions from contact with plants, and those related to sunlight exposure. Given a specific plant-related history, the diagnoses to be considered include irritant contact dermatitis and allergic contact dermatitis. If sun exposure alone precedes the eruption, the etiology may be a medication-induced phototoxic or photoallergic reaction, polymorphous light eruption, lupus erythematosus, pellagra, or suntan. Without a history of plant, perfume, or light exposure, hyperpigmentation may be due to melasma. Other distinctions to be made are between phytophotodermatitis and other vesiculobullous diseases such as

Fig. 50.39. Brown hyperpigmentation caused by application of topical psoralens and indiscriminate exposure to sunlight (see also Plate 50.14, pp. 494–495).

erythema multiforme, bullous pemphigoid, pemphigus vulgaris, and porphyria cutanea tarda. Trauma from physical abuse must be considered in children with linear inflammatory plaques and vesicles (Barradell et al., 1993; Weber, 1999). These patterned skin lesions may look like burns or hand prints. Therefore, it is important to identify phytophotodermatitis by its uniformly dark color, absence of definite dermatomal pattern, and its distribution within the sunlight exposed areas of the skin. Fungal infections and bacterial cellulitis are other diagnoses to rule out. Paederus dermatitis created by crushing the causative beetle over the skin can also mimic phytophotodermatitis (Uslular et al., 2002; Weber et al., 1999). Irritant contact dermatitis from plants may present acutely as vesiculobullous, erythematous, or urticarial lesions. However, chronic hyperpigmentation is not a prominent feature. Unlike phytophotodermatitis, the eruption does not require UVA light exposure (Barradell et al., 1993). Allergic contact dermatitis from plants, unlike phytophotodermatitis, requires prior sensitization. The source of allergen is most commonly the Rhus family of plants. Minimal contact is sufficient to elicit the allergic reaction, while phytophotodermatitis occurs only when significant doses are absorbed. The clinical pattern may be similar, but allergic contact dermatitis may occasionally have more diffuse patchy erythema. Pruritus is almost always present. Medication-induced phototoxic reactions appear similar to an exaggerated sunburn. During the acute phase they may resemble phytophotodermatitis, but prolonged hyperpigmentation is not a feature of drug-induced phototoxic reactions. Drugs implicated in this reaction include amiodarone, doxycycline, thiazides, furosemide, sulfonamides, and phenothiazines. Photoallergic reactions caused by medications may appear identical to phytophotodermatitis. In contrast to berloque dermatitis, prolonged skin tanning in the affected sites is not a feature of this condition. Berloque dermatitis presenting with hyperpigmentation alone may resemble a suntan because of the similar brown 949

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patches on sun-exposed skin. However, the two are clinically different in several ways. A suntan has an immediate pigment darkening effect, followed by a delayed darkening which peaks at about 72 hours. Berloque dermatitis pigmentation may appear between 24 and 48 hours, but persists as long as six months. Whereas suntan darkens all exposed skin diffusely, psoralen and UVA light produces well-circumscribed lesions, often with bizarre shapes. Lentigines are tan to dark brown macules which are suninduced and may resemble the lesions of berloque dermatitis. However, each lentigo is rarely larger than 20 mm; multiple lentigines are not confluent. Once present, they do not fade. Melasma is the brown “mask of pregnancy” frequently seen in women either during pregnancy or while taking oral contraceptives. These well-defined macules or patches can be aggravated by sunlight. In contradiction to phytophotodermatitis, melasma appears commonly on the face and has a more reticulated pattern.

Pathology Light Microscopy The histologic picture in the acute, inflammatory stage is that of contact dermatitis. There is epidermal spongiosis with intraepidermal vesicles and a dermal infiltrate. Very mild lesions may show only single-cell epidermal necrosis. In the later phase, there may be hyperkeratosis, slight epidermal thinning, dermal–epidermal junction vacuolization, and parakeratosis. A perivascular lymphohistiocytic infiltrate is present. Hyperpigmentation can be seen in the epidermis and dermis. Pigmentary incontinence in the form of dermal melanin is found both extracellularly and within melanophages (Egan and Sterling, 1993; Heskel et al., 1983; Webb and Brooke, 1995). Increased epidermal melanin, much like that seen with a suntan, is seen in the chronic cutaneous hyperpigmentation phase of berloque dermatitis.

Pathogenesis The plant families known to cause phytophotodermatitis include the Umbelliferae, the Rutaceae, the Moraceae, and the Leguminosae. These include edible vegetables such as celery, parsnip, parsley, dill, carrots, citrus fruits such as limes and oranges, and other plants such as giant hogweed, berryrue, and wild rhubarb (Egan and Sterling, 1993; Tunget et al., 1994). Tobacco, garlic, fig, mustard, buttercup, corn, fennel, angelica, masterwort, atrillal, hot peppers, daffodil bulbs, and hyacinth may also contain natural psoralens (Adams 1998; Bergeson and Weiss 2000; Bollero et al., 2001; Bosch 1997). Psoralens may be found in certain brands of perfumes and cosmetics as well as Saint John’s Wort (Adams, 1998). In experimental studies, high furocoumarin concentrations cause photohemolysis of red blood cells (Bollero et al., 2001). Rarely, reports of wound sepsis and retinal hemorrhages can complicate the course of the illness (Bollero et al., 2001). Psoralens may be phytoalexins, protective compounds made by the plant in response to stress or infection. Skin contact with the plant causes exposure to psoralens, which when activated by UVA light leads to skin inflam950

mation and increased melanocyte activity, producing increased cutaneous pigmentation. Furocoumarins are a group of molecules related to coumarin. They have a double-ringed structure attached to a furan ring. At least 28 furocoumarins are found naturally in plants, but only three are commonly implicated in phytophotodermatitis. These are 8-methoxypsoralen (xanthotoxin or methoxsalen), 5-methoxypsoralen (bergapten), and 4,5¢,8trimethylpsoralen (TMP) (Harber and Baer, 1972; Yurkow and Laskin, 1991). Furocoumarins are chromophores, molecules which absorb radiation. Absorption of one photon increases the energy of the molecule, exciting one electron into a higher orbit, which creates a transiently reactive species. This process photoactivates the furocoumarin. The absorption spectrum specific for each furocoumarin molecule is within the UVA spectrum (320–400 nm). The action spectrum, which specifies the wavelength necessary to provoke furocoumarin phototoxicity, is close to the absorption spectrum, ranging from 312 nm to 406 nm for different molecules (Musajo and Rodighiero, 1970; Yurkow and Laskin, 1991). Skin injury occurs after the furocoumarins traverse the stratum corneum and enter the keratinocytes. Inside the nuclei of these cells, the furocoumarins react with DNA. In the dark, they intercalate between adjacent base pairs in the double helix with weak chemical bonds (Musajo and Rodighiero, 1970). In this circumstance, no permanent reaction takes place. However, a UVA light-activated furocoumarin can bind specifically and covalently to pyrimidine bases. Two monoadducts are possible via this reaction. Cycloaddition can take place between the 5,6positions on thymine or uracil and either the 4¢,5¢- or the 3,4diene on the psoralen. With the addition of a second photon, 3,4-monoadducts covalently cross-link between strands of DNA, forming a bifunctional adduct. This is structurally analogous to interstrand thymine dimerization, preventing strand separation for DNA replication (Harber and Baer, 1972; Musajo and Rodighiero, 1970). This inhibits DNA synthesis and may cause cell death (Ena et al., 1989). In addition to interacting with nucleic acids, psoralens may also bind to cell proteins and membrane lipids. Two types of reactions are postulated to occur: type I, or anoxic reactions, produce DNA and amino acid interactions; type II, or oxygendependent reactions, produce reactive oxygen species, causing membrane lipid damage and cellular destruction. This inflammatory response is not an immune-mediated process, and immunocompetence is not required to develop phytophotodermatitis. Neither ultraviolet light nor psoralen alone produces these effects. Phytophotodermatitis is a dose-dependent reaction. A sufficient psoralen concentration must be present with adequate exposure to UVA light. The dose relationship between UV and psoralens may be synergistic rather than additive (Yurkow and Laskin, 1991). Psoralen absorption influences whether this reaction takes place; this is facilitated by moisture, a thin stratum corneum, and minimal epidermal melanin (Izumi and Dawson, 2002). UV light must be of a wavelength com-

ACQUIRED EPIDERMAL HYPERMELANOSES

patible with the action spectrum of the psoralens. At suberythemogenic doses, the result is hyperpigmentation without inflammation. The hyperpigmentation of berloque dermatitis is caused by stimulation of melanocytes in the basal epidermis and hair follicle infundibulum, inducing melanogenesis. It also causes melanocyte migration into affected areas (Levine, 1993). The mechanism of this effect is unknown. In more severe cases, postinflammatory dermal melanin deposition may be an element of the increased pigmentation as well (Gould et al., 1995).

Treatment Management of acute phytophotodermatitis is aimed at diminishing symptoms and inflammation. Topical corticosteroids are effective in most cases. Cool compresses as well as nonsteroidal anti-inflammatory medications may help reduce pain and erythema. Systemic corticosteroids are rarely required. Hyperpigmentation does not respond to topical corticosteroid therapy (Egan and Sterling, 1993; Tunget et al., 1994). The discoloration does tend to diminish gradually over several months. Long-term prevention requires knowledge of offending substances and avoidance of contact. Perfumes containing oil of bergamot should be avoided when sun exposure is anticipated. Certain work situations, such as grocery store checkout stands, may predispose employees to exposure to celery or limes. Individuals working in these environments may benefit from wearing protective gloves; stores can also institute more protective packaging (Webb and Brooke, 1995).

Prognosis The prognosis for patients who develop phytophotodermatitis is excellent. Spontaneous resolution usually occurs without scarring in three to five days. The chronic sequela consists of sustained hyperpigmentation. This fades after weeks to months (Barradell et al., 1993; Webb and Brooke, 1995). Rarely, hypertrophic scarring can result from severe involvement (Bollero et al., 2001).

References Adams, S. P. Dermacase: Phytophotodermatitis. Can. Fam. Physician 44:503–509, 1998. Barradell, R., A. Addo, A. J. McDonagh, M. J. Cork, and J. K. Wales. Phytophotodermatitis mimicking child abuse. Eur. J. Pediatr. 152:291–292, 1993. Bergeson, P. S., and J. C. Weiss. Picture of the month. Phytophotodermatitis. Arch. Pediatr. Adolesc. Med. 154:201–202, 2000. Bollero, D., M. Stella, A. Rivolin, P. Cassano, D. Risso, and M. Vanzetti. Fig leaf tanning lotion and sun-related burns: case reports. Burns 27:777–779, 2001. Bosch, J. J. Phytophotodermatitis. J. Pediatr. Health Care 11:84, 97–98, 1997. Camm, E., H. W. Buck, and J. C. Mitchell. Phytophotodermatitis from Heracleum mantegazzianum. Contact Dermatitis 2:68–72, 1976. Egan, C. L., and G. Sterling. Phytophotodermatitis: a visit to Margaritaville. Cutis 51:41–42, 1993. Ena, P., G. Dessi, F. Chiarolini, and P. Fabbri. Phytophotodermatitis from a Cachrys species. Contact Dermatitis 20:144–145, 1989.

Finkelstein, E., U. Afek, E. Gross, N. Aharoni, L. Rosenberg, and S. Halevy. An outbreak of phytophotodermatitis due to celery. Int. J. Dermatol. 33:116–118, 1994. Gawkrodger, D. J., and J. A. Savin. Phytophotodermatitis due to common rue (Ruta graveolens). Contact Dermatitis 9:224, 1983. Gould, J. W., M. G. Mercurio, and C. A. Elmets. Cutaneous photosensitivity diseases induced by exogenous agents. J. Am. Acad. Dermatol. 33:551–573, 1995. Harber, L. C., and R. L. Baer. Pathogenic mechanisms of drug-induced photosensitivity. J. Invest. Dermatol. 58:327–342, 1972. Heskel, N. S., R. B. Amon, F. J. Storrs, and C. R. White Jr. Phytophotodermatitis due to Ruta graveolens. Contact Dermatitis 9:278–280, 1983. Izumi, A. K. and K. L. Dawson. Zabon phytophotodermatitis: first case reports due to Citrus maxima. J. Am. Acad. Dermatol. 46(5 Suppl): S146–147, 2002. Levine, N. Pigmentation and Pigmentary Disorders. Boca Raton, FL: CRC Press, 1993. Moller, H. Phototoxicity of Dictamnus alba. Contact Dermatitis 4:264–269, 1978. Musajo, L., and G. Rodighiero. Studies on the photo-C4-cycloaddition reactions between skin-photosensitizing furocoumarins and nucleic acids. Photochem. Photobiol. 11:27–35, 1970. Rook, A., D. S. Wilkinson, and F. J. G. Ebling. Textbook of Dermatology, 5th ed. London: Blackwell Scientific Publications, 1992. Tunget, C. L., S. G. Turchen, A. S. Manoguerra, R. F. Clark, and D. E. Pudoff. Sunlight and the plant: a toxic combination: severe phytophotodermatitis from Cneoridium dumosum. Cutis 54:400– 402, 1994. Uslular, C., H. Kavukcu, D. Alptekin, M. A. Acar, Y. G. Denli, H. R. Memisioglu, and H. Kasap. An epidemicity of Paederus species in Cukurova region. Cutis 69:277–279, 2002. Webb, J. M., and P. Brooke. Blistering of the hands and forearms. Arch. Dermatol. 131:833–834, 1995. Weber, I. C., C. P. Davis, and D. M. Greeson. Phytophotodermatitis: the other “lime” disease. J. Emerg. Med. 17:235–237, 1999. Yurkow, E. J., and J. D. Laskin. Mechanism of action of psoralens: isobologram analysis reveals that ultraviolet light potentiation of psoralen action is not additive but synergistic. Cancer Chemother. Pharmacol. 27:315–319, 1991.

Polyneuropathy, Organomegaly, Endocrinopathy, M Protein, and Skin Changes: POEMS Syndrome James J. Nordlund

Historical Background The first report of a plasma cell dyscrasia associated with polyneuritis was published in 1968 (Shimpo, 1968).

Synonyms The proper name for this syndrome is “Polyneuropathy, Organomegaly, Endocrinopathy, M protein with Skin changes”. The acronym POEMS syndrome (Bardwick et al., 1980) has become the common name.

Epidemiology The first case of POEMS syndrome was reported in the Japanese literature (Shimpo, 1968). It was thought that this 951

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syndrome most commonly affected Japanese males. However review of the literature does not seem to substantiate this suggestion (Bardwick et al., 1980). Males and females seem to be affected about equally with some predilection for males (Fam et al., 1986). The median age of 38 patients with POEMS was 51 years (range 32–83 years) (Miralles et al., 1992).

Clinical Description This syndrome is defined as the association of polyneuropathy, organomegaly, endocrinopathy, the presence of an M protein in the serum, and skin changes. Each of the five features has an array of manifestations. The polyneuropathy affects peripheral nerves of both extremities (Bardwick et al., 1980; Ku et al., 1995; Miralles et al., 1992; Romas et al., 1992). The cranial nerves tend to be spared but edema of the optic nerve and papilledema have been reported (Bardwick et al., 1980; Bolling and Brazis, 1990; Romas et al., 1992). One patient reported in the literature had all features of this syndrome but did not have neuropathy (Morrow et al., 1982). Polyneuropathy is not a common feature of most disorders associated with plasma cell dyscrasias. Typically the neuropathy of plasma cell disorders is caused by deposition of amyloid in the nerve (Miralles et al., 1992). Patients with POEMS syndrome have a focal or segmental demyelinating disorder with axonal degeneration (Romas et al., 1992). Both sensory and motor nerves are involved (Ku et al., 1995) and the patient can be disabled from resultant muscle degeneration (Bardwick et al., 1980). Painful dysesthesias and paresthesias are common. Nerve biopsies confirm the loss of myelinated fibers with some nerves showing wallerian degeneration. A small number of axonal fibers showed grossly thickened myelin sheaths. Amyloid was not observed (Romas et al., 1992). Organomegaly most commonly is manifested by hepatomegaly, splenomegaly, and/or lymphadenopathy. The number of affected individuals who have one or more organs enlarged has been reported to be as low as 25% (Miralles et al., 1992) to as high as 67% (Fam et al., 1986). Liver biopsies on some patients were normal in appearance (Bardwick et al., 1980). Endocrine abnormalities are numerous in type. In one series of 38 patients, hypothyroidism, elevated serum prolactin levels, low serum testosterone, impotence, gynecomastia, and/or elevated gonadotropin levels were noted (Miralles et al., 1992). These abnormalities were also noted in a carefully studied two patients (Bardwick et al., 1980). About 65% of affected individuals have some form of endocrine disorder (Fam et al., 1986). Almost all patients reported had a plasma cell abnormality (Miralles et al., 1992). These included plasmacytomas in the skin (Feddersen et al., 1989), lytic or sclerotic tumors in bone (Bardwick et al., 1980; Chan et al., 1990; Miralles et al., 1992; Puig et al., 1985; Romas et al., 1992; Shelley and Shelley, 1987), or benign gammopathies (Miralles et al., 1992; Morrow et al., 1982; Rongioletti et al., 1994). The most

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Fig. 50.40. Hyperpigmentation of the leg (see also Plate 50.15, pp. 494–495).

Fig. 50.41. Diffuse hyperpigmentation of the dorsum of hands (see also Plate 50.16, pp. 494–495).

common gammopathy is of the IgA form but IgG, l-light chain, and indeterminate monoclonal peaks have been noted. Skin changes were also of multiple types. The most common is a diffuse brown hyperpigmentation observed in about half

ACQUIRED EPIDERMAL HYPERMELANOSES

Fig. 50.42. Close-up view of the pigmentary changes.

Fig. 50.43. Glomeruloid angiomata characteristic of POEMS. Note the diffuse hyperpigmentation commonly observed in patients with POEMS (see also Plate 50.17, pp. 494–495).

of affected individuals (Bardwick et al., 1980; Feddersen et al., 1989; Ishikawa et al., 1987; Miralles et al., 1992; Morrow et al., 1982; Romas et al., 1992; Shelley and Shelley, 1987). The discoloration affects the entire integument including the palms and genitalia (Figs 50.40 and 50.41). There are many other cutaneous abnormalities. The skin has been noted to be thickened and almost sclerodermoid in quality (Fig. 50.42). Hypertrichosis also is common, especially over skin not typically hirsute (Miralles et al., 1992; Shelley and Shelley, 1987). The skin is a common site for tumors of vascular origin. These include angiomata (Kanitakis et al., 1988; Puig et al., 1985), glomeruloid hemangiomata (Fig. 50.43) (Chan et al., 1990; Del Rio et al., 1994; Hudnall et al., 2003; Ishikawa et al., 1987; Kanitakis et al., 1988; Kingdon et al., 2001; Longo et al., 1999; Rongioletti et al., 1994; Obermoser et al., 2003; Scheers et al., 2002; Tsai et al., 2001), and miscellaneous other vascular neoplasms (Tsang et al., 1991). The glomeruloid angiomata have been attributed to herpesvirus 8 by some investigators (Belec et al., 1999a, b; Papo et al., 1999; Tohda et al., 2001; Hudnall et al., 2003). Others have not found this virus in the angiomata (Obermoser et al., 2003; Zumo and Grewal 2002).

often lower than normal. Levels of luteinizing hormone or prolactin are elevated. The serum in most individuals will have an M protein. No one finding is specific. No feature is found in all individuals although neuropathy and the presence of a plasma cell dyscrasia with an associated M protein are very common clinical features.

Histology

References

The histologic features of the pigmentation are not specific. There seems to be increased melanin in the basilar layer of the epidermis (Feddersen et al., 1989; Tsang et al., 1991). Changes related to deposition of mucin-like proteins and the various vascular neoplasms are found in dermatopathology textbooks.

Laboratory Findings There are many laboratory abnormalities because of the multiplicity of the tissues and organs involved by this syndrome. Nerve conduction studies are typical of a demyelinating disorder. Serum levels of sex steroids such as testosterone are

Pathogenesis Elevated levels of interleukin (IL)-1b (Gherardi et al., 1994; Tartour et al., 1994) and IL-6 have been reported (Fukatsu et al., 1992; Gherardi et al., 1994; Mandler et al., 1992; Tartour et al., 1994). The kidneys also have been found to have increased quantities of IL-6 in some of the cells of the glomerulus (Fukatsu et al., 1992). This has led some to propose that elevated cytokines, particularly IL-6, are major factors in causing this syndrome. More recently vascular endothelial growth factor (VEGF) has been identified in the blood of patients with POEMS syndrome (Hashiguchi et al., 2000; Minamitani et al., 2002; Niimi et al., 2000; Soubrier et al., 1999; Watanabe et al., 1996, 1998). It has been proposed as a factor for the formation of the angiomatous lesions in the skin.

Bardwick, P. A., N. J. Zvaifler, G. N. Gill, D. Newman, G. D. Greenway, and D. L. Resnick. Plasma cell dyscrasia with polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes: the POEMS syndrome. Report on two cases and a review of the literature. Medicine 59:311–322, 1980. Belec, L., F. J. Authier, A. S. Mohamed, M. Soubrier, and R. K. Gherardi. Antibodies to human herpesvirus 8 in POEMS (polyneuropathy, organomegaly, endocrinopathy, M protein, skin changes) syndrome with multicentric Castleman’s disease. Clin. Infect. Dis. 28:678–679, 1999a. Belec, L., A. S. Mohamed, F. J. Authier, M. C. Hallouin, A. M. Soe, S. Cotigny, P. Gaulard, and R. K. Gherardi. Human herpesvirus 8 infection in patients with POEMS syndrome-associated multicentric Castleman’s disease. Blood 93:3643–3653, 1999b.

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CHAPTER 50 Bolling, J. P., and P. W. Brazis. Optic disk swelling with peripheral neuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS syndrome). Am. J. Ophthalmol. 109:503–510, 1990. Chan, J. K., C. D. Fletcher, G. A. Hicklin, and J. Rosai. Glomeruloid hemangioma: a distinctive cutaneous lesion of multicentric Castleman’s disease associated with POEMS syndrome. Am. J. Surg. Pathol. 14:1036–1046, 1990. Del Rio, R., M. Alsina, J. Monteagudo, D. Torremorell, U. Gonzalez, J. Luelmo, and J. M. Mascaro. POEMS syndrome and multiple angioproliferative lesions mimicking generalized histiocytomas. Acta Derm. Venereol. 74:388–390, 1994. Fam, A. G., J. D. Rubenstein, and D. H. Cowan. POEMS syndrome. Study of a patient with proteinuria, microangiopathic glomerulopathy, and renal enlargement. Arthritis Rheum. 29:233–241, 1986. Feddersen, R. M., W. Burgdorf, K. Foucar, L. Elias, and S. M. Smith. Plasma cell dyscrasia: a case of POEMS syndrome with a unique dermatologic presentation. J. Am. Acad. Dermatol. 21:1061–1068, 1989. Fukatsu, A., Y. Ito, Y. Yuzawa, F. Yoshida, M. Kato, K. Miyakawa, and S. Matsuo. A case of POEMS syndrome showing elevated serum interleukin 6 and abnormal expression of interleukin 6 in the kidney. Nephron 62:47–51, 1992. Gherardi, R. K., L. Belec, G. Fromont, M. Divine, D. Malapert, P. Gaulard, and J. D. Degos. Elevated levels of interleukin-1 beta (IL-1 beta) and IL-6 in serum and increased production of IL-1 beta mRNA in lymph nodes of patients with polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes (POEMS) syndrome. Blood 83:2587–2593, 1994. Hashiguchi, T., K. Arimura, K. Matsumuro, R. Otsuka, O. Watanabe, M. Jonosono, Y. Maruyama, I. Maruyama, and M. Osame. Highly concentrated vascular endothelial growth factor in platelets in CrowFukase syndrome. Muscle Nerve 23:1051–1056, 2000. Hudnall, S. D., T. Chen, K. Brown, T. Angel, M. R. Schwartz, and S. K. Tyring. Human herpesvirus-8-positive microvenular hemangioma in POEMS syndrome. Arch. Pathol. Lab. Med. 127:1034–1036, 2003. Ishikawa, O., Y. Nihei, and H. Ishikawa. The skin changes of POEMS syndrome. Br. J. Dermatol. 117:523–526, 1987. Kanitakis, J., H. Roger, M. Soubrier, J. J. Dubost, B. Chouvet, and P. Souteyrand. Cutaneous angiomas in POEMS syndrome. An ultrastructural and immunohistochemical study. Arch. Dermatol. 124:695–698, 1988. Kingdon, E. J., B. B. Phillips, M. Jarmulowicz, S. H. Powis, and M. P. Vanderpump. Glomeruloid haemangioma and POEMS syndrome. Nephrol. Dial. Transplant. 16:2105–2107, 2001. Ku, A., E. Lachmann, R. Tunkel, and W. Nagler. Severe polyneuropathy: initial manifestation of Castleman’s disease associated with POEMS syndrome. Arch. Phys. Med. Rehabil. 76:692–694, 1995. Longo, G., G. Emilia, and U. Torelli. Skin changes in POEMS syndrome. Haematologica 84:86, 1999. Mandler, R. N., D. P. Kerrigan, J. Smart, W. Kuis, P. Villiger, and M. Lotz. Castleman’s disease in POEMS syndrome with elevated interleukin-6. Cancer 69:2697–2703, 1992. Minamitani, S., S. Ohfuji, S. Nishiguchi, S. Shiomi, M. Ogami, T. Matsuo, M. Ohsawa, K. Ohta, and M. Hino. An autopsy case of POEMS syndrome with a high level of IL-6 and VEGF in the serum and ascitic fluid. Intern. Med. 41:233–236, 2002. Miralles, G. D., J. R. O’Fallon, and N. J. Talley. Plasma-cell dyscrasia with polyneuropathy. The spectrum of POEMS syndrome. N. Engl. J. Med. 327:1919–1923, 1992. Morrow, J. S., E. J. Schaefer, D. P. Huston, and S. W. Rosen. POEMS syndrome: studies in a patient with an IgG-kappa M protein but no polyneuropathy. Arch. Intern. Med. 142:1231–1234, 1982. Niimi, H., K. Arimura, M. Jonosono, T. Hashiguchi, M. Kawabata,

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M. Osame, and I. Kitajima. VEGF is causative for pulmonary hypertension in a patient with Crow-Fukase (POEMS) syndrome. Intern. Med. 39:1101–1104, 2000. Obermoser, G., C. Larcher, J. A. Sheldon, N. Sepp, and B. Zelger. Absence of human herpesvirus-8 in glomeruloid haemangiomas associated with POEMS syndrome and Castleman’s disease. Br. J. Dermatol. 148:1276–1278, 2003. Papo, T., M. Soubrier, A. G. Marcelin, V. Calvez, B. Wechsler, J. M. Huraux, J. C. Piette, and P. Cacoub. Human herpesvirus 8 infection, Castleman’s disease and POEMS syndrome. Br. J. Haematol. 104:932–933, 1999. Puig, L., A. Moreno, P. Domingo, E. Llistosella, and J. M. de Moragas. Cutaneous angiomas in POEMS syndrome. J. Am. Acad. Dermatol. 12:961–964, 1985. Romas, E., E. Storey, M. Ayers, and E. Byrne. Polyneuropathy, organomegaly, endocrinopathy, M-protein and skin change (POEMS) syndrome with IgG kappa paraproteinemia. Pathology 24:217–220, 1992. Rongioletti, F., C. Gambini, and R. Lerza. Glomeruloid hemangioma: a cutaneous marker of POEMS syndrome. Am. J. Dermatopathol. 16:175–178, 1994. Scheers, C., A. Kolivras, A. Corbisier, P. Gheeraert, C. Renoirte, A. Theunis, N. de Saint-Aubain, J. Andre, U. Sass, and M. Song. POEMS syndrome revealed by multiple glomeruloid angiomas. Dermatology 204:311–314, 2002. Shelley, W. B., and E. D. Shelley. The skin changes in the Crow-Fukase (POEMS) syndrome: a case report. Arch. Dermatol. 123:85–87, 1987. Shimpo, S. Solitary plasmacytoma with polyneuritis and endocrine disturbances. Nippon Rinsho 26:2444–2447, 1968. Soubrier, M., C. Sauron, B. Souweine, C. Larroche, B. Wechsler, L. Guillevin, J. C. Piette, H. Rousset, and P. Deteix. Growth factors and proinflammatory cytokines in the renal involvement of POEMS syndrome. Am. J. Kidney Dis. 34:633–638, 1999. Tartour, E., D. Adams, J. F. Besancenot, W. H. Fridman, and M. Schlumberger. Poems syndrome with high interleukin (IL) 6 and IL1 beta serum levels, in a patient with thyroid carcinoma and melanoma. Eur. J. Cancer 30A:893–894, 1994. Tohda, S., N. Murakami, and N. Nara. Human herpesvirus 8 DNA in HIV-negative Japanese patients with multicentric Castleman’s disease and related diseases. Int. J. Mol. Med. 8:549–551, 2001. Tsai, C. Y., C. H. Lai, H. L. Chan, and Tt. Kuo. Glomeruloid hemangioma — a specific cutaneous marker of POEMS syndrome. Int. J. Dermatol. 40:403–406, 2001. Tsang, W. Y., J. K. Chan, and C. D. Fletcher. Recently characterized vascular tumours of skin and soft tissues. Histopathology 19:489–501, 1991. Watanabe, O., K. Arimura, I. Kitajima, M. Osame, and I. Maruyama. Greatly raised vascular endothelial growth factor (VEGF) in POEMS syndrome. Lancet 347: 702, 1996. Watanabe, O., I. Maruyama, K. Arimura, I. Kitajima, H. Arimura, M. Hanatani, K. Matsuo, T. Arisato, and M. Osame. Overproduction of vascular endothelial growth factor/vascular permeability factor is causative in Crow-Fukase (POEMS) syndrome. Muscle Nerve 21:1390–1397, 1998. Zumo, L., and R. P. Grewal. Castleman’s disease-associated neuropathy: no evidence of human herpesvirus type 8 infection. J. Neurol. Sci. 195:47–50, 2002.

Urticaria Pigmentosum and Mastocytosis James J. Nordlund

Historical Background The first report of urticaria pigmentosum was published by

ACQUIRED EPIDERMAL HYPERMELANOSES

Nettleship who suggested that this syndrome was a form of urticaria or hives (Nettleship and Tay, 1869). It was Sangster who later coined the term urticaria pigmentosum (Sangster, 1878). Unna (1887) first suggested that this disorder was an abnormality of cutaneous mast cells. Sézary suggested the term mastocytosis (Sézary et al., 1936) [excerpted from The Skin in Mastocytosis (Soter, 1991)].

Table 50.3. Classification of mastocytosis by organ involvement (Longley et al., 1995).

Synonyms

Group II: Extracutaneous mastocytosis Hematopoietic Gastrointestinal Hepatic Reticuloendothelial Osseous Other

Mastocytosis, bullous mastocytosis, diffuse cutaneous mastocytosis, mastocytoma, systemic mastocytosis, telangiectasia macularis eruptiva perstans.

Epidemiology

Group 1: Cutaneous mastocytosis Solitary mastocytoma Urticaria pigmentosum Diffuse cutaneous mastocytosis Telangiectasia macularis eruptiva perstans

The prevalence of mast cell disease is not known. It has been estimated that less than 1:1000 patients coming to a dermatology clinic have mast cell disease in some form (Longley et al., 1995). There seems to be no predilection for either gender. Many cases are sporadic but there are other reports suggesting an autosomal dominant inheritance (Longley et al., 1995; Soter, 1991). Recent studies on the pathogenesis of the systemic forms suggest that the disease in at least some individuals is caused by a mutation in the c-kit protooncogene and thus inheritable (Boissan et al., 2000; Buttner et al., 1998; Longley and Metcalfe, 2000; Longley et al., 1993, 1996; Nagata et al., 1995; Valent et al., 1999). Gene expression has been studied and several genes and gene products are consistently overexpressed including a tryptase, b tryptase and carboxypeptidase A (Roberts and Oates, 1991; D’Ambrosio et al., 2003).

Clinical Manifestations Urticaria pigmentosum and/or mastocytosis represent a group of disorders caused by abnormalities in mast cells of the skin or other organs. Mast cell disease can be mild, debilitating or fatal. Mast cell disease that affects infants commonly is selflimiting and spontaneously regresses by adolescence. Mast cell disease beginning during adulthood persists and can produce severe systemic signs and symptoms (for reviews see Azana et al., 1994; DiBacco and DeLeo, 1982; Kettelhut and Metcalfe, 1991; Lazarus et al., 1991; Longley et al., 1995; Metcalfe, 1991a; Monheit et al., 1979; Soter, 1991). Many forms of this disease cause hyperpigmentation of the epidermis. There are several classifications of mast cell disorders based on age of onset, severity of symptoms, and organs involved (Azana et al., 1994; Longley et al., 1995; Metcalfe, 1991a; Soter, 1991). The classification by organ systems of Longley will be used for this discussion (Table 50.3). The mastocytoma occurs typically during infancy and presents as an isolated nodule either at birth or developing shortly after. The lesion is typically 1 to several centimeters in diameter and is typically reddish brown or yellowish brown in color. Gentle stroking of the lesion causes it to become erythematous and often edematous, a phenomenon called Darier sign. Occasionally a blister forms following trauma to the

Fig. 50.44. Urticaria pigmentosa with hyperpigmented macules and papules (see also Plate 50.18, pp. 494–495).

lesion. The lesion is benign and may regress spontaneously although some seem to persist into adolescence. Urticaria pigmentosum of childhood typically presents as brownish macules (Fig. 50.44) or papules occurring during infancy. Typically the lesions first appear before the age of 1 955

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year (Azana et al., 1994; Kettelhut and Metcalfe, 1991) although they have been noted to form as late as 11 years. The brown hue can vary from reddish to yellow. These spots like the mastocytoma exhibit a Darier sign. The typical lesion is 3–10 mm in diameter. The number of spots can vary from few to hundreds. The child may have itching and occasional blisters (Azana et al., 1994; Longley et al., 1995; Soter, 1991). The lesions are typically on the trunk and spare the extremities and face. They regress spontaneously (Czarnetzki et al., 1988). Adult onset urticaria pigmentosum differs significantly from the childhood form. The lesions first appear during the third or fourth decade of life although the range of onset is throughout adult life. The lesions are similar to those observed in the infantile form of this disease. These lesions tend to persist and cause symptoms of pruritus in many patients and many adults have evidence of systemic involvement, especially bone marrow (Czarnetzki et al., 1988) and liver (Mican et al., 1995) and other organs (Metcalfe, 1991b). Telangiectasia macularis eruptiva perstans is a variant of adult disease characterized by prominent telangiectases typically on the trunk. The lesions are not pigmented but the skin exhibits marked dermographism, i.e., development of hives following stroking of the skin. Like those with diffuse mastocytosis, the skin color can have a mildly pigmented hue. Both adults and children can have diffuse cutaneous mastocytosis (Fig. 50.45). The infant is born with a thickening of the skin (Fig. 50.46). Gentle trauma such as picking up the child can provoke localized urticaria, blisters (Oranje et al., 1991), and systemic symptoms such as wheezing, diarrhea, and hypotension thought to be related to sudden release of large amounts of histamine, prostaglandin D2, heparin, and other mast cell mediators (Roberts and Oates, 1991). The skin often is diffusely pigmented (Fig. 50.47).

Fig. 50.45. Marked hyperpigmentation in a boy with diffuse mastocytosis (see also Plate 50.19, pp. 494–495).

Histology The typical lesions show a dense infiltrate of polygonal, eosinophilic mast cells in the dermis (Anstey et al., 1991; Haas et al., 1995; Oku et al., 1990; Oranje et al., 1991). The mast cells exhibit metachromasia when stained with the toluidine blue technique. The mast cells also stain with a variety of histochemical and immunohistochemical stains including those for tryptase, chymase, and the ligand for c-kit receptor (Haas et al., 1995; Longley et al., 1993). Electron microscopy confirms the abnormalities found by light microscopy (Anstey et al., 1991). The epidermis shows moderate to large amounts of melanin compared to the surrounding normal skin, easily demonstrated by staining the affected and normal skin with the Fontana-Masson silver stain for melanin (Figs 50.48 and 50.49). The number of melanocytes per unit area appears to be normal when epidermal sheets are stained by the dopa techniques (Fig. 50.50). These data suggest that the hyperpigmentation is caused by stimulation of melanocyte function and not alterations in proliferation of the melanocytes.

956

Fig. 50.46. Generalized or diffuse mastocytosis. Note the skin thickening associated with hyperpigmentation (same patient as in Fig. 50.45) (see also Plate 50.20, pp. 494–495).

Laboratory Findings The most important findings are histologic. However analyses of urinary excretory products for histamine and its metabolites, prostaglandin D2 and its metabolites (Roberts and Oates,

ACQUIRED EPIDERMAL HYPERMELANOSES

Fig. 50.49. Fontana-Masson stain of epidermis overlying a mastocytoma. The epidermis contains increased amounts of melanin.

Fig. 50.47. Hyperpigmentation of the face in a patient with diffuse mastocytosis and silver blond hair and very fair skin (see also Plate 50.21, pp. 494–495). A

Fig. 50.48. Fontana-Masson stain of normal skin. B

1991) can be useful. Liver function studies and biopsies (Metcalfe, 1991b; Mican et al., 1995) have been used to confirm systemic disease. The bone marrow often confirms systemic disease (Czarnetzki et al., 1988). X-rays and scans of

Fig. 50.50. (A, B) Dopa stain of epidermis of normal and affected skin. The affected skin has normal numbers of melanocytes but excessive melanin, an indicator of melanogenesis without proliferation of the pigment cells.

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the bones can confirm marrow involvement (Andrew and Freemont, 1993; Chen et al., 1994).

Criterion for Diagnosis The histologic finding of increased mast cells in the skin confirms the diagnosis of urticaria pigmentosum.

Differential Diagnosis The generalized macules must be distinguished from ephelides that typically occur on sun-exposed skin in contrast with urticaria pigmentosum on the trunk. Nevi and lentigines do not have a positive Darier sign but histology will distinguish easily between the various disorders. Xanthomata and nevoxanthoendothelioma might be difficult to distinguish from urticaria pigmentosum but the histology is definitive for all these disorders. Solitary mastocytoma might be confused with a bug bite although the latter is transient and the former persistent. The systemic mastocytosis with blistering has been confused with Staphylococcus scalded skin syndrome (Oranje et al., 1991).

Pathogenesis The disease is one of mast cells. The signs and symptoms can be attributed to excessive numbers of mast cells in the skin and other organs (Akiyama et al., 1991; DiBacco and DeLeo, 1982; Lazarus et al., 1991; Longley et al., 1995). The precise abnormality producing abnormalities in the mast cell is not well identified. The c-kit protooncogene located on chromosome 12 (Grabbe et al., 1994) codes for a transmembrane tyrosine kinase receptor (Nagata et al., 1995) found on both melanocytes and mast cells (Dippel et al., 1995; Grabbe et al., 1994). The ligand for this receptor is called stem cell factor or SCF. The factor has many functions for hematopoietic cells (Grabbe et al., 1994). Its precise function for melanocytes is not well defined but is capable of stimulating melanogenesis (Longley et al., 1993). However mutations in the c-kit receptor (Longley et al., 1996; Nagata et al., 1995; Valent et al., 1999) have been identified in patients with systemic mastocytosis. An abundance of stem cell factor and its mRNA have been observed in similar patients. Similar studies on urticaria pigmentosum show different staining patterns for stem cell factor but the results do not definitively establish a role for c-kit receptor or stem cell factor in all forms of mastocytosis (Hamann et al., 1995) although c-kit and SCF are well established as causative in some patients with urticaria pigmentosum (Longley et al., 1996; Buttner et al., 1998; Valent et al., 1999; Boissan et al., 2000; Longley and Metcalfe, 2000).

Animal Models A disorder resembling human mastocytosis has been described in both a goat (Khan et al., 1995) and in a group of dogs (O’Keefe et al., 1987).

Treatment There is no good treatment for the pigmentary abnormality 958

associated with this syndrome. In infants the pigmented spots tend to disappear by the middle of teenage years (Soter, 1991). For those with systemic mastocytosis or the adult forms, treatment is directed at controlling the signs and symptoms related to release of mast cell mediators and include antihistamines, cromolyn, and similar agents (Gasior-Chrzan and Falk, 1992; Horan et al., 1990; Metcalfe, 1991c). Photochemotherapy has been used to degranulate the mast cells and prevent symptoms (Smith et al., 1990).

References Akiyama, M., Y. Watanabe, and T. Nishikawa. Immunohistochemical characterization of human cutaneous mast cells in urticaria pigmentosa (cutaneous mastocytosis). Acta Pathol. Jpn. 41:344–349, 1991. Andrew, S. M., and A. J. Freemont. Skeletal mastocytosis. J. Clin. Pathol. 46:1033–1035, 1993. Anstey, A., D. G. Lowe, J. D. Kirby, and M. A. Horton. Familial mastocytosis: a clinical, immunophenotypic, light and electron microscopic study. Br. J. Dermatol. 125:583–587, 1991. Azana, J. M., A. Torrelo, I. G. Mediero, and A. Zambrano. Urticaria pigmentosa: a review of 67 pediatric cases. Pediatr. Dermatol. 11:102–106, 1994. Boissan, M., F. Feger, J. J. Guillosson, and M. Arock. c-Kit and c-kit mutations in mastocytosis and other hematological diseases. J. Leukoc. Biol. 67:135–148, 2000. Buttner, C., B. M. Henz, P. Welker, et al. Identification of activating c-kit mutations in adult-, but not in childhood-onset indolent mastocytosis: a possible explanation for divergent clinical behavior. J. Invest. Dermatol. 111:1227–1231, 1998. Chen, C. C., M. P. Andrich, J. M. Mican, and D. D. Metcalfe. A retrospective analysis of bone scan abnormalities in mastocytosis: correlation with disease category and prognosis. J. Nucl. Med. 35:1471–1475, 1994. Czarnetzki, B. M., G. Kolde, A. Schoemann, S. Urbanitz, and D. Urbanitz. Bone marrow findings in adult patients with urticaria pigmentosa. J. Am. Acad. Dermatol. 18:45–51, 1988. D’Ambrosio, C., C. Akin, Y. Wu, M. K. Magnusson, and D. D. Metcalfe. Gene expression analysis in mastocytosis reveals a highly consistent profile with candidate molecular markers. J. Allergy Clin. Immunol. 112:1162–1170, 2003. DiBacco, R. S., and V. A. DeLeo. Mastocytosis and the mast cell. J. Am. Acad. Dermatol. 7:709–722, 1982. Dippel, E., N. Haas, J. Grabbe, D. Schadendorf, K. Hamann, and B. M. Czarnetzki. Expression of the c-kit receptor in hypomelanosis: a comparative study between piebaldism, naevus depigmentosus and vitiligo. Br. J. Dermatol. 132:182–189, 1995. Gasior-Chrzan, B., and E. S. Falk. Systemic mastocytosis treated with histamine H1 and H2 receptor antagonists. Dermatology 184:149– 152, 1992. Grabbe, J., P. Welker, E. Dippel, and B. M. Czarnetzki. Stem cell factor, a novel cutaneous growth factor for mast cells and melanocytes. Arch. Dermatol. Res. 287:78–84, 1994. Haas, N., K. Hamann, J. Grabbe, B. Algermissen, and B. M. Czarnetzki. Phenotypic characterization of skin lesions in urticaria pigmentosa and mastocytomas. Arch. Dermatol. Res. 287:242– 248, 1995. Hamann, K., N. Haas, J. Grabbe, and B. M. Czarnetzki. Expression of stem cell factor in cutaneous mastocytosis. Br. J. Dermatol. 133:203–208, 1995. Horan, R. F., A. L. Sheffer, and K. F. Austen. Cromolyn sodium in the management of systemic mastocytosis. J. Allergy Clin. Immunol. 85:852–855, 1990. Kettelhut, B. V., and D. D. Metcalfe. Pediatric mastocytosis. J. Invest. Dermatol. 96:15S-18S, 1991.

ACQUIRED EPIDERMAL HYPERMELANOSES Khan, K. N., J. E. Sagartz, G. Koenig, and K. Tanaka. Systemic mastocytosis in a goat. Vet. Pathol. 32:719–721, 1995. Lazarus, G. S., C. Guzzo, R. M. Lavker, G. F. Murphy, and N. M. Schechter. Urticaria pigmentosum: nature’s experiment in mast cell biology. J. Dermatol. Sci. 2:395–401, 1991. Longley, B. J., and D. D. Metcalfe. A proposed classification of mastocytosis incorporating molecular genetics. Hematol. Oncol. Clin. North Am. 14:697–701, viii, 2000. Longley, B. J., Jr., G. S. Morganroth, L. Tyrrell, T. G. Ding, D. M. Anderson, D. E. Williams, and R. Halaban. Altered metabolism of mast-cell growth factor (c-kit ligand) in cutaneous mastocytosis. N. Engl. J. Med. 328:1302–1307, 1993. Longley, B. J., L. Tyrrell, S. Z. Lu, Y. S. Ma, K. Langley, T. G. Ding, T. Duffy, P. Jacobs, L. H. Tang, and I. Modlin. Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm. Nat. Genet. 12:312–314, 1996. Longley, J., T. P. Duffy, and S. Kohn. The mast cell and mast cell disease. J. Am. Acad. Dermatol. 32:545–561, 1995. Metcalfe, D. D. Classification and diagnosis of mastocytosis: current status. J. Invest. Dermatol. 96:2S-4S, 1991a. Metcalfe, D. D. The liver, spleen, and lymph nodes in mastocytosis. J. Invest. Dermatol. 96:45S-46S, 1991b. Metcalfe, D. D. The treatment of mastocytosis: an overview. J. Invest. Dermatol. 96:55S-56S (discussion 56S-59S), 1991c. Mican, J. M., A. M. Di Bisceglie, T. L. Fong, W. D. Travis, D. E. Kleiner, B. Baker, and D. D. Metcalfe. Hepatic involvement in mastocytosis: clinicopathologic correlations in 41 cases. Hepatology 22:1163–1170, 1995. Monheit, G. D., T. Murad, and M. Conrad. Systemic mastocytosis and the mastocytosis syndrome. J. Cutan. Pathol. 6:42–52, 1979. Nagata, H., A. S. Worobec, C. K. Oh, B. A. Chowdhury, S. Tannenbaum, Y. Suzuki, and D. D. Metcalfe. Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder. Proc. Natl. Acad. Sci. U. S. A. 92:10560–10564, 1995. Nettleship, E., and W. Tay. Rare forms of urticaria. Br. Med. J. 2:323–324, 1869. O’Keefe, D. A., C. G. Couto, C. Burke-Schwartz, and R. M. Jacobs. Systemic mastocytosis in 16 dogs. J. Vet. Intern. Med. 1:75–80, 1987. Oku, T., H. Hashizume, R. Yokote, T. Sano, and M. Yamada. The familial occurrence of bullous mastocytosis (diffuse cutaneous mastocytosis). Arch. Dermatol. 126:1478–1484, 1990. Oranje, A. P., W. Soekanto, A. Sukardi, V. D. Vuzevski, A. van der Willigen, and H. M. Afiani. Diffuse cutaneous mastocytosis mimicking staphylococcal scalded-skin syndrome: report of three cases. Pediatr. Dermatol. 8:147–151, 1991. Roberts, L. J., and J. A. Oates. Biochemical diagnosis of systemic mast cell disorders. J. Invest. Dermatol. 96:19S-24S (discussion 24S– 25S), 1991. Sangster, A. An anomalous mottled rash, accompanied by pruritus, factitious urticaria and pigmentation, urticaria pigmentosa(?). Trans. Clin. Soc. London 11:161–163, 1878. Sezary, A., G. Levy-Coblentz, and P. Chauvillon. Dermographisme et mastocytose. Bull. Soc. Franc. Dermatol. Syphiligr. 43:359–361, 1936. Smith, M. L., P. W. Orton, H. Chu, and W. L. Weston. Photochemotherapy of dominant, diffuse, cutaneous mastocytosis. Pediatr. Dermatol. 7:251–255, 1990. Soter, N. A. The skin in mastocytosis. J. Invest. Dermatol. 96:32S–38S (discussion 38S–39S), 1991. Unna, P. G. Beitrage zur Anatomie und Pathogenese der Urticaria simplex und Pigmentosa. Mschr. Prakt. Dermatol. Suppl. Dermatol. Stud. 3:9, 1887. Valent, P., L. Escribano, R. M. Parwaresch, V. Schemmel, L. B. Schwartz, K. Sotlar, W. R. Sperr, and H. P. Horny. Recent advances

in mastocytosis research. Summary of the Vienna Mastocytosis Meeting 1998. Int. Arch. Allergy Immunol. 120:1–7, 1999.

Poikiloderma of Civatte Vlada Groysman and Norman Levine

Historical Background Civatte originally described this disorder appearing in the sunexposed areas of the lateral neck and upper chest as a type of postinflammatory alteration (Civatte, 1923). Over the past several years, it has been noted that this disease is characterized by interfollicular erythema and pigmentation without inflammation.

Synonyms Menopausal solar dermatitis and atrophic degenerative pigmentary dermatitis and Berkshire neck are other names used for this syndrome.

Epidemiology This entity is more common in middle-aged women and in outdoor workers of either gender, particularly those with fair skin (Graham, 1989).

Clinical Findings Poikiloderma of Civatte presents gradually with retiform hyperpigmentation and telangiectasia with progressive epidermal atrophy over the sun-exposed portions of the lateral neck (Fig. 50.51) and upper chest. The submandibular and submental areas are spared. Milder forms are common and those patients often do not seek medical advice. It is usually asymptomatic but occasionally causes burning, itching, and discomfort. Reddish brown reticulated pigmentation with telangiectasia and interspersed atrophic pale spots are found in fairly symmetric pattern on the lateral cheeks and the sides of the neck but sparing the area shaded by the chin. Occasionally, the

Fig. 50.51. Poikilodermatous patches on the lateral aspect of the neck and face (see also Plate 26.1, pp. 494–495).

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condition becomes so extensive that there is significant cosmetic disfigurement.

Associated Disorders This may occur in the context of other chronic photodermatosis such as actinic keratoses, nonmelanoma skin cancer and photoaging changes elsewhere in sun-exposed skin.

Pathology The epidermis shows atrophy and irregular hyperpigmentation of the basal cell layer. Occasional liquefaction degeneration in the basal cell layer and mild perivascular lymphocytic infiltration in the upper dermis is noted. Pigment incontinence and dermal melanophages are frequently seen. Moderate basophilic degeneration of collagen and solar elastosis are evident.

hormonal milieu in postmenopausal women and genetic predisposition. Photodynamic substances in fragrances and cosmetics have also been implicated in pathogenesis. Kathon CG is a common component of cosmetic products and is made of mixture of methylchloroisothiazolinone and methylisothiazolinone 3:1. In low concentrations, Kathon CG is a cosmetic allergen that plays a role in initiation or continuation of poikiloderma of Civatte (Kumar, 2001; Lee and Lam, 1999). A delayed contact hypersensitivity reaction initiated by allergens may cause basal layer deterioration and melanin incontinence, resulting in reticular hyperpigmentation (Katoulis, 2001). Genetically determined predisposition is also postulated in the pathogenesis of poikiloderma of Civatte as shown by increased susceptibility to UV radiation in certain families. It presents as an autosomal dominant trait with variable penetrance (Katoulis et al., 1999).

Laboratory Findings None.

Criteria for Diagnosis Physical examination and exclusion of other photosensitivity diseases are usually sufficient to make the diagnosis.

Differential Diagnosis The differential diagnosis of poikiloderma of Civatte includes other skin conditions which produce lesions in sun-exposed skin and those which cause poikilodermatous change. Lupus erythematosus may have atrophic lesions in sun-exposed skin. However, the lesions are not as extensive and do not limit themselves to the neck area. Dermatomyositis may have similar lesions to those of lupus erythematosus. Atrophy and telangiectasias may occur on the neck and chest wall. However, there are usually facial lesions, periorbital edema, and a violaceous discoloration of the upper eyelids. Riehl melanosis may occur on the lateral neck and have dyspigmentation. There is no atrophy or telangiectasia in the lesions, however. Berloque dermatitis commonly occurs on the neck, especially after application of certain fragrances. However, the lesions are hyperpigmented without atrophy or telangiectasias. Poikiloderma atrophicans vasculare occurs in the context of peripheral T-cell lymphoma or as an isolated finding and has all of the physical elements of poikiloderma of Civatte, namely, hyperpigmentation, telangiectasia, and atrophy. However, the lesions do not limit themselves to sunexposed areas and are generally much smaller in size. Two pediatric syndromes, Bloom syndrome and Rothmund–Thomson syndrome, produce lesions with poikiloderma. Both present with lesions in early life and have many other cutaneous and systemic findings that allow easy differentiation from poikiloderma of Civatte.

Pathogenesis Chronic exposure to sunlight is an important pathogenic factor (Goldberg and Altman, 1984), as evidenced by distribution on face and neck, in combination with changes in

960

Animal Models None.

Treatment Meticulous sunscreen protection and avoidance of sun exposure is recommended. Topical corticosteroids are not effective in treatment of poikiloderma of Civatte, and should be avoided to prevent the aggravation of cutaneus atrophy. There is also evidence of clinical improvement in patients after complete avoidance of perfumes and cosmetics (Katoulis et al., 2002). Studies indicate that pulsed tunable dye laser (PDL) at 585 nm produces reduction in telangiectasia-induced erythema (Clark and Jiminez-Acosta, 1994; Geronemus, 1990; Haywood and Monk, 1998; Wheeland and Applebaum, 1990). Complications with use of PDL include posttreatment purpura, hypopigmentation, and in some cases scarring. Treatment with potassium titanyl phosphate laser (KTP) at 532 nm offers reduction in both telangiectatic and pigmented lesions (Batta et al., 1999). Treatment of poikilodenma of Civatte with intense pulsed light source, which delivers multiple wavelengths with software managed pulse durations, allows some reduction in both vascular and pigmented lesions with minimal side effects (Goldman, 2001). Argon lasers have also been used in management; however, there is significant risk of scarring (Oldbricht, 1987).

Prognosis Poikiloderma of Civatte progresses with increasing exposures to sunlight. The lesions persist permanently and no spontaneous regression has been reported. In severe cases, there is cosmetic disfigurement.

References Batta, K., C. Hindson, J. A. Cotterill, and I. S. Foulds. Treatment of poikiloderma of Civatte with the potassium titanyl phosphate laser. Br. J. Dermatol. 146:1191–1192, 1999. Clark, R. E., and F. Jiminez-Acosta. Poikiloderma of Civatte. Resolu-

ACQUIRED EPIDERMAL HYPERMELANOSES tion after treatment with pulsed dye laser. N. Carolina Med. J. 55:234–235, 1994. Civatte, A. Poikilodermie reticulee pigmentaire du visage et du col. Ann. Dermatol. Syphiligr. (Paris) 6:605–620, 1923. Geronemus, R. Poikiloderma of Civatte. Arch. Dermatol. 126:547– 548, 1990. Goldberg, L. H., and A. Altman. Benign skin changes associated with chronic sunlight exposure. Cutis 34:33–38, 1984. Goldman, M. P., and R. A. Weiss. Treatment of poikiloderma of Civatte on the neck with an intense pulsed light source. Plast. Reconstr. Surg. 107:1376–1381, 2001. Graham, R. What is poikiloderma of Civatte? Practitioner 233:1210, 1989. Haywood, R. M., and B. E. Monk. Treatment of Poikiloderma of Civatte with the pulsed dye laser: a series of seven cases. J. Cutan. Laser Ther. 1:45–48, 1999. Katoulis, A. C., N. G. Stavrianeas, A. Katsarou, C. Antoniou, S. Georgala, D. Rigopoulos, E. Koumantaki, G. Avgerinou, and A. D. Katsambas. Evaluation of the role of contact sensitization and photosensitivity in the pathogenesis of poikiloderma of Civatte. Br. J. Dermatol. 147:493–497, 2002. Katoulis, A. C., N. G. Stavrianeas, A. Katsarou, C. Antoniou and J. D. Stratigos. Familial cases of poikiloderma of Civatte: genetic implications in its pathogenesis? Clin. Exp. Dermatol. 24:385–387, 1999. Lee, T. Y., and T. H. Lam. Allergic contact dermatitis due to Kathon CG in Hong Kong. Contact Dermatitis 41:41–42, 1999. Raulin, C., B. Greve, and H. Grema. Laser. Surg. Med. 32:78–87, 2003. Sahoo, B., and B. Kumar. Role of methylchloroisothiazolinone/ methylisothiazolinone (Kathon CG) in poikiloderma of Civatte. Contact Dermatitis 44:249, 2001. Wheeland, R. G., and J. Applebaum. Flashlamp-pumped pulsed dye laser therapy for poikiloderma of Civatte. J. Dermatol. Surg. Oncol. 16:12–16, 1990. Fig. 50.52. Pigmented contact dermatitis (Riehl’s melanosis).

Riehl’s Melanosis Scott Bangert and Norman Levine

Historical Background

Clinical Findings

In Vienna during the First World War, Riehl (1917) first described several patients with a gray-brown reticular facial pigmentation, which he attributed to contact with “noxious substances” of wartime living conditions. Hoffman and Habermann (1918) reported similar cases they dubbed “melanodermatitis toxica,” which they believed to be due to an unknown photosensitizing agent in mineral oil. During World War II and afterward, cases of Riehl melanosis were reported in France (Granier, 1943), Austria (Meischer, 1949), and Argentina (Peirini, 1952). Tar exposure producing a low-grade contact dermatitis was believed to be the causative agent. Some 20 years later, a number of cases were reported in Japan. Nakayama (1976) linked this outbreak to cosmetic ingredients and proposed the term “pigmented contact dermatitis.”

Riehl’s melanosis typically affects middle-aged women. Dark skin and ability to tan also appear to be risk factors. Patients typically present with a rapid onset of diffuse, reticular patches of brown-gray hyperpigmentation. The face and neck are primarily involved (Fig. 50.52), especially the forehead, temple, and zygomatic area, although the ears, nape of the neck, and scalp can be involved as well. In addition, use of optical whiteners can produce hyperpigmentation of the hands, forearms, and trunk, although typically this is not as pronounced (Osmundsen, 1970; Pinol-Agiade et al., 1971). Occasionally, patients may demonstrate mild erythema, scaling, or pruritus, although such symptoms are usually absent, and hyperpigmentation occurs without preceding symptoms (Nakagawa et al., 1984). The time course of the condition is variable and may become progressively worse if the causative agent is not removed.

Synonyms

Histopathology

Riehl’s melanosis is also known as pigmented contact dermatitis or female facial melanosis.

The dominant histologic feature of Riehl’s melanosis is liquefaction degeneration of the basal layer of the epidermis

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resulting in pigment incontinence into the dermis. The papillary dermis contains an infiltrate of lymphocytes and macrophages containing large amounts of melanin, and the basal lamina is prominently thickened. Epidermal melanin may be increased or decreased. While the epidermal change and lymphohistiocytic infiltrate are not as marked, the vacuolar changes and dermal melanosis of Riehl’s melanosis are comparable to other lichenoid dermatoses, including lichen planus and lupus erythematosus (Nakagawa et al., 1984). Such findings suggest that Riehl’s melanosis fits on a continuum of lichenoid dermatoses (Masahiro et al., 2003). It differs from typical contact dermatitis by showing liquefaction rather than spongiosis and by having a less pronounced dermal infiltrate.

Differential Diagnosis Hyperpigmentation produced by other causes can resemble Riehl’s melanosis, including melasma, acquired bilateral nevus of Ota-like macules, pigmented actinic lichen planus, photosensitivity reactions, and berloque dermatitis. Melasama can closely mimic Riehl’s melanosis, and coexistence of the two entities is possible, as cosmetic agents used to cover up the melasma can exacerbate the Riehl condition. In distinguishing between these two entities, it should be noted that melasma typically affects the central face, including the chin, nose, upper lip, and forehead, while Riehl’s melanosis is more prominent in the lateral areas of the face. Pigmented actinic lichen planus is more violaceous in color, and typically presents with overlying scale and elevated borders. Acquired bilateral nevus of Ota-like macules typically usually involve the forehead, eyelids, cheeks, and nose, and may be differentiated from Riehl’s melanosis based on histology. A history of photosensitizer use can help distinguish photosensitivity reactions and berloque dermatitis from Riehl melanosis. Poikiloderma of Civatte, erythrose péribuccale pigmentaire of Brocq, and erythromelanosis follicularis are three facial hyperpigmentation disorders that at one time were considered variants of Riehl’s melanosis, but are now regarded as distinct entities (Katoulis et al., 2002). All three entities present with more erythema than Riehl melanosis. Poikiloderma of Civatte presents with telangiectasias and atrophy, while erythromelanosis has a papular component not present in Riehl’s melanosis. Erythrose péribuccale pigmentaire of Brocq can further be differentiated based on its perioral location.

Pathogenesis While the pathogenesis of Riehl’s melanosis is not entirely clear, it is believed to be primarily due to contact sensitivity to chemical agents found in perfumes and cosmetics. Darkskinned individuals are more commonly affected, possibly because signs of inflammation upon contactant exposure are less visible clinically in these patients (Cotterill et al., 1987). Another explanation for the lack of clinical inflammation of Riehl’s melanosis suggests that the offending agents have an affinity for melanin or react in the presence of melanin, producing a localization of inflammatory cells to melanocytes 962

(Hayakawa et al., 1987). It is not clear how such agents would damage only the basilar epidermal layer without producing other signs of cutaneous injury. Studies to isolate the most common offending agents have suggested several chemicals found in cosmetics including Brilliant Lake Red, I. Sudan, hydroxycitronellal, benzyl salicylate, jasmine, canaga oil, lemon oil, and geraniol (Nakagawa et al., 1984; Rorsman, 1982). It has also been postulated that UV radiation is a pathogenic factor, since combinations of chemicals and UV light produce histologic findings similar to Riehl’s melanosis, and the lesions are typically often distributed in sun-exposed skin. Optic whiteners containing pyrazolone derivatives are believed to be responsible for producing the truncal distribution sometimes seen in this condition (Rorsman, 1982), although this has become less of a problem following its removal from most consumer products (Serrano et al., 1989). Though chemical agents have been seen as causative since Riehl first described it, new cases have been described not linked to chemical exposure. Histologically, Riehl melanosis shows lichenoid changes similar to lichen planus and lupus erythematosus, and it is possible that all three are pathogenically linked. Masahiro et al. (2003) reported a patient with lichen planus and Riehl’s melanosis in separate locations. This patient was patch test negative and no causative agent could be identified, and the authors suggested a common, possibly extrinsic factor, in both conditions. Similarly, Miyoshi and Kodama (1997) reported a patient with concurrent Sjögren syndrome and Riehl’s melanosis. Again, no cause was found for the Riehl’s melanosis, and the patient’s condition improved despite continuing the same cosmetics. The authors suggested the possibility that Riehl’s melanosis could be a cutaneous manifestation of Sjögren syndrome. Both cases suggest that Riehl’s melanosis fits on a continuum of lichenoid immune reactions and may be caused by intrinsic as well as extrinsic factors.

Treatment Removal of the offending agent is necessary to reverse the hyperpigmentation of Riehl’s melanosis. This often necessitates a screen of the ingredients of all cosmetics and perfumes used by the patient, and even then a cause may not be found (Nakagawa et al., 1984). Gradual resolution occurs over the course of one to two years, although use of hydroquinone and tretinoin or glycolic acid may speed the process (Perez-Bernal, 2000). Although lasers have been used effectively in the treatment of melasma, to date, no published reports have studied their use in Riehl’s melanosis. While facial lesions could be expected to respond well, use for the neck lesions would likely produce less satisfactory results.

References Cotterill, J. A., K. S. Ryatt, and R. Greenwood. Prurigo pigmentosa. Br. J. Dermatol. 105:707–710, 1981. Garnier, G. Les pigmentations cervico-faciales de guerre (melanose de Riehl Poikilodermie). Presse Med. August 14:435, 1943. Hayakawa, R., K. Matsunaga, and Y. Arima. Airborne pigmented contact dermatitis due to musk ambrette in incense. Contact Dermatitis 16:96–98, 1987.

ACQUIRED EPIDERMAL HYPERMELANOSES Katoulis, A. C., N. G. Stavrianeas, A. Katsarou, C. Antoniou, S. Georgala, D. Rigopoulos, E. Koumantaki, G. Avgerinou, and A. D. Katsambas. Evaluation of the role of contact sensitization and photosensitivity in the pathogenesis of poikiloderma of Civatte. Br. J. Dermatol. 147:493–497, 2002. Lautenschlager, S., and P. H. Itin. Reticulate, patchy and mottled pigmentation of the neck. Dermatology 197:291–296, 1998. Lee, C. S., and H. W. Lim. Cutaneous diseases in Asians. Dermatol. Clin. 21:669–678, 2003. Meischer, G. Ausspra Uber Melanosis Riehl. Arch. Dermatol. Syphil. 189:301, 1949. Miyoshi, K., and H. Kodama. Riehl’s melanosis-like eruption associated with Sjögren’s syndrome. J. Dermatol. 24:784–786, 1997. Mosher, D. B., T. B. Fitzpatrick, J. P. Ortonne, and Y. Hori. Hypomelanoses and hypermelanoses. In: Dermatology in General Medicine. 5th ed., I. M. Freedberg, A. Z. Eisen, K. Wolff, K. F. Austen, L. A. Goldsmith, S. I. Katz, and T. B. Fitzpatrick (eds). New York: McGraw-Hill, 1999, pp. 1008–1009. Nakagawa, H., S. Matsuo, R. Hayakawa, K. Takahashi, T. Shigematsu, and S. Ota. Pigmented cosmetic dermatitis. Int. J. Dermatol. 23:299, 1984. Osmundsen, P. E. Pigmented contact dermatitis. Br. J. Dermatol. 83:296–298, 1970. Peirini, L. E. Melanosis de Riehl. Arch. Argent. Dermatol. 2:315, 1952. Perez-Bernal, A., M. A. Munoz-Perez, and F. Camacho. Management of facial hyperpigmentation. Am. J. Clin. Dermatol. 1:261–268, 2000. Pinol-Agiade, J., F. Grimalt, and C. Ramaguera. Dermatitis porblanqueadores opticos. Med. Critanea 5:249, 1971. Riehl, G. Veber ein egenaartige melanose. Wien. Klin. Wochenschr. 30:780–781, 1917. Rorsman, H. Riehl’s melanosis. Int. J. Dermatol. 2:75–78, 1982. Seike, M., Y. Hirose, M. Ikeda, and H. Kodama. Coexistence of Riehl’s melanosis and lichen planus. J. Dermatol. 30:132–134, 2003. Serrano, G., C. Pujol, J. Cuadia, S. Gallo, and A. Aliaga. Riehl’s melanosis: pigmented contact dermatitis caused by fragrances. J. Am. Acad. Dermatol. 21:1057–1060, 1989.

Atrophoderma of Pasini et Pierini James J. Nordlund, Norman Levine, Charles S. Fulk, and Randi Rubenzik

Historical Background The first patients with atrophoderma were reported by Pasini in 1923 and Pierini in 1936 (Pierini, 1936; Pierini and Vivoli, 1936). The name atrophoderma of Pasini and Pierini was used by Canizares et al. (1958) when they reviewed a number of additional cases. Since that time, many cases with similar clinical characteristics have been reported.

Synonyms Atrophoderma of Pasini and Pierini is also known as morphea plana atrophica, progressive idiopathic atrophoderma, atrophic scleroderma d’emblée, dyschromic and atrophic scleroderma, atrophoscleroderma superficialis circumscripta (Musgnug, 1995).

Clinical Characteristics This syndrome is characterized by atrophy of the dermis man-

Fig. 50.53. Pigmented atrophoderma of Pasini and Pierini (see also Plate 50.22, pp. 494–495).

ifested by a slight depression of the skin with a very abrupt edge, termed a cliff sign. The atrophic area has normal epidermis which is hyperpigmented in many but not all individuals (Buechner and Rufli, 1994; Iriondo et al., 1987; Poche, 1980). The lesions typically develop during the teenage years or early adulthood. The largest group of patients studied, 34 patients, consisted of 21 females and 13 males (Buechner and Rufli, 1994). The age of onset of the lesions for this group ranged from 7 years to 66 years. The average age was 26 years (males) or 33 years (females). Women are more commonly affected than are men (Buechner and Rufli, 1994). It has never been reported in Asians and only one case of this condition in black people has been described (Murphy et al., 1990). The skin in this syndrome can have a normal color (Iriondo et al., 1987). Typically the lesions are hyperpigmented, often brown in color (Fig. 50.53), suggestive of epidermal hyperpigmentation (Berman et al., 1988; Buechner and Rufli, 1994; Wakelin and James, 1995). The individual lesions are often palm sized, but they may range from a few centimeters in diameter to one where almost the entire back is covered. They are round or oval and are arranged with the long axis parallel to the planes of cleavage. The plaques may be gray, violaceous, or brown (Figs 50.54 and 50.55). In some patients the color has been noted to be bluish to violaceous, which suggests dermal melanosis (Berman et al., 1988; Heymann, 1994; Poche, 1980; Pullara et al., 1984). Deeper blood vesels are faintly visible as if seen through tinted glass (Miller, 1965). Most patients eventually develop multiple lesions, but many may have one or two lesions initially. Usually the lesions affect the trunk and are bilateral, but zosteriform and unilateral distributions have been described (Bourgiois-Spinasse and Grupper, 1969; Buechner and Rufli, 1994; Iriondo et al., 1987; Murphy et al., 1990; Wakelin and James, 1995). Individual lesions may coalesce into oddly shaped plaques. The most common site affected is the back (Buechner and Rufli, 1994). Other areas where lesions are observed are the chest, arms, and abdomen. The face is never involved (Poche, 1980). 963

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Associated Disorders Atrophoderma of Pasini and Pierini is often observed in individuals who are otherwise healthy in other respects. However, it also has been observed in association with morphea (Murphy et al., 1990; Pullara et al., 1984; Wakelin and James, 1995) or lichen sclerosis et atrophicus (Heymann, 1994; Wakelin and James, 1995). These associations have been the basis for some authors suggesting that this syndrome is a variant of morphea [for reviews see Poche (1980) and Pullara et al. (1984)].

Histology Fig. 50.54. Typical lesion of the right scapular area (see also Plate 50.23, pp. 494–495).

The histology is not diagnostic and the findings not specific. The epidermis is usually normal but increased amounts of basilar melanin may be noted. The dermis has a mononuclear cell infiltrate. Melanophages may be present. The collagen is decreased in quantity. Elastic fibers seem to be increased but might be due to condensation of the fibers from the loss of collagen (Buechner and Rufli, 1994; Lever and SchaumbergLever, 1991).

Differential Diagnosis The main differential diagnosis is morphea.

Treatment and Prognosis There is no treatment for atrophoderma of Pasini and Pierini. The lesions usually persist for the life of the individual. Some have been noted to resolve spontaneously.

References

Fig. 50.55. Extensive involvement of the arm and shoulder.

On palpation, the plaques feel like normal skin, without any suggestion of sclerosis or a leathery feel (Poche, 1980). Occasionally, there may be some sclerosis in very old lesions. The sclerosis has suggested to some that atrophoderma is a mild variant of morphea (see Morphea and Scleroderma below). 964

Berman, A., G. D. Berman, and R. K. Winkelmann. Atrophoderma (Pasini-Pierini). Findings on direct immunofluorescent, monoclonal antibody, and ultrastructural studies. Int. J. Dermatol. 27:487–490, 1988. Bourgiois-Spinasse, J., and H. Grupper. Atrophodermie de PasiniPierini en band unilaterale. Bull. Soc. Franc. Dermatol. Syphiligr. 76:494–498, 1969. Buechner, S. A., and T. Rufli. Atrophoderma of Pasini and Pierini. Clinical and histopathologic findings and antibodies to Borrelia burgdorferi in thirty-four patients. J. Am. Acad. Dermatol. 30:441–446, 1994. Canizares, O., P. M. Sachs, L. Jaimovich, and V. M. Torres. Idiopathic atrophoderma of Pasini and Pierini. Arch. Dermatol. 77:42–60, 1958. Heymann, W. R. Coexistent lichen sclerosus et atrophicus and atrophoderma of Pasini and Pierini. Int. J. Dermatol. 33:133–134, 1994. Iriondo, M., R. F. Bloom, and K. H. Neldner. Unilateral atrophoderma of Pasini and Pierini. Cutis 39:69–70, 1987. Lever, W. F., and G. Schaumberg-Lever. Histopathology of the Skin. Philadelphia: J. B. Lippincott, 1991. Miller, R. F. Idiopathic atrophoderma. Arch. Dermatol. 92:653–660, 1965. Murphy, P. K., S. R. Hymes, and N. A. Fenske. Concomitant unilateral idiopathic atrophoderma of Pasini and Pierini (IAPP) and morphea. Observations supporting IAPP as a variant of morphea. Int. J. Dermatol. 29:281–283, 1990. Musgnug, R. H. Atrophoderma of Pasini and Pierini. In: Clinical Dermatology, D. J. Demis (ed.). Philadelphia: Lippincott-Raven Publishers, 1995, pp. 21–25.

ACQUIRED EPIDERMAL HYPERMELANOSES Pasini, A. Atrophodermia idiopatica progressiva. G. Ital. Dermatol. Sifilol. 58:785–809, 1923. Pierini, L. E. Atrophoderma idiopathica progressive. Rev. Argent. Dermatsif. 19:322, 1936. Pierini, L. E., and D. Vivoli. Atrofodermia idiopahica progressiva (Pasini). G. Ital. Dermatol. 77:403–409, 1936. Poche, G. W. Progressive idiopathic atrophoderma of Pasini and Pierini. Cutis 25:503–506, 1980. Pullara, T. J., C. W. Lober, and N. A. Fenske. Idiopathic atrophoderma of Pasini and Pierini [review]. Int. J. Dermatol. 23:643–645, 1984. Wakelin, S. H., and M. P. James. Zosteriform atrophoderma of Pasini and Pierini. Clin. Exp. Dermatol. 20:244–246, 1995.

Hyperpigmentation Associated with Scleromyxedema and Gammopathy Kazunori Urabe, Juichiro Nakayama, and Yoshiaki Hori Scleromyxedema was recognized as a distinct entity by Buschke in 1902. It is characterized by woody, nonpitting induration of the skin. The induration is caused by deposition of mucin in the dermis. Scleromyxedema is often associated with a monoclonal gammopathy. The gammopathy is usually an IgG although IgA and IgM have also been reported. The light chains can be either of the kappa or lambda type (Fleishmajer, 1993). Two cases in which abnormal skin pigmentation was noted have been reported. One had localized hyperpigmentation, the other generalized hyperpigmentation. In both cases the mechanism for the excessive production of pigmentation was not identified. The first case described by Kovary (1981) was a 65-yearold woman with high levels of serum IgG. Her skin abnormality began as induration and stiffness of the neck. Within the first year the infiltration had spread to the face, shoulders, upper part of the arms, and thorax. On physical examination of these lesions, nonpitting induration was observed. Localized hyperpigmentation was present on the face and the “V” of the neck. Microscopic examination of the skin biopsy showed a normal epidermis but an acellular fibrosis of the dermis reaching to the subcutaneous fat. Direct immunofluorescent staining did not disclose deposition of immunoglobulins within the affected skin. The second patient (McFadden et al., 1987) was a 56year-old man with hyperlipoproteinemia and cardiovascular disease. He had IgG lambda paraproteinemia without evidence of multiple myeloma. His skin was markedly indurated over the neck and the upper aspect of the trunk, arms, thighs, and lower aspect of the trunk. The legs and feet appeared less involved. He had generalized, light brown pigmentation of the skin that was more pronounced in the indurated skin and less pronounced over the hands, feet, and pubic and genital areas. Leukonychia involving all fingernails was noted. Light microscopic examination of the lesional skin biopsy specimen showed normal epidermis and an increased amount of pigmentation in the basal cell layer. The dermis was notably thickened and fibrotic with separation and swelling of the collagen tissue. Direct immunofluorescent staining failed to show any immunoglobulin or complement deposition in the lesional

skin. Ultrastructural examination showed normal-appearing melanocytes with increased transfer of melanosomes to basal keratinocytes. The melanocytes had morphologic features suggestive of active melanogenesis. Melanosomes in all four stages of melanization were present. In the dermis, melanophages with melanosome complexes were noted. Lipid droplets were seen in the cytoplasm of every third basal keratinocyte or melanocyte.

References Fleishmajer, R. Scleredema and papular mucinosis. In: Dermatology in General Medicine, 4th ed., T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M. Freedberg, and K. F. Austen (eds). New York: McGraw Hill, 1993, pp. 2186–2187. Kovary, P. M., F. Vakilzadeh, E. Macher, H. Zaun, H. Merk, and G. Goerz. Monoclonal gammopathy in scleredema. Arch. Dermatol. 117:536–539, 1981. McFadden, N., K. Ree, E. Soyland, and T. E. Larceny. Scleredema adultorum associated with a monoclonal gammopathy and generalized hyperpigmentation. Arch. Dermatol. 123:629–632, 1987.

Ichthyosis Nigricans, Keratoses, and Epidermal Hyperplasia James J. Nordlund

Introduction The color of the skin is determined by the quantity of melanin in the epidermis. The thickness of the epidermis can alter the quantity of melanin in the epidermis. If the epidermis is acanthotic, the skin will appear darker (see Chapters 27 and 28 on normal and abnormal skin color). This discoloration is observed in a variety of conditions not of pigment cell origin. They include the ichthyoses, hyperkeratoses, and epidermal hyperplasia. In addition the stratum corneum is often abnormal. Light striking the aberrant stratum corneum is not reflected normally. This physical phenomenon also alters the color of such skin and gives it a gray or blue-gray hue.

Clinical Features There are many forms of ichthyoses (Fig. 50.56), all characterized by an excessively thick stratum corneum. This skin has an abnormally dark appearance. The discoloration is present from birth (Figs 50.57–50.59). The epidermis can become thicker at later times in life. The typical adult develops seborrheic keratoses (Figs 50.60 and 50.61). These are discrete papules and plaques that are usually located on the trunk. The lesions vary in size from 1 mm to several centimeters. The lesions have discrete edges and are raised with a velvety, verrucous, or scaly surface. Many are irregularly pigmented (see Figs 50.60 and 50.61). One very pigmented type of seborrheic keratosis is the melanoacanthoma (Lever and Schaumberg-Lever, 1991; Pinkus and Mehregan, 1981) (see section on melanoacanthoma earlier in this chapter). Skin chronically rubbed or scratched thickens. This condi965

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Fig. 50.58. Typical clinical appearance of ichthyosis nigricans.

Fig. 50.56. Prominent and extensive hyperpigmentation in a patient with ichthyosis hystrix (see also Plate 50.24, pp. 494–495).

Fig. 50.57. Hyperpigmentation due to epidermal thickening of ichthyosis in a newborn child (see also Plate 50.25, pp. 494–495).

tion is called lichen simplex chronicus. Typically the patient complains of incessant itch. The skin has thickened skin lines. It is hyperpigmented, usually brown in color indicative of epidermal hypermelanosis. However on occasion there is dermal melanosis and the discoloration has a gray hue. 966

Fig. 50.59. Thickened stratum corneum manifested as hyperpigmented scales.

Fig. 50.60. Seborrheic keratosis markedly hyperpigmented.

Histology The histopathology of all ichthyoses is characterized by hyperkeratosis or a thickened stratum corneum (Lever and Schaumberg-Lever, 1991; Pinkus and Mehregan, 1981). Seborrheic keratoses exhibit a great variety in clinical and histo-

ACQUIRED EPIDERMAL HYPERMELANOSES

Fig. 50.61. Heterogeneous pigmentation in a seborrheic keratosis.

logic appearances. All exhibit hyperkeratoses, acanthosis, and papillomatosis (Lever and Schaumberg-Lever, 1991; Pinkus and Mehregan, 1981). Melanocytes can be found in aberrant places. Typically they are located at the dermoepidermal junction. In the seborrheic keratosis, dendritic melanocytes can be seen in the mid-epidermis where they likely contribute to the deeply pigmented appearance. Lichen simplex chronicus exhibits similar histologic features, hyperkeratosis and acanthosis along with other features of dermatitis. It is likely that the hyperkeratosis and acanthosis are responsible for the discoloration typical of this condition. Fig. 50.62. Isolated hyperpigmented morphea.

Treatment and Pathogenesis These conditions are not primarily pigmentary disorders and the discoloration disappears when the epidermal thickening is corrected. For approach to treatment, standard textbooks of dermatology should be consulted.

ratio is about 3 : 1 (Krafchik, 1992; Serup, 1986; Uziel et al., 1994).

References

Clinical Findings

Lever, W. F., and G. Schaumberg-Lever. Histopathology of the Skin. Philadelphia: J. B. Lippincott, 1991. Pinkus, H., and A. H. Mehregan. A Guide to Dermatohistopathology. New York: Appleton-Century-Crofts, 1981.

Morphea and Scleroderma James J. Nordlund

Synonyms Morphea is also labeled localized scleroderma or circumscribed scleroderma.

Epidemiology The prevalence of morphea in the general population is estimated to be about 2–5 per million adults (Uziel et al., 1994). There are no good data for prevalence of morphea in children. Females seem to be affected more commonly than males; the

Morphea, or localized scleroderma, can affect children or adults (Krafchik, 1992; Serup, 1986; Uziel et al., 1994). Five clinical varieties have been described (Lever and SchaumbergLever, 1991): plaque, guttate, linear, segmental, and generalized. The plaque type (Fig. 50.62) is the most common and is characterized by indurated, often pigmented, depressed plaques on the trunk. Guttate lesions often surround the plaques. Widespread lesions on the trunk and extremities are termed generalized morphea (Fig. 50.63). It must be distinguished from scleroderma, which is not generalized morphea but a different disease. The linear form occurs most commonly on the extremities and on the scalp and face. The segmental form affects the face and causes atrophy of the skin, subcutaneous tissue, and muscles. The face is severely deformed. The early lesion of morphea is an erythematous plaque often indurated to palpation. There is a characteristic violet or lilac halo around the edge (Fig. 50.64). As the lesion evolves, the skin atrophies. The epidermis shows minimal changes other 967

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in facial hemiatrophy. The affected tissue can be very distorted. Atrophic, bound-down lesions across a joint can interfere with the motion of the limb. In one study of 30 children affected with morphea, 26 had the linear form. The lesions were on the extremity in 19 children and on the face in 7 children. Three had plaque morphea and one had generalized morphea (Uziel et al., 1994). In another study of 58 adults, 38 had the plaque or generalized form and only 20 had lesions on the extremities. Facial hemiatrophy was observed in eight of the adults (Serup, 1986). There seems to be a significant difference in the type of lesions observed in adults and children. There are many pigmentary changes. The earliest lesions have the lilac halo typical of the inflammatory stage of the disease. Hyperpigmentation, usually brown, which is suggestive of epidermal hypermelanosis, is common in all forms of morphea or localized scleroderma. It is especially common in the plaque and generalized variants and in the linear form on the extremities (Serup, 1986). Pigmentary changes are not common in the linear scleroderma on the face (en coup d’sabre). Gray hyperpigmentation suggestive of dermal melanosis and hypopigmentation have also been described (Krafchik, 1992). Pigmentary changes, both hyper- and hypopigmentation, have been associated with progressive systemic sclerosis (scleroderma) (Fig. 50.65).

Associated Findings

Fig. 50.63. Generalized hyperpigmented morphea.

Morphea should be distinguished from scleroderma which is a systemic disorder that clinically differs greatly from morphea. The histologic changes of the two are similar. Lichen sclerosis et atrophicus, a condition that results in atrophy of the epidermis and destruction of the melanocytes, is sometimes found in association with morphea. Some have considered the two disorders to be variable expressions of the same disorder (Tremaine et al., 1990; Uitto et al., 1980). Others consider morphea and lichen sclerosis et atrophicus to be different and not associated (Patterson and Ackerman, 1984). Atrophoderma of Pasini and Pierini is thought by others to be a variant of morphea (Kencka et al., 1995).

Histology

Fig. 50.64. Prominent atrophy and lilac ring.

than pigmentation (described later). There might be mild atrophy. The dermis is firm and the subcutaneous tissue atrophic. The mature plaque is depressed. There can be involvement of the muscles and connective tissue such as seen

968

The histology of early inflammatory lesions differs from that of late sclerotic lesions. Early lesions show thickened collagen and moderately severe inflammatory infiltrates usually composed of mononuclear cells. In later lesions the inflammatory infiltrate is gone and collagen deposition is marked (Lever and Schaumberg-Lever, 1991). The epidermis can be normal or show atrophy. In pigmented lesions there are increased quantities of melanin, which might be apparent only with special stains and when compared to biopsies from uninvolved skin. Lipid droplets are present in the cytoplasm of melanocytes but not in keratinocytes or Langerhans cells. This finding is not unique to morphea (Ortonne et al., 1980). The number of mast cells in the dermis is fewer than normal (Nishioka et al., 1987).

ACQUIRED EPIDERMAL HYPERMELANOSES

Fig. 50.65. Diffuse hyperpigmentation with depigmentation on the neck and upper trunk.

Pathogenesis The cause of morphea is not known. Attempts to document a role for Borrelia burgdorferi have been inconclusive but the weight of evidence seems to suggest that this organism does not have a role in morphea’s cause (Abele and Anders, 1990; Aberer et al., 1991; Meis et al., 1993; Schempp et al., 1993). Organic solvents (Yamakage and Ishikawa, 1982) and eosinophilic fasciitis (Hulshof et al., 1992; Mensing and Schmidt, 1985; Valentini et al., 1988) have been implicated.

Therapy There is no good therapy for morphea. Epidermal pigmentation can be treated with standard lightening agents such as hydroquinone, steroids, and retinoids. There are no published reports on the success of therapy.

References Abele, D. C., and K. H. Anders. The many faces and phases of borreliosis II [review]. J. Am. Acad. Dermatol. 23:401–410, 1990. Aberer, E., H. Klade, and G. Hobisch. A clinical, histological, and

immunohistochemical comparison of acrodermatitis chronica atrophicans and morphea. Am. J. Dermatopathol. 13:334–341, 1991. Hulshof, M. M., B. W. Boom, and B. A. Dijkmans. Multiple plaques of morphea developing in a patient with eosinophilic fasciitis [letter]. Arch. Dermatol. 128:1128–1129, 1992. Kencka, D., M. Blaszczyk, and S. Jablonska. Atrophoderma PasiniPierini is a primary atrophic abortive morphea. Dermatology 190:203–206, 1995. Krafchik, B. R. Localized cutaneous scleroderma [review]. Semin. Dermatol. 11:65–72, 1992. Lever, W. F., and G. Schaumberg-Lever. Histopathology of the Skin. Philadelphia: J. B. Lippincott, 1991. Meis, J. F., R. Koopman, B. van Bergen, G. Pool, and W. Melchers. No evidence for a relation between Borrelia burgdorferi infection and old lesions of localized scleroderma (morphea) [letter]. Arch. Dermatol. 129:386–387, 1993. Mensing, H., and K. U. Schmidt. Diffuse fasciitis with eosinophilia associated with morphea and lichen sclerosus et atrophicus. Acta Derm. Venereol. 65:80–83, 1985. Nishioka, K., Y. Kobayashi, I. Katayama, and C. Takijiri. Mast cell numbers in diffuse scleroderma. Arch. Dermatol. 123:205–208, 1987. Ortonne, J. P., H. Perrot, D. Schmitt, and P. Bioulac. Cutaneous hypermelanosis and intramelanotic lipid droplets. Arch. Dermatol. 116: 301–306, 1980. Patterson, J. A., and A. B. Ackerman. Lichen sclerosus et atrophicus is not related to morphea. A clinical and histologic study of 24 patients in whom both conditions were reputed to be present simultaneously. Am. J. Dermatopathol. 6:323–335, 1984. Schempp, C., H. Bocklage, R. Lange, H. W. Kolmel, C. E. Orfanos, and H. Gollnick. Further evidence for Borrelia burgdorferi infection in morphea and lichen sclerosus et atrophicus confirmed by DNA amplification. J. Invest. Dermatol. 100:717–720, 1993. Serup, J. Assessment of epidermal atrophy in localized scleroderma (morphea). Dermatologica 172:205–208, 1986. Tremaine, R., J. E. Adam, and M. Orizaga. Morphea coexisting with lichen sclerosus et atrophicus. Int. J. Dermatol. 29:486–489, 1990. Uitto, J., D. J. Santa Cruz, E. A. Bauer, and A. Z. Eisen. Morphea and lichen sclerosus et atrophicus. Clinical and histopathologic studies in patients with combined features. J. Am. Acad. Dermatol. 3:271–279, 1980. Uziel, Y., B. R. Krafchik, E. D. Silverman, P. S. Thorner, and R. M. Laxer. Localized scleroderma in childhood: a report of 30 cases. Semin. Arthritis Rheum. 23:328–340, 1994. Valentini, G., R. Rossiello, L. Gualdieri, G. Tirri, J. C. Gerster, and E. Frenck. Morphea developing in patients previously affected with eosinophilic fasciitis. Report of two cases. Rheumatol. Int. 8:235–237, 1988. Yamakage, A., and H. Ishikawa. Generalized morphea-like scleroderma occurring in people exposed to organic solvents. Dermatologica 165:186–193, 1982.

Pigmentary Changes Associated with Addison Disease Cindy L. Lamerson and James J. Nordlund

Historical Background In 1855 Thomas Addison published a monograph entitled On the Constitutional and Local Effects of Disease of the SupraRenal Gland, which presented the characteristics of what is now known as Addison disease.

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Fig. 50.66. Hyperpigmentation of the dorsum of the feet.

Fig. 50.67. Hyperpigmented palmar creases.

Synonyms Addison disease is also known as hypocorticism, Addison syndrome, suprarenal insufficiency, and hypoadrenalism.

Epidemiology Addison disease is caused by a deficiency of adrenocortical hormones including glucocorticoids such as cortisone, and mineralocorticoids such as aldosterone. Addison is an uncommon disorder affecting approximately 1 in 10 000 individuals (Kong and Jeffcoate, 1994). The characteristic features of the disorder include a generalized brown hyperpigmentation, hypotension, weakness, nausea, vomiting, and diarrhea. There is no age, race, or sexual predilection.

Clinical Findings One of the most striking cutaneous changes in Addison disease is hyperpigmentation of the skin and mucous membranes (Plates 50.26–50.28; Kong and Jeffcoate, 1994; Mulligan and Sowers, 1985; Whitehead et al., 1989; Zelissen et al., 1995). In 20–40% of patients the hyperpigmentation is the presenting symptom of their disease. The pigmentation is a brown or bronze color suggestive of a deep suntan. The hyperpigmentation is generalized in distribution but more accentuated in sun-exposed areas. Often this brown hyperpigmentation is referred to as the persistence of a summer tan. The pigmentation is usually darker in areas prone to trauma (Fig. 50.66) such as palmar creases (Fig. 50.67 and Plate 28.5, pp. 494–495), knees, and elbows, which are subjected to pressure and friction. There is also darker pigmentation in scars (Fig. 50.68). Common areas for deeper pigmentation are the areola (Fig. 50.69), axilla, perineum, and genitalia. Moreover, the linea nigra may become prominent in parous women. Hyperpigmentation of the buccal and gingival mucosa as well as the tongue (Fig. 50.70) may be present as patchy macules and may be a gray blue color. In addition to the hyperpigmentation of the normal skin, melanocytic nevi may darken and lentigines may appear (Braverman, 1981; Nerup, 1974). The hair may darken and gray hairs may regain pigmentation. Longitudinal pigmented bands may be prominent in 970

Fig. 50.68. Hyperpigmented surgical scar.

the nails (Allenby and Snell, 1966; Sterling et al., 1988). The bands are similar to those normally occurring in the nails of individuals with type V or type VI skin. In addition to the pigmentary changes, individuals with Addison disease may have other cutaneous changes, including loss of body hair, especially in the axilla, and fibrosis and calcification of the pinna (Graner, 1985; Haegy, 1982; Ketterer and Frenk, 1995; Kim, 1988; Shimao, 1971).

Associated Disorders Adrenal atrophy is rarely a single disorder. Currently, adrenal disease is most commonly caused by an autoimmune mechanism in which antibodies are thought to destroy the gland. Such individuals commonly have other endocrine abnormalities caused by antibodies. These include thyroid disease, diabetes mellitus, pernicious anemia, and/or gonadal failure (Appel and Holub, 1976; Chen et al., 1996; Inaba and Morii, 1995; Nomura et al., 1994; Zelissen et al., 1995). Other individuals have tuberculosis, systemic fungal infec-

ACQUIRED EPIDERMAL HYPERMELANOSES

Histology The histology of pigmentary changes of Addison disease is not diagnostic. There is no increase in the number of melanocytes. The melanocytes are more active and produce increased amounts of melanin. Increased melanin is visible in the basal layer of the epidermis and upper layers, including the stratum corneum. Melanophages may be present in the upper dermis (Lerner, 1955; Montgomery and O’Leary, 1930).

Pathogenesis

A

B Fig. 50.69. Hyperpigmentation of areolae before treatment (A) that resolved after treatment (B) (see also Plate 50.26A and 50.26B, pp. 494–495).

The adrenal cortex fails to synthesize glucocorticoids appropriately in individuals with Addison disease. Adrenocortical failure is most often a result of atrophy of the gland (Kong and Jeffcoate, 1994; Zelissen et al., 1995), although very rarely the patient might have a defect in the pituitary (Yamamoto et al., 1992). These latter individuals do not exhibit hyperpigmentation. The adrenal gland may be destroyed by a number of mechanisms, including autoimmune antibodies (Chen et al., 1996; Inaba and Morii, 1995; Kong and Jeffcoate, 1994; Nomura et al., 1994; Zelissen et al., 1995), tuberculosis, deep fungi (histoplasmosis and paracoccidiomycosis) (Kong and Jeffcoate, 1994; Nomura et al., 1994; Zelissen et al., 1995), or metastatic carcinoma (Kong and Jeffcoate, 1994). In healthy individuals the adrenal gland is stimulated by adrenocorticotropic hormone (ACTH) secreted by the pituitary gland. ACTH is derived from its precursor, proopiomelanocortin. The adrenal gland synthesizes cortisone that serves to suppress the pituitary function, a feedback mechanism. When the gland is destroyed by any mechanism, there is a disruption of the normal cortisol–pituitary–hypothalamic feedback, and ACTH levels rise. Along with ACTH, other peptides from proopiomelanocortin are also produced in increased quantities. These include b-lipotropin (LPH), g-melanotropin, b-melanotropin, and a-melanotropin. It is thought that the molecules of melanotropin give rise to the pigmentation (Shizume, 1985).

Criteria for Diagnosis and Laboratory Abnormalities

Fig. 50.70. Typical hyperpigmentation of Addison disease (see also Plate 50.27, pp. 494–495).

tions, or metastatic carcinoma (Kong and Jeffcoate, 1994; Nomura et al., 1994; Zelissen et al., 1995). Adrenoleukodystrophy is a cause of adrenal disease in young boys (Kong and Jeffcoate, 1994; Laureti et al., 1996).

Addison disease cannot be diagnosed in the absence of adrenal hypofunction. The clinical manifestations are not specific (Marlette, 1975; Runcie et al., 1986; Strakosch and Gordon, 1978; Whitehead et al., 1989). ACTH stimulation test is used to diagnose adrenal cortical failure. Serum cortisol levels are measured following stimulation with ACTH. If the cortisol levels remain low despite ACTH stimulation, the diagnosis of Addison disease is made. Early in the course of the disease, the test may be negative, so repeat testing may be necessary. Moreover, partially compensated hypoadrenalism may present with skin hyperpigmentation (Whitehead et al., 1989). In approximately 50% of patients, autoantibodies directed against the adrenal gland are present in the patient’s serum (Chen et al., 1996; Nomura et al., 1994; Whitehead et al., 1989; Zelissen et al., 1995). Many other metabolic abnormalities are associated with 971

CHAPTER 50 Table 50.4. Differential diagnosis of generalized brown hyperpigmentation. Exogenous administration of ACTH Nelson syndrome AIDS (resulting from glucocorticoid deficiency at target tissues) (Norbiato et al., 1994) Hyperthyroidism Carcinoid Pheochromocytoma Cirrhosis POEMS syndrome Siemerling–Creuzfeldt disease; adrenoleukodystrophy Drug-induced hyperpigmentation Methotrexate 5-fluorouracil Congenital polychlorinated biphenyl (PCB) poisoning (Miller, 1985)

adrenal insufficiency. Many of these are related to the deficiency of mineralocorticoids such as aldosterone. Such abnormalities include hypoglycemia, hyponatremia, and hyperkalemia, as well as an elevated blood urea nitrogen.

Differential Diagnosis The differential diagnosis of diffuse brown hyperpigmentation of Addison disease is presented in Table 50.4.

Treatment Replacement therapy permits the pigment to fade gradually. Without such treatment the disease will be progressive and lead to the death of the individual. The usual therapy is prednisone and a mineralocorticoid such as fludrocortisone. If treatment is initiated appropriately, the prognosis for the disease is excellent (Shimao, 1971).

References Addison, T. On the Constitutional and Local Effects of Disease of the Supra-Renal Capsules. London: Samuel Highley, 1855, p. 43. Allenby, C. F., and P. H. Snell. Longitudinal pigmentation of the nails in Addison’s disease. Br. Med. J. 5503:1582–1583, 1966. Appel, G. B., and D. A. Holub. The syndrome of multiple endocrine gland insufficiency. Am. J. Med. 61:129–133, 1976. Braverman, I. M. Skin Signs of Systemic Disease, 2nd ed. Philadelphia: W. B. Saunders, 1981, p. 366. Chen, S., J. Sawicka, C. Betterle, M. Powell, L. Prentice, M. Volpato, B. Rees Smith, and J. Furmaniak. Autoantibodies to steroidogenic enzymes in autoimmune polyglandular syndrome, Addison’s disease, and premature ovarian failure. J. Clin. Endocrinol. Metab. 81:1871–1876, 1996. Graner, J. L. Addison, pernicious anemia and adrenal insufficiency. Can. Med. Assoc. J. 133:855–857, 1985. Haegy, J. M. Acute adreno-cortical insufficiency. Eight case reports. Semaine Hopitaux 58:143–147, 1982. Inaba, M., and H. Morii. Polyglandular autoimmune syndrome [review; in Japanese]. Nippon Rinsho 53:974–978, 1995.

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Ketterer, R., and E. Frenk. Skin changes in endocrine disorders (with the exception of diabetes) [review; in German]. Ther. Umsch. 52:269–274, 1995. Kim, H. W. Generalized oral and cutaneous hyperpigmentation in Addison’s disease. Odonto-Stomatol. Tropicale 11:87–90, 1988. Kong, M. F., and W. Jeffcoate. Eighty-six cases of Addison’s disease. Clin. Endocrinol. 41:757–761, 1994. Laureti, S., G. Casucci, F. Santeusanio, G. Angeletti, P. Aubourg, and P. Brunetti. X-linked adrenoleukodystrophy is a frequent cause of idiopathic Addison’s disease in young adult male patients. J. Clin. Endocrinol. Metab. 81:470–474, 1996. Lerner, A. B. Melanin pigmentation. Am. J. Med. 19:902–924, 1955. Marlette, R. H. Generalized melanoses and nonmelanotic pigmentations of the head and neck. J. Am. Dent. Assoc. 90:141–147, 1975. Miller, R. W. Congenital PCB poisoning: a reevaluation. Environ. Health Perspec. 60:211–214, 1985. Montgomery, H., and P. A. O’Leary. Pigmentation of the skin in Addison’s disease, acanthosis nigricans and hemochromatosis. Arch. Dermatol. Syphil. 21:970–984, 1930. Mulligan, T. M., and J. R. Sowers. Hyperpigmentation, vitiligo, and Addison’s disease. Cutis 36:317–318, 322, 1985. Nerup, J. Addison’s disease: a review of some clinical, pathological and immunological features. Dan. Med. Bull. 21:201–207, 1974. Nomura, K., H. Demura, and T. Saruta. Addison’s disease in Japan: characteristics and changes revealed in a nationwide survey. Intern. Med. 33:602–606, 1994. Norbiato, G., M. Galli, V. Righini, and M. Moroni. The syndrome of acquired glucocorticoid resistance in HIV infection. Ballieres Clin. Endocrinol. Metab. 8:777–787, 1994. Runcie, C. J., C. G. Semple, and S. D. Slater. Addison’s disease without pigmentation. Scott. Med. J. 31:111–112, 1986. Shimao, S. Addison’s disease and problems in hormone therapy: skin hyperpigmentation and hormone therapy. Horumon to Rinsho Clin. Endocrinol. 19:103–107, 1971. Shizume, K. Thirty five years of progress in the study of MSH. Yale J. Biol. Med. 58:561–570, 1985. Sterling, G. B., L. F. Libow, and M. E. Grossman. Pigmented nail streaks may indicate Laugier-Hunziker syndrome. Cutis 42: 325–326, 1988. Strakosch, C. R., and R. D. Gordon. Early diagnosis of Addison’s disease: pigmentation as sole symptom. Aust. N. Z. J. Med. 8:189– 190, 1978. Whitehead, E. M., A. B. Atkinson, D. R. Hadden, J. Weaver, and B. Sheridan. Partially compensated hypoadrenalism presenting with persistent skin pigmentation. J. Endocrinol. Invest. 12:187–191, 1989. Yamamoto, T., J. Fukuyama, K. Hasegawa, and M. Sugiura. Isolated corticotropin deficiency in adults. Report of 10 cases and review of literature [review]. Arch. Intern. Med. 152:1705–1712, 1992. Zelissen, P. M., E. J. Bast, and R. J. Croughs. Associated autoimmunity in Addison’s disease. J. Autoimmun. 8:121–130, 1995.

Pigmentary Changes Associated with Cutaneous Lymphomas Debra L. Breneman

Historical Background The term mycosis fungoides was introduced by Alibert to

ACQUIRED EPIDERMAL HYPERMELANOSES

characterize a patient with a desquamating rash who developed tumors resembling mushrooms. Bazin (1870) described a natural progression from a premycotic phase to tumors. An erythrodermic phase was later described by Besnier and Hallopeau (1892). Sézary and Bouvrain (1938) described the triad of erythroderma, leukemia composed of large mononuclear cells with convoluted nuclei, and lymphadenopathy. This subsequently became known as Sézary syndrome (Taswell and Winkelmann, 1961). It was later shown that some patients with mycosis fungoides developed involvement of the blood and viscera (Clendenning et al., 1964; Epstein et al., 1972). Studies using cytogenetics and cellular immunology showed these diseases represented neoplasms of T lymphocytes (Broome et al., 1973; Crossen et al., 1971). In the vast majority of cases the abnormal cells were found to have functional and phenotypic features of helper T cells (Berger et al., 1979; Kung et al., 1981).

Synonyms The designation, cutaneous T-cell lymphoma, represents a group of neoplasms of T-cells that present with the skin as the initial site of involvement. Mycosis fungoides is the most common type of cutaneous T-cell lymphoma. Other types of cutaneous T-cell lymphoma include Sézary syndrome, adult T-cell leukemia/lymphoma, CD30+ large cell lymphoma, angiocentric T-cell lymphoma, and subcutaneous T-cell lymphoma.

Epidemiology Mycosis fungoides is increasing in incidence. The incidence has been reported to be as high as 0.9 per 100 000 (Chuang et al., 1990). The cause is unknown. Environmental, infectious, and genetic factors have all been postulated to play a role in the development of this disease but these proposals have not been proved (Tuyp et al., 1987).

General Clinical Findings Mycosis fungoides has been reported to occur mostly in older individuals and is rare in children and adolescents (Peters et al., 1990). It has been reported to occur more frequently in blacks and is also more common in men, with male/female ratios variably being reported at 2:1 to 3:1 (Chuang et al., 1990).

Clinical Description of Disease The natural history of mycosis fungoides is variable. Most patients have a protracted preclinical phase and a prolonged survival with little morbidity. Others may have a fulminate course with rapid development of systemic dissemination and death (Bunn and Lamberg, 1979). There are three distinct cutaneous phases which occur in mycosis fungoides (Edelson, 1980). In the patch stage the lesions are flat and nonpalpable. They are often erythematous and may be scaly. They tend to occur on covered portions of

Fig. 50.71. Multiple patches of hypopigmented mycosis fungoides.

the body, and unusual shapes are common. In a variant of the patch phase, poikiloderma vasculare atrophicans, the skin is atrophic with mottled pigmentation and telangiectasia. In the plaque phase of mycosis fungoides the lesions become elevated above the surrounding skin surface. They may be erythematous or violaceous. In heavily pigmented individuals the plaques may be hyperpigmented. They may ulcerate and become secondarily infected. In the tumor phase the lesions become further elevated. The tumors may arise de novo or in pre-existing plaques. Mycosis fungoides may also progress to an exfoliative erythroderma in which the skin is diffusely red and scaly and there is severe pruritus. Mycosis fungoides may disseminate to extracutaneous sites. The sites of dissemination are varied and disease has been identified in virtually every organ system (Rappaport and Thomas, 1974). The lymph nodes are the most common site of extracutaneous involvement. Gross involvement of the peripheral blood generally occurs relatively late in the disease. There is a strong association between gross lymph node or blood involvement and visceral disease. Dissemination may occur late in the disease to the bone marrow, liver, spleen, lungs, bones, central nervous system, and other organs.

Hypopigmentation and Mycosis Fungoides Cutaneous pigmentary changes are well recognized in mycosis fungoides but usually occur either in association with poikiloderma vasculare atrophicans or following treatment and regression of the cutaneous lesions, with a resultant mixed pattern of hypo- and hyperpigmentation (Smith and Sammon, 1978). Additionally, untreated plaques of mycosis fungoides in heavily pigmented individuals commonly are hyperpigmented (Zackheim et al., 1982). Hypopigmented mycosis fungoides (Figs 50.71 and 50.72) was initially described in 1973 (Ryan et al., 1973). To date,

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lymphoma mimicking porokeratosis have been reported (Hsu et al., 1992). These patients had widespread sharply demarcated macules that clinically resembled disseminated superficial actinic porokeratosis. In one patient the macules were hypopigmented and in the other they were hyperpigmented.

Associated Disorders

Fig. 50.72. Hypopigmented mycosis fungoides characterized by round to irregular hypopigmented patches.

there have been 29 cases reported of hypopigmented mycosis fungoides (Breathnach et al., 1982; Cooper et al., 1992; Dabski et al., 1985; Goldberg et al., 1986; Handfield-Jones et al., 1992; Lambroza et al., 1995; Misch et al., 1987; Ryan et al., 1973; Rustin et al., 1986; Sigal et al., 1987; Smith and Sammon, 1978; Volkenandt et al., 1993; Whitmore et al., 1994; Zackheim et al., 1982). The age of onset of reported cases ranges from 4 to 72 years with most cases beginning before the fourth decade. Eleven cases have been reported with onset of hypopigmented mycosis fungoides under the age of 20 years. This is in contrast to mycosis fungoides in general which more commonly presents in older adults. Hypopigmented mycosis fungoides occurs with equal frequency in men and women (Lambroza et al., 1995). Only one case of hypopigmented mycosis fungoides has been reported in a white patient (Sigal et al., 1987). All other cases have been reported in individuals with brown or black skin (Lambroza et al., 1995). Hypopigmented mycosis fungoides presents clinically as circular or oval areas of hypopigmentation with distinct or indistinct borders. There may be some degree of erythema, scaling, and infiltration. Small islands of normal pigmentation may be present in their center. Patients may present with macular hypopigmentation only or there may be associated erythematous patches, plaques, and papules as well as poikiloderma. Only a few lesions may be present, or most of the body surface may be involved. The hypopigmentation generally involves the trunk and extremities, and only rarely is the face involved. Areas of involvement are often bilaterally symmetric and there may be a progressive loss of pigment over time. Hypopigmented macules appear de novo and are not preceded by erythema, scaling, or induration. Pruritus within lesions is variable. Hypopigmented areas have been reported to be present for up to 20 years prior to presentation. Areas of pigmentary loss are generally hypopigmented but occasionally may appear depigmented. Additionally, two black patients with cutaneous T-cell 974

Poikiloderma vasculare atrophicans and large plaque parapsoriasis both may precede mycosis fungoides, and are felt by some authors to be indistinguishable from early mycosis fungoides (Lindae et al., 1988; Sanchez and Ackerman, 1979). Poikiloderma vasculare atrophicans is characterized by atrophic patches containing hypo- and hyperpigmented macules and telangiectasia. A progression to overt mycosis fungoides may be associated with some degree of induration. The clinical findings of large plaque parapsoriasis may be identical to those found in patch stage mycosis fungoides. These lesions are erythematous to light brown patches which may be atrophic and have a fine superficial scale. Both conditions have a predilection for covered areas of the body. Lymphomatoid papulosis is an entity consisting of recurrent self-healing papulo-nodular lesions that resemble pityriasis lichenoides et varioliformis acuta clinically but lymphoma histologically. Lymphomatoid papulosis most commonly has a benign clinical course lasting for months to decades (Thomsen and Wantzin, 1987). Approximately 25% of patients with lymphomatoid papulosis develop lymphoma with mycosis fungoides being the most commonly associated type (Wantzin et al., 1985). Mycosis fungoides may precede, develop concurrently, or appear after the onset of lymphomatoid papulosis. Follicular mucinosis is a condition of unknown etiology consisting of follicular papules or indurated plaques with hair loss. Histologically there is accumulation of acid mucopolysaccharides in the pilosebaceous unit. A coexisting lymphoma, most commonly mycosis fungoides, occurs in 15% of patients with follicular mucinosis (Lancer et al., 1984). Patients with coexisting follicular mucinosis and mycosis fungoides are generally in the fourth to seventh decades of life.

Other Pigmentary Abnormalities in Mycosis Fungoides Diffuse progressive hyperpigmentation as a manifestation of mycosis fungoides has been reported in one patient (David et al., 1987). The patient, who presented for evaluation of hyperpigmentation and pruritus of simultaneous onset, had widespread, poorly defined, dark brown macules over much of the skin surface. Histologically the hyperpigmented macules showed typical findings of mycosis fungoides. The patient, who had tumor stage disease and lymph node involvement, died of complications of staphylococcal sepsis. In addition to histologic changes typical of mycosis fungoides, a large amount of melanin in the basal layer and giant melanin granules in focal areas throughout the spinous layer were noted.

ACQUIRED EPIDERMAL HYPERMELANOSES

One patient who was subsequently shown to have mycosis fungoides presented with well-defined, blue macules within what appeared to be an eczematous dermatitis (Neuman, 1984). Histologic examination of a blue macule, in addition to changes characteristic of mycosis fungoides, showed marked incontinence of pigment. This may have led to a Tyndall effect producing the bluish discoloration. Four patients have been reported who developed pigmented purpuralike eruptions which progressed to mycosis fungoides (Barnhill and Braverman, 1988; Waisman, 1976). These patients presented with petechial patches with varying degrees of scale, pigmentation, and lichenification. Initial biopsies were consistent with pigmented purpuric dermatosis, and the diagnosis of mycosis fungoides was made following serial skin biopsies.

Routine Histology, Histochemistry, and Immunochemistry

Fig. 50.73. Lower trunk and thighs of patient with mycosis fungoides showing reticulated hyperpigmentation (see also Plate 50.28, pp. 494–495).

Electron microscopy revealed large lymphocytes with cerebriform nuclei in the dermis and epidermis. Melanin granules were seen within the cytoplasm of tumor cells infiltrating the dermis, in typical Lutzner cells, and in nontumoral macrophages. In the epidermis, keratinocytes of the suprabasal layer contained multiple dispersed melanin granules with several giant granules within the cytoplasm. Langerhans cells in the epidermis showed a few aggregated melanin granules. A patient with florid reticulate pigmentation (see Fig 50.73) as a manifestation of mycosis fungoides has been reported (McDonagh et al., 1991). The patient, who had tumors involving the chest and back, died of unrelated causes. In other patients with mycosis fungoides reported with reticulate pigmentation the pigmentary abnormality has been more limited in extent and severity (Brehmer-Andersson, 1976). Histologically within the hyperpigmented areas in this patient, in addition to features typical of mycosis fungoides, pigmentary incontinence and numerous melanophages within the dermis were noted.

The histologic appearance of mycosis fungoides varies with the cutaneous stage of the disease (Nickoloff, 1988). The histologic diagnosis of the patch stage is often difficult and early biopsies may be described as showing a nonspecific chronic dermatitis. In the patch and plaque stages, epidermotropism of lymphocytes is present, often with minimal spongiosis. Infiltration of the epidermis by mononuclear cells without evident spongiosis is strongly suggestive of early cutaneous T-cell lymphoma. Lymphocytes may appear to line up along the dermal–epidermal junction. In the patch stage there is a sparse infiltrate of mononuclear cells in the upper dermis. The lymphocytes may or may not appear to be pleomorphic or hyperchromatic or contain convoluted nuclei. As the clinical lesions become thicker, forming definite plaques, there is a tendency for epidermotropic cells to form Pautrier microabscesses, which are clusters of lymphocytes in the epidermis. A lichenoid and/or perivascular infiltrate is often present. The mononuclear cells may show deeply indented nuclei along with a mixed inflammatory response. In the tumor stage the infiltrate is denser and extends into the subcutaneous fat, and there may be a loss of epidermotropism. There may be varying degrees of transformation with mononuclear cells containing large hyperchromatic nuclei, prominent nucleoli, and frequent mitotic figures. In hypopigmented mycosis fungoides, exocytosis is often moderate and may be so extreme as to simulate pagetoid reticulosis (Lambroza et al., 1995). Dermal involvement may be minimal. Melanin may be reduced or absent, and mild to marked pigmentary incontinence may be seen. Immunocytochemistry studies in lesions of mycosis fungoides generally show predominance of CD4 antigen positive cells. Partial loss of the CD7 antigen is common (Whitmore et al., 1994).

Other Investigations Electron microscopy of hypopigmented mycosis fungoides, in addition to showing focal invasion of the epidermis by atypical lymphocytes, may show enlargement of epidermal 975

CHAPTER 50

intercellular spaces suggestive of intraepidermal edema, and degenerative changes in adjacent melanocytes and keratinocytes in some cases (Breathnach et al., 1982; Misch et al., 1987; Sigal et al., 1987). Degenerative changes present within melanocytes may include extensive cytoplasmic vacuolization, marked dilatation of the rough endoplasmic reticulum, and swelling of mitochondria with loss of cristae. Melanocytes may be incompletely melanized and occasionally may be undergoing disintegration. Occasionally the overall numbers of melanocytes may be reduced. Numbers of melanosomes within keratinocytes may be normal or decreased. Melanin-containing macrophages may be seen in the papillary dermis (Goldberg et al., 1986; Lambroza et al., 1995). By using a specific molecular probe for the malignant lymphoid clone in a patient with mycosis fungoides, hypopigmented lesions, which were previously unclassifiable by conventional microscopy, were found to contain tumor clonespecific DNA. This evidence suggested that the hypopigmented lesions were actually early manifestations of mycosis fungoides (Volkenandt et al., 1993). In another patient with hypopigmented mycosis fungoides, T-cell receptor gene rearrangement analysis was negative (Lambroza et al., 1995). However, gene rearrangement studies are frequently negative in early stage mycosis fungoides.

Criteria for Diagnosis The diagnosis of mycosis fungoides is generally made by light microscopy of hematoxylin and eosin stained sections from involved skin (Nickoloff, 1988). Multiple biopsies obtained over months or years are sometimes necessary to establish the diagnosis. Immunoperoxidase studies and T-cell receptor gene rearrangement analysis are sometimes useful in confirming the diagnosis. Further evaluation includes a thorough physical examination with emphasis on examination of the lymph nodes, liver, and spleen. Peripheral blood studies should include a complete blood count with determination of the white blood cell count and examination of a peripheral smear for the presence of atypical lymphocytes. Additional testing for blood involvement may include lymphocyte flow cytometry and T-cell receptor gene rearrangement analysis in some cases. Other baseline tests usually include serum chemistries and lactic dehydrogenase and a chest X-ray. Additional staging procedures useful in selected patients include computed tomography (CT) scan of the abdomen and pelvis, lymph node biopsy, and bone marrow biopsy (Sausville et al., 1988). Further evaluation of specific organ systems is indicated when involvement is suggested by history, physical examination, or baseline test results.

Differential Diagnosis Mycosis fungoides is a great mimicker and it has been reported to imitate numerous other cutaneous diseases. It is most commonly mistaken for chronic dermatitis. The differential diagnosis of hypopigmented mycosis fungoides includes vitiligo, 976

idiopathic guttate hypomelanosis, tinea versicolor, pityriasis alba, sarcoidosis, leprosy, pityriasis lichenoides chronica, and postinflammatory hypopigmentation.

Pathogenesis Mycosis fungoides represents a monoclonal expansion of helper T cells which in the initial stages have a strong affinity for the epidermis (Heald and Edelson, 1993). The disease may initially remain localized to one area of the skin but with time can progress to involve multiple regions. Subclones that are biologically more malignant exhibit less affinity for the epidermis. They enter a vertical growth pattern and form tumors. Subclones with decreased affinity for the skin may lead to spread to lymph nodes and viscera. The development of hypopigmented mycosis fungoides lesions may be related to melanocyte degeneration and abnormal melanogenesis caused by a nonspecific response to cell injury associated with inflammation (Breathnach et al., 1982). A defect in melanosome transfer has also been postulated as an etiology for the reduced pigment (Goldberg et al., 1986). More than one cause for this pigmentary defect may be operational. A spectrum of abnormal melanogenesis may exist, with pigment cell death as the most extreme expression (Lambroza et al., 1995).

Animal Models A cutaneous lymphoma similar to mycosis fungoides has been described in dogs (Fivenson et al., 1994). Canine mycosis fungoides is a T-cell lymphoma that mimics mycosis fungoides immunophenotypically and ultrastructurally.

Treatments There is an often bewildering array of therapeutic options available for the treatment of mycosis fungoides. Much of the treatment is directed towards skin involvement. Especially in early stage patients, there is no evidence that systemic chemotherapy alters the prognosis. A number of topical therapies are used in treating patients with mycosis fungoides. Very little has been published on the use of topical corticosteroids, although they are routinely used in clinical practice in patients with early stage skin disease or as adjunctive therapy. Two topical chemotherapeutic agents, mechlorethamine hydrochloride and carmustine, are used routinely in mycosis fungoides. Topical mechlorethamine has been used for this disease for more than 30 years, is effective in early disease, and has little toxicity (Vonderheid et al., 1989). Mechlorethamine may be applied either as an aqueous or ointment preparation. Carmustine (BCNU), like mechlorethamine hydrochloride, is an alkylating agent. Carmustine is generally applied in solution form but may also be applied as an ointment. Efficacy rates are similar to those attained with topical mechlorethamine (Zackheim et al., 1992). Both UVB and PUVA phototherapy have been used in treating mycosis fungoides. UVB phototherapy is generally

ACQUIRED EPIDERMAL HYPERMELANOSES

most useful for patients with patch phase skin disease (Resnick and Vonderheid, 1993). PUVA is most useful in patients with patch or plaque phase skin disease (Gilchrest, 1979). Relapse is common when treatment is discontinued. Because neoplastic cells of mycosis fungoides are radiosensitive, radiation is useful in the treatment of this disease. Total skin irradiation allows delivery of high energy electrons to a limited depth of the entire skin surface and prevents systemic toxicity. Total skin irradiation provides the highest complete response rate in patients with early skin disease that can be achieved with any treatment modality (Hoppe et al., 1969). Local radiotherapy is commonly used in treating individual tumors or ulcerating plaques. Advanced mycosis fungoides often requires systemic therapy. Objective response rates of 60–70% have been reported with single agent cytotoxic chemotherapy, but median duration of response is less than six months (Holloway et al., 1992). Single agents which have been used include mechlorethamine, cyclophosphamide, chlorambucil, methotrexate, cisplatin, prednisone, VP16, vincristine, adriamycin, and bleomycin. Newer agents include fludarabine monophosphate, deoxycoformycin, and 2-chlorodeoxyadenosine. Combination chemotherapy utilizes multiple drugs to inhibit different phases of the cell cycle. Objective response rates of 60–100% have been reported in patients with mycosis fungoides treated with combination chemotherapy (Holloway et al., 1992). Other systemic agents which are useful in the treatment of mycosis fungoides include the interferons and the retinoids. Photophoresis, also known as extracorporeal photochemotherapy, is effective in some erythrodermic patients (Holloway et al., 1992). Patients with hypopigmented mycosis fungoides have been reported to develop perifollicular repigmentation within hypopigmented patches with PUVA therapy. This may be followed by clearing both clinically and histologically (Breathnach et al., 1982; Handfield-Jones et al., 1992; Lambroza et al., 1995; Whitmore et al., 1994). Partial or complete repigmentation of hypopigmented mycosis fungoides may result from the use of carmustine (Zackheim et al., 1982), topical mechlorethamine (Goldberg et al., 1986), total skin irradiation, or adjuvant chemotherapy (Lambroza et al., 1995). However, interruption of therapy may result in recurrence of hypopigmentation. Of note, hyperpigmentation may occur as a result of some topical agents used in treating mycosis fungoides. Particularly in dark complexioned persons, hyperpigmentation may follow the topical application of carmustine or mechlorethamine hydrochloride. Hyperpigmentation may also occur with ultraviolet B or PUVA phototherapy (Zackheim et al., 1982).

Prognosis Patients with mycosis fungoides may be divided into three distinct prognostic groups (Sausville et al., 1988). Patients with the best prognosis are those with patch or plaque phase skin disease without involvement of lymph nodes, blood, or

viscera. These patients have a median survival of greater than 12 years. An intermediate prognostic group consists of patients with tumors or erythroderma or those with early involvement of the lymph nodes or blood but without visceral involvement. These patients have a median survival of five years. Poor risk patients are those with visceral disease or effaced lymph nodes. These patients have a median survival of 2.5 years. The long-term prognosis of patients with hypopigmented mycosis fungoides is unknown, particularly since many of these patients present at a younger age than do patients with typical mycosis fungoides. Most patients reported with hypopigmented mycosis fungoides have had indolent disease over limited follow-up periods (Whitmore et al., 1994). One patient, however, developed cutaneous tumors and involvement of axillary lymph nodes, and subsequently died of bone marrow aplasia and septicemia, despite topical and systemic chemotherapy and electron beam therapy (Sigal et al., 1987). Of note, this patient was white, in contrast with most patients with hypopigmented mycosis fungoides who are dark skinned.

References Alibert, J. L. Description des maladies de la peau: Observees à l-hopital St. Louis et exposition des eilleurs methodes Suivies pour leur traitement. Paris: Barrois l’aine et Fils; p. 157, 1806. Barnhill, R. L., and I. M. Braverman. Progression of pigmented purpura-like eruptions to mycosis fungoides: Report of three cases. J. Am. Acad. Dermatol. 19:25–31, 1988. Bazin, E. Leçons sur le traitement des maladies chroniques en général affections de la peau en particulier par l’emploi comparé des eaux minérales de l’hydrothérapie et des moyens pharmaceutiques. Paris: Adrien Delahaye; p. 425, 1870. Berger, C. L., D. Warburton, J. Raafeet, P. LoGerfo, and R. L. Edelson. Cutaneous T-cell lymphoma: Neoplasm of T-cells with helper activity. Blood 53:642–651, 1979. Besnier, E., and H. Hallopeau. On the erythrodermia of mycosis fungoides. J. Cutan. Genito. Urin. Dis. 10:453, 1892. Breathnach, S. M., P. H. McKee, and N. P. Smith. Hypopigmented mycosis fungoides: Report of five cases with ultrastructural observation. Br. J. Dermatol. 106:643–649, 1982. Brehmer-Andersson, E. Mycosis fungoides and its relationship to Sezary syndrome, lymphomatoid papulosis, and primary cutaneous Hodgkin’s disease. Acta Derm. Venereol. Suppl. (Stockh.) 56:1– 142, 1976. Broome, J. D., D. Zucker-Franklin, M. S. Weiner, C. Bianco, and V. Nussenweig. Leukemic cells with membrane properties of thymusderived (T) lymphocytes in a case of Sezary syndrome: morphologic and immunologic studies. Clin. Immunol. Immunopathol. 1:319– 329, 1973. Bunn, P. A., and S. I. Lamberg. Report of the committee on staging and classification of cutaneous T-cell lymphomas. Cancer Treatment Rep. 63:725–728, 1979. Chuang, T.-Y., D. Su, and S. A. Muller. Incidence of cutaneous T-cell lymphoma and other rare skin cancers in a defined population. J. Am. Acad. Dermatol. 23:254–256, 1990. Clendenning, W. E., G. Brecher, and E. J. Van Scott. Mycosis fungoides: Relationship to malignant cutaneous reticulosis and the Sezary syndrome. Arch. Dermatol. 89:785–792, 1964. Cooper, D., M. Jacobson, and R. S. Bart. Hypopigmented macules. Hypopigmented mycosis fungoides (MF). Arch. Dermatol. 128: 1266–1267,1269–1270, 1992.

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CHAPTER 50 Crossen, P. E., J. E. Mellor, A. G. Finley, R. B. M. Ravich, P. C. Vincent, and F. W. Gunz. The Sezary syndrome: Cytogenetic studies and identification of the Sezary cell as an abnormal lymphocyte. Am. J. Med. 50:24–34, 1971. Dabski, K., H. L. Stoll, and H. Milgrom. Unusual clinical presentation of epidermotropic cutaneous lymphoma: small hypopigmented macules. Int. J. Dermatol. 24:108–115, 1985. David, M., A. Shanon, B. Hazaz, and M. Sandbank. Diffuse progressive hyperpigmentation: An unusual skin manifestation of mycosis fungoides. J. Am. Acad. Dermatol. 16:257–260, 1987. Edelson, R. L. Cutaneous T-cell lymphoma: Mycosis fungoides, Sezary syndrome, and other variants. J. Am. Acad. Dermatol. 2:89–106, 1980. Epstein, E. H., D. L. Levin, J. D. Croft, and M. A. Lutzner. Mycosis fungoides. Survival, prognostic features, response to therapy and autopsy findings. Medicine (Baltimore) 51:61–72, 1972. Fivenson, D. P., G. M. Saed, E. R. Beck, R. W. Dunstan, and P. F. Moore. T-cell receptor cell gene rearrangement in canine mycosis fungoides. Further support for a canine model of cutaneous T-cell lymphoma. J. Invest. Dermatol. 102:227–230, 1994. Gilchrest, B. A. Methoxsalen photochemotherapy for mycosis fungoides. Cancer Treat. Rep. 63:663–667, 1979. Goldberg, D. J., R. S. Schinella, and P. Kechijian. Hypopigmented mycosis fungoides. Speculation about the mechanism of hypopigmentation. Am. J. Dermatopathol. 8:326–330, 1986. Handfield-Jones, S. E., N. P. Smith, and S. M. Breathnach. Hypopigmented mycosis fungoides. Clin. Exp. Dermatol. 17:374–375, 1992. Heald, P. W., and R. L. Edelson. Lymphomas, pseudolymphomas, and related conditions. In: Dermatology in General Medicine, 4th ed., T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M. Freedberg, and K. F. Austen (eds). New York: McGraw-Hill, Inc., 1993, pp. 1285– 1307. Holloway, K. B., F. P. Flowers, and F. A. Ramos-Caro. Therapeutic alternatives in cutaneous T-cell lymphoma. J. Am. Acad. Dermatol. 27:367–378, 1992. Hoppe, R. T., R. S. Cox, Z. Fuks, N. M. Price, M. A. Bagshaw, and E. M. Farber. Electron beam therapy for mycosis fungoides: The Stanford University experience. Cancer Treat. Rep. 63:691–700, 1969. Hsu, W. T., M. B. Toporcer, G. R. Kantor, E. C. Vonderheid, and M. E. Kadin. Cutaneous T-cell lymphoma with porokeratosislike lesions. J. Am. Acad. Dermatol. 27(2 Pt 2):327–330, 1992. Kung, P. C., C. L. Berger, G. Goldstein, P. LoGerfo, and R. L. Edelson. Cutaneous T-cell lymphoma: Characterization by monoclonal antibody. Blood 57:261–266, 1981. Lambroza, E., S. R. Cohen, R. Phelps, M. Lebwohl, I. M. Braverman, and D. DiConstanzo. Hypopigmented variant of mycosis fungoides: Demography, histopathology, and treatment of seven cases. J. Am. Acad. Dermatol. 32:987–993, 1995. Lancer, H. A., B. R. Bronstein, H. Nakagawa, A. K. Bhan, and M. C. Mihm. Follicular mucinosis. J. Am. Acad. Dermatol. 10:760–768, 1984. Lindae, M. L., E. A. Abel, R. T. Hoppe, and G. S. Wood. Poikilodermatous mycosis fungoides and atrophic large plaque parapsoriasis exhibit similar abnormalities of T-cell antigen expression. Arch. Dermatol. 124:366–372, 1988. McDonagh, A. J., D. J. Gawkrodger, A. E. Walker, and P. B. Gray. Reticulate pigmentation in mycosis fungoides. Int. J. Dermatol. 30:658–659, 1991. Misch, K. J., J. A. Maclennan, and R. A. Marsden. Hypopigmented mycosis fungoides. Clin. Exp. Dermatol. 12:53–55, 1987. Neuman, K. Blue polka-dots: An unusual presentation of mycosis fungoides. Cutis 33:373–374, 1984. Nickoloff, B. Light microscopic assessment of 100 patients with

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patch/plaque stage mycosis fungoides. Am. J. Dermatopathol. 10: 469–477, 1988. Peters, M. S., S. N. Thibodeau, J. W. White, and R. A. Winkelmann. Mycosis fungoides in children and adolescents. J. Am. Acad. Dermatol. 22:1011–1018, 1990. Rappaport, H., and L. B. Thomas. Mycosis fungoides. The pathology of extracutaneous involvement. Cancer 34:1198–1229, 1974. Resnick, K. S., and E. C. Vonderheid. Home UV phototherapy of early mycosis fungoides. Long term follow-up observations in 31 patients. J. Am. Acad. Dermatol. 29:73–77, 1993. Rustin, M. H., M. Griffiths, and C. M. Ridley. The immunopathology of hypopigmented mycosis fungoides. Clin. Exp. Dermatol. 11:332–339, 1986. Ryan, E. A., K. V. Sanderson, P. N. Bartak, and P. D. Sammon. Can mycosis fungoides begin in the epidermis? A hypothesis. Br. J. Dermatol. 88:419–429, 1973. Sanchez, J. L., and A. B. Ackerman. The patch stage of mycosis fungoides. Criteria for histologic diagnosis. Dermatol. Clin. 1:5–26, 1979. Sausville, E. A., J. L. Eddy, R. W. Makuch, A. B. Fischmann, G. P. Schechter, M. Matthews, E. Glatstein, D. C. Ihde, F. Kaye, S. R. Veach, R. Phelps, T. O’Conner, J. B. Trepel, J. D. Kotelingam, A. F. Gazzar, J. D. Minna, and P. A. Bunn. Histopathologic staging at initial diagnosis of mycosis fungoides and the Sezary syndrome. Ann. Intern. Med. 109:372–382, 1988. Sézary, A., and Y. Bouvrain. Erythrodermia avec presence de cellules monstreuses uses dans le derme et le sang circulant. Soc. Dermatol. Syphil. 13:254–260, 1938. Sigal, M., M. Grossin, L. LaRoche, F. Basset, G. Aitkin, J. L. Haziza, and S. Belaich. Hypopigmented mycosis fungoides. Clin. Exp. Dermatol. 12:453–454, 1987. Smith, N. P., and P. D. Sammon. Mycosis fungoides presenting with areas of cutaneous hypopigmentation. Clin. Exp. Dermatol. 3:213– 215, 1978. Taswell, H. F., and R. K. Winkelmann. Sezary syndrome — a malignant reticulemic erythroderma. J. Am. Med. Assoc. 177:465–472, 1961. Thomsen, K., and G. L. Wantzin. Lymphomatoid papulosis. A followup study of 30 patients. J. Am. Acad. Dermatol. 17:632–636, 1987. Tuyp, E., A. Borgoyne, T. Aitchison, and R. MacKie. A case-control study of possible causative factors in mycosis fungoides. Arch. Dermatol. 123:196–200, 1987. Volkenandt, M., H. P. Soyer, L. Cerroni, O. M. Koch, J. Atzpodien, and H. Kerl. Molecular detection of clone-specific DNA in hypopigmented lesions of a patient with early evolving mycosis fungoides. Br. J. Dermatol. 28:423–428, 1993. Vonderheid, E. C., E. T. Tan, A. F. Kantor, L. Shrager, B. Micaily, and E. J. Van Scott. Long term efficacy, curative potential, and carcinogenicity of topical mechlorethamine chemotherapy in cutaneous T-cell lymphoma. J. Am. Acad. Dermatol. 20:416–428, 1989. Waisman, M. Lichen aureus. Arch. Dermatol. 112:696–697, 1976. Wantzin, G. L., K. Thomsen, F. Brandrup, and J. K. Larsen. Lymphomatoid papulosis. Development into cutaneous T-cell lymphoma. Arch. Dermatol. 121:792–794, 1985. Whitmore, S. E., E. Simmons-O’Brien, and F. S. Rotter. Hypopigmented mycosis fungoides. Arch. Dermatol. 130:476–480, 1994. Zackheim, H. S., E. H. Epstein, D. A. Grekin, and N. S. McNutt. Mycosis fungoides presenting as areas of hypopigmentation. J. Am. Acad. Dermatol. 6:340–345, 1982. Zackheim, H. S., E. H. Epstein, and W. R. Crain. Topical carmustine (BCNU) for cutaneous T-cell lymphoma: A 15 year experience in 143 patients. J. Am. Acad. Dermatol. 22:802–810, 1992.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Hypermelanosis Associated with Gastrointestinal Disorders Sections Porphyria Cutanea Tarda Eun Ji Kwon and Victoria P. Werth Cronkhite–Canada Syndrome Eun Ji Kwon, James J. Nordlund, and Victoria P. Werth Hemochromatosis and Hemosiderosis Joerg Albrecht and Victoria P. Werth Primary Biliary Cirrhosis Joerg Albrecht and Victoria P. Werth Inflammatory Bowel Disease and Pigmentation Joerg Albrecht and Victoria P. Werth Pellagra Eun Ji Kwon and Victoria P. Werth Peutz–Jeghers Syndrome Nancy Burton Esterly, Eulalia Baselga, and Beth A. Drolet

Porphyria Cutanea Tarda

“hereditary porphyria” (Gawkrodger, 1994; Harber and Held, 1993).

Eun Ji Kwon and Victoria P. Werth

Epidemiology Introduction The porphyrias are a group of disorders with a defect in biosynthesis of heme, the oxygen carrier in hemoglobin. While numerous types of porphyria exist, porphyria cutanea tarda (PCT) is the most common and characterized by hyperpigmentation of the integument. The term PCT includes several related disorders, all of which are characterized by uroporphyrinogen decarboxylase (URO-D) deficiency and subsequent systemic accumulation of porphyrin by-products of the heme biosynthesis pathway.

Historical Background In a 1911 publication, Günther originally classified PCT as hematoporphyria chronica. Waldenstrom, in 1937, first used the term porphyria cutanea tarda to describe the findings (Bickers and Frank, 2003). Brunsting (1951) further described the condition in a report out of the Mayo Clinic. The enzyme responsible for all types of PCT is URO-D, which was identified in the 1970s. More recently it has been shown that more than one type of PCT exists (Harber and Held, 1993). The association between porphyrins and photosensitivity has been recognized since 1912, when the German physician Meyer-Betz developed severe phototoxic reaction after self-injection with hematoporphyrin (Sarkany, 2001).

Synonyms Porphyria Some of “acquired phyria,” “acquired

cutanea tarda has been given a number of names. these are “symptomatic cutaneous porphyria,” hepatic porphyria,” “cutaneous hepatic por“hepatic porphyria,” “sporadic porphyria,” porphyria,” “symptomatic porphyria,” and

PCT is the most common of the porphyrias. Its exact prevalence is unknown but estimates of 1 in 25 000 in North America to 1 in 5000 in Czechoslovakia have been proposed (Elder, 1990). There is no racial predilection (Gawkrodger, 1994) and the male to female incidence is about equal (Grossman et al., 1979). PCT has been most often seen in males who consumed heavy amounts of alcohol prior to the use of estrogencontaining birth control pills (de Salamanca et al., 1982). Type I, or sporadic PCT, accounts for about 80% whereas type II, or familial PCT, is responsible for the remaining 20% of patients with this disorder (Gawkrodger, 1994). A third type of PCT has been described in four families in Spain (Gawkrodger, 1994). Type II PCT, (familial), presents at any age, including childhood, whereas the more common type I PCT (sporadic) most often begins in middle age (Gawkrodger, 1994).

Clinical Findings PCT is a photosensitivity disorder in which the most characteristic findings are noted on the exposed skin. Most often a subtle increased skin fragility is noted especially on the dorsal aspects of the hands, although the feet can also be involved (Harber and Held, 1993; Bickers and Frank, 2003). Vesicles and bullae develop on areas of sun exposure and repeated trauma. They frequently rupture and become infected (Figs 51.1 and 51.2). Healing is slow, with scarring, thickening of the skin, and milia formation (Hurley, 1992). Mottled areas of reddish to purple discoloration may remain (Harber and Held, 1993). Pigmentary changes are characteristic clinical signs of PCT (see Fig. 51.3). More frequently in females the face may have 979

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Fig. 51.1. Hyperpigmentation and scars on the dorsum of the hand.

Fig. 51.3. Hyperpigmentation and hypertrichosis of the face (see also Plate 51.1, pp. 494–495).

sun-exposed surfaces. These lesions present as yellow to white, waxy, indurated plaques that clinically and histologically resemble morphea or scleroderma (Bickers and Frank, 1993). Calcium may be deposited within these plaques (Harber and Held, 1993).

Associated Disorders

Fig. 51.2. Blister and hyperpigmentation.

a melasmalike periocular mottled dyspigmentation (Harber and Held, 1993). Individuals with PCT usually develop a photodistributed and sometimes widespread brown hyperpigmentation (Harber and Held, 1993). The “tan” may persist into the winter months, and this hyperpigmentation may indicate a flare in disease activity (Held et al., 1989). Rarely the hyperpigmentation may involve only the lower extremities (Held et al., 1989). Other cutaneous findings associated with PCT include hypertrichosis, which is most apparent along the temples and cheeks (Fig. 51.3) (Bickers and Frank, 2003). The pathogenesis of the hypertrichosis is unknown and androgen levels are reported to be normal (Bickers and Frank, 2003). Some patients present with a reddish-purple heliotrope-like coloration of the central face, which may be most prominent periorbitally. Scarring alopecia and onycholysis are other cutaneous findings (Grossman and Poh-Fitzpatrick, 1986). Fluorescence of the teeth and erythrocytes may be noted in some patients (Lim and Poh-Fitzpatrick, 1984). Sclerodermoid plaques have been noted in patients with PCT and they can involve both sun-exposed and non– 980

Iron overload with elevated plasma iron levels can occur in a third to half of individuals with PCT. Increased amounts of iron identified by staining is present in Kupffer cells and hepatocytes (Grossman et al., 1979). Ferrokinetic studies in these individuals have been shown to be normal (Bickers and Frank, 2003). There are numerous hypotheses that suggest a role for iron in the pathogenesis of PCT. However, the mechanism for the development of porphyria and the role of iron remain unclear (Bickers and Frank, 2003). Recent evidence demonstrates an association between PCT and mutations in the hereditary hemochromatosis gene HFE (hemochromatosis, ferrum/iron). Prevalence of the hemochromatosis C282Y mutation in the HFE gene has been found to be increased among British patients with sporadic PCT and North American sporadic and familial PCT patients (Bulaj et al., 2000; Roberts et al., 1997). PCT patients with homozygous genotype for the C282Y mutation have considerably higher iron overload than those heterozygous for the mutation or without the mutation (Bulaj et al., 2000). Homozygosity for the C282Y HFE mutation is a significant susceptibility factor in sporadic and familial PCT, whereas heterozygosity has less effect on the development of PCT (Brady et al., 2000; Bulaj et al., 2000). Increased prevalence of a second milder mutation of the HFE gene, H63D, was found among southern European PCT patients (Sampietro et al., 1998). The role of this mutation in PCT pathogenesis is uncertain. Association between PCT and hepatitis C infection has been made in recent years. Approximately 50% of PCT patients worldwide are infected with HCV (Gisbert et al., 2003), but there is considerable geographical variation in this association. The exact role of HCV infection in PCT is unclear.

HYPERMELANOSIS ASSOCIATED WITH GASTROINTESTINAL DISORDERS

With genotyping at the HFE locus now available through diagnostic laboratories, screening PCT patients for HFE mutations and hepatitis C infection may be useful for early treatment of these PCT-associated conditions. Avoidance of alcohol and estrogen should be encouraged (Bonkovsky et al., 1998; Bulaj et al., 2000; Mendez et al., 1998). Abnormal glucose tolerance tests have been reported in 25–50% of patients with PCT, a third of whom require treatment (Bickers and Frank, 2003; Grossman et al., 1979). Association between HIV and PCT has been described (Cohen et al., 1990; Drobacheff et al., 1998; Gafa et al., 1992; Wissel et al., 1987). PCT has also been associated with benign and malignant hepatic tumors (Gawkrodger, 1994; Grossman and Poh-Fitzpatrick, 1986). Associations with subacute cutaneous and systemic lupus erythematosus, hemodialysis, and Klinefelter syndrome have also been described (Callen and Ross, 1981; Epstein et al., 1973; Kelly, 1992).

Histology Routine staining of a PCT blister generally reveals a noninflammatory subepidermal separation in which the dermal papillae often extend irregularly from the blister floor into the blister cavity. This is termed festooning but is not specific for PCT. Superficial perivascular homogeneous, pale, eosinophilic deposits are noted. PAS staining is recommended because it may reveal thickening of the basement membrane zone around capillaries. In areas of sclerosis the collagen bundles are thickened (Lever and Schaumburg-Lever, 1990). Histology of the hyperpigmented skin has been studied carefully. Hyperpigmented skin tends to have increased epidermal melanin deposits, but no specific or diagnostic changes for PCT. None of the skin biopsies showed fluorescence due to porphyrin or significant hemosiderosis (Tsega et al., 1980). Pigmentary incontinence is not a feature of PCT. Some electron micrograph studies found a tendency for dispersion of single, larger melanosomes. This pattern is similar to black or tanned skin. This is unusual for light-colored skin, which has usually smaller melanosomes dispersed in groups in keratinocytes (Konrad and Wolff, 1973) (see Chapters 27 and 28 on normal and abnormal color of skin). However, other studies have not supported this finding (Held et al., 1989). Direct immunofluorescence often reveals positive staining of IgG and complement in the blood vessel wall and the basement membrane zone. This is thought to result from trapping of this material rather than by immunological mechanisms (Lever and Schaumburg-Lever, 1990).

Laboratory Findings Elevated iron stores and abnormal glucose tolerance test are frequently observed in PCT patients. Occasionally erythrocytosis and cryoglobulinemia are also noted (Bickers and Frank, 2003). Wood’s lamp examination of urine may reveal a characteristic pinkish-red fluorescence due to excretion of increased levels of uroporphyrin (Bickers and Frank, 2003). The porphyrin pattern in PCT has three main features: (1) increased urinary excretion of 8, 7, 6, 5, and 4-carboxy por-

phyrins; (2) a distinctive pattern of excretion of isomer series I and III; and (3) increased fecal excretion of isocoproporphyrin (Harber and Held, 1993). The ratio of uroporphyrin to coproporphyrin in urine is usually greater than 3 to 1 (Bickers and Frank, 2003).

Criteria for Diagnosis PCT diagnosis is suspected due to the presence of the abovementioned typical clinical signs and symptoms. Wood’s light examination of the urine supports these findings. The definitive diagnosis of PCT is confirmed, however, by examination of the porphyrin excretion profile (Harber and Held, 1993).

Differential Diagnosis The differential diagnosis of PCT includes variegate porphyria, pseudoporphyria, hepatoerythropoietic porphyria, hereditary coproporphyria, porphyrin secreting hepatic tumors, and epidermolysis bullosa acquisita. Other conditions which can be confused with PCT are hydroa aestivale, hydroa vacciniforme, scleroderma, morphea, neurotic excoriations, polymorphous light eruption, pemphigus vulgaris, and bullous pemphigoid (Bickers and Frank, 2003; Harber and Held, 1993). Some medications cause fragility of sun-exposed skin. Rarely widespread hyperpigmentation may resemble Addison disease (see Chapter 50) or hemochromatosis (Grossman and Poh-Fitzpatrick, 1986).

Pathogenesis The enzyme responsible for all types of PCT is URO-D. In type I (sporadic) PCT, extrahepatic URO-D levels are normal, but hepatic levels are decreased (Bickers and Frank, 2003). There is no family history of the disorder. Type I PCT is frequently precipitated by environmental factors such as alcohol, estrogens, iron, and polyhalogenated cyclic hydrocarbons (Gawkrodger, 1994). Although alcohol and estrogens appear to trigger PCT in 80% of the cases with type I disorder, the exact mechanism by which these agents cause PCT is not known (Elder, 1990). Acute and chronic alcohol ingestion has been shown to decrease activity of URO-D and ethanol stimulates hepatic aminolevulinic acid (ALA) synthase in PCT patients (Bickers and Frank, 2003; McColl et al., 1981). Also, chronic alcoholism results in increased iron absorption, possibly associated with inherited HFE mutations mentioned above (Bickers and Frank, 2003). Ferrous ions have been shown in vitro to inhibit URO-D activity (Kushner et al., 1972, 1975). It has been suggested that estrogens and chlorinated hydrocarbons (e.g., hexachlorobenzene), acting in a manner similar to that of ferrous ions, inhibit URO-D activity (Bickers and Frank, 2003; Grossman and Poh-Fitzpatrick, 1986). In Type II (familial) PCT, the mode of transmission is autosomal dominant. However, penetrance of type II PCT is low (approximately 20%), and most individuals with inherited defects in URO-D do not develop PCT (Bickers and Frank, 2003; Gawkrodger, 1994). In contrast to type I PCT, there is a reduction in both hepatic and extrahepatic URO-D activity 981

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in type II PCT (Gawkrodger, 1994). Like sporadic PCT, decreased URO-D activity is confined to the liver in type III PCT. However, type III PCT is characterized by positive family history (Held et al., 1989). In PCT uroporphyrins and 7-carboxyl porphyrins have been shown to accumulate in the skin (Bickers and Frank, 2003; Grossman and Poh-Fitzpatrick, 1986), but porphyrin may also be synthesized in the skin (Bickers et al., 1977). These cutaneous porphyrins absorb light energy maximally in the Soret band (400–410 nm), which penetrates the epidermis. The absorbed energy is transferred to surrounding molecules and results in hydrogen peroxide or lipid peroxide (Grossman and Poh-Fitzpatrick, 1986). The peroxides damage cellular membranes and disrupt their integrity. This leads to the release of secondary mediators. Repeated insults eventually lead to the chronic skin changes. However, the pathophysiology of the hypertrichosis, sclerodermalike changes, skin fragility, and pigmentation abnormalities are not well understood. Some data have suggested that uroporphyrin I, a porphyrin upregulated in PCT via downstream URO-D deficiency, may contribute to sclerodermalike skin changes because it stimulates collagen synthesis in human fibroblasts (Bickers and Frank, 2003; Grossman and Poh-Fitzpatrick, 1986; Held et al., 1989).

the depletion of excess iron stores often seen in PCT. Depletion of iron may produce improvement in the clinical signs and symptoms by decreasing ALA synthase activity and porphyrin synthesis, reducing inhibition of URO-D activity, lowering iron-overload induced hepatic lipid peroxidation, and minimizing the oxidation of the uroporphyrinogen substrate to nonmetabolizable porphyrins (Bickers and Frank, 2003). In patients where phlebotomy is contraindicated, chloroquine can be used. Chloroquine appears to form a watersoluble complex with porphyrin, thus allowing for excretion (Grossman and Poh-Fitzpatrick, 1986). A recent study indicates PCT patients homozygous for the C282Y HFE mutation respond poorly to chloroquine treatment, while C282Y heterozygosity and compound heterozygosity (C282Y/H63D) do not lessen therapeutic response to chloroquine (Stolzel et al., 2003). Other treatments which have been used include plasmapheresis, cholestyramine, iron chelators, and activated charcoal (Grossman and Poh-Fitzpatrick, 1986; Grossman et al., 1979; Miyauchi et al., 1983). Coexisting HCV and/or HIV infection should be managed appropriately. Treatment with highly active antiretroviral therapy (HAART) alone caused resolution of PCT lesions in a case report of a PCT patient with AIDS and HCV infection (Rich et al., 1999).

Animal Models A zebrafish model with homozygous mutation in the URO-D gene shows a photosensitive porphyria syndrome which can be reversed by transient and germline expression of the wildtype URO-D allele (Wang et al., 1998). Type I PCT has been induced in iron-loaded mice exposed to hexachlorobenzene (Smith and Francis, 1983). Using homologous recombination, Phillips et al. (2001) produced mice with disruption of one allele of the URO-D gene. Increased hepatic porphyrins and hepatic URO-D activity reduction was seen upon treating the URO-D+/- mice with iron-dextran and d-ALA. By breeding HFE-/- mice with URO-D+/- mice, the same authors generated mice heterozygous URO-D and homozygous null HFE genotype. These animals showed PCT-like phenotype without ALA supplementation and dramatically decreased URO-D activity, suggesting HFE-mutation-induced iron overload alone can reduce URO-D activity in URO-D heterozygous mice (Phillips et al., 2001).

Treatment Identification and avoidance of precipitating factors such as alcohol, estrogen, and iron, along with sun avoidance and use of sunscreens, are general measures which should be included in the treatment of PCT (Gawkrodger, 1994). Removal of environmental precipitants alone can result in a slow improvement of the clinical features (Grossman and Poh-Fitzpatrick, 1986). However, most PCT patients receive repeated phlebotomy and oral antimalarials to treat the disease (Bickers and Frank, 2003). Phlebotomy is a widely used and safe procedure for these patients. The efficacy of phlebotomy has been attributed to 982

Prognosis The prognosis of patients with PCT is dependent on treatment and any underlying associated condition (hepatic cancer, HIV, HCV, diabetes, etc.). The pigment disorder slowly clears with treatment with marked improvement after 18–24 months (Grossman et al., 1979).

References Bickers, D. R., and J. Frank. The porphyrias. In: Fitzpatrick’s Dermatology in General Medicine, 6th ed., I. M. Freedberg, A. Z. Eisen, K. Wolff, K. F. Austen, L. A. Goldsmith, and S. Katz (eds). New York: McGraw Hill, Inc., 2003, pp. 1435–1466. Bickers, D. R., L. Keogh, A. B. Rifkind, L. C. Harber, and A. Kappas. Studies in porphyria. VI. Biosynthesis of porphyrins in mammalian skin and in the skin of porphyric patients. J. Invest. Dermatol. 68:5–9, 1977. Bonkovsky, H. L., M. Poh-Fitzpatrick, N. Pimstone, J. Obando, A. Di Bisceglie, C. Tattrie, K. Tortorelli, P. LeClair, M. G. Mercurio, and R. W. Lambrecht. Porphyria cutanea tarda, hepatitis C, and HFE gene mutations in North America. Hepatology 27:1661–1669, 1998. Brady, J. J., H. A. Jackson, A. G. Roberts, R. R. Morgan, S. D. Whatley, G. L. Rowlands, C. Darby, E. Shudell, R. Watson, J. Paiker, M. W. Worwood, and G. H. Elder. Co-inheritance of mutations in the uroporphyrinogen decarboxylase and hemochromatosis genes accelerates the onset of porphyria cutanea tarda. J. Invest. Dermatol. 115:868–874, 2000. Brunsting, L. A., H. L. Mason, and R. A. Aldrich. Adult form of chronic porphyria with cutaneous manifestations. J. Am. Med. Assoc. 146:1207–1212, 1951. Bulaj, Z. J., J. D. Phillips, R. S. Ajioka, M. R. Franklin, L. M. Griffen, D. J. Guinee, C. Q. Edwards, and J. P. Kushner. Hemochromatosis genes and other factors contributing to the pathogenesis of porphyria cutanea tarda. Blood 95:1565–1571, 2000.

HYPERMELANOSIS ASSOCIATED WITH GASTROINTESTINAL DISORDERS Callen, J. P., and L. Ross. Subacute cutaneous lupus erythematosus and porphyria cutanea tarda. J. Am. Acad. Dermatol. 5:269–273, 1981. Cohen, P. R., S. M. Suarez, and V. A. DeLeo. Porphyria cutanea tarda in human immunodeficiency virus-infected patients. J. Am. Med. Assoc. 264:1315–1316, 1990. de Salamanca, R. E., D. Mingo, S. Chinarro, J. J. Munoz, and J. Perpina. Porphyrin-excretion in female estrogen-induced porphyria cutanea tarda. Arch. Dermatol. Res. 274:179–184, 1982. Drobacheff, C., C. Derancourt, H. Van Landuyt, D. Devred, B. de Wazieres, B. Cribier, D. Rey, J. M. Lang, C. Grosieux, B. Kalis, and R. Laurent. Porphyria cutanea tarda associated with human immunodeficiency virus infection. Eur. J. Dermatol. 8:492–496, 1998. Elder, G. H. Porphyria cutanea tarda: A multifactorial disease. In: Recent Advances in Dermatology, vol. 8, R. H. Champion, and R. J. Pye (eds). Edinburgh: Churchill Livingstone, 1990, pp. 55–69. Epstein, J. H., D. L. Tuffanelli, and W. L. Epstein. Cutaneous changes in the porphyrias. Arch. Dermatol. 107:689–698, 1973. Gafa S., A. Zannini, and C. Gabrielli. Porphyria cutanea tarda and HIV infection: effect of zidovudine treatment on a patient. Infection 20:373–374, 1992. Gawkrodger, D. J. The porphyrias. In: Textbook of Dermatology, 5th ed., A. Rook, D. S. Wilkinson, and F. J. G. Ebling (eds). Cambridge: Blackwell Scientific Publications, Inc., 1994, pp. 2304–2308. Gisbert, J. P., L. Garcia-Buey, J. M. Pajares, and R. Moreno-Otero. Prevalence of hepatitis C virus infection in porphyria cutanea tarda: systematic review and meta-analysis. Hepatology 39:620–627, 2003. Grossman, M. E., and M. B. Poh-Fitzpatrick. Porphyria cutanea tarda. Dermatol. Clin. 4:297–309, 1986. Grossman, M. E., D. R. Bickers, M. B. Poh-Fitzpatrick, V. A. Deleo, and B. C. Harber. Porphyria cutanea tarda. Am. J. Med. 67:277–286, 1979. Harber, L. C., and J. L. Held. Porphyria cutanea tarda. In: Clinical Dermatology, 20th ed., D. J. Demis (ed.). Philadelphia: JB Lippincott, 1993, pp. 1–13. Held, J. L., D. N. Silvers, and M. E. Grossman. Hyperpigmentation of the lower extremities associated with porphyria cutanea tarda. Arch. Dermatol. 125:297–298, 1989. Hurley, H. J. The porphyrias. In: Dermatology, 3rd ed., S. L. Moschella, and H. J. Hurley (eds). Philadelphia: WB Saunders, 1992, pp. 1667–1681. Kelly, W. N. Textbook of Internal Medicine, 2nd ed. Philadelphia: JB Lippincott, 1992. Konrad, K., and K. Wolff. Hyperpigmentation, melanosome size, and distribution patterns of melanosomes. Arch. Dermatol. 107:853– 860, 1973. Kushner, J. P., G. R. Lee, and S. Nacht. The role of iron in the pathogenesis of porphyria cutanea tarda. J. Clin. Invest. 51:3044–3051, 1972. Kushner, J. P., D. P. Steinmuller, and G. R. Lee. The role of iron in the pathogenesis of porphyria cutanea tarda. J. Clin. Invest. 56:661– 667, 1975. Lever, W. F., and G. Schaumburg-Lever. Histopathology of the Skin, 7th ed. Philadelphia: JB Lippincott, 1990. Lim, H. W., and M. B. Poh-Fitzpatrick. Hepatoerythropoietic porphyria: A variant of childhood-onset porphyria cutanea tarda. J. Am. Acad. Dermatol. 11:1103–1111, 1984. McColl, K. E., M. R. Moore, G. G. Thompson, and A. Goldberg. Abnormal haem biosynthesis in chronic alcoholics. Eur. J. Clin. Invest. 11:461–468, 1981. Mendez, M., L. Sorkin, M. V. Rossetti, K. H. Astrin, A. M. del C. Batlle, V. E. Parera, G. Aizencang, and R. J. Desnick. Familial porphyria cutanea tarda: characterization of seven novel uroporphyrinogen decarboxylase mutations and frequency of common hemochromatosis alleles. Am. J. Hum. Genet. 63:1363– 1375, 1998.

Miyauchi, S., S. Shiraishi, and Y. Miki. Small volume plasmapheresis in the management of porphyria cutanea tarda. Arch. Dermatol. 119:752–755, 1983. Phillips, J. D., L. K. Jackson, M. Bunting, M. R. Franklin, K. R. Thomas, J. E. Levy, N. C. Andrews, and J. P. Kushner. A mouse model of familial porphyria cutanea tarda. Proc. Natl. Acad. Sci. U. S. A. 98:259–264, 2001. Rich, J. D., E. Mylonakis, R. Nossa, and R. M. Chapnick. Highly active antiretroviral therapy leading to resolution of porphyria cutanea tarda in a patient with AIDS and hepatitis C. Dig. Dis. Sci. 44:1034–1037, 1999. Roberts, A. G., S. D. Whatley, R. R. Morgan, M. Worwood, and G. H. Elder. Increased frequency of the haemochromatosis Cys282Tyr mutation in sporadic porphyria cutanea tarda. Lancet 349:321– 323, 1997. Sampietro, M., A. Piperno, L. Lupica, C. Arosio, A. Vergani, N. Corbetta, I. Malosio, M. Mattioli, A. L. Fracanzani, M. D. Cappellini, G. Fiorelli, and S. Fargion. High prevalence of the His63Asp HFE mutation in Italian patients with porphyria cutanea tarda. Hepatology 27:181–184, 1998. Sarkany, R. P. E. The management of porphyria cutanea tarda. Clin. Exp. Dermatol. 26:225–232, 2001. Smith, A. G., and J. E. Francis. Synergism of iron and hexachlorobenzene inhibits hepatic uroporphyrinogen decarboxylase in inbred mice. Biochem. J. 214:909–913, 1983. Stolzel, U., E. Kostler, D. Schuppan, M. Richter, U. Wollina, M. O. Doss, C. Wittekind, and A. Tannapfel. Hemochromatosis (HFE) gene mutations and response to chloroquine in porphyria cutanea tarda. Arch. Dermatol. 139:309–313, 2003. Tsega, E., B. Damtew, J. W. Landells, A. Besrat, and E. Seyoum. Hyperpigmentation of the face and hands without blisters: porphyria cutanea tarda. Br. J. Dermatol. 103:187–190, 1980. Wang, H., Q. Long, S. D. Marty, S. Sassa, and S. Lin. A zebrafish model for hepatoerythropoietic porphyria. Nat. Genet. 20:239–243, 1998. Wissel, P. S., P. Sordillo, K. E. Anderson, S. Sassa, R. L. Savillo, and A. Kappas. Porphyria cutanea tarda associated with the acquired immune deficiency syndrome. Am. J. Hematol. 25:107–113, 1987.

Cronkhite–Canada Syndrome Eun Ji Kwon, James J. Nordlund, and Victoria P. Werth

Historical Background In 1955 Cronkhite and Canada presented two women with debilitating diarrhea, generalized hair loss, hyperpigmentation of the skin, and onychotrophia. The backs of the hands, fingers, and the body folds were especially pigmented although there was melanin deposition in the oral cavity and much of the integument. The second patient was thought also to have vitiligo. Both patients had polyposis of the intestinal tract (Cronkhite and Canada, 1955).

Synonyms Intestinal polyposis with hyperpigmentation; intestinal polyposis with nail loss

Epidemiology Cronkhite–Canada syndrome is a rare disorder which was first described by Cronkhite and Canada in 1955. As of 2002, only 467 cases have been reported worldwide, with approximately 75% of cases reported from Japan (Goto, 1995; Takakura et al., 2004). The reason for this distribution is not known. The 983

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Fig. 51.4. Total alopecia and diffuse hyperpigmentation. (Courtesy of Dr. J. Bazex.)

Fig. 51.5. Extensive hyperpigmentation of the trunk and hands with nail changes (see also Plate 51.2, pp. 494–495). (Courtesy of Dr. J. Bazex.)

estimated male to female ratio is 3:2, with a mean age at onset of 59 years (Daniel et al., 1982). Most cases are sporadic and there seems to be no familial or genetic basis for this syndrome.

1987; Allbritton et al., 1998; Bianchi et al., 1984; Bruce et al., 1999; Casalnuovo et al., 1991; Cronkhite and Canada, 1955; Goto, 1994, 1995; Herzberg and Kaplan, 1990; Lin et al., 1987; Pal et al., 1990). The cause of this pigmentation is not known. There is an increased amount of melanin in the epidermis (Herzberg and Kaplan, 1990; Ortonne et al., 1985), although dermal melanosis has also been described (Ortonne et al., 1985). In one case, the pigmentation began on the pelvic girdle but rapidly spread to other parts of the skin, but in other patients patchy vitiligo has also been reported (Cronkhite and Canada, 1955). Dyspigmentation may persist or resolve after therapy or spontaneously (Daniel et al., 1982).

Clinical Features The main feature is the development of polyps that form throughout the intestinal tract and affecting the mucosa of the stomach, the small and large bowel (Shibuya, 1972). Most of the original cases were adults, although children with intestinal polyposis have been described (Kucukaydin et al., 1992; Scharf et al., 1986). Adults in the ninth decade of life who developed this syndrome have been described (Jones and Paone, 1984). The patient presents with watery diarrhea, protein-losing enteropathy, malnutrition, electrolyte unbalance, hypogeusia, and weight loss. The diarrhea and malabsorption are caused by the loss of fluid from the polyps (Harned et al., 1995; Jenkins et al., 1985). There are many cutaneous manifestations of this syndrome. Patchy hair loss that progresses to almost complete alopecia is common (Fig. 51.4) (Aanestad et al., 1987; Allbritton et al., 1998; Bianchi et al., 1984; Bruce et al., 1999; Casalnuovo et al., 1991; Cronkhite and Canada, 1955; Goto, 1994, 1995; Ortonne et al., 1985; Takakura et al., 2004). The eyebrows, eye lashes, and other body hair can be lost. The hair loss might be due to anagen effluvium. The fingernails and toenails become thin and brittle and later are shed from the ends of the fingers and toes (Fig. 51.5). Subepidermal blisters, keratoacanthoma, and oral erosions also have been observed. Acquired ichthyosis, purpura and ecchymoses, psoriasiform plaques and pruritus all have been observed in the Cronkhite–Canada syndrome (Bianchi et al., 1984). Hair and nail regrowth may occur after treatment, during remission, or spontaneously despite continued active disease (Daniel et al., 1982). A common feature is diffuse distribution of hyperpigmented light-to-dark brownish macules and patches affecting all parts of the integument (see Figs 51.4 and 51.5) (Aanestad et al., 984

Associated Disorders A number of other disorders have been described in association with this syndrome. They include lupus erythematosus (Kubo et al., 1986), hypothyroidism (Pal et al., 1990), tuberculosis (Lin et al., 1987), cataracts (Hutnik and Nichols, 1998; Simcock et al., 1996), portal thrombosis with high titer of antinuclear antibodies and membranous glomerulonephritis (Takeuchi et al., 2003), and zinc deficiency-related taste disturbances (Yoshida and Tomita, 2002). Although gastric and colorectal cancers have been reported among patients with Canada–Cronkhite syndrome (Egawa et al., 2000; Goto and Shimokawa, 1994; Katayama et al., 1985; Malhotra and Sheffield, 1988; Murai et al., 1993; Nagata et al., 2003; Nakatsubo et al., 1997; Rappaport et al., 1986; Satoh et al., 2000; Takeuchi et al., 2003; Watanabe et al., 1999; Yamaguchi et al., 2001), whether Canada–Cronkhite polyps carry malignant potential remains controversial.

Histology The epidermis of the pigmented skin is usually slightly thickened with compact hyperkeratosis and has increased amounts of melanin with normal or slightly increased numbers of melanocytes (Herzberg and Kaplan, 1990; Ortonne et al., 1985). By electron microscopy the melanocytes appear to be activated and have increased melanogenic activity. The

HYPERMELANOSIS ASSOCIATED WITH GASTROINTESTINAL DISORDERS

epidermis may also be atrophic, with only a few hair follicles (Kindblom et al., 1977; Jarnum and Jensen, 1966) and hair shaft miniaturization, as well as follicular dilatation and extensive glycosaminoglycan deposits in the reticular dermis (Allbritton et al., 1998). In the dermis melanophages and some perivascular mononuclear cell infiltrate can be found.

et al., 1993; Scharf et al., 1986, Takakura et al., 2004), but to date the treatment is still empirical.

Prognosis

Radiological or endoscopic examination of the intestinal tract including the stomach, small and large intestines, and the rectum will demonstrate the presence of diffuse polyposis (Harned et al., 1995; Rossi et al., 1981). These changes may cause abnormal gastrointestinal absorption. Patients with serious disease may demonstrate mild to moderate anemia, electrolyte disturbances, and a decrease in total serum protein (Daniel et al., 1982).

Canada–Cronkhite syndrome has traditionally carried a poor prognosis (Canada and Cronkhite, 1955). However, nutritional therapy has been reported to achieve remission for more than 5 years (Daniel et al., 1982), but complete remission with medical treatment is rare (Ward et al., 2002). Surgical treatment is also not universally successful and the longest reported survival after surgical treatment is 15–17.5 years (Daniel et al., 1982). The major causes of death include gastrointestinal bleeding, intussusception, gastrointestinal prolapse, thromboembolic episodes, malnutrition, and massive fluid, protein, and electrolyte losses (Daniel et al., 1982). In some patients the disease is associated with neoplasms in the intestinal tract (Murai et al., 1993) or other organs.

Criteria for Diagnosis

References

Laboratory Findings

The association of diffuse polyposis, protein-losing enteropathy, loss of nails and/or hair with or without hyperpigmentation establishes the diagnosis.

Differential Diagnosis The differential diagnosis includes all syndromes characterized by intestinal polyps (Finan and Ray, 1989; Harned et al., 1995). These include the Peutz–Jeghers syndrome (see later), Gardner syndrome, Bannayan–Riley–Ruvalcaba syndrome, and the Ruvalcaba–Myhre–Smith syndrome.

Pathophysiology The etiology of the disease is unclear. So far no consistent inheritance pattern has been observed. In Japan mental stress and physical fatigue have been identified as important risk factors (Goto, 1995). Reports of patients with antinuclear antibodies (Murata et al., 2000; Takeuchi et al., 2003) and positive response to corticosteroids in many cases suggest an underlying immunological abnormality in the pathogenesis of Canada–Cronkhite syndrome (Canada and Cronkhite, 1955; Daniel et al., 1982). In their first description Cronkhite and Canada attributed the cutaneous changes to severe malnutrition secondary to diarrhea and malabsorption. However, cutaneous changes may precede the gastrointestinal manifestations and may develop independently of gastrointestinal disease activity (Daniel et al., 1982).

Treatment Currently there are no specific therapies for the hyperpigmentation. The bowel disease has been treated surgically. A multitude of medical treatments have been used in Cronkhite–Canada syndrome, including corticosteroids, anabolic steroids, antibiotics, and H1- and H2-receptor blockers, antiplasmin and mesalazine as well as nutritional supplementation (Allbritton et al., 1998; Casalnuovo et al., 1991; Canada and Cronkhite, 1955; Daniel et al., 1982; Jones and Paone, 1984; Kaneko et al., 1991; Kubo et al., 1986; Murai

Aanestad, O., N. Raknerud, S. T. Aase, and G. Narverud. The Cronkhite-Canada syndrome: Case report. Acta. Chirurg. Scand. 153:143–145, 1987. Allbritton, J., E. Simmons-O’Brien, D. Hutcheons, and S. E. Whitmore. Cronkhite-Canada Syndrome: report of two cases, biopsy findings in the associated alopecia, and a new treatment option. Cutis 61:229–232, 1998. Bianchi, C. A., A. Garcia Garcia, and O. Stringa. [Cutaneous manifestations of the malabsorption syndrome]. Med. Cutanea IberoLat-Am. 12:227–235, 1984. Bruce, A., S. N. Ng, H. C Wolfsen, R. C. Smallfridge, and D. P. Lookingbill. Cutaneous clues to Cronkhite-Canada syndrome: a case report. Arch. Dermatol. 135:212, 1999. Casalnuovo, C., M. Ghigliani, A. Avagnina, B. Elsner, and G. Palau. [Cronkhite-Canada syndrome: Report of a case]. Medicina (B. Aires) 51:155–160, 1991. Cronkhite, L. S., and W. J. Canada. Generalized intestinal polyposis: an unusual syndrome of polyposis, pigmentation, alopecia and onychotrophia. N. Engl. J. Med. 252:1011–1015, 1955. Daniel, E. S., S. L. Ludwig, K. J. Lewin, R. M. Ruprecht, G. M. Rajacich, and A. D. Schwabe. The Cronkhite-Canada Syndrome: an analysis of clinical and pathologic features and therapy in 55 patients. Medicine. 61:293–309, 1982. Egawa, T., T. Kubota, Y. Otani, N. Kurihara, S. Abe, M. Kimata, J. Tokuyama, N. Wada, K. Suganuma, Y. Kuwano, K. Kumai, Y. Sugino, M. Mukai, and M. Kitajima. Surgically treated CronkhiteCanada syndrome associated with gastric cancer. Gastric Cancer 3:156–160, 2000. Finan, M. C., and M. K. Ray. Gastrointestinal polyposis syndromes. Dermatol. Clin. 7:419–434, 1989. Goto, A. [Cronkhite-Canada syndrome]. Nippon Rinsho Suppl 6:23–26, 1994. Goto, A. Cronkhite-Canada syndrome: epidemiological study of 110 cases reported in Japan. Nippon Geka Hokan 64:3–14, 1995. Goto, A., and K. Shimokawa. Cronkhite-Canada syndrome associated with lesions predisposing to development of carcinoma. J. Jpn. Soc. Cancer Ther. 29: 1767–77, 1994. Harned, R. K., J. L. Buck, and L. H. Sobin. The hamartomatous polyposis syndromes: clinical and radiologic features. Am. J. Roentgenol. 164:565–571, 1995. Herzberg, A. J., and D. L. Kaplan. Cronkhite–Canada syndrome: Light and electron microscopy of the cutaneous pigmentary abnormalities. Int. J. Dermatol. 29:121–125, 1990.

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CHAPTER 51 Hutnik, C. M., and B. D. Nichols. Cataracts in systemic diseases and syndromes. Curr. Opin. Ophthalmol. 9:14–19, 1998. Jarnum, S., and H. Jensen. Diffuse gastrointestinal polyposis with ectodermal changes. Gastroenterology 50: 107–118, 1966. Jenkins, D., P. M. Stephenson, and B. B. Scott. The Cronkhite-Canada syndrome: an ultrastructural study of pathogenesis. J. Clin. Pathol. 38:271–276, 1985. Jones, A. F., and D. B. Paone. Canada-Cronkhite syndrome in an 82year-old woman. Am. J. Med. 77:555–557, 1984. Kaneko, Y., H. Kato, Y. Tachimori, H. Watanabe, K. Ushio, H. Yamaguchi, M. Itabashi, and M. Noguchi. Triple carcinomas in Cronkhite-Canada syndrome. Jpn. J. Clin. Oncol. 21:194–202, 1991. Katayama, Y., M. Kimura, and M. Konn. Cronkhite-Canada syndrome associated with a rectal cancer and adenomatous changes in colonic polyps. Am. J. Surg. Pathol. 9:65–71, 1985. Kindblom, L. G., L. Angervall, B. Santesson, and S. Selander. Cronkhite-Canada syndrome. Cancer 39:2651–2657, 1977. Kubo, T., S. Hirose, S. Aoki, T. Kaji, and M. Kitagawa. CanadaCronkhite syndrome associated with systemic lupus erythematosus. Arch. Intern. Med. 146:995–996, 1986. Kucukaydin, M., T. E. Patiroglu, H. Okur, and M. Icer. Infantile Cronkhite-Canada syndrome? Case report. Eur. J. Pediatr. Surg. 2:295–297, 1992. Lin, H. J., Y. T. Tsai, S. D. Lee, K. H. Lai, W. W. Ng, T. N. Tam, H. C. Lin, L. B. Liou, and S. H. Tsay. The Cronkhite-Canada syndrome with focus on immunity and infection: Report of a case. J. Clin. Gastroenterol. 9:568–570, 1987. Malhotra, R., and A. Sheffield. Cronkhite-Canada syndrome associated with colon carcinoma and adenomatous changes in C-C polyps. Am. J. Gastroenterol. 83:772–776, 1988. Murai, N., T. Fukuzaki, T. Nakamura, H. Hayashida, M. Okazaki, K. Fujimoto, and T. Hirai. Cronkhite-Canada syndrome associated with colon cancer: report of a case. Surg. Today 23:825–829, 1993. Murata, I., I. Yoshikawa, M. Endo, M. Tai, C. Toyoda, S. Abe, Y. Hirano, and M. Otsuki. Cronkhite-Canada syndrome: report of two cases. J. Gastroenterol. 35: 706–11, 2000. Nagata, J., H. Kijima, K. Hasumi, T. Suzuki, T. Shirai, and T. Mine. Adenocarcinoma and multiple adenomas of the large intestine, associated with Cronkhite-Canada syndrome. Dig. Liver Dis. 35:434–438, 2003. Nakatsubo, N., R. Wakasa, K. Kiyosaki, K. Matsui, and F. Konishi. Cronkhite-Canada syndrome associated with carcinoma of the sigmoid colon: report of a case. Surg. Today 27:345–348, 1997. Ortonne, J. P., J. Bazex, and P. Berbis. [Cronkhite-Canada disease. Discussion apropos of a case and study of the pigmentation]. Ann. Dermatol. Venereol. 112:951–958, 1985. Pal, A., S. Sen, S. Ghosh, R. Sarkar, and K. N. Jalan. CronkhiteCanada syndrome with hypothyroidism. Indian J. Gastroenterol. 9:229–230, 1990. Rappaport, L. B., H. V. Sperling, and A. Stavrides. Colon cancer in the Cronkhite-Canada syndrome. J. Clin. Gastroenterol. 8:199– 202, 1986. Rossi, A., G. Marconi, U. Camellini, and G. Marchitelli. [Radiological aspect of Peutz-Jeghers syndrome: considerations on a case]. Acta. Biomed. Ateneo. Parmense 52:237–244, 1981. Satoh, H., K. Togashi, F. Konishi, K. Sakuma, and H. Nagai. Diagnosis of neoplastic changes by magnifying colonoscopy in a patient with Cronkhite-Canada syndrome: report of a case. Dig. Endosc. 12:255–258, 2000. Scharf, G. M., J. H. Becker, and N. J. Laage. Juvenile gastrointestinal polyposis or the infantile Cronkhite-Canada syndrome. J. Pediatr. Surg. 21:953–954, 1986. Shibuya, C. An autopsy case of Cronkhite-Canada’s syndrome — generalized gastrointestinal polyposis, pigmentation, alopecia and onychotrophia. Acta. Pathol. Jpn. 22:171–183, 1972.

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Simcock, P. R., H. J. Zambarakji, and B. R. Muller. Cataract formation in the Cronkhite-Canada syndrome. J. Cataract Refract. Surg. 22:1125–1126, 1996. Takakura, M., A. Hitomi, N. Tsuchihashi, E. Miyazaki, Y. Yoshioka, K. Yoshida, F. Oryo, and T. Sawada. A case of Cronkhite-Canada syndrome markedly improved with mesalazine therapy. Dig. Endosc. 16:74–78, 2004. Takeuchi, Y., M. Yoshikawa, N. Tsukamoto, A. Shiroi, Y. Hoshida, Y. Enomoto, T. Kimura, K. Yamamoto, H. Shiiki, E. Kikuchi, and H. Fukui. Cronkhite–Canada syndrome with colon cancer, portal thrombosis, high titer of antinuclear antibodies, and membranous glomerulonephritis. J. Gastroenterol. 38:791–795, 2003. Ward, E., H. C. Wolfsen, and C. Ng. Medical management of Cronkhite-Canada syndrome. South Med. J. 95:272–274, 2002. Watanabe, T., M. Kudo, H. Shirane, H. Kashida, S. Tomita, A. Orino, A. Todo, and T. Chiba. Cronkhite-Canada syndrome associated with triple gastric cancers: a case report. Gastrointest. Endosc. 50:688–691, 1999. Yamaguchi, K., Y. Ogata, Y. Akagi, T. Sasatomi, K. Ozaki, A. Ohkita, H. Ikeda, and K. Shirouzu. Cronkhite-Canada syndrome associated with advanced rectal cancer treated by a subtotal colectomy: report of a case. Surg. Today 31:521–526, 2001. Yoshida, S., and H. Tomita. A case of Cronkhite–Canada syndrome whose major complaint, taste disturbance, was improved by zinc therapy. Acta. Otolaryngol. Suppl. 546:154–158, 2002.

Hemochromatosis and Hemosiderosis Joerg Albrecht and Victoria P. Werth Hemochromatosis is an iron-storage disorder due to an inappropriately high absorption of iron from the duodenum. This leads to iron deposition in various organs with eventual impairment, especially of the liver, pancreas, heart, and pituitary gland. The term hemochromatosis denotes generally genetic hemochromatosis, whereas other diseases associated with iron overload, like thalassemia or sideroblastic anemia, are referred to as secondary iron overload. The term hemosiderosis describes the histologic appearance of increased stainable tissue iron. Hemosiderosis may be associated with secondary iron overload in any tissue, however, the focus of this section will be on cutaneous hemosiderosis.

Hereditary Hemochromatosis Introduction Hemochromatosis is a genetic metabolic disease characterized by an increased iron absorption that causes primary iron overload with organ damage. In the skin hemochromatosis is associated with a bronze or grayish melanocytic dermal and epidermal hyperpigmentation.

Historical Background The disease is also called Troisier–Hanot–Chauffard syndrome after three of its first describers in 1865 (Pietrangelo, 2003). The term hemochromatosis was coined by von Recklinghausen in 1899 to describe a pigmentation which was thought to be derived from the blood.

Epidemiology Hemochromatosis has frequently been described as a disease

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predominantly of the male adult population. However, a recent, large French study has found that the onset of the disease is not later in women, but the authors concede that homozygous hemochromatosis is slightly underexpressed in women (Moirand et al., 1997). The prevalence of the heterozygotic hemochromatosis gene is about 9.2%, and homozygotic carriers make up 0.4% in a northern European population with similar numbers occurring in the white population in North America or Australia. The disease is much rarer in nonwhite patients (Bomford, 2002). Overall only 50% of the homozygous patients show clinical features of hemochromatosis (Olynyk et al., 1999). In spite of this, the number of patients diagnosed has increased significantly over the last decades due to increased awareness and ease of diagnosis (Niederau et al., 1996) and this may influence the epidemiological estimates, which are likely to be corrected upward.

Clinical Description of Disease/Associated Disorders Clinical diagnosis of hemochromatosis is difficult, because the early symptoms are usually nonspecific (Ryan et al., 2002). According to a recent series, around 30% of the diagnosed patients currently present without any clinical symptoms (Adams et al., 1991, 1997; Niederau et al., 1996), but this figure was up to 70% in an American series (Bacon and Sadiq, 1997). Most patients present with nonspecific symptoms like arthralgia, fatigue, abdominal pain, weight loss, or impotence (Adams et al., 1997; Niederau et al., 1996), indicating that gonadal, adrenal or pituitary damage may be early events. The classic clinical tetrad of hemochromatosis (liver disease, diabetes mellitus, cardiac failure, and hyperpigmentation) was only found in 10–20% of patients in large series (Adams et al., 1997). Liver fibrosis rarely develops in patients younger than 40 years. Exceptions are patients who have predisposing cofactors like excessive alcohol consumption (Tavill, 1999). Due to the lack of specific early symptoms hemochromatosis is underdiagnosed and a high incidence of clinical suspicion may be necessary for diagnosis. However, early diagnosis and treatment is vital to improve the prognosis and to avoid liver cirrhosis. Therefore family screening is important to identify family members who may have the disease (Ryan et al., 2002). In spite of the genetic nature of hemochromatosis, the diagnosis is dependent on the phenotype rather than the genotype (Pietrangelo, 2003). Of patients who present with characteristic symptoms, more than 90% have a discoloration of their skin. Because of its insidious onset (Chevrant-Breton et al., 1977) or the mildness of the discoloration (Milder et al., 1980) the skin is usually not the reason medical attention is sought. Patients have a characteristic grayish hue that may seem metallic and has given the disease the name bronze diabetes. The pigmentation is usually diffuse or generalized but may be more pronounced on the face, neck, extensor aspects of the lower forearm, dorsa of the hands, lower legs, genital region, and in scars. Because discolorations may be most prominent in the sun-exposed areas hemochromatosis may be mistaken for a suntan.

More than 95% of symptomatic patients have hepatomegaly at presentation, many of these without laboratory abnormalities. Those patients who develop hepatic cirrhosis have a 30% chance of developing hepatic carcinoma and have a clinical course and clinical symptoms similar to patients with other forms of hepatic cirrhosis. Diabetes mellitus occurs in about 30% of the patients (Niederau et al., 1996), with an increased likelihood in patients who have a family history of diabetes. This suggests that direct damage to the pancreatic islets occurs in combination with a genetic predisposition. Arthropathy has a varying course and age of presentation. Usually it involves the joints of the hand first. The joints to be affected are usually second and third metacarpophalangeal joints, and a progressive polyarthritis involving wrists, hips, ankles, and knees may follow. The arthropathy tends to progress in spite of iron removal or appropriate treatment. Between 5% and 15% of the patients have cardiac involvement (Niederau et al., 1996), which may present with electrocardiographic changes and cardiac arrhythmia. Particularly in young adults cardiac involvement may present as congestive heart failure that quickly leads to death. If other overt manifestations of the underlying disease are absent, the diffuse cardiac enlargement may be misdiagnosed as idiopathic cardiomyopathy. Hypogonadism may be a presenting symptom also. Iron deposits may harm the hypothalamic–pituitary function and lead to reduced production of gonadotropins. Primary testicular dysfunction has been observed as well. The overwhelming number of patients with hereditary hemochromatosis will have the above clinical characteristics, but different forms of hemochromatosis have been described. A number of Italian cases showed a vastly accelerated type of hemochromatosis, called type 2, which primarily affects the heart and the endocrine system, leading to death before the thirtieth birthday (Andrews, 1999; Camaschella et al., 1997). Neonatal hemochromatosis is even more fulminant and leads to neonatal death due to liver failure (Andrews, 1999).

Histology Due to the importance of laboratory test for the diagnosis of hemochromatosis, skin biopsy is now considered only for confirmatory purposes. The site for skin biopsy is of minor relevance since iron deposits can be found in nonpigmented areas as well. It is recommended to avoid biopsy of the legs because venous stasis or prior inflammation may cause ferritin deposits which are independent of hemochromatosis. The most common stain used to detect iron deposits in histologic examinations is Perls Prussian blue, sometimes called Perls acid or ferrocyanide reaction. The pigmentation of hemochromatosis is a combination of hemosiderin and melanin. The epidermis normally is normal in thickness, but atrophy and ichthyosis may be observed. Increased melanin can be found in the basilar and suprabasilar layers. Aside from free hemosiderin around the blood vessels, in the upper dermis, hemosiderin is also found in macrophages and in the basement membrane of eccrine sweat 987

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glands. Rarely hemosiderin is observed in the epidermis or within eccrine secretory cells (Chevrant-Breton et al., 1977).

Laboratory Findings Hemochromatosis is most reliably diagnosed by “transferrin saturation” testing. This test can be done in patients older than 1 year. For randomly drawn specimens a saturation of >50% should raise the suspicion of hemochromatosis and should be repeated, although the cut-off point is disputed and may be as low as 45% (Durupt et al., 2000). Fasting saturation values >62% are highly likely to indicate hemochromatosis and are recommended to verify randomly drawn results. Due to diurnal variations the test should be conducted in the morning, after the patient has fasted overnight and consumed no vitamin or mineral supplements for at least 24 hours. Vitamin C supplements or general consumption is particularly relevant since it increases iron absorption in the intestine (Felitti, 1999). Serum ferritin has a good correlation with iron excess in tissue, but its specificity is low because many factors, like inflammation or cytolysis, can influence the results (Pietrangelo, 2003).

Diagnosis The diagnosis of the disease may be based on the classic tetrad of liver cirrhosis, cardiac failure, diabetes, and hyperpigmentation, but in more and more patients the diagnosis is made prior to the development of characteristic symptoms at a stage when the disease presents with uncharacteristic symptoms like weakness. In most patients the diagnosis is probably never made (Nichols and Bacon, 1989). If hemochromatosis is suspected genetic testing should be performed, but due to the low penetrance of the disease the tests will not confirm the diagnosis in the absence of other findings. Additionally liver biopsy or preferably SQUID (supraconducting quantum interference device) may identify increased iron deposition in the liver.

Differential Diagnosis The nonspecific clinical presentation of the disease may indicate a large number of differential diagnoses. Laboratory results indicating an iron storage disease limit this number significantly. Secondary siderosis can develop after multiple transfusions, e.g., due to congenital anemia, or due to alcohol abuse in patients with porphyria. Some of these patients with secondary siderosis may be heterozygotic C282Y gene carriers and this defect may exacerbate their iron storage disease.

Pathogenesis Iron causes the grayish hue that has been described as bronze, but the characteristic tan color of the patients’ skin is caused by increased melanin (Felitti, 1999). Melanocytes seem to be stimulated by the iron in hemochromatosis, but the exact mechanism of this stimulation is currently unknown. It has

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been postulated that melanocyte stimulation is secondary to tissue injury caused by the iron deposit. Mouse experiments demonstrated increased melanin production when exogenous iron was injected into hairless mice (Tsuji, 1980). Secondary changes of the skin due to hormonal disturbances and liver cirrhosis are frequently observed as well. Hemochromatosis is caused by an increased absorption of iron from the duodenum due to a mutation in the gene controlling the intestine’s mucosal barrier to iron absorption. It can be caused by mutations of multiple genes and at least 5 types of genetic variations have been identified. Type 1 or hereditary hemochromatosis is by far the most common form. It is an autosomal recessive human leukocyte antigen (HLA)linked disorder caused by a mutation of the HFE gene, that leads to a substitution of a tyrosine residue in place of a cysteine at position 282 (C282Y), a mutation that is also common in PCT (see section on PCT earlier in this chapter). More than 80% of the European patients with hemochromatosis are homozygous for this mutation, which is thought to be 2000 years old (Rochette et al., 1999). One other mutation relevant to type 1 hemochromatosis is H63D. This mutation is more widespread outside Europe and seems to be due to a higher proportion of spontaneous mutations and has less penetrance than the C282Y mutation (Bomford, 2002). Type 2 or juvenile hemochromatosis is a rare, autosomal recessive condition caused by an unidentified locus on chromosome 1q. It affects men and women equally. Type 3 is an autosomal recessive trait first described in Southern Italy. Type 4 is associated with the gene ferroportin 1, also known as IREG1 or MTP1, which encodes an intestinal transport molecule. It has been described in three families (Bomford, 2002). Type 5 is a form of autosomal dominant overload that has first been described in Japan. The mutation for this gene encodes the H-subunit of familial iron overload. Other forms of hemochromatosis have been described but not genetically characterized (Bomford, 2002). In spite of these advances in the genetic characterization of hemochromatosis the disease is poorly understood. It is especially puzzling that the variation of phenotype of homozygous patients with a C282Y mutation cannot be explained currently.

Animal Models To date there are two mouse models of hemochromatosis: one is based on a b2-microglobulin knockout (Rothenberg and Voland, 1996) and the second is based on a mutation of the murine analog of the HFE gene (Zhou et al., 1998). Increased iron storage disease has been observed regularly in captive lemurs. The increased incidence of iron storage disease in these animals seems to be due to a relative lack of ironbinding food components like tannins in captive diets (Wood et al., 2003).

Treatment The main aim of the treatment is depletion of the iron stores of the body. This is done by repeated phlebotomy. Initially

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500 mL blood may be drawn weekly until the ferritin is below 20 mg/L. Thereafter four times a year is frequently sufficient. The goal is to keep the hemoglobin about 11–12 g/dL and the ferritin below 50 mL/L. In order to reduce protein losses associated with phlebotomy, erythropheresis is recommended. In general patients are advised to reduce iron absorption, e.g., by drinking black tea with their meals: and to reduce alcohol consumption since alcohol has shown to worsen the prognosis of the disease (Scotet et al., 2003). However, due to the complexity and difficulty of keeping a diet with a low iron content, patients are not usually given specific dietary advice. Regular large doses of vitamin C should be avoided since it increases iron absorption. Deferoxamine is less effective than phlebotomy and is mostly recommended for secondary siderosis after transfusions.

Prognosis The principal causes of death of patients with untreated hemochromatosis are cardiac failure (30%), hepatocellular failure or portal hypertension (25%), and hepatocellular carcinoma (30%). However, untreated cases are rare and patients who are treated before liver damage has occurred have a normal life expectancy.

References Adams, P. C., Y. Deugnier, R. Moirand, and P. Brissot. The relationship between iron overload, clinical symptoms, and age in 410 patients with genetic hemochromatosis. Hepatology 25:162–166, 1997. Adams, P. C., A. E. Kertesz, and L. S. Valberg. Clinical presentation of hemochromatosis: a changing scene. Am. J. Med. 90:445–449, 1991. Andrews, N. C. Disorders of iron metabolism. N. Engl. J. Med. 341:1986–1995, 1999. Bacon, B. R., and S. A. Sadiq. Hereditary hemochromatosis: presentation and diagnosis in the 1990s. Am. J. Gastroenterol. 92:784–789, 1997. Bomford, A. Genetics of haemochromatosis. Lancet 360:1673–1681, 2002. Camaschella, C., A. Roetto, M. Cicilano, P. Pasquero, S. Bosio, L. Gubetta, F. Di Vito, D. Girelli, A. Totaro, M. Carella, A. Grifa, and P. Gasparini. Juvenile and adult hemochromatosis are distinct genetic disorders. Eur. J. Hum. Genet. 5:371–375, 1997. Chevrant-Breton, J., M. Simon, M. Bourel, and B. Ferrand. Cutaneous manifestations of idiopathic hermochromatosis. Study of 100 cases. Arch. Dermatol. 113:161–165, 1977. Durupt, S., I. Durieu, R. Nove-Josserand, L. Bencharif, H. Rousset, and Durand D. Vital. [Hereditary hemochromatosis]. Rev. Med. Interne 21:961–971, 2000. Felitti, V. J. Hemochromatosis: A common, rarely diagnosed disease. Perm. J. 3:10–22, 1999. Milder, M. S., J. D. Cook, S. Stray, and C. A. Finch. Idiopathic hemochromatosis, an interim report. Medicine (Baltimore) 59:34–49, 1980. Moirand, R., P. C. Adams, V. Bicheler, P. Brissot, and Y. Deugnier. Clinical features of genetic hemochromatosis in women compared with men. Ann. Intern. Med. 127:105–110, 1997. Nichols, G. M., and B. R. Bacon. Hereditary hemochromatosis: pathogenesis and clinical features of a common disease. Am. J. Gastroenterol. 84:851–862, 1989.

Niederau, C., R. Fischer, A. Purschel, W. Stremmel, D. Haussinger, and G. Strohmeyer. Long-term survival in patients with hereditary hemochromatosis. Gastroenterology 110:1107–1119, 1996. Olynyk, J. K., D. J. Cullen, S. Aquilia, E. Rossi, L. Summerville, and L. W. Powell. A population-based study of the clinical expression of the hemochromatosis gene. Gastroenterology 341:718–724, 1999. Pietrangelo, A. Haemochromatosis. Gut 52 Suppl 2:ii23–ii30, 2003. Rochette, J., J. J. Pointon, C. A. Fisher, G. Perera, M. Arambepola, D. S. Arichchi, S. De Silva, J. L. Vandwalle, J. P. Monti, J. M. Old, A. T. Merryweather-Clarke, D. J. Weatherall, and K. J. Robson. Multicentric origin of hemochromatosis gene (HFE) mutations. Am. J. Hum. Genet. 64:1056–1062, 1999. Rothenberg, B. E., and J. R. Voland. Beta2 knockout mice develop parenchymal iron overload: A putative role for class I genes of the major histocompatibility complex in iron metabolism. Proc. Natl. Acad. Sci. U. S. A. 93:1529–1534, 1996. Ryan, E., V. Byrnes, B. Coughlan, A. M. Flanagan, S. Barrett, J. C. O’Keane, and J. Crowe. Underdiagnosis of hereditary haemochromatosis: lack of presentation or penetration? Gut 51:108–112, 2002a. Scotet, V., M. C. Merour, A. Y. Mercier, B. Chanu, T. Le Faou, O. Raguenes, G. Le Gac, C. Mura, J. B. Nousbaum, and C. Ferec. Hereditary hemochromatosis: effect of excessive alcohol consumption on disease expression in patients homozygous for the C282Y mutation. Am. J. Epidemiol. 158:129–134, 2003. Tavill, A. S. Clinical implications of the hemochromatosis gene. N. Engl. J. Med. 341:755–757, 1999. Tsuji, T. Experimental hemosiderosis: relationship between skin pigmentation and hemosiderin. Acta. Derm. Venereol. 60:109–114, 1980. Wood, C., S. G. Fang, A. Hunt, W. J. Streich, and M. Clauss. Increased iron absorption in lemurs: quantitative screening and assessment of dietary prevention. Am. J. Primatol. 61:101–110, 2003. Zhou, X. Y., S. Tomatsu, R. E. Fleming, S. Parkkila, A. Waheed, J. Jiang, Y. Fei, E. M. Brunt, D. A. Ruddy, C. E. Prass, R. C. Schatzman, R. O’Neill, R. S. Britton, B. R. Bacon, and W. S. Sly. HFE gene knockout produces mouse model of hereditary hemochromatosis. Proc. Natl. Acad. Sci. U. S. A. 95:2492–2497, 1998.

Hemosiderosis Introduction Hemosiderosis is defined as the histologic equivalent of secondary iron overload, which may be local, i.e., restricted to one organ, or generalized.

Epidemiology The incidence and prevalence of hemosiderosis is unknown due to the multiplicity of associated diseases.

Clinical Description of Disease/Associated Disorders A comprehensive description of changes that lead to cutaneous hemosiderosis is not possible due to the variety of causative conditions. Essentially any condition that is associated with erythrocyte extravasation will cause some hemosiderosis (Fig. 51.6). Therefore any vasculitis or malformation of cutaneous vessels, like pseudo-Kaposi sarcomas or arteriovenous malformations, are histologically associated with iron deposits of variable extent and duration. Medical treatments with ironcontaining drugs, e.g., Monsel solution, may lead to cutaneous

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hemosiderosis as well (Amazon et al., 1980; Wood and Severin, 1980). The following discussion is therefore not comprehensive but reflects those conditions that have been discussed in the dermatology literature due to their association with hemosiderosis. The most common condition associated with cutaneous hemosiderosis is probably actinic purpura, also called senile or Bateman purpura. This purpura is due to damages of the microvasculature, which are caused by ultraviolet radiationinduced dermal tissue atrophy. Atrophy renders the skin and microvasculature more susceptible to the effects of minor trauma and shearing forces which leads to extravasation of blood components and secondary hemosiderosis (Arya and Kihiczak, 2002; Gilchrest 1996). Local extravasation of erythrocytes also frequently occurs in relation to varicose veins and venous ulcers. This discoloration is associated with the acute disease and usually subsides after the underlying cause has been treated. Varicose dyschromia is a combination of increased melanin production and deposition of hemosiderin. While compression therapy reduces melanocytic pigmentation reliably, the resolution of hemosiderin discoloration is quite variable (Cuttell and Fox, 1982). Hemosiderin deposits usually disappear completely after one year but may persist for more than five years after successful sclerotherapy (Georgiev, 1990; Goldman et al., 1987). Sequential biopsies from chronic venous ulcers have not only shown hemosiderin deposits around the margins but also demonstrated that these are reduced during healing (Herrick et al., 1992). Another disorder associated with cutaneous hemosiderin deposits is targetoid hemosiderotic hemangioma. These hemangiomas are rare benign vascular tumors, usually smaller than 1 cm in diameter which may be annular (Saga, 1981). The lesions may fluctuate and can be swollen and tender. Histologically the differential diagnosis is Kaposi sarcoma (Perrin et al., 1995). Confusingly, given the name, lack of hemosiderin deposits does not exclude the diagnosis of targetoid hemosiderotic hemangioma (Ly et al., 1998). Congenital histiocytosis is a self-healing condition, associated with brown cutaneous papulonodules in newborns which regress within two to four months. The disease may present with massive hemosiderosis, which may give the impression of strong pigmentation (Belajouza-Noueiri et al., 2001). Hemosiderotic fibrohistiocytic lipomatous lesion is a recently described heavily pigmented spindle cell proliferation which occurs within a lipoma and can grow to impressive sizes. The lesions in this small series occurred primarily on the ankles of female patients in their fifties and developed mostly after local trauma. They tend to recur after excision (Marshall-Taylor and Fanburg-Smith, 2000). Hemosiderosis as a generalized secondary iron overload syndrome has a multiplicity of causes and clinical presentations that can be seen in Table 51.1. Hemosiderosis chiefly occurs in the lungs and kidneys and is the result of other disease processes. Pulmonary hemosiderosis caused by recurrent pulmonary hemorrhage occurs as an idiopathic entity 990

Fig. 51.6. Cutaneous hemosiderosis secondary to fracture of the arm.

(Milman and Pedersen, 1998), as part of Goodpasture syndrome, or in severe mitral stenosis. Occasionally, the blood loss from these episodes of hemorrhage into the lungs causes iron-deficiency anemia. Renal hemosiderosis can result from extensive intravascular hemolysis caused by trauma to red blood cells (e.g., chronic disseminated intravascular coagulation, defective or torn heart valve leaflets, prosthetic mechanical heart valves) or in paroxysmal nocturnal hemoglobinuria. In renal hemosiderosis, free hemoglobin is filtered at the glomerulus, e.g., due to hemolysis. Haptoglobin usually binds the free hemoglobin, but when haptoglobin is saturated renal iron deposition occurs. Usually the renal parenchyma is not damaged by these iron deposits, but severe hemosiderinuria may result in iron deficiency (Andrews, 1999; Lim et al., 2000).

Histology Hemosiderosis is not a disease entity but rather a condition in which iron, usually in its ferric state (Fe3+), is deposited in tissue. The most common stain used to detect ferric iron in histology is Perls Prussian blue, sometimes called Perls acid or ferrocyanide reaction.

Laboratory Findings In order to distinguish localized hemosiderosis, e.g., due to actinic purpura, from generalized hemosiderosis, e.g., secondary to multiple infusions, the iron stores of the body have to be evaluated. This is most easily done using serum ferritin as a screening test. In cases of tissue damage and inflammation the increase in ferritin may be misleadingly high, while in scorbutic patients the ferritin may be misleadingly low (Porter, 2001). However, an increased transferrin saturation and serum iron will confirm a diagnosis of iron overload.

Pathogenesis The pathogenesis of hemosiderosis is iron overload caused by an underlying disease or external causes. It may be iatrogenic, e.g., after multiple blood transfusions (Hb SS, thalassemia); or

HYPERMELANOSIS ASSOCIATED WITH GASTROINTESTINAL DISORDERS Table 51.1. Classification of hemosiderosis and hemochromatosis. Primary genetic hemochromatosis Iron overload Congenital hemolytic anemias Increased parental iron intake (rare) Repeated transfusions, e.g., due to defective Hb synthesis (sickle cell disease, thalassemia) Iron dextran IM Increased intestional iron absorption African iron overload Kashin–Bek disease with hemosiderosis (Beers and Berkow, 1999) Localized hemosiderosis Pulmonary (idiopathic) Renal Hepatic (porphyria cutanea tarda) Cutaneous (e.g., actinic purpura)

self-inflicted due to African iron overload (Andrews, 1999). In case of localized hemosiderosis the pathogenesis may be related to extravasation of red blood cells, e.g., after vasculitis or due to chronic actinic vascular damage as in actinic purpura or the application of iron-containing drugs. Trauma or intoxication lead to hemosiderosis due to iron-containing objects, e.g., nails or ammunition. Two patterns of iron overload are usually observed. Iron overload in which the plasma iron content exceeds the transferrin binding capacity will lead to deposits of iron in the parenchyma cells of the liver, the heart, and a subgroup of endocrine tissues. The most typical disease with this type of iron deposition is hereditary hemochromatosis. Iron overload that results from increased catabolism of erythrocytes leads to accumulation of iron in the reticuloendothelial macrophages, which only later spills over to the parenchymal cells. This parenchymal iron overload causes fibrosis and tissue damage that may characterize some forms of hemosiderosis. Iron accumulation in the macrophages only is usually not dangerous (Andrews, 1999).

Animal Models A specific animal model for hemosiderosis does not exist, however, systemic hemosiderosis has been induced in SpragueDawley rats by subcutaneous injection of elemental iron as iron–dextran complex (Pelot et al., 1998). Another way to induce systemic hemosiderosis is through infection and hemolysis. Cullen and Levine (1987) developed a model of Syrian hamsters who were infected by Babesia microti, a malaria-like protozoan usually associated with rodents. The animals did not die but the disease caused intravascular and extravascular hemolysis and marked renal hemosiderosis.

Treatment Treatment of hemosiderosis-associated pigmentary changes will depend on whether the discoloration is primarily due to

hemosiderin or melanin and whether the pigmentation is dermal or epidermal in origin (Briganti et al., 2003). The different treatment options are discussed in more detail in Chapters 59–64 of this book. Transfusion iron overload and iron overload that occurs due to defective erythropoiesis (e.g., in congenital hemolytic anemias or hemoglobinopathies) is usually obvious from the clinical history. Because the transfusions are given due to anemia, phlebotomy may not be possible. Desferrioxamine can be given to effectively reduce iron stores for symptomatic systemic hemosiderosis. The drug is given slowly subcutaneously because boluses are associated with acute systemic side effects. Toxic effects resulting from desferrioxamine therapy are now rare provided the recommended dosing schedules are not exceeded. However, baseline examinations of ears and eyes are recommended. Efficacy must continually be evaluated (usually by urine iron measurement) due to tachyphylaxis that may develop with desferrioxamine therapy. Alternatively, salmon- or rusty colored urine confirms >50 mg/day of iron in the urine (Porter, 2001).

Prognosis The prognosis of hemosiderosis is dependent on the underlying condition.

References Amazon, K., M. J. Robinson, and A. M. Rywlin. Ferrugination caused by Monsel’s solution. Clinical observations and experimentations. Am. J. Dermatopathol. 2:197–205, 1980. Andrews, N. C. Disorders of iron metabolism. N. Engl. J. Med. 341:1986–1995, 1999. Arya V., and G. Kihiczak. Actinic purpura. 2002, www.emedecine. com (accessed February 28, 2004). Belajouza-Noueiri, C., M. Denguezli, H. Selmi, M. Mokni, B. Jomaa, and R. Nouira. [Intense hemosiderin deposits in a case of selfhealing congenital histiocytosis]. Ann. Dermatol. Venereol. 128:238–240, 2001. Briganti, S., E. Camera, and M. Picardo. Chemical and instrumental approaches to treat hyperpigmentation. Pigment Cell Res. 16:101– 110, 2003. Cullen, J. M., and J. F. Levine. Pathology of experimental Babesia microti infection in the Syrian hamster. Lab. Anim. Sci. 37:640– 643, 1987. Cuttell, P. J., and J. A. Fox. The aetiology and treatment of varicose pigmentation. Phlebologie 35:381–389, 1982. Georgiev, M. Postsclerotherapy hyperpigmentations: a one-year follow-up. J. Dermatol. Surg. Oncol. 16:608–610, 1990. Gilchrest, B. A. A review of skin ageing and its medical therapy. Br. J. Dermatol. 135:867–875, 1996. Goldman, M. P., R. P. Kaplan, and D. M. Duffy. Postsclerotherapy hyperpigmentation: a histologic evaluation. J. Dermatol. Surg. Oncol. 13:547–550, 1987. Herrick, S. E., P. Sloan, M. McGurk, L. Freak, C. N. McCollum, and M. W. Ferguson. Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers. Am. J. Pathol. 141:1085–1095, 1992. Lim, Y. K., A. Jenner, A. B. Ali, Y. Wang, S. I. Hsu, S. M. Chong, H. Baumman, B. Halliwell, and S. K. Lim. Haptoglobin reduces renal oxidative DNA and tissue damage during phenylhydrazine-induced hemolysis. Kidney Int. 58:1033–1044, 2000.

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CHAPTER 51 Ly, S., J. Versapuech, B. Vergier, M. Beylot-Barry, and C. Beylot. Guess what! Targetoid hemosiderotic hemangioma. Eur. J. Dermatol. 8:583–585, 1998. Marshall-Taylor, C., and J. C. Fanburg-Smith. Hemosiderotic fibrohistiocytic lipomatous lesion: ten cases of a previously undescribed fatty lesion of the foot/ankle. Mod. Pathol. 13:1192–1199, 2000. Milman, N., and F. M. Pedersen. Idiopathic pulmonary haemosiderosis. Epidemiology, pathogenic aspects and diagnosis. Respir. Med. 92:902–907, 1998. Pelot, D., X. J. Zhou, P. Carpenter, and N. D. Vaziri. Effects of experimental hemosiderosis on pancreatic tissue iron content and structure. Dig. Dis. Sci. 43:2411–2414, 1998. Perrin, C., S. Rodot, J. P. Ortonne, and J. F. Michiels. [Targetoid hemosiderotic hemangioma]. Ann. Dermatol. Venereol. 122:111– 114, 1995. Porter, J. B. Practical management of iron overload. Br. J. Haematol. 115:239–252, 2001. Saga, K. Annular hemosiderotic histiocytoma. J. Cutan. Pathol. 8:251–255, 1981. Wood, C., and G. L. Severin. Unusual histiocytic reaction to Monsel’s solution. Am. J. Dermatopathol. 2:261–264, 1980.

Primary Biliary Cirrhosis Joerg Albrecht and Victoria P. Werth

Introduction Primary biliary cirrhosis (PBC) is a chronic non–pusproducing destructive cholangitis, which is also less commonly named “primary biliary hepatitis (PBH),” “chronic nonsuppurative destructive cholangitis,” and “primary autoimmune cholangitis.”

Epidemiology PBC affects all races but seems to cluster within specific geographic areas. The male/female ratio is 9:1 with a median age of onset of 20 years, with a range from 20 to 90 years. The annual incidence and prevalence for the disease ranges from 2 and 24 cases to 19 and 240 cases per million population (Jones, 2003; Kim et al., 2000; Talwalkar and Lindor, 2003). The reported incidence is highest in areas of the UK and lowest in Canada and Australia, with almost no cases in subSaharan Africa or India. This range of prevalence is probably partly due to environmental factors that may influence disease expression, however, no distinct factors have been identified, although an association with infections has been suggested (Feld and Heathcote, 2003; Gish and Mason, 2001; Prince et al., 2001).

Historical Background In 1857 Thomas Addison (1785–1860) and William Gull described the first cases of what is now recognized as the autoimmune liver disease, PBC. Thomas Addison was born in an area with one of the highest prevalences of PBC yet described, and there is some suggestion from contemporary sources that he himself succumbed to the liver disease (Jones, 2003). 992

Fig. 51.7. Woman with biliary cirrhosis and diffuse hyperpigmentation more pronounced on sun-exposed areas (see also Plate 51.3, pp. 494–495).

Clinical Description of Disease/Associated Disorders Patients with PBC show diffuse darkening of the skin, with accentuation in the sun-exposed areas (Fig. 51.7). The hyperpigmentation is associated with pruritus, jaundice, and subcutaneous lipid deposits around the eyes (xanthelasma) or on joints or tendons (xanthomas). Patients with PBC may also show the typical pigmented corneal rings that are usually associated with Wilson disease (Fleming et al., 1975; Goldstein, 1976). Occasionally PBC is associated with other connective tissue diseases like rheumatoid arthritis, thyroiditis (Hashimoto), or Sjögren syndrome. In 10% the disease shows clinical overlap with CREST syndrome or autoimmune hepatitis, which may distort the clinical picture (Bolognia and Braverman, 2001; Nishio et al., 2000). Currently most patients are diagnosed as having PBC during routine screening. Suspicion may be raised by a twofold or greater level of serum alkaline phosphatase levels with concurrent elevation of the serum 5¢-nucleotidase activity and the g-glutamyl transpeptidase levels, while serum bilirubin is still

HYPERMELANOSIS ASSOCIATED WITH GASTROINTESTINAL DISORDERS

normal. Patients are usually asymptomatic and may remain so for prolonged periods of time; 90% of the patients with symptoms are women between 35 and 60 years. Patients have intense pruritus and fatigue independent of the extent of their liver involvement. The pruritus may initially be limited to the palms and the soles. The other symptoms of the disease are largely related to impaired bile excretion and subsequent hepatocellular failure, and may not occur in patients with a protracted course of the disease. Clinical changes usually associated with PBC are jaundice, hepatomegaly, maldigestion, and portal hypertension with its associated morbidity and mortality.

mately 50% of patients have antinuclear antibodies, sometimes against very specific proteins (nuclear pore membrane protein gp210, transcriptional activator Sp100, inner nuclear membrane protein LBR). Absence of an elevated serum IgM concentration and/or specific autoantibodies argues against PBC. The clinical signs of liver disease are accompanied by characteristic changes in the cholestatic enzymes, such as an increase in alkaline phosphatase, decrease in leukocyte alkaline phosphatase (LAP), as well as increases in bilirubin and g-glutamyl transpeptidase (GGT or g-GT and GGTP or gGTP). Patients usually show hypercholesterolemia as a further consequence of liver disease.

Histology

Diagnosis

Skin histology is rarely warranted, in spite of the hyperpigmentation that is observed in patients with PBC. A histologic and ultrastructural study of PBC has demonstrated that cutaneous pigmentation in PBC is due to the presence of increased amounts of melanin, widely dispersed throughout both epidermis and dermis. The authors observed no deposits of stainable iron, but found unusually high levels of melanosomes in the epidermis. These melanosomes were packaged in large membrane-bound clusters. It remains unclear whether the excess melanin in PBC results from increased melanogenesis or from defective melanin degradation. The authors did not demonstrate hormonal (b-melanocyte-stimulating hormone and adrenocorticotropic hormone) or chemical (bile salt irritation) stimuli that may have increased melanogenesis. The melanocyte to keratinocyte ratio was not significantly higher in PBC when compared to skin from matched sites from control patients with alcoholic cirrhosis or no abnormalities of pigmentation (Mills et al., 1981). Histologic staging of liver disease in PBC is not always straightforward, although a four-stage morphologic classification has been established. Frequently different stages of the disease can be observed in one patient at the same time. Small biopsies may miss this variety of presentations and thus risk misclassification. As a general rule the highest stage in any one patient is considered to represent the clinical stage. Stage I is the earliest recognizable stage and termed nonsuppurative destructive cholangitis. It is a necrotizing inflammatory process of the portal triads characterized by destruction of medium and small bile ducts, and a dense infiltrate of acute and chronic inflammatory cells. These inflammatory cells become less prominent when the number of bile ducts has been reduced and small new bile ducts proliferate (stage II). The progression of the disease leads to a decrease in interlobular ducts, loss of liver cells, and expansion of periportal fibrosis into a network of connective tissue scars (stage III). Stage IV documents liver cirrhosis (Chung and Podolski, 2001).

The diagnosis of primary biliary cirrhosis is only rarely based on the skin changes. Indicators of cholestasis and increases in IgM may point towards the disease, as do positive antimitochondrial antibody (AMA) titers. False-positive AMA results occur, and a number of patients may never develop AMA titers. In some of these latter patients antinuclear or smooth muscle antibodies are present and the disease is described as autoimmune cholangitis, although the histology is identical to PBC and the natural history seems to be similar. Alternatively AMA-negative patients may have primary sclerosing cholangitis and the biliary tract should be evaluated to exclude this condition (Chung and Podolski, 2001). If the diagnosis of PBC has been established the patient should be screened for other associated autoimmune diseases like Sjögren syndrome, thyroiditis, CREST syndrome, or scleroderma, and metabolic disease including bone problems and vitamin deficiency should be investigated (Gish and Mason, 2001).

Differential Diagnosis Based on the hyperpigmentation the most relevant differential diagnosis is POEMS [polyneuropathy: organomegaly (spleen, liver, lymph nodes); endocrinopathies (impotence, gynecomastia); M-protein; and skin changes]. The skin changes of POEMS syndrome include hyperpigmentation, skin thickening, hypertrichosis, and angiomas (Bolognia and Braverman, 2001). The clinical differential diagnosis for diseases of the liver include intrahepatic cholestasis, as seen in cholestatic viral hepatitis or drug-induced jaundice, or extrahepatic cholestasis seen in cholelithiasis, tumors, maw-worms, liver flukes, or stenosis of the common bile duct after laparoscopic cholecystectomy. Pruritus as a basis for differential diagnosis can be associated with diseases of the skin, allergies, PBC or PSC, a variety of intestinal parasites, diabetes, lymphomas, renal insufficiency, polycythemia vera, iron deficiency, senile pruritus, or associated with psychiatric diseases.

Laboratory Findings More than 90% of patients have autoantibodies against specific mitochondrial proteins (AMA), most often the E2 subunits of the oxo-acid dehydrogenase complexes. Approxi-

Pathogenesis PBC is a complex autoimmune disease. More than 90% of the patients have IgG antimitochondral antibodies in the serum 993

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that are particularly directed at the pyruvate dehydrogenase complex (PDC); specifically the autoantibody response is directed at the enzyme 2, dihydrolipoamide acetyl transferase (E2), and enzyme 3 building protein (E3BP) components of PDC. It is thought likely that CD8+ cytotoxic T cells with selfPDC derived epitopes are responsible for target cell damage, i.e., the apoptosis of biliary epithelial cells, although direct cytotoxic activity has not been demonstrated. As a reflection of this process lymphocytes are prominent in the portal regions and surround damaged bile duct. These histologic changes associated with PBC resemble graft-versus-host disease and suggest that damage to the bile ducts may be biologically mediated. The pathognomonic liver damage is caused by impaired biliary excretion due to fibrosing obliteration of intrahepatic bile ductules, which causes progressive fibrosis of the liver (Jones, 2003).

gression in individual patients several mathematical models based on clinical, laboratory, and histological criteria have been developed, the most popular of these based on the Mayo Clinic patient population and available on the web (Dickson et al., 1989). In general, the development of portal hypertension indicates a poor prognosis, as does anti-M4 or anti-M8. The serum bilirubin concentration is the best prognostic indicator of all laboratory values. Once the serum bilirubin concentration exceeds 145 mmol/L or there is evidence of ascites, hepatic encephalopathy, or variceal bleeding, patients should be considered for liver transplantation. Prognosis after liver transplantation is excellent with 85–90% survival after one year. Thereafter survival rates are similar to those of healthy patients matched for age and sex (Nishio et al., 2000).

Animal Models

Beswick, D. R., G. Klatskin, and J. L. Boyer. Asymptomatic primary biliary cirrhosis. A progress report on long-term follow-up and natural history. Gastroenterology 89:267–271, 1985. Bolognia, J. L., and I. M. Braverman. Skin manifestations of internal disease. In: Harrison’s Principles of Internal Medicine, 15th ed. E. Braunwald, A. S. Fauci, D. L. Kasper, S. L. Hauser, D. L. Longo, and J. L. Jameson (eds). New York: McGraw-Hill, 2001. Chung, R. T., and D. K. Podolski. Cirrhosis and its complications. In: Harrison’s Principles of Internal Medicine, 15th ed. E. Braunwald, A. S. Fauci, D. L. Kasper, S. L. Hauser, D. L. Longo, and J. L. Jameson (eds). New York: McGraw-Hill, 2001. Dickson, E. R., P. M. Grambsch, T. R. Fleming, L. D. Fisher, and A. Langworthy. Prognosis in primary biliary cirrhosis: model for decision making. Hepatology 10:1–7, 1989. Feld, J. J., and E. J. Heathcote. Epidemiology of autoimmune liver disease. J. Gastroenterol. Hepatol. 18:1118–1128, 2003. Fleming, C. R., E. R. Dickson, R. W. Hollenhorst, N. P. Goldstein, J. T. McCall, and A. H. Baggenstoss. Pigmented corneal rings in a patient with primary biliary cirrhosis. Gastroenterology 69:220–225, 1975. Gish, R. G., and A. Mason. Autoimmune liver disease. Current standards, future directions. Clin. Liver Dis. 5:287–314, 2001. Goldstein, N. P. Letter: Pigmented corneal rings in a patient with primary biliary cirrhosis. Arch. Neurol. 33:372–1976. Jones, D. E. Addison’s other disease: primary biliary cirrhosis as a model autoimmune disease. Clin. Med. 3:351–356, 2003. Jones, D. E., J. M. Palmer, J. A. Kirby, D. J. De Cruz, G. W. McCaughan, J. D. Sedgwick, S. J. Yeaman, A. D. Burt, and M. F. Bassendine. Experimental autoimmune cholangitis: a mouse model of immune-mediated cholangiopathy. Liver 20:351–356, 2000. Kim, W. R., K. D. Lindor, G. R. Locke, III, T. M. Therneau, H. A. Homburger, K. P. Batts, B. P. Yawn, J. L. Petz, L. J. Melton, III, and E. R. Dickson. Epidemiology and natural history of primary biliary cirrhosis in a US community. Gastroenterology 119:1631–1636, 2000. Levy, C., and K. D. Lindor. Current management of primary biliary cirrhosis and primary sclerosing cholangitis. J. Hepatol. 38(Suppl 1):S24–S37, 2003. Mills, P. R., C. J. Skerrow, and R. M. MacKie. Melanin pigmentation of the skin in primary biliary cirrhosis. J. Cutan. Pathol. 8:404–410, 1981. Nishio, A., E. B. Keeffe, H. Ishibashi, and E. M. Gershwin. Diagnosis and treatment of primary biliary cirrhosis. Med. Sci. Monit. 6:181–193, 2000.

Recently an animal model for PBC, i.e., experimental autoimmune cholangitis (EAC) has been developed. To acquire the disease female SJL/J mice have to be sensitized with PDC, i.e., its purified E2/E3BP component. Within five weeks the mice have high titer antibodies and splenic T-cell proliferation, and after 30 weeks a high proportion of the mice have EAC (Jones et al., 2000).

Treatment While there is no specific therapy for PBC, ursodiol has been shown to be beneficial to relieve symptoms. Classic immunosuppressive medications like glucocorticoids, methotrexate, colchicine, azathioprine, ciclosporin, and tacrolimus have been disappointing in controlled trials. Unfortunately the ultimate treatment for PBC still is liver transplantation. The patients have excellent survival, but about 25% will show histologic changes of PBC after five years, which is often asymptomatic (Nishio et al., 2000). While the traditional treatment dogma has been to slow or prevent disease progression, it is now appreciated that at least equal effort should be given to treating symptoms such as fatigue and pruritus, which can dramatically improve the patient’s quality of life (Jones, 2003). A detailed description of symptomatic treatment is beyond the scope of this chapter and the reader is referred to the current medical literature (Levy and Lindor, 2003).

Prognosis PBC is a progressive disease that leads to cirrhosis and liver failure. The time from diagnosis to end-stage liver disease can range from a few months to 20 years, depending upon when the diagnosis is first made. Many patients have a chronic and indolent course of the disease; in the majority of usually elderly patients the risk of developing the disease is very low and these patients may have a normal life expectancy (Jones, 2003; Nishio et al., 2000). However, association with other autoimmune disease generally predicts a worse prognosis (Beswick et al., 1985). In order to calculate the disease pro994

References

HYPERMELANOSIS ASSOCIATED WITH GASTROINTESTINAL DISORDERS Prince, M. I., A. Chetwynd, P. Diggle, M. Jarner, J. V. Metcalf, and O. F. James. The geographical distribution of primary biliary cirrhosis in a well-defined cohort. Hepatology 34:1083–1088, 2001. Talwalkar, J. A., and K. D. Lindor. Primary biliary cirrhosis. Lancet 362:53–61, 2003.

Inflammatory Bowel Disease and Pigmentation Joerg Albrecht and Victoria P. Werth Inflammatory bowel diseases, i.e., Crohn disease and ulcerative colitis, are associated with a number of cutaneous findings of varying frequency (Apgar, 1991). However, the association between inflammatory bowel disease and pigmentation is a tenuous one. There have been repeated reports of correlation between the activity of Crohn disease and vitiligo. In one case the two conditions only coexisted (Monroe, 1976), in a second the diseases showed parallel activity and were both suppressed by the steroid treatment for Crohn disease (McPoland and Moss 1988). A recent epidemiologic study confirmed that the incidence of inflammatory bowel diseases is approximately twice as high in patients with vitiligo as in the general population (Alkhateeb et al., 2003). This, however, is not a very strong association between two autoimmune diseases and it does not imply that vitiligo and inflammatory bowel diseases are associated in a clinically meaningful way.

References Alkhateeb, A., P. R. Fain, A. Thody, D. C. Bennett, and R. A. Spritz. Epidemiology of vitiligo and associated autoimmune diseases in Caucasian probands and their families. Pigment Cell Res. 16:208–214, 2003. Apgar, J. T. Newer aspects of inflammatory bowel disease and its cutaneous manifestations: a selective review. Semin. Dermatol. 10:138–147, 1991. McPoland, P. R., and R. L. Moss. Cutaneous Crohn’s disease and progressive vitiligo. J. Am. Acad. Dermatol. 19:421–425, 1988. Monroe, E. W. Vitiligo associated with regional enteritis. Arch. Dermatol. 112:833–834, 1976.

Pellagra Eun Ji Kwon and Victoria P. Werth

Historical Background Pellagra, derived from the Italian pelle meaning “skin” and agra meaning “dry,” was first described by Gaspar Casal in the Asturias region of Spain in 1735 (Sebrell, 1981). Called “mal de la rosa” among the Asturians for its characteristic erythematous rash on the skin, the disease struck poor peasants who subsisted on a maize-based diet lacking meat intake. Casal speculated that this was a nutritional disease after noting that adding animal products to the peasants’ diet ameliorated symptoms. The nutritional basis of pellagra was established during the 1910s by Joseph Goldberger of the

United States Public Health Service. Through a series of clinical trials on prisoners, Goldberger proved that a diet poor in animal protein caused pellagra and that an animal–proteinrich diet could treat and prevent pellagra. The exact nutritional deficiency was not identified until 1937, when Conrad Elvehjem of the University of Wisconsin demonstrated conclusively that niacin was the key pellagra-preventive factor (Roe, 1973).

Epidemiology Although pellagra is rare in industrialized countries, it is still observed in areas of the world where malnutrition continues to be a problem. In endemic areas it affects individuals of all ages, typically first appearing in children after breastfeeding ceases. Endemic areas include India where millet and corn are primary dietary staples and Asia and Africa where corn (containing a nonabsorbable bound form of niacin) is the primary staple (Nieves and Goldsmith, 2003; Stratigos and Katsambas, 1977). Multiple outbreaks of pellagra have occurred since 1988 in refugee programs in Angola, Ethiopia, Malawi, Nepal, Swaziland, the former Zaire, and Zimbabwe (World Health Organization, 2000). Sporadic cases of pellagra have been reported in developed nations, especially in association with chronic alcoholism, malabsorption disorders, and nutrient–drug interactions (Nieves and Goldsmith, 2003).

Clinical Description of Disease/Associated Disorders Pellagra is characterized by the triad of three Ds — dermatitis, diarrhea, and dementia. The cutaneous lesions are symmetric and appear on sun-exposed areas such as the dorsum of the hands, wrists, and forearms (Fig. 51.8), face, neck, anterior chest, knees, shoulders, and elbows (Figs 51.9 and 51.10) (Miller, 1989). The lesions are precipitated by sunlight, heat, or trauma (Miller, 1989). The disease begins as a dermatitis with well-defined, painful or burning areas of erythema and edema. Erythema on the face may resemble the “butterfly” rash of lupus erythematosus. These lesions may resemble early sunburn and the initial erythema changes to cinnamon-brown in color. However, the rate of tanning is slower than is characteristic in sunburn (Hegyi et al., 2004). Vesicles or bullae can also occur. Later, the lesions, sometimes described as “gooselike,” become hyperpigmented, hyperkeratotic, scaly, variably fissured and crusted (see Fig. 51.10) (Miller, 1989). The dorsal surfaces of the hands are most frequently involved, and referred to as the “glove” or “gauntlet” of pellagra. Lesions on the feet and leg may resemble a “boot.” Casal’s “necklace” refers to the lesions that occur as a wide band around the neck and on the anterior chest. With advanced deficiency state, the skin may become covered with black crusts due to hemorrhages (Nieves and Goldsmith, 2003). Angular cheilosis and stomatitis, glossitis (“beefy” red, smooth or black, atrophic tongue), proctitis, vulvovaginitis, and scrotal dermatitis are common in individuals with pellagra (Miller, 1989). Infants may present with a diaper dermatitis. However, there is evidence suggesting angular 995

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Fig. 51.8. Hyperpigmentation scaly fissured dermatitis on the sunexposed areas (see also Plate 51.4, pp. 494–495).

cheilitis and nasolabial seborrhea without other pellagra skin lesions, as well as pellagrous vulvovaginitis and perianal and scrotal lesions are secondary to riboflavin deficiency, rather than niacin insufficiency (Sebrell, 1979). Healing of pellagra lesions occurs centrifugally, as desquamation begins from the center of the lesion and spreads towards the periphery where there is still active inflammation (Nieves and Goldmisth, 2003). Other gastrointestinal symptoms include dysphagia, abdominal pain, nausea, and vomiting (Miller, 1989; Spivak and Jackson, 1977). Neuropsychiatric symptoms include headache, dizziness, weakness, anorexia, depression, anxiety, insomnia, apathy, memory impairment, hallucinations, delusions, and encephalopathy (Miller, 1989; Spivak and Jackson, 1977). In severe and untreated patients, signs of peripheral nerve, posterolateral cord, and pyramidal tract involvement may occur, and patients may enter a stuporous and comatose state before their eventual death (Hegyi et al., 2004; Miller, 1989; Spivak and Jackson, 1977).

Fig. 51.9. Scaly dermatitis involving the face and V of neck in patient with pellagra. The eruption rapidly resolved with proper dietary intake (see also Plate 51.5, pp. 494–495).

Histology The histology of pellagra is nonspecific (Moore et al., 1942). Early lesions may show ballooning degeneration of the epidermis that results in an intraepidermal vesicle or bulla, or marked papillary dermal edema with resultant subepidermal vesicle or bulla (Hendricks, 1991). Later lesions may show hyperkeratosis and parakeratosis, acanthosis that may be psoriasiform, and increased melanin along the basal layer (Moore et al., 1942). The spinous layer may contain vacuolar changes, similar to those observed in acrodermatitis enteropathica and necrolytic migratory erythema. Atrophic sebaceous glands may be seen (Nieves and Goldsmith, 2003). The infiltrate is mononuclear and located in the superficial dermis (Moore et al., 1942). In the papillary dermis there may be dilated blood vessels and red blood cell extravasation (Hendricks, 1991).

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Fig. 51.10. Pellagra. Scaly dermatitis on the arms of the man in Figure 51.10 (see also Plate 51.6, pp. 494–495).

HYPERMELANOSIS ASSOCIATED WITH GASTROINTESTINAL DISORDERS

Laboratory Findings Patients with pellagra have a low 24-hour urinary excretion of one or both metabolites of niacin, i.e., N1methyl nicotinamide and its 2-pyridone derivative (Russell, 2001). A combined excretion of N1methyl nicotinamide and 2-pyridone lower than 1.5 mg in 24 hours is suggestive of severe niacin deficiency (Hegyi et al., 2004). They may also have increased serum serotonin and its metabolite 5-hydroxyindoleacetic acid. Hypoalbuminemia, macrocytic anemia, hyperuricemia, hypercalcemia, hypokalemia, hypophosphatemia, abnormal liver function tests, and increased serum porphyrin levels may also be present (Spivak and Jackson, 1977; Hegyi et al., 2004).

Diagnosis Diagnosis is made by the presence of typical cutaneous lesions and history of diet deficiencies with or without gastrointestinal and neurologic symptoms. The diagnosis is confirmed if the syndrome disappears when niacin is introduced into the diet.

Differential Diagnosis Drug eruptions, photodermatitis, atopic dermatitis, subacute lupus erythematosus, polymorphous light eruption, pemphigus vulgaris, PCT, and porphyria variegata and deficiencies of other nutrients, such as zinc, may resemble pellagra (Hegyi et al., 2004; Hendricks, 1991). Although kwashiorkor may also mimic pellagra, kwashiorkor is more common in children, and unlike pellagra, involves the hair and nails (Karthikeyan and Thappa, 2002). It is also important to identify pellagratriggering conditions, such as anorexia nervosa, malabsorptive disorders, alcoholism, carcinoid tumor, Hartnup syndrome, and HIV/AIDS (Murray et al., 2001; Pitche et al., 1999; Prousky, 2003; Russell, 2001).

Pathogenesis Pellagra is caused by a deficiency of niacin (nicotinic acid, vitamin B3, pellagra-preventing vitamin) or nicotinamide. Niacin is a substituted pyridine derivative (Henderson, 1983). Nicotinamide is a derivative of nicotinic acid that contains an amide instead of a carboxyl group. It is nutritionally equivalent to nicotinic acid since it is readily deaminated in the body. Sources of niacin include diet and conversion from tryptophan in vivo. Foods abundant in niacin include lean meats, liver, fish, poultry, unrefined and enriched grains and cereals, milk, nuts, and legumes (Stratigos and Katsambas, 1977). Conversion of tryptophan to niacin occurs when there is an abundance of amino acids in the diet. It requires as cofactors vitamin B1, B2, and B6. In the presence of vitamins B2 and B6, 1 mg of niacin is formed from 60 mg of tryptophan. Nicotinic acid and nicotinamide play important roles in metabolism. They are key components of coenzyme I (nicotinamide adenine dinucleotide, or NAD+) and coenzyme II (nicotinamide adenine dinucleotide phosphate, or NADP+.)

These coenzymes participate in oxidation–reduction reactions, and thus participate in carbohydrate, amino acid, and fatty acid metabolism. NAD and NADP are also active in DNA repair and calcium mobilization involving adenosinediphosphate–ribose transfer reactions. Therefore, niacin deficiency has profound negative effects on cellular function throughout the body, especially in tissues with high energy requirements and/or high cellular turnover, such as the brain, intestinal mucosa, and skin (Hendricks, 1991; Russell, 2001). It is hypothesized that niacin deficiency may trigger phototoxic reactions in the skin through the accumulation of kynurenic acid, a photosensitizing by-product of the tryptophan–niacin conversion pathway that is downregulated by nicotinamide (Hendricks, 1991). In addition, NADPH deficiency (secondary to niacin/nicotinamide insufficiency) may lead to increased oxidative damage to the skin by decreasing concentrations of reduced glutathione needed to detoxify H2O2 to H2O (Hendricks, 1991). Decreased levels of reduced glutathione may also be associated with hyperpigmentation. There is evidence in mammalian models that low levels of reduced glutathione and glutathione reductase activity in the melanocyte milieu stimulate eumelanin-type pigment synthesis (Benedetto et al., 1981, 1982; Halprin and Ohkawara, 1966). Inadequate dietary intake of niacin, nicotinamide, or tryptophan can produce pellagra (Stratigos and Katsambas, 1977). Individuals living in cultures with diets rich in maize have historically been at risk for developing pellagra. Although niacin is present in maize in significant amounts, it is in a nonabsorbable form that is bound to peptides and complex carbohydrates. However, niacin in maize may be released in an alkaline environment. In Mexico, where the practice of washing maize with limewater is common, pellagra has not been a public health issue even among the indigent rural population (Stratigos and Katsambas, 1977). Other causes of nutrition deficiency, such as chronic alcoholism, psychiatric disorders, malabsorption (e.g., jejunoileitis, Crohn disease, gastroenterostomy, or subtotal gastrectomy), and eating disorders also place individuals at risk for pellagra (Nieves and Goldsmith, 2003; Prousky, 2003). In addition, increased dietary leucine (as seen in regions in India with leucine-rich millet-based diet) may precipitate pellagra by inhibiting the conversion from tryptophan to niacin (Stratigos and Katsambas, 1977). Pellagra may be secondary to systemic conditions. Carcinoid tumors often precipitate clinical pellagra by converting a large proportion of tryptophan to serotonin, depleting tryptophan available for niacin production. Tryptophan malabsorption is also seen in Hartnup disease, an inherited disorder of intestinal and kidney tryptophan transport (Russell, 2001). In addition, decreased serum levels of tryptophan and pellagralike manifestations have been described among individuals with HIV infection (Murray et al., 2001; Pitche et al., 1999). Deficiencies in other essential vitamins and nutrients, as commonly seen in malnourished individuals, also contribute

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to pellagra. For example, deficiency in B2 and B6 (cofactors in conversion reaction from tryptophan to niacin) may cause pellagra (Stratigos and Katsambas, 1977). Therefore, it is important to include replacement of all essential nutrients when treating pellagra. Various medications have also been shown to cause pellagra. Isoniazid and 5-fluorouracil are known to inhibit the conversion of tryptophan to niacin. 6-Mercaptopurine triggers pellagra by inhibiting NAD phosphorylase and NAD production (Miller, 1989). Other pellagragenic medications include pyrazinamide, hydantoins, ethionamide, phenobarbital, azathioprine, and chloramphenicol (Hegyi et al., 2004).

Animal Models “Black tongue,” an experimental analog of pellagra in dogs, was cured and prevented, when their diet was supplemented with nicotinamide or niacin (Elvehjem et al., 1938; Goldberger and Tanner, 1922). Dietary niacin deficiency has also been induced in Japanese quails and Weanling Long-Evans rats (Boyonoski et al., 2000; Koh et al., 1998).

Treatment Oral nicotinamide or nicotinic acid supplementation of 100– 200 mg three times daily for five days is used to treat the clinical manifestations of pellagra. Nicotinamide is preferred over niacin because it does not produce a flushing reaction. Parenteral nicotinamide may be used for patients with poor oral toleration (Hendricks, 1991; Russell, 2001). Diet rich in proteins and essential nutrients should accompany nicotinamide supplementation for complete recovery. Identification and management of underlying causes of secondary pellagra is also crucial. The minimal daily requirement of niacin is 4.4 mg/1000 kcal and the recommended daily allowance of niacin is 6.6 mg/1000 kcal (Food and Nutrition Board and National Research Council, 1989). Therefore, multivitamins with at least 15 mg of nicotinamide should be given daily as preventive therapy along with a high-protein diet (Hendricks, 1991). The response to therapy is dramatic. Early cutaneous lesions and cutaneous, gastrointestinal, and neurologic symptoms respond within 24 hours.

Prognosis Prognosis is good with treatment. If untreated, the course is chronic with seasonal exacerbations, and death can occur as a consequence of dehydration or central nervous system involvement (Hendricks, 1991).

References Benedetto, J. P., J. P. Ortonne, C. Voulot, C. Khatchadourian, G. Prota, and J. Thivolet. Role of thiol compounds in mammalian melanin pigmentation. Part I. Glutathione and related enzymatic activities. J. Invest. Dermatol. 77:402–405, 1981. Benedetto, J. P., J. P. Ortonne, C. Voulot, C. Khatchadourian, G. Prota, and J. Thivolet. Role of thiol compounds in mammalian melanin pigmentation. II. Glutathione and related enzymatic activities. J. Invest. Dermatol. 79:422–424, 1982.

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Boyonoski, A. C., L. M. Gallacher, M. M. ApSimon, R. M. Jacobs, G. M. Shah, G. G. Poirier, and J. B. Kirkland. Niacin deficiency in rats increases the severity of ethylnitrosourea-induced anemia and leukopenia. J. Nutr. 130:1102–1107, 2000. Elvehjem, C. A., R. J. Madden, F. M. Strong, and D. W. Woolley. Isolation and identification of anti-black tongue factor. J. Biol. Chem. 123:137–149, 1938. Goldberger, J., and W. F. Tanner. Amino acid deficiency is probably the primary etiologic factor in pellagra. Public. Health Rep. 37:462–486, 1922. Food and Nutrition Board, and National Research Council. Recommended daily allowances, 10th ed. Washington DC: National Academy of Sciences, 1989. Halprin, K. M., and A. Ohkawara. Glutathione and human pigmentation. Arch. Dermatol. 94:355–357, 1966. Hegyi, J., R. A. Schwartz, and V. Hegyi. Pellagra: Dermatitis, dementia and diarrhea. Int. J. Dermatol. 43:1–5, 2004. Henderson, L. M. Niacin. Annu. Rev. Nutr. 3:289–307, 1983. Hendricks, W. M. Pellagra and pellagralike dermatoses: etiology, differential diagnosis, dermatopathology and treatment. Semin. Dermatol. 10:282–292, 1991. Karthikeyan, K., and D. M. Thappa. Pellagra and skin. Int. J. Dermatol. 41:476–481, 2002. Koh, Y. H., K. H. Choi, and I. K. Park. Effects of niacin deficiency on the levels of soluble proteins and enzyme activities in various tissues of Japanese quail. Int. J. Biochem. Cell. Biol. 30:943–953, 1998. Miller, S. J. Nutritional deficiency and the skin. J. Am. Acad. Dermatol. 21:1–30, 1989. Moore, R. A., T. D. Spies, and Z. K. Cooper. Histopathology of the skin in pellagra. Arch. Dermatol. 46:100–111, 1942. Murray, M. F., M. Langan, and R. R. MacGregor. Increased plasma tryptophan in HIV-infected patients treated with pharmacologic doses of nicotinamide. Nutrition 17:654–656, 2001. Nieves, D. S., and L. A. Goldsmith. Cutaneous changes in nutritional disease. In: Fitzpatrick’s Dermatology in General Medicine, 6th ed. I. M. Freedberg, A. Z. Eisen, K. Wolff, K. F. Austen, L. A. Goldsmith, and S. Katz (eds). New York: McGraw Hill, Inc., 2003, pp. 1399–1411. Pitche, P., K. Kombate, and K. Tchangai-Walla. Prevalence of HIV infection in patients with pellagra and pellagra-like erythemas. Med. Trop. (Mars.) 59:365–367, 1999. Prousky, J. E. Pellagra may be a rare secondary complication of anorexia nervosa: a systematic review of the literature. Altern. Med. Rev. 8:180–185, 2003. Roe, D. A. A Plague of Corn: The Social History of Pellagra. Ithaca, NY: Cornell University Press, 1973. Russell, R. M. Vitamin and trace mineral deficiency and excess. In: Harrison’s Principles of Internal Medicine, 15th ed. E. Braunwald, A. S. Fauci, D. L. Kasper, S. L. Hauser, D. L. Longo, and J. L. Jameson (eds). New York: McGraw-Hill, Inc., 2001, pp. 461–469. Sebrell, W. H. Identification of riboflavin deficiency in human subjects. Fed. Proc. 38:2694, 1979. Sebrell, W. H., Jr. History of pellagra. Fed. Proc. 40:1520–1522, 1981. Spivak, J. L., and D. L. Jackson. Pellagra: an analysis of 18 patients and review of the literature. Johns Hopkins Med. J. 140:295–309, 1977. Stratigos, J. K., and A. Katsambas. Pellagra: a still existing disease. Br. J. Dermatol. 96:99–106, 1977. World Health Organization. Nutrition for Health and Development: A global agenda for combating malnutrition. World Health Organization, 2000. www.who.int//mipfiles/2231/ NHDprogressreport2000.pdf (accessed February 11, 2004).

HYPERMELANOSIS ASSOCIATED WITH GASTROINTESTINAL DISORDERS

Peutz–Jeghers Syndrome Nancy Burton Esterly, Eulalia Baselga, and Beth A. Drolet

Historical Background Peutz–Jeghers syndrome consists of macular mucocutaneous pigmentation and hamartomatous polyps of the gastrointestinal tract. Although delineation of this syndrome has been credited to two physicians of last century, it was actually first described in twin sisters by Sir Jonathan Hutchinson in 1896. Both of these girls had pigmented lesions on the lips. One died of intestinal intussusception at the age of 20 years and the other died of breast cancer at 52 years (Jeghers et al., 1949). Peutz in 1921 reported on seven individuals in three generations of a Dutch family who had pigmented lesions of the lips and oral mucosa as well as intestinal polyposis. He stressed the association of these two findings (Peutz, 1921). Several incomplete descriptions of patients who probably had this disorder were published subsequently. Then in 1949, Jeghers, McKusick, and Katz collected ten of these patients and, in a landmark paper published in the New England Journal of Medicine, they established this disorder as a specific entity distinct from other disorders of abnormal pigmentation and gastrointestinal polyposis.

Fig. 51.11. Multiple pigmented macules on the lower lip of a patient with Peutz–Jeghers syndrome (see also Plate 51.7, pp. 494–495).

Synonyms This syndrome has also been called Peutz–Touraine–Jeghers syndrome, intestinal polyposis type II and periorificial lentiginosis. The last name is an erroneous term because the pigmented lesions are not lentigines.

Epidemiology and Genetics Peutz–Jeghers syndrome has been described throughout the world in individuals of many ethnic groups. It is inherited as a simple mendelian dominant trait with nearly 100% penetrance and variable expressivity (Foley et al., 1988). It has been estimated that 42–64% of cases arise from spontaneous mutations (Salmon and Frieden, 1995). There is no sex predilection (Jeghers et al., 1949). It is said to occur in 1 in 8300–29 000 births (Kitagawa et al., 1995).

Clinical Findings Mucocutaneous pigmented macules are usually the first sign of the disorder and develop in infancy or childhood. Rarely they are present at birth (Jeghers et al., 1949) or develop as late as the seventh decade of life (McKenna et al., 1994). The macules are brown (Fig. 51.11), black or less commonly blueblack. They range in size from 1 mm to 5 mm and have a round, ovoid or irregular configuration and sometimes a stippled appearance (Jeghers et al., 1949). They occur most often on the lips, particularly the lower lip, and on the buccal mucosa (Fig. 51.12). The gingivae, hard palate and, rarely, the tongue are also sites of predilection. The pigmented macules developing on the cutaneous surfaces are most numerous in the perioral area (Fig. 51.13) but the forehead, paranasal, and periorbital skin can also be involved (Kitagawa et al., 1995).

Fig. 51.12. Typical pigmented macules of the oral mucosa in Peutz–Jeghers syndrome (see also Plate 51.8, pp. 494–495).

In these sites the macules may be smaller and darker in color. At times the macules are distributed over the bridge of the nose in a butterfly pattern (Yosowitz et al., 1974). Elsewhere on the body, sites of predilection include the dorsum of the hands and feet, the fingertips (Fig. 51.14) and toe tips, the umbilicus, and the anus (Jeghers et al., 1949; Kitagawa et al., 1995; Lucky, 1988). Longitudinal pigmented bands may be seen in the nails. The cutaneous pigmentation tends to fade in adulthood but the mucosal pigment persists indefinitely. The pigmented macules are considered markers for the more troublesome gastrointestinal polyps. However, both findings can occur independently and the number of skin lesions does not predict the severity of bowel involvement. The intestinal polyps vary in size, distribution, and number but are multiple in 90% of cases (Yosowitz et al., 1974) and can be sessile or pedunculated. They are believed to be hamartomatous and have a specific pathologic pattern consisting of benign glan999

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are often bilateral and multifocal (Burdick and Prior, 1982; Dreyer et al., 1994; Giardiello et al., 1987; Hizawa et al., 1993; Spigelman et al., 1989; Trau et al., 1982; Wilson et al., 1986) and appear at a younger age than expected in the general population (Spigelman et al., 1989).

Histology

Fig. 51.13. Perioral pigmented macules.

Fig. 51.14. Small pigmented macules on the fingertips.

dular structures surrounded by branching bundles of smooth muscle that project towards the surface of the polyp forming a treelike pattern (Hizawa et al., 1993; Perzin and Bridge, 1982). The small bowel, particularly the jejunum and ileum, are the most common sites, but polyps also occur in the stomach and colon. Rarely, polyps have been documented in the gallbladder, kidney, bladder, ureter, and nasopharynx (Salmon and Frieden, 1995). Recurrent bouts of colicky abdominal pain due to obstruction or intussusception, bleeding and chronic iron-deficiency anemia due to fecal blood loss are frequent findings. Although the gastrointestinal polyps are benign, there is a 2–3% risk for malignant transformation. The gastric and duodenal mucosa are the most frequent sites for the development of malignancies (Cordts and Chabot, 1983; Hizawa et al., 1993; Perzin and Bridge, 1982). In addition to gastrointestinal malignancies, which may arise from normal mucosa as well as from polyps, patients with Peutz–Jeghers syndrome are predisposed to develop other types of cancer, particularly breast, lung, and pancreatic cancer, adenoma maligna of the uterine cervix and gonadal tumors such as Sertoli cell testicular tumors and ovarian sex cord tumors with annular tubules. These tumors

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There are only a few reports of the histologic findings in the pigmented lesions. The cutaneous macules are characterized by mild acanthosis, elongation of the rete ridges, increased melanin in the epidermal basal cell layer, particularly at the tips of the rete ridges, as well as in melanophages in the subpapillary dermis (Kitagawa et al., 1995; Uno and Hori, 1987). Trau described increased numbers of melanocytes in the basal cell layer as well (Trau et al., 1982). However, Yamada, using dopa stains (Yamada et al., 1981), found approximately the same number of melanocytes in affected skin as in normal skin. Yamada et al. (1981) and Kitagawa et al. (1995) agree that the number of melanocytes in the oral lesions is not increased. Banse-Kupin and Douglass (1986) found an increased number of melanocytes with long dendrites filled with melanosomes in the acral macules but did not describe the histologic findings in oral mucosal lesions. Ultrastructural studies of the oral mucosal macules demonstrated large, deeply pigmented melanosomes singly dispersed within keratinocytes (Yamada et al., 1981). Melanophages in the dermis contained partially degraded melanosomes. In contrast, pigmented macules on nonmucosal surfaces contained numerous melanosomes and melanin granules within the dendrites of the melanocytes but few within the keratinocytes, suggesting a pigment blockade. The dendrites of the melanocytes were noted to be exceptionally long and branched compared to those in the normal skin (Yamada et al., 1981). Uno and Hori (1987) described similar findings in macules of both the lips and fingers. The melanocytes were filled with large numbers of fully melanized melanosomes and had elongated dendritic processes. Portions of the dendritic processes were found among the upper epidermal keratinocytes supporting the concept of pigment blockade as characteristic of this disorder. Giant melanosomes have never been reported in Peutz–Jeghers syndrome.

Diagnosis The criteria for diagnosis of Peutz–Jeghers syndrome are clinical because there are no pathognomonic laboratory studies. Approximately 50% of patients will have a positive family history (Kitagawa et al., 1995; Yosowitz et al., 1974). The findings of typical pigmented macules in characteristic locations along with gastrointestinal polyps, recurrent intussusceptions, episodic bouts of colicky abdominal pain, intestinal obstruction, gastrointestinal hemorrhage, and anemia are highly suggestive of the diagnosis (Jeghers et al., 1949; McAllister and Richards, 1977; Yosowitz et al., 1974). The diagnosis is confirmed by radiologic and endoscopic studies of the gastrointestinal tract that demonstrate multiple polyps which have

HYPERMELANOSIS ASSOCIATED WITH GASTROINTESTINAL DISORDERS

distinctive histologic features. In the infant, the identifying mucocutaneous pigmentation may be absent, posing a considerable diagnostic challenge (Howell et al., 1981).

Differential Diagnosis Differential diagnosis includes juvenile polyposis, which is frequently manifest by gastrointestinal bleeding and secondary iron-deficiency anemia but rarely by colicky pain. This syndrome is not characterized by associated pigmented macules (Yosowitz et al., 1974). Two acquired conditions that overlap with Peutz–Jeghers syndrome are Cronkhite–Canada syndrome and Laugier–Hunziker syndrome. In the former, there is gastrointestinal polyposis, but the hyperpigmented macules affect the skin and not the mucosa. In addition there is nail dystrophy and alopecia and onset is usually over the age of 50 years (Daniel et al., 1982). Laugier–Hunziker syndrome is characterized by hyperpigmented macules of the oral mucosa and lips, hyperpigmented streaks in the nails and absence of gastrointestinal polyps (Koch et al., 1987).

Pathogenesis The pathogenesis is poorly understood. It has been suggested that the gene in Peutz–Jeghers syndrome controls growth and differentiation of cells in the gastrointestinal tract because of the development of the hamartomas and complicating neoplasms (Spigelman et al., 1989). The mechanism for the development of increased mucocutaneous pigmentation is unclear.

Treatment Most authors advise conservative management for the bouts of abdominal pain including bed rest, mild sedation and nasogastric suction. Spontaneous reduction of intussusception is common in these patients. However, continued bleeding or progression to obstruction or strangulation of the bowel demands immediate surgical intervention. Multiple enterostomies will permit removal of large numbers of polyps. Resection should be reserved for short segments of bowel containing large or very numerous polyps to preserve as much bowel as possible (McAllister and Richards, 1977; Yosowitz et al., 1974). Because the risk of malignancy is relatively low in the hamartomatous polyps, prophylactic resection of bowel is not indicated (Howell et al., 1981). More aggressive management consisting of endoscopic polypectomy of as many polyps as possible, particularly those in the stomach, duodenum, and colon, and routine radiologic surveillance studies have been advocated by some authors (Foley et al., 1988). The increased risk for extraintestinal malignancy mandates routine mammography, breast examination, and gynecologic surveillance (to include pelvic ultrasound and cervical smears) for early detection of breast, cervical, and ovarian neoplasms in women with this syndrome. Testicular and pancreatic tumors in particular must be watched for in affected males (Foley et al., 1988; Giardiello et al., 1987; Hizawa et al., 1993; Spigelman et al., 1989). According to relative risk analysis, patients with Peutz–

Jeghers syndrome have a significantly greater risk for the development of gastrointestinal and extra-intestinal cancer and death at a relatively young age (Giardiello et al., 1987; Salmon and Frieden, 1995). Premature death from complications of the gastrointestinal polyps may also occur.

References Banse-Kupin, L. A., and M. C. Douglass. Localization of PeutzJeghers macules to psoriatic plaques. Arch. Dermatol. 122:679– 683, 1986. Burdick, D., and J. T. Prior. Peutz-Jeghers syndrome. A clinicopathologic study of a large family with a 27-year follow-up. Cancer 50:2139–2146, 1982. Cordts, A. E., and J. R. Chabot. Jejunal carcinoma in a child. J. Pediatr. Surg. 18:180–181, 1983. Daniel, E. S., S. I. Ludwig, and K. J. Lewin. The Cronkhite–Canada syndrome: an analysis of clinical and pathologic features and therapy in 55 patients. Medicine 61:293–309, 1982. Dreyer, L., W. K. Jacyk, and D. J. du Plessis. Bilateral large-cell calcifying Sertoli cell tumor of the testes with Peutz-Jeghers syndrome: a case report. Pediatr. Dermatol. 11:335–337, 1994. Foley, T. R., T. J. McGarrity, and A. B. Abt. Peutz-Jeghers syndrome: A clinicopathologic survey of the “Harrisburg family” with a 49year follow-up. Gastroenterology 95:1535–1540, 1988. Giardiello, F. M., S. B. Welsh, S. R. Hamilton, G. J. A. Offerhaus, A. M. Gittelsohn, S. V. Booker, A. J. Krush, J. H. Yardley, and G. D. Luk. Increased risk of cancer in the Peutz-Jeghers syndrome. N. Engl. J. Med. 116:1511–1514, 1987. Hizawa, K., M. Iida, T. Matsumoto, N. Kohrogi, H. Kinoshita, T. Yao, and M. Fujishima. Cancer in Peutz-Jeghers syndrome. Cancer 72:2777–2781, 1993. Howell, J., K. Pringle, B. Kirschner, and J. D. Burrington. PeutzJeghers polyps causing colocolic intussusception in infancy. J. Pediatr. Surg. 16:82–84, 1981. Hutchinson, J. Pigmented spots on the lips of twins. Arch. Surg. 7:290, 1896. Jeghers, H., V. A. McKusick, and K. A. Katz. Generalized intestinal polyposis and melanin spots of the oral mucosa, lips and digits: A syndrome of diagnostic significance. N. Engl. J. Med. 241:993– 1005,1031–1036, 1949. Kitagawa, S., B. L. Townsend, and A. A. Hebert. Peutz-Jeghers syndrome. [Review]. Dermatol. Clin. 13:127–133, 1995. Koch, S. E., P. E. LeBoit, and R. B. Odom. Laugier-Hunziker syndrome. J. Am. Acad. Dermatol. 16:431–434, 1987. Lucky, A. W. Pigmentary abnormalities in genetic disorders. Dermatol. Clin. 6:193–203, 1988. McAllister, A. J., and K. F. Richards. Peutz-Jeghers syndrome: Experience with twenty patients in five generations. Am. J. Surg. 134: 717–720, 1977. McKenna, K. E., M. Y. Walsh, and D. Burrows. Pigmentation of Peutz-Jeghers syndrome occurring in psoriatic plaques. Dermatology 189:297–300, 1994. Perzin, K. H., and M. F. Bridge. Adenomatous and carcinomatous changes in hamartomatous polyps of the small intestine (PeutzJeghers syndrome): report of a case and review of the literature. Cancer 49:971–983, 1982. Peutz, J. L. A very remarkable case of familial polyposis of mucous membrane of intestinal tract and nasopharynx accompanied by peculiar pigmentations of skin and mucous membrane. Nederl. Maatsch. N. F. 10:134–146, 1921. Salmon, J. K., and I. J. Frieden. Congenital and genetic disorders of hyperpigmentation. Curr. Probl. Dermatol. 7:145–196, 1995. Spigelman, A. D., V. Murday, and R. K. S. Phillips. Cancer and the Peutz-Jeghers syndrome. Gut 30:1588–1590, 1989.

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CHAPTER 51 Trau, H., M. Schewach-Millet, B. K. Fisher, and H. Tsur. Peutz-Jeghers syndrome and bilateral breast carcinoma. Cancer 50:788–792, 1982. Uno, A., and Y. Hori. Disturbance of melanosome transfer in pigmented macules of Peutz-Jeghers syndrome. In: Brown Melanoderma: Biology and Disease of Epidermal Pigment, T. B. Fitzpatrick, M. M. Wick, and K. Toda (eds). New York: Columbia University Press, 1987, pp. 173–278. Wilson, D. M., W. C. Pitts, R. L. Hintz, and R. G. Rosenfeld. Testic-

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ular tumors with Peutz-Jeghers syndrome. Cancer 57:2238–2240, 1986. Yamada, K., A. Matsukawa, Y. Hori, and A. Kukita. Ultrastructural studies on pigmented macules of Peutz-Jeghers syndrome. J. Dermatol. 8:367–377, 1981. Yosowitz, P., R. Hobson, and F. Ruymann. Sporadic Peutz-Jeghers syndrome in early childhood. Am. J. Dis. Child. 128:709–712, 1974.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Acquired and Congenital Dermal Hypermelanosis Sections Sacral Spot of Infancy Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im Nevus of Ota Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im Nevus of Ito Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im Phakomatosis Pigmentovascularis Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im Other Congenital Dermal Melanocytosis Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im Acquired Dermal Melanocytosis Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im Carleton–Biggs Syndrome Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im Acquired Bilateral Nevus of Ota-like Macules (ABNOM) Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im Blue Macules Associated with Progressive Systemic Sclerosis Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im

Sacral Spot of Infancy Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im

Historical Background Professor E. Balz (1885), a German who taught internal medicine at the University of Tokyo, first described blue-black macules on the buttocks of Japanese children and called them Mongolian spots because he believed them to be characteristic of the Asian peoples. The term Mongolian spot persists in common usage although it has no relation to mongolism (Down syndrome). Anthropologic theories have attempted to explain the origin of this spot, and suggest that it was derived from the racial admixture resulting from the Mongolian invasion of Europe (Cordova, 1981). This benign, blue-gray macule is also called the sacral spot or blue-gray macule of infancy.

Clinical Findings Mongolian spots are present at birth or appear soon after. They usually are located over the sacrum (Figs 52.1 and 52.2). The Mongolian spot, a form of dermal melanocytosis, is the most common congenital pigmented lesion. It is present in approximately 80–100% of newborns of Asian and black origin. About 94% of Korean newborns have this spot (Kim and Herr, 1986). They are also seen in about 1 to 15% of Caucasians (Hori and Takayama, 1988; Leung, 1988). The incidence is slightly higher in males than females. Lesions vary in size from 1–2 cm to extensive areas covering most of the

sacrum, buttocks, and back. In the majority of individuals they occupy less than 5% of the body surface area. Lesions are completely macular and usually have indistinct borders. Their color varies from gray to grayish blue to grayish black. Such variability may be noted in a single patient. The most common location is the sacrococcygeal area followed by the lumbar area, buttock, and back. Less common locations include the thorax and abdomen, leg, arm, and shoulder (Leung, 1988) (Fig. 52.3). Rarely, Mongolian spots occur in the scalp area (Leung and Kao, 1999). Occasionally, in extrasacral regions they are termed aberrant Mongolian spots or macular blue nevus. These macules are usually darker than usual lumbosacral spots and sometimes increase in intensity of color with age. These macules should be called macular type of blue nevus rather than “persistent” Mongolian spots (Hori and Takayama, 1988). Some of the macular types of blue nevi have prominent hair growth within them. Ikeda (1981) labeled macular type of blue nevus as the plaque type of blue nevus. White halo may develop around the sacral spot (Bart and Olson, 1991). The natural history of most sacral spots is the intensification of color in the first year of life. They reach maximum size by 2 years of age, followed by gradual disappearance during the first 6–7 years of life. By 10 years of age almost all Mongolian spots have disappeared. The frequency of persistent lesions has been estimated to be 3–4% (Kikuchi, 1982; Leung, 1988).

Associated Disorders In a condition as common as the Mongolian spot, one must 1003

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52.1

52.2

Figs. 52.1 and 52.2. Mongolian spots at sacral region (see also Plate 52.1, pp. 494–495).

be cautious in labeling associated abnormalities as more than a coincidence. Nevertheless, a true association appears to exist between extensive Mongolian spots and metabolic disorders. An abnormality of sphingolipid metabolism, GM1 type 1 gangliosidosis, is caused by a deficiency of the enzyme bgalactosidase. Severe developmental delay, facial and peripheral edema, unusual facial features, hepatosplenomegaly, and cherry-red spots of the retina have been observed in these patients. The skin findings in those cases consist of widespread multiple confluent and individual bluish macules varying in size from a few millimeters to several centimeters. The diagnosis is made by demonstration of absence of b-galactosidase activity in peripheral leukocytes and/or skin fibroblasts (Beratis et al., 1989; Selsor and Lesher, 1989; Weissbluth et al., 1981). Grant et al. (1998) investigated a 9-month-old black girl in whom evaluation for extensive Mongolian spots led to an early diagnosis of Hurler syndrome. Hurler syndrome is recognized as the most common and severe form of the mucopolysaccharidoses in infancy. The molecular basis for Hurler syndrome involves a series of specific mutations on the short arm of chromosome 4. These mutations cause an absence of the lysosomal glycosidase a-L-iduronidase, which

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is required in the sequential process of dismantling glycosaminoglycans. The partially degraded glycosaminoglycans heparan sulfate and dermatan sulfate are deposited in various organs, producing a wide array of clinical manifestations, including coarse facial features, hypertrichosis, cloudy corneas, progressive mental retardation, and hepatosplenomegaly (Grant et al., 1998). A clinical correlation was suggested between extensive Mongolian spots and Hunter syndrome (Ochiai et al., 2003). Hunter syndrome is an Xlinked disorder characterized by a deficiency of lysosomal iduronate-2-sulfatase, and resultant accumulation of dermatan and heparin sulfate. Both Hurler and Hunter syndromes are progressive in nature, and thus the importance of early diagnosis should be emphasized to prevent progressive, irreversible organ damage. Extensive Mongolian spots is one of the cutaneous findings that provides the clue (Grant et al., 1998; Ochiai et al., 2003). A meningeal tumor of the spine, which was found to be a benign melanocytoma, was described in an adult patient with a persistent Mongolian spot on the back. Another case of neurocutaneous melanosis has been reported in association with extensive slate-gray lesions of dermal melanocytosis and

ACQUIRED AND CONGENITAL DERMAL HYPERMELANOSIS

Fig. 52.3. Aberrant Mongolian spot (macular type of blue nevus) (see also Plate 52.2, pp. 494–495).

a nevus flammeus (Novotny and Urich, 1986). A Hispanic infant with lesions clinically identical to Mongolian spots was found to have a myelomeningocele as well as hydrocephalus. Biopsy of the skin, however, demonstrated an increase in epidermal as well as dermal melanocytes which differed from the usual histology of Mongolian spots (Schwartz et al., 1986). Several papers reported an association of dermal melanocytosis with cleft lip (Bart and Olson, 1991; Inoue et al., 1982; Kurata et al., 1989; Mori et al., 1975) and it is called a “cleft lip-Mongolian spot” (Igawa et al., 1994). A patient with an adult onset sacral spot was reported as “adult onset Mongolian spot” (Carmichael et al., 1993).

Histopathology The etiology of this birthmark is unknown. During fetal life, dermal melanocytes migrate from the neural crest to their designated site at the dermoepidermal junction. Dermal melanocytes are present in the dermis of embryos beginning in

the tenth week, and between the eleventh and fourteenth week these migrate to the epidermis. They gradually disappear from the dermis after the twentieth week. A sacral spot of infancy is thought to be due to a failure of migration of dermal melanocytes to the epidermis and their delayed disappearance from the dermis (Leung et al., 1988). A Japanese anatomist, Morooka, examined fetuses and noted that dermal melanocytes typical of the spots were present at three months of fetal life. Lesions were recognizable macroscopically by seven months gestation (Kikuchi, 1982). Histopathologically the lesions consist of dendritic melanocytes containing melanin granules lying scattered between the collagen bundles in the lower one half to two thirds of the dermis. By electron microscopy these melanocytes contain numerous melanized melanosomes. Also, in most white children, dermal melanocytes can be found histologically in the lumbosacral region but they possess incompletely melanized melanosomes and are not visible clinically. The regression of Mongolian spots is a unique event not seen in other forms of dermal melanocytosis. The mechanism for their disappearance is poorly understood. The ultrastructural examinations show an extracellular sheath enclosing dermal melanocytes. It is less well developed in the Mongolian spot than in the nevus of Ito (Okawa et al., 1979). Inoue et al. (1982) noted that persistent Mongolian spots were enveloped in a fibrous sheath, which is not present in regressing lesions. Halo Mongolian spots have been described rarely. Histologic examination shows a lack of inflammatory infiltrate (Bart and Olson, 1991). Very extensive Mongolian spots may regress more slowly and portions may never disappear completely. Persistent Mongolian spots, also called dermal melanocytic hamartoma, occur in approximately 3–4% of Japanese adults. They are probably analogous to the nevus of Ota or Ito. Persistent lesions are also seen in the nevus pigmentovascularis.

Differential Diagnosis and Management The differential diagnosis of Mongolian spots includes traumatic ecchymosis. Occasionally parents of affected infants have erroneously been accused of child abuse (Smialek, 1980; Dungy, 1982). Other diagnostic considerations include nevi of Ota and Ito as well as large congenital melanocytic nevi. Nevi of Ota and Ito appear from birth to adolescence and usually persist in life. The histology of the Mongolian spot is similar to that of nevi of Ota and Ito. They show wavy, bipolar melanocytes lying parallel to the surface and scattered among the collagen bundles. The melanocytes are located predominantly in the upper dermis in nevi of Ota and Ito, but in the lower dermis in Mongolian spots. Mongolian spots are almost always self-limited and of no clinical importance. No treatment is necessary.

References Balz, E. Die korperlichen Eigneschaften der Japaner. Mitteheil d. deutsch Gesell. f. Natur-u Volkerkunde Ostasiens Bd. 4:H. 32, 1885 (as cited in Morooka, 1931).

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CHAPTER 52 Bart, B. J., and C. L. Olson. Congenital halo mongolian spot. J. Am. Acad. Dermatol. 6:1082–1083, 1991. Beratis, N. G., A. Varvarigou-Frimas, S. Beratis, and S. L. Sklower. Angiokeratoma corporis diffusum in GM1 type gangliosidosis, type 1. Clin. Genet. 36:59–64, 1989. Carmichael, A. J., C. Y. Tan, and S. M. Abraham. Adult onset Mongolian spot. Clin. Exp. Dermatol. 18:72–74, 1993. Cordova, A. The mongolian spot. Clin. Pediatr. (Phila.) 20:714–719, 1981. Dungy, C. I. Mongolian spots, day care centers, and child abuse. Pediatrics 69:672, 1982. Grant, B. P., J. S. Beard, F. Castro, M. C. Guiglia, and B. D. Hall. Extensive Mongolian spots in an infant with Hurler syndrome. Arch. Dermatol. 134:108–109, 1998. Hori, Y., and O. Takayama. Circumscribed dermal melanoses: Classification and histologic features. Dermatol. Clin. 6:315–326, 1988. Igawa, H. H., T. Ohura, T. Sugihara, T. Ishikawa, and M. Kumakiri. Cleft lip Mongolian spot: Mongolian spot associated with cleft lip. J. Am. Acad. Dermatol. 30:566–569, 1994. Ikeda, S., A. Makita, and K. Tajima. Clinical and histologic study of blue nevus. In: Biology and Diseases of Dermal Pigmentation, T. B. Fitzpatrick, A. Kukita, F. Morikawa, M. Seiji, A. J. Sober, and K. Toda (eds). Tokyo: University of Tokyo Press, 1981, pp. 95– 106. Inoue, S., I. Kukuchi, and T. Ono. Dermal melanocytosis associated with cleft lip. Arch. Dermatol. 118:443–444, 1982. Kikuchi, I. What is a mongolian spot? Int. J. Dermatol. 21:131–133, 1982. Kim, J. H., and H. Herr. A statistical study of mongolian spot. Korean J. Dermatol. 24:373–379, 1986. Kurata, S., Y. Ohara, S. Itami, Y. Inoue, H. Ichikawa, and S. Takayasu. Mongolian spots associated with cleft lip. Br. J. Plast. Surg. 42:625–627, 1989. Leung, A. K. C. Mongolian spots in Chinese children. Int. J. Dermatol. 27:106–108, 1988. Leung, A. K. C., R. B. Lowry, I. Mitchell, S. Martin, and D. M. Cooper. Klippel-Trenaunay and Sturge-Weber syndrome with extensive Mongolian spots, hypoplastic larynx and subglottic stenosis. Clin. Exp. Dermatol. 13:128–132, 1988. Leung, A. K. C., and C. P. Kao. Extensive Mongolian spots with involvement of the scalp. Pediatr. Dermatol. 16:371–372, 1999. Mori, T., T. Onizuka, and T. Akagawa. Cleft lip nevus. Jpn. J. Plast. Reconstr. Surg. 18:526–527, 1975. Novotny, E. J., and H. Urich. The coincidence of neurocutaneous melanosis and encephalofacial angiomatosis. Clin. Neuropathol. 5:246–251, 1986. Ochiai, T., K. Ito, T. Okada, M. Chin, H. Shichino, and H. Mugishima. Significance of extensive Mongolian spots in Hunter’s syndrome. Br. J. Dermatol. 148:1173–1178, 2003. Okawa, Y., R. Yokota, and A. Yamauchi. On the extracellular sheath of dermal melanocytes in nevus fuscocaeruleus acromiodeltoideus (Ito) and Mongolian spot: an ultrastructural study. J. Invest. Dermatol. 73:224–230, 1979. Schwartz, R. A., N. Cohen-Addad, M. W. Lambert, and W. C. Lambert. Congenital melanocytosis with myelomeningocele and hydrocephalus. Cutis 37:37–39, 1986. Selsor, L. C., and J. L. Lesher. Hyperpigmented macules and patches in a patient with GM1 type gangliosidosis. J. Am. Acad. Dermatol. 20:878–882, 1989. Smialek, J. E. Significance of Mongolian spots. J Pediatr. 97:504–505, 1980. Weissbluth, M., N. B. Esterly, and W. A. Caro. Report of an infant with GM1 type gangliosidosis type 1 and extensive and unusual mongolian spots. Br. J. Dermatol. 104:195–200, 1981.

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Nevus of Ota Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im

Historical Background Nevus of Ota is a form of benign melanocytosis located over the first (ophthalmic) and second (maxillary) branches of the trigeminal nerve. It was first described by Ota and Tanino (1939) as naevus fuscocaeruleus ophthalmomaxillaris. Later Fitzpatrick et al. (1956) suggested the term oculodermal melanocytosis. The term nevus of Ota is commonly used to refer to this anomaly.

Epidemiology Most cases of nevus of Ota have been reported in Asia including Japan and Korea. Nevus of Ota occurs in approximately 1 out of 500 people (Hidano et al., 1967). Eighty percent of all reported cases are of females (Geronemus, 1992; Hidano et al., 1967; McDonald and Georgouras, 1992). The ratio of males to females in Korea is 1:2.67 (Lee, 1986). In Japan, 0.4–1% of all patients seen in dermatologic clinics have nevus of Ota (Balmaceda et al., 1993; Hidano et al., 1967). The general prevalence of nevus of Ota in Korea was reported as 0.03% in adolescents. In dermatology clinics in Korea the prevalence was reported as 0.24% (Lee, 1986). It also occurs in Chinese, East Indians, and black and white people (Watanabe and Takahashi, 1994). Nevus of Ota is typically congenital but can be acquired (Balmaceda et al., 1993; Whitmore et al., 1991). Inherited cases are rare. To date only five familial cases of nevus of Ota have been reported (Trese et al., 1981).

Clinical Findings There is a bimodal age of onset: the first (about 50–60% of all cases) in early childhood before the age of 1 year, with the majority present at birth; and the second (40 to 50%) around puberty (Hidano et al., 1967; Lynn et al., 1993; RuizVillaverde et al., 2003). The remainder arise during the first four decades of life (Hidano et al., 1967; Whitmore et al., 1991). The female to male ratio is 4.8:1 in Japan. Some have speculated that a hormonal influence on melanogenesis may play a role in unmasking the nevus and may contribute to the female predominance and bimodal peak age of onset. The intensity of pigmentation may be influenced by fatigue, menstruation, insomnia, and weather (Hidano et al., 1967). In approximately 5% of cases the onset of the nevus is noted after minor trauma such as a contusion or sunburn. However, the relationship between these events and dermal melanocytosis remains unclear (Kopf and Weidman, 1962; Okawa et al., 1979; Whitmore et al., 1991). Clinically an ill-defined, brown, blue-gray or blue-black patchy hyperpigmentation intermingled with small flat brown spots is observed in the distribution of the maxillary and ophthalmic branches of the fifth cranial nerve. Rarely, it involves the third branch of the trigeminal nerve. In 5% of cases, the lesions are bilateral (Hidano, 1985; Hori et al., 1984; Reinke et al., 1974) although bilateral involvement occurs in 5–13%

ACQUIRED AND CONGENITAL DERMAL HYPERMELANOSIS

Fig. 52.5. Ocular hyperpigmentation in a patient with nevus of Ota.

Fig. 52.4. Slate-blue hyperpigmentation of the cheek.

of affected individuals (Kopf and Weidman, 1962). There are individuals with a unilateral nevus of Ito and a bilateral nevus of Ota (Furukawa et al., 1970; Hidano et al., 1965). The nevus of Ito differs from the nevus of Ota only by its distribution. The nevus of Ito affects the supraclavicular, scapular, and deltoid regions. It may occur alone or in association with an ipsilateral or bilateral nevus of Ota (Hidano et al., 1965; Mishima and Mevorah, 1961). The pigmentation is usually poorly demarcated, macular or slight raised and mottled with colors that vary from black to purple, blue-black, slate-blue (Fig. 52.4), purple-brown, or brown (Fitzpatrick et al., 1987; Hidano et al., 1967; Hori and Takayama, 1988; Liesegang, 1994). Slightly raised or nodular areas identical to blue nevi may be present (Dorsey and Montgomery, 1954; Kopf and Weidman, 1962; McDonald and Georgouras, 1992). Focal cellular blue nevi that enlarge can be mistaken for a malignant melanoma (Kersting and Caro, 1956; Yeschua et al., 1975). There are no abnormalities of vascularization or hair (Kopf and Weidman, 1962; Okawa et al., 1979). Ocular involvement in the nevus of Ota occurs approximately in two-thirds of patients (Patel et al., 1998). Hyper-

pigmentation may occur on the sclera (Fig. 52.5), cornea, iris, optic nerve, fundus, extraocular muscles, retrobulbar fat, and periosteum (Balmaceda et al., 1993; Bhattacharya et al., 1973; Hidano et al., 1967; Hori and Takayama, 1988; Liesegang, 1994; McDonald and Georgouras, 1992). The melanocytic infiltration of the iris may manifest as heterochromia iridis. Similar pigment changes can rarely be seen in the mucosal membranes of the head and neck such as pharynx, nasal mucosa, buccal mucosa, tympanic membrane, and hard palate (Balmaceda et al., 1993; Bhattacharya et al., 1973; Hidano et al., 1967; Hori and Takayama, 1988; Liesegang, 1994; McDonald and Georgouras, 1992). The lesion can be found on the eustachian tube, the tympanic membrane (55%), nasal mucosa (28%), pharynx (24%), or on the palate (18%) (Hidano et al., 1967). Pigmented melanocytes also may be found in underlying periosteum, bone, dura mater, and cerebral cortex, the orbicularis and temporalis muscles, or maxillary sinus. Many patients report fluctuation of pigment intensity associated with fatigue, menstruation, insomnia, and weather changes (Hidano et al., 1967; Yoshida, 1952). The brown spots are caused by epidermal hyperpigmentation. The blue-black or slate-gray macules are caused by the presence of dermal melanocytes. Spontaneous regression does not occur. The nevus of Ota has been classified by the extent of the anomaly into four types (Tanino, 1939). Types I–III are unilateral. Type IA or mild orbital type is characterized by light brown or slate spotted pigmentation limited to the upper and lower eyelids. Type IB (mild zygomatic) has discrete brownish purple spots limited to the zygomatic region. Type II (moderate) exhibits deep slate- to brown-purple, relatively dense spotted pigmentation on the eyelids, zygomatic region, and base of the nose. Type III (extensive) is characterized by deep blue to bluish-purple, densely spotted, or almost diffuse pigmentation on the eyelids, zygomatic region, base of the nose and nasal ala, forehead, external ear, post auricular region, and anterior scalp. Type IV (bilateral) involves both sides of the face. Extracutaneous pigmentation is common and affects 1007

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the conjunctiva, sclera, and tympanic membrane. Less frequently, the nasal mucosa, palate, pharynx, cornea, iris, uveal tract, and fundus are affected. There are two main complications associated with nevus of Ota: ocular complications and malignancy. The nevus of Ota can develop a number of ocular complications such as melanosis oculi (Teekhasaence et al., 1990), ocular hypertension (Kitagawa et al., 2003), and glaucoma of the involved eye (Kopf and Weidman, 1962; Sang et al., 1977). Abnormalities of ocular pigmentation occur in 49–65% of patients with nevus of Ota (Hidano et al., 1967; Kopf and Weidman, 1962; Tanino, 1939). It is almost always unilateral. Only one case of bilateral ocular involvement has been reported (Dompmartin et al., 1989). In cases of ocular involvement, ipsilateral sclera is always involved, most commonly in the temporal or superior quadrants (Cowan and Balistocky, 1962). Other sites of ocular involvement are the iris (50%), the conjunctiva (40%), the choroid (18%), and the optic nerve and its meningeal sheath (5%) (Cowan and Balistocky, 1962; Tanino, 1940). Melanocytosis of the cornea, the ciliary body, extraocular muscles, orbital fat, and periorbital and orbital bones has been described. Ocular hypertension has been found in patients with nevus of Ota and it is thought to be due to goniodysgenesis, abnormally thick cornea, or infiltration of melanocytic cells into the trabecular meshwork (Kitagawa et al., 2003; Teekhasaenee et al., 1990). Ocular melanocytosis with occlusion of the canal of Schlemm that results in secondary glaucoma has been observed in seven patients (Reinke et al., 1974; Teekhasaenee et al., 1990). Melanoma associated with nevus of Ota was first described by Hulke in 1861. In general, nevus of Ota does not often give rise to malignant melanoma. However, malignant transformation has occurred in 4.6% of 670 cases of nevus of Ota reported in the literature (Miller, 1994; Shaffer et al., 1992). The median age at which the melanoma was detected was 51 years (range 15–72). Melanoma occurs more frequently in white people. Of the 32 reported cases, 28 occurred in white patients, 3 in Japanese and 1 in a black patient. Dutton and colleagues (1984) estimated the incidence of malignancy to be up to 25% for white patients, 1% for black patients, and 0.5% for Asians. The most common site for melanoma is the choroid, followed by the uveal tract (Bordon et al., 1995; Dutton et al., 1984; Hagler and Brown, 1966; Haim et al., 1982; Hartmann et al., 1989; Jay, 1965; Liesegang, 1994; Speakman and Phillips, 1973). Seventeen of 39 individuals had a choroidal melanoma (Albert and Scheie, 1963; Croxatto et al., 1981; Frezzotti et al., 1968; Gonder et al., 1981, 1982; Halasa, 1970; Hulke, 1861; Makley and King, 1967; Mohandessan et al., 1979; Nik et al., 1982; Ray and Schaeffer, 1967; Singh et al., 1988; Ticho et al., 1989; Velazquez and Jones, 1983; Yamamoto, 1969). Conjunctival melanoma has not been reported (Liesegang, 1994). Melanoma can also arise in the skin (Croxatto et al., 1981; Dompmartin et al., 1989; Hartmann et al., 1989; Kopf and Bart, 1982).

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Fig. 52.6. Bipolar melanocytes in the dermis cause this abnormality.

A few cases of intracranial melanoma have been reported (Balmaceda et al., 1993; Horsey et al., 1980; Theunissen et al., 1993). Primary melanoma developed in the central nervous system in 23% of affected individuals (Botticelli et al., 1983; Enriquez et al., 1972; Hartmann et al., 1989; Horsey et al., 1980; Sang et al., 1977; Shaffer et al., 1992; Willis, 1967; Yanoff and Zimmerman, 1967). These have involved the dura (Horsey et al., 1980), Meckel’s cave (Botticelli et al., 1983), pineal gland (Enriquez et al., 1973), cortical surfaces (Sang et al., 1977; Willis, 1967), and the optic chiasm (Kojima et al., 1959). Nevus of Ota has also been reported with an ipsilateral plexiform neuroma (Gupta et al., 1986). The cause of the melanoma has not been determined. A causal relationship between nevus of Ota and melanoma has not yet been established.

Histology The epidermis is typically normal. There is a dense population of bipolar melanocytes in the dermis (Hirayama and Suzuki, 1991; Hori et al., 1982; Ishibashi et al., 1990) (Fig. 52.6). These melanocytes are weakly dopa positive, an indication that they retain some capacity to synthesize melanin. On electron microscopy the dermal melanocytes are noted to contain many singly dispersed melanosomes 0.4–0.8 mm in size (Hori et al., 1982). Occasionally giant melanosomes may be seen (Ticho et al., 1989). Most melanosomes are fully melanized. Partially melanized melanosomes are observed rarely if at all. The melanosomes in melanocytes in the basal layer of the epidermis overlying the nevus are small, 0.2–0.3 mm in longest dimension. A lamina lucida and lamina densa can be found around some dermal melanocytes. The dermal melanocytes are surrounded by an extracellular sheath of various thicknesses. The extracellular sheaths are PAS negative and are composed of fine filaments, 2–4 nm in diameter and have a feltlike structure. The thickness of extracellular sheaths differs according to the age of the patient and thickens as the patient gets older. This pattern is similar to that in Mongolian spots

ACQUIRED AND CONGENITAL DERMAL HYPERMELANOSIS

A

B

Fig. 52.7. Nevus of Ota showing intense blush color (A). Total disappearance of the lesion after laser treatment (B) (see also Plate 52.3A and B, pp. 494–495).

except that in nevus of Ota it tends to be more superficial (Maize and Ackerman, 1987; Lever and Schaumburg-Lever, 1983). Merkel cells have also been reported in these lesions (Ono et al., 1984). Although most of the melanocytes lie in the upper third of the reticular dermis, melanocytes also can be found in the papillary dermis and the subcutis tissue around adnexa (Hidano et al., 1965). Hirayama and Suzuki (1991) classified nevus of Ota into five types according to the distribution of dermal melanocytes. These are the superficial (type S), superficial dominant (type SD), diffuse (type Di), deep dominant (type DD), and deep (type De). The histologic types correlated well with the visible color of the nevus. The more brownish lesions represented type S or type SD. The most bluish lesions were type Di, DD or De. Melanophages are rarely observed. The slightly raised or infiltrated areas within the nevus of Ota and Ito show a larger number of elongated, dendritic melanocytes than do non-infiltrated areas. These areas mimic the pathology of a common or cellular blue nevus (Kopf and Weidman,

1962). In the oral mucosa, a more dense concentration of melanocytes are found in the dermis (Mishima and Mevorah, 1961). In ocular tissue, melanocytes may be seen in the episclera, deeper layers of scleral stroma, conjunctiva, iris, and choroid (Fitzpatrick et al., 1956).

Associated Disorders Nevus of Ota has been associated with other disorders such as vascular malformations and/or hemiparesis (Ono et al., 1984). This syndrome is classified as nevus pigmentovascularis (Benson and Rennie, 1992; Reed and Sugarman, 1974). Other reported associations include the Sturge–Weber syndrome (Noreiga-Sanchez et al., 1972), neurosensory hearing loss (Reed and Sugarman, 1974; Alvarez-Cuesta et al., 2002), spinocerebellar degeneration (Whyte and Dekaban, 1976), the Klippel–Trenaunay–Weber syndrome (Furukawa et al., 1970), and intracranial vascular malformations (Keinke et al., 1974). A number of other congenital anomalies including multiple gastrointestinal hemangiomas, intracranial arteriovenous mal-

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formation, enlargement of the posterior cranial fossa, von Recklinghausen disease, ipsilateral sensorineural deafness, Down syndrome with unilateral congenital cataract and ipsilateral hemiatrophy of the upper extremity, and familial cerebellar degeneration have been reported in association with the nevus of Ota. Whether these associations are causally related to the nevus of Ota or are incidental findings is not clear.

Pathogenesis Nevus of Ota is thought to represent disorder of neural crest migration (Benson and Rennie, 1992). Melanoblasts normally arise from the neural crest and migrate to the skin, leptomeninges, ocular structures and the internal ear. Deafness has been associated with nevus of Ota (Reed and Sugarman, 1974). Some cells ascend to the dermal–epidermal junction to become branched melanocytes producing melanin. In the nevus of Ota, blue nevus and Mongolian spots, melanocytes are thought to be aberrant and become arrested during migration, remaining in the dermis. This results in a persistent discoloration and cosmetic disfigurement (Maize and Ackerman, 1987; Zimmerman and Becker, 1959).

Treatment The lesion of nevus of Ota persists throughout life. There are no reports of spontaneous regression. The Mongolian spot typically disappears during childhood. With time, the nevus of Ota/Ito may increase in intensity, particularly within the first year of life. Rarely, the lesions may become less apparent during childhood. If ocular pigmentation is present, patients should be followed periodically by an ophthalmologist. It is unnecessary to remove prophylactically stable cutaneous lesions (Kopf and Weidman, 1962). Several aggressive therapies have been used in the past such as surgical excision with or without skin grafting, dermabrasion, and cryotherapy. Surgical treatment causes scarring, but some surgeons have demonstrated good results with a microsurgical technique (Furukawa et al., 1970). Cryotherapy can be effective but success depends on the site of the lesion. It is not reliable and may cause atrophy or scarring if it is applied excessively. Lasers in recent years have also been used to treat successfully the nevus of Ota (Apfelberg et al., 1981). The monochromatic, focused light beam from Q-switched ruby or Q-switched neodymium:yttrium aluminum garnet (Nd:YAG) lasers have been shown to interact selectively with melanin killing the melanocytes (Fig. 52.7). Several reports have proved the safety and efficacy of laser treatments (Geronemus, 1992; Lowe et al., 1993; Taylor et al., 1994; Watanabe and Takahashi, 1994). The treatment of choice is laser therapy with the Q-switched ruby (Charles et al., 1994), Q-switched Nd:YAG, or Q-switched alexandrite layers (al Deeb and Bahou, 1993; Geronemus, 1992; Sawada et al., 1990; Watanabe and Takahashi, 1994; Zimmerman and Becker, 1959). These are helpful in reducing the intensity of pigment with minimal risk of scarring. Watanabe treated a nevus of Ota on 114 patients with good results. Of the 35 patients who 1010

received four or five treatments, 33 had an excellent response (lightening of 80% or more) and 2 had a good response (lightening of 40–80%). The laser treatment of nevus of Ota in children can achieve a good result in fewer sessions and at a lower complication rate than later treatment (Kono et al., 2003). The risk of recurrence after laser treatment is estimated to be between 0.6% and 1.2% (Chan et al., 2001). Dermabrasion can be helpful in some cases. Because most nevi are macular, many patients feel opaque covering agents yield cosmetically acceptable results.

References Albert, D. M., and H. G. Scheie. Naevus of Ota with malignant melanoma of the choroid: Report of a case. Arch. Ophthalmol. 69:774–777, 1963. al Deeb, S. M., and Y. Bahou. A new neurocutaneous syndrome possibly related to Ota’s nevus. J. Neurol. Sci. 118:92–96, 1993. Alvarez-Cuesta, C. C., C. Raya-Aguado, F. Vazquez-Lopez, P. B. Carcia, and N. Perez-Olova. Nevus of Ota associated with ipsilateral deafness. J. Am. Acad. Dermatol. 47:S257–S259, 2002. Apfelberg, D. B., M. R. Moser, H. Lash, and J. Rivers. The argon laser for cutaneous lesions. J. Am. Med. Assoc. 245:2073–2075, 1981. Balmaceda, C. M., M. R. Fetell, J. Powers, J. L. O’Brien, and E. H. Housepian. Nevus of Ota and leptomeningeal melanocytic lesions. Neurology 43:381–386, 1993. Benson, M. T., and I. G. Rennie. Hemi-naevus of Ota: perturbation of neural crest differentiation as a likely mechanism. Graefes Arch. Klin. Exp. Ophthalmol. 230:226–229, 1992. Bhattacharya, S. K., H. S. Girgla, and G. Singh. Nevus of Ota. Int. J. Dermatol. 12:344–347, 1973. Bordon, A. F., M. L. Wray, R. Belfort, I. W. McLean, and M. Burnier. Choroidal malignant melanoma in association with oculodermal melanocytosis in a black patient. Br. J. Ophthalmol. 79:191–192, 1995. Botticelli, A. R., M. Villani, P. Angiori, and L. Peserice. Meningeal melanocytoma of Meckel’s cave associated with ipsilateral Ota nevus. Cancer 51:2304–2310, 1983. Chan, H. H., L. K. Lam, D. S. Wong, R. S. Leung, S. Y. Ying, C. F. Lai, W. S. Ho, and J. K. Chua. Nevus of Ota: a new classification based on the response to laser treatment. Lasers Surg. Med. 28:267–272, 2001. Charles, R. T., J. F. Thomas, G. William, and A. Rox. Treatment of nevus of Ota by Q-switched ruby laser. J. Am. Acad. Dermatol. 30:743–751, 1994. Cowan, T. H., and M. Balistocky. The nevus of Ota or oculodermal melanocytosis. The ocular changes. Arch. Ophthalmol. 65:483– 492, 1962. Croxatto, J. W., D. E. Charles, and E. S. Mallran. Neurofibromatosis associated with nevus of Ota and choroidal melanoma. Am. J. Ophthalmol. 92:578–580, 1981. Dompmartin, A., D. Leroy, D. Labbe, J. B. Letessier, and J. C. Mandard. Dermal malignant melanoma developing from a nevus of Ota. Int. J. Dermatol. 28:535–536, 1989. Dorsey, C. S., and H. Montgomery. Blue nevus and its distinction from Mongolian spot and the nevus of Ota. J. Invest. Dermatol. 22:225–236, 1954. Dutton, J. J., R. L. Anderson, R. L. Schelper, J. J. Purcell, and D. T. Tse. Orbital malignant melanoma and oculodermal melanocytosis: Report of two cases and review of the literature. Ophthalmology 91:497–507, 1984. Enriquez, R., B. Egbert, and J. Bullock. Primary malignant melanoma of central nervous system: Pineal involvement in a patient with nevus of Ota and multiple pigmented skin nevi. Arch. Pathol. 95:392–395, 1972.

ACQUIRED AND CONGENITAL DERMAL HYPERMELANOSIS Enriquez, R., B. Egbert, and J. Bullock. Primary malignant melanoma of the central nervous system: pineal involvement in a patient with nevus of Ota and multiple pigmented skin nevi. Arch. Pathol. 95:392–395, 1973. Fitzpatrick, T. B., R. Zeller, A. Kukita, and H. Kitamura. Ocular and dermal melanocytosis. Arch. Ophthalmol. 56:830–832, 1956. Fitzpatrick, T. B., A. Z. Eisen, K. Wolff, I. M. Freedburg, and K. F. Austen. Dermatology in General Medicine, 3rd ed. New York: McGraw Hill, Inc., 1987, pp. 979–981. Frezzotti, R., R. Guerra, G. P. Dragoni, and P. Bonamsi. Malignant melanoma of the choroid in a case of naevus of Ota. Br. J. Ophthalmol. 52:922–924, 1968. Furukawa, T., A. Igata, Y. Toyokura, and S. Ikeda. Sturge-Weber and Klippel-Trenaunay syndrome with nevus of Ota and Ito. Arch. Dermatol. 102:640–645, 1970. Geronemus, R. G. Q-switched ruby laser therapy of nevus of Ota. Arch. Dermatol. 128:1618–1622, 1992. Gonder, J. R., J. A. Shields, and D. M. Albert. Malignant melanoma of the choroid associated with oculodermal melanocytosis. Ophthalmology 88:372–376, 1981. Gonder, J. R., E. B. Shields, D. M. Albert, J. J. Augsburger, and P. T. Lavin. Uveal malignant melanoma associated with ocular and oculodermal melanocytosis. Ophthalmology 89:953–959, 1982. Gupta, A., J. Ram, and I. S. Jain. Nevus of Ota associated with neurofibromatosis. Ann. Ophthalmol. 18:154–155, 1986. Hagler, W. S., and C. C. Brown. Malignant melanoma of the orbit arising in the nevus of Ota. Trans. Am. Acad. Ophthalmol. Otolaryngol. 70:817–822, 1966. Haim, T., E. Meyer, H. Kerner, and S. Zonis. Oculodermal melanocytosis and orbital malignant melanoma. Ann. Ophthalmol. 14:1132– 1136, 1982. Halasa, A. Malignant melanoma in a case of bilateral nevus of Ota. Arch. Ophthalmol. 84:176–178, 1970. Hartmann, L. C., G. F. Oliver, R. K. Winkleman, T. V. Colby, T. M. Sundt, and B. P. O’Neill. Blue nevus and nevus of Ota associated with dural melanoma. Cancer 64:182–186, 1989. Hidano, A. Acquired, bilateral nevus of Ota-like macules [letter]. J. Am. Acad. Dermatol. 12:368–369, 1985. Hidano, A., H. Kajima, and Y. Endo. Bilateral nevus of Ota associated with nevus of Ito. Arch. Dermatol. 91:357, 1965. Hidano, A., H. Kajima, S. Ikeda, H. Mizutani, H. Miyasato, and M. Niimura. Natural history of nevus of Ota. Arch. Dermatol. 95:187–195, 1967. Hirayama, T., and T. Suzuki. A new classification of Ota’s nevus based on histopathological features. Dermatologica 183:169–172, 1991. Hori, Y., and O. Takayama. Circumscribed dermal melanoses: Classification and histologic features. Dermatol. Clin. 6:315–326, 1988. Hori, Y., K. Oohara, and M. Niimura. Electron microscopy: ultrastructural observations of the extracellular sheath of dermal melanocytes in the nevus of Ota. Am. J. Dermatopathol. 4:245–251, 1982. Hori, Y., M. Kawashima, and K. Oohara. Acquired, bilateral nevus of Ota-like macules. J. Am. Acad. Dermatol. 10:961–964, 1984. Horsey, W. J., J. M. Bilbae, J. Nethencott, R. Myers, and H. J. Hoffman. Oculodermal melanosis complicated by multiple intracranial tumors. Can. J. Neurol. Sci. 7:101–107, 1980. Hulke, J. W. Series of cases of carcinoma of eyeball (case 2). Ophthalmol. Hosp. Res. 3:279–286, 1861. Ishibashi, A., K. Kimura, and A. Kukita. Plaque-type blue nevus combined with lentigo (nevus spilus). J. Cutan. Pathol. 17:241–245, 1990. Jay, B. Malignant melanoma of the orbit in a case of oculodermal melanosis. Br. J. Ophthalmol. 49:359–363, 1965. Keinke, T., K. Kaber, and A. Tosselson. Ota nevus, multiple heman-

giomas, and Takayasu arteritis. Arch. Dermatol. 110:442–450, 1974. Kersting, D. W., and M. R. Caro. Cellular blue nevus of Ota followed for twenty-two years. Arch. Dermatol. 74:59–62, 1956. Kitagawa, K., S. Hayasaka, and Y. Nagaki. Falsely elevated intraocular pressure due to an abnormally thick cornea in a patient with nevus of Ota. Jpn. J. Ophthalmol. 47:142–144, 2003. Kojima, K., C. Natsume, and A. Honda. A melanoma of the optic chiasm occurs in a case of Ota’s nevus and melanosis bulbi. Jpn. J. Clin. Ophthalmol. 13:502–504, 1959. Kono, T., H. H. Chan, A. R. Ercocen, Y. Kikuchi, S. Uezono, S. Iwasaka, T. Isago, and M. Nozaki. Use of Q-switched ruby laser in the treatment of nevus of Ota in different age groups. Lasers Surg. Med. 32:391–395, 2003. Kopf, A. W., and R. S. Bart. Tumor Conference No 42. J. Dermatol. Surg. Oncol. 8:442–445, 1982. Kopf, A. W., and A. I. Weidman. Nevus of Ota. Arch. Dermatol. 85:195–207, 1962. Lee, J. H. A clinical observation about the nevi involving melanocytes in the Korea youth. Korean J. Dermatol. 24:73–78, 1986. Lever, W. F., and G. Schaumburg-Lever. Histopathology of the Skin, 6th ed. Philadelphia: JB Lippincott Co., 1983, pp. 699–700. Liesegang, T. J. Pigmented conjunctival and scleral lesions. Mayo Clin. Proc. 69:151–161, 1994. Lowe, N. J., J. M. Wieder, D. Sawcer, P. Burrows, and M. Chalet. Nevus of Ota: Treatment with high energy fluences of the Qswitched ruby laser. J. Am. Acad. Dermatol. 29:997–1001, 1993. Lynn, A., S. J. Brozena, C. G. Espinoza, and N. A. Fenske. Nevus of Ota acquisita of late onset. Cutis 51:194–196, 1993. Maize, J. C., and B. A. Ackerman. Pigmented Lesions of the Skin. Philadelphia: Lea and Febiger, 1987, pp. 134–137. Makley, T. A., Jr., and C. M. King. Malignant melanoma of the choroid in melanosis oculi. Trans. Am. Acad. Ophthalmol. Otolaryngol. 71:638, 1967. McDonald, R. R., and K. E. Georgouras. Skin disorders in IndoChinese immigrants. Med. J. Aust. 156:847–853, 1992. Miller, M. Follow-up to case report: invasive naevus of Ota. Pathology 26:76, 1994. Mishima, Y., and B. Mevorah. Nevus Ota and nevus Ito in American Negroes. J. Invest. Dermatol. 36:133, 1961. Mohandessan, M., C. Fetkanhour, and R. O’Grady. Malignant melanoma of the choroid in a case of nevus of Ota. Ann. Ophthalmol. 11:189–192, 1979. Nik, N. A., W. B. Glew, and L. E. Zimmerman. Malignant melanoma of the choroid in the nevus of Ota of a black patient. Arch. Ophthalmol. 100:1641–1643, 1982. Noreiga-Sanchez, A., O. N. Markand, and J. H. Herndon. Oculocutaneous melanosis associated with the Sturge-Weber syndrome. Neurology 22:256–262, 1972. Okawa, Y., R. Yokota, and A. Yamauchi. On the extracellular sheath of dermal melanocytes in nevus fuscocaeruleus acromiodeltoideus (Ito) and Mongolian spot: an ultrastructural study. J. Invest. Dermatol. 73:224–230, 1979. Ono, T., K. Mah, and F. Hu. Dermal Merkel cells in the nevus of Ota and leopard syndrome. J. Am. Acad. Dermatol. 11:245–249, 1984. Ota, M., and H. Tanino. The naevus fusco-caeruleus opthalmomaxillaris and its relationship to pigmentary changes in the eye. Jikeikai Med. J. 63:1243–1244, 1939. Patel, B. C., C. A. Egan, R. W. Lucius, J. W. Gerwels, N. Mamalis, and R. L. Anderson. Cutaneous malignant melanoma and oculodermal melanocytosis (nevus of Ota): report of a case and review of the literature. J. Am. Acad. Dermatol. 38:862–865, 1998. Ray, P. E., and E. M. Schaeffer. Nevus of Ota and choroidal melanoma. Surg Ophthalmol 12:130–131, 1967. Reed, W. B., and G. I. Sugarman. Unilateral nevus de Ota with sensorineural deafness. Arch. Dermatol. 109:881–883, 1974. Reinke, T., K. Haber, and A. Josselson. Ota nevus, multiple heman-

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CHAPTER 52 gioma, and Takayasu arteritis. Arch. Dermatol. 110:447–450, 1974. Ruiz-Villaverde, R., J. B. Melguizo, A. B. Eisman, and S. S. Ortega. Bilateral Ota naevus. J. Eur. Acad. Dermatol. Venereol. 17:437– 439, 2003. Sang, D. N., D. M. Albert, A. J. Sober, and T. O. McMeekin. Nevus of Ota with contralateral cerebral melanoma. Arch. Ophthalmol. 95:1820–1824, 1977. Sawada, Y., M. Iwata, and Y. Mitsusashi. Nevus pigmentovascularis. Ann. Plast. Surg. 25:142–145, 1990. Shaffer, D., K. Walker, and G. R. Weiss. Malignant melanoma in a Hispanic male with nevus of Ota. Dermatology 185:146–150, 1992. Singh, M., B. Kaur, and H. M. Annuar. Malignant melanoma of the choroid in nevus of Ota. Br. J. Ophthalmol. 72:131–133, 1988. Speakman, J. S., and M. J. Phillips. Cellular and malignant blue nevus complicating oculodermal melanosis. Can. J. Ophthalmol. 8:539–547, 1973. Tanino, H. Uber eine in Japan Haufig vorkommende Navus form: naevus fusco-caeruleus ophthal-maxillaris Ota. Jpn. J. Dermatol. Urol. 46:107–111, 1939. Tanino, H. Uber eine in Japan Haufig vorkommende Navusform: naevus fusco-caeruleus ophthal-maxillaris Ota. Jpn. J. Dermatol. Urol. 47:51–53, 1940. Taylor, C. R., T. J. Flotte, W. Gange, and R. R. Anderson. Treatment of nevus of Ota by Q-switched ruby laser. J. Am. Acad. Dermatol. 30:743–751, 1994. Teekhasaenee, C., R. Ritch, U. Rutnin, and N. Leelawongs. Ocular findings in oculodermal melanocytosis. Arch. Ophthalmol. 108: 1114–1120, 1990. Theunissen, P., G. Spincemaille, M. Pannebakker, and J. Lambers. Meningeal melanoma associated with nevus of Ota: case report and review. J. Clin. Neuropathol. 12:125–129, 1993. Ticho, B. H., M. O. Tso, and S. Kishi. Diffuse iris nevus in oculodermal melanocytosis: A light and electron microscopic study. J. Pediatr. Ophthalmol. Strabismus 26:244–250, 1989. Trese, M. T., T. H. Pettit, R. Y. Foos, and J. Hofbauer. Familial nevus of Ota. Ann. Ophthalmol. 13:855–857, 1981. Velazquez, N., and I. S. Jones. Ocular and oculodermal melanocytosis associated with uveal melanoma. Ophthalmology 90:1472– 1476, 1983. Watanabe, S., and H. Takahashi. Treatment of nevus of Ota with the Q-switched ruby laser. N. Engl. J. Med. 331:1745–1750, 1994. Whitmore, S. E., B. B. Wilson, and P. H. Cooper. Late-onset nevus of Ota. Cutis 48:213–216, 1991. Whyte, M. P., and A. S. Dekaban. Familial cerebellar degeneration with slow eye movements, mental deterioration and incidental nevus of Ota. Dev. Med. Child. Neurol. 18:373–379, 1976. Willis, R. A. Pathology of Tumors. London: Butterworth, 1967, pp. 930. Yamamoto, T. Malignant melanoma of the choroid in nevus of Ota. Ophthalmologica 151:1–10, 1969. Yanoff, M., and L. E. Zimmerman. Histogenesis of malignant melanomas of the uvea. Arch. Ophthalmol. 77:331–336, 1967. Yeschua, R., M. R. Wexler, and Z. Newman. The nevus of Ota: case report. Plast. Reconstr. Surg. 55:229–233, 1975. Yoshida, K. Nevus fusco-caeruleus ophthalmo-maxilaris Ota. Tohoku J. Exp. Med. 55(suppl. 1):34–43, 1952. Zimmerman, A. A., and S. W. Becker. Melanocytes and melanoblasts in fetal negro skin. In: Illinois Monographs in Medical Science. Urbana, IL: University of Illinois Medical Press, 1959, pp. 1–59.

Nevus of Ito Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im The nevus of Ito, first reported by Ito in 1954, is a nevoid 1012

A

B Fig. 52.8. (A, B) Typical clinical appearance of nevus of Ito.

pigmentary anomaly similar to the nevus of Ota in clinical appearance but located on the skin innervated by the posterior supraclavicular and lateral brachiocutaneous nerves (Fig. 52.8). Nevus of Ito typically has flat blue-black or slate-gray macules intermingled with small, flat brown spots. The brown

ACQUIRED AND CONGENITAL DERMAL HYPERMELANOSIS

spots are caused by localized epidermal hyperpigmentation and blue-black or slate-gray macules are caused by the presence of dermal melanocytes. It is usually congenital but may appear in early childhood. It does not disappear with time. It may be associated with nevus of Ota or nevus of flammeus. The latter association is called phakomatosis pigmentovascularis (Furukawa et al., 1970). Histologically the epidermis is normal and the dermis contains numerous bipolar melanocytes. By electron microscopy, the dermal melanocytes contain singly dispersed, fully melanized melanosomes (Hori and Takayama, 1988). The melanocytes are typically surrounded by a sheath of variable thickness. Malignant transformation has been observed in nevus of Ito (van Krieken et al., 1988).

References Furukawa, T., A. Igata, Y. Toyokura, and S. Ikeda. Sturge-Weber and Klippel-Trenaunay syndrome with nevus of Ota and Ito. Arch. Dermatol. 102:640–645, 1970. Hori, Y., and O. Takayama. Circumscribed dermal melanoses: Classification and histologic features. Dermatol. Clin. 6:315–326, 1988. van Krieken, J. H., B. W. Boom, and E. Scheffer. Malignant transformation in a naevus of Ito. A case report. Histopathology 12: 100–102, 1988.

Phakomatosis Pigmentovascularis Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im

Historical Background A pigmented nevus of various types and a vascular nevus commonly occur together (Gilliam et al., 1993; Hasegawa and Yasuhara, 1985; Sawada et al., 1990). Nevus pigmentovascularis, first described in 1920, is a large melanocytic nevus associated with vascular nevus (Fig. 52.9). In 1947, Ota et al. (Hasegawa and Yasuhara, 1985; Sawada et al., 1990) reported a case of nevus pigmentovascularis. He noted the association of nevus flammeus (port wine stain) and pigmented nevi such as a persistent Mongolian spot (dermal melanocytosis), nevus spilus, zosteriform lentiginous nevus, or nevus pigmentosus et verrucous. Some affected individuals might have a nevus anemicus. These anomalies have been associated with multiple granular cell tumors (Gilliam et al., 1993; Guiglia and Prendiville, 1991; Hasegawa and Yasuhara, 1985; Sawada et al., 1990).

Type 1: A combination of a nevus flammeus and nevus pigmentosus and verrucous epidermal nevus. Type 2: A combination of a nevus flammeus and a Mongolian spot with or without nevus anemicus. Type 3: A combination of a nevus flammeus and nevus spilus with or without nevus anemicus. Type 4: A combination of a nevus flammeus, nevus anemicus, and a Mongolian spot with or without nevus anemicus. Recently, the classification of a combination of a cutis marmorata telangiectatica congenita and aberrant Mongolian spots as a new entity in the form of phakomatosis pigmentovascularis (type 5) has been proposed (Enjolras and Mulliken, 2000; Torrelo et al., 2003). Nearly 200 cases have been reported, most of them in Japanese literature. Among 60 cases, 85% were type 2; 5% were type 3; 10% were type 4. However, there has been no Japanese report of type 1 (Happle, 1991; Hasegawa and Yasuhara, 1985; Libow, 1993; Ruiz-Maldonado et al., 1987; Vidaurri-de la Cruz et al., 2003). Phakomatosis pigmentovascularis is frequently observed in females. The male to female ratio is 1:1.34 (Vidaurri-de la Cruz et al., 2003). Although patients may have lesions limited to the skin, extracutaneous abnormalities have been frequently described. Leptomeningeal angiomatosis has been detected in individuals with a port wine stain in the characteristic trigeminal nerve distribution. Glaucoma has been noted in individuals with a facial port wine stain in a trigeminal distribution or with a nevus of Ota affecting the eye. Other abnormalities include soft-tissue hypertrophy, hypoplastic larynx and subglottic stenosis, scoliosis, anemia, venous hypoplasia from the inferior vena cava to the superficial femoral vein, renal anomaly, renal angiomas, moyamoya disease, malignant polyposis, and mental disturbance. Melanosis oculi with mammillations resembling Lisch nodules have also been described (Di Landro et al., 1999; Gilliam et al., 1993; Guiglia and Prendiville, 1991; Huang and Lee, 2000; Happle, 1991; Hasegawa and Yasuhara, 1985; Libow, 1993; Park et al., 2003; Sawada et al., 1990; Tsuruta et al., 1999).

Differential Diagnosis

Synonym Phakomatosis pigmentovascularis pigmentovascularis.

and Yasuhara (1985) have subdivided nevus pigmentovascularis into four subtypes. Each type has been further classified into two subdivisions: (a) cutaneous involvement only, and (b) cutaneous and systemic involvement.

is

also

called

nevus

Clinical Findings Patients with this syndrome have a distinctive association of an extensive nevus flammeus with melanocytic (Figs 52.9– 52.13) or epidermal nevi. The melanocytic lesions may be persistent aberrant Mongolian spots or nevus spilus. Hasegawa

Other syndromes can resemble phakomatosis pigmentovascularis. Patients with type IIb phakomatosis pigmentovascularis can have a nevus flammeus, intracranial and visceral angiomata, and seizure disorders. Such individuals fulfill the clinical criteria for Sturge–Weber syndrome. Patients with phakomatosis pigmentovascularis type IIb may appear to have the Klippel–Trenaunay syndrome. Such individuals have an extensive nevus flammeus associated with lymphangioma and/or hemihypertrophy of the limbs. 1013

52.9

52.11

52.10

52.13

52.12

Figs. 52.9–52.13. Phakomatosis pigmentovascularis illustrating both dermal melanocytosis and nevus flammeus (see also Plates 52.4–52.7, pp. 494–495).

ACQUIRED AND CONGENITAL DERMAL HYPERMELANOSIS

Pathogenesis Like other rare congenital disorders, the pathogenesis of phakomatosis pigmentovascularis has not been clarified. A hypothesis was proposed that a pathogenic factor (drugs, virus, or substances toxic to the nervous system) could have an irritating effect and cause some clones of angioblasts and melanoblasts to proliferate in an aberrant form (Ortonne et al., 1978; Ruiz-Maldonado et al., 1987). Another possible explanation is the “twin spot theory.” Twin spotting is presumed to be caused by different recessive heterozygous mutations whose loci are located on the same chromosome. During embryogenesis, each of two different recessive mutations on one chromosome becomes homozygous in each of the daughter cells by somatic crossing over or false chromosomal cleavage. Therefore, one daughter cell is homozygous for one of two recessive phenotypes, whereas the other is homozygous for the other phenotype. Because the coexistence of nevus flammeus and nevus anemicus is compatible with the “twin spot theory,” phakomatosis pigmentovascularis could be explained by genetic abnormalities in the vasomotor nerve cells and melanocytes, both of which derive from a common neuroectodermal precursor (Happle, 1991, 1999; Park et al., 2003; Torrelo et al., 2003).

References Di Landro, A., G. L. Tadini, L. Marchesi, and T. Cainelli. Phakomatosis pigmentovascularis: A new case with renal angiomas and some considerations about the classification. Pediatr. Dermatol. 16:25–30, 1999. Enjolras, O., and J. B. Mulliken. Vascular malformations. In: Textbook of Pediatric Dermatology, J. Harper (ed.). Oxford: Blackwell Science, 2000, pp. 975–996. Gilliam, A. C., N. K. Ragge, M. I. Perez, and J. L. Bolognia. Phakomatosis pigmentovascularis type IIb with iris mammillations. Arch. Dermatol. 129:340–342, 1993. Guiglia, M. C., and J. S. Prendiville. Multiple granular cell tumors associated with giant speckled lentiginous nevus and nevus flammeus in a child. J. Am. Acad. Dermatol. 24:359–363, 1991. Huang, C., and P. Lee. Phakomatosis pigmentovascularis IIb with renal anomaly. Clin. Exp. Dermatol. 25:51–54, 2000. Happle, R. Allelic somatic mutations may explain vascular twin nevi. Hum. Genet. 86:321–322, 1991. Happle, R. Loss of heterozygosity in human skin. J. Am. Acad. Dermatol. 40;318–321, 1999. Hasegawa, Y., and M. Yasuhara. Phakomatosis pigmentovascularis type IVa. Arch. Dermatol. 121:651–655, 1985. Libow, L. F. Phakomatosis pigmentovascularis type IIIb. J. Am. Acad. Dermatol. 29:305–307, 1993. Ortonne, J. P., D. Flored, J. Coiffet, and X. Cottin. Syndrome de Sturge-Weber associé à une mélanose oculo-cutanée: étude clinique, histologique et ultrastructurale d’ un cas. Ann. Dermatol. Venerol. 105:1019–1031, 1978. Park, J. G., K. Y. Roh, H. J. Lee, S. J. Ha, J. Y. Lee, S. S. Yun, K. W. Lim, K. S. Song, and J. W. Kim. Phakomatosis pigmentovascularis IIb with hypoplasia of the inferior vena cava and the right iliac and femoral veins causing recalcitrant stasis leg ulcers. J. Am. Acad. Dermatol. 49:S167-S169, 2003. Ruiz-Maldonado, R., L. Tamayo, A. M. Laterza, G. Brawn, and A. Lopez. Phakomatosis pigmentovascularis: A new syndrome? Report of four cases. Pediatr. Dermatol. 4:189–196, 1987. Sawada, Y., M. Iwata, and Y. Mitsusashi. Nevus pigmentovascularis. Ann. Plast. Surg. 25:142–145, 1990.

Torrelo, A., A. Zambrano, and R. Happle. Cutis marmorata telangiectatica congenita and extensive Mongolian spots: type 5 phacomatosis pigmentovascularis. Br. J. Dermatol. 148:342–345, 2003. Tsuruta, D., K. Fukai, M. Seto, K. Fujitani, K. Shindo, T. Hamada, and M. Ishii. Phakomatosis pigmentovascularis type IIIb associated with moyamoya disease. Pediatr. Dermatol. 16:35–38, 1999. Vidaurri-de la Cruz., H, L. Tamayo-Sanchez, C. Duran-McKinster, L. Orozco-Covarrubias Mde, and R. Ruiz-Maldonado. Phakomatosis pigmentovascularis II A and II B: clinical findings in 24 patients. J. Dermatol. 30:381–388, 2003.

Other Congenital Dermal Melanocytosis Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im Dermal melanocytosis is characterized by the presence of ectopic melanocytes in the dermis (Jimenez et al., 1994). Dermal melanocytosis is seen in various congenital conditions, mostly in children, and occasionally in adulthood (Hidano and Kaneko, 1991). Congenital dermal melanosis has several morphologic forms, including the Mongolian spot, the blue nevus, the nevus of Ota, and the nevus of Ito (Fukuda et al., 1993). However, a few cases of congenital dermal melanocytosis do not fit into any of the above mentioned entities. This section focuses on the rare variants of congenital dermal melanosis.

Clinical Findings Dermal melanocytosis typically occurs on the face, in the supraclavicular region (Lever, 1976), over the upper part of the back, the neck, breasts, and abdomen (Cole et al., 1950; El Bahrawy, 1922), and less commonly over the sacrum. Other cases do not fit this picture. In reported congenital generalized dermal melanocytosis, a baby girl was born with blue-gray discoloration of almost the entire integument (Fig. 52.13). The face, palms, soles, genital area, and nipples were spared. The pigmentation varied in intensity from one area to another with a few nonpigmented areas on the thigh and abdomen. The margins of the pigmented regions were ill defined (Bashiti et al., 1981). Velez et al. (1992) described a 28-year-old woman with speckled grayblue pigmentation in a segmental pattern on the right side of the trunk, involving several thoracic dermatomes, that was termed congenital segmental dermal melanocytosis. Grezard et al. (1999) reported a 45-year-old woman who was born with grey-blue pigmentation on both sides of the back that subsequently spread along the dermatomes. The latter case of congenital bilateral dermal melanocytosis resembles the former, but with bilateral involvement (Grezard et al., 1999). Krishnan et al. (2003) reported a 31-year-old man with a congenital presentation of a solitary 10 cm ¥ 8 cm sized speckled blue-brown patch.

Histopathology Light microscopic examination of pigmented skin shows 1015

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numerous stellate and fusiform melanocytes diffusely distributed throughout the dermis. The cells tend to be oriented parallel to the skin surface and contain particulate brown pigment. Melanocytes are numerous in the middle and lower dermis and occasionally extend into the subcutaneous tissue. There are no melanophages in the dermis. The overlying epidermal melanocytes appear normal in number and morphology. The dopa reaction and the Fontana-Masson stain are positive in virtually all the dermal and epidermal melanocytes. Electron microscopic examination shows the pigmented cells to be typical dermal melanocytes. The cells contained premelanosomes and melanosomes with varying degree of maturation within a same cell. A narrow band of extracellular filamentous material surrounds each melanocyte. In the biopsy specimen from nonpigmented areas, the epidermal melanocytes were present in seemingly normal numbers and the melanin pigment was normal (Bashiti et al., 1981; Grezard et al., 1999; Velez et al., 1992).

Pathogenesis The pathogenesis of persistent dermal melanocytes is uncertain. Dermal melanocytes are presumed by analogy with epidermal melanocytes to arise in the neural crest and migrate onto the skin. It has been suggested that dermal melanocytes are simply melanocytes destined for the epidermis that have remained in the dermis. Because of the lack of information on the determinants of normal migration of epidermal melanocytes, our understanding of these lesions is poor (Bashiti et al., 1981).

References Bashiti, H. M., J. D. Blair, R. A. Triska, and L. Keller. Generalized dermal melanocytosis. Arch. Dermatol. 117:791–793, 1981. Cole, H. N., Jr., W. R. Hubler, and H. J. Lund. Persistent aberrant mongolian spot. Arch. Dermatol. 61:244–260, 1950. El Bahrawy, A. A. Ueba den mongolenfleck bei. Eur. Arch. Dermatol. Syphil. 141:172–192, 1922. Fukuda, M., J. Kitajima, H. Fushida, and T. Hamada. Acquired dermal melanocytosis of the hand: a new clinical type of dermal melanocytosis. J. Dermatol. 20:561–565, 1993. Grezard, P., F. Berard, B. Balme, and H. Perrot. Congenital bilateral dermal melanocytosis with a dermatomal pattern. Dermatology 198:105–106, 1999. Hidano, A., and K. Kaneko. Acquired dermal melanocytosis of the face and extremities. Br. J. Dermatol. 124:96–99, 1991. Jimenez, E., P. Valle, and P. Villegas. Unusual acquired dermal melanocytosis. J. Am. Acad. Dermatol. 30:277–278, 1994. Krishnan, R. S., T. R. Roark, and S. Hsu. Isolated patch of speckled, congenital, pigmented dermal melanocytosis outside the face or acromioclavicular regions. J. Eur. Acad. Dermatol. Venereol. 17:238–239, 2003. Jimenez, E., P. Valle, and P. Villegas. Unusual acquired dermal melanocytosis. J. Am. Acad. Dermatol. 30:277–278, 1994. Lever, W. F. Histopathology of the Skin. Philadelphia: JB Lippincott Co., 1976, p. 777. Velez, A., C. Fuente, I. Belinchon, N. Martin, V. Furio, and E. Sanchez Yus. Congenital segmental dermal melanocytosis in an adult. Arch. Dermatol. 128: 521–525, 1992.

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Acquired Dermal Melanocytosis Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im

Historical Background Acquired dermal melanocytosis initially was described by Mevorah et al. in 1977. They reported a 32-year-old Portuguese woman with a bluish spot on her right hand. The lesion had appeared at the age of 11 years.

Clinical and Histological Findings Acquired dermal melanocytosis can occur in adult life. Acquired dermal melanocytosis can be classified into generalized, linear, and localized types on clinical grounds. One case of acquired generalized dermal melanocytosis was a 55-yearold man with a leiomyosarcoma of the rectum and multiple metastatic lesions in the liver. The initial lesions were noticed on the face at the age of 53 years. They extended to the upper extremities and back. On physical examination, there were extensive and multiple blue-gray macules on the face, upper extremities, shoulders, and back. Gray flecks on both palpebral conjunctivae, blue-gray flecks on the palate, and blue and brown pigmentation on the gingiva were also noted (Ono et al., 1991). Pariser and Bluemink (1982) described a case of acquired linear dermal melanocytosis. The lesion began after blunt trauma on the posterior aspect of the lower part of the leg and extended distally for several months. There was no history of an eruption preceding the onset of the hyperpigmentation and the patient had taken no systemic medication before or after the onset of the lesion. The lesion was composed of irregularly shaped 2–8 mm brown macules arranged in a linear fashion with many “skip” areas. The sural nerve innervated the affected area. The authors suggested that trauma preceding the onset of the lesion might have induced nerve reactivity that stimulated melanocytes in the area. Histologically, cells with granular brown cytoplasmic pigment located in the papillary dermis were observed. Pigment granules were stained black by the Fontana-Masson stain. Electron microscopic study showed that dendritic melanocytes were present in the papillary dermis (Pariser and Bluemink, 1982). In 1977, Mevorah et al. described a 32-year-old Portuguese woman with a bluish spot on her right hand that had appeared when she was 11 years. The lesion involved the second finger, part of the dorsum of the hand, and the adjoining distal third of the palm. Clinically this lesion was similar to an aberrant, persistent Mongolian spot. However histologically there was a significant number of melanophages accompanying the dermal melanocytes, features that are not characteristic of the Mongolian spot (Mevorah et al., 1977). Acquired dermal melanocytoses are sometimes classified according to their characteristic clinical appearances. Hori et al. (1984) described cases involving only the face as acquired, bilateral nevus of Ota-like macules. Hidano and Kaneko (1991) described acquired dermal melanocytosis of the face

ACQUIRED AND CONGENITAL DERMAL HYPERMELANOSIS

and extremities. Several cases of acquired dermal melanocytosis distributed only on the extremities without involving the face were described (Fukuda et al., 1993; Kuniyuki, 1997; Mevorah et al., 1977). Ono et al. (1991) reported seven Japanese males with acquired dermal melanocytosis on the upper back as an upper back variant of late onset dermal melanocytosis. Ono et al. (1991) described a male with acquired dermal melanocytosis disseminated on the face, upper limbs, and trunk as generalized type of acquired dermal melanocytosis.

Pathogenesis The pathogenesis of acquired dermal melanocytosis has been proposed to be the reactivation of pre-existing dermal melanocytes or the manifestation of latent dermal melanocytosis triggered by dermal inflammation, atrophy, or degeneration of the epidermis and/or dermis (Hori et al., 1984). Ultraviolet irradiation may increase MSH or endothelins, increasing tyrosinase activity and thus inducing melanogenesis, especially when the lesions are distributed on sun-exposed areas of the face and extremities (Imokawa et al., 1995; Kuniyuki, 1997). Kunachak et al. (1996) postulated that female hormones affect the occurrence of acquired dermal melanocytosis because of the high percentage of women exhibiting these lesions. Recently, Rubin et al. (2001) reported a case of acquired dermal melanocytosis appearing during pregnancy, and suggested estrogen and progesterone may induce onset of acquired dermal melanocytosis.

References Fukuda, M., J. Kitajima, H. Fushida, and T. Hamada. Acquired dermal melanocytosis of the hand: A new clinical type of dermal melanocytosis. J. Dermatol. 20:561–565, 1993. Hidano, A., and K. Kaneko. Acquired dermal melanocytosis of the face and extremities. Br. J. Dermatol. 124:96–99, 1991. Hori, Y., M. Kawashima, K. Oohara, and A. Kukita. Acquired, bilateral nevus of Ota-like macules. J. Am. Acad. Dermatol. 10:961–964, 1984. Imokawa, G., M. Miyagishi, and Y. Yada. Endothelin-1 as a new melanogen: Coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis. J. Invest. Dermatol. 105:32–37, 1995. Kunachak, S., S. Kunachakr, V. Sirikulchayanonta, and P. Leelaudomniti. Dermabrasion is an effective treatment for acquired bilateral nevus of Ota-like macules. Dermatol. Surg. 22:559–562, 1996. Kuniyuki, S. Acquired dermal melanocytosis on the wrist. J. Dermatol. 24:120–124, 1997. Mevorah, B., E. Frenk, and J. Delacretaz. Dermal melanocytosis: Report of an unusual case. Dermatologica 154:107–114, 1977. Ono, S., M. Hori, and K. Yamashita. Generalized type of acquired dermal melanocytosis. Jpn. J. Dermatol. 101:965–971, 1991. Ono, T., K. Egawa, K. Kayashima, and M. Kitoh. Late onset dermal melanocytosis: An upper back variant. J. Dermatol. 18:97–103, 1991. Pariser, R. J., and G. G. Bluemink. Acquired linear dermal melanocytosis: nerve course distribution. Arch. Dermatol. 118:125–128, 1982. Rubin, A. I., S. Van Laborde, and M. J. Stiller. Acquired dermal melanocytosis: Appearance during pregnancy. J. Am. Acad. Dermatol. 45:609–613, 2001.

Carleton–Biggs Syndrome Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im Acquired dermal hyperpigmentation is caused either by the presence of dermal melanocytes (dermal melanocytosis) or by the presence of free melanin in the dermis or within dermal macrophages (dermal melanosis). The etiology of acquired dermal melanocytosis is thought to be reactivation of latent dermal melanocytes or migration of melanocytes into the dermis from the hair bulb. Acquired dermal melanosis occurs in many inflammatory disorders of the epidermis such as lichen planus. In 1948, Carleton and Biggs described a 14-year-old girl who was normal apart from her skin, which was covered with widespread bluish spots. These lesions were not congenital although at birth there was pigmentation on the sclera and iris of the left eye. The girl also had thickening of the skull behind the ears and pronounced ballooning of the posterior cranial fossa. At 3 years of age, a blue spot appeared on her back. At 8 years of age another appeared on her nose and more appeared around the age of 9. Metastatic malignant melanoma was found in the liver and the lymph nodes at the age of 43 years (Levene, 1979). The primary melanoma was not identified. At the autopsy, widespread dermal, visceral, and cranial melanoses were observed. Melanophages were present in visceral and cranial tissue, and melanocytes and melanophages in the dermis. Histologic examination showed the presence of dendritic or bipolar melanocytes scattered through the dermis around blood vessels and sweat ducts.

References Carleton, A., and R. Biggs. Diffuse mesodermal pigmentation with congenital cranial abnormality. Br. J. Dermatol. 60:10–13, 1948. Levene, A. Disseminated dermal melanocytosis terminating in melanoma. Br. J. Dermatol. 101:197–205, 1979.

Acquired Bilateral Nevus of Ota-like Macules (ABNOM) Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im

Clinical and Histologic Findings Acquired bilateral nevus of Ota-like macules (ABNOM), or Hori’s macules, are characterized by development of bilateral blue-brown or slate-gray macules on the face (Fig. 52.14). Blue-brown macules can be found on the sides of the forehead, temples, eyelids, malar areas, and root and alae of the nose. Women are affected more commonly than men (Hori et al., 1984). The ocular and mucosal membranes are not involved. Histologically, bipolar or oval dermal melanocytes that are slightly dopa-positive are found scattered in the upper and middle portions of the dermis. On electron microscopy these dermal melanocytes contain many singly dispersed

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ECS

1 mm Fig. 52.15. Electron micrograph of a dermal melanocyte from acquired, bilateral nevus of Ota-like macule. ECS, extracellular sheath, uranyl acetate and lead citrate stain.

References Hori, Y., K. Oohara, and M. Niimura. Electron microscopy: ultrastructural observations of the extracellular sheath of dermal melanocytes in the nevus of Ota. Am. J. Dermatopathol. 4:245– 251, 1982. Hori, Y., M. Kawashima, and K. Oohara. Acquired, bilateral nevus of Ota-like macules. J. Am. Acad. Dermatol. 10:961–964, 1984. Okawa, Y., R. Yokota, and A. Yamauchi. On the extracellular sheath of dermal melanocytes in nevus fuscocaeruleus acromiodeltoideus (Ito) and Mongolian spot: an ultrastructural study. J. Invest. Dermatol. 73:224–230, 1979. Fig. 52.14. Acquired facial blue macules resembling a bilateral nevus of Ota (see also Plate 52.8, pp. 494–495).

Blue Macules Associated with Progressive Systemic Sclerosis Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im

melanosomes that are 0.4–0.8 mm in size and are in stages II, III, and IV of melanization (Fig. 52.15). These melanocytes are surrounded by an extracellular sheath (Hori et al., 1982; Okawa et al., 1979). The epidermis is normal.

Differential Diagnosis These macules can be differentiated clinically and histologically from congenital bilateral nevus of Ota (type IV nevus of Ota), female facial melanosis, Riehl melanosis, and dermal melasma. Congenital bilateral nevus of Ota is usually present at or soon after birth or develops around adolescence. The conjunctivae and the mucosal membrane of the mouth and nose or the tympanic membrane are commonly involved by the nevus of Ota. Dermal melanocytes are not found in facial melanosis, Riehl melanosis, and melasma. The dermal melanocytes of acquired bilateral nevus of Otalike macules may be attributed to the migration of hair bulb melanocytes into the dermis or to the reactivation of latent dermal melanocytes in the affected areas. The latter hypothesis is supported by recent observations confirming the existence of melanoblasts in the dermis of the adult skin. 1018

In progressive systemic sclerosis, brownish hyperpigmentation and depigmentation of the involved areas of the skin are commonly observed and are considered highly suggestive of scleroderma (See Fig. 50.65, Chapter 50, section on Morphea and scleroderma). Blue macules have also been observed, however, on the back, shoulders, and one or both upper portions of the arms in the first four years in the course of the disease. The blue macules consisted mostly of linear blue streaks along with some small blue patches (Hori et al., 1976). Histologically, spindle-shaped and brown pigment-bearing dermal melanocytes can be recognized in the upper two-thirds of the dermis. These dermal melanocytes are widely spaced in the dermis between the connective and elastic tissue fibers. The dopa reaction of the dermal melanocytes in the blue macule is positive. Electron microscopically, many fully melanized melanosomes are present in these dermal melanocytes. A few immature melanosomes may be found. Almost all melanosomes, whatever the stage of melanization, are singly distributed in the cytoplasm (Hori and Takayama, 1988; Stanford and Georgouras, 1996).

ACQUIRED AND CONGENITAL DERMAL HYPERMELANOSIS

References Hori, Y., S. Miyazawa, and S. Nishiyama. “Naevus caeruleus tardus” in association with progressive systemic scleroderma. In: Pigment Cell, V. Riley (ed.). Basel: S Karger, 1976, pp. 273–283.

Hori, Y., and O. Takayama. Circumscribed dermal melanoses: classification and histologic features. Dermatol. Clin. 6:315–326, 1988. Stanford, D. G., and K. E. Georgouras. Dermal melanocytosis: a clinical spectrum. Australas. J. Dermatol. 37:19–25, 1996.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Mixed Epidermal and Dermal Hypermelanoses and Hyperchromias Sections Melasma Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im Melanosis from Melanoma Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im

Melasma Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im

Clinical Findings Melasma is defined as light to dark brown, purple, irregular hypermelanosis of the face (Fig. 53.1). It develops slowly and is usually symmetric (Newcomer et al., 1961). Although it may occur in men (Vazquez et al., 1988), it is far more common in women particularly those of Hispanic origin (Sanchez et al., 1981) and Asians. Dark-skinned races living in India, Pakistan, and the Middle East tend to develop this problem in the first decade of life (Pathak et al., 1986) but patients from most other races note the onset at puberty or later. The condition may last for many years with relapses during the summer and relative remissions during the winter. The lesions of melasma are strictly limited to sun-exposed skin. The arcuate or polycyclic lesions are brown, gray, or blue macules which coalesce into irregular patches. The lesions may be linear and have a starburst distribution. There are three patterns of distribution of the hyperpigmentation (Sanchez et al., 1981). The most common presentation is in the central facial area observed in about two-thirds of affected individuals. These individuals also have lesions on the forehead, nose, chin, and medial aspects of the cheeks. The “malar pattern” appears in about 20% of patients. Their lesions are limited to the cheeks and nose. Melasma over the ramus of the mandible, called the “mandibular pattern,” occurs in about 15% of patients. Areas off of the face, particularly the forearms, may be involved along with any of these patterns. The hyperpigmentation of melasma can be of two forms. The first is due to increase of epidermal melanin and the color of the affected areas is brown. The other is caused by increased epidermal melanin and deposition of melanin in the dermis. The color is gray-brown or slate. The various types of melasma can be differentiated by Wood’s light examination. Four distinct patterns are identifiable (Sanchez et al., 1981). In the epidermal type, Wood’s light produces an enhancement of the color contrast between affected and normal skin (Gilchrest et al., 1977). This is the most common pattern. In one study 72% of patients mani1020

fested this type of discoloration. Melasma of the dermal type shows no enhancement of color contrast under Wood’s light. There is a mixed dermal–epidermal type in which the Wood’s light examination shows enhancement but no color accentuation in different areas on the same patient. Finally, in very dark-skinned individuals, one cannot see the lesions of melasma under Wood’s light. The light emitted by Wood’s light is absorbed by melanin. In the epidermal type of melasma, the affected sites appear darker because the extra melanin absorbs all the light. For those with dermal pigmentation, there is scattering and reflection of the light from the epidermis that hides the hyperpigmentation visible under normal lighting (Findlay, 1970).

Histopathology There are two types of microscopic finding: an epidermal pattern and a dermal pattern (Sanchez et al., 1981). In the epidermal type, melanin deposition is mainly increased in the basal and suprabasal layers of the epidermis. There may be increased quantities of pigment throughout the epidermis including the stratum corneum. In addition, there is vacuolar degeneration of the basal cells. The dermal type of pigment deposition is characterized by melanin deposition in perivascular macrophages around both the superficial and deep dermal vessels (Sanchez et al., 1981). However, melanophages were present both in melasma and in normal facial skin in some of melasma patients, and melanophages were present in the normal dermis of Japanese skin. Thus, some researchers suggest that the melanophages cannot be a hallmark of the dermal type of melasma (Kang et al., 2002; Ohkuma, 1991). Pigment deposition in the dermal type of melasma is much more prominent than that in the epidermal type. Dopa staining of epidermal preparations shows increased melanin production in epidermal melanocytes in both the epidermal and dermal patterns. The dendrites of the melanocytes contain an increased number of melanosomes (Sanchez et al., 1981). Dopa-positive melanocytes do not increase in number.

Differential Diagnosis Many different conditions can produce increased pigmentation in sun-exposed skin. These include medications such as

MIXED EPIDERMAL AND DERMAL HYPERMELANOSES AND HYPERCHROMIAS

Fig. 53.1. Melasma.

amiodarone, phenothiazines, and heavy metals. However, the pattern of pigmentation is not arcuate or polycyclic as observed in melasma. Inflammatory insults, particularly in dark-skinned people, can lead to postinflammatory hyperpigmentation. Cutaneous lupus erythematosus, other photosensitivity reactions, skin infections, and severe atopic dermatitis all may result in hyperpigmentation but the inflammatory phase is almost always easily noted. This clinical feature differentiates postinflammatory hyperpigmentation from melasma, which has no inflammatory phase. Poikiloderma of Civatte can result in reticulated hyper- and hypopigmented atrophic plaques on the neck and upper chest. This may resemble melasma but the atrophy and telangiectasia seen in this condition are not noted in melasma.

Pathogenesis The cause of melasma is not known but there are a number of factors that may be involved in causing or aggravating the condition. The related condition chloasma occurs during pregnancy and in women on oral contraceptives (Cook et al., 1961; Esoda, 1963; Resnick, 1967). This is the so-called “mask of pregnancy.” The hyperpigmentation often decreases or disappears completely after parturition but may not regress

in women on oral contraceptives until the medication is discontinued (Resnick, 1967). In one study, 87% of patients who developed melasma while on birth control pills also had this problem during a subsequent pregnancy, an observation suggesting that these are individuals susceptible to hormonal stimulation of melanocytes (Sanchez et al., 1981). Both estrogen and progesterone are likely to be involved in inducing melasma. There is a report that a melanocyte stimulating hormone (a-MSH) antigen was highly expressed in the lesional keratinocytes of melasma. Locally produced a-MSH is known to be involved in epidermal skin pigmentation by stimulating tyrosinase activity and melanin synthesis in vivo. It is suggested that a-MSH is involved in the hyperpigmentation of melasma skin (Im et al., 2002). However, it is possible that other hormones such as b-lipotropin, which is secreted by the pituitary gland and is a melanotropic peptide, could play some role. Plasma levels of this hormone do not differ in patients with or without melasma (Smith et al., 1977). There is also a report that thyroid disorders are associated with melasma in women whose pigmentation develops during pregnancy or after ingestion of oral contraceptive drugs (Lutfi et al., 1985). Results of some studies suggest that melasma may be a type of photocontact dermatitis. The allergens were found to be ingredients in cosmetics. Indoor lighting at low intensity over long periods of time seems to elicit a photosensitivity reaction (Verallo-Rowell et al., 2001). Photocontact dermatitis might be a mechanism by which release of a-MSH from keratinocytes is stimulated. The most important exacerbating factor for melasma is exposure to ultraviolet light. In one study of 76 patients, all reported that sun exposure selectively darkened the areas already hyperpigmented by melasma (Sanchez et al., 1981). Individuals whose condition cleared up with therapy often note a recurrence after subsequent sun exposure. Ultraviolet light normally increases melanogenic activity in melanocytes with resulting hyperpigmentation (Pathak et al., 1962). In addition to direct ultraviolet light exposure, accumulated sun exposure is also important to develop melasma. Increased solar elastosis is found in the lesional skin of melasma compared to adjacent perilesional normal skin, suggesting chronic sun exposure (Kang et al., 2002). There may be genetic factors, which make one more prone to develop melasma. Aside from the disease occurring more frequently in certain racial groups, there have been many cases of familial melasma (Sanchez et al., 1981). There is also a report that two identical twins had melasma precipitated by the same trigger factors, i.e., oral contraceptive pills and pregnancy. Both noted accentuation following exposure to ultraviolet light although other members of their family did not have similar problems (Hughes, 1987). It is commonly believed that certain cosmetics can produce hyperpigmentation. Virtually all women who have melasma report the use of cosmetics (Sanchez et al., 1981). These agents have been impugned as causative or at least as contributory factors (Montagna et al., 1980). No specific ingredient in 1021

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cosmetic preparations has been implicated and there has never been an experimental reproduction of melasma after the use of any topically applied chemical. Many individuals with melasma attempt to improve their appearance by using coverup makeup. It is quite likely that makeup use is a coincidental event which has no etiologic relationship to the skin disease. A likely scenario for the production of melasma is that clones of melanocytes are activated by ultraviolet light and functionally altered by other factors such as hormones which increase the synthesis of melanin.

Treatment Before embarking on therapy, it is important to determine the clinical/histologic type of melasma. Wood’s lamp examination usually is sufficient to make this determination. If not, a biopsy might be necessary. If the patient has the dermal form of the disease, there is little one can do to bleach the pigment. Since sunlight exacerbates the problem, potent, broad-spectrum sun protection is important to minimize sun-induced hyperpigmentation (Pathak et al., 1986; Vazquez and Sanchez, 1983). Since ultraviolet A (UVA) can enhance melanogenesis, sunscreens that block in that spectrum as well as in the ultraviolet B (UVB) range are necessary. For those patients with the epidermal type of melasma, bleaching agents are useful when used for prolonged periods. Hydroquinone-containing agents in a concentration of 1–4% are the most commonly used bleaching products (Arndt and Fitzpatrick, 1965) (Fig. 53.2A, B). Hydroquinone is not always helpful because of a high incidence of contact dermatitis and postinflammatory hyperpigmentation (Jimbow et al., 1974). To prevent these side effects, corticosteroids are often mixed with hydroquinone as one of the combination therapies (Kligman and Willis, 1975). The combination of 2% hydroquinone and 0.05% or 0.1% tretinoin (retinoic acid) has been used successfully for melasma (Pathak et al., 1986). The combination may be more effective than either 2% hydroquinone or tretinoin alone although irritation is still problematic. Topical tretinoin (0.1%) alone is reported to be effective in fair-skinned women (Griffiths et al., 1993) and black (African-American) patients (Kimbrough-Green et al., 1994), but the time to clinical improvement is long, at least 24 weeks (Griffiths et al., 1993). Combination products containing hydroquinone 4%, fluocinolone acetonide 0.01% and tretinoin 0.025% are available commercially (TriLuma®) and lighten epidermal melasma within two to three months in many patients. The mechanism of action of tretinoin in lightening melasma is not understood although reduction in epidermal melanin perhaps as a result of inhibition of the melanin-forming enzyme tyrosinase (Orlow et al., 1990) might be a factor in clinical improvement. Jimbow (1991) has reported marked improvement without irritation in 9 of 12 cases of melasma treated with topical 4% N-acetyl-4-cysteaminylphenol. Two other novel compounds, arbutin and kojic acid, have been described as beneficial (Maeda and Fukuda, 1991; Mishima, 1992). 1022

A

B Fig. 53.2 Extensive melasma of the cheeks (A). Same patient showing almost complete regression of hyperpigmentation after hydroquinone treatment (B) (see also Plates 53.1A and 53.1B, pp. 494–495).

In the epidermal form of melasma if one can selectively decrease the population of affected melanocytes, skin color can be lightened. This can be accomplished with physical modalities such as liquid nitrogen cryotherapy, CO2 laser vaporization, chemical peeling, and superficial dermabrasion. The risk with all of these procedures is that the resultant skin color will not be a perfect match to the surrounding unaffected skin.

References Arndt, K., and T. Fitzpatrick. Topical use of hydroquinone as a depigmentating agent. J. Am. Med. Assoc. 194:965–967, 1965. Cook, H. H., C. J. Gamble, and A. P. Saherthwaite. Oral contraception by norethynodrel. Am. J. Obstet. Gynecol. 88:437–445, 1961. Esoda, E. C. J. Chloasma from progestational oral contraceptives. Arch. Dermatol. 87:486, 1963. Findlay, G. Blue skin. Br. J. Dermatol. 83:127–134, 1970. Gilchrest, B. A., T. B. Fitzpatrick, R. R. Anderson, and J. A. Parrish. Localization of melanin pigmentation in the skin with Wood’s lamp. Br. J. Dermatol. 96:245–248, 1977. Griffiths, C. E., L. J. Finkel, C. M. Ditre, T. A. Hamilton, C. N. Ellis, and J. J. Voorhees. Topical tretinoin (retinoic acid) improves

MIXED EPIDERMAL AND DERMAL HYPERMELANOSES AND HYPERCHROMIAS melasma. A vehicle-controlled, clinical trial. Br. J. Dermatol. 129:415–421, 1993. Hughes, B. R. Melasma occurring in twin sisters. J. Am. Acad. Dermatol. 17:841, 1987. Im, S., J. Kim, W. Y. On, and W. H. Kang. Increased expression of alpha-melanocyte-stimulating hormone in the lesional skin of melasma. Br. J. Dermatol. 146:165–167, 2002. Jimbow, K. N-acetyl-4-S-cysteaminylphenol as a new type of depigmenting agent for the melanoderma of patients with melasma. Arch. Dermatol. 127:1528–1534, 1991. Jimbow, K., H. Obata, M. A. Pathak, and T. B. Fitzpatrick. Mechanism of depigmentation by hydroquinone. J. Invest. Dermatol. 62:436–449, 1974. Kang, W. H., K. H. Yoon, E. S. Lee, J. Kim, K. B. Lee, H. Yim, S. Sohn, and S. Im. Melasma: histopathological characteristics in 56 Korean patients. Br. J. Dermatol. 146:228–237, 2002. Kimbrough-Green, C. K., C. E. Griffiths, L. J. Finkel, T. A. Hamilton, S. M. Bulengo-Ransby, C. N. Ellis, and J. J. Voorhees. Topical retinoic acid (tretinoin) for melasma in black patients. A vehiclecontrolled clinical trial. Arch. Dermatol. 130:727–733, 1994. Kligman, A. M., and I. Willis. A new formula for depigmenting human skin. Arch. Dermatol. 111:40–48, 1975. Lutfi, R. J., M. Fridmanis, A. L. Misiunas, O. Pafume, E. A. Gonzalez, J. A. Villemur, M. A. Mazzini, and H. Niepomniszcze. Association of melasma with thyroid autoimmunity and other thyroidal abnormalities and their relationship to the origin of the melasma. J. Clin. Endocrinol. Metab. 61:28–31, 1985. Maeda, K., and K. Fukuda. In vitro effectiveness of several whitening cosmetic components in human melanocytes. J. Soc. Cosmet. Chem. 42:361, 1991. Mishima, Y. A post melanosomal era: control of melanogenesis and melanoma growth. Pigment Cell Res. Suppl 2:3–16, 1992. Montagna, W., F. Hu, and K. Carlisle. Reinvestigation of solar lentigines. Arch. Dermatol. 116:1151–1154, 1980. Newcomer, V. D., M. C. Lindbert, and T. H. Stenbert. A melanosis of the face (“chloasma”). Arch. Dermatol. 83:284–297, 1961. Ohkuma, M. Presence of melanophages in the normal Japanese skin. J. Am. Acad. Dermatol. 13: 32–37, 1991. Orlow, S. J., A. K. Chakraborty, and J. M. Pawelek. Retinoic acid is a potent inhibitor of inducible pigmentation in murine and hamster melanoma cell lines. J. Invest. Dermatol. 94:461–464, 1990. Pathak, M. A., F. C. Riley, and T. B. Fitzpatrick. Melanogenesis in human skin following exposure to long-wave ultraviolet and visible light. J. Invest. Dermatol. 39:435–443, 1962. Pathak, M. A., T. B. Fitzpatrick, and E. W. Kraus. Usefulness of retinoic acid in the treatment of melasma. J. Am. Acad. Dermatol. 15:894–899, 1986. Resnick, S. Melasma induced by oral contraceptive drugs. J. Am. Med. Assoc. 199:95, 1967. Sanchez, N. P., M. A. Pathak, S. Sato, T. B. Fitzpatrick, J. L. Sanchez, and M. C. Mihm Jr. Melasma: a clinical, light microscopic, ultrastructural and immunofluorescence study. J. Am. Acad. Dermatol. 4:698–710, 1981. Smith, A. G., S. Shuster, A. J. Thody, and M. Peberky. Chloasma, oral contraceptives, and plasma immunoreactive b-melanocytestimulating hormone. J. Invest. Dermatol. 68:169–170, 1977. Vazquez, M., and J. L. Sanchez. The efficacy of a broad-spectrum sunscreen in the treatment of melasma. Cutis 32:92–96, 1983. Vazquez, M., H. Maldonado, C. Benmaman, and J. L. Sanchez. Melasma in men. A clinical and histologic study. Int. J. Dermatol. 27:25–27, 1988. Verallo-Rowell, V. M., D. Villacarlos-Buatista, N. S. Oropeza, B. Gepaya-Resurreccion, and S. A. Mendoza. Indoor lights used in the photopatch testing of a case-controlled group of melasma and non-melasma patients. In: Skin in the Tropics: Sunscreens and the Hyperpigmentations, V. M. Verallo-Rowell (ed.). Manila: Anvil Press, 2001, pp. 102–131.

Melanosis from Melanoma Sang Ju Lee, Seung-Kyung Hann, and Sungbin Im

Historical Background In 1864 the German pathologist, Ernst Wagner, described a man in his thirties who had developed a melanoma in congenital nevus and subsequently acquired a generalized uniformly bluish-gray discoloration. That paper might have been the first on this subject.

Epidemiology The incidence of noticeable cutaneous melanosis in patients with metastatic melanoma is probably around 1–2%, a figure confirmed by Rorsman et al., who observed three cases in a group of 161 patients in Sweden (Agrup et al., 1979). A truly striking melanosis occurs at a lower frequency. Because the condition is so rare, most publications report only one patient. To date, about 60 cases have been reported in the literature (Böhm et al., 2001; Carruth et al., 2002; Lerner and Moellmann, 1993; Manganoni et al., 1993; Murray et al., 1999; Pec et al., 1993; Tsukamoto et al., 1998).

Clinical Findings By definition this form of discoloration occurs only in individuals with metastatic melanoma. The initial discoloration is usually noted as slate blue, even cerulean blue (Pack and Scharnagel, 1951), and is probably missed in many cases of widely disseminated metastatic melanoma. It develops insidiously. This early bluish coloration is an indication that the melanin deposition begins in the deeper layers of the skin, i.e., the dermis. Later, in those few patients who live to develop a full-blown melanosis, pigment deposition progresses rapidly until the patient is a dark slate color at the time of death (Fig. 53.3). Lerner and Moellmann reviewed the 42 patients reported in the literature: 20 males, 14 females, and 8 whose gender was not recorded. The age range of the women was 20–67 years (mean 40, median 41) and of the men 31–70 years (mean 50, median 49). Three women were pregnant. All patients had widespread metastatic disease. Metastases in the liver, skin, lymph node, spleen, heart, kidney, bone marrow, and gastrointestinal tract were observed with high frequency. Some patients had spectacular showers of hundreds of cutaneous metastases that ranged in size from less that 1 mm2 to larger than 1 cm2. In nearly all patients, light-exposed areas were considerably darker than the covered parts. The conjunctiva and oral mucosa may be involved and even the hair may darken. The melanosis also affects internal epithelia and connective tissues. The urine is usually black or turns black on exposure to air and light. There were no reports of patients with vitiligo (Lerner and Moellmann, 1993). The location of the primary lesions varied (Lerner and Moellmann, 1993). In at least 7, perhaps 10, of the 35 patients for whom the site of origin of the melanoma was reported, the malignant transformation had occurred in congenital nevi, 1023

CHAPTER 53

Increases in the epidermal pigment are not as common. When present, it is located predominantly in the keratinocytes of the stratum basale and the skin color tends toward brown or black. The appearance is that of a normal epidermis, albeit one more darkly pigmented than would be expected on the basis of genetic skin type. Melanocytes may or may not appear to be turned on to produce more melanin. The melanocyte density is thought to be normal.

Differential Diagnosis Some rare conditions are similar in appearance. Legg (1884), more than a century ago, wondered whether his patient with metastatic melanosis had ingested silver nitrate because this man looked as if he were afflicted with argyria. The color of individuals with argyria resembles closely that of individuals afflicted with melanomatosis. Mild melanosis is clearly distinguishable from jaundice. Widespread metastases of the liver can produce a superimposed jaundice. If the adrenal glands have been destroyed by metastases the individual has three causes for hyperpigmentation, excessive melanotropin secretion from the adrenal insufficiency, melanomatosis, and jaundice. Patients with adrenal insufficiency without melanoma do not have melanuria and their skin darkens to brown without developing a bluish hue.

Pathogenesis

Fig. 53.3. Diffuse hypermelanosis in a patient with metastatic malignant melanoma (courtesy of Dr. Aaron Lerner). See also Plate 53.2, pp. 494–495.

none larger than 5–7 cm in its broadest dimension. Two, and perhaps four, patients had unknown primaries. In the two questionable cases, the disease was said to have arisen in one or both adrenal glands. Only one patient had a primary melanoma of the eye. In the remaining cases, the melanoma had developed in an acquired nevus or independently of an existing melanocytic lesion.

Histopathology The findings can be divided into two groups according to the location of the excess pigment, i.e., dermal alone and dermal plus epidermal. Dermal pigment is the hallmark of all melanoma-associated cases of cutaneous melanosis and the cause of the blue hue (Findlay, 1970). The pigment is primarily in macrophages, especially those located in the vicinity of microvessels. In addition, melanin may be found free among the connective tissue fibers, as part of cellular debris or in dermal fibroblasts, endothelial cells, and disseminated melanoma cells when such are present. Melanoma cells have been noted singly or in microscopic tumors in the dermis and in the lumina of dermal blood and lymphatic capillaries (Murray et al., 1999). 1024

In a hypothesis published in 1954, Fitzpatrick et al. suggested that the darkening in generalized melanomatosis in patients with metastatic disease was due to an increase of soluble precursors of melanin produced by the malignant melanocytes (Fitzpatrick et al., 1954). These intermediates diffuse into the circulation, are deposited in the skin and other tissues to produce the melanosis. Some are excreted by the kidneys and darken the urine. Rorsman et al. made the more specific suggestion that trichochromes, and possibly also polymers containing 6-hydroxy-5-methoxyindole-2-carboxylic acid (Rorsman et al., 1986), are deposited in tissues and cause the melanosis (Agrup et al., 1978, 1979; Tsukamoto et al., 1998). Trichochromes are pheomelanic pigments formed from 5-Sand 2-S-cysteinyldopa and are markedly elevated in the sera of patients with generalized melanosis due to melanoma. Silberberg et al. were the first to examine melanotic skin with an electron microscope (Silberberg et al., 1968). They determined that copious amounts of pigment were present in particulate form and that the epidermal component of the melanosis in their patient was due to increased melanogenic activity in melanocytes, possibly assisted by epidermal cytostasis secondary to chemotherapy. A hypothesis proposed in 1974/1980 by Wolff and coworkers suggested that the melanosis may be due in part to pigmented single cell metastases in the dermis and/or epidermis (Konrad and Wolff, 1974; Schuler et al., 1980). A solitary nest of malignant melanocytes has been seen within a small dermal vessel, probably a lymphatic (Murray et al., 1999). However, observations reported by others are not consistent with this

MIXED EPIDERMAL AND DERMAL HYPERMELANOSES AND HYPERCHROMIAS

view (Adrian et al., 1981; Gebhart and Kokoschka, 1981; Rorsman et al., 1986; Steiner et al., 1991). Lerner (1993) hypothesized that the anomalous presence of growth factors that stimulate the proliferation and melanogenic differentiation of normal and malignant melanocytes play a major role in producing the clinical event. Böhm et al. observed the blood levels of a-MSH, hepatocyte growth factor, and endothelin-1 were significantly elevated in the patient with melanosis from melanoma. And they suggested aberrant production of these factors may be responsible not only for activation of the pigment system in diffuse melanosis of metastatic melanoma, but also for increased proliferation, motility, and pigment incontinence of normal and malignant melanocytes (Böhm et al., 2001).

References Adrian, R. M., G. F. Murphy, S. Sato, R. D. Granstein, T. B. Fitzpatrick, and A. J. Sober. Diffuse melanosis secondary to metastatic malignant melanoma. Light and electron microscopic findings. J. Am. Acad. Dermatol. 5:308–318, 1981. Agrup, G., C. Lindbladh, G. Prota, H. Rorsman, A.-M. Rosengren, and E. Rosengren. Trichochromes in the urine of melanoma patients. J. Invest. Dermatol. 70:90–91, 1978. Agrup, G., P. Agrup, C. Hansson, H. Rorsman, and E. Rosengren. Diffuse melanosis and trichochromuria in malignant melanoma. Acta Derm. Venereol. 59:456–457, 1979. Böhm, M., M. Schiller, D. Nashan, R. Stadler, T. A. Luger, and D. Metze. Diffuse melanosis arising from metastatic melanoma: pathogenetic function of elevated melanocyte peptide growth factors. J. Am. Acad. Dermatol. 44:747–754, 2001. Carruth, M. R., G. D. Goldstein, and D. K. Tillman. Localized melanosis in an immunocompromised patient with local metastatic melanoma. Dermatol. Surg. 28:241–3, 2002. Findlay, G. Blue skin. Br. J. Dermatol. 83:127–134, 1970. Fitzpatrick, T. B., H. Montogomery, and A. B. Lerner. Pathogenesis of generalized dermal pigmentation secondary to malignant melanoma and melanuria. J. Invest. Dermatol. 22:163, 1954. Gebhart, W., and E. M. Kokoschka. Generalized diffuse melanosis

secondary to malignant melanoma. In: Pathology of Malignant Melanoma, A. B. Ackerman (ed.). New York: Masson, 1981, pp. 243–249. Konrad, K., and K. Wolff. Pathogenesis of diffuse melanosis secondary to malignant melanoma. Br. J. Dermatol. 91:635, 1974. Legg, J. W. Multiple melanotic sarcomata beginning in the choroid, followed by pigmentation of the skin of the face and hands. Trans. Path. Soc. 35:367–372, 1884. Lerner, A. B., and G. Moellmann. Two rare manifestations of melanomas: generalized cutaneous melanosis and rapid solar induction of showers of small pigmented lesions. A critical review of the literature and presentation of two additional cases. Acta Derm. Venereol. 73:241–250, 1993. Manganoni, A. M., F. Facchetti, A. Lonati, P. Calzavara, and G. De Panfilis. Generalized melanosis associated with malignant melanoma: unusual histologic appearance. Cutis 52:93–94, 1993. Pack, G. T., and I. M. Scharnagel. The prognosis for malignant melanoma in the pregnant woman. Cancer 4:324–334, 1951. Pec, J., L. Plank, E. Minarikova, E. Palencarova, Y. Rollova, Z. Lazarova, S. Auxtova, and L. Lauko. Generalized melanosis with malignant melanoma metastasizing to skin — a pathological study with S-100 protein and HMB-45. Clin. Exp. Dermatol. 18:454–457, 1993. Rorsman, H., P. Agrup, B. Carlen, C. Hansson, N. Jonsson, and E. Rosengren. Trichochromuria in melanosis of melanoma. Acta Derm. Venereol. 6:468–473, 1986. Schuler, G., H. Honigsmann, and K. Wolff. Diffuse melanosis in metastatic melanoma. Further evidence for disseminated single cell metastases in the skin. J. Am. Acad. Dermatol. 3:363–369, 1980. Silberberg, I., A. W. Kopf, and S. L. Gumport. Diffuse melanosis in malignant melanoma. Arch. Dermatol. 97:671, 1968. Steiner, A., K. Rappersberger, V. Groh, and H. Pehamberger. Diffuse melanosis in metastatic malignant melanoma. J. Am. Acad. Dermatol. 24:625–628, 1991. Tsukamoto, K., M. Furue, Y. Sato, O. Takayama, R. Akasu, N. Ohtake, K. Wakamatsu, S. Ito, K. Tamaki, and S. Shimada. Generalized melanosis in metastatic malignant melanoma: the possible role of DOPAquinone metabolites. Dermatology 197:338–342, 1998. Wagner, E. Fall von Combination eines Pigmentkrebses mit einer reinen Pigmentgeschwulst. Arch. Heilkunde 5:280–284, 1864.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

54

Drug-induced or -related Pigmentation Peter A. Lio and Arthur J. Sober

Cutaneous Pigmentation

Fixed Drug Eruption

Reflection, absorption, and back-scattering of incident light determine the remittance spectrum or color of human skin (Anderson and Parrish, 1981; Findlay, 1970; Jeghers, 1944). Approximately 5% of incident light is reflected off normal stratum corneum. The major visible light chromophores present in human skin include epidermal melanin, intravascular hemoglobin, bilirubin, and carotene. Deposition of strong chromophores into the epidermis (quinacrine) or superficial dermis (tattoo inks) alters the absorption of certain visible light wavelengths and, thus, skin color. Absorptive subtraction of specific wavelengths from the total remittance spectrum is straightforward; however, dermal interactions with incident light are more complex (see Chapters 27 and 28). Visible light that escapes corneal reflection, absorption by epidermal melanin, and epidermal remittance is largely scattered by dermal collagen fibers and absorbed by native intravascular chromophores. Shorter wavelengths of light are backscattered at more superficial dermal layers than longer wavelengths. Deposition of broadly absorbing pigments (melanin, melanin–drug complexes) into deeper layers of the dermis can change the color of dermal remittance. A portion of the penetrating blue light is backscattered in superficial dermal layers, while forward-scattered blue light and longer wavelengths are absorbed by deeply deposited dermal pigments. Therefore, preferential dermal backscattering of shorter wavelengths and deep dermal pigment absorption of longer wavelengths imparts a blue hue to dermal remittance in a phenomenon known as Rayleigh or Tyndall scattering (Bohnert et al., 2000; Prum and Torres, 2003). Increased epidermal melanization absorbs a large percentage of incident and scattered light and diminishes the effect of differential dermal backscattering on skin color. Diffuse dermal deposition of black/brown pigment also masks differential spectral scattering and results in dark-brown, gray, or black coloration. Drugs or chemicals which perturb cutaneous spectral remittance comprise the focus of the ensuing discussion. This section expands upon information previously presented in several reviews (Granstein and Sober, 1981; Kang et al., 1993; Lerner and Sober, 1986, 1988; Levantine and Almeyda, 1973; Sober and Granstein, 1981).

Historical Background

1026

A case of fixed drug eruption was first documented by Bourns in 1889 when he described a patient who developed sharply demarcated hyperpigmented lesions on the lips and tongue after ingesting 20 grains of antipyrine (phenazone). The term fixed drug eruption, l’eruption erythémato-pigmentée fixé, was coined five years later by Brocq when he described three cases of erythematous and pigmented fixed eruptions following administration of antipyrine (Brocq, 1894). In 1919, Goldenberg and Chargin reported the first case of fixed drug eruption in the American literature entitled “Dermatitis medicamentosa (arsphenamin) with pigmentation” (Korkij and Soltani, 1984).

Epidemiology In a series of 446 inpatients with drug eruptions, Kauppinen (1984) showed that 92 patients (21%) manifested fixed drug eruptions, second only to exanthematous eruptions. Although fixed drug eruption can occur at any age, it is most commonly seen in 21–40-year-old adults; fixed drug eruptions occur more often in men than women (Chan, 1984a; Shukla, 1981). A genetic predisposition to fixed drug eruption was suggested by Pellicano et al. (1994) after finding a significantly higher prevalence of B22 and Cw1 major histocompatibility complex (MHC) antigens in 36 unrelated patients with fixed drug eruption. They proposed that a biochemical interaction between offending drugs and these two MHC-I molecules on epidermal cells may render individuals susceptible to fixed drug eruptions. Further research in this vein revealed a strong relation between trimethoprim– sulfamethoxazole-induced fixed drug eruption and the HLA-A30 antigen (Ozkaya-Bayazit and Akar, 2001). These investigators suggested that perhaps susceptibility to fixed drug eruptions is conferred by an as yet unrecognized gene near the HLA-A locus.

Clinical Findings Clinically, fixed drug eruption produces well-circumscribed lesions that recur at fixed sites after each challenge with the offending agent. Although some patients may present with multiple lesions, fixed drug eruption classically appears as a

DRUG-INDUCED OR -RELATED PIGMENTATION

Fig. 54.1. The vesicular phase of fixed drug eruption (see also Plate 54.1, pp. 494–495).

Fig. 54.2. Late stage of fixed drug eruption showing residual brown to black hyperpigmentation (see also Plate 54.2, pp. 494–495).

solitary erythematous macule that subsequently evolves into an edematous dusky red plaque. Vesicles and bullae (Fig. 54.1) with crusting and/or desquamation may develop at a later stage (Korkij and Soltani, 1984). Pruritus and burning are common symptoms; associated constitutional symptoms are rare but have been reported (Sehgal and Gangwani, 1987). The acute phase inflammation spontaneously resolves leaving a brown to violet-brown (Fig. 54.2) to black (Fig. 54.3) hyperpigmented macule that persists for weeks to months and becomes more pronounced with each fixed drug eruption recurrence (Korkij and Soltani, 1984; Sehgal and Gangwani, 1987). Repeated attacks of fixed drug eruption may both increase the size of existing lesions and induce new lesions (Korkij and Soltani, 1984). Fixed drug eruption can occur at any mucocutaneous site; however, predilected areas include the lips, arms, legs, trunk, sacral regions, and genitalia, particularly the glans penis (Sehgal and Gangwani, 1987). Palms, soles, and nail folds can also be involved (Baran and Perrin, 1991; Korkij and Soltani, 1984). Sites of prior pathology, such as insect bites, vascular lesions, herpes zoster, or previous cellulitis may predetermine the site of fixed drug

Fig. 54.3. Blue-black hyperpigmentation from chronic fixed drug eruption.

eruption (Korkij and Soltani, 1984). Recent work by OzkayaBayazit (2003) elucidated preferential site involvement for several specific agents. Notably, co-trimoxazole most frequently produced lesions on genital mucosa, while naproxen induced lesions on the lips with a highly significant association. Fixed drug eruption typically presents one to two weeks after initiating drug therapy, but sensitization may require a few weeks to several years of drug exposure (Korkij and Soltani, 1984). Once a patient is sensitized to a drug, reexposure will cause an eruption within 30 minutes to 8 hours, with a mean of 2.1 hours (Korkij and Soltani, 1984; Sehgal and Gangwani, 1987). Patients with fixed drug eruption may demonstrate a refractory period of weeks to several months during which the offending drug fails to induce a reaction (Korkij and Soltani, 1984). In contrast to the classic form of fixed drug eruption, there are patients who develop a generalized bullous fixed eruption. Also, not all fixed drug eruptions result in hyperpigmentation (Shelley and Shelley, 1987b). The drugs associated with nonpigmenting fixed drug eruptions include pseudoephedrine hydrochloride, tetrahydrozoline hydrochloride, piroxicam, diflunisal, thiopental, iothalamate, and arsphenamine, although case reports exist for other individual agents as well (Benson et al., 1990; Krivda and Benson, 1994; Roetzheim et al., 1991; Shelley and Shelley, 1987b).

Histology Histopathologic features of fixed drug eruption include hydropic degeneration with subsequent pigmentary incontinence, scattered dyskeratotic keratinocytes, and subepidermal bullae (Lever and Schaumburg-Lever, 1990; Masu and Seiji, 1983). The upper dermis usually reveals vascular dilatation, marked edema, and a perivascular inflammatory cell infiltrate consisting of histiocytes, polymorphonuclear leukocytes, and mast cells (Sehgal and Gangwani, 1987). Masu and Seiji have proposed the following mechanism for the development of pigmentary incontinence: (1) lymphocytes migrate into the 1027

CHAPTER 54 Table 54.1. Substances producing fixed drug eruption.* Antimicrobials

Analgesics/antipyretics

Metals/halides

Miscellaneous

Acetarsone Acriflavine

Antimony potassium tartrate Arsenicals

Anthralin Belladonna alkaloids

Amodiaquine Amoxicillin

Acetanilide Acetaminophen (paracetamol) (Zemtsov et al., 1992) Aminopyrine Antipyrine

Bismuth subsalicylate Bromides

Ampicillin (Chan, 1984b)

Aspirin

Gold sodium thiosulfate

Chlorhexidine (Moghadam et al., 1991) Chloroquine phosphate Dapsone Erythromycin (Pigatto et al., 1984) Fluconazole (Morgan and Carmichael, 1994) Griseofulvin (Feinstein et al., 1984)

Cincophen

Iodine

8-Chlorotheophylline Chlorphenesin carbamate (Coskey, 1982) Dextromethorphan (Stubb and Reitamo, 1990b) Diethylstilbestrol

Phenacetin Salicylates Nonsteroidal anti-inflammatory drugs (NSAIDs) Diflunisal Ibuprofen

Mercurial salts Antihistamines Cyclizine

Disulfiram Eosin Ephedrine Epinephrine Ergot

Levamisole Methenamine Metronidazole (Shelley and Shelley, 1987a)

Meclofenamic acid Mefenamic acid (Long et al., 1992) Naproxen (Habbema and Bruynzeel, 1987)

Nystatin p-Aminosalicylic acid Penicillin G

Oxyphenbutazone Phenylbutazone Piroxicam (Stubb and Reitamo, 1990a) Sulindac (Bruce and Odom, 1986) Tolmentin

Dimenhydrinate Diphenhydramine (Dwyer and Dick, 1993) Sedatives/hypnotics Barbiturates Carbamazepine (Shuttleworth and Graham-Brown, 1974) Chloral hydrate Chlordiazepoxide Chlormezanone (Mohamed and Bahru, 1983) Ethchlorvynol (Auerbach, 1965) Lormetazepam (Jafferany and Haroon, 1988) Meprobamate Methaqualone (Slazinski and Knox, 1984) Temazepam (Archer and English, 1988)

Pipemidic acid (Miyagawa et al., 1991) Quinacrine Quinine Rifampin (Mimouni et al., 1990) Streptomycin

Tolphenamic acid (Autio and Stubb, 1993) Cardiac agents Digitalis

Sulfonamides

Hydralazine (Sehgal and Gangwani, 1986) Pentaerythritol tetranitrate

Tetracyclines (Minkin et al., 1969) Trimethoprim (Hughes et al., 1987)

Quinidine Tetraethylammonium

Erythrosin Formaldehyde Heroin (Westerhof et al., 1983) Ipecac (emetine) Isoaminile citrate Iothalamic acid Karaya gum Legumes Oral contraceptives Oxyphenisatin acetate Pamabrom (Nedorost et al., 1991) Papaverine (Kirby et al., 1994) Phenolphthalein Phenothiazine Pseudoephedrine Saccharin Sodium cacodylate Sulfobromophthalein sodium Strychnine sulfate

*Table modified from Korkij and Soltani (1984).

epidermis and attack and injure keratinocytes; (2) dermal macrophages migrate to the epidermis and phagocytose dyskeratotic keratinocytes and their melanin stores; (3) the macrophages return to the dermis and digest all keratinocyte remnants except the melanosomes, which resist degradation and tattoo the dermis (Masu and Seiji, 1983).

Pathogenesis Numerous drugs and substances have been reported to cause 1028

fixed drug eruption (Table 54.1). Fixed drug eruptions have been most commonly associated with analgesics, barbiturates, salicylates, sulfonamides, tetracyclines, antipyrine, chlordiazepoxide, dapsone, oxyphenbutazone, phenolphthalein, and quinine (Olumide, 1979; Shukla, 1981). Although fixed drug eruption is usually due to a single drug, there are patients in whom fixed drug eruption develops following the ingestion of chemically related drugs (cross sensitivity) or unrelated drugs (polysensitivity) (Kawada et al., 1994; Korkij and

DRUG-INDUCED OR -RELATED PIGMENTATION

Soltani, 1984; Mishra et al., 1990). Oral rechallenge is the gold standard for definitive diagnosis of fixed drug eruption; some authors have raised theoretical concerns that peroral provocation could potentially induce either a generalized bullous eruption or an anaphylactic reaction (Alanko et al., 1987; Sehgal and Gangwani, 1987). Occasionally, patch, scratch, or intracutaneous testing is helpful; positive patch tests are frequently seen with phenazone derivatives (antipyrine), barbiturates, and sulfonamides (Alanko et al., 1987; Granstein and Sober, 1981; Korkij and Soltani, 1984; Pigatto et al., 1984; Stubb and Reitamo, 1990b). The pathogenetic mechanism(s) and the factor(s) that contribute to the preferential localization of fixed drug eruption lesions to certain skin sites remains unknown. Studies have failed to demonstrate the presence of the offending drug or its metabolite in affected skin. Several in vivo and in vitro studies implicate an immune pathogenesis. One study reports elevated serum immunoglobulin G (IgG) and IgA, yet normal serum IgM, levels two days after the reappearance of fixed drug eruption. These immunoglobulins return to normal levels three weeks after the disappearance of the lesions (Theodoridis et al., 1977). Immunofluorescence microscopic studies have shown the presence of IgG, C3 and fibrin in intercellular spaces of affected epidermis (Korkij and Soltani, 1984). Wyatt et al. reported that serum samples obtained from active fixed drug eruption could elicit an inflammatory response if injected into a quiescent prior fixed drug eruption site (Wyatt et al., 1972). This serum factor was thermolabile and was thought to represent a protein-conjugated drug derivative; drug alone failed to produce a reaction at either normal or prior fixed drug eruption sites. A study by GimenezCamarasa et al. (1975) supported the presence of a thermolabile serum factor in vitro with a lymphocyte blast transformation assay. Exposure to causative drug alone produced no lymphocyte blast transformation; however, autologous serum from patients with active fixed drug eruption did induce blast transformation, which increased when the offending drug was added to the lymphocyte culture. The clinical course of fixed drug eruption suggests that a cutaneous factor possessing memory function is also involved. Hindsén et al. (1978) found CD8-positive T cells in the epidermis during both the acute and healing phase of fixed drug eruptions, thus suggesting that memory T suppressor/ cytotoxic cells may play an important role in mediating the epidermal damage seen in the fixed drug eruption. Teraki et al. (1994) observed in two patients that within 1.5 hours, oral drug rechallenge induced intense surface expression of intercellular adhesion molecule (ICAM)-1 in only lesional keratinocytes and vascular endothelium. The intensity of ICAM1 expression correlated with the degree of T cell invasion into the epidermis. In vitro studies using skin organ culture showed that tumor necrosis factor a or interferon g stimulated lesional keratinocytes and endothelium to express ICAM-1 more rapidly. This suggests that nonspatially specific drug-induced cytokine expression induces focal, rapid, and intense ICAM1 expression by keratinocytes and endothelial cells that may

activate and home disease-specific T cells to fixed cutaneous sites. Using flow cytometry in both active and inactive lesions, Teraki and Shiohara (2003) proposed that CD8-positive T cells producing interferon g act as effector cells in the epidermal injury process, while CD4-positive T cells produce interleukin (IL)-10 to play a regulatory role. These authors hypothesized that it is the balance between the CD4- and CD8-positive cells which determines activity of the fixed drug eruption. Autotransplantation experiments have yielded variable results. Full-thickness skin transplanted from a fixed lesion into normal skin retains its ability to react for several weeks before reverting to normal; normal skin transplanted into a fixed drug eruption lesion initially does not react but later becomes positive. Since grafted skin eventually adopts the characteristics of its new environment, this would suggest the presence of fixed yet transferable etiologic factors within the cutis or subcutis of reaction sites (Knowles et al., 1936; Korkij and Soltani, 1984).

Treatment Discontinuation of the offending agent is obviously the best treatment for fixed drug eruptions. Corticosteroid therapy may diminish the intensity of the reaction in fixed drug eruption without affecting the temporal course of the disease (Stitzler and Kopf, 1960). Antihistamines have been found to have no effect on fixed drug eruption (Feinstein et al., 1984).

Discoloration of Skin Caused by Metals and Drugs Metals Iron Ferric salt solutions used to treat Rhus dermatitis (Traub and Tennen, 1936), Monsel solution (ferric subsulfate) (Danau and Grosshans, 1981; Olmstead et al., 1980; Wood and Severin, 1979), and industrial exposure to a “pickling” fluid that contained ferric salts dissolved in hydrochloric acid (Hare, 1951) have all caused iron tattooing of the dermis. In these instances, acute spongiotic dermatitis, surgical wounding, or acid exposure disrupted the epidermal barrier and permitted penetration of iron salts. Perls stain highlights iron encrustation of collagen fibers and deposits in dermal macrophages. Cutaneous hyperpigmentation can complicate sclerotherapy of superficial veins. Extravasated red blood cells decompose and release their iron stores that become sequestered as hemosiderin within dermal macrophages. The vast majority of post–sclerotherapy-induced hyperpigmentation resolves within six months to two years. Some patients experience persistent pigmentation which may respond to treatment with the copper vapor laser (Thibault and Wlodarczyk, 1992). Red blood cells can extravasate through the capillary walls when the latter are inflamed. This condition is called capillaritis. Typically the patient notes pinpoint red macules on the skin of the lower legs (Fig. 54.4). The red lesions turn red1029

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Fig. 54.4. Hemosiderin pigmentation from chronic capillaritis.

Fig. 54.5. Extensive lichen aureus from iron deposits in the skin (see also Plate 54.3, pp. 494–495).

Fig. 54.6. Slate-gray color most prominent on sun-exposed skin (normal hand for comparison) (see also Plate 54.4, pp. 494–495).

tattoos which have been more problematic (Tope and Shellock, 2002). orange in color and persist indefinitely. If this process is extensive, the skin can acquire an orange color in a disorder known as lichen aureus (Fig. 54.5). The stain or orange color is due to the oxidation of iron to the ferric state. A single case of orange-brown staining of the toenail plates has been reported in a patient whose feet were exposed to rural well water with a high iron content (Olsen and Jatlow, 1984). Prussian blue staining and spectroscopic analysis of nail plate samples confirmed the presence of increased iron. Since the nail discoloration followed the pattern of the proximal nail fold, not the lunula, the authors concluded that iron had penetrated the nail plate from the outside. There was no evidence of systemic iron overload in this patient. Several cases of burning or pain within iron tattoos have been reported during magnetic resonance imaging (MRI) procedures (e.g., Kreidstein et al., 1997). This phenomenon has prompted some radiologists to refuse to perform MRI procedures on individuals with permanent cosmetic tattoos such as eyeliner or blush. A large survey study rebuffs this stance and attempts to delineate between cosmetic tattoos and decorative 1030

Platinum In one case, administration of the chemotherapeutic agent, cisplatin, produced a gray-white line at the gingival margin (Ettinger and Freeman, 1979). Cutaneous pigmentation has not been noted with this drug.

Silver Silver compounds, ingested orally or applied to mucosal surfaces were once popular for the treatment of gastrointestinal, upper respiratory tract, and genitourinary infections. Generalized, slate-gray pigmentation (Figs 54.6 and 54.7) characteristic of argyria often appeared after a few months to 25 years of silver compound use; however, the majority of cases manifested after two to three years of continuous or intermittent administration (Hill and Montgomery, 1941). Generalized argyria tends to be most prominent in sun-exposed areas; the nails (Fig. 54.8), mucous membranes, and sclerae may also be discolored. Occupational exposure to silver refinery furnace fumes has

DRUG-INDUCED OR -RELATED PIGMENTATION

A

B Fig. 54.7. A silvery color to the skin of a man with marked argyria. Discoloration is most noticeable in sun-exposed skin. The hand exhibits normal skin color for comparison (see also Plate 54.5, pp. 494–495).

Fig. 54.8. Azure discoloration of the nail plate from silver deposition (see also Plate 54.6, pp. 494–495).

Fig. 54.9 Routine histologic examination of a specimen from a patient with argyria showing an eccrine gland (A). Polarized demonstrating silver particles (B) especially prominent around the eccrine apparatus.

resulted in significant pulmonary absorption and the eventual development of generalized argyria (Bleehen et al., 1981). Localized pigmentary changes have been noted in both an ungloved hand chronically exposed to silver-containing photographic solution (Buckley, 1963) as well as a burn wound treated with silver sulfadiazine (Dupuis et al., 1985). Biopsy of a pigmented area of the skin of a photo-processing technician demonstrated silver deposition only within the papillary dermis adjacent to eccrine ducts, thus implying that the sweat ducts served as the conduit for percutaneous absorption. Histopathology reveals finely granular black pigment scattered throughout the dermis yet prominently aggregated within the membrana propria of eccrine glands (Fig. 54.9A, B) (Hill and Montgomery, 1941; Mehta et al., 1966). Less striking concentrations of silver grains appear at the dermoepidermal junction, around sebaceous glands and hair follicles, and in the walls of blood vessels. Dark field microscopy renders the silver granules brilliantly refractile. Electron 1031

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Fig. 54.11. Discoloration of the hands from chrysiasis; normal hands shown for comparison (see also Plate 54.8, pp. 494–495).

Fig. 54.10. A patient with rheumatoid arthritis treated with gold. Her skin has a lilac discoloration most visible in sun-exposed skin (see also Fig. 54.11 and Plate 54.7, pp. 494–495).

microscopy reveals electron-dense deposits within lysosomal structures of macrophages (Bleehen et al., 1981) and fibroblasts (Mehta et al., 1966). Electron-dense granules have also been found in association with elastic and collagen fibers (Bleehen et al., 1981; Mehta et al., 1966; Prose, 1963). X-ray microanalysis has definitively identified elemental silver in argyric lesions (Bleehen et al., 1981).

Gold The first descriptions of chrysiasis appeared in the 1920s and referred to gold-treated tuberculosis patients who developed blue-gray discoloration of sun-exposed skin (Schmidt, 1941). Today, gold therapy is used primarily as a treatment alternative for rheumatoid arthritis. Chrysiasis typically presents as a photodistributed, blue-gray hyperpigmentation over the face (Fig. 54.10), neck, upper chest, arms, and hands (Fig. 54.11); (Beckett et al., 1982; Schmidt, 1941)]. Gold tattooing of the dermis directly contributes to the cutaneous pigmentation noted in chrysiasis patients. Some data suggest that gold also increases cutaneous melanization (Leonard et al., 1986). Why the pigmentation is most prominent in sun-exposed areas is 1032

not clear although ultraviolet light has been reported to enhance local gold deposition (Leonard et al., 1986; Schmidt, 1941). Administration of greater than 50–150 mg/kg of cumulative gold sodium thiosulfate (20–60 mg/kg gold) predisposes to the development of chrysiasis after a latent phase of months to years. Lower cumulative doses of chrysotherapy result in microscopic deposition of gold in the skin but not gross pigmentary changes (Jeffrey et al., 1975). There are some data to suggest that certain individuals are more susceptible to chrysiasis. Rodriguez-Perez et al. (1994) demonstrated that, among patients with rheumatoid arthritis receiving gold therapy, human leukocyte antigen (HLA)-B27 antigen is associated with the development of chrysiasis. Gold therapy was linked to yellow discoloration of the toenails in a patient who received a cumulative dose of 2.5 g of gold sodium thiomalate (Fam and Paton, 1984). Unlike cutaneous pigmentation which may be permanent (Beckett et al., 1982; Granstein and Sober, 1981), nail discoloration clears as the nail plate grows distally. Light microscopy reveals finely granular black pigment around appendages, vessels, and nerves but not within appendages, the epidermis, or basement membranes (Millard et al., 1988; Schultz Larsen et al., 1984). Epipolarized light microscopy enhances the detection of gold particles. Transmission electron microscopy demonstrates dense particles primarily within macrophage lysosomes. Gold particles appear as both filaments and microtubules in fine crystalline patterns, as dense small and rod-like particles, and as larger irregular crystalline masses. Laser microprobe mass spectroscopy, X-ray microanalysis, and backscattered electron imaging have definitively identified gold within biopsy samples (Millard et al., 1988).

Mercury Early in this century, topical preparations of inorganic mercury compounds, such as ammoniated mercury, mercurous chlo-

DRUG-INDUCED OR -RELATED PIGMENTATION

ride, and mercurous oxide, were applied both as bleaching agents and psoriasis treatments (Burge and Winkelmann, 1970; Young, 1960). Pigmentary changes caused by topical mercurial creams were reported first in 1922 and most recently in 1990 (Burge and Winkelmann, 1970; Dyall-Smith and Scurry, 1990; Goeckermann, 1922; Hollander and Baer, 1929; Kern, 1969; Lamar and Bliss, 1966; Lang, 1988). Industrial cutaneous exposure to mercurial salts has caused not only cutaneous hyperpigmentation but also signs and symptoms of systemic mercury intoxication or hydrargyrosis (Kennedy et al., 1977). Rarely, prolonged application of topical mercurial vanishing creams has resulted in significant systemic absorption. One woman who had applied a relatively high percentage 17.5% mercuric ammonium chloride preparation daily for 18 years presented with perifollicular hyperpigmentation, elevated blood and urine mercury levels, and possible neuropsychiatric toxicity (Dyall-Smith and Scurry, 1990). Mercury-induced hyperpigmentation arises in areas directly contacted by mercurial salts. This pigmentation is most pronounced in skin creases, the periorbital region, and around hair follicles (Burge and Winkelmann, 1970; Dyall-Smith and Scurry, 1990; Kennedy et al., 1977; Lamar and Bliss, 1966). Mercury penetrates the epidermis via its appendages and then crosses the appendageal epithelium into the dermis by an unclear mechanism (Scott, 1959; Witten, 1957). Once in the dermis, mercury ions, presumably, inhibit melanin synthesis by displacing copper from tyrosinase, thus rendering this apoenzyme inactive (Lerner, 1952). However, contrary to mercury’s purported therapeutic indication, long-term use of mercurial creams induces hyperpigmentation as a consequence of heavy metal tattooing of the dermis in conjunction with increased epidermal melanin deposition (Burge and Winkelmann, 1970; Lamar and Bliss, 1966). Routine histopathology and electron microscopy have localized upper dermal metallic granules, free in the dermis, in association with elastic fibers, and within macrophages (Burge and Winkelmann, 1970). Silver stains have revealed increased basal layer epidermal melanin content (Burge and Winkelmann, 1970). Dark field microscopy demonstrates brilliantly refractile granules within the upper dermis as well as in the upper stratum corneum if mercury exposure were relatively recent (Burge and Winkelmann, 1970; Kennedy et al., 1977). Both neutronactivation and analytical electron microscopy have confirmed the presence of elemental mercury in specimens derived from hyperpigmented patients (Burge and Winkelmann, 1970; Kennedy et al., 1977). While neutron-activation spectroscopy must be performed blindly on a bulk sample, analytical electron microscopy allows for simultaneous structural and elemental analysis of discrete particles. Once within dermal macrophages the mercury tattooed areas are unlikely to resolve spontaneously; however, perifollicular deposits may rapidly disappear after stopping topical mercurials (Dyall-Smith and Scurry, 1990). Slate-gray dermal mercury tattoos can be treated with dermabrasion although iatrogenic postinflammatory hyperpigmentation is always a concern (Lang, 1988).

Fig. 54.12. Hyperchromatic margins of the gums from deposition of lead (see also Plate 54.9, pp. 494–495).

Lead Ingestion or inhalation of lead-bearing compounds present in the home or at work accounts for the vast majority of lead intoxication (Bruggenkate et al., 1975). The symptoms of plumbism include lethargy, gastrointestinal distress, peripheral neuritis, and encephalopathy. Eighty percent of patients with chronic lead intoxication present with a characteristic bluegray, Burton line over the marginal gingivae (Fig. 54.12) [(Bruggenkate et al., 1975; Dummett, 1964)]. Bacterially generated hydrogen sulfide may precipitate lead in the gingival tissues. Gingival biopsy has demonstrated dark-brown pigment within the subepithelial connective tissue. Qualitative electron microprobe analysis has confirmed the presence of lead within the Burton lines (Bruggenkate et al., 1975). Gingival pigmentation may be the sole clue to chronic lead toxicity, as reported in an institutionalized patient with precedent mental retardation and pica syndrome (Lockhart, 1981). Calcium ethylenediamine tetraacetic acid, dimercaptoethanol (BAL), and D-penicillamine represent chelating agents employed in the treatment of lead toxicity. Elimination of lead exposure, chelation therapy, and improved oral hygiene may result in the slow resolution of gingival pigmentation (Bruggenkate et al., 1975). While pigmentation of the skin has not been reported with lead intoxication, nail hyperpigmentation has been reported in an infant treated daily with Tao Dan® powder, a leadbearing absorbent compound (Shengda and Dean, 1989). This infant had a significantly elevated blood lead level. Energy dispersive X-ray microanalysis confirmed the presence of lead within the nail plate. Nail plate proteins contain numerous sulfhydryl groups that can avidly bind lead. Discontinuance of the powder resulted in restoration of normal nail plate color.

Bismuth Bismuth compounds have been used to treat venereal disease, psoriasis, lichen planus, and inflammatory gastrointestinal 1033

CHAPTER 54

disorders (Dummett, 1964). Long-term oral or parenteral bismuth administration has produced generalized slate-gray hyperpigmentation, also known as bismuthia, which is often accentuated over the face, neck, and dorsa of the hands (Leuth et al., 1936; Zala et al., 1993). Gingival and oral mucosal hyperpigmentation may accompany cutaneous discoloration. As proposed for lead deposition, oral bacterial flora produce hydrogen sulfide that may precipitate bismuth in the marginal gingivae (Dummett, 1964). Bismuth-induced pigmentation is permanent and may present without the nephropathy, stomatitis, vomiting, diarrhea, and neurologic disorders typical of bismuth poisoning. Chelating agents, such as BAL, are indicated only in patients demonstrating systemic bismuth toxicity and blood bismuth levels greater than 200–300 mg/L (Zala et al., 1993). Biopsy reveals finely granular, non-doubly refractile, dark pigment scattered in the papillary and reticular dermis (Leuth et al., 1936; Zala et al., 1993). Basic qualitative chemical analysis has been used to prove the presence of bismuth in bulk biopsy specimens (Leuth et al., 1936). The staining method of Christeller and Komaya has identified bismuth particles as brown granules on fixed tissue sections (Zala et al., 1993). Although not reported, analytical electron microscopy or laser probe spectroscopy could be used to demonstrate the presence of bismuth.

Semimetals Arsenic Inorganic trivalent arsenic preparations, such as Fowler solution (liquor potasii arsenites or potassium arsenite), have been prescribed for the treatment of psoriasis, folliculitis, lichen planus, pityriasis rubra pilaris, and dermatitis herpetiformis (Cuzick et al., 1982; Levantine and Almeyda, 1973). Injected pentavalent organic arsenic compounds were employed as treatments for syphilis (Sutton, 1923). Water supplies may also be contaminated with arsenic (Cebrian et al., 1983). Prolonged ingestion of inorganic arsenic can result in the development of diffuse macular hyperpigmentation, particularly over the trunk. Within the hyperpigmented macules reside several small islands of normally pigmented skin, which produce a “rain drop” appearance (Fig. 54.13). Arsenical keratoses, particularly over the hands and feet (Fig. 54.14), often accompany the cutaneous hyperpigmentation. One study of patients who had been exposed to arsenic-tainted well water estimated the threshold cumulative dose necessary to induce hyperpigmentation to be 3 g (Cebrian et al., 1983). The latent period between exposure and appearance of pigmentary changes ranges from 1 to 20 years. Bartolome et al. (1999) reported a case of acute arsenic ingestion of between 8 g and 16 g of sodium arsenite by a psychotic woman who subsequently developed erythroderma within five days. Biopsy of the eruption revealed the presence of multiple small pigment granules in and around histiocytes and throughout the collagen fibers of the papillary dermis. These granules stained positively with Fontana-Masson and negatively with periodic

1034

Fig. 54.13. Leucomelanoderma from ingestion of arsenic. Note both hyperpigmented skin and small hypopigmented macules (courtesy of Dr. Claire Beylot).

acid-Schiff and HMB-45, supporting the notion that they represented arsenic deposition at an early stage. Routine histopathologic examination classically reveals basal layer and dermal melanin deposition (Montgomery and Ornsby, 1954). Osborne’s method of staining has demonstrated arsenic-containing crystals within the dermis and epidermis (Montgomery and Ornsby, 1954). Arsenic may promote melanin synthesis by binding sulfhydryl groups, thus liberating copper ions to activate tyrosinase (Levantine and Almeyda, 1973).

Tellurium Tellurium is a heavy element which is used in the vulcanizing of rubber, as a diagnostic test for diphtheria, as an additive to cast iron, steel, and copper, as a glass coloring agent, and as a chemical catalyst (Blackadder and Manderson, 1975). Laboratory exposure of two workers to tellurium hexafluoride gas resulted in characteristic signs of tellurium toxicity, the most distinctive of which is a strong garliclike odor of breath and excreta. Additionally, these exposed workers manifested

DRUG-INDUCED OR -RELATED PIGMENTATION

A

B

Fig. 54.14. Punctate keratoses on the palms (A) and soles (B) of the man with a history of arsenic ingestion taken as a tonic during childhood.

hyperpigmented patches over their face, neck, and finger webs (Blackadder and Manderson, 1975). The skin discoloration faded over several weeks. Biopsy samples were not analyzed.

Tattoos Tattooing involves the implantation of pigmented particles into the dermis (Fig. 54.15) (Morgan, 1974). Commonly used chromophores include: carbon (black), cinnabar, a combination of mercuric and cadmium sulfide (red) (Fig. 54.16), chromium oxide (green) (Figs 54.17 and 54.18), cadmium sulfide (yellow), cobalt (blue) (see Figs 54.17 and 54.18), ferric salts (brown/pink), manganese (purple), and titanium oxide (white). Photoallergic reactions to cadmium sulfide and manganese have been reported (Goldstein, 1967; Nguyen and Allen, 1979). Eczematous and granulomatous reactions to mercury, cobalt, and chromium salts may also complicate cutaneous tattoos (Fig. 54.19) (Beerman and Lane, 1957; Fregert and Rorsman, 1966; Hirsh and Johnson, 1984; McGrouther et al., 1977; Schmidt and Christensen, 1978). Most tattoos are intentional. However, accidental cutaneous

injury may introduce a variety of substances into the dermis. Abrasions from falling on roads or streets often are contaminated with tiny particles of dirt, which may persist indefinitely in the skin, thus producing a traumatic tattoo. For example, a piercing pencil injury may deposit carbon/graphite particles into the dermis and cause a permanent tattoo. Gunpowder (Fig. 54.20) ejected from the end of a gun barrel can penetrate the skin and form a spray of small (1 mm) blue macules and papules. Before the advent of laser therapy, tattoo ablation options were limited to surgical excision, deep dermabrasion, salabrasion, or destruction with topical acids (Morgan, 1974). Use of the Q-switched ruby and neodymium:yttrium aluminum garnet (Nd:YAG) lasers allows for removal of tattoos with reduced dermal scarring (Kilmer and Anderson, 1993; Taylor et al., 1990). Recently, it has been reported that laser treatment of tattoos may trigger local and generalized allergic reactions by liberating pigment antigens from within histiocytes and exposing them to the immune system (Ashinoff et al., 1995). In patients with history of laser-triggered allergy to tattoo pigments, pretreatment with systemic corticosteroids

1035

CHAPTER 54

Fig. 54.15. Professional tattoo.

Fig. 54.16. Professional tattoo illustrating yellows and reds (see also Plate 54.10, pp. 494–495).

and antihistamines can prevent subsequent allergic reactions (Ashinoff et al., 1995). Imiquimod, a topical immune-response modifier, has been shown to be effective in tattoo removal in a guinea pig model when applied soon after application of the tattoo (Solis et al., 2002). Thus, further study may determine a role for this modality, perhaps as an adjunct or alternative to laser tattoo removal.

Carotenoids b-Carotene Carotene, the precursor of vitamin A (provitamin A), is a yellowish lipochrome found in several green, yellow, and orange fruits and vegetables, such as leafy greens, broccoli, carrots, squash, sweet potatoes, oranges, apricots, cantaloupe, mango, and papaya (Lescari, 1981). Cooking, pureeing, or mashing of vegetables liberates carotene crystals from within the plant cell walls and, thus, significantly increases its bioavailability (Lescari, 1981; Micozzi et al., 1988). Carotene is also found in butter, eggs, milk, certain vegetable oils, and purified supplements. About one-third of ingested carotene is absorbed. Some of the absorbed carotene is converted in the small intestine and liver to vitamin A. Since this conversion is slow, carotenemia does not lead to hypervitaminosis A. 1036

Fig. 54.17. Professional tattoo illustrating pinks, blues, greens, and light purples (see also Plate 54.11, pp. 494–495).

Carotenemia refers to a yellow discoloration of the skin (Fig. 54.21) most commonly due to ingestion of excess carotene-bearing foods. Hypothyroidism, diabetes mellitus, hyperlipidemias, and nephrotic syndrome manifest elevated b-lipoproteins, the major carriers of serum carotene, and

DRUG-INDUCED OR -RELATED PIGMENTATION

Fig. 54.18. Artistic tattoo showing multiple colors and shades (see also Plate 54.12, pp. 494–495).

predispose to carotenemia (Lescari, 1981; Stack et al., 1988; Walton et al., 1965). Rare cases of carotenemia are due to inborn errors of metabolism that interfere with conversion of carotene to vitamin A (Jones and Black, 1994; McLaren and Zekian, 1971; Sharvill, 1970). Excess serum carotene is deposited in the skin, with a predilection for the subcutaneous fat and stratum corneum (Greenberg et al., 1958; Lescari, 1981). Sweat, sebum, and urine may contain significant quantities of carotene. Nonkeratinized epithelia do not have an affinity for carotene; thus, unlike jaundice, the sclerae and oral mucous membranes do not display yellow discoloration in carotenemia. Serum carotene concentration above 250 µg/dL results in obvious skin yellowing, first appearing over the palms, soles, nasolabial folds, tip of the nose, and forehead, then progressing to involve the dorsal digits, buttocks, breasts, abdomen, chin, and postauricular areas (Lescari, 1981; Stack et al., 1988). A careful history and physical examination should be able to distinguish carotenemia from jaundice. Serum carotene and bilirubin levels should be measured in equivocal cases. Addition of alcohol to a serum sample will result in an organic

Fig. 54.19. Tattoo with granuloma in skin bearing red colors due to an allergic reaction to cinnabar, a compound made from mercury. The red areas are inflamed.

Fig. 54.20. Gunpowder tattoo.

phase that is yellow in hyperbilirubinemia but clear in carotenemia. Conversely, petroleum ether added to a serum sample will create a yellow organic layer in carotenemia yet a clear organic phase in hyperbilirubinemia (Stack et al., 1988). 1037

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Fig. 54.21. Yellow-colored hands from ingestion of beta-carotene causing carotenoderma. Normal hand included for comparison (see also Plate 54.13, pp. 494–495).

Fig. 54.22. Blue-black chromophore deposits in sun-exposed areas of the face of a man taking amiodarone (see also Plate 54.14, pp. 494–495).

In the short term, carotenemia is harmless; however, longstanding carotenemia has been associated with weakness, weight loss, hepatomegaly, neutropenia, hypotension, and amenorrhea. Yellow skin color will return to normal within two to six weeks after dietary retinol restriction. The differential diagnosis of xanthoderma includes lycopenemia (see below) and ingestion or percutaneous absorption of quinacrine, saffron, picric acid, tetryl, and dinitrophenol (Lescari, 1981).

agent does not cause hypervitaminosis A. Retinopathy, hepatitis, cutaneous hypersensitivity, and aplastic anemia have been associated with use of this agent as a tanning pill (Bluhm et al., 1990).

Lycopene Lycopene is an isomer of b-carotene that is found in beets, tomatoes, rose hips, bittersweet berries, and chili beans (Baran and Perrin, 1991). Although the pigmentary change induced by lycopene can mimic carotenemia, lycopenemia usually imbues an orange hue. Since lycopene is a more intensely colored pigment, staining of the skin and fat can occur at serum concentrations lower than that necessary to induce carotenemia. Lycopenemia manifests over the palms and soles, spares the sclerae, but may involve the hard palate. Simultaneous lycopenemia and carotenemia has been reported in patients who ingest large amounts of tomatoes and yellow vegetables (Hughes and Wooten, 1966).

Canthaxanthin Canthaxanthin is a carotenoid present in crustaceans, fish, bird feathers, mushrooms, algae, food dyes, and yellow and orange vegetables (Jackson, 1981). When ingested and deposited in the subcutaneous fat and epidermis, this orangecolored chromophore simulates a tan. Preparations of this pigment, such as Orobronze, have been distributed contrary to the US Food and Drug Administration (FDA) recommendations as oral tanning agents; however, the skin coloration produced by this agent is not protective against ultraviolet radiation. Canthaxanthin has also been used in the management of photosensitivity disorders and vitiligo (Gupta et al., 1988). Since canthaxanthin is not a vitamin A precursor, this 1038

Amiodarone Amiodarone is a diiodinated benzofuran used to suppress both supraventricular and ventricular cardiac arrhythmias. After approximately four months of amiodarone therapy, 30–76% of patients have experienced photosensitivity, predominantly to UVA but some to UVB (Chalmers et al., 1982; Harris et al., 1983a, b; Rappersberger et al., 1989; Roupe et al., 1987; Waitzer et al., 1987; Zachary et al., 1984). Of patients taking amiodarone, 1–10% will develop cutaneous, slate-gray hyperpigmentation after an average of 20 months and a minimum of 160 g of amiodarone therapy (Bucknall et al., 1986; Casillas et al., 1978; Harris et al., 1983a, b; Matheis, 1972; Rappersberger et al., 1989). Over several months to two years after discontinuing amiodarone, the pigmentary changes tend to fade (Matheis, 1972; Rappersberger et al., 1989; Trimble et al., 1983). One patient who had amiodarone-induced facial hyperpigmentation for four years noted complete clearance of this discoloration approximately six months after lowering the dose to 200 mg/day. Subsequently, the patient continued to take amiodarone at this dose for two years without developing recurrent hyperpigmentation (Beukema and Grayboys, 1988). Amiodarone-induced hyperpigmentation is most prominent in sun-exposed areas (Figs 54.22 and 54.23) (Weiss et al., 1984) and may be initiated by a phototoxic reaction (Rappersberger et al., 1989; Trimble et al., 1983; Wanet et al., 1971). Ultraviolet radiation-induced free radical generation may predispose to amiodarone accumulation within dermal cells in sun-exposed areas (Alinovi et al., 1985; Miller and McDonald, 1984). High-performance liquid chromatography has demonstrated levels of amiodarone and its major metabolite, desethylamidarone, to be approximately ten times greater

DRUG-INDUCED OR -RELATED PIGMENTATION

Fig. 54.24. The tibial surface of a patient taking minocycline showing blue discoloration from deposition of the drug chelated with metal ions (see also Plate 54.16, pp. 494–495).

tion with the Q-switched ruby laser. This yielded complete resolution of the pigment after one treatment, with no recurrence after a year of follow-up, despite continuing on amiodarone.

Tetracyclines Minocycline

Fig. 54.23. Close-up of the patient in Fig. 54.22, showing blueblack discoloration of the skin (see also Plate 54.15, pp. 494–495).

in sun-exposed pigmented tissue compared with those in nonexposed skin (Adams et al., 1985; Zachary et al., 1984). Histologically, biopsies disclose yellow-brown refractile granules in the dermis, especially around vessels. Sudan black stain accents the lipid-laden dermal deposits. Electron microscopy localizes electron-dense material to membranebound, lysosomal-like structures within histiocytes, endothelial cells, smooth muscle cells, fibroblasts, pericytes, polymorphonuclear leukocytes, and keratinocytes (Adams et al., 1985; Miller and McDonald, 1984; Trimble et al., 1983; Waitzer et al., 1987; Zachary et al., 1984). Four types of lysosomal deposits have been described: (1) lamellar or myelin figurelike inclusion (most typical), (2) electron dense, (3) membrane-bound electronlucent, and (4) mixed (Waitzer et al., 1987). Within these lysosomal inclusions, electron probe microscopy has disclosed definite iodine peaks, suggesting the presence of amiodarone or a metabolite. Thus, it has been hypothesized that amiodarone accumulation in lysosomes represents a drug-induced lipidosis (Trimble et al., 1983; Waitzer et al., 1987). Although the pigmentation tends to self-resolve after cessation of amiodarone, for some patients this is not possible. Karrer et al. (1999) successfully treated the hyperpigmenta-

Minocycline is a tetracycline derivative frequently employed to treat acne vulgaris. Oxidation turns yellow crystalline minocycline black. At doses of 200 mg/day, vestibular toxicity is the most frequent side effect. However, continuous administration can result in localized blue-black hyperpigmentation of inflamed or previously scarred skin (type I), bluegray pigmentation of previously normal extremity skin (type II) (Fig. 54.24), or diffuse muddy brown discoloration of sunexposed areas (type III) (Argenyi et al., 1987; Basler and Kohnen, 1978; Basset-Séguin et al., 1995; McGrae and Zelickson, 1980; Pepine et al., 1993; Ridgway et al., 1982; Sauer, 1979; Simons and Morales, 1980). Face, lower extremities, and arms are common targets for minocycline-induced pigmentary changes. Nail beds, sclerae, mucous membranes (Fig. 54.25) and teeth (Fig. 54.26) may also be affected (Pepine et al., 1993). Minocycline has been associated with extracutaneous pigmentation of thyroid, cartilage, bones, and brain in humans (Attwood and Dennett, 1976; Gordon et al., 1984) and laboratory animals (Benitz et al., 1967). Pigmentary changes from minocycline have been noted as early as nine weeks after starting minocycline at a dose of 100 mg twice per day (cumulative dose = 12.1 g) (Ridgway et al., 1982). After discontinuing minocycline, the induced pigmentation tends to slowly fade over months to years. Histopathologic findings vary depending on the type of minocycline-induced pigmentation (Argenyi et al., 1987). Biopsies of type I pigmentation have revealed both intra- and extracellular, iron-positive pigment granules within the dermis (Basler and Kohnen, 1978; Fenske et al., 1980). Electron microscopic analysis of the type I pigment granules identifies hemosiderin- and ferritin-like membrane-bound particles as 1039

CHAPTER 54

Similar to the case with amiodarone-induced pigmentation, Q-switched lasers have also proved to be effective at treating minocycline-related pigmentation, particularly that of type II (Green and Freedman, 2001).

Tetracycline

Fig. 54.25. Hyperpigmentation of the oral mucosa from minocycline (see also Plate 54.17, pp. 494–495).

Osteoma cutis is a rare manifestation of acne vulgaris and appears as small blue-gray nodules over the face. Some reports have blamed tetracycline treatment for the appearance of these lesions (Basler et al., 1974; Walter and Macknet, 1979). One acne patient developed osteoma cutis after four years of tetracycline therapy and fluorescence microscopy identified a chromophore consistent with tetracycline (Walter and Macknet, 1979). Furthermore, no new lesions of osteoma cutis developed after tetracycline was discontinued. Whether this association between tetracycline and osteoma cutis is etiologic or coincidental remains to be discerned. In addition, tetracycline has been reported to produce yellow lunulae (Hendricks, 1980).

Methacycline

Fig. 54.26. Discoloration of the teeth (see also Plate 54.18, pp. 494–495).

well as non–membrane-bound inclusions. Type II biopsies have demonstrated predominantly intracellular pigment granules within the dermis and subcutis (Argenyi et al., 1987; McG1rae and Zelickson, 1980; Ridgway et al., 1982). Perls and Fontana-Masson stains show the pigment granules to have characteristics of both iron and melanin. Electron microscopy has identified electron-dense, iron-containing particles in siderosomes, within dermal histiocytes, free within the cytoplasm, or, rarely, scattered among dermal collagen fibers (Sato et al., 1981). Since electron microscopy has not revealed increased numbers of melanosomes and the pigment does not bleach with hydrogen peroxide, it has been hypothesized that the Fontana-Masson stain is detecting an oxidized minocycline metabolite, which has chelated iron and, to a lesser degree, calcium (Argenyi et al., 1987). X-ray analysis of types I and II specimens has found evidence of iron, calcium, sulfur, and chlorine with the pigment granules (Argenyi et al., 1987; Ridgway et al., 1982; Sato et al., 1981). Type III biopsies are remarkable for increased basal layer and dermal melanization without evidence of increased iron deposition (Simons and Morales, 1980). 1040

Methacycline is another tetracycline derivative that has been reported to produce gray-black pigmentation of light-exposed areas of the skin and yellow-brown pigmentation of the conjunctiva in 7/250 patients studied (Walter and Macknet, 1979). Cumulative doses ranged from 420 g to 1575 g over 2–7.5 years. Salient histologic features included moderate basal layer pigmentation, elastotic changes of the upper and intermediate dermis, pigmented macrophages that variably stained positive with Fontana-Masson stain, and refractile dermal granules that stained with von Kossa stain. Electron microscopy defined the basal layer and macrophage pigment as consistent with melanin. Dermal elastic fibers were found to contain streaks of osmiophilic, irregularly shaped granules.

Ochronosis Ochronosis refers to tissue deposition of microscopic, ochrecolored pigment, which when present in the dermis imparts a blue-black hue to the skin. Inherited deficiency of homogentisic acid oxidase results in accumulation of polymerized homogentisic acid in skeletal, cardiovascular, genitourinary, respiratory, ocular, and cutaneous tissues. This inborn error of amino acid metabolism defines alkaptonuria or endogenous ochronosis (Albers et al., 1992). Hydroquinone, phenol, resorcinol, and antimalarial agents represent pharmacologic agents that may induce exogenous ochronosis (Figs 54.27and 54.28).

Phenolic Compounds Topical preparations of hydroquinone serve as popular treatments for epidermal pigmentary disorders, such as melasma (Engasser and Maibach, 1981). Chronic application of hydroquinones in concentrations as low as 2% may result in localized dermal pigmentation that typically presents as well-demarcated, blue-black pigmented facial macules, which are often permanent (Bentley-Philips and Bayles, 1975; Cullison et al., 1983; Dogliotti and Leibowitz, 1979;

DRUG-INDUCED OR -RELATED PIGMENTATION

Fig. 54.27. Localized ochronosis of the face from excessive use of hydroquinone (see also Plate 59.4, pp. 494–495).

Hardwick et al., 1989; Howard and Furner, 1990; Lawrence et al., 1988). Hydroquinone creams have also caused nail plate pigmentation, possibly due to photopolymerization of absorbed hydroquinone into a brown pigment (Arndt and Fitzpatrick, 1965; Mann and Harman, 1983). This nail discoloration disappeared one month after discontinuing hydroquinone creams (Mann and Harman, 1983). Phenol-containing medicaments have produced pigmentary changes in cartilage, skin, sclerae, and urine, that are nearly indistinguishable from endogenous ochronosis (Beddard and Plumtre, 1912; Berry and Peat, 1931; Cullison et al., 1983). Carbolic acid (phenol) dressings used for the treatment of chronic skin ulcers have induced cutaneous ochronosis (Brogren, 1952). Hair tonics containing phenolic compounds have been linked to hyperpigmentation of the scalp and palms (Forman, 1975). Topically applied resorcinol (Howard and Furner, 1990) in acne preparations, in Castellani’s paint, or as a consequence of industrial exposure (Dupre et al., 1979) as well as topical picric acid (Cullison et al., 1983) burn treatments have caused localized ochronosis. Biopsy of hyperpigmented ochronotic areas has revealed dermal deposits of ochre (yellow-brown) pigment as fine gran-

Fig. 54.28. Patient in Fig. 54.27 showing discoloration of the chin from localized ochronosis due to excessive application of hydroquinone.

ules both free and within macrophages (Albers et al., 1992; Attwood and Dennett, 1976; Findlay et al., 1975). Larger crystalline masses of similar pigment are interposed between collagen bundles. These irregularly shaped, crystalline masses represent degenerated collagen fibers interspersed with ochronotic pigment. Pigment granules also have been found within elastic fibers. Larger pigment clumps were more common in areas of solar elastosis, suggesting that solar radiation promoted pigment deposition. By analogy to endogenous ochronosis, it has been proposed that the pigment of exogenous ochronosis also represents polymerized homogentisic acid. Both endogenous and exogenous ochronosis are indistinguishable under the microscope as both manifest as golden brown (ochre) pigment deposits that stain positive with Fontana-Masson and negative with Perls reagents. Unlike melanin, neither endogenous nor exogenous ochronosis bleaches with hydrogen peroxide treatment (Attwood and Dennett, 1976). The phenolic compounds, hydroquinone, picric acid, and phenol may be oxidized and enzymatically converted to hydroxyindole molecules which can polymerize and form ochronotic pigment (Cullison et al., 1041

CHAPTER 54

Fig. 54.29. Patient with atabine-induced lichen planus-like eruption. Note the brown-violaceous discoloration of the skin of the abdomen and the yellow color of the extremities (see also Plate 54.19, pp. 494–495).

1983). Alternatively, it has been suggested that these phenolic compounds may inhibit homogentisic acid oxidase, thus mimicking the mechanism of endogenous ochronosis (Howard and Furner, 1990). Treatment modalities are limited although Kramer et al. (2000) showed some success with the Q-switched ruby laser.

Antimalarials Antimalarial compounds, such as chloroquine, hydroxychloroquine, quinine, quinacrine, amodiaquine (Fig. 54.29) may induce cutaneous hyperpigmentation in 8–25% of patients taking these medicines for longer than three to four months (Bruce et al., 1986; Campbell, 1960; Leigh et al., 1979; Levy, 1982; Ludwig et al., 1963; Lutterloh and Shallenberger, 1946; Sugar and Waddell, 1946; Tuffanelli et al., 1963). Typically, the ochronosis-like, gray to blue-black pigmentation is distributed over the anterior tibiae, face, hard palate, gums, sclerae, and subungual areas. Discoloration of the hard palate is usually the initial manifestation. Quinine shots have produced localized pigmentary changes over the buttocks (Bruce et al., 1986). Discontinuance of the offending agents has led to decreased intensity, but not complete resolution, of the hyperpigmentation. Hydroxychloroquine has been noted to bleach hair in a smaller percentage of individuals (Fig. 54.30). The bleaching is reversible when the medication is discontinued. Histopathologic examination of antimalarial ochronosis has revealed classic ochronotic changes as well as focal staining for melanin and hemosiderin. Since chloroquine has a high affinity for melanin (Sams and Epstein, 1965), it has been proposed that melanin is the pigmented polymer responsible for cutaneous discoloration. In a patient with localized, quinine shot-induced cutaneous pigmentation, reverse-phase highpressure liquid chromatography analysis of a biopsy specimen revealed an oxidized metabolite of quinine (Bruce et al., 1986). The time interval between sampling and the last dose of quinine was approximately 70 years. 1042

Fig. 54.30. Patient taking chloroquine exhibits loss of hair color (see also Plate 54.20, pp. 494–495).

In another patient who developed anterior tibial pigmentation secondary to Atabrine, direct tissue analysis found almost twofold higher levels in a pigmented area than in a nonpigmented area. In addition, fluorescence microscopy demonstrated that pigment granules fluoresced at the same wavelength as pure Atabrine. Thus, several lines of evidence suggest that melanin–antimalarial drug complexes and hemosiderin account for the pigment composition. The presence of melanin within this pigment creates a semantical argument against assigning antimalarial drug-induced pigmentation as a form of ochronosis which, classically, refers to a nonmelanin polymer of phenolic metabolite monomers (Cullison et al., 1983). More commonly than it induces hyperpigmentation, quinacrine imbues a lemon yellow hue to the skin secondary to deposition in the epidermis (Lutterloh and Shallenberger, 1946; Sokol et al., 1982). The discoloration becomes noticeable within 3–10 days of administration. Wood’s lamp examination of the skin and urine demonstrates yellow-green fluorescence. In contrast to jaundice, quinacrine negligibly discolors the sclerae. Yellow discoloration of the skin disappears one to four months after discontinuing the drug.

Clofazimine Clofazimine is a reddish blue, substituted phenazine dye employed to combat multibacillary leprosy as well as other mycobacterial infections, rhinoscleroma, and inflammatory dermatoses (Arbiser and Moschella, 1995). This drug is highly lipophilic and is consumed avidly by macrophages. Within two weeks of taking clofazimine, patients may develop a reddish hue which represents deposition of clofazimine into the skin (Browne, 1965). Due to their ample population of macrophages, active inflammatory lesions absorb clofazimine to a greater degree than normal tissue and, thus, can display a deep red-blue hue (Figs 54.31 and 54.32) (Job et al., 1990; Kossard et al., 1987)]. Initial studies of clofazimine pigmentation revealed subtle evidence of drug deposition in routinely

DRUG-INDUCED OR -RELATED PIGMENTATION

Fig. 54.31. Clofazimine hyperpigmentation. The lighter arm is normal and included for comparison with the deep brown color of the arm of a patient taking clofazimine (see also Plate 54.21, pp. 494–495).

Fig. 54.33. Patient taking thorizine who developed a mauve discoloration on her cheeks and sclera (see also Plate 54.23, pp. 494–495).

processed skin biopsies (Levy and Randall, 1970; Sakurai and Skinsnes, 1970, 1977). However, analysis of frozen sections, which does not expose tissue to organic solvents that can extract clofazimine, demonstrated prominent deposition of vivid red crystals concentrated around vessels in the reticular dermis (Kossard et al., 1987). Chronic administration of clofazimine for months commonly results in a generalized pink-brown discoloration that is accentuated in sun-exposed areas (Mackey and Barnes, 1974). Light microscopy of biopsies from brown areas showed focal, dermal collections of foamy macrophages with diffusely distributed brownish granular pigment (Job et al., 1990). Lipofuscin stains highlighted these pigment granules; melanin and iron stains were negative. Electron microscopy displayed numerous multivesiculated phagolysosomes. These intralysosomal vesicles were lined by a trilamellar membrane and contained electron-dense amorphous granular material and lamellar structures characteristic of lipofuscin. Both subacute and chronic clofazimine-induced cutaneous pigmentation gradually resolve once the drug is discontinued (Job et al., 1990).

Psychotropic Drugs Phenothiazines

Fig. 54.32. The brown arm on the right is that of a patient treated with clofazimine. The normal-colored arm on the left is for comparison (see also Plate 54.22, pp. 494–495).

Daily administration of 125 mg of phenothiazine for six months has been shown to be associated with the development of cutaneous hyperpigmentation (Ban et al., 1985), however, most cases appear after daily doses of at least 300 mg for two years (Bond and Yee, 1980; Greiner and Berry, 1964; Haddad, 1977; Zelickson and Zeller, 1964). This pigmentation begins as golden-brown pigmentation over sun-exposed areas and can progress to slate gray-blue–black discoloration. Darker pigmentation most commonly occurs over the face and has been called “magot chinois” (Ey and Rappart, 1956) and “visage mauve” (Fig. 54.33) (Perrot and Bourjala, 1962). Increased epidermal melanin has been described in unexposed 1043

CHAPTER 54

areas (Robins, 1975). Nail bed pigmentation has also been ascribed to phenothiazine use (Satanove, 1965). Lenticular and corneal pigment deposits may be detectable on slit-lamp examination (Bond and Yee, 1980). It has been reported that pigmentary changes are more common in women than in men (Greiner and Berry, 1964), but this contention has been challenged (Ban et al., 1985). The incidence of overall pigmentary changes has been reported to be 1.2–2.9% in patients on chronic phenothiazine therapy (Ananth et al., 1972; Ban et al., 1985; Greiner and Berry, 1964); however, less than 1% develop slate gray-blue–black discoloration (Mathalone, 1965; Robins, 1975; Tredici et al., 1965). Chlorpromazine has been implicated as the most frequent phenothiazine inducer of hyperpigmentation, but haloperidol and levomepromazine have also been blamed (Ban et al., 1985). Initially, phenothiazine-induced pigmentation was thought to be permanent (Gibbard and Lehman, 1966). More recent reports claim that chlorpromazine-induced blue-black pigmentary changes are reversible (Ewing and Einarson, 1981; Lal et al., 1993). One study followed the persistence of blue-black pigmentary changes after chlorpromazine had been discontinued and replaced with other neuroleptics, including alternative phenothiazines. The study demonstrated that 14/15 patients had complete clearing within six months to five years; the remaining patient displayed marked improvement (Lal et al., 1993). D-penicillamine has been used to treat phenothiazine-induced pigmentation (Gibbard and Lehman, 1966), but its side-effect profile, modest efficacy, and recent evidence suggesting the spontaneous reversibility of pigmentation preclude its use. Histopathologic examination reveals golden-brown pigment granules located predominantly around vessels in the papillary and upper reticular dermis (Benning et al., 1988; Satanove, 1965). This pigment stained with Fontana-Masson but not with Perls reagent. Electron microscopy demonstrated membrane-bound, electron-dense inclusions within macrophages, endothelial cells, and Schwann cells (Benning et al., 1988; Hashimoto et al., 1966; Zelickson, 1965). Increased epidermal melanin has also been noted (Hashimoto et al., 1966). Energy-dispersive X-ray microanalysis has detected a significant sulfur peak, which implied the presence of chlorpromazine within these dense inclusions (Benning et al., 1988). Darkly pigmented skin from some affected patients contained a purple substance with a reflectance curve that differed from that of melanin (Satanove, 1965). Perry observed that the major metabolite of chlorpromazine, 7-hydroxychlorpromazine, turns purple on exposure to ultraviolet light, thus potentially explaining why pigmentary changes are most prominent in sun-exposed skin (Perry, 1964). In vitro, chlorpromazine has been shown to bind strongly to melanin (Potts, 1964). Although the exact nature of the intracellular “chlorpromazine bodies” is not known, several points of evidence suggest a composition of drug-metabolite–melanin complexes (Benning et al., 1988). 1044

Fig. 54.34. Localized hyperpigmentation after topical treatment with nitrogen mustard (see also Plate 54.24, pp. 494–495).

Tricyclic Antidepressants Both desipramine (Steele and Ashby, 1993) and imipramine (Hare, 1970; Hashimoto et al., 1991) have been reported to cause slate-gray pigmentation in sun-exposed areas over the face, hands, arms, and neck. Imipramine dosages ranged from 150 mg/day for five years to 300 mg/day for ten years. Desipramine induced pigmentation when given as 450 mg/day for seven years. Biopsies of affected areas demonstrated golden-brown granules in the upper dermis (Hashimoto et al., 1991; Steele and Ashby, 1993). Electron microscopy of a specimen from a patient with imipramine-induced pigmentation demonstrated both increased phagocytosed dermal melanin as well as electron-dense inclusions within dermal histiocytes, fibroblasts, and dendrocytes (Hashimoto et al., 1991). The pigmentation induced by tricyclic antidepressants appears to be caused by both increased dermal melanin as well as distinct inclusions that may represent drug metabolite complexes (Steele and Ashby, 1993).

Chemotherapeutic Agents Several chemotherapeutic agents such as nitrogen mustard (Fig. 54.34) and bleomycin (Figs 54.35 and 54.36) are able to produce mucocutaneous hyperpigmentation by unknown mechanism(s). The pigment responsible for this pigmentation is melanin, with the exception of cisplatin, in which deposition of platinum has been implicated (Ettinger and Freeman, 1979). Recent evidence suggests that fragments of nucleic acid, modeled to mimic repair by-products of ultraviolet–lightinduced DNA damage, can stimulate melanogenesis (Eller et al., 1994). Similarly, it is possible that chemotherapy-induced damage to cutaneous cellular DNA could produce signals that promote melanin synthesis and transport. Chemotherapy can enhance sensitivity to ionizing and ultraviolet radiation and reduce the radiation dose necessary to induce hyperpigmentation (Falkson and Schultz, 1962). Direct melanocyte toxicity may trigger melanogenesis or

DRUG-INDUCED OR -RELATED PIGMENTATION

alter melanosome transport. Some evidence suggests that certain cytotoxic agents can alter keratinocyte melanosome aggregation and, thus alter skin color (Flaxman et al., 1973). The individual agents reported to cause hyperpigmentation are listed in Table 54.2. Note that azidothymidine (AZT) is included in this table. Although AZT is used as an antiretroviral drug, it was originally developed as a chemotherapeutic agent and probably induces hyperpigmentation in a manner similar to other members of this group (Adrian et al., 1980). Discrete hyperpigmentation that correlates to areas covered

by adhesive tape or electrocardiographic leads occurs in chemotherapy patients (Horn et al., 1989; Singal et al., 1991). Thiotepa, cyclophosphamide, etoposide, and carboplatin may be secreted in the sweat, accumulate under occlusion, and influence melanogenesis or melanosome dispersion (Kang et al., 1993).

Fig. 54.35. Flagellate distribution of bleomycin-induced hyperpigmentation (see also Plate 54.25, pp. 494–495).

Fig. 54.36. Pathognomonic clinical appearance of bleomycininduced hyperpigmentation (see also Plate 54.26, pp. 494–495).

Miscellaneous Drugs and Chemicals The details of pigmentary changes induced by additional drugs, chemicals, and dyes are included in Tables 54.3 and 54.4.

Table 54.2. Chemotherapeutics and pigmentation. Agent

Clinical appearance

Ametantrone

Diffuse blue-gray pigmentation

Histopathology/comment

Drug is blue-black aminoanthraquinone dye, which may be responsible for hyperpigmentation Occurs in all patients who receive more than 135 mg/m2 per injection (Loesch et al., 1983) Azidothymidine Mucocutaneous and nail plate pigmentation (Bendick et al., Increased melanin within basal keratinocytes and dermal (AZT) 1989; Furth and Kazakis, 1987; Greenberg and Berger, macrophages 1990) Two weeks of high-dose AZT induced pigmentation in mouse model; increased melanosomes in epidermal melanocytes noted (Obuch et al., 1992) 1,3-Bis(chloroethyl)- Localized hyperpigmentation at site of application Increased number of melanocytes and pigmented 1-nitrosourea keratinocytes (Hilger et al., 1974) (BCNU) Causes hyperpigmentation only when applied topically; first noted on hands of health professionals administering BCNU (Frost and Devita, 1966) May represent postinflammatory hyperpigmentation; however, precedent erythema is not essential (Frost and Devita, 1966) Pigmentation reproduced in hairless mice (Hilger et al., 1974) Bleomycin Linear, “flagellate” pigmented bands over chest, back, finger High bleomycin concentration in skin (Werner and joints, palms, elbows, knees (Lowitz, 1975; Sonntag, Thornberg, 1976) 1972) Associated with minor trauma (Moulin et al., 1970) Increased epidermal melanin, little dermal pigment incontinence (Perrot and Ortonne, 1978)

1045

CHAPTER 54 Table 54.2. Continued. Agent

Busulfan

Cisplatin Cyclophosphamide (Cytoxan)

Clinical appearance

Histopathology/comment

Reticulate pattern in one patient. (Also received cyclophosphamide, vincristine, adriamycin, and methotrexate) (Wright et al., 1990) Pigmented bands in nail bed (one patient also received vinblastine) (Shetty, 1973; Sonntag, 1972) One case localized to fresh striae distensae (also received cisplatin and vincristine) (Tsuji and Sawabe, 1993) 3/19 (Galton, 1953) and 39/788 (Kyle et al., 1961) CML patients developed diffuse brownish pigmentation over face, forearms, trunk, dorsal hands; resolved in three months (Galton, 1953) #207] May mimic Addison disease (Kyle et al., 1961)

Dopa staining of an epidermal sheet has revealed larger melanocytes with complex dendrites and increased tyrosinase activity (Granstein and Sober, 1981) 8–20% incidence of hyperpigmentation (Blum et al., 1973)

Gray-white line at gingival margin (Ettinger and Freeman, 1979) Local or generalized skin and nail plate/bed hyperpigmentation (Romankiewics, 1974; Shah et al., 1978; Solidoro and Saenz, 1966) Brown pigmentation of gingival margin in one patient (Harrison and Wood, 1972) Supravenous hyperpigmentation was noted in a patient who received combination chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone (Schulte-Huermann et al., 1994)

Dactinomycin

Generalized hyperpigmentation, most prominent over face (Bronner and Hood, 1983; Ma et al., 1971)

Daunorubicin

Transverse brown-black bands in nail plate (de Marins et al., 1978) Cutaneous hyperpigmentation (not specified)

Dibromomannitol Doxorubicin (Adriamycin)

5-Fluorouracil (systemic)

1046

Brown-black nail pigmentation: horizontal bands (Morris et al., 1977), longitudinal bands (Priestman and James, 1975), and diffuse (Orr and McKernan, 1980) Appears to involve nail plate (Morris et al., 1977) Can involve dorsal finger joints (Orr and McKernan, 1980; Pratt and Shanks, 1974) as well as palms, soles, buccal mucosa (Rothberg et al., 1974), and tongue (Rao et al., 1976) Uniform hyperpigmentation over sun-exposed areas often preceded by erythema (Falkson and Schultz, 1962) One report of hyperpigmentation over infused vein (Hrushesky, 1976) Localized hyperpigmentation of dorsal hands (Perlin and Ahlgren, 1991; Reed and Morris, 1984), palms, soles, and trunk (Cho et al., 1988) Unusual serpentine hyperpigmented streaks over the trunk (Vukelja et al., 1991) Five reported cases of transient corneal striate melanokeratosis following subconjunctival injection (Stank et al., 1990)

Onset of pigmentation after 90–285 mg (Cohen et al., 1973) Increased melanin in basal keratinocytes and dermal macrophages

Speculation that busulfan inactivates sulfhydryl groups, liberates copper, and thus activates tyrosinase (Kyle et al., 1961) Topical busulfan fails to produce hyperpigmentation (Granstein and Sober, 1981) Hyperpigmentation is more likely to be induced in darkskinned patients (Burns et al., 1971)

In five cases of black nail pigmentation, cumulative dose = 1.2–12.3 g over 10 days to 26 weeks 20% of treated neuroblastoma patients manifested hyperpigmentation (Thurman et al., 1964) Pigmentation starts in proximal nail fold suggesting that matrical melanocytes are stimulated (Bronner and Hood, 1983) Resolution 6–12 months after end of treatment (Bronner and Hood, 1983) Incidence of hyperpigmentation reported to be occasional (Karnofsky, 1968) to universal — 18/18 patients (Ma et al., 1971)

5/11 CML patients treated intravenously developed hyperpigmentation (Canellos et al., 1975) Increased epidermal melanization and number of melanocytes (Rothberg et al., 1974) More common in dark-skinned patients (Pratt and Shanks, 1974; Rothberg et al., 1974)

Increased basal layer melanin, scattered dyskeratotic keratinocytes 2–42% treated patients affected (Bateman et al., 1971; Falkson and Schultz, 1962; Hrushesky, 1976) 5-fluorouracil can reduce threshold of ionizing- and ultraviolet-induced cutaneous hyperpigmentation (Falkson and Schultz, 1962; Hum and Bateman, 1975)

DRUG-INDUCED OR -RELATED PIGMENTATION Table 54.2. Continued. Agent

Clinical appearance

Histopathology/comment

Hydroxyurea

Increased pigmentation over back and pressure points that tends to be reversible (Majumdar et al., 1990) Longitudinal band nail hyperpigmentation (Beylot-Barry et al., 1995; Pirard et al., 1994; Vomvouras et al., 1991) Hyperpigmentation tends to be diffuse but is more intense in lesional skin (Epstein and Ugel, 1970; Mandy et al., 1971; Van Scott and Winters, 1970) Pigmentation fades after treatment is discontinued; lesional discoloration is slow to resolve

Lichenoid eruption with secondary hyperpigmentation (Kennedy et al., 1975)

Mechlorethamine

Disaggregation of melanosomes, increased numbers of melanocytes and keratinocyte melanosomes (Flaxman et al., 1973) No alteration in melanosome size

Table 54.3. Miscellaneous inducers of pigmentation. Drug or chemical

Clinical appearance

Ciclosporin

Progressive hyperpigmentation in a renal transplant patient (Brady and Wing, 1989) Brown-gray pigmentation in sun-exposed areas 90% of 122 patients directly exposed to PCBcontaminated cooking oil demonstrated mucocutaneous pigmentation: nose, conjunctiva, oral mucosa, nails (Wong et al., 1982) Prenatal exposure due to contaminated cooking oil has caused congenital pigmentation of face, perineum, genitalia, nail bed and plate, and palms (Gladen et al., 1990) Rare congenital diffuse brown pigmentation (Rogan, 1982) Nail pigmentation in workers exposed to PCBs (Gladen et al., 1990; Reggiani and Bruppacher, 1985) Diffuse hyperpigmentation

Dioxin/polychlorinated biphenyls

Levodopa

Hydantoins (phenytoin and methoin)

Oxprenolol Pefloxacin/norfloxacin

Phenacetin Rifampicin

10% develop hyperpigmentation of face and neck resembling chloasma; no significant associated endocrine abnormalities (Kuske and Krebs, 1964; Levantine and Almeyda, 1972) Prenatal exposure to phenytoin associated with nail hyperpigmentation (Johnson and Goldsmith, 1981) Pigmentation in light- and pressure-exposed areas (Richards, 1985) Blue-black pigmentation of the legs secondary to pefloxacin; resolved in 45 days; recurred after taking closely related norfloxacin (Le Cleach et al., 1995)

Diffuse brown pigmentation (Nanra et al., 1978) Orange-red discoloration of skin, urine, tears after accidental ingestion of 2 g in 18-month-old; resolved by one month (Salazar de Souza et al., 1987)

Histopathology/comments

Basal layer pigmentation (Kikuchi, 1984) Gas chromatography has detected PCBs in skin and subcutaneous fat, perhaps contributing to pigmentation Transplacental exposure possible (Brodkin and Schwartz, 1984; Gladen et al., 1990)

Sign of significant dioxin exposure (Dunagin, 1984)

Deeper color in women than in men; thought that estrogens may increase tyrosinase utilization of levodopa which is a melanogen (Robins, 1979)

Pigmentary abnormalities resolve upon discontinuation Focal dense, dermal, black pigment Perls stain positive Electron microscopy reveals electron-dense particles within dermal histiocytes X-ray microanalysis shows that pigment particles contain iron No dermal melanosomes Lipofuscin pigment (Gloor, 1978)

Modified from Kang et al. (1993) and Lerner and Sober (1986). PCB, polychlorinated biphenyl.

1047

CHAPTER 54 Table 54.4. Nitro compounds, dyes, and tar. Dye

Clinical appearance

Comments

Aniline dyes (Orange II)

Pigmented contact dermatitis of the face

Azo dyes (Sudan I)

Pigmented contact dermatitis of the face

Danthron (Dorbanex)

Red-brown discoloration of skin in perianal and thigh areas (Barth et al., 1984) Yellow-orange pigmentation

Pigment incontinence and hydropic degeneration of the basal layer (Rorsman, 1982) Pigment incontinence and hydropic degeneration of the basal layer (Rorsman, 1982) Staining occurs from this anthraquinone, which is used as a laxative Color change does not involve melanin. Postulated mechanism of action involves reduction of sulfhydryl groups in keratin. Used as a “tanning” agent Chemical was used to treat obesity by means of its mild central nervous system stimulatory action (Cutting et al., 1933) Used in serum amylase determination (Fregert and Trulson, 1980) Henna is derived from Lawsonia inermis and other members of this family. Henna may react with amino acids in the stratum corneum Pigmentation is presumably secondary to direct tissue staining The localization of this yellow dye in the skin has not been determined (Alano and Webster, 1970). Used as urinary tract analgesic Taken by malingering soldiers in World War I to produce a clinically jaundiced appearance. Of note, some of the nitro compounds can cause true chemical hepatitis Staining secondary to nitric acid reacting with aromatic amino acids in proteins (Fregert et al., 1980) Pigment laden dermal macrophages, perivascular infiltrate. Affects workers exposed to tar, oil, pitch and other tar products

Dihydroxyacetone

2,4-Dinitrophenol

Dinitrosalicylic acid

Yellow pigmentation of exposed areas on contact and jaundiced appearance on oral use (Jeghers, 1944) Yellow-stained palms and fingers

Henna (2,4-dihydroxynaphthoquinone)

Reddish-orange coloration

Nitrazepam

Yellow cheeks and soles (Fregert et al., 1980)

Phenazopyridine (Pyridium)

Picric acid

Diffuse yellow color resembling jaundice with scleral pigmentation (Engle and Schoolwerth, 1981) Jaundiced appearance

Sodium nitrate

Yellow staining

Tar melanosis (melanodermatitis toxica)

Postinflammatory reticulate hyperpigmentation secondary to a combination of irritation and phototoxicity (Mosher et al., 1987; Sugai et al., 1977) Canary yellow staining from contact Yellow staining topically

Tetryl Trinitrotoluene, nitric acid, styphnate hexanitrodiphenylamine

Used in explosives (Jeghers, 1944)

Modified from Kang et al. (1993) and Lerner and Sober (1986).

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Sauer, G. Muddy skin from minocycline. Schoch Letter 29:3, 1979. Schmidt, H., and H. Christensen. Red poster paint tattoo granuloma. Arch. Dermatol. 114:965–966, 1978. Schmidt, O. Chrysiasis. Arch. Dermatol. Syphil. 44:446–452, 1941. Schulte-Huermann, P., M. Zumdick, and T. Ruzicka. Supravenous hyperpigmentation in association with CHOP chemotherapy of a CD30 (Ki-1)-positive anaplastic large-cell lymphoma. Dermatology 191:65–67, 1994. Schultz Larsen, F., H. Boye, and E. Hage. Chrysiasis: electron microscopic studies and X-ray microanalysis. Clin. Exp. Dermatol. 9:174–180, 1984. Scott, A. The behaviour of radioactive mercury and zinc after application to normal and abnormal skin. Br. J. Dermatol. 71:181–189, 1959. Sehgal, V., and M. Gangwani. Hydralazine induced fixed drug eruption. Int. J. Dermatol. 25:394, 1986. Sehgal, V., and O. Gangwani. Fixed drug eruption. Current concepts. Int. J. Dermatol. 26:67–74, 1987. Shah, P., K. Rao, and A. Patel. Cyclophosphamide induced nail pigmentation. Br. J. Dermatol. 98:675–680, 1978. Sharvill, D. Familial hypercarotenemia and hypovitaminosis A. Proc. R. Soc. Med. 63:605–606, 1970. Shelley, W., and E. Shelley. Fixed drug eruption due to metronidazole. Cutis 39:393–394, 1987a. Shelley, W., and E. Shelley. Nonpigmenting fixed drug eruption as a distinctive reaction pattern: Examples caused by sensitivity to pseudoephedrine hydrochloride and tetrahydrozoline. J. Am. Acad. Dermatol. 17:403–407, 1987b. Shengda, H., and D. Dean. Hyperpigmentation of the nail from lead deposition. Int. J. Dermatol. 28:273–275, 1989. Shetty, M. Case of pigmented banding of the nail caused by bleomycin. Cancer Treatment Rep. 61:501–502, 1973. Shukla, S. Drugs causing fixed drug eruptions. Dermatologica 163:160–163, 1981. Shuttleworth, D., and R. Graham-Brown. Fixed drug eruption due to carbamazepine. Clin. Exp. Dermatol. 9:424–426, 1974. Simons, J., and A. Morales. Minocycline and generalized cutaneous pigmentation. J. Am. Acad. Dermatol. 3:244, 1980. Singal, R., W. W. Tunnessen Jr., J. M. Wiley, and A. F. Hood. Discrete pigmentation after chemotherapy. Pediatr. Dermatol. 8:231–235, 1991. Slazinski, L., and D. Knox. Fixed drug eruption due to methaqualone. Arch. Dermatol. 120:1073–1075, 1984. Sober, A., and R. Granstein. Chemical and pharmacologic agents causing dermal pigmentation. In: Biology and Diseases of Dermal Pigmentation, T. Fitzpatrick, A. Kukita, F. Morikawa, M. Seiji, A. Sober, and K. Toda (eds). Tokyo: University of Tokyo Press, 1981, pp. 117–126. Sokol, R., P. Lichtenstein, and M. Farrell. Quinacrine hydrochlorideinduced yellow discoloration of the skin in children. Pediatrics 2:232–231, 1982. Solidoro, A., and R. Saenz. Effects of cyclophosphamide on 127 patients with malignant lymphoma. Cancer Treat. Rep. 50:265–270, 1966. Solis, R. R., D. G. Diven, M. I. Colome-Grimmer, N. Snyder, and R. F. Wagner. Experimental nonsurgical tattoo removal in a guinea pig model with topical imiquimod and tretinoin. Dermatol. Surg. 28:83–86, 2002. Sonntag, G. Bleomycin (NSC-125066): phase I clinical study. Cancer Chemother. Rep. 36:197–205, 1972. Stack, K., M. Churchwell, and R. Skinner. Xanthoderma: Case report and differntial diagnosis. Cutis 41:100–102, 1988. Stank, T., T. Krupin, and M. Feitl. Subconjunctival 5-fluorouracilinduced transient striate melanokeratosis. Arch. Ophthalmol. 108:1210, 1990. Steele, T. E., and J. Ashby. Desipramine-related slate-gray skin pigmentation [letter]. J. Clin. Psychopharmacol. 13:76–77, 1993.

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CHAPTER 54 Stitzler, C., and A. Kopf. Fixed drug eruption caused by 8-chlorotheophylline in dramamine with clinical and histologic changes. J. Invest. Dermatol. 34:319–330, 1960. Stubb, S., and S. Reitamo. Fixed drug eruption caused by piroxicam. J. Am. Acad. Dermatol. 22:1111–1112, 1990a. Stubb, S., and S. Reitamo. Fixed drug eruption due to dextromethorphan. Arch. Dermatol. 126:970–971, 1990b. Sugai, T., Y. Tahahashi, and T. Takagi. Pigmented cosmetic dermatitis and coal tar dyes. Contact Dermatitis 3:249–256, 1977. Sugar, H., and W. Waddell. Ochronosis-like pigmentation associated with the use of Atabrine. Ill. Med. J. 89:234–239, 1946. Sutton, R. Diseases of the Skin. St. Louis: Mosby, 1923, pp. 880– 884. Taylor, C., R. Gange, J. Dover, T. Flotte, E. Gonzalez, N. Michaud, and R. Anderson. Treatment of tattoos by Q-switched ruby laser. Arch. Dermatol. 126:893–899, 1990. Teraki, Y., N. Moriya, and T. Shiohara. Drug-induced expression of intercellular adhesion molecule-1 on lesional keratinocytes in fixed drug eruption. Am. J. Pathol. 145:550–560, 1994. Teraki, Y., and T. Shiohara. IFN-gamma-producing effector CD8+ T cells and IL-10-producing regulatory CD4+ T cells in fixed drug eruption. J. Allergy Clin. Immunol. 112:609–615, 2003. Theodoridis, A., R. Tsabaos, C. Sivenas, and J. Capetanakis. Serum immunoglobulins in fixed drug eruption. Acta Derm. Venereol. 57:507–508, 1977. Thibault, P., and J. Wlodarczyk. Postsclerotherapy hyperpigmentation. The role of serum ferritin levels and the effectiveness of treatment with the copper vapor laser. J. Dermatol. Surg. Oncol. 18:47–52, 1992. Thurman, W., D. Fernbach, and M. Sullivan. Cyclophosphamide therapy in childhood neuroblastoma. N. Engl. J. Med. 270:1336–1342, 1964. Tope, W. D., and F. G. Shellock. Magnetic resonance imaging and permanent cosmetics (tattoos): survey of complications and adverse events. J. Magn. Reson. Imaging 15:180–184, 2002. Traub, E., and J. Tennen. Permanent pigmentation following application of iron salts. J. Am. Med. Assoc. 106:1711–1712, 1936. Tredici, I., B. Schiele, and W. McClanahan. The incidence and management of chlorpromazine skin-eye syndrome. Minn. Med. 48:569–574, 1965. Trimble, J., D. Mindelson, B. Fetter, P. Ingram, J. Gallagher, and J. Shelburne. Cutaneous pigmentation secondary to amiodarone therapy. Arch. Dermatol. 119:914–918, 1983. Tsuji, T., and M. Sawabe. Hyperpigmentation in striae distensae after bleomycin treatment. J. Am. Acad. Dermatol. 28:503–505, 1993. Tuffanelli, D., R. Abraham, and E. Dubois. Pigmentation from antimalarial therapy. Arch. Dermatol. 88:419–426, 1963. Van Scott, E., and P. Winters. Responses of mycosis fungoides to intensive external treatment with nitrogen mustard. Arch. Dermatol. 102:507–514, 1970. Vomvouras, S., A. S. Pakula, and J. M. Shaw. Multiple pigmented nail bands during hydroxyurea therapy: an uncommon finding. J. Am. Acad. Dermatol. 24(6 Pt 1):1016–1017, 1991.

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Vukelja, S. J., M. W. Bonner, M. McCollough, P. W. Cobb, D. A. Gaule, P. J. Fanucchi, and J. H. Keeling. Unusual serpentine hyperpigmentation associated with 5-fluorouracil. Case report and review of cutaneous manifestations associated with systemic 5-fluorouracil. J. Am. Acad. Dermatol. 25(5 Pt 2):905–908, 1991. Waitzer, S., J. Butany, L. From, W. Hanna, C. Ramsay, and E. Downar. Cutaneous ultrastructural changes and photosensitivity associated with amiodarone activity. J. Am. Acad. Dermatol. 16:779–787, 1987. Walter, J., and K. Macknet. Pigmentation of osteoma cutis caused by tetracycline. Arch. Dermatol. 115:1087–1088, 1979. Walton, K., D. Campbell, and E. Tonks. The significance of alterations in serum lipids in thyroid dysfunction. Clin. Sci. 29:199–215, 1965. Wanet, J., G. Achten, and G. Barchewitz. Amiodarone et dépôts: Etude clinques et histologique. Ann. Dermatol. Venereol. 98:131–140, 1971. Weiss, S., H. Lim, and G. Curtis. Slate-gray pigmentation of sunexposed skin induced by amiodarone. J. Am. Acad. Dermatol. 11:898–900, 1984. Werner, Y., and B. Thornberg. Cutaneous side effects of bleomycin therapy. Acta. Derm. Venereol. 56:155–158, 1976. Westerhof, W., E. Wolters, J. Brookbakker, R. Boelen, and M. Schipper. Pigmented lesions of the tongue in heroin addicts: Fixed drug eruption. Br. J. Dermatol. 109:605–610, 1983. Witten, V. Studies on the mechanism of eczematous contact dermatitis. I. Findings on human skin with radioactive bichloride of mercury. J. Invest. Dermatol. 28:339–346, 1957. Wong, C.-K., C.-J. Chen, P.-C. Cheng, and P.-H. Chen. Mucocutaneous manifestations of polychlorinated biphenyls (PCB) poisoning: a study of 122 cases in Taiwan. Br. J. Dermatol. 107:317–323, 1982. Wood, C., and G. Severin. Unusual histiocytic reactions to Monsel’s solution. Am. J. Dermatopathol. 2:261–264, 1979. Wright, A., S. Bleehen, and A. Champion. Reticulate pigmentation due to bleomycin: light and electron-microscopic studies. Dermatologica 180:255–257, 1990. Wyatt, E., M. Greaves, and J. Sondergaard. Fixed drug eruption (phenolphthalein). Arch. Dermatol. 106:671–673, 1972. Young, E. Ammoniated mercury poisoning. Br. J. Dermatol. 72:449–455, 1960. Zachary, C., D. Slater, D. W. Holt, G. Storrey, and D. MacDonald. The pathogenesis of amiodarone-induced pigmentation and photosensitivity. Br. J. Dermatol. 110:451–456, 1984. Zala, L., T. Hunziker, and L. R. Braathen. Pigmentation following long-term bismuth therapy for pneumatosis cystoides intestinalis. Dermatology 187:288–289, 1993. Zelickson, A. Skin pigmentation and chlorpromazine. J. Am. Med. Assoc. 194:670–672, 1965. Zelickson, A., and H. Zeller. A new and unusual reaction to chlorpromazine. J. Am. Med. Assoc. 188:394–396, 1964. Zemtsov, A., D. Yanase, A. Boyd, and B. Shehata. Fixed drug eruption to Tylenol: Report of two cases and review of the literature. Cutis 50:281–282, 1992.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Disorders of Pigmentation of the Nails and Mucous Membranes

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

55

The Melanocyte System of the Nail and its Disorders Robert Baran, Christophe Perrin, Luc Thomas, and Ralph Braun

Introduction The presence of melanocyte in normal nails was first demonstrated in 1968 by Higashi and confirmed by the ultrastructural study of Hashimoto in 1971, who identified many dendritic melanocytes containing mature and immature melanosomes in the matrix of the nails of Japanese and black people.

Anatomy The nail is the permanent product of the nail matrix. Its normal appearance and growth depends on the integrity of several components: the surrounding tissues or perionychium and the bony phalanx. Together, these form the nail apparatus or nail unit (Fig. 55.1).

The Nail Melanin Unit Distribution of Melanocytes According to Type of Epithelium The nail apparatus consists of three types of epithelium: 1 an epidermal-type of epithelium localized at the folded dorsal and ventral aspect of the proximal nail fold, the lateral nail folds, and the hyponychium; 2 a stratified epithelium keratinizing via a granular layer but different from the epidermis by virtue of its thinness, and the lack of papillomatosis and sweat glands. This epithelium, called eponychium, covers the ventral aspect of the proximal nail fold; and 3 a stratified epithelium without a granular layer covering both the matrix and the nail bed. The elective and sequential expression of hair keratins at the keratogenous zone of the matrix is responsible for the generation of the nail plate (Perrin et al., 2004). The nail epidermal-type epithelium contains active melanocytes whose distribution and density are identical to those observed in the neighboring epidermis. The nail epithelium corresponding to eponychium, matrix, and the nail bed contains a scattering of quiescent melanocytes that are not detectable with Fontana-Masson stain.

Bicompartmentalization of Melanocytes of the Eponychium Matrix and Nail Bed White people show two types of melanocytic distribution (Perrin et al., 1997): ∑ A compartment of quiescent melanocytes unable to synthesize melanin under normal conditions (Figs 55.2 and 55.3A). These amelanotic melanocytes (dopa-negative) have been demonstrated by immunohistochemical techniques: this reacts with the monoclonal (Mo) antibodies (Ab) TMH1 and MoAb B8G3, which recognize tyrosinase-related protein (TRP) 1, but not with MoAb 5C12 and MoAb 2B7, which recognize tyrosinase. ∑ A compartment of functionally differentiated melanocytes which were first described by Higashi and Saito (1969) as dopa-positive melanocytes (Fig. 54.3B). Immunohistochemical studies (Perrin et al., 1997; Tosti et al., 1994b) have demonstrated that these melanocytes contain a multifunctional enzyme tyrosinase, which catalyzes the initial events of melanogenesis and the other regulatory proteins and is known as tyrosinase-related protein (TRP) I and II. These melanocytes consequently express all the key enzymes necessary for melanin synthesis. In contrast with previously held views (Baran and Kechijian, 1989; Tosti et al., 1994b), the melanocytes of the distal matrix are not more numerous than those of the proximal matrix. Their density is nevertheless low (Perrin et al., 1997): 217/mm2 compared to that of the epidermis. The difference between these matricial zones is solely in the distribution of dormant and potentially active compartments. The melanocytes of the proximal matrix are mainly dormant. In the distal matrix the quiescent melanocytic compartment is reduced whereas the group of functionally differentiated melanocytes is dominant. The melanocytes of both divisions, unlike the epidermal melanocytes, express MoAb HMB-45, which recognizes the glycoprotein P mel-17 present in the immature melanosome. Together these immunohistochemical data suggest that the matrix melanocytes contain premelanosomes and melanosome types 1 and 2 with little or no active production of mature melanosomes. This is in accordance with ultrastructural studies (Hashimoto, 1971), which demonstrated the scarcity of recognizable melanosomes in the Caucasian nail. Perrin et al. have also defined a smaller population of nail bed dormant melanocytes, with approximately 25% of the 1057

CHAPTER 55

Sagittal section through finger nail

Proximal matrix Proximal nail fold Eponychium Cuticle Lunula Nest of pigment producing cells Nail plate Nail

Distal matrix

bed

Hyponychium Distal groove

A

B

A

Fig. 55.1. (A) Anatomy of the nail apparatus. (B) Formation of longitudinal melanonychia (see also Plate 55.1, pp. 494–495).

NP

KZ

B Fig. 55.3. (A) Split epithelial sheet of the nail matrix. Note the arboreal pattern in the proximal matrix (MoAb TMH1). (B) The increased parallel pattern of the ridges in the distal matrix and the beginning of the nail bed (dopa reaction). (Original magnification ¥100.) A

NP

KZ

B Fig. 55.2. (A, B) Frozen vertical sections of proximal matrix stained with (A) MoAb TMH1 reactive to TRP1 and (B) MoAb HMB-45. Note the heterogeneous suprabasal distribution of melanocytes. (Original magnification ¥250.) NP, nail plate; KZ, keratogenous zone.

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number of melanocytes formed in the matrix (Fig. 55.4). This differs from De Berker et al.’s (1996a) observation where the nail bed was noted to lack melanocyte markers. This discrepancy may be due to differences in investigation techniques, one of which was the split-skin preparation. The melanocytes of the eponychium are mainly composed of dormant cells. Their density is close to that found in the matrix (C. Perrin, personal data). There is only one ultrastructural study comparing the nail melanocytes according to the skin color type (Hashimoto, 1971). The melanocytes in Japanese nails contained all gradation of maturing melanosomes (the majority being of an immature variety) and transferred melanosomes (often just half-filled with melanin) were regularly seen within the keratinocytes. In the nails of black subjects, most of the melanosomes including transferred melanosomes were mature.

THE MELANOCYTE SYSTEM OF THE NAIL AND ITS DISORDERS

Fig. 55.4. Split epithelial sheet of the nail bed. The long epithelial ridges are aligned longitudinally (MoAb TMH1; original magnification ¥100).

Intraepithelial Distribution of Melanocytes in Eponychium, Matrix, and Nail Bed Melanocytes of the proximal matrix and, to a lesser degree, the distal matrix have a basal as well as suprabasal distribution (Higashi, 1968; Perrin et al., 1997; Tosti et al., 1994b). These false images of migration are explained by the thickness of the basal keratinocytic compartment composed of two to ten layers of basaloid cells (see Fig. 55.2B). This suprabasal location can rise very high, immediately beneath the keratogenous zone and such dendritic melanocytes, expressing HMB45, may pose a problem for interpretation where there is a suspicion of melanoma. Two main aspects should be highlighted (Perrin et al., 1997): (1) there are no melanocytes in the keratogenous zone; and (2) these melanocytes remain isolated from each other without organization into nevus cell nests. We have not found the “small clusters” gathered together as described by Tosti (1994b) neither on normal nail unit specimens nor on the biopsies of longitudinal melanonychia associated histologically with simple melanocytic activation (C. P. personal data). The melanocytes of the hyponychium and of the nail bed retain a basal distribution because of the monocellular nature of the basal layer in these two anatomic regions.

Distinguishing Features between the Nail and Follicular Unit Like the melanocytes of the eponychium and nail bed, melanocytes of the outer root sheath (ORS) of the follicles are L-dopa negative and contain a negligible amount of tyrosinase. However the latter differs from the former by the lack of expression of TRP1 and TRP2 (Horikawa et al., 1996). In addition, although the antigens recognized by HMB-45 and NK-1/beted are the product of the same gene (the silver locus gene, P mel-17), melanocytes of the ORS react with NK1/beted but not with HMB-45 (Horikawa et al., 1996). These data suggest that the melanocytes of eponychium and nail bed have a greater ability to be activated than those of the ORS.

Fig. 55.5. Longitudinal melanonychia (single band) (see also Plate 55.2, pp. 494–495).

Melanocytes of the follicular bulb are different from matrix melanocytes: they have a cyclic activity corresponding to a single type of melanocyte for each stage (Commo and Bernard, 2000). It is likely that the degree of activation of the nail melanocytes is largely due to the varying thicknesses of nail epithelium, the sun protection of the nail plate, and the proximal nail fold. Eponychium and proximal matrix are protected from the sun by the connective and epithelial axis of the proximal nail fold and the nail plate. The latter protects the distal matrix and the nail bed in the same way; the existence of a contingent of potentially active melanocytes in the distal matrix and not in the nail bed is in a large part due to the reduced thickness of the epithelium of the distal matrix compared to the deep epithelial ridges of the nail bed.

Melanonychia The term melanonychia, which means black nail, encompasses a brown to black coloration due to melanin, whereas nonmelanotic hyperplasia refers to coloration due to other substances or blood pigment. Longitudinal melanonychia is characterized by the presence of a single (Fig. 55.5) or 1059

CHAPTER 55 Table 55.1. Drug- and toxin-induced melanonychia. Chemotherapeutics Bleomycin sulfate Busulfan Cyclophosphamide Dacarbazine Daunorubicin hydrochloride Doxorubicin Etoposide 5-fluorouracil Hydroxyurea Melphalan hydrochloride Methotrexate Nitrogen mustard Nitrosurea Tegafur

Fig. 55.6. Longitudinal melanonychia (multiple bands) (see also Plate 55.3, pp. 494–495).

multiple pigmented streaks (Fig. 55.6), tan, brown, black, or blue (Smith et al., 2003), within the nail plate. Transverse melanonychia is not nearly as common (Quinlan et al., 2005). Active melanocytes produce melanin-rich melanosomes, which are transferred via dendrites to keratinocytes of the matrix. As the keratinocytes of the matrix differentiate into nail plate onychocytes, melanin will continuously be enclosed in the growing nail plate to give rise to a longitudinal band. The more proximal the origin, the more superficial is the melanin within the nail. It is therefore possible to identify the site of origin of pigmentation in longitudinal melanonychia within the matrix by staining distal nail plate clippings with Fontana-Masson argentaffin reaction. Longitudinal melanonychia is most common in frequently used fingers.

Topography of the Melanin Pigment within the Nail Plate Longitudinal melanonychia originates more often in the distal matrix (Baran and Kechijian, 1989) because only the distal matrix contains potentially active melanocytes (Perrin et al., 1997). This distal location allows nail surgery without dys1060

Miscellaneous Antimalarials Arsenic, thallium, mercury, PCB Clofazimine Clomipramine, cyclines, roxithromycin, sulfonamide Gold salts Ibuprofen Ketoconazole, fluconazole, amorolfine Fluoride Lamivudine, zidovudine Mepacrine, amodiaquine, chloroquine Phenothiazine Phenytoin Psoralen Steroids, ACTH, MSH Sulfonamide Timolol

trophic sequelae. The biopsy takes into account distal keratin examination after Fontana-Masson stain. Pigment located within the lower third indicates a distal matrix origin. Pigment invading the whole thickness of the nail plate indicates a proximal pigmentary source. There are many causes of longitudinal melanonychia and the differential diagnosis includes numerous conditions (Baran and Kechijian, 1989). The most frequent are listed in Tables 55.1 and 55.2.

Differential Diagnosis of Nail Pigmentation Besides the common causes of endogenous melanin deposition, pigmentation of the nail may be due to bacterial infection, hematoma, etc. ∑ Exogenous hyperchromia (Haneke and Baran, 2001): Usually the brown to black hue does not present as longitudinal melanonychia. Most of the causes such as tobacco, potassium permanganate, silver nitrate, can easily be scratched off. ∑ Bacterial hyperchromia (Haneke and Baran, 2001): Subjects involved in “wet work” are vulnerable to bacterial growth on the nail apparatus. Most bacteria producing a gray to black color belong to the group of Gram-negative pathogens

THE MELANOCYTE SYSTEM OF THE NAIL AND ITS DISORDERS Table 55.2. Nondrug-induced melanonychia due to melanocyte activation. Endocrine diseases

HIV infection Inflammatory nail disorders

Laugier syndrome Peutz–Jeghers syndrome Nonmelanocytic neoplasms

Nutritional Trauma (acute or repeated)

Physical agents

Addison disease, acromegaly, Cushing syndrome Hyperthyroidism, Nelson syndrome, pregnancy Lichen planus, lichen striatus, pustular psoriasis, Hallopeau acrodermatitis Onychomycosis, radiodermatitis, localized scleroderma Systemic lupus erythematosus

Bowen disease, basal cell carcinoma, myxoid pseudocyst Subungual fibrous histiocytoma, warts Folate deficiency, malnutrition, vitamin B12 deficiency Frictional melanonychia, onychotillomania, occupational melanonychia Surgery, cryosurgery, phototherapy, radiation

Idiopathic

(Pseudomonas, Klebsiella and Proteus spp.). The pigmentation usually originates under the junction of the lateral and proximal nail folds or in the lateral nail groove. The color rapidly returns when scraped off. Bacterial culture will confirm the diagnosis. ∑ Subungual hematoma (Haneke and Baran, 2001): This occurs due to single acute and heavy trauma, has a typical history, and does not reach the free edge of the nail. The extravasated erythrocytes from the capillary vessels of the nail bed are incorporated within the growing nail. The dark pigment is positive for blood with the pseudo-peroxidase reaction (Hemostix test). However, an underlying tumor may bleed spontaneously and yield a positive blood test. Repeated minor trauma (friction of shoes) may take on a longitudinal elliptical shape. If the nail is notched with the scalpel at the proximal margin of the spot, distal migration of the hematoma can be accurately measured as the nail plate grows. However, nonmigrating hematomas and foreign bodies do not follow this rule and require more extensive evaluation.

How to Distinguish between Melanin Pigmentation and Nonmelanin Hyperchromia Melanin pigment is visible after hematoxylin and eosin (H&E) stain as brown deposits with multiple granules within onychocytes. Hematic deposits are different and present as patches, more or less pronounced, depending on whether they are due to splinter hemorrhages or a large hematoma. They

are red-brown on H&E stain, greenish on toluidine blue, and negative on Fontana-Masson staining. Due to the lack of hemosiderin, Perls stain is negative. Rarely, examination of the distal nail keratin may show numerous atypical melanocytes scattered among the onychocytes (Haneke and Binder, 1978; Kerl et al., 1984; Kuchelmester et al., 2000). They are the signature of a nail melanoma. Linear melanonychia due to subungual keratosis of the nail bed is a new entity (Baran and Perrin, 1999). After nail avulsion, it appears as a single, prominent, longitudinal keratinized and pigmented mass.

Fungal Melanonychia Histologic examination facilitates the distinction between two main categories: the pigmented dermatophytes and the dematiaceous-type molds, such as Scytalidium dimidiatum, Wangiella dermatitidis, Fusarium solani, Alternaria spp. The dermatophytes, especially Trichophyton rubrum, with a diffusible black pigment produce a mycelium whose hyphae are of homogeneous caliber and regular septae. Often, they are long and straight and are mixed with globular monomorphic arthrospores. Fontana-Masson stains their cytoplasm while their walls remain hyaline (Perrin and Baran, 1997). The dematiaceous molds have irregular morphology and caliber. Their walls are thick and more or less loaded with melanin. They are stained by the Fontana-Masson, which does not stain the cytoplasm (Russel-Bell, 1983). Several types of fungus, especially Trichophyton soudanense and Candida, can produce a pigmentation of the nail by activating melanocytes in the matrix and/or nail bed. However, the black pigmentation sometimes associated with nail Candida infection may remain unexplained. In most cases, clinical examination may differentiate between these entities but dermoscopy is very helpful in difficult cases. It facilitates the identification of small melanin inclusions in the nail plate which resemble tiny gray-black granules on dermoscopic examination. These granules are not seen in hematomas or fungal infections. The latter is frequently associated with a nail dystrophy, which clinically is already suggestive of this diagnosis. Dermoscopically, the pseudopigmentation in fungal infections of the nail is homogeneous and does not always show melanin inclusions. As about two-thirds of ungual melanomas start with brown to black nail pigmentation, the diagnosis of subungual melanonychia must always be included in the differential diagnosis of longitudinal melanonychia.

When Should the Clinician be Suspicious? (Baran and Kechijian, 1989) When longitudinal melanonychia: ∑ begins in a single digit of a person during the fourth to sixth decade of life or later. However, melanonychia due to subungual melanoma has even been observed in children; ∑ develops abruptly in a previously normal nail plate; ∑ becomes suddenly darker or wider (Fig. 55.7); ∑ occurs in the thumb, or index finger, or great toe; 1061

CHAPTER 55

Fig. 55.7. Proximal widening of longitudinal melanonychia (melanoma in situ) (see also Plate 55.4, pp. 494–495).

Fig. 55.8. Melanonychia associated with Hutchinson sign (melanoma) (see also Plate 55.5, pp. 494–495). (Courtesy C. Beylot, France.)

∑ occurs in a person who gives a history of digital trauma;

mandatory. It provides useful information that could aid in determining whether a nail apparatus biopsy should be done.

∑ occurs singly in the digit of a dark-skinned patient, partic-

ularly if the thumb or great toe is affected; ∑ demonstrates blurred, rather than sharp, lateral borders; ∑ occurs in a person who gives a history of malignant

melanoma; ∑ occurs in a person in whom the risk of melanoma is increased (e.g., dysplastic nevus syndrome); ∑ is accompanied by nail dystrophy, such as partial nail destruction or disappearance of the nail plate; ∑ is associated with the Hutchinson sign (Fig. 55.8). This periungual spread of pigmentation is the most important indicator of subungual melanonychia provided the conditions accompanied by the pseudo-Hutchinson sign (Baran and Kechijian, 1996; Fig. 55.9) have been ruled out (Table 55.3); ∑ bands that do not extend distally to the free edge of the nail are unlikely to represent subungual melanoma. However, they may represent metastatic melanoma (Fig. 55.10) or longitudinal melanonychia arising from the nail bed. Nevertheless, despite meticulous evaluation, the etiology of longitudinal melanonychia may remain obscure, perplexing to the physician, and distressing for the adult patient. Dermoscopic examination of longitudinal melanonychia is, therefore,

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Dermoscopic Examination of Longitudinal Melanonychia (Ronger et al., 2002) Dermoscopy is a noninvasive method for the differential diagnosis of pigmented lesions of the skin and the early diagnosis of melanoma. It has been shown to increase diagnostic accuracy compared to clinical visual inspection. Dermoscopy uses an immersion technique to render the stratum corneum translucent, and optical magnification. The handheld devices (dermoscopes) are easy to use and relatively inexpensive. Recently, dermoscopy has been found to be useful for the assessment and diagnosis of longitudinal melanonychia. For the examination of the nail apparatus we recommend the use of a gel (cosmetic gel, ultrasound gel) as an immersion liquid. Due to its viscosity, the gel does not wash off and fills the space ideally between the curved nail surface and the plane contact plate of the handheld dermoscope. For evaluation of the pigmentation it may be useful to vary the focus of the device. With dermoscopy the following criteria can be evaluated:

THE MELANOCYTE SYSTEM OF THE NAIL AND ITS DISORDERS

Fig. 55.10. Longitudinal melanonychia that does not reach the free edge (melanoma metastasis) (see also Plate 55.7, pp. 494–495).

Fig. 55.9. Subungual hemorrhage associated with the pseudoHutchinson sign (see also Plate 55.6, pp. 494–495). (Courtesy R. Sinclair, Australia.)

∑ Homogeneous grayish lines with gray pigmentation of the

background: This pattern is usually due to an epithelial hyperpigmentation without melanocytic hyperplasia (lentigo, drug induced pigmentation, ethnic pigmentation, etc.). ∑ Brown background pigmentation: A brown background pigmentation is usually associated with overlying lines (which can be regular or irregular) and is due to prominent melanocytic hyperplasia. ∑ Regular pattern: Brown longitudinal parallel lines with regular coloration, spacing, and thickness (Fig. 55.11). This pattern is usually associated with a brown homogeneous coloration of the background. The color of the lines varies from light brown to black. However the same shade of brown is consistent within the lesion. The spacing between the bands is regular and the thickness of the bands is similar throughout the lesion. ∑ Irregular pattern: Longitudinal brown to black lines with spacing of irregular thickness, spacing and/or coloration, and disruption of the parallelism (Fig. 55.12). This pattern is also associated with a homogeneous brown pigmentation of the background, but in this case the color of the lines varies from light brown to black and in most cases many different colors and shades of brown can be seen in the same nail pigmenta-

tion. The lines vary in their thickness and spacing. In many cases the lines that are supposed to run parallel lose their parallelism and cross each other. Dermoscopy has changed the management of nail pigmentation because it facilitates the differentiation of melanocytic hyperplasia and epithelial pigmentation. If the pattern is irregular, complete excisional biopsy is always recommended for both diagnosis and treatment.

Appropriate Surgical Intervention in Longitudinal Melanonychia No single biopsy method meets the needs of all patients with longitudinal melanonychia. The type of biopsy selected (Baran and Kechijian, 1989) is determined by a range of factors: (1) periungual pigmentation; (2) the location of the band within the nail (median or lateral); (3) the origin of the band in the matrix (proximal or distal); (4) the width of the band. Periungual pigmentation indicates a high risk of malignancy. If there are no other factors to account for this pigmentation, the whole nail apparatus should be removed “en bloc” down to the bone with a 1 mm margin of normal tissue and without cosmetic consideration. When the lateral third of the nail plate is involved, lateral longitudinal excisional biopsy is the preferred method, since the cosmetic outcome is reasonable. When the mid-portion of the nail plate is involved the potential of postoperative dystrophy may be important. To perform the optimal biopsy, it is necessary to identify the origin of melanocytes responsible for the band in the matrix. This may be determined by sampling the free edge of the nail

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CHAPTER 55 Table 55.3. Conditions accompanied by pseudo-Hutchinson sign. Condition Benign Illusory pigmentation Ethnic pigmentation

Clinical features

Regressing nevoid melanosis in childhood Subungual hematoma Silver nitrate

Dark color traverses the transparent cuticle and thin nail fold Pigmentation of proximal nail fold in dark-skinned persons; lateral nail folds not involved; longitudinal melanonychia not always present; often exaggerated in thumbs Macular pigmentation of lips, mouth, and genitalia; one or several fingers involved Hyperpigmentation of fingers and toes; macular pigmentation of buccal mucosa and lips Diffuse tanning of both exposed and nonexposed portions of the body; bluish-black discoloration of the mucous membranes of the lips and mouth Reported after treatment of finger dermatitis, psoriasis, and chronic paronychia Polydactylous involvement Polydactylous involvement Polydactylous involvement Polydactylous involvement; zidovudine produces similar pigmentation Friction, nail biting and picking, and boxing Pigment recurrence after biopsy of longitudinal melanonychia in acquired and congenital melanocytic nevi; often striking cytologic atypia after biopsy Monodactylous; initial increase in dyschromia followed by subsequent pigment regression; perplexing disorder Exceptionally, blood spreads to nail folds and hyponychia area For treatment of granulation tissue; may produce a black halo

Malignant Bowen disease

Monodactylous or polydactylous with longitudinal melanonychia

Laugier syndrome Peutz–Jeghers syndrome Addison disease Radiation therapy Malnutrition Minocycline Amlodipine Patients with AIDS Trauma-induced congenital nevus Congenital or acquired nevus

plate and using Masson-Fontana stain. Pigment in the lower part of the nail reflects a distal matrix origin with good cosmesis. In contrast, pigment in the upper portion of the nail reflects an origin in the proximal matrix and carries a high risk of scarring with secondary nail dystrophy following excision. The type of biopsy needed will depend on the width of the longitudinal melanonychia: ∑ For a band less than 3 mm in width, a 3 mm punch biopsy is advised. ∑ For a band 3–6 mm in width, if the pigment arises from distal matrix, a transverse matrix biopsy can be performed while proximal matrix pigment requires an “en bloc” removal and repair, using a U-flap. ∑ For a band greater than 6 mm in width, matrix punch or transverse biopsy is usually adequate as a preliminary investigation. Recently, tangential matrix excision has been suggested (Haneke, 1999). It gives a specimen less than 1 mm thick. This technique may avoid the risk of postoperative nail dystrophy. If the lesion turns out to be malignant, total nail ablation may be performed three days later.

Benign Melanonychia Benign melanonychia presents two problems for the pathologist.

How to Distinguish Simple Melanocyte Activation from Lentigo? The histologic definition of lentigo classically rests on two cri1064

teria: a regular epidermal papillomatosis and an increase of the number of melanocytes arranged in solitary units; it is termed lentigo-nevus when at least one nest is present. The lack of nevus nests and discrete melanocytic hyperplasia are often observed in the setting of a benign melanonychia. The difficulty in distinguishing a simple melanocyte activation from a lentigo in the nail matrix is a classic problem in the literature (Goettmann-Bonvallot et al., 1999; Molina and Sanchez, 1995). In addition, the value of a possible epithelial hyperplasia is debatable because of the high physiologic variability of the matrix papillomatosis. In our practice (C. P. personal data), when the number of melanocytes is close to normal — the most common situation — we use criteria adjusted to the topography and morphology of the melanocytes. Melanocyte activation differs from lentigo by the dendritic aspect of the melanocytes and their suprabasal topography (Fig. 55.13). The lentigo corresponds to melanocytes scattered along the basal membrane. They have a round appearance often with a clear halo of cytoplasmic retraction. The dendritic shapes of the melanocytes in melanocyte activation are sometime well visible when the dendrites are overloaded with melanin granules. However, very often only Fontana-Masson stain or HMB-45 immunoreactivity can display them. The suprabasal topography of these melanocytes provides evidence of their physiologic characteristics which are really different from the basal hamartomatous location of the lentigo. In the proximal matrix, the dendritic melanocytes are found between the second and tenth layer of basaloid cells whereas in the

THE MELANOCYTE SYSTEM OF THE NAIL AND ITS DISORDERS

Fig. 55.11. Benign melanocytic nevus of the thumbnail of a 6-yearold male patient. The background pigmentation is brown, indicating the presence of melanocytic hyperplasia and the pattern of the lines is regular with regard to coloration, spacing, thickness, and parallelism (see also Plate 55.8, pp. 494–495).

Fig. 55.12. Acral lentiginous melanoma of the hallux in a 54-yearold female patient (Clark level II, 0.32 mm). The background pigmentation is also brown, but the lines are irregular in thickness, spacing, and coloration, and show areas of disruption of parallelism (see also Plate 55.9, pp. 494–495).

distal matrix they are limited to the first suprabasal layer.

Characteristic Histologic Features of the Nail Apparatus Nevi Like the plantar nevi, the ungual nevi may mimic a nail melanoma (Clemente et al., 1995). Classically the features that can be mistaken for malignancy are: lentiginous manner of growth; suprabasal migration with nail plate involvement; and a dermal inflammatory response. Two further characteristics of this region should be mentioned: ∑ The symmetric nature of the nevus is often difficult to evaluate because of the frequent extension of the matrix nevi to the ventral aspect of the proximal nail fold and more rarely to the nail bed and to the hyponychium (Coskey et al., 1983, Goettmann-Bonvallot et al., 1999; Tosti et al., 1996). These various regions have differing anatomic architecture, which makes comparative analysis of the lesional edges barely reliable (Fig. 55.14).

Fig. 55.13. Melanocyte activation (HMB-45 staining; original magnification ¥250).

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CHAPTER 55

Fig. 55.14. Nail matrix nevus. Note the extension to the eponychium (arrowheads).

In some junctional nevi there is persistence of a basal and suprabasal physiologic dendritic melanocyte contingent (C. P. personal data). This dendritic aspect is regarded as the earliest histologic sign of acral lentiginous melanoma. However, it should be carefully interpreted in the nail apparatus and always linked with nuclear atypias.

Melanoma in Situ The histologic diagnosis of melanoma in situ is sometimes difficult because of the poor quality of the biopsy or because of the inappropriate site of origin. The acral lentiginous type predominates. The presence of nests and/or migration throughout the epithelium and the nail plate indicates an advanced stage and one should look for an invasive focus on serial sections (Kuchelmester et al., 2000). Improvements in the clinical criteria for assessment of the longitudinal melanonychia and in biopsy techniques has enabled the detection of a larger number of incipient acral lentiginous melanomas. The best diagnostic criteria, in this type, are the cytonuclear atypias and the confluence, even focal, of these atypical melanocytes. Melanoma in situ, initially pagetoid, is rare in the nail apparatus whereas a pagetoid contingent within an acral lentiginous melanoma is frequent and may present in up to 50% of cases (Takematsu et al., 1985). Tomizawa (2000) has reported a variant of pagetoid melanoma in situ characterized by a very limited number of melanocytes scattered throughout the whole thickness of the basal compartment, which could be generated from suprabasal melanocytes in the matrix. In fact, serial sections disclose in this variant rare atypical melanocytes in the keratogenous zone and in the nail plate (Perrin, 2000). Nuclear atypias do not pose a problem when they are present as hyperchromatic or clear nuclei of varying size and with prominent nucleoli. Sometimes, the distinctions are more subtle. They may be limited to a minimal increase in nuclei which are of the same size as the keratinocytes. Useful distinguishing features are their angular shape, dense chromatin, 1066

Fig. 55.15. Isolated atypical melanocyte diagnosed at the time of the biopsy as atypical melanocyte hyperplasia and corresponding to acral lentiginous melanoma when the monobloc excision of the whole band was performed (HES ¥400).

polymorphism, and variation in size in different fields. Sometimes the atypical nuclei are so discrete that it is difficult to make a diagnosis with certainty. In these cases greater weight must be given to the clinical features. If there is a resemblance to a melanoma or if there is a widening of the longitudinal band, it is better to use the descriptive term “atypical melanocytic hyperplasia” (Fig. 55.15) and consider a monobloc excision of the band. The age of the patient is important as even slight atypias in a middle-aged patient are much more significant than those in a child (GoettmannBonvallot et al., 1999).

Invasive Melanoma Ungual melanomas are not restricted to the acral lentiginous type. All the types observed elsewhere in the epidermis may be seen. However, the assessment of the level of invasion does not follow the classical rules used for the skin. Clark levels are modified for two reasons: ∑ Because of the unique fibrotendinous structure of the nail dermis, it is often difficult to determine accurately the interface between papillary and reticular dermis. Consequently, the incidence of levels III and IV is increased. ∑ In addition, because of a lack of an anatomic hypodermal layer, level V corresponds to an invasion of the osteocartilaginous structures (Patterson and Helwig, 1980). The Breslow index is also modified by two factors (Perrin, 2000). The epithelium of the matrix and nail bed is devoid of a granular layer. In melanoma an epidermal metaplasia is frequently present which facilitates the calculation of this index. However, this metaplasia may be partial and its thickness very variable from one plane to another. In the physiologic state and compared to the epidermis, the epithelium of the proximal nail fold and of the distal matrix is much thinner. These features are only visible on transverse sections. Longitudinal sections, which are usually performed,

THE MELANOCYTE SYSTEM OF THE NAIL AND ITS DISORDERS

Fig. 55.16. Young child presenting with longitudinal melanonychia — progressive fading — disappearance at age 18. (Courtesy E. Grosshans, France.)

are more difficult to orientate in a vertical plane and the thickness of the epithelium is apparently excessively increased because some sections are tangential. The nail bed epithelium has a papillary design which is more pronounced than that of the epidermis in case of invasion. This architecture of the nail bed may lead to features similar to verrucous melanoma. The Breslow index, used on the epidermis, is not entirely transferable to the nail apparatus because of microanatomic particularities of the nail and sectional artifacts. Further studies are still necessary to ratify the prognosis and therapeutic value of the Breslow index on the nail unit.

Spontaneous Regression of Melanocytic Lesions Longitudinal melanonychia, as a sign of the “nevoid nail area melanosis” observed in Japanese children, may demonstrate spontaneous regression and even disappearance (Kikuchi et al., 1983). The histologic features of these pigmented lesions are lacking. Longitudinal melanonychia from a matrix nevus was also observed to fade with time (Tosti et al., 1994a). Grosshans (1994) reported the complete disappearance of longitudinal melanonychia occurring in a 4-year-old white patient after 14 years (Fig. 55.16).

References Baran, R., and P. Kechijian. Longitudinal melanonychia. Diagnosis and management. J. Am. Acad. Dermatol. 21:1165–1175, 1989. Baran, R., and P. Kechijian. Hutchinson’s sign: A reappraisal. J. Am. Acad. Dermatol. 34:87–90, 1996. Baran, R., and C. Perrin. Linear melanonychia due to subungual keratosis of the nail bed: a report of 2 cases. Br. J. Dermatol. 140:730–733, 1999. Clemente, C., S. Zurrida, C. Bartoli, A. Bono, P. Collini, and F. Rilke. Acrallentiginous naevus of plantar skin. Histopathology 27;549–555, 1995.

Commo, S., and B. A. Bernard. Melanocyte subpopulation turnover during the human hair cycle: an immunohistological study. Pigment Cell Res. 13:253–259, 2000. Coskey, R. J., T. D. Magnell, and E. G. Bernacki. Congenital subungual nevus. J. Am. Acad. Dermatol. 9:747–751, 1983. De Berker, D., R. P. R. Dawber, A. Thody, and A. Graham. Melanocytes are absent from normal nail bed; the basis of a clinical dictum. Br. J. Dermatol. 134:564, 1996.

Goettmann-Bonvallot, S., J. André, and S. Belaich. Longitudinal melanonychia in children: A clinical and histopathologic study of 40 cases. J. Am. Acad. Dermatol. 41:17–22, 1999. Grosshans E. Mélanines, mélanomes. Nouv. Dermatol. 13:497–498, 1994. Haneke E. Operative therapie akraler und subungualer melanoma. In: Operative und onkologische Dermatologie. Fortschritte der operativen und onkologischen Dermatologie 15, R. Rompel, and J. Petres (eds). Berlin: Springer, 1999, pp. 210–144. Haneke, E., and D. Binder. Subuguales Melanome mit streifiger Nagelpigmentierung. Hautarzt 29:389–391, 1978. Haneke, E., and R. Baran. Longitudinal melanonychia. Dermatol. Surg. 27:580–584, 2001. Hashimoto, K. Ultrastructure of the human toenail. J. Invest. Dermatol. 56:235–246, 1971. Higashi, N. Melanocytes of nail matrix and nail pigmentation. Arch. Dermatol. 97:570–574, 1968. Higashi, N., and D. Saito. Horizontal distribution of the dopapositive melanocytes in the nail matrix. J. Investig. Dermatol. 53:163–165, 1969. Horikawa, T., D. A. Norris, T. W. Johnson, T. Zekman, N. Dunscomb, S. D. Bennion, R. L. Jackson, and J. G. Morelli. DOPA-negative melanocytes in the outer root sheath of human hair follicles express premelanosomal antigens but not a melanosomal antigen of the melanosome-associated glycoproteins tyrosinase, TRP-1, and TRP-2. J. Invest. Dermatol. 106:28–35, 1996. Kerl, H., H. Trau, and A. B. Ackerman. Differentiation of melanocytic nevi from malignant melanomas in palms, soles and nail bed solely by signs in the cornified layer of the epidermis. Am. J. Dermatopathol. 6:159–161, 1984. Kikuchi, I., S. Inoue, E. Sakaguchi, and T. Ono. Regressing nevoid nail area melanosis in childhood. Dermatology 186:88–93, 1993. Kuchelmester, C., G. Schaumburg-Lever, and C. Garbe. Acral cutaneous melanoma in Caucasians: clinical features, histopathology and prognosis in 112 patients. Br. J. Dermatol. 143:275–280, 2000. Molina, D., and J. L. Sanchez. Pigmented longitudinal bands of the nail. Am. J. Dermatopathol. 17:539–541, 1995. Patterson, R. H., and E. B. Helwig. Subungual malignant melanoma: A clinical-pathologic study. Cancer 46:2074–2087, 1980. Perrin, C., and R. Baran. Longitudinal melanonychia caused by Trichophyton rubrum. J. Am. Acad. Dermatol. 31:311–316, 1994. Perrin, C., J. F. Michiels, A. Pisani, and J. P. Ortonne. Anatomic distribution of melanocytes in normal nail unit. Am. J. Dermatopathol. 19:462–467, 1997. Perrin, C., L. Langbein, and J. Schweizer. Expression of hair keratins in the adult nail unit: an immunohistochemical analysis of the onychogenesis in the proximal nail fold, matrix and nail bed. Br. J. Dermatol. 151:362–371, 2004. Perrin, Ch. Anatomie microscopique de l’appareil unguéal: histologie et histopathologie. In: L’ongle. Monographie du GEM N° 27, C. Dumontier ed. Paris: Elsevier, 2000, pp. 19–28. Quinlan, K. E., J. J. Janiga, R. Baran, and H. W. Lim. Transverse melanonychia secondary to total skin electron beam therapy: a report of 3 cases. J. Am. Acad. Dermatol. 53:S112–114, 2005. Ronger, S., S. Touzet, C. Ligeron, B. Balme, A. M. Viallard, D. Barrut, C. Colin, and L. Thomas. Dermoscopic examination of nail pigmentation. Arch. Dermatol. 138:1327–1333, 2002. Russel-Bell, B. Characterization of pigmented fungi by melanin staining. Am. J. Dermatopathol. 5:77–81, 1983. Smith, D. F., M. B. Morgan, and M. S. Bettencourt. Longitudinal melanonychia. Arch. Dermatol. 139:1209–1214, 2003. Takematsu, H., M. Obata, Y. Tomita, T. Kato, M. Takahashi, and R. Abe. Subungual melanoma. Cancer 55:2725–2731, 1985. Tomizawa, K. Early malignant melanoma manifested as longitudinal

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CHAPTER 55 melanonychia: subungual melanoma may arise from suprabasal melanocytes. Br. J. Dermatol. 143:431–434, 2000. Tosti, A., R. Baran, R. Morelli, P. A. Fanti, and A. Peserico. Progressive fading of longitudinal melanonychia due to a nail matrix melanocytic nevus in a child. Arch. Dermatol. 130:1076–1077, 1994a. Tosti, A., N. Cameli, B. M. Piraccini, P. A. Fanti, and J. P. Ortonne.

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Characterization of nail matrix melanocytes with anti-PEP1, antiPEP8, TMH-1, and HMB-45 antibodies. J. Am. Acad. Dermatol. 31:193–196, 1994b. Tosti, A., R. Baran, B. M. Piraccini, N. Cameli, and P. A. Fanti. Nail matrix nevi: A clinical and histopathologic study of twenty-two patients. J. Am. Acad. Dermatol. 34:765–771, 1996.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Pigmentary Abnormalities and Discolorations of the Mucous Membranes John C. Maize, Jr. and John C. Maize, Sr.

Introduction Pigmentary abnormalities and discolorations of the oral and anogenital mucosa, like those of the cutaneous surface, have a variety of causes and implications. Causes range from benign physiologic pigmentation which may be an incidental finding or a cosmetic concern for an individual to malignant neoplasms that portend a grave prognosis to the patient. This chapter discusses alterations in pigmentation (both hyperpigmentation and hypopigmentation) and color that affect the oral, genital, and anal surfaces, as well as the causes, associated conditions, and treatments.

Normal Melanocyte Numbers in Mucosae

Genital Melanocyte numbers on genital skin are at the upper end of the range of variation of melanocyte density observed regionally on the cutaneous surface in general and is in keeping with the density of melanocytes observed in oral mucosal epithelium and external anal epithelium (Freedberg et al., 1999).

Anal Melanocyte density on external anal skin and the squamous zone of the anal canal is similar to the density of melanocytes elsewhere on the cutaneous surface. Melanocytes are present in transitional zone epithelium of the anal canal but are decreased in number compared with the squamous zone. Melanocytes are present but exceedingly rare in normal colorectal mucosa (Clemmensen and Fenger, 1991).

Hyperpigmentation

Oral Melanocyte number and distribution within the epithelium of the oral mucosa is similar to the epidermis of the cutaneous surface in general. In the epidermis from the cutaneous surface, melanocytes vary in density depending upon the site, with more melanocytes per unit area on skin of the face (1 melanocyte per 4 keratinocytes within the basal epidermis in routine hematoxylin and eosin stained sections) than on the trunk (1:14) (Hicks and Flaitz, 2000). Within the oral gingival mucosa, the melanocyte to keratinocyte ratio is 1:15 (Cicek and Ertas, 2003; Hicks and Flaitz, 2000). The number of melanocytes within the mucosa in light and darkerskinned races is the same (Cicek and Ertas, 2003). Melanocytes in the oral mucosa produce melanin pigment within melanosomes in the cytoplasm as they do elsewhere, but the amount of melanin pigment production is decreased relative to melanocytes in normal skin (Cicek and Ertas, 2003). Variations in pigmentation of the mucosa among racial groups is due to differences in pigment production by melanocytes and to the type and distribution of melanosomes within melanocytes (Cicek and Ertas, 2003). The morphology of normal melanocytes in the mucosa on routinely stained sections is similar to that of melanocytes in the epidermis, with a round, dark nucleus, inconspicuous nucleolus, and often a clear halo around the nucleus. Cytoplasm is usually difficult to appreciate.

Oral There is an array of etiologies for clinically appreciable pigmentation of the oral mucous membranes. Causes include: endogenous chromophores such as blood and hemoglobin, exogenous chromophores (medications, metals, dental amalgam), the pigment melanin, neoplasms (lentigines, nevi, melanomas, benign and malignant vascular neoplasms), and genetic or systemic disorders [acromegaly, Addison disease, Albright syndrome, hemochromatosis, Laugier–Hunziker syndrome, and Peutz–Jeghers syndrome (PJS)]. Each of these categories will be discussed in this section.

Endogenous Chromophores (Blood and Heme) Benign Hemangiomas (Capillary Hemangiomas; Hemangiomas of Infancy; Strawberry Nevi) Hemangiomas are benign proliferative vascular lesions and are the most common tumors of infancy. Girls are more often affected than boys, and premature infants are the most susceptible to developing a hemangioma. Fifty percent of lesions are present congenitally, with the remainder usually presenting in the month after birth. Early lesions may appear telangiectatic or ecchymotic, and rarely, appear to be a hypochromic patch clinically. Lesions subsequently enlarge and become dark red to deep purple nodules or tumors. Lesions can grow rapidly or slowly over a 1069

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period of months up to one year of age before stabilizing and then slowly involuting over the next few years to one decade. Lesions on the lip as well as a few other sites such as the nose or parotid gland may not involute. Intraoral hemangiomas can occur and, early on, be confused with a pigmented lesion. With cervicofacial hemangiomas in the beard area of the face including the lip, involvement of the airway can be problematic and may require a tracheotomy. Exact mechanisms controlling lesion development and devolution are not known. Treatment may involve the use of intralesional or systemic corticosteroids, lasers, surgery, or systemic interferon. The mode of therapy depends largely on the exact lesion location as well as size, growth rate, and risk to the patient (Metry and Hebert, 2000). Histopathology Evolving lesions show a proliferation of endothelial cells which may be enlarged and display numerous mitoses. Cells may be arranged in solid aggregates or strands, and vascular lumina may be small, slitlike, and not easily visualized. Later, lesions develop a more obvious lobular pattern of vascular spaces with flattened endothelial cells lining the lumina. Endothelial cells label with standard endothelial cell markers, including CD31 and CD34. Immunoperoxidase staining for GLUT-1 differentiates these lesions from vascular malformations that have no tendency for involution (Weedon, 2002a). Port Wine Stain (Nevus Flammeus) Port wine stains are uncommon congenital vascular malformations usually of the head and neck area, which persist throughout life in most cases. Involvement of the face in the distribution of the V1 branch of the trigeminal nerve is a component of the Sturge–Weber syndrome. Enlargement of the lips can be especially disfiguring as a result of vascular stasis and soft tissue hypertrophy (macrocheilia) (Del Pozo et al., 2004). Intraoral involvement can appear as a dark red–purple patch or plaque and is usually in addition to involvement of the face. Isolated oral involvement must be exceedingly rare if it occurs at all. Histopathology Early lesions demonstrate slight dilation of the vessels of the superficial vascular plexus. With time, vascular channels may become notably distended and congested by erythrocytes. Vessel walls are thin and there is no proliferation of vascular structures (Weedon, 2002b). Trauma (Hemoglobin/Hemosiderin) Hyperchromic lesions on the oral mucosa may sometimes follow trauma with submucosal hemorrhage. Breakdown of red blood cells liberates hemoglobin, which is phagocytized by tissue macrophages. Within the lysosomes of macrophages, hemoglobin is converted by the action of various enzymes into hemosiderin. Siderophages may persist within the area of injury for a prolonged period of time (many months) and be appreciable clinically as a discoloration.

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Fig. 56.1. Venous lake of the upper lip. Diascopy will often reveal the vascular nature of these lesions; however, when they thrombose, they may not blanch with pressure and may simulate a melanocytic lesion.

Venous lakes Clinical Venous lakes are common lesions of aged individuals and often affect the lower lip as well as other areas of the head and neck (Fig. 56.1). They are solitary papules usually less than 1 cm in diameter and are clinically dark-blue to black in appearance. A simple clinical test will confirm the diagnosis in many instances: firm digital pressure will compress the lesion and evacuate it; the lesion will then refill with blood slowly when pressure is released. If the lesion is thrombosed, however, it may be firm and noncompressible, clinically mimicking a melanocytic lesion, especially malignant melanoma. Histopathology Biopsy reveals a dilated endothelial-lined vascular space with a single lumen in the upper dermis. The lumen usually contains many erythrocytes (Fig. 56.2). Thrombus may partly or completely fill the space in some lesions (Weedon, 2002c). Malignant Kaposi sarcoma Kaposi sarcoma (KS) is a herpes virusinduced malignant vascular endothelial proliferation caused by human herpes virus-8 (HHV-8) (Drago and Rebora, 1999). There are multiple clinical settings for development of KS: (1) classic type — lesions occurring on the lower legs of elderly men of Mediterranean heritage; (2) endemic type — seen mostly in adult men in central Africa; (3) iatrogenic type — seen in patients with organ transplants or patients who have undergone chemotherapy for lymphoproliferative diseases; and (4) HIV-associated (Drago and Rebora, 1999). Involvement of the oral mucosa is usually seen in patients with HIV-associated KS (Newland et al., 1988), and may be

PIGMENTARY ABNORMALITIES AND DISCOLORATIONS OF THE MUCOUS MEMBRANES

Fig. 56.2. Venous lake histopathology. There is a dilated vascular space congested by erythrocytes. There is partial thrombosis at one edge of the lumen.

Fig. 56.3. Intraoral Kaposi sarcoma. KS on the mucosa may simulate a melanocytic neoplasm. Mucosal KS is most often seen in the setting of human immunodeficiency virus (HIV) disease (see also Plate 56.1, pp. 494–495).

the presenting sign of illness on occasion. Early, or patchstage, lesions, are flat, often irregular, and are dark red or purple in color (Fig. 56.3), whereas nodular lesions of KS may be large, elevated, indurated, and hemorrhagic or black in appearance. Lesions at any stage may be confused clinically with a melanocytic neoplasm, and biopsy may be needed to correctly establish the diagnosis. Histopathology The microscopic features of the neoplasm depend on the stage of development of the lesion. Early lesions of KS are composed of delicate — at times insidious — ramifying vascular channels which make the dermis appear subtly more cellular than normal at scanning magnification.

Fig. 56.4. Kaposi sarcoma histopathology. There is a proliferation of spindled endothelial cells which lack significant cytologic atypia. There are many erythrocytes within the irregular vascular channels formed by the endothelial cells as well as within the intracytoplasmic lumens in some cells.

The mucosal epithelium in patch-stage lesions is unaltered. Delicate spindled endothelial cells may closely hug the dermal collagen bundles, and intraluminal erythrocytes in the vessels between collagen bundles may be mistaken for extravasation if care is not taken. The presence of neoplastic vascular channels which enclose pre-existing vessels may make them appear to protrude or “float” within an empty space, a change classically referred to as the “promontory sign.” As lesions advance, they become more cellular and may even consist of solid-appearing zones of spindled cells (Fig. 56.4). The surface may ulcerate. Cytologically, the spindled cells are not notably atypical in most cases. Intracytoplasmic lumens containing erythrocytes may be appreciated and hyalinized eosinophilic round globules which represent effete erythrocytes may be seen intracellularly and extracellularly. Hemosiderin deposits are common in lesions at all stages of development. The spindled cells label with the typical immunoperoxidase markers of endothelium: CD31, CD34, and factor VIII-related antigen (Newland et al., 1988). Immunoperoxidase staining for human herpes virus 8 (KS herpes virus) is a sensitive and specific confirmatory test (Patel et al., 2004). Treatment In recent years with advances in HIV treatment using highly active antiretroviral therapy (HAART), specific treatment for KS has been largely obviated by the immune reconstitution which HAART therapy promotes, though improvement does take time once therapy is initiated. Breakthrough or nonresponsive cases do still occur. Depending upon the severity of disease, therapy may include the topical retinoid gel alitretinoin, surgery, radiotherapy, interferon or interleukin-2 infusions, or intralesional or systemic chemotherapy with vinblastine. Other chemotherapeutic agents have also

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which occurs in Addison disease (see the following section on Addison disease). Hyperpigmentation in acromegaly is due to increased melanin production by melanocytes in response to increased a-melanocyte stimulating hormone (a-MSH) produced by the pituitary (Lenane and Powell, 2000).

Fig. 56.5. Physiologic (racial) pigmentation. There are relatively symmetric hyperpigmented patches on the gingiva of this black patient.

been successfully used as single-drug treatments including chlorambucil, cyclophosphamide, and actinomycin (Sterling and Kurtz, 1998).

Melanin and Melanocytes Benign Physiologic (Racial) Pigmentation Hyperpigmentation of the oral mucosa often occurs as a variant of normal. Clinically appreciated pigmentation of the oral mucosa is rare in white people, but common in darker-skinned individuals (Fig. 56.5). Onset of this so-called physiologic pigmentation is during infancy or puberty (Eisen and Voorhees, 1991). Physiologic pigmentation is present to different degrees among individuals within the same race and within a given individual in different areas of the mouth. The labial gingiva is more commonly affected than the buccal, palatal, or lingual gingiva, and, more specifically, the attached gingiva is most frequently affected, followed in decreasing order by the papillary gingiva, the marginal gingiva, and the alveolar mucosa (Cicek and Ertas, 2003). On physical examination, physiologic pigmentation is almost always bilateral and symmetrical (Dummet and Bolden, 1964). Microscopically, the cause of the pigmentation is attributable to increased pigment production within melanocytes in the hyperpigmented areas and not to a difference in numbers of melanocytes in those areas. Treatment is not required, but may be desired for cosmetic reasons. Surgical, cryosurgical, and laser techniques may result in improvement (Almas and Sadig, 2002; Tal, 1991; Tal et al., 2003). Acromegaly Acromegaly is a disease caused by excessive secretion of human growth hormone by the pituitary, usually due to a hormone-secreting tumor. Roughly 40% of patients with the disease will also develop hyperpigmentation of the face, oral and genital mucosa, scars, and palmar creases. The pattern of hyperpigmentation is very similar to the pattern 1072

Addison Disease One potentially life-threatening cause of mucosal hyperpigmentation is Addison disease. Addison disease is rare, estimated to occur in one person per million population, is more common in women, and is caused by lack of production of cortisol and other steroid hormones by the adrenal glands. Causes of primary hypocortisolism (adrenal failure) can include autoimmune destruction of the adrenal cortex (most common), infections such as tuberculosis or histoplasmosis, or metastatic disease involving the adrenals with destruction of glands. Mucosal pigmentation often occurs in the setting of generalized hyperpigmentation or “tanning,” but may occur in isolation or in the presence of only subtle or slowly developing generalized hyperpigmentation. Other signs and symptoms of Addison disease include hypotension, fatigue/loss of energy, weight loss, and electrolyte imbalances. Pigmentation is due to increased pigment production by melanocytes secondary to stimulation by aMSH, and biopsy would reveal only hyperpigmentation of the basal epidermis without an increase in melanocyte number. aMSH is formed by cleavage of proopiomelanocortin which is released from the pituitary gland in response to low circulating levels of cortisol (failure of negative inhibition). Clinically, the oral pigmentation most commonly presents as multiple blue-black macular pigmented areas involving any of the mucosal surfaces within the mouth. Other signs include accentuation of pigmentation within established scars, development of genital hyperpigmentation, and pigmentation of the nails and palmar creases (Erickson et al., 2000; Lamey et al., 1985). Albright Syndrome (McCune–Albright Syndrome) The features of this syndrome include fibrous dysplasia of one or more bones with associated pain, deformity, and pathological fractures; large hyperpigmented patches with irregular, jagged outlines (“coast of Maine” café-au-lait macules), and, in girls only, precocious puberty. Hyperpigmented macules are most frequent on the trunk and extremities, but they may affect the face and, sometimes, the lip. Biopsy shows a normal number of melanocytes, some of which have macromelanosomes, and hyperpigmentation of the basal epidermis (Champion et al., 1998; Lenane and Powell, 2000). Carney Complex (LAMB Syndrome; NAME Syndrome) Clinical In 1973, Rees and others first reported a patient with numerous brown macules on the skin and a left atrial “endotheliomyxoma” (Rees et al., 1973). Atherton and coworkers (1980) later described a 10-year-old boy with generalized pigmented macules, blue nevi, congenital melanocytic nevi, myxoid cutaneous lesions, and bilateral atrial myxomas. They designated this compilation of findings the NAME syndrome for Nevi, Atrial myxomas, Myxomas of the skin, and

PIGMENTARY ABNORMALITIES AND DISCOLORATIONS OF THE MUCOUS MEMBRANES

Ephelides. In 1984, Rhodes and coworkers described an additional patient with both cutaneous and cardiac lesions. This patient had brown and black macules on the face and vulva that were documented to have increased melanocyte numbers. The authors felt that, in the light of this finding, “lentigines” was a more appropriate designation for these lesions than the term “ephelides” utilized in the acronym NAME, and they proposed the acronym LAMB instead for Lentigines, Atrial myxomas, Mucocutaneous myxomas, and Blue nevi (Rhodes et al., 1984). Carney and coworkers in 1985 reported a syndrome of hyperpigmented macules, cardiac myxomas, and endocrine abnormalities. Carney published several subsequent reports on this syndrome, and the term Carney complex (CNC) has supplanted the terms “LAMB” and “NAME” for this multiple neoplasia syndrome. CNC type 1 has been linked to a mutation in the PRKAR1A gene on chromosome 17q in a large percentage of affected persons, while other cases (type 2) have been linked to a locus on chromosome 2p16 (McKusick, 1986a). Patients with CNC develop brown or black macules of the face, lips and genital skin, multiple blue nevi, atrial myxomas (in up to 75% of cases) and myxomas of the mucosa and skin. The pigmented macules may develop in early childhood. Psammomatous melanotic schwannoma is a distinctive tumor that has been noted to occur in the skin as well as spinal nerve roots, the gastrointestinal tract, and bone of patients with CNC. A variety of endocrine abnormalities may occur in CNC, including acromegaly (in up to 10% of patients), and tumors of various endocrine glands (adrenals, gonads, thyroid) (Pandolfino et al., 2001). The presence of genital pigmented macules, especially black ones, helps distinguish CNC from Peutz–Jeghers syndrome (Rhodes et al., 1984). Also, with Peutz–Jeghers syndrome, pigmented macules appear predominantly on perioral skin and oral mucosa, eyelids, palms, and soles (Rhodes et al., 1984).

entirely dermal collections of two types of melanocytes. One type is heavily pigmented fusiform melanocytes and the other type is large melanocytes with a polygonal shape, a central round nucleus, and abundant cytoplasm. Melanocytes do not demonstrate maturation with dermal descent. Misdiagnosis of these lesions as malignant melanoma is a danger. The melanocytes of EBN, though large, are not atypical (Izquierdo et al., 2001).

Histopathology Biopsy of pigmented macules of the face, lips, or genital skin can show a spectrum of findings. In some instances, there is hyperpigmentation of the basal zone only without an appreciable increase in melanocytes; in other words, they are ephelides. Other lesions may demonstrate not only hyperpigmentation of the basal epidermis, but also a slight increase in melanocyte numbers and are better termed lentigines, though they may lack epidermal changes of elongation and clubbing of rete ridges seen commonly in simple lentigines. Still other lesions have been described with features of junctional or compound melanocytic nevi. One lesion in particular deserves a brief discussion here, and that is the epithelioid blue nevus (EBN). This lesion was originally described as occurring in the context of CNC but has since been noted to occur sporadically in the absence of other stigmata of CNC. EBN tends to occur on the upper back, shoulders, and extremities and clinically the nevi are indistinguishable from other types of blue nevi. Several cases of sporadic EBN have been described on genital mucosa in both men and women. Microscopically, EBN is comprised of

Histopathology Fixed drug eruptions are microscopically distinctive and a specific diagnosis may be rendered if the reaction is captured during an active phase. Because of the acuity of the reaction, there is a basket-woven stratum corneum. There is a bandlike infiltrate of lymphocytes with admixed eosinophils and scattered neutrophils which abuts and may obscure the dermal–epidermal interface. Necrotic keratinocytes are numerous along the junction and melanophages may be sparse or plentiful depending on the skin type of the affected individual and the length of time the reaction has been ongoing. There is also an accompanying mid to deep dermal perivascular infiltrate which is helpful in distinguishing fixed drug reaction from erythema multiforme (Fig. 56.7). Lesions sampled late in the course may demonstrate postinflammatory pigmentary alteration only (Ackerman et al., 1997).

Fixed Drug Eruption Fixed drug eruption is a peculiar hypersensitivity reaction to a medication or ingested compound which recurs repeatedly in the same specific location(s) after each exposure to the offending agent, though new sites in addition to the older ones may be affected each time the reaction recurs. Sensitization to the offending agent is required and the condition may onset after several weeks of exposure initially and more rapidly (within hours) with subsequent exposures. Lesions start as discrete, round to oval erythematous macules or patches but may become indurated or bullous. They often develop a distinctive red-brown or purple-brown color. Although the lesions do not appear initially as pigmented lesions, they do progress to a stage of hyperpigmentation after the acute reaction wanes. Lesions may affect any part of the body, but are particularly common on mucosal surfaces, including the lips (Fig. 56.6), the genital mucosa, and the perianal skin. Lesions are frequently single but may be multiple. The hyperpigmentation is due to melanin incontinence from the interface reaction that occurs in fixed drug eruptions. Repeated exposure to the drug may result in progressive darkening over time. The most common causative agents have been reported to be sulfonamides, tetracyclines, penicillins, nonsteroidal anti-inflammatory agents, and barbiturates. Phenolphthalein was a common offending agent in the past before its removal from laxative preparations and some beverages (English et al., 1997).

Freckles Clinical Freckles are common lesions, and may occur on the vermilion of the lip as they do elsewhere on sun-exposed skin. 1073

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Fig. 56.7. Fixed drug eruption histopathology. In this acute lesion, there is confluent epidermal necrosis beneath a basket-weave stratum corneum and there is a bandlike mixed inflammatory infiltrate in the dermis. Note the large number of melanophages.

opment of hepatocellular carcinoma, hyperpigmentation, and diabetes mellitus. Up to 25% of patients may have blue-gray macular hyperpigmentation of the hard palate and attached gingiva. The pigmentation is due to increased melanin pigmentation in the basal layer and not to iron deposition within the tissue (Lenane and Powell, 2000; McKusick, 1986b). Fig. 56.6. Fixed drug eruption. The red-brown pigmentation of the eruption involves the upper cutaneous lip and extends onto the vermilion.

The lower lip is more commonly affected than the upper lip (see Figs 54.1–54.3 and Plates 54.1 and 54.2). Freckles are usually tan to brown macules several millimeters in maximal diameter, but they may be larger. The hallmark of a freckle clinically is that it darkens with increased exposure to sunlight, and fades if exposure is limited (Maize and Ackerman, 1987). Histopathology Freckles show hyperpigmentation of the basal layer of the epidermis. Unlike in solar lentigines, there is no elongation or clubbing of rete ridges. There is no acanthosis of the epidermis which is typically seen in labial melanotic macules (see below). Melanocytes are not increased in number, but they may be mildly enlarged with more prominent dendritic processes in the melanocytes of adjacent unaffected skin (Maize and Ackerman, 1987). Hemochromatosis Hemochromatosis in its classic form is an autosomal recessive disease due to defect in the HFE gene on chromosome 6p21.3. The result is systemic iron overload, which, if untreated, can lead to cirrhosis of the liver and devel1074

Labial Melanotic Macules Clinical Labial melanotic macules are not uncommon brown to blue-black flat lesions which usually occur on the vermilion of the lower lip, but may occur on the upper lip as well (Ho et al., 1993). Although they have also been referred to as labial lentigines, they are morphologically distinct from traditional lentigines and do not evolve into nevi as do some lentigines; therefore, the term melanotic macule is preferred for these lesions (Maize, 1988). Patients with melanotic macules of the lip are prototypically white women about 40 years of age with a slowly appearing (over months to years) asymptomatic macule on the lower lip (Fig. 56.8) (Ho et al., 1993). Men less commonly develop melanotic macules, and these lesions have been noted in black patients as well. Many times, these lesions possess a benign clinical morphology, but some lesions may display features such as asymmetry, border irregularity, color variation, and large size which may simulate melanoma in situ. For this reason, they are a cause for clinical concern and biopsy. Oral and labial melanotic macules have been reported in association with human immunodeficiency virus (HIV) disease (Cohen and Callen, 1992). Some authors have proposed the unitarian view that isolated oral/labial melanotic macules are but one manifestation of Laugier–Hunziker syndrome (Dupre and Viraben, 1990) (see discussion on Laugier–Hunziker syndrome below).

PIGMENTARY ABNORMALITIES AND DISCOLORATIONS OF THE MUCOUS MEMBRANES

Fig. 56.8. Labial melanotic macule. Lesions may be asymmetrical and possess an irregular border, simulating melanoma clinically (see also Plate 56.2, pp. 494–495).

Fig. 56.9. Labial melanotic macule histopathology. There is acanthosis of the epidermis with broad rete ridges which show prominent basilar hyperpigmentation. Melanocytes are not increased. Parakeratosis often overlies these lesions as in this example.

Histopathology Melanotic macules classically show hyperpigmentation of the basal layer principally at the tips of elongated and broadened rete ridges. At times, however, pigmentation may be confluent within the basal epidermis. Epithelial changes include compact orthohyperkeratosis, mild parakeratosis, and hyperplasia manifested by widened and elongated rete ridges. In the lamina propria, features such as telangiectasia, enlarged fibroblasts, and melanophages are characteristically present (Fig. 56.9). Although most sources report that melanotic macules are due to increased melanin pigment alone and not to an increase in melanocytes, some studies have demonstrated a twofold

Fig. 56.10. Laugier–Hunziker syndrome. There are multiple tan to brown macules on the vermilion of the lower lip (see also Plate 56.3, pp. 494–495).

increase in the number of melanocytes in these lesions compared to normal lip mucosa (Sexton and Maize, 1987). Melanocytes in melanotic macules also possess morphologic differences over normal mucosal melanocytes. In the case of melanocytes in melanotic macules, individual melanocytes display a network of long, fine, branching dendrites which intercalate among the keratinocytes of the adjacent basal layer and overlying spinous layer. These dendrites are heavily melanized. Dendritic melanocytic proliferations of the mucosal surfaces must be viewed with caution, as melanomas in situ of the mucosa will at times manifest as a lentiginous proliferation of single units with a predominantly dendritic morphology. In contrast to melanotic macules, however, melanomas in situ display long, coarse dendrites which may extend into the upper reaches of the spinous layer. Also, cytologic atypia is not a feature of the melanocytes in melanotic macules; in melanomas in situ, however, atypia manifested by overall cellular and nuclear enlargement, nuclear hyperchromasia and pleomorphism, and nucleolar enlargement may be marked. It should be understood that some areas within a lesion of melanoma may be deceptively benign appearing and small samples may therefore miss the telltale features required to make the diagnosis. Therefore, any biopsy specimen submitted to exclude melanoma should include areas of induration, erosion, or ulceration if such areas are present in a given lesion (Maize, 1988). Laugier–Hunziker Syndrome Clinical In 1970, Laugier and Hunziker described a condition they termed “essential lenticular melanotic pigmentation of the oral cavity and the lips.” The condition predominantly affects Caucasians and onset is during adulthood. Affected persons can display multiple discrete pigmented macules on virtually any aspect of the oral mucosa and often on multiple surfaces (Figs 56.10 and 56.11). The syndrome has been expanded to include pigmented macules 1075

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Fig. 56.12. Peutz–Jeghers syndrome. There are subtle tan macules on the lower lip (see also Plate 56.4, pp. 494–495).

Fig. 56.11. Laugier–Hunziker syndrome. The tongue is also involved in this patient.

or patches on the neck, chest, abdomen, fingers, soles, penis, vulva, vagina, cervix, perineum, and perianal skin as well. Pigmented longitudinal streaks of the nail or pigmentation of the whole nail complete with the Hutchinson sign (pigmentation extending onto the proximal nail fold) have been described. No other organ involvement has been reported in addition to skin and mucosal pigmentation (Veraldi et al., 1991). The unitarian view has been proposed that stipulates that the term “Laugier disease” be used whenever there is a clinically hyperpigmented macule with the histopathologic features of a melanotic macule. Proponents of this view believe that isolated labial melanotic macules, penile lentigines, vulvar melanosis, some lesions of linear melanonychia (those not due to a true melanocytic proliferation), and volar melanotic macules of the palms and soles are merely variant expressions of the classic form of Laugier–Hunziker syndrome (Dupre and Viraben, 1990). Histopathology Biopsy reveals acanthosis of the epidermis, basal layer hyperpigmentation, and pigment incontinence, findings indistinguishable from those of isolated melanotic macules of the lip or genitalia. Melanocytes are normal in number, arrangement within the epidermis, and morphology. Elongated intracytoplasmic melanosomes are present within keratinocytes of the basal epidermis, and it is postulated that there is some functional alteration in melanocytes with respect to melanosome synthesis and transfer to keratinocytes (Veraldi et al., 1991). Treatment Therapy is not required once the diagnosis is established. Laser therapy utilizing lasers effective on melanin 1076

pigment has been successfully performed for improvement of cosmesis (Papadavid and Walker, 2001). Peutz–Jeghers Syndrome PJS is an autosomal dominantly inherited disease, although about 40% of cases are new mutations. The defective gene is SKT11 on chromosome 19p13.3 which encodes a serine-threonine kinase (McKusick, 2004). Patients develop pigmented macules of the oral mucosa, especially the lower lip (Fig. 56.12) and buccal mucosa, smaller pigmented macules (usually less than 1 mm) on the face near the mouth, and pigmented macules on the palms and soles, sometimes with pigmented longitudinal streaking of the nails. The pigmented lesions most commonly arise in the early years of life, though they may be congenital or develop during adulthood. Typically, facial and acral lesions fade over time, though intraoral pigmentation usually persists. In addition to the cutaneous findings, patients develop gastrointestinal polyps mostly in the small intestines, but other areas of the gastrointestinal tract may also be affected. Histopathology Microscopically, the pigmented lesions demonstrate hyperpigmentation of the basal epidermis without an increase in melanocytes. The polyps are usually benign hamartomas, though adenocarcinomas can occur in 2–3% of patients, some of which develop independent of the hamartomatous polyps. There is an increased risk of malignancy developing outside the gastrointestinal tract as well in patients with PJS, especially the breast and ovary. Treatment The skin lesions serve as a hallmark of disease, but they do not require treatment. Lasers effective for skin lesions due to increased melanin pigment have been successfully employed to eliminate lesions in some patients (Kato et al., 1998). Persons affected by PJS must be followed by a gastroenterologist for regular surveillance. Polyps may have to be removed, especially if there are symptoms. Large numbers of

PIGMENTARY ABNORMALITIES AND DISCOLORATIONS OF THE MUCOUS MEMBRANES

Fig. 56.13. Melanoacanthoma. Note the irregular and stippled brown-black macules on the labial mucosa.

Fig. 56.14. Melanoacanthoma histopathology. A scanning view demonstrating the marked hyperplasia of the mucosal epithelium (H&E).

polyps may pose a management problem. Prophylactic colectomy has been employed in some such cases (Champion et al., 1998; Lenane and Powell, 2000). LEOPARD Syndrome (Multiple Lentigines Syndrome) This is an autosomally dominant inherited cardiocutaneous syndrome. The acronym LEOPARD stands for Lentigines, Electrocardiographic conduction abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retardation of growth, and Deafness. Lentigines are distributed over the lower central face including the lips, neck, trunk, extremities (including palms and soles), and genitalia, but are not present inside the mouth. Cardiac problems may include obstructive cardiomyopathy and mitral regurgitation in addition to electrocardiographic abnormalities. The genetic defect has been identified as a mutation in the gene encoding proteintyrosine phosphatase, nonreceptor type, 11 (PTPN11) on chromosome 12q24.1. Different areas of the same gene are mutated in Noonan syndrome, and, therefore, Noonan syndrome and LEOPARD syndrome are allelic variants of one another (McKusick, 1986c). Histopathology Biopsy of the pigmented lesions shows an increase in single unit melanocytes in the basal epidermis in addition to increased pigmentation of the keratinocytes in the basal layer, i.e., the lesions are true lentigines. In addition, melanocytes contain an abundance of melanosomes which may be substantially enlarged (macromelanosomes) (Nordlund et al., 1973; Weiss and Zelickson, 1977). Melanoacanthoma (Melanoacanthosis) of the Oral Mucosa Clinical Melanoacanthomas are benign pigmented lesions of the oral mucosa which, with rare exception, affect black patients. Lesions more commonly occur in women than in men, and they tend to arise in patients in their teens to late thirties/early forties. The buccal mucosa is most commonly affected in just over half of cases, with the labial mucosa comprising approximately another fifth (Fig. 56.13). Lesions are

brown, black or blue-black in appearance, often with a rough surface, and may simulate nevi or melanoma clinically. Individual lesions may be strikingly large, measuring up to five centimeters in diameter and, rarely, they can diffusely affect the mucosal surfaces. Lesions are often asymptomatic, but they may be painful or pruritic. The etiology of these lesions is not known, but there may be a role for chronic irritation due to physical trauma (e.g., cheek biting, ill-fitting dentures) or cigarette smoking. Lesions usually regress in a matter of weeks after biopsy or after resolution of the trauma or smoking cessation. It has been postulated that black patients with physiologic hyperpigmentation are likely predisposed to reactive changes involving melanocytes inhabiting mucosal epithelium in addition to the more universal reactive changes in keratinocytes as a response to chronic trauma and/or irritation (Maize, 1988; Tomich and Zunt, 1990). Histopathology Lesions demonstrate a hyperplastic epidermis with large, dendritic melanocytes scattered throughout the thickened mucosal epithelium (Figs 56.14–56.17). These melanocytes are fairly evenly distributed within the spinous layer and do not form nests. Melanocytes have abundant melanin pigment granules within their cytoplasm, including within the long, thin dendritic processes extending between keratinocytes of the epithelium. Few pigment granules are transferred to the keratinocytes and it seems as though there is a defect in melanosome transfer from melanocytes to the adjacent keratinocytes. Melanocytes are not increased in number in the basal epidermis and are not atypical in appearance (Tomich and Zunt, 1990). Nevi Melanocytic nevi occur within the mouth as they do elsewhere on the cutaneous surface, though they are uncommon, occurring in about 0.1% of people. All age groups can 1077

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Fig. 56.15. Melanoacanthoma histopathology. At higher power, single unit melanocytes are discernable within the thickened spinous layer of the epithelium (H&E).

Fig. 56.17. Melanoacanthoma histopathology. Note the delicate, markedly elongated dendrites of the melanocytes within this melanoacanthoma (Warthin–Starry).

Fig. 56.18. Oral blue nevus. A blue-black papule on the hard palate (see also Plate 56.5, pp. 494–495). Fig. 56.16. Melanoacanthoma histopathology. In sections stained with a silver stain, many more melanocytes are evident scattered as single cells throughout the epithelium.

be affected, but lesions are most commonly noted in young to middle-aged adults. Palatal, gingival, and labial mucosa can be affected, with the palate being the most common location. Most lesions are only several millimeters in diameter, though nevi one centimeter or more in diameter are encountered on occasion. Lesions may be macular, but up to two thirds of lesions are papular. Up to a fifth of mucosal nevi are nonpigmented (Buchner and Hansen, 1979, 1980b). Approximately a third of nevi from oral mucosa are common blue nevi (Figs 55.18–55.20). Congenital pattern nevi, combined nevi, and Spitz nevi have all been reported to occur in the mouth. Microscopic features of these lesions are identical to those occurring elsewhere. 1078

Fig. 56.19. Oral blue nevus histopathology. At scanning power, there is heavy pigmentation of the submucosa.

PIGMENTARY ABNORMALITIES AND DISCOLORATIONS OF THE MUCOUS MEMBRANES

Fig. 56.20. Oral blue nevus histopathology. Higher power reveals the pigment to be within bipolar melanocytes situated between and parallel to collagen bundles of the submucosa.

Smoker’s Melanosis Individuals who smoke tobacco have been observed to have increased pigmentation of the oral mucosal surfaces in general as compared with nonsmokers. However, discrete focal areas of macular hyperpigmentation usually less than 1 cm in diameter on the oral mucosal surface in persons who smoke are referred to as smoker’s melanosis or smoker’s stains, though “melanosis” seems most appropriate since the cause is increased melanin pigment within the mucosal epithelium. The usual locations are the attached labial gingiva and the mandibular interdental papillae. Incidence increases with increasing tobacco use and is seen more commonly in women over 30 years old (Cicek and Ertas, 2003). Malignant Oral Melanoma The subject of oral melanoma has been extensively discussed in numerous articles and texts and will be only briefly reviewed herein for the sake of completeness. Readers are referred to other sources for more detailed discussion (Bongiorno and Arico, 2002; Eisen and Voorhees, 1991; Hicks and Flaitz, 2000; Rapini et al., 1985; Umeda et al., 2002). Clinical Melanoma of the oral cavity is an uncommon neoplasm, though in reported series, the incidence varies from 0.1% to 8% of all melanomas. In some populations, such as the Japanese, oral melanomas constitute a higher percentage of all melanomas than in whites (Umeda et al., 2002). The most common sites of involvement within the mouth are the palate (40%) and the maxillary gingiva (Fig. 56.21). Presentation is quite variable: lesions may develop slowly or quickly; change subtly or dramatically; be pigmented or amelanotic. Most lesions are asymptomatic, but lesions may ulcerate, bleed, and cause pain. The vast majority of oral melanomas develop in patients over 40 years of age and are extremely rare

Fig. 56.21. Intraoral melanoma. The maxillary gingiva is the most common site of development of intraoral melanoma (see also Plate 56.6, pp. 494–495).

prior to age 20 (Eisen and Voorhees, 1991; Rapini et al., 1985). Biopsy of all oral lesions of unknown origin, especially pigmented lesions which are new or changing, is in the patient’s best interest. The prognosis of melanoma occurring in the oral cavity is much more dismal than melanomas on the cutaneous surface, with a survival rate of only 5–15% after 5 years (Bongiorno and Arico, 2002). Whether the particularly poor prognosis of the lesions is due to inherent genetically programmed behavioral differences in melanomas arising on the mucosa, to a delay in detection due to the location, to inadequate surgical treatment, or to a combination of these factors has not been determined. Histopathology Histopathologic features of melanoma in the mouth are variable as they are in the skin. Melanocyte morphology may be spindled, epithelioid, dendritic, or mixed. Single unit melanocytes may predominate and display confluence and pagetoid upward migration. Nests and/or sheets of melanocytes may also be present. Architectural features such as size, symmetry, and circumscription are important in evaluating melanocytic neoplasms of the mucosa as they are in the skin. It is worth mentioning one specific pattern of oral melanoma that is uncommon in cutaneous melanoma (except acral lentiginous types), namely, the dendritic intraepithelial pattern. Caution must be used when prominent dendritic processes are encountered in lesions with increased melanocytes confined to the basal layer of the epithelium. Variability in the thickness of the dendritic processes and the length of the processes can be a hallmark of in situ melanoma at times. Melanocytic processes which extend substantially above the basal and parabasal layers are abnormal and, when coupled with variability in the thickness and length of the dendrites, can be a clue to the diagnosis of in situ melanoma (Figs 56.22 and 56.23). Prognosis of lesions based on depth of invasion as measured 1079

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Fig. 56.22. Intraoral melanoma histopathology. This melanoma in situ shows a marked increase in single unit melanocytes within the basal layer of the mucosal epithelium. Note the enlarged and hyperchromatic nuclei and the extension to the portion of the epithelium overlying the papillae of the lamina propria (H&E).

Fig. 56.23. Intraoral melanoma histopathology. Melanocytic proliferations on mucosal surfaces which demonstrate marked dendrites as pictured here should raise suspicion for melanoma in situ (Mart-1).

in millimeters has not been studied as rigorously as in the skin, but some small studies corroborate a worse survival for deeper lesions. No large series have compared survival of patients with oral melanoma versus survival of patients with cutaneous melanoma thickness for thickness. Clark level measurements do not apply to oral melanomas due to the absence of the delineation of a papillary and a reticular dermis as exists in the skin (Eisen and Voorhees, 1991).

Exogenous Chromophores Amalgam Tattoo Dental amalgam used as a filler material in restoration proce1080

Fig. 56.24. Amalgam tattoo. There is dark blue-black macular pigmentation of the buccal mucosa adjacent to a decayed tooth (see also Plate 56.7, pp. 494–495).

Fig. 56.25. Amalgam tattoo histopathology. There are variably sized aggregates of amalgam material in the submucosa with a moderately dense mixed inflammatory infiltrate (H&E).

dures may be traumatically introduced into the tissues of the mouth and result in clinically appreciated blue-black or gray macules (Fig. 56.24). Lesions are most commonly located on the gingiva and alveolar mucosa, but the buccal mucosa can also be affected. Lesions vary in size from 1 mm to 2 cm (Eisen and Voorhees, 1991). The amalgam material may be viewed as radio-opaque material in the tissue on X-ray of the affected area. Although the diagnosis is most often easily made based on the location, appearance and dental history, sometimes biopsy is necessary to confirm the clinical impression. Biopsy reveals the presence of dark granular material within histiocytes, fibroblasts, and endothelial cells and associated with collagen bundles, nerves, or around minor salivary glands (Fig. 56.25). In some instances, the amalgam may be situated

PIGMENTARY ABNORMALITIES AND DISCOLORATIONS OF THE MUCOUS MEMBRANES Table 56.1. Drugs causing discoloration of the oral mucosa, skin, and nails. Drug name

Oral dyschromia (Abdollahi and Radfar, 2003)

Amodiaquine Azidothymidine (AZT; zidovudine) Betel Bleomycin Busulfan Chloroquine Clofazimine Cyclophosphamide Doxorubicin Estrogen/Progestins Heroin (inhaled) Hydroxychloroquine Ketoconazole Mepacrine (Quinacrine) Methyldopa Minocycline Nicotine Nitrogen mustard Phenothiazines Quinidine Quinine

+ + + + + + + + + + + + + + + + + + + + +

(Palate) (Soft palate, gingiva, lips, tongue)

(Palate)

(Mucosa, tongue) (Tongue) (Palate) (Palate) (Tongue)

(Palate)

as large clumps within the lamina propria or the submucosa. Foreign body reactions are sometimes observed (Buchner and Hansen, 1980a). Medications There are a number of drugs which are well documented to cause discoloration of the oral mucosa. Such dyschromic lesions often accompany discolorations of the skin and nails. (See Table 56.1 for a listing of drugs which cause oral mucosal dyschromia.) Azidothymidine (AZT), a medication used in the treatment of HIV infection, can cause rapid onset of mucosal pigmentation as soon as a few weeks after the start of therapy. AZT directly stimulates melanin production in mucosal melanocytes and is not due to medication deposition. AZT pigmentation of oral mucosa, skin, and nails predominantly occurs in dark-skinned patients (Greenberg and Berger, 1990). All of the commonly used antimalarials agents (hydroxychloroquine, chloroquine, and quinacrine) can cause a blue-black hyperchromia of the skin which is accentuated in areas of sun exposure, as well as discoloration of the palatal mucosa and nail (Sontheimer and Provost, 1996). Heavy Metals Ingestion of heavy metals may result in deposition of the metals within the tissues of the oral mucosa and cause visible dyschromia. The classic example is the gingival “lead line” of lead toxicity (plumbism), but there are others. Linear metal deposits within the gingiva have also been described with

Skin dyschromia

Nail dyschromia (Scher and Daniel, 1997)

+ (Sontheimer and Provost, 1996) + (Greenberg and Berger, 1990)

+ +

+ (Bork, 1988) + (Bork, 1988) + (Sontheimer and Provost, 1996) + (Bork, 1988) + (Bork, 1988) + (Bork, 1988) + (Bork, 1988)

+ + +

+ (Sontheimer and Provost, 1996) + + (Sontheimer and Provost, 1996)

+

+ (Bork, 1988) +

+ +

+ (Bork, 1988) + (Conroy et al., 1996) + (Bork, 1988)

+

+ +

+

bismuth, copper, mercury, and thallium. Other metals may also cause oral dyschromia in different locations of the oral mucosa, including arsenic, gold, iron, manganese, silver, tin, and vanadium (Abdollahi and Radfar, 2003).

Genital Benign Vulvar Hyperpigmentation Racial variation of pigmentation may be observed on the genital mucosa as it is in the oral mucosa and is regarded as a normal variant. A number of other causes may result in the benign increase in melanin pigment in genital skin. Hormonal influences such as pregnancy, Cushing disease, Addison disease, and exogenous androgens may cause increased mottled or diffuse macular hyperpigmentation of the genital skin without an increase in melanocytes. Common causes of pigmentary changes elsewhere on the skin may also occur on the genital skin, such as post-inflammatory hyper- (or hypo-) pigmentation (Rock, 1992). Vulvar Lentigines In two studies assessing frequency of pigmented lesions of the vulva, simple lentigines were the most common pigmented lesion seen, being present in 3.5–7% of the study population. Clinically, lentigines are usually less than 5 mm in diameter, are well circumscribed, and may occur on the vulvar skin or the mucosa. Typically, there are a small number of lentigines occurring on the vulva of a given patient, but at times there 1081

Fig. 56.26. Vulvar melanosis. Hyperpigmented brown-black irregular patches are situated near the vaginal introitus (see also Plate 56.8, pp. 494–495).

Fig. 56.27. Vulvar melanosis histopathology. Note the bulbous contour of the rete ridges of the hyperplastic epidermis (H&E).

may be numerous lesions. Microscopically, there are elongated and club-shaped rete ridges and there is hyperpigmentation of the basal epidermis. Increased numbers of melanocytes may be appreciated, but melanocyte atypia is not a feature. Lesions indistinguishable from simple lentigines may also occur in the Carney complex and in the LEOPARD syndrome. Patients with neurofibromatosis may have freckling in the genital area which may be clinically similar to lentiginosis (Rock, 1992). Vulvar Melanosis Hyperpigmented macules which are similar in many ways to the oral labial melanotic macule occur on the vulva of women. Identical lesions have also been reported within the vagina and on the uterine cervix. These lesions tend to occur in women who are between menarche and menopause, though there has been no reported association with abnormal hormonal states or exogenous hormone use. Lesions are often large, being between 10 mm and 45 mm in one reported case series (SisonTorre and Ackerman, 1985), and malignant melanoma is often a clinical consideration in the differential diagnosis of these lesions. Unlike with labial melanotic macules, however, lesions may be multiple in a substantial percentage of affected persons. Involvement of the labia minora is most frequently observed, but lesions may also occur on the labia majora as well as the introitus and perineum (Fig. 56.26). Microscopic features are like those of oral melanotic macules, with epidermal hyperplasia and hyperpigmentation of the basal zone (Figs 56.27 and 56.28). Some reports have mentioned a mild increase in melanocytes which may have prominent, thin, arborizing dendrites, but no nests of melanocytes and no cytologic atypia (Rudolph, 1990). Once the diagnosis is correctly established, treatment is not necessary (Sison-Torre and Ackerman, 1985). Penile Lentigo/Atypical Penile Lentigo Men may have lesions involving the glans penis or penile shaft 1082

Fig. 56.28. Vulvar melanosis histopathology. Hyperpigmentation of the basal epidermis is most prominent at the tips of the broadened rete. There is a mild infiltrate which contains melanophages (H&E).

which clinically and histopathologically are similar to vulvar melanosis and oral melanotic macules (Figs 56.29–56.31). These hyperpigmented macular lesions have been reported under the terms penile lentigo and atypical penile lentigo. Several reports have described slightly increased numbers of single unit melanocytes with dendritic morphology in the basal layer of epidermis. Barnhill and coworkers noted that penile and vulvar lesions differed with respect to the presence of melanocytes with a dendritic morphology. Specifically, all penile lesions they reviewed displayed prominent dendritic melanocytes, whereas vulvar lesions showed little in the way of dendritic melanocytes (Barnhill et al., 1990). In the lesion reported as atypical penile lentigo, melanocytic atypia of some melanocytes was observed. The reported incidence of these

PIGMENTARY ABNORMALITIES AND DISCOLORATIONS OF THE MUCOUS MEMBRANES

Fig. 56.29. Penile lentigo. A markedly irregular patch on the glans penis with variegated tan to dark brown pigmentation (see also Plate 56.9, pp. 494–495).

Fig. 56.31. Penile lentigo histopathology. There is no melanocytic proliferation, merely basilar hyperpigmentation.

Fig. 56.30. Penile lentigo histopathology. There is epidermal hyperplasia with broad rete ridges with hyperpigmentation at the tips of the rete (H&E).

lesions is low and the natural history is not known. As with vulvar melanosis and labial melanotic macules, knowledge of these lesions is important to avoid overtreatment (Maize, 1988). Genital Nevi (Including “Milk Line” Nevi) On the vulva, melanocytic nevi are the second most frequent pigmented lesion after lentigines, occurring in 2% of white women in some studies. All types of nevi which occur on the torso may occur on the vulva: junctional, compound, and intradermal nevi, including atypical (Clark, “dysplastic”) nevi (Rock, 1992). The histopathology of most nevi on the vulva is not unique to the site, but there are special features which can occur in nevi in this location which are important to recognize. The concept of milk line nevi or so-called atypical nevi of special anatomic sites has been propagated fairly recently in the medical literature after the initial description of the

“atypical melanocytic nevus of the genital type” by Wallace Clark and others (Clark et al., 1998), though the recognition of distinctive features of melanocytic nevi occurring on the genitalia in particular predates their work by many years (Maize and Ackerman, 1987). Knowledge of these distinctive changes is important because melanocytic nevi on the genitalia and other special sites such as breast, axilla, and umbilicus may simulate melanoma in situ. The melanocytes within the epidermis in nevi of the genitalia tend to be large with large round or oval nuclei with an open chromatin pattern, and nuclei may show some pleomorphism. There is often a population of melanocytes with abundant, pale staining cytoplasm reminiscent of the dusty melanin pigment observed in some lesions of melanoma. Nests within the epidermis vary in size and shape, but they are often relatively large and are closely spaced (Figs 56.32 and 56.33). Pagetoid scatter may be a minor feature and does not extend beyond the last nest defining the periphery of the lesion. Despite some unsettling cytologic and architectural features, these lesions overall are small, well circumscribed, symmetrical, and lack atypia of the dermal component as do nevi from other locations (Fig. 56.34) (Maize and Ackerman, 1987; Zhou and Crowson, 2003). 1083

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Fig. 56.32. Vulvar melanocytic nevus histopathology. There is a compound melanocytic proliferation. Within the epidermis, nests of melanocytes vary in size and shape, and there are zones of confluence (H&E).

Fig. 56.34. Vulvar melanocytic nevus histopathology. Dermal melanocytes are small and uniform in appearance, a reassuring finding (H&E).

elongated and hyperpigmented rete ridges (Lenane and Powell, 2000).

Fig. 56.33. Vulvar melanocytic nevus histopathology. The melanocytes within the epidermis are large with abundant pale cytoplasm (H&E).

Bannayan–Riley–Ruvalcaba Syndrome (BRRS) BRRS is usually an autosomally dominant inherited disease, though sporadic cases do occur. Patients are predominantly male and may have macrocephaly, hypotonia, hyporeflexia, developmental delay, lipomas (cutaneous and/or visceral), hemangiomas, hamartomatous polyps of the intestine, Hashimoto thyroiditis, and pigmented macules of the penis or vulva. Familial cases have been linked to mutations in the PTEN gene on chromosome 10q23.31, the same gene locus as Cowden syndrome. It has been postulated that these two syndromes are merely different phenotypic expressions of a single syndrome, and, therefore, patients with both syndromes should be carefully followed for the development of cancer (McKusick, 1986d). Biopsy of the pigmented genital macules shows a slight increase in melanocytes in the basal layer with 1084

Vulvar Dowling–Degos Disease (Reticulate Pigmented Anomaly of the Flexures) Dowling–Degos disease is usually an autosomally dominant inherited condition which affects the flexural areas including initially the axillae and groin but may later involve the neck, inframammary folds, natal cleft, upper arms, and inner thighs. There have been several cases of Dowling–Degos disease affecting exclusively the vulva. One 55-year-old Japanese woman developed pruritus vulvae which preceded by several years the development of “innumerable pigmented macules on her bilateral vulva.” These were brown to black in color, less than 1 mm in diameter, and symmetrically distributed. Histopathology of lesions of Dowling–Degos disease resembles a reticulated seborrheic keratosis with elongated, hyperpigmented downgrowths from the epidermis and extensions emanating from follicular infundibula. Horn pseudocysts and dilated follicular opening may be seen. There are scattered melanophages in the papillary dermis (Maize and Ackerman, 1987). Laugier–Hunziker Syndrome See discussion of this condition in the section on pigmentary abnormalities of the oral mucosa.

Malignant Vulvar Melanoma Approximately 8–11% of vulvar malignancies are melanomas, and these comprise roughly 2–5% of all melanomas. White people, followed closely by Asians, are most commonly affected, with black people being uncommonly affected. The disease strikes predominantly postmenopausal women aged 50–80 years. Clinical appearance and symptomatology of melanomas of the vulva is not different than melanomas

PIGMENTARY ABNORMALITIES AND DISCOLORATIONS OF THE MUCOUS MEMBRANES

from other body sites, but the depth of invasion of vulvar melanomas is typically greater at discovery. Survival estimates for patients diagnosed with vulvar melanoma range from 36% to 54%. It is not known whether this is attributable entirely to delayed diagnosis by virtue of the hidden and private nature of the location preventing patient knowledge of a new or changing lesion or resulting in a delay in presentation to or examination by a healthcare provider, or if there is also a distinct biologic difference in behavior of genital melanomas (Rock, 1992). Microscopic criteria for a diagnosis of melanoma of the vulva are the same as for elsewhere. Lesions of the genital mucosa may show atypical lentiginous melanocytic proliferation of single unit melanocytes, sometimes with prominent dendrites, as in melanoma of the oral mucosa. Penile Melanoma As in the case of vulvar melanoma, melanoma of the penis is rare: only 1% of all penile malignancies are melanomas. Most patients are between 50 and 70 years of age, with rare cases reported in younger individuals. Two-thirds of cases present as lesions on the glans penis, and ulceration is a frequent finding. Metastasis is present in up to 60% of cases at diagnosis. Lesions may be misdiagnosed as an infection or an inflammatory process initially, delaying appropriate diagnosis and treatment. Clinical and histopathologic criteria for diagnosis of melanoma in general also apply to penile melanoma (English et al., 1997). Pigmented Genital Carcinomas Squamous cell carcinoma in situ (SCCIS), invasive squamous cell carcinoma, and basal cell carcinoma can all occur with pigmented variants. SCCIS as a cause of a pigmented lesion on the vulva occurred with a frequency roughly equal to that of melanocytic nevi in one series (Rock, 1992). Extensive carcinoma in situ of the vulva has been reported which presented as diffuse macular hyperpigmentation with focal ulceration (Rock, 1992). Full-thickness atypia of squamous keratinocytes with enlarged and pleomorphic nuclei, nuclear crowding and loss of polarity, atypical mitotic figures, scattered individually necrotic keratinocytes, and parakeratosis of the stratum corneum are prototypical findings in SCCIS. In pigmented variants, there is abundant melanin pigment in the neoplastic keratinocytes. The microscopic differential diagnosis includes bowenoid papulosis which may be indistinguishable from SCCIS. However, the clinical appearance is distinct, as bowenoid papulosis presents as multiple verrucous tan or brown papules on the genitalia often indistinguishable clinically from condylomata acuminata (Maize and Ackerman, 1987).

Anal Benign Laugier–Hunziker Syndrome See discussion of this condition in the section on pigmentary abnormalities of the oral mucosa.

Malignant Anal Melanoma Melanoma of the anus comprises less than 5% of all anal malignancies and less than 1% of all melanomas. However, the anus is the most frequent site of development of melanoma of the gastrointestinal tract. The most frequently affected group is white women between the ages of 50 and 80 years, although anal melanoma may make up a higher proportion of all melanomas in darker-skinned populations. Melanoma of the anus often causes bleeding or a mass which prompts the patient to seek attention. Importantly, up to 80% of anal melanomas are not appreciably pigmented clinically, and a fifth are amelanotic microscopically as well. Because of this absence of notable pigmentation in many cases, melanomas may be misdiagnosed and treated as hemorrhoids for a while before the correct diagnosis is rendered. Anal melanomas are thicker at diagnosis than melanomas from non-anogenital sites, typically being greater than 1 mm Breslow thickness. The five-year survival rate is under 20% with the median survival between 8 and 23 months. Management of the disease consists of surgical removal of the neoplasm with appropriate margins of normal-appearing tissue. In the past, aggressive surgical management with wide excision and node dissection was advocated, but these procedures did not afford any clear survival benefit over local excision alone. Radiation therapy has been proposed as a reasonable adjunctive measure after surgical removal of the tumor (Kim et al., 2004; Moozar et al., 2003).

Hypopigmentation Genital Vitiligo Vitiligo is an acquired disease due to loss of melanocytes which results in depigmentation of the affected skin. Although the cause is not precisely known, there is evidence supporting an immune-mediated mechanism of melanocyte destruction (Lepe et al., 2003). The disease occurs in all ages and races, and in both sexes and it occurs with greater frequency in individuals with a family history of vitiligo. The disease is especially stigmatizing in darker-skinned individuals and certain cultural groups. Other autoimmune diseases are seen with greater frequency in patients with vitiligo versus patients without vitiligo, including thyroid disease, diabetes mellitus, pernicious anemia, and Addison disease (Alkhateeb et al., 2003; Kenney, 1988). The reasons behind the development of a cytotoxic immune response against melanocytes are not fully understood. Any part of the body may be affected, including the genitalia, and, in some instances there may be exclusive involvement of the genitalia. In such instances, the clinical differential diagnosis may include lichen sclerosus. Chemical leukoderma caused by exposure to agents that are toxic to melanocytes, such as monobenzyl ether of hydroquinone and phenolic compounds, may present with genital depigmentation in some instances and must be considered in the 1085

CHAPTER 56

differential diagnosis as well. The diagnosis is made by careful history. Histopathology The microscopic findings in vitiligo depend upon the stage of disease progression sampled. Early lesions may demonstrate diminished basal layer pigmentation within the epidermis and even some residual melanocytes. Melanophages are present in the papillary dermis and there may be a sparse superficial perivascular inflammatory infiltrate of lymphocytes. Rarely, there may be exocytosis of lymphocytes into the epidermis. In fully developed lesions, there is loss of both melanocytes in the lesional skin and loss of melanin pigment within keratinocytes of the basal layer. Special staining techniques for melanocytes using immunoperoxidase markers such as Melan-A/Mart-1 and the Fontana-Masson stain for melanin pigment may be useful in confirming a loss of melanocytes and a reduction or absence in melanin pigment. Melanophages may or may not be present (Ackerman et al., 1997). Chemical leukoderma is histologically indistinguishable from vitiligo. Treatment Treatment of vitiligo may be most successful if initiated early in the disease process. Topical steroids have been a first-line therapy for many years. Newer topical immune modulators such as topical tacrolimus have shown recent promise in vitiligo therapy (Lepe et al., 2003). Psoralens with ultraviolet A light (PUVA) (both with topically applied psoralens and orally administered psoralens) and narrow-band UVB have been successfully employed for vitiligo therapy, though such treatments may not be practical for genital skin. Treatment employing a 308 nm xenon chloride excimer laser has been useful in some cases of limited vitiligo (Baltas et al., 2002). Melanocyte transfer and skin grafting techniques may restore pigmentation to affected skin (Gupta et al., 2004). Camouflage of affected areas using stains, self-tanning creams, waterproof cosmetics, and tattooing have been utilized to minimize the appearance of lesional skin.

Lichen Sclerosus Clinical White women who are postmenopausal are most commonly affected by lichen sclerosus (LS), but the disease is seen in a wide range of age and in all races. LS often presents as a maddeningly pruritic patch that is erythematous and inflamed during the early stages and eventuates in a depigmented — classically, porcelain white — and atrophic plaque. There are often superimposed secondary changes of lichenification or excoriation. At times, lesions are strikingly purpuric or surmounted by hemorrhagic vesicles and bullae. The vulva surrounding the vaginal introitus is usually the epicenter of the disease, but it may spread centrifugally to surround not only the vaginal opening but also the anus in a pattern likened to a “figure-of-eight.” In advanced disease, there may be extensive scarring and fusion of the vulva with attendant constric1086

Fig. 56.35. Lichen sclerosus of the penis (balanitis xerotica obliterans).

tion of the introitus and concealment of the clitoris. In men, LS (historically termed “balanitis xerotica obliterans” or BXO) is usually confined to the glans penis and foreskin and presents with pruritus, pain, and phimosis. Involvement of the perineum and perianal skin are unusual. LS may occur on nongenital skin also as guttate papules or as large plaques. The clinical appearance of the lesions is similar, with a finely wrinkled, atrophic surface and porcelain-white coloration (Fig. 56.35). Lesions may co-occur with classic lesions of morphea. Rare cases of LS of the oral mucosa have been described. The etiology of the disease is not known. Longstanding genital lesions of LS carry an approximately 5% risk of developing squamous cell carcinoma (Lorenz et al., 1998). Histopathology Lesions of LS share identical histopathologic features irrespective of the site of occurrence. Early lesions demonstrate a bandlike infiltrate of lymphocytes which abuts and focally obscures the dermal–epidermal junction. Injury to the epidermis is seen as vacuolization and squamatization of the basal layer with scattered necrotic keratinocytes along the junction. Exocytosis of lymphocytes into the epidermis during the early phases of disease may simulate mycosis fungoides. Fully developed lesions demonstrate veritable sclerosus of the papillary dermis, often with pronounced edema. The overlying epidermis shows thinning with a flattened rete ridge pattern and

PIGMENTARY ABNORMALITIES AND DISCOLORATIONS OF THE MUCOUS MEMBRANES

there is compact orthohyperkeratosis of the cornified layer. There may be plugging of adnexal openings. A subtle lichenoid infiltrate may be present subjacent to the zone of sclerosus and there is usually a superficial perivascular infiltrate of lymphocytes. Clinically apparent hypopigmentation is due to damage to the basal layer resulting in loss of melanin pigment from basal keratinocytes. Melanophages are often noted in the sclerotic papillary dermis (Carlson et al., 1998). Treatment Ultra-potent topical steroids (class I) such as 0.05% clobetasol propionate are considered first-line therapy for the management of genital LS. Intensive therapy initiated early in the course of disease may prevent untoward sequelae such as scarring and labial fusion. Close monitoring of patients utilizing strong steroids on genital skin is required to prevent unintended side effects. One treatment protocol involves application twice daily for 1 month, then once daily at bedtime for one month, then twice a week at bedtime for three months, then once or twice a week as needed to control symptoms (Lorenz et al., 1998). Less potent steroids may be employed once the disease is under control. Newer therapeutic options such as topical tacrolimus ointment have shown some promise (Ruzicka et al., 2003). Topical testosterone is no longer recommended. For men with phimosis, circumcision is often required.

Melanocytic Nevi in Lichen Sclerosus A rare but potentially vexing problem arises when LS occurs in an area containing a melanocytic neoplasm. When this occurs, melanocytic nevi may clinically, dermoscopically, and microscopically mimic malignant melanoma. Because the disease process in LS affects both the epidermis with damage to the epidermal–dermal interface and the papillary dermis with sclerosis of the collagen bundles, the process affects melanocytic neoplasms in much the same way as partial biopsy or external trauma does: the nevus may take on characteristics of persistent (recurrent) nevi, so-called “pseudomelanoma” change. The criteria which apply to correctly identifying a persistent nevus over a persistent melanoma still apply in this scenario, though the background scarring in a typical persistent nevus is replaced by a background of the classic appearance of LS (pale-pink homogenization of collagen within the papillary dermis, a variable degree of vacuolar alteration of the epidermal–dermal junction with lymphocytes along the junction, and a compact cornified layer with slight to marked hyperplasia). The criteria for identifying nevi within an area of LS are as follows: crisp lateral demarcation of the epidermal component, limited pagetoid scatter, simultaneous presence of zones mimicking melanoma in situ, dermal fibrosis, and areas identifiable as melanocytic nevus with small, uniform and orderly melanocytes within the dermis beneath the area of papillary dermal sclerosus, absence of mitoses in dermal melanocytes, HMB-45 staining limited to epidermal melanocytes and those within the zone of sclerosus (not deeper melanocytes), and a MIB-1(Ki-67) labeling index of less than

10% (Carlson et al., 2002). Care must be taken to avoid diagnosis of these lesions as melanoma with regression. The small size of the process, usually less than 6 mm, is helpful in this regard (Zhou and Crowson, 2003).

Contact Leukoderma Depigmentation of the skin which may be difficult or impossible to distinguish from vitiligo may occur after contact with certain chemical compounds which are cytotoxic to melanocytes. These include monobenzyl ether of hydroquinone and phenolic compounds. History of exposure to these chemical agents is required for diagnosis. Biopsy shows loss of melanocytes and absence of melanin pigmentation within the epidermis as in vitiligo (Reitschel and Pine, 1995).

Leukoderma Syphiliticum Syphilis is a known cause of depigmentation of the skin since ancient times, though leukoderma syphiliticum is not observed frequently today. Leukoderma syphiliticum typically occurs in the skin following the resolution of classic papular lesions of secondary syphilis. The depigmentation classically persists throughout life. Rare cases of leukoderma syphiliticum have been described affecting only the genitalia. On routine microscopy, melanocytes are roughly normal in number and morphologic appearance. On electron microscopy, melanosomes are smaller and less melanized than in normal skin, and they are not distributed throughout the cell in a typical fashion, being more clustered within the cell body and less dispersed within the dendritic processes. Spirochetes are not detectable (Frithz et al., 1982).

Extramammary Paget Disease A rare cause of hypopigmented patches involving the genitalia is extramammary Paget disease. Usually this disease presents as velvety, erythematous or eroded plaques in the groin or anogenital area. Sometimes the plaques are hyperpigmented or, rarely, may present as hypopigmented macules alone or in conjunction with more classic-appearing lesions. Microscopically, there are pale, atypical cells within the epidermis in pagetoid array. These cells label with immunoperoxidase staining methods for carcinoembryonic antigen as well as low molecular-weight keratin (e.g., CAM 5.2). In the hypopigmented areas, melanin pigment is reduced or absent and there are papillary dermal melanophages in these areas. There may or may not be an associated internal malignancy, and appropriate clinical and radiological studies must be performed to exclude the possibility of associated malignancy in all cases (Kakinuma et al., 1994).

References Abdollahi, M., and M. Radfar. A review of drug-induced oral reactions. J. Contemp. Dent. Pract. 4:10–31, 2003. Ackerman, A. B., N. Chongchitnant, J. Sanchez, Y. Guo, B. Bennin, M. Reichel, and M. B. Randall. Histologic Diagnosis of Inflammatory Skin Diseases: An Algorithmic Method Based on Pattern Analysis, Second Edition. Baltimore: Williams & Wilkins, 2003.

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CHAPTER 56 Alkhateeb, A., P. R. Fain, A. Thody, D. C. Bennett, and R. A. Spritz. Epidemiology of vitiligo and associated autoimmune diseases in Caucasian probands and their families. Pigment Cell Res. 16: 208–214, 2003. Almas, K., and W. Sadig. Surgical treatment of melanin-pigmented gingiva: an esthetic approach. Indian J. Dent. Res. 13:70–73, 2002. Atherton, D. J., D. W. Pitcher, R. S. Wells, and D. M. MacDonald. A syndrome of various cutaneous pigmented lesions, myxoid neurofibromata and atrial myxoma: the NAME syndrome. Br. J. Dermatol. 103:421–429, 1980. Baltas, E., Z. Csoma, F. Ignacz, A. Dobozy, and L. Kemeny. Treatment of vitiligo with the 308-nm xenon chloride excimer laser. Arch. Dermatol. 138:1619–1620, 2002. Barnhill, R. L., L. S. Albert, S. K. Shama, M. A. Goldenhersh, A. R. Rhodes, and A. J. Sober. Genital lentiginosis: a clinical and histopathologic study. J. Am. Acad. Dermatol. 22:453–460, 1990. Bongiorno, M. R., and M. Arico. Primary malignant melanoma of the oral cavity: case report. Int. J. Dermatol. 41:178–181, 2002. Bork, K. Cutaneous Side Effects of Drugs. Philadelphia: W. B. Saunders, 1988, p. 422. Buchner, A., and L. S. Hansen. Pigmented nevi of the oral mucosa: a clinicopathologic study of 32 new cases and review of 75 cases from the literature. Part I. A clinicopathologic study of 32 new cases. Oral Surg. Oral Med. Oral Pathol. 48:131–142, 1979. Buchner, A., and L. S. Hansen. Amalgam pigmentation (amalgam tattoo) of the oral mucosa. A clinicopathologic study of 268 cases. Oral Surg. Oral Med. Oral Pathol. 49:139–147, 1980a. Buchner, A., and L. S. Hansen. Pigmented nevi of the oral mucosa: a clinicopathologic study of 32 new cases and review of 75 cases from the literature. Part II. Analysis of 107 cases. Oral Surg. Oral Med. Oral Pathol. 49:55–62, 1980b. Carlson, J. A., P. Lamb, J. Malfetano, R. A. Ambros, and M. C. Mihm., Jr. Clinicopathologic comparison of vulvar and extragenital lichen sclerosus: histologic variants, evolving lesions, and etiology of 141 cases. Mod. Pathol. 11:844–854, 1998. Carlson, J. A., X. C. Mu, A. Slominski, K. Weismann, A. N. Crowson, J. Malfetano, V. G. Prieto, and M. C. Mihm, Jr. Melanocytic proliferations associated with lichen sclerosus. Arch. Dermatol. 138:77–87, 2002. Champion, R. H., J. L. Burton, D. A. Burns, and S. M. Breathnach (eds). Rook/Wilkinson/Ebling Textbook of Dermatology. Malden, Massachusetts: Blackwell Science, 1998. Cicek, Y., and U. Ertas. The normal and pathological pigmentation of oral mucous membranes: a review. J. Contemp. Dent. Pract. 4:76–86, 2003. Clark, W. H., Jr., A. F. Hood, M. A. Tucker, and R. M. Jampel. Atypical melanocytic nevi of the genital type with a discussion of reciprocal parenchymal-stromal interactions in the biology of neoplasia. Hum. Pathol. 29(1 Suppl 1):S1–24, 1998. Clemmensen, O. J., and C. Fenger. Melanocytes in the anal canal epithelium. Histopathology 18:237–241, 1991. Cohen, L. M., and J. P. Callen. Oral and labial melanotic macules in a patient infected with human immunodeficiency virus. J. Am. Acad. Dermatol. 26:653–654, 1992. Conroy, E. A., M. O. Liranzo, J. McMahon, W. D. Steck, and R. J. Tuthill. Quinidine-induced pigmentation. Cutis. 57:425–427, 1996. Del Pozo, J., J. M. Pazos, and E. Fonseca. Lower lip hypertrophy secondary to port-wine stain: combined surgical and carbon dioxide laser treatment. Dermatol Surg. 30(2 Pt 1):211–214, 2004. Drago, F., and A. Rebora. The new herpesviruses: emerging pathogens of dermatological interest. Arch. Dermatol. 135:71–75, 1999. Dummet, C. O., and T. E. Bolden. Melanoblasts in gingival irritation. Mil. Med. 129:1191–1197, 1964. Dupre, A., and R. Viraben. Laugier's disease. Dermatologica. 181:183–186, 1990. Eisen, D., and J. J. Voorhees. Oral melanoma and other pigmented

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lesions of the oral cavity. J. Am. Acad. Dermatol. 24:527–537, 1991. English, J. C., 3rd, R. A. Laws, G. C. Keough, J. L. Wilde, J. P. Foley, and D. M. Elston. Dermatoses of the glans penis and prepuce. J. Am. Acad. Dermatol. 37:1–24, 1997. Erickson, Q. L., E. J. Faleski, M. K. Koops, and D. M. Elston. Addison’s disease: the potentially life-threatening tan. Cutis 66:72–74, 2000. Freedberg, I. M., A. Z. Eisen, K. Wolff, K. F. Austen, L. A. Goldsmith, S. I. Katz, and T. B. Fitzpatrick (eds). Fitzpatrick’s Dermatology in General Medicine. New York: McGraw-Hill, 1999. Frithz, A., Lagerholm, B., and T. Kaaman. Leukoderma syphiliticum: ultrastructural observations on melanocyte function. Acta Derm. Venereol. 62:521–525, 1982. Greenberg, R. G., and T. G. Berger. Nail and mucocutaneous hyperpigmentation with azidothymidine therapy. J. Am. Acad. Dermatol. 22(2 Pt 2):327–330, 1990. Gupta, S., K. Sandhu, A. Kanwar, and B. Kumar. Melanocyte transfer via epidermal grafts for vitiligo of labial mucosa. Dermatol Surg. 30:45–48, 2004. Hicks, M. J., and C. M. Flaitz. Oral mucosal melanoma: epidemiology and pathobiology. Oral Oncol. 36:152–169, 2000. Ho, K. K., Dervan, P., O’Loughlin, S., and F. C. Powell. Labial melanotic macule: a clinical, histopathologic, and ultrastructural study. J. Am. Acad. Dermatol. 28:33–39, 1993. Izquierdo, M. J., M. A. Pastor, L. Carrasco, C. Moreno, H. Kutzner, O. P. Sangueza, and L. Requena. Epithelioid blue naevus of the genital mucosa: report of four cases. Br. J. Dermatol. 145:496–501, 2001. Kakinuma, H., U. Iwasawa, N. Kurakata, and H. Suzuki. A case of extramammary Paget’s disease with depigmented macules as the sole manifestation. Br. J. Dermatol. 130:102–105, 1994. Kato, S., J. Takeyama, Y. Tanita, and K. Ebina. Ruby laser therapy for labial lentigines in Peutz-Jeghers syndrome. Eur. J. Pediatr. 157:622–624, 1998. Kenney, J. A., Jr. Vitiligo. Dermatol. Clin. 6:425–434, 1988. Kim, K. B., A. M. Sanguino, C. Hodges, N. E. Papadopoulos, O. Eton, L. H. Camacho, L. D. Broemeling, M. M. Johnson, M. T. Ballo, M. I. Ross, J. E. Gershenwald, J. E. Lee, P. F. Mansfield, V. G. Prieto, and A. Y. Bedikian. Biochemotherapy in patients with metastatic anorectal mucosal melanoma. Cancer 100:1478–1483, 2004. Lamey, P. J., F. Carmichael, and C. Scully. Oral pigmentation, Addison's disease and the results of screening for adrenocortical insufficiency. Br. Dent. J. 158:297–298, 1985. Lenane, P., and F. C. Powell. Oral pigmentation. J. Eur. Acad. Dermatol. Venereol. 14:448–465, 2000. Lepe, V., B. Moncada, J. P. Castanedo-Cazares, M. B. Torres-Alvarez, C. A. Ortiz, and A. B. Torres-Rubalcava. A double-blind randomized trial of 0.1% tacrolimus vs 0.05% clobetasol for the treatment of childhood vitiligo. Arch. Dermatol. 139:581–585, 2003. Lorenz, B., R. H. Kaufman, and S. K. Kutzner. Lichen sclerosus. Therapy with clobetasol propionate. J. Reprod. Med. 43:790–794, 1998. Maize, J. C. Mucosal melanosis. Dermatol. Clin. 6:283–293, 1988. Maize, J. C., and A. B. Ackerman. Pigmented Lesions of the Skin. Philadelphia: Lea & Febiger, 1987, p. 328. McKusick, V. A. Carney complex, type 1; CNC1. (3/20/2002) http://www. ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=160980, 1986a. McKusick, V. A. Hemochromatosis; HFE. (5/30/04) http://www.ncbi.nlm. nih.gov/entrez/dispomim.cgi?id=235200, 1986b. McKusick, V. A. LEOPARD syndrome. (8/16/2002) http://www.ncbi.nlm. nih.gov/entrez/dispomim.cgi?id=151100, 1986c. McKusick, V. A. Macrocephaly, multiple lipomas, and hemangiomata.

(4/19/2001) http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id= 153480, 1986d. McKusick, V. A. Peutz–Jeghers syndrome. (1/15/2004) http://www. ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=175200, 2004.

PIGMENTARY ABNORMALITIES AND DISCOLORATIONS OF THE MUCOUS MEMBRANES Metry, D. W., and A. A. Hebert. Benign cutaneous vascular tumors of infancy: when to worry, what to do. Arch. Dermatol. 136: 905–914, 2000. Moozar, K. L., C. S. Wong, and J. Couture. Anorectal malignant melanoma: treatment with surgery or radiation therapy, or both. Can. J. Surg. 46:345–349, 2003. Newland, J. R., D. P. Lynch, and N. G. Ordonez. Intraoral Kaposi’s sarcoma: a correlated light microscopic, altrastructural and immunohistochemical study. Oral Surg. Oral Med. Oral Pathol. 66:48–58, 1988. Nordlund, J. J., A. B. Lerner, I. M. Braverman, and J. S. McGuire. The multiple lentigines syndrome. Arch. Dermatol. 107:259–261, 1973. Pandolfino, T. L., S. Cotell, and R. Katta. Pigmented vulvar macules as a presenting feature of the Carney complex. Int. J. Dermatol. 40:728–730, 2001. Papadavid, E., and N. P. Walker. Q-switched Alexandrite laser in the treatment of pigmented macules in Laugier-Hunziker syndrome. J. Eur. Acad. Dermatol. Venereol. 15:468–469, 2001. Patel, R. M., J. R. Goldblum, and E. D. Hsi. Immunohistochemical detection of human herpes virus-8 latent nuclear antigen-1 is useful in the diagnosis of Kaposi sarcoma. Mod. Pathol. 17:456–460, 2004. Rapini, R. P., L. E. Golitz, R. O. Greer, Jr., E. A. Krekorian, and T. Poulson. Primary malignant melanoma of the oral cavity. A review of 177 cases. Cancer 55:1543–1551, 1985. Rees, J. R., F. G. Ross, and G. Keen. Lentiginosis and left atrial myxoma. Br. Heart J. 35:874–876, 1973. Reitschel, R. L., and J. W. Pine (eds). Fisher’s Contact Dermatitis. Baltimore: Williams & Wilkins, 1995. Rhodes, A. R., R. A. Silverman, T. J. Harrist, and A. R Perez-Atayde. Mucocutaneous lentigines, cardiomucocutaneous myxomas, and multiple blue nevi: the “LAMB” syndrome. J. Am. Acad. Dermatol. 10:72–82, 1984. Rock, B. Pigmented lesions of the vulva. Dermatol. Clin. 10:361–370, 1992. Rudolph, R. I. Vulvar melanosis. J. Am. Acad. Dermatol. 23 (5 Pt 2):982–984, 1990. Ruzicka, T., T. Assmann, and M. Lebwohl. Potential future dermatological indications for tacrolimus ointment. Eur. J. Dermatol. 13:331–342, 2003.

Scher, R. K., and C. R. Daniel. Nails: Therapy, Diagnosis, Surgery, 2nd ed. Philadelphia: W. B. Saunders Company, 1997:205–207. Sexton, F. M., and J. C. Maize. Melanotic macules and melanoacanthomas of the lip. A comparative study with census of the basal melanocyte population. Am. J. Dermatopathol. 9:438–444, 1987. Sison-Torre, E. Q., and A. B. Ackerman. Melanosis of the vulva. A clinical simulator of malignant melanoma. Am. J. Dermatopathol. 7 Suppl:51–60, 1985. Sontheimer, R. D., and T. T. Provost (eds). Cutaneous Manifestations of Rheumatic Diseases. Baltimore: William & Wilkins, 1996. Sterling, J. C., and J. B. Kurtz. In: Rook/Wilkinson/Ebling Textbook of Dermatology, vol. 2. R. H. Champion, J. L. Burton, D. A. Burns, and S. M. Breathnach (eds). Malden, Massachusetts: Blackwell Science, 1998, pp. 1063–1064. Tal, H. A novel cryosurgical technique for gingival depigmentation. J. Am. Acad. Dermatol. 24(2 Pt 1):292–293, 1991. Tal, H., Oegiesser, D., and M. Tal. Gingival depigmentation by erbium:YAG laser: clinical observations and patient responses. J. Periodontol. 74:1660–1667, 2003. Tomich, C. E., and S. L. Zunt. Melanoacanthosis (melanoacanthoma) of the oral mucosa. J. Dermatol. Surg. Oncol. 16:231–236, 1990. Umeda, M., H. Komatsubara, Y. Shibuya, S. Yokoo, and T. Komori. Premalignant melanocytic dysplasia and malignant melanoma of the oral mucosa. Oral Oncol. 38:714–722, 2002. Veraldi, S., S. Cavicchini, C. Benelli, and G. Gasparini. LaugierHunziker syndrome: a clinical, histopathologic, and ultrastructural study of four cases and review of the literature. J. Am. Acad. Dermatol. 25:632–636, 1991. Weedon, D. Skin Pathology, 2nd ed. Philadelphia: Churchill Livingstone, 2002a, p. 1009. Weedon, D. Skin Pathology, 2nd ed. Philadelphia: Churchill Livingstone, 2002b, p. 1003. Weedon, D. Skin Pathology, 2nd ed. Philadelphia: Churchill Livingstone, 2002c, p. 1007. Weiss, L. W., and A. S. Zelickson. Giant melanosomes in multiple lentigines syndrome. Arch. Dermatol. 113:491–494, 1977. Zhou, J. H., and A. N. Crowson. Pathologic quiz case: pigmented lesion on the mons pubis of a 17-year-old girl. Compound nevus with features of milk mine nevus. Arch. Pathol. Lab. Med. 127:e391–e392, 2003.

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Benign Neoplasms of Melanocytes

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Common Benign Neoplasms of Melanocytes Sections Pigmented Spindle Cell Nevi Julie V. Schaffer and Jean L. Bolognia Speckled Lentiginous Nevus (Nevus Spilus) Julie V. Schaffer and Jean L. Bolognia Melanocytic (Nevocellular) Nevi and Their Biology Julie V. Schaffer and Jean L. Bolognia

Pigmented Spindle Cell Nevi Julie V. Schaffer and Jean L. Bolognia

Historical Background In 1975, Reed et al. first proposed that the pigmented spindle cell nevus was a benign melanocytic neoplasm distinct from the usual epithelioid and spindle cell (Spitz) nevus. Distinguishing features included its capacity to produce melanin and its growth in an expansive fashion rather than in an infiltrating pattern (Fig. 57.1) (Reed et al., 1975).

Synonyms Additional names for pigmented spindle cell nevus include pigmented spindle cell nevus of Reed (Cochran et al., 1993), pigmented spindle cell tumor (Ainsworth et al., 1979; Kolde and Vakilzadeh, 1987; Schubert, 1986), pigmented spindle cell tumor of Reed (Gartmann, 1981; Smith, 1983, 1987), Reed’s nevus (Abramovits and Gonzalez-Serva, 1993), Reed’s pigmented spindle cell nevus (Wistuba and Gonzalez, 1990), and pigmented spindle cell nevus of non-Spitz type (Christiansen et al., 1985).

1981; Kolde and Vakilzadeh, 1987; Requena and Sanchez Yus, 1990; Sagebiel et al., 1984; Sau et al., 1993; Schubert, 1986; Smith, 1987) [(Barnhill et al., 1991a) plexiform variant excluded] 184 (48%) were found in this location. The upper extremity represents the next most common site with 25% of pigmented spindle cell nevi occurring in this site (95/386). In one institution, the diagnosis of Spitz nevus was ten times more common than that of pigmented spindle cell nevus (Gartmann, 1981). However, in a retrospective analysis of 247 epithelioid and/or spindle cell nevi excised between 1974 and 1993 at the University of Milan (Dal Pozzo et al., 1997), 21% were pigmented spindle cell nevi, 50% were pigmented Spitz (epithelioid and spindle cell) nevi, and the remaining 29% were “nonpigmented” Spitz nevi. Approximately onehalf of the lesions in the first two groups were located on the lower extremities, whereas those in the third group favored the head and neck; the mean age of patients with pigmented lesions (n = 177, including the 52 pigmented spindle cell nevi) was 14 years, compared with 9 years for those with domeshaped, nonpigmented lesions (n = 70) (Dal Pozzo et al., 1997).

Clinical Presentation Epidemiology/Clinical Findings The vast majority of pigmented spindle cell nevi are acquired and in one study of 40 patients with such lesions, they were reportedly present for 1 month to 3 years with a mean of 9.7 months (Smith, 1987). A mean duration of 6 years was reported in a second series of 22 patients (Requena and Sanchez Yus, 1990). In the six largest series of patients to date (Barnhill et al., 1991a; Gartmann, 1981; Requena and Sanchez Yus, 1990; Sagebiel et al., 1984; Sau et al., 1993; Smith, 1987), these nevi were observed more commonly in women than in men (243 women/395 total) with a combined female : male ratio of 1.6:1. The age at diagnosis ranged from 2 to 60 years (Sagebiel et al., 1984; Sau et al., 1993), but the average age ranged from 16 to 25 years (Barnhill et al., 1991a; Requena and Sanchez Yus, 1990; Sagebiel et al., 1984; Sau et al., 1993; Smith, 1987). The lower extremity is the most common site for pigmented spindle cell nevi and in a total of 386 patients (Barnhill and Mihm, 1989; Gartmann,

The classic presentation for a pigmented clinical cell nevus is a single, darkly pigmented macule or papule on the lower extremity of a young female patient (Cochran et al., 1993; Requena and Sanchez Yus, 1990; Smith, 1987). These nevi can vary in size from 1 mm to 11 mm (Gartmann, 1981; Kolde and Vakilzadeh, 1987; Requena and Sanchez Yus, 1990), but are usually 3 mm to 6 mm in diameter (Requena and Sanchez Yus, 1990; Sau et al., 1984; Smith, 1987). There may be a history of fairly rapid growth (Ainsworth et al., 1979). Pigmented spindle cell nevi tend to be sharply demarcated from the normal surrounding skin (Requena and Sanchez Yus, 1990; Sagebiel et al., 1984; Smith, 1987), although occasionally a pigmented macular halo may be seen (Ainsworth et al., 1979). The well-defined border is a clinical reflection of circumscribed nests of melanocytes and a lack of “trailing off” of single melanocytes as is seen in atypical nevi. The color of pigmented spindle cell nevi can vary from dark brown to black to intense gray or blue (Reed et al., 1093

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1975; Sau et al., 1984) and their topography has been described as smooth and slightly dome-shaped (Requena and Sanchez Yus, 1990; Smith, 1987). A clinical diagnosis of cutaneous melanoma is fairly common given the dark brown to black color (Barnhill and Mihm, 1989; Requena and Sanchez Yus, 1990), and in two series, the clinical diagnosis of pigmented cell nevus was suspected in up to 10% of cases (Requena and Sanchez Yus, 1990; Smith, 1987). In addition to cutaneous lesions, a pigmented spindle cell nevus of the bulbar conjunctiva has been described, with a clinical differential diagnosis that included ocular melanoma (Seregard, 2000). Lastly, there is one report of agminated lesions (both Spitz and pigmented spindle cell nevi) on the plantar surface of the foot in a 12-year-old African-American girl (Abramovits and Gonzalez-Serva, 1993).

Associated Disorders None.

Histology

Spitz nevus

Melanoma Fig. 57.1. Schematic representation of the growth pattern and histologic features of pigmented spindle cell nevus, Spitz nevus, and melanoma. (Adapted from N. P. Smith. The pigmented spindle cell tumor of Reed: an underdiagnosed lesion. Sem. Diagn. Pathol. 4:75–87, 1987, with permission of the publisher.)

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Rather large and elongated compact nests of melanocytes are seen at the dermoepidermal junction and less often in the papillary dermis, overall demonstrating a plate-like architectural pattern (Fig. 57.2A; see also Fig. 57.1A) (Reed et al., 1975; Requena and Sanchez Yus, 1990). In one series of 40 pigmented spindle cell nevi, 18 (45%) were junctional nevi and 22 (55%) were compound nevi (Smith, 1987). It is unusual for the nevus cells to involve the reticular dermis. In one review of 91 cases, only seven had a depth of invasion greater than 0.6 mm (Sagebiel et al., 1984). The individual cells are spindle shaped and highly melanized (Fig. 57.2B and C) (Cochran et al., 1993). In typical pigmented spindle cell nevi, a symmetrical pattern of proliferation is observed (Smith, 1987) and the lateral demarcation is rather sharp (Requena and Sanchez Yus, 1990; Sau et al., 1993; Smith, 1987). Occasionally, a portion of the nevus will be composed of isolated single spindle cells at the dermoepidermal junction (Requena and Sanchez Yus, 1990). Junctional nests have been observed in the dermal portion of eccrine ducts as well as in the infundibulum of hair follicles (Requena and Sanchez Yus, 1990; Sagebiel et al., 1984; Sau et al., 1993; Smith, 1987). Of note, sparse upward growth of individual cells into the overlying epidermis was observed by Sagebiel et al. (1984) in 25 of their 91 cases (27%). Requena and Sanchez Yus (1990) described single cells or small nests in the overlying epidermis in 19 of 22 cases. However, they saw no extension of solitary melanocytes laterally beyond the body of the nevus. Pagetoid melanocytosis, defined as the upward discontinuous extension of melanocytes into the superficial epidermis, was observed by Haupt et al. (1995) in 20% (2/10) of the pigmented spindle cell nevi in their series. In contrast, several authors have stated that there is no pagetoid spread of single melanocytes in these lesions (Cochran et al., 1993; Reed et al., 1975). Cochran et al. (1993) believe that keratinocytes in overlying epidermis that contain abundant melanin may have an appearance similar to pagetoid cells. Multinucleated giant cells with small

COMMON BENIGN NEOPLASMS OF MELANOCYTES

Fig. 57.2. (A) Photomicrograph of pigmented spindle cell nevus demonstrating symmetry, expansile growth pattern, and superficial nature of the nevus (H&E, ¥40). (B, C) Proliferation of uniformappearing, spindle-shaped cells, some of which contain obvious melanin; melanophages are seen at the base of the lesion as well as admixed with the nevus cells (H&E, B, ¥100; C, ¥400).

uniform nuclei have been observed in up to half of the lesions (Sau et al., 1993). In typical pigmented cell nevi, pure collections of large epithelioid cells are not seen nor is pleomorphism or cytologic atypia (Cochran et al., 1993; Requena and Sanchez Yus, 1990; Sagebiel et al., 1984). For example, Sagebiel et al. (1984)

reported uniform nuclei in 97% of their cases with prominent nucleoli in 23%. Although atypical variants of pigmented spindle cell nevi with cytologic atypia and prominent lentiginous melanocytic hyperplasia have been described (Barnhill and Mihm, 1989; Barnhill et al., 1991a), some authors believe that such lesions are better classified as pigmented Spitz nevi (Requena and Sanchez Yus, 1990). In the compound pigmented spindle cell nevi, a decrease in cellular size and cytoplasm content, i.e., maturation, can be seen in the lower portions of the lesions (Barnhill and Mihm, 1989; Requena and Sanchez Yus, 1990; Sagebiel et al., 1984). The presence of typical mitotic figures has been noted by several groups of investigators, but their frequency has varied. In a series of 40 pigmented spindle cell nevi (Smith, 1987) more than one mitosis per 5 high power fields was seen in only four lesions (10%) whereas Sau (1993) reported an average of three mitoses per 10 high-power fields (n = 95). To date, no atypical mitotic figures have been observed (Guillen and Murphy, 1985; Sagebiel et al., 1984; Sau et al., 1993). Some degree of hyperplasia of the epidermis is observed frequently (Cochran et al., 1993; Requena and Sanchez Yus, 1990). Epidermal hyperplasia was observed in 92.5% (37 of 40) of the lesions in one series (Smith, 1987). Pigment is readily apparent in the nests of melanocytes and the keratinocytes of the surrounding epidermis (Wistuba and Gonzalez, 1990). In the darkly pigmented lesions, melanin is seen within macrophages in the papillary dermis as well as in the stratum corneum (Imber, 1990; Requena and Sanchez Yus, 1990). Celleno and Massi (2002) described a variant of junctional pigmented spindle cell nevus with particularly abundant melanophages resembling the “tumoral melanosis” that can be seen upon regression of a cutaneous melanoma. Eosinophilic globules (Kamino bodies) have been identified in pigmented spindle cell nevi and their frequency varies from 10% [4 of 40 (Smith, 1987)] to 32% [7 of 22 (Requena and Sanchez Yus, 1990)] to 80% [8/10 (Wistuba and Gonzalez, 1990)]. Occasionally, the Kamino bodies may be darkly pigmented (Requena and Sanchez Yus, 1990). Some authors state that a lymphoid infiltrate is unusual (Cochran et al., 1993) whereas others state that the majority of pigmented spindle cell nevus have an infiltrate of mononuclear cells at the base of the lesion (Requena and Sanchez Yus, 1990; Sagebiel et al., 1984; Sau et al., 1993; Smith, 1987).

Laboratory Findings and Investigations The measurement of 5-S-cysteinyl dopa levels in a variety of pigmented lesions has revealed a significant difference between pigmented spindle cell nevi and melanomas with levels of £50 ng/mg in pigmented spindle cell nevi and ≥100 ng/mg in melanomas (Takeuchi and Morishima, 1990). Additional information can be gained by staining histologic sections with silver to visualize nucleolar organizer regions. Pigmented spindle cell nevus have fewer nucleolar organizer regions and the majority are organized within well-defined nucleolar clusters (Evans et al., 1991). In contrast, the nucleolar organizer regions in melanomas are widely 1095

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dispersed within the nucleus and are often in an extranucleolar location. Several clinicians have advocated the use of epiluminescence microscopy to distinguish pigmented Spitz nevi from cutaneous melanoma. Clues to the diagnosis of pigmented Spitz nevus as opposed to melanoma include a monomorphous appearance, a regular pigment network, bizarre retiform depigmentation in the center of the lesion (i.e., a negative pigment network), a regular distribution of brown globules and black dots, and the lack of pseudopods or radial streaming (Pehamberger et al., 1987; Steiner et al., 1987, 1992). A superficial black network (corresponding to areas of pigmented parakeratosis) represents an additional dermoscopic feature that is seen in approximately 10% of pigmented Spitz nevi (Argenziano et al., 2001). Rubegni et al. (2002) used digital dermoscopic analysis to determine the most useful variables in differentiating pigmented Spitz nevi from cutaneous melanoma, and found that measurements of entropy, red color, and peripheral black areas as well as diameter tended to be higher in the latter lesions. Argenziano et al. (1999) further classified the dermoscopic patterns of pigmented Spitz nevi into three main groups: (1) a starburst pattern characterized by radial streaks on a background of diffuse gray-blue to black pigmentation (50%; particularly common in lesions with classic histologic features of pigmented spindle cell nevi); (2) a globular pattern demonstrating central brown to gray-blue pigmentation and a peripheral rim of large brown globules (25%); and (3) an atypical pattern with irregular pigmentation and structures indistinguishable from those seen in melanoma (25%). Evolution from a globular pattern to a starburst pattern over a period of months has been described in several pigmented Spitz nevi (Piccolo et al., 2003; Pizzichetta et al., 2002). One important pitfall in distinguishing pigmented Spitz nevi from melanomas via dermoscopy is the observation that melanomas occasionally exhibit the globular and starburst patterns (Argenziano et al., 1999). Nevertheless, in a study of 54 pigmented Spitz nevi, the use of dermoscopy improved the accuracy of clinical diagnosis from 56% to 93% (Steiner et al., 1992), and in another study of 26 Spitz nevi, observation via digital videomicroscopy increased clinical diagnostic accuracy from 15% to 58% (Pellacani et al., 2000). Dermoscopy thus represents a useful tool, helping to further characterize individual melanocytic lesions in the context of a thorough history and total cutaneous examination. A definitive diagnosis, however, requires histologic examination (Peris et al., 2002).

Criteria for Diagnosis Elongated compact nests of spindle shaped and heavily melanized melanocytes are seen at the dermoepidermal junction and less often in the papillary dermis with a tendency for neighboring nests to fuse (Reed et al., 1975; Requena and Sanchez Yus, 1990). The growth pattern is expansive as opposed to infiltrating (Reed et al., 1975). In typical pigmented spindle cell nevus, there are uniform nuclei, a uniform cytologic pattern, few if any large epithelioid cells, and no 1096

lateral pagetoid spread of single melanocytes (Reed et al., 1975). Atypical variants can have cellular atypia and prominent lentiginous melanocytic hyperplasia, but retain a symmetric and orderly growth pattern (Barnhill and Mihm, 1989).

Differential Diagnosis Clinically, the differential diagnosis includes atypical nevus and cutaneous melanoma while the major entities in the histologic differential diagnosis are spindle and epithelioid cell (Spitz) nevus and cutaneous melanoma. Although hyperplasia of the epidermis is seen in pigmented spindle cell nevus, it is not to the extent as in a Spitz nevus (Ainsworth et al., 1979; Reed et al., 1975). In addition, infiltration of the reticular dermis in an interstitial pattern is seen in Spitz nevi but not pigmented spindle cell nevi (Imber, 1990). Pigment production is usually more striking in a pigmented spindle cell nevus and substantial numbers of plump epithelioid cells are not seen except in atypical variants (Barnhill et al., 1991a). Pigmented spindle cell nevus can have eosinophilic globules (Kamino bodies), but there is rarely edema in the papillary dermis, a marked vascular proliferation, or a desmoplastic stroma (Gartmann, 1981; Requena and Sanchez Yus, 1990; Sau et al., 1993; Smith, 1987). Also, the cells within a pigmented spindle cell nevus are thought to be more uniform throughout the lesion than the cells of a Spitz nevus (Smith, 1987). However, there are pathologists who believe that the pigmented spindle cell nevus is simply a variant of the Spitz nevus (Echevarria and Ackerman, 1967; Sagebiel et al., 1984; Wistuba and Gonzalez, 1990). In comparison to atypical nevi, the epidermal and dermal components of typical pigmented spindle cell nevus are coterminous (Cochran et al., 1993) and these nevi lack a junctional “shoulder.” There are also more well-defined lateral margins and less stromal changes such as lamellar fibroplasia in pigmented spindle cell nevi (Guillen and Murphy, 1985; Sagebiel et al., 1984). The deep penetrating or plexiform spindle cell nevus is another benign pigmented lesion that is composed of pigmented spindle cells (Barnhill et al., 1991b; Cooper, 1992; Seab et al., 1989). However, as its names imply, the nevus cells extend into the deep reticular dermis and even the subcutis and, because the fascicles involve neurovascular structures and adnexae, a plexiform appearance is observed. In contrast to pigmented spindle cell nevus, cutaneous melanomas are usually asymmetric histologically and not sharply demarcated (Guillen and Murphy, 1985). Typical pigmented spindle cell nevus have no pleomorphism or cellular atypia whereas the atypical variants can have “uniform but worrisome hyperchromatic nuclei” (Reed et al., 1975). However, the cellular atypia is never as marked as in melanoma (Barnhill and Mihm, 1989), i.e., a wide range of nuclear changes is not observed (Reed et al., 1975). If a dermal component is present, maturation of the cells is observed in a pigmented spindle cell nevus as opposed to the complete absence of cytologic maturation at the base of a melanoma (Guillen and Murphy, 1985). Lateral extension of single melanocytes beyond the body of the nevus is characteristic of melanoma,

COMMON BENIGN NEOPLASMS OF MELANOCYTES

but not typical pigmented spindle cell nevus (Requena and Sanchez Yus, 1990). Regression and surface ulceration are also not characteristic features of pigmented spindle cell nevus (Sagebiel et al., 1984; Sau et al., 1993; Smith, 1987). One area of concern is the number of pigmented spindle cell nevi that have been diagnosed as melanoma by pathologists, e.g., greater than 43% (10/23) in one series (Smith, 1987), 50% (5/10) in a second (Wistuba and Gonzalez, 1990), and 27% (23/85) in a third (Sau et al., 1993). The controversial form of cutaneous melanoma known as minimal deviation melanoma has a pigmented spindle cell variant that shares some features with pigmented spindle cell nevus including a monotonous and uniform proliferation of melanocytes, but in this type of minimal deviation melanoma, the tumors are deeper, there is an absence of cellular maturation, and numerous mitoses may be seen (Mérot and Mihm, 1985; Reed et al., 1975). In the spindle cell variants of melanoma, e.g., desmoplastic melanoma, a proliferation of atypical spindle cells is seen in the dermis, but the pattern is infiltrative and is often composed of single cells (Smith, 1987). Lastly, Reed (1985) believes that there is a malignant counterpart to the pigmented spindle cell tumor.

Pathogenesis Unknown.

Animal Models None.

Treatment Given their dark color and frequent location on the lower extremities of women, biopsies of pigmented spindle cell nevus are obtained routinely. However, the clinician must decide whether the entire lesion should be removed surgically. One argument for complete local excision is the need for a thorough histologic examination of the lesion given the number of malignant tumors with spindle cell differentiation (Guillen and Murphy, 1985). Dellon and Farmer (1988) recommended margins of 3 mm to 5 mm of adjacent skin to allow histologic evaluation of the transition zone of normal skin to nevus and to include the few lesions with indistinct borders. Currently, the general consensus is to recommend complete excision (Sau et al., 1993).

Prognosis The pigmented spindle cell nevus is considered a benign neoplasm of melanocytes (Requena and Sanchez Yus, 1990) and, to date, there have been no reports of the development of metastases or a significant local recurrence rate (Guillen and Murphy, 1985). Sau et al. (1993) reported no local recurrences or distant spread in 57 patients followed for an average of 6 years whereas Smith (1987) and Sagebiel et al. (1984) had the same experience with 30 patients followed for a mean of 3 years and 38 patients followed an average of 14 months, respectively. Even in 15 patients (9 of whom had atypical pigmented spindle cell nevus) followed for a mean of 8.8 years,

there were no recurrences (Barnhill et al., 1991a). However, the fact that the majority of lesions were completely excised does influence these results. To date, pigmented spindle cell nevus have not been associated with an increased incidence of a personal or family history of melanoma or the presence of atypical nevi (Barnhill et al., 1991a; Smith, 1987).

References Abramovits, W., and A. Gonzalez-Serva. Multiple agminated pigmented Spitz nevi (mimicking acral lentiginous malignant melanoma and dysplastic nevus) in an African-American girl. Int. J. Dermatol. 32:280–285, 1993. Ainsworth, A. M., R. Folberg, R. J. Reed, and W. H. Clark Jr. Melanocytic nevi, melanocytomas, melanocytic dysplasias and uncommon forms of melanoma. In: Human Malignant Melanoma, W. H. Clark Jr., L. I. Goldman, and M. J. Mastrangelo (eds). New York: Grune and Stratton, 1979, pp. 179–182. Argenzaino, G., M. Scalvenzi, S. Staibano, B. Brunetti, D. Piccolo, M. Delfino, G. de Rosa, and H. P. Soyer. Dermatoscopic pitfalls in differentiating pigmented Spitz naevi from cutaneous melanoma. Br. J. Dermatol. 141:788–793, 1999. Argenziano, G., H. P. Soyer, G. Ferrara, D. Piccolo, R. HofmannWellenhof, K. Peris, S. Staibano, and S. Chimenti. Superficial black network: an additional dermoscopic clue for the diagnosis of pigmented spindle and/or epithelioid cell nevus. Dermatology 203:333–335, 2001. Barnhill, R. L., and M. C. Mihm Jr. Pigmented spindle cell naevus and its variants: distinction from melanoma. Br. J. Dermatol. 121:717– 726, 1989. Barnhill, R. L., M. A. Barnhill, M. Berwick, and M. C. Mihm Jr. The histologic spectrum of pigmented spindle cell nevus: a review of 120 cases with emphasis on atypical variants. Hum. Pathol. 22:52–58, 1991a. Barnhill, R. L., M. C. Mihm Jr., and C. M. Magro. Plexiform spindle cell naevus: a distinctive variant of plexiform melanocytic naevus. Histopathology 18:243–247, 1991b. Celleno, L., and G. Massi. A variant of junctional naevus of epithelioid and spindle cell type rich in melanophages. Acta Dermatol. Venereol. 82:456–459, 2002. Christiansen, P., S. Oster, and H. Sogaard. Pigmented spindle cell nevus of non-Spitz type. Differential diagnosis from malignant melanoma. Ugeskr. Laeger 147:3910–3911, 1985. Cochran, A. J., C. Bailly, P. Eberhard, and D. Dolbeau. Nevi, other than dysplastic and Spitz nevi. Semin. Pathol. 10:3–17, 1993. Cooper, P. H. Deep penetrating (plexiform spindle cell) nevus. J. Cutan. Pathol. 19:172–180, 1992. Dal Pozzo, V., C. Benelli, L. Restano, R. Gianotti, and B.M. Cesana. Clinical review of 247 case records of Spitz nevus (epithelioid cell and/or spindle cell nevus). Dermatology 194:20–25, 1997. Dellon, A. L., and E. R. Farmer. Pigmented Spitz nevus in an adult. Ann. Plast. Surg. 20:128–132, 1988. Echevarria, R., and L. V. Ackerman. Spindle and epithelioid cell nevi in the adult: clinicopathologic report of 26 cases. Cancer 20:175– 189, 1967. Evans, A. T., J. M. Orrell, and A. Grant. Re-evaluating silver-stained nucleolar organizer regions (AgNORs) in problematic cutaneous melanocytic lesions: A study with quantitation and pattern analysis. J. Pathol. 165:61–67, 1991. Gartmann, H. Der pigmentierte Spindelzellentumor (PSCT). Z. Hautkrankh. 56:862–876, 1981. Guillen, F. J., and G. F. Murphy. Capsule dermatopathology: Nevomelanocytic lesions with spindle cell differentiation. J. Dermatol. Surg. Oncol. 11:225–230, 1985. Haupt, H. M., and J. B. Stem. Pagetoid melanocytosis; Histologic features in benign and malignant lesions. Am. J. Surg. Pathol. 19:792–797, 1995.

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CHAPTER 57 Imber, M. J. Benign cutaneous lesions potentially misdiagnosed as malignant neoplasms. Semin. Diagn. Pathol. 7:139–145, 1990. Kolde, G., and F. Vakilzadeh. Der pigmentierte Spindelzellentumor. Hautarzt 38:743–745, 1987. Mérot, Y., and M. C. Mihm Jr. Aspect inhabituel et méconnu du mélanome malin cutané: Le mélanome malin a déviation minime. Etude rétrospective de 45 cas. Ann. Dermatol. Venereol. 112:325–336, 1985. Pehamberger, H., A. Steiner, and K. Wolff. In vivo epiluminescence microscopy of pigmented skin lesions. I. Pattern analysis of pigmented skin lesions. J. Am. Acad. Dermatol. 17:571–583, 1987. Pellacani, G., A. M. Cesinaro, and S. Seidenari. Morphological features of Spitz naevus as observed by digital videomicroscopy. Acta Dermatol. Venereol. 80:117–121, 2000. Peris, K., A. Ferrari, G. Argenziano, H. P. Soyer, and S. Chimenti. Dermoscopic classification of Spitz/Reed nevi. Clin. Dermatol. 20:259–262, 2002. Piccolo, D., A. Ferrari, and K. Peris. Sequential dermoscopic evolution of pigmented Spitz nevus in childhood. J. Am. Acad. Dermatol. 49:556–558, 2003. Pizzichetta, M. A., G. Argenziano, G. Grandi, C. de Giacomi, G. Trevisan, and H. P. Soyer. Morphologic changes of a pigmented Spitz nevus assessed by dermoscopy. J. Am. Acad. Dermatol. 47:137–139, 2002. Reed, R. J. The histological variance of malignant melanoma: The interrelationship of histological subtype, neoplastic progression, and biological behaviour. Pathology 17:301–319, 1985. Reed, R. J., H. Ichinose, W. H. Clark, and M. C. Mihm Jr. Common and uncommon melanocytic nevi and borderline melanomas. Semin. Oncol. 2:119–147, 1975. Requena, L., and E. Sanchez Yus. Pigmented spindle cell naevus. Br. J. Dermatol. 123:757–763, 1990. Rubegni, P., A. Ferrari, G. Cevenini, D. Piccolo, M. Burroni, R. Perotti, K. Peris, P. Taddeucci, M. Biagioli, G. Dell’Eva, S. Chimenti, and L. Andreassi. Differentiation between pigmented Spitz naevus and melanoma by digital dermoscopy and stepwise logistic discriminant analysis. Melanoma Res. 11:37–44, 2001. Sagebiel, R. W., E. K. Chinn, and B. M. Egbert. Pigmented spindle cell nevus: Clinical and histologic review of 90 cases. Am. J. Surg. Pathol. 8:645–653, 1984. Sau, P., J. H. Graham, and E. B. Helwig. Pigmented spindle cell nevus. Arch. Dermatol. 120:1615, 1984. Sau, P., J. H. Graham, and E. B. Helwig. Pigmented spindle cell nevus: a clinicopathologic analysis of ninety-five cases. J. Am. Acad. Dermatol. 28:565–571, 1993. Schubert, E. Der pigmentierte Spindelzelltumor (PSCT). Z. Hautkrankh. 61:827–829, 1986. Seab, J. A., Jr., J. H. Graham, and E. B. Helwig. Deep penetrating nevus. Am. J. Surg. Pathol. 13:39–44, 1989. Seregard, S. Pigmented spindle cell naevus of Reed presenting in the conjunctiva. Acta Ophthalmol. Scand. 78:104–106, 2000. Smith, N. P. The pigmented spindle cell tumour of Reed — an underrecognized lesion. Br. J. Dermatol. 109:39, 1983. Smith, N. P. The pigmented spindle cell tumor of Reed: an under diagnosed lesion. Semin. Diagn. Pathol. 4:75–87, 1987. Steiner, A., H. Pehamberger, and K. Wolff. In vivo epiluminescence microscopy of pigmented skin lesions. II. Diagnosis of small pigmented skin lesions and early detection of malignant melanoma. J. Am. Acad. Dermatol. 17:584–591, 1987. Steiner, A., H. Pehamberger, M. Binder, and K. Wolff. Pigmented Spitz nevi: improvement of the diagnostic accuracy by epiluminescence microscopy. J. Am. Acad. Dermatol. 27:697–701, 1992. Takeuchi, M., and T. Morishima. Pigmented spindle cell nevus and pigmented Spitz nevus — clinical and histopathological study on pigmented Spitz nevus, and its differentiation from early melanoma by fluorescence method and measurement of 5-S-CD level in the

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lesion [ Japanese]. Nippon Hifuka Gakkai Zasshi 100:1153–1165, 1990. Wistuba, I., and S. Gonzalez. Eosinophilic globules in pigmented spindle cell nevus. Am. J. Dermatopathol. 12:268–271, 1990.

Speckled Lentiginous Nevus (Nevus Spilus) Julie V. Schaffer and Jean L. Bolognia

Historical Background For this particular pigmented lesion, there is a long history of disagreement regarding nomenclature, i.e., “speckled lentiginous nevus” versus nevus spilus. This controversy over terminology dates back to the use of the term “nevus spilus” in the late nineteenth and early twentieth centuries to describe a uniformly pigmented brown patch. Several writings from this period (Besnier et al., 1902; Bulkley, 1842; Duhring, 1883; Hyde and Montgomery, 1901; Kaposi, 1887) are frequently cited to support the argument that use of the term nevus spilus creates confusion (Altman and Banse, 1992; Misago et al., 1993; Pritchett and Pritchett, 1982; Stewart et al., 1978; Vion et al., 1985). These early references contain relatively nonspecific descriptions of the clinical characteristics of a nevus spilus, with no mention of superimposed, more darkly pigmented macules or papules. For example, Bulkley (1842) classified ephelis and nevus spilus as macules, his definition of a macule being a “permanent alteration of the color of the skin, either of its whole surface or some of it only, without elevation or desquamation.” Kaposi (1887) referred to a nevus spilus as a birthmark with a smooth, soft surface without any other skin changes, and Besnier et al. (1902) stated that the description of this lesion was completely confused with that of a lentigo. In 1952, Ito and Hamada reintroduced the term nevus spilus to refer to a hyperpigmented patch with superimposed darker macules. Biopsies were performed in 4 of 21 cases, and no nevus cells were seen (Ito and Hamada, 1952). However, the exact sites within the nevus spilus that were biopsied were not mentioned, and two of the cases may have represented Becker melanosis. Benedict et al. (1968) subsequently defined a nevus spilus as a single light brown patch with a diameter of 1 cm to 20 cm and a clinical appearance similar to that of a caféau-lait macule. Two years later, Cohen et al. (1970) reported 17 patients with the clinical diagnosis of nevus spilus whose lesions were similar in appearance to those described by Ito and Hamada (1952); however, biopsy specimens from both the background hyperpigmentation and the more darkly pigmented speckles contained nevus cells. Then, in the mid1970s, the term “speckled lentiginous nevus” first appeared as a name for pigmented lesions with a background of macular hyperpigmentation containing smaller, darker macules and/or papules (Stewart et al., 1978). It is easy to understand from this historical account why there has been so much confusion over terminology. In a review of MEDLINE citations over the past decade

COMMON BENIGN NEOPLASMS OF MELANOCYTES

(1994–2004), the two terms have been used in a ratio of approximately 1.5 nevus spilus to 1 speckled lentiginous nevus. Proponents of the term nevus spilus point to the Greek root spilos, the translation of which is “spot” (Dorland, 1994; Leider and Rosenblum, 1976), and interpret this word to mean or imply “spotty” (Cohen et al., 1970; Rhodes and Mihm, 1990). The authors and others, however, favor the term speckled lentiginous nevus not only from a historical perspective or because of the correct translation of spilos, but as a means of emphasizing the biologic behavior of this pigmented lesion. A speckled lentiginous nevus can be likened to a melanocytic garden within which a variety of lesions can grow, from junctional nevi to blue nevi to melanoma (Cramer, 2001; Schaffer et al., 2001a). Unfortunately, as recently as 15–20 years ago, a nevus spilus was incorrectly viewed as a lesion with no malignant potential (Rhodes, 1983), and the histologic features of the spots of a nevus spilus are still often described simply as those of a junctional nevus (Elder and Elenitsas, 1997; Kurban et al., 1992). Several authors have pointed out that use of the term speckled lentiginous nevus serves to broaden both the clinical and histologic spectrum (Aloi et al., 1995; Altman and Banse, 1992; Misago et al., 1993; Schaffer et al., 2001a). Lastly, the practice of referring to a smaller lesion as a nevus spilus and a larger lesion as a speckled lentiginous nevus only serves to add to the confusion.

Synonyms Although the majority of cases are described as either speckled lentiginous nevus (Aloi et al., 1995; Betti et al., 1994, 1997a, b, 1999; Ceylan et al., 2003; Crosti and Betti, 1994; Guiglia and Prendiville, 1991; Hofmann et al., 1998; Langenbach et al., 1998a; Marchesi et al., 1993; Misago et al., 1993; Nguyen et al., 1982; Paik et al., 1996; Schaffer et al., 2001a; Stewart et al., 1978; Torrelo et al., 2003a; Vásquez-Doval et al., 1995; Wagner and Cottel, 1989) or nevus spilus (Bielsa et al., 1998; Borrego et al., 1994; Breitkopf et al., 1996; Breuillard et al., 1991; Cohen et al., 1970; Cox et al., 1997; Cramer, 1977; Falo et al., 1994; Frenk et al., 1975; Gerardo et al., 2001; Gold et al., 1999; Grevelink et al., 1997; Hori et al., 1978; Hwang et al., 1997; Ishibashi et al., 1990; Keine et al., 1995; Konrad et al., 1974; Kopf et al., 1985a, b; Krahn et al., 1992; Langenbach et al., 1998b; Mang et al., 2003; Page and Windhorst, 1972; Park et al., 1999; Roma et al., 2000; Rütten and Goos, 1990; Sigg and Pelloni, 1989; Sigg et al., 1990; Skogh and Moi, 1978; Takahashi, 1976; Toppe and Haas, 1988; Weinberg et al., 1998; Welch and James, 1993; Zvulunov, 1995), when the lesion is linear or covers large areas of the body, the terms zosteriform speckled lentiginous nevus (Altman and Banse, 1992; Bolognia, 1991; Pritchett and Pritchett, 1982), speckled zosteriform lentiginous nevus (Simoes, 1981; Stern et al., 1990), zosteriform lentiginous nevus (Johnson, 1973; Matsudo et al., 1973; Ruth et al., 1980; van der Horst and Dirksen, 1981; Vignale et al., 1994), and mosaic speckled lentiginous nevus (Nguyen et al., 1982) have been used. Additional synonyms are nevus on nevus (Guillot

et al., 1991) which is also known as naevus sur naevus (Brufau et al., 1986; Hidano, 1986; Vion et al., 1985) or nevus sobre nevus (Piñol-Aguadé and Peyri Rey, 1973), speckled nevus spilus (Vion et al., 1985), speckled nevus (Nguyen et al., 1982), segmental nevus spilus (Moreno-Arias et al., 2001), nevus spilus zoniforme (Ito and Hamada, 1952), nevus spilus en nappe (Ito and Hamada, 1952), and spotty nevus (Vion et al., 1985). The term dysplastic nevus spilus (Grinspan et al., 1997; Kurban et al., 1992; Rhodes and Mihm, 1990) has been used to describe a speckled lentiginous nevus in which the intraepidermal melanocytes in the speckles have cellular atypia and pleomorphism.

Epidemiology/Clinical Findings In clinical studies involving children and adults, speckled lentiginous nevi have been shown to have a prevalence in the general population similar to that of congenital melanocytic nevi. For example, in a series of 601 consecutive patients who were primarily adults, 2.3% were noted to have a speckled lentiginous nevus >1.5 cm in diameter on total body skin examination (Kopf et al., 1985b). The prevalence rates of speckled lentiginous nevi in three series of children aged 8–16 years (n = 939), 6–15 years (n = 1123), and 6–18 years (n = 1592) were 2.1% (Sigg et al., 1990), 1.8% (Rivers et al., 1995), and 1.3% (McLean and Gallagher, 1995), respectively. However, much lower prevalence rates have been reported in series of newborns. Rhodes and Mihm (1990) observed that only 2/1118 newborns had a clinically evident speckled lentiginous nevus, and no speckled lentiginous nevi were noted in two studies assessing birthmarks in 4641 (Alper and Holmes, 1983) and 1058 (Walton et al., 1976) newborns. Based on these findings, Rhodes and Mihm (1990) concluded that speckled lentiginous nevi are acquired lesions. In contrast, of 26 patients described in four series (Brufau et al., 1986; Cohen et al., 1970; Stewart et al., 1978; Vion et al., 1985), 21 (81%) had lesions that were present at birth or noted during early infancy; these series included small lesions, with at least 40% measuring <6 cm in diameter. One explanation for the controversy regarding whether speckled lentiginous nevi are congenital or acquired is based on their natural history. These lesions often appear at birth as a lightly colored café-au-lait macule and may initially be subtle in their clinical presentation, with the more obvious pigmented macules and papules developing over a period of months to years to decades (Aloi et al., 1995; Cohen et al., 1970; Falo et al., 1994; Matsudo et al., 1973; Page and Windhorst, 1972; Stewart et al., 1978; Vion et al., 1985). There are a number of arguments in favor of the contention that speckled lentiginous nevi represent a subtype of congenital melanocytic nevus (Table 57.1). These include: the percentage of lesions noted at birth in several series (as discussed above); patterns of distribution reflecting embryonic development (e.g., sharp demarcation at the midline, lesions in a blocklike pattern or following the lines of Blaschko, and a divided form involving both upper and lower eyelids); hamartomatous behavior with various types of nevi (e.g., junctional, 1099

CHAPTER 57 Table 57.1. Speckled lentiginous nevi (SLN): subtype of congenital melanocytic nevus (CMN) versus acquired lesions. Arguments that SLN are a subtype of CMN Usually noted at birth or soon after (21/25 patients in four series) Initial hyperpigmented patches may be subtle, developing characteristic “spots” over time Patterns of distribution that reflect embryonic development Sharp demarcation at the midline Lesions along Blaschko lines Divided form involving both upper and lower eyelids Hamartomatous behavior “Spots” that develop can vary over time, e.g., Spitz nevi followed by blue nevi Presence of satellite CMN Transition from SLN to “classic” CMN Hybrid lesions of SLN and “classic” CMN Histologic features of CMN within spots Arguments that SLN are acquired SLN noted in 0.2% of newborns versus 2.3% of adults No SLN described in two large series of 4641 and 1058 newborns Adapted from Schaffer et al., 2000a.

blue, and Spitz nevi) presenting in the same lesion over time; and the presence of satellite congenital melanocytic nevi (Schaffer et al., 2001a). Recently, Schaffer et al. (2001a) described ten patients with congenital pigmented lesions that had the clinical appearance of speckled lentiginous nevi in whole or in part. These lesions either evolved over time to acquire an appearance more suggestive of “classic” congenital nevi, or existed as “hybrid” lesions with portions having the clinical features of a “classic” congenital nevus either admixed with or adjacent to portions having the appearance of a speckled lentiginous nevus. Additional cases with features of the admixed hybrid type of speckled lentiginous nevus have been reported (Langenbach et al., 1998b; Saraswat et al., 2003; Torrelo et al., 2003a). Lastly, histologic examination of biopsy specimens from spots within conventional speckled lentiginous nevi (Betti et al., 1994, 1997b; Casanova et al., 1996; Kopf et al., 1985b; Langenbach et al., 1998b; Maize and Ackerman, 1987; Mang et al., 2003; Saraswat et al., 2003; Weinberg et al., 1998) as well as the nevi reported by Schaffer et al. (2001a) has revealed features of congenital melanocytic nevi. Together, these observations provide strong support for the hypothesis that speckled lentiginous nevi are a subtype of congenital melanocytic nevus. Speckled lentiginous nevi have no sexual predilection. In a study of Canadian children, the prevalence in those of Asian (n = 378) and white European (n = 1145) origin was the same (McLean and Gallagher, 1995). Although speckled lentiginous nevi can appear on any area of the body, in one study of 14 patients, the lesions were concentrated on the trunk and lower extremities (Kopf et al., 1985b).

Clinical Description A number of red to brown to blue to black macules and 1100

papules are seen within a patch of hyperpigmentation (Stewart et al., 1978; Woerdeman, 1984). There is a wide spectrum of clinical findings when it comes to the spots, because the macular and papular lesions can vary from lentigines to junctional, compound, and dermal nevi to Spitz and blue nevi (Akasaka et al., 1993; Aloi et al., 1995; Betti et al., 1997a, b, 1999; Hidano, 1986; Hofmann et al., 1998; HofmannWellenhof et al., 1994; Hori et al., 1978; Ishibashi et al., 1990; Keine et al., 1995; Korting, 1967; Langenbach et al., 1998a; Maize and Ackerman, 1987; Marchesi et al., 1993; Misago et al., 1993; Park et al., 1999; Simoes, 1981; Toppe and Haas, 1988; Vion et al., 1985; Woerdeman, 1984) (Figs 57.3–57.5). Their size can range from 1 mm to >9 mm (Hofmann-Wellenhof et al., 1994), but they are often 2 mm to 3 mm in diameter. It is also important to remember that different types of nevi may appear at different points in time. For example, at age 3 years, one child was described as having multiple Spitz nevi within his speckled lentiginous nevus (Prose et al., 1983), but when examined at age 13 years, he had multiple blue and compound nevi within the lesion (personal communication, S. J. Orlow). Occasionally, papules or plaques with the clinical appearance of classic congenital melanocytic nevi may be found admixed with or adjacent to areas with the appearance of a typical speckled lentiginous nevus (i.e., “hybrid” lesions) (Langenbach et al., 1998b; Mang et al., 2003; Saraswat et al., 2003; Schaffer et al., 2001a; Torrelo et al., 2003a) (Fig. 57.6); in such cases, satellite congenital melanocytic nevi may be present (Schaffer et al., 2001a; Torrelo et al., 2003a). Areas with an increased number of terminal hairs have been described within both hybrid (see Fig. 57.6) and conventional speckled lentiginous nevi (Grinspan et al., 1997; Langenbach et al., 1998b; Mang et al., 2003; Prose et al., 1983; Saraswat et al., 2003; Schaffer et al., 2001a). The background hyperpigmentation can range in size from 1 cm to >60 cm in diameter (Crosti and Betti, 1994), but is often 3 cm to 6 cm in diameter (Stewart et al., 1978). In one study of 14 patients with nevus spilus >1.5 cm in diameter, the average size was 4.3 ± 3.5 cm (Kopf et al., 1985b). The color of the background pigmentation can vary from light tan (Stewart et al., 1978) to dark brown, and may darken following sun exposure (Falo et al., 1994; Matsudo et al., 1973). When the background pigmentation is a light tan color, clustering of the speckles may be overlooked (Bolognia, 1991) or a misdiagnosis made, such as agminated nevi or partial unilateral lentiginosis (Figs 57.7 and 57.8). Most commonly, the shape of a speckled lentiginous nevus is oval, but it can be blocklike in configuration with a sharp demarcation at the midline as well as linear. The latter have been described as zosteriform (Altman and Banse, 1992; Bolognia, 1991; Johnson, 1973; Matsudo et al., 1973; Pritchett and Pritchett, 1982; Ruth et al., 1980; Simoes, 1981; Stern et al., 1990; Stewart et al., 1978; van der Horst and Dirksen, 1981) and the linear lesions may follow the lines of Blaschko (Bolognia et al., 1994; Langenbach et al., 1998b; Welch and James, 1993) (Fig. 57.9). Extensive areas of the

COMMON BENIGN NEOPLASMS OF MELANOCYTES

Fig. 57.3. The background hyperpigmentation in this speckled lentiginous nevus was present since early infancy. A biopsy specimen of the largest “speckle” showed a pigmented dermal nevus.

Fig. 57.4. A speckled lentiginous nevus in which the largest erythematous papule had the histologic features of a Spitz nevus. (Courtesy of Yale Residents’ Slide Collection.)

Fig. 57.5. An extensive speckled lentiginous nevus in which junctional, compound, and Spitz nevi developed. (Courtesy of New York University Department of Dermatology Slide Collection.)

Fig. 57.6. Hybrid speckled lentiginous nevus in which plaques with the appearance of classic congenital melanocytic nevi are admixed with smaller macules and papules, all within a tan background patch. Note the areas with an increased density of terminal hairs.

body can be involved in these cases (Betti et al., 1994; Crosti and Betti, 1994; Davis and Shaw, 1964; Hofmann et al., 1998; Nguyen et al., 1982; Tadini et al., 1998; Torrelo et al., 2003a; Welch and James, 1993). Lastly, scleral pigmentation similar to that seen in nevus of Ota can occur when lesions containing blue nevi are located in the periocular area (Brufau et al., 1986; Hofmann et al., 1998), and a divided form of speckled lentiginous nevus that involved both the upper and lower eyelids has been described (Sato et al., 1979). This latter phenomenon reflects the probable timing of nevus formation during embryonic development, occurring after eyelid fusion in the eighth to ninth week of gestation but before eyelid reopening in the sixth month of gestation.

Associated Disorders There have been at least 22 reports of cutaneous melanoma developing within a speckled lentiginous nevus (Bolognia, 1991; Borrego et al., 1994; Breitkopf et al., 1996; Breuillard and Duthoit, 1991; Brufau et al., 1986; Cox et al., 1997; Grinspan et al., 1997; Guillot et al., 1991; Kopf et al., 1985a; Krahn et al., 1992; Kurban et al., 1992; Perkinson, 1957; Rhodes and Mihm, 1990; Rütten and Goos, 1990; Stern et al., 1990; Tadini et al., 1998; Vásquez-Doval et al., 1995; Vignale et al., 1994; Vion et al., 1985; Wagner and Cottel, 1989; Weinberg et al., 1998) (Fig. 57.10). These melanomas were diagnosed at a mean age of 52 years (range 25–79 years), and two patients also fulfilled the clinical criteria for neurofibromatosis type 1 (NF-1) (Perkinson, 1957; Rütten and Goos, 1990). In a few cases, the melanomas have been fatal (Bolognia, 1991; Brufau et al., 1986; Stern et al., 1990; Weinberg et al., 1998) and in one, there were two separate primaries (Krahn et al., 1992). The background speckled lentiginous nevi varied in size from 3 cm in diameter (Wagner and Cottel, 1989) to extensive zosteriform lesions (Bolognia, 1101

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Fig. 57.8. A subtle speckled lentiginous nevus that might be overlooked by the clinician.

Fig. 57.7. A. Although the initial clinical impression was agminated nevi, Wood’s lamp examination demonstrated background hyperpigmentation and pointed to the diagnosis of speckled lentiginous nevus; the lesion had been present since birth. B. Agminated compound nevi arising on a background of normally pigmented skin even under Wood’s lamp examination.

1991; Borrego et al., 1994; Guillot et al., 1991; Stern et al., 1990; Tadini et al., 1998), and some had speckles with atypical cytologic features (Bolognia, 1991; Grinspan et al., 1997; Kurban et al., 1992; Rhodes and Mihm, 1990). It has yet to be determined whether the surface area of the speckled lentiginous nevus, the number or type of superimposed nevi, or the presence of atypical cytologic features in the speckles correlates with the risk of developing cutaneous melanoma. Recently, the level of concern regarding speckled lentiginous nevi has become similar to that assigned to classic congenital 1102

Fig. 57.9. An extensive unilateral speckled lentiginous nevus with midline demarcation; note that the upper edge of the nevus follows the lines of Blaschko.

COMMON BENIGN NEOPLASMS OF MELANOCYTES

Fig. 57.10. Speckled lentiginous nevus that extends from the midline of the back (A) onto the left upper arm within the lines of Blaschko (B). The scar from the previous excision of the primary melanoma is seen at the base of the neck (A). A histologic section from one of the atypical nevi within the speckled nevus demonstrates lentiginous hyperplasia of the rete ridges, proliferation of atypical nevus cells, melanophages, and a superficial perivascular infiltrate (C). From Bolognia, J.L. Fatal melanoma arising in a zosteriform speckled lentiginous nevus. Arch. Dermatol. 1991; 127:1240–1241, copyright 1991, American Medical Association.

nevi. From (1992) suggested that speckled lentiginous nevi may actually present a greater risk, noting that of more than 2000 melanomas seen at her pigmented lesion clinic during a 15-year period, more arose from speckled lentiginous nevi (n = 3) than from large classic congenital melanocytic nevi (n = 1). In a study of 105 patients with melanoma and 601 controls, the prevalence of speckled lentiginous nevi >1.5 cm in diameter was not significantly higher in the melanoma group (4.8%) compared with the control group (2.3%) (Kopf et al., 1985a). Based upon the cutaneous findings, there are four major categories within the syndrome complex known as phako-

matosis pigmentovascularis (PPV) (Hasegawa and Yasuhara, 1985): type I, nevus flammeus and epidermal nevus; type II, nevus flammeus and dermal melanocytosis (nevus of Ota, nevus of Ito or aberrant Mongolian spots), with or without nevus anemicus; type III, nevus flammeus and nevus spilus, with or without nevus anemicus; and type IV, nevus flammeus, dermal melanocytosis, and nevus spilus, with or without nevus anemicus. More recently, a fifth category (type V) consisting of cutis marmorata telangiectatica congenita (CMTC) and dermal melanocytosis was proposed (Enjolras and Mulliken, 2000; Torrelo et al., 2003b); a patient with both CMTC and a nevus spilus has also been described (Kudo et al., 1986). 1103

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Patients with a nevus spilus, nevus anemicus, and primary lymphedema either with (Steijlen and Kuiper, 1990) or without (Bielsa et al., 1998) an associated capillary malformation have been reported; Bielsa et al. suggested that lymphatic malformations be included in the spectrum of vascular findings in PPV. Each of the PPV categories is subdivided into either cutaneous disease only (a) or cutaneous plus systemic disease (b). Signs and symptoms of the Sturge–Weber syndrome, Klippel–Trenaunay syndrome, and oculodermal melanosis can be seen in the latter group (Guiglia and Prendiville, 1991; Hasegawa and Yasuhara, 1985; Libow, 1993; Sawada et al., 1990; Sigg and Pelloni, 1989; Tsuruta et al., 1999). However, given the confusion over terminology, it should come as no surprise that in at least three reports of PPV, the “nevus spilus” was an extensive, uniformly pigmented patch without nevus cells (Hasegawa and Yasuhara, 1985; Sawada et al., 1990; Tsuruta et al., 1999), while in other reports, the lesions were speckled nevi (Guiglia and Prendiville, 1991; Prigent et al., 1991; Happle and Steijlen, 1989; Libow, 1993; Sigg and Pelloni, 1989; Zahorcsek et al., 1988). In 1996, Happle et al. coined the term “phakomatosis pigmentokeratotica” for a specific “twin nevus” syndrome in which a speckled lentiginous nevus (typically distributed in a “checkerboard” pattern) is associated with an organoid nevus with sebaceous differentiation (most often arranged in a linear pattern following the Blaschko lines); extracutaneous features can include musculoskeletal and neurologic anomalies such as hemiatrophy with muscular weakness, scoliosis, segmental dysesthesia and/or hyperhidrosis, mild mental retardation, and seizures (Boente et al., 2000; Descamps et al., 2000; Hermes et al., 1997; Hill et al., 2003; Kinoshita et al., 2003; Langenbach et al., 1998a; Saraswat et al., 2003; Tadini et al., 1998; Torrelo and Zambrano, 1998). At least three of the patients reported to date had organoid nevus-associated hypophosphatemic vitamin D-resistant rickets (Goldblum and Headington, 1993; Saraswat et al., 2003; Tadini et al., 1998). Several cases that fit the definition of phakomatosis pigmentokeratotica had been previously reported as epidermal or sebaceous nevus syndrome (Goldberg et al., 1987; Goldblum and Headington, 1993; Kopf and Bart, 1980; Misago et al., 1994; Stein et al., 1972; Wollenberg et al., 2002), Feuerstein– Mims neuroectodermal syndrome (Wauschkuhn and Rohde, 1971), and “nevus on nevus” (Brufau et al., 1986; PiñolAguadé and Peyri Rey, 1973). More recently, Happle (2002) defined the “speckled lentiginous nevus syndrome” as the association of this skin lesion with ipsilateral sensory and/or motor neuropathy (Holder et al., 1994; Piqué et al., 1995), spinal muscular atrophy (Hofmann et al., 1998), or muscular hypertrophy (Brufau et al., 1986). Of note, two of these cases were originally reported as partial unilateral lentiginosis, one with clinically apparent background hyperpigmentation more consistent with a diagnosis of speckled lentiginous nevus (Piqué et al., 1995) and the other with no obvious background hyperpigmentation, but multiple blue nevi as well as lentigines within a discrete area on the affected limb (Hofmann et al., 1104

1998). Happle (2002) proposed that this complex of neurologic and musculoskeletal abnormalities, distinct from the findings observed in association with organoid nevi in Schimmelpenning syndrome, is specific to the speckled lentiginous nevus “half” of phakomatosis pigmentokeratotica. Skin lesions with the clinical appearance of zosteriform speckled lentiginous nevi represent the cutaneous findings in the “FACES” syndrome, which is characterized by facial features (e.g., bifid tip of the nose), anorexia, cachexia, eye and skin anomalies (Friedman and Goodman, 1984). This syndrome was described in a mother and two daughters from a family with consanguinity. Additional clinical features included a high-pitched, nasal voice, severe muscle wasting, and musculoskeletal abnormalities. Of note, the skin lesions first appeared at age 7 years in the proband. There is a report of both ichthyosis (presumably X-linked) and a congenital speckled lentiginous nevus in a young man and his maternal grandfather (Crosti and Betti, 1994). Other males (maternal first cousins) in the family had only ichthyosis. In addition, a girl with phakomatosis pigmentokeratotica was noted to have diffuse, ichthyosis-like hyperkeratosis (Tadini et al., 1995). An extensive nevus spilus was also observed in a patient with Ebstein’s disease, which is the eponym for congenital downward displacement of the tricuspid valve into the right ventricle (Hori et al., 1978). Other reported associations with speckled lentiginous nevi include corneal snowflake dystrophy [2/38 patients in one series (Meretoja, 1985)] and adult-onset hearing loss (Nguyen et al., 1982). Although there are at least four reports of patients with segmental NF-1 plus either a speckled lentiginous nevus (Paik et al., 1996) or segmental lentiginosis (Allegue et al., 1989; Roth et al., 1987; Wong et al., 1997), two additional reports of segmental NF-1 in cases with a speckled lentiginous nevus are problematic because the diagnosis of segmental NF-1 is questionable (Finley and Kolbusz, 1993; Selvaag et al., 1994). In one patient, the diagnosis was based solely on the histologic features of a papule present within the speckled lentiginous nevus (Finley and Kolbusz, 1993); i.e., there were no other stigmata of NF. A collection of spindle cells was observed within a fine fibrocollagenous background in the reticular dermis, and the pathologic diagnosis was neurofibroma (no immunohistochemical studies were performed). As pointed out by Trattner and Hodak (1994), the lesion could have been simply a neurotized melanocytic nevus, a type of lesion that has been reported to develop within speckled lentiginous nevi (Hwang et al., 1997; van der Horst and Dirksen, 1981). In the second patient, a malformation of the fifth lumbar vertebra and shortening of the left extremity were seen in association with a speckled lentiginous nevus involving the left lower trunk and lower extremity (Selvaag et al., 1994). Again, there were no other signs of neurofibromatosis, and perhaps the diagnosis was actually SLN syndrome.

Histology In the areas of background hyperpigmentation, a variable

COMMON BENIGN NEOPLASMS OF MELANOCYTES

degree of lentiginous hyperplasia of the epidermis is observed, as is increased pigmentation of the basal layer, i.e., histologic findings similar to those of a lentigo simplex (Altman and Banse, 1992; Betti et al., 1997a; Crosti and Betti, 1994; Hermes et al., 1997; Hofmann et al., 1998; Pritchett and Pritchett, 1982; Stewart et al., 1978; Tadini et al., 1998). An increase in the number of active melanocytes also has been reported (Crosti and Betti, 1994; Hofmann et al., 1998; Matsudo et al., 1973; Ruth et al., 1980; Stewart et al., 1978; Takahashi, 1976). Occasionally, early nesting of nevus cells is seen within biopsy specimens of the background hyperpigmentation, which is sometimes referred to as nevus incipiens (Altman and Banse, 1992; Cohen et al., 1970; Prose et al., 1983). Histologic findings in biopsy specimens of the speckles can vary from lentiginous melanocytic hyperplasia and junctional theques at the tips of elongated rete ridges (macular lesions) to ordinary compound and dermal nevi (Altman and Banse, 1992; Betti et al., 1994, 1997a; Bielsa et al., 1998; Ceylan et al., 2003; Cramer, 1977; Crosti and Betti, 1994; Hermes et al., 1997; Langenbach et al., 1998a; Maize and Ackerman, 1987; Misago et al., 1994; Nguyen et al., 1982; Pock et al., 1991; Rhodes and Mihm, 1990; Roma et al., 2000; Simoes, 1981; Stewart et al., 1978; Tadini et al., 1998; Torrelo et al., 2003a). Collections of nevus cells around and within adnexal structures, nerves and/or blood vessels as well as between collagen bundles in the reticular dermis, representing characteristic findings of congenital melanocytic nevi, also may be present (Betti et al., 1994, 1997a; Casanova et al., 1996; Kopf et al., 1985b; Langenbach et al., 1998b; Maize and Ackerman, 1987; Mang et al., 2003; Saraswat et al., 2003; Schaffer et al., 2001a; Weinberg et al., 1998). In addition, the histologic features of Spitz nevus (Akasaka et al., 1993; Aloi et al., 1995; Betti et al., 1997b; Hofmann-Wellenhof et al., 1994; Vion et al., 1985; Woerdeman, 1984), dysplastic nevus (Bolognia, 1991; Grinspan et al., 1997; Kurban et al., 1992; Rhodes and Mihm, 1990; Wollenberg et al., 2002), or blue nevus (Betti et al., 1997a, 1999; Hofmann et al., 1998; Hori et al., 1978; Ishibashi et al., 1990; Korting, 1967; Langenbach et al., 1998a; Marchesi et al., 1993; Misago et al., 1993, 1994; Park et al., 1999; Pock et al., 1991; Toppe and Haas, 1988) can be seen. There are several reports of neurotized nevi developing within a speckled lentiginous nevus (Finley and Kolbusz, 1993; Hwang et al., 1997; van der Horst and Dirksen, 1981). Dermal melanophages also may be seen in the absence of an inflammatory infiltrate (Nguyen et al., 1982; Ruth et al., 1980). Speckled lentiginous nevi that contain blue nevi may occasionally have additional hamartomatous components other than melanocytic nevi, e.g., hyperplastic eccrine glands and ducts in the papillary dermis [Betti et al., 1997a; see section on Pilar neurocristic hamartoma (Chapter 58)], basaloid proliferation (Betti et al., 1999), and hyperplasia of arrector pili muscles (Park et al., 1999). These observations suggest that, as neural crest derivatives, melanocytes in the dermis may potentially have a “mesenchymal” role contributing to the

induction of epithelial proliferations. Cases in which a nevus sebaceus was completely superimposed on a speckled lentiginous nevus have also been described (Kopf and Bart, 1980; Langenbach et al., 1998a; Misago et al., 1994); these met the definition of phakomatosis pigmentokeratotica, and pigmented basal cell carcinomas eventually developed in two of the lesions (Langenbach et al., 1998a; Misago et al., 1994).

Laboratory Findings and Investigations Electron microscopic studies have shown the junctional theques in speckled lentiginous nevi to be composed of Mishima B-type cells with primarily fully melanized melanosomes and a few premelanosomes (Matsudo et al., 1973; Ruth et al., 1980). In addition, macromelanosomes have been seen within the melanocytes and keratinocytes of speckled lentiginous nevus (Frenk et al., 1975; Konrad et al., 1974; Pritchett and Pritchett, 1982; Rhodes and Mihm, 1990). Analysis by flow cytometry of a “dysplastic” nevus spilus in which a melanoma arose showed aneuploidy in the DNA of the melanoma as well as one of the pigmented lesions (a lentiginous junctional dysplastic nevus with slight atypia) within the nevus spilus (Kurban et al., 1992). The background hyperpigmentation and the remainder of the speckles (junctional and compound dysplastic nevi with slight to focally moderate atypia) were diploid. A systemic evaluation is recommended if the patient has other cutaneous features suggestive of PPV III or IV, e.g., nevus flammeus, nevus anemicus, and dermal melanocytosis (Guiglia and Prendiville, 1991; Happle and Steijlen, 1989; Hasegawa and Yasuhara, 1985; Libow, 1993; Sigg and Pelloni, 1989; Toda, 1986; Zahorcsek et al., 1988). The presence or absence of signs and symptoms such as seizures, mental retardation, glaucoma, and limb hypertrophy determines whether the diagnosis is PPV IIIa or IVa (cutaneous disease only) versus PPV IIIb or IVb (cutaneous plus systemic disease). If the patient has an extensive speckled lentiginous nevus and/or an associated organoid nevus, an evaluation focusing on the neurologic and musculoskeletal systems is warranted to assess for features of the speckled lentiginous nevus syndrome and phakomatosis pigmentokeratotica (see above) (Happle, 2002; Tadini et al., 1998).

Criteria for Diagnosis Diagnosis is based on the presence of a patch of hyperpigmentation which contains a variable number of smaller macules and/or papules that are usually more darkly pigmented. Biopsy specimens of the speckles demonstrate lentiginous melanocytic hyperplasia, collections of nevus cells, or dermal melanocytes. Of note, nevus cells may be present in biopsy specimens of the background hyperpigmentation (Altman and Banse, 1992; Cohen et al., 1970; Prose et al., 1983); this argues against using the histologic features of the background as a primary criterion for the diagnosis of this pigmented lesion (Kurban et al., 1992). 1105

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Differential Diagnosis The major entity in the differential diagnosis of the earliest stage of a speckled lentiginous nevus, the patch of hyperpigmentation, is a café-au-lait macule (Cohen et al., 1970). Agminated melanocytic nevi may be confused with a speckled lentiginous nevus, but in the former the background skin is not hyperpigmented. The clinician can use a Wood’s lamp to confirm the presence or absence of background hyperpigmentation. In partial unilateral lentiginosis, multiple lentigines are grouped together within an area of normal skin, and there is often a sharp demarcation at the midline (Cappon, 1948; Micali et al., 1994; Pickering, 1973; Schaffer et al., 2001b; Thompson and Diehl, 1980; Trattner and Metzker, 1993); the pigmented macules typically appear during childhood and undergo a gradual wavefront extension over months to years. Biopsy specimens demonstrate the histologic features of lentigines, e.g., an increase in the melanin content and lentiginous hyperplasia of the epidermis. Occasionally, small nests of nevus cells may be seen at the tips of the rete ridges (Marchesi et al., 1992), forming a ‘‘jentigo” pattern. One problem is the use by a few authors of the term “partial unilateral lentiginosis” to describe clustered lentigines within a patch of hyperpigmentation (Piqué et al., 1995). This may be due to the misconception that the speckles in a speckled lentiginous nevus cannot be lentigines. There are a few reports of patients with both types of lesions, speckled lentiginous nevus and segmental lentiginosis (Betti et al., 1997b; Crosti and Betti, 1994). When the histologic features of the speckles are those of Spitz nevi, one must be aware of the confusion in the literature regarding terminology. The term “agminated Spitz nevi” has been used to refer to clusters of nevi in an area of normally pigmented skin (Abramovits and Gonzalez-Serva, 1993; Krakowski et al., 1981; Orita et al., 1986) as well as clusters of nevi on a background of hyperpigmentation (Böer et al., 2001; Brownstein, 1972; Bullen et al., 1995; Duperrat and Dufourmentel, 1959; Goldberg et al., 1989; Grupper and Tubiana, 1955; Herd et al., 1994; Kopf and Andrade, 1966; Lancer et al., 1983; Palazzo and Duray, 1988; Quirino et al., 1996; Sourriel et al., 1969), even when the latter are admixed with other types of melanocytic nevi (Renfro et al., 1989). Several authors have advanced the notion that multiple Spitz nevi within a hyperpigmented patch represent a subtype of speckled lentiginous nevus (Akasaka et al., 1993; HofmannWellenhof et al., 1994; Woerdeman, 1984), and the present authors agree with this unifying concept, reserving the term “agminated Spitz nevi” to a cluster of nevi arising within an area of normally pigmented skin. The eccrine-centered nevus and the spotted grouped pigmented nevus (types I and II) represent variants of congenital melanocytic nevi in which speckles occur in a perifollicular and/or perieccrine distribution (Drut et al., 1999; Mishima, 1973; Morishima et al., 1976) (Fig. 57.11). Confusion has occurred because the term “spotted grouped pigmented nevus” has been used to describe speckled lentiginous nevi (Morishima et al., 1976; Sato et al., 1979). However, these 1106

Fig. 57.11. A speckled lentiginous nevus that overlaps clinically with a spotted grouped nevus.

two forms of nevi may simply represent ends of a spectrum since, in our opinion, both are subtypes of congenital nevi. There is also an example of a patient with a widespread speckled lentiginous nevus in which some of the grouped nevi appeared on a tan background patch, while others did not (Betti et al., 1994). In a biopsy specimen of a brown papule from normally pigmented skin, nevus cells were observed around and within adnexal structures, nerves, and blood vessels as well as splayed between collagen bundles, as in a congenital nevus (Betti et al., 1994). Additional confusion arises from the use of the terms “zosteriform melanocytic nevus” to describe large café-au-lait lesions that histologically lack nevus cells (Alper and Holmes, 1983) and “zosteriform lentiginous nevus” to describe lesions more compatible with linear and whorled nevoid hypermelanosis or epidermal nevi (Port et al., 1978). Also, the term speckled lentiginous nevus has been used to describe agminated nevi that are not within a tan or brown background patch (Maize and Ackerman, 1987). Although scattered lentigines within areas of postinflammatory hyperpigmentation can appear as psoriatic plaques, resolve, and resemble nevus spilus clinically (Burrows et al., 1994; Helland and Bang, 1980; Hofmann et al., 1977; Skogh and Moi, 1978), there is usually no difficulty separating these two entities.

Pathogenesis The speckled lentiginous nevus has been described as a “field defect.” Rhodes and Mihm (1990) suggested that speckled lentiginous nevi may be due to a defect in the melanoblasts that populate a localized area of the skin, and considered the possible influence of environmental and genetic factors. Other authors have raised the possibility of mosaicism as an explanation for zosteriform speckled lentiginous nevi (Davis and Shaw, 1964). Both extensive speckled lentiginous nevi and large classic congenital melanocytic nevi typically have a mosaic pattern of distribution, with the former lesions tending to either have a blocklike configuration (“checkerboard”; type

COMMON BENIGN NEOPLASMS OF MELANOCYTES

2) or follow Blaschko lines (type 1), and the latter usually having a patchy pattern without midline separation (type 4) (Happle, 1993). Interestingly, there are a few reports of familial occurrence of speckled lentiginous nevi (Crosti and Betti, 1994; Suzuki et al., 1990) and classic congenital nevi (Danarti et al., 2003). Recently, the concept of paradominant inheritance has been proposed to explain the genetic basis of speckled lentiginous nevi, classic congenital melanocytic nevi, and other conditions (e.g., Klippel–Trenaunay syndrome, Sturge–Weber syndrome, nevus anemicus, and organoid nevi) that are characterized by a mosaic distribution of lesions and occurrence that is primarily sporadic, but occasionally familial (Danarti et al., 2003; Happle, 2002). Zygotes that are homozygous for a paradominant mutation die during early embryogenesis, whereas individuals who are heterozygous are phenotypically normal; this allows a mutant gene to be transmitted without clinical expression through many generations. The trait only becomes manifest when a somatic mutation in a developing embryo results in loss of heterozygosity, thereby forming a population of cells that is homozygous for the mutation. Happle and Steijlen (1989) and Tadini et al. (1998) proposed the genetic concept of twin spotting (didymosis) for patients with PPV or PPK who have a nevus flammeus plus a speckled lentiginous nevus or an organoid nevus plus a speckled lentiginous nevus, respectively. The presumption is that the nevus flammeus (or the organoid nevus) and the speckled lentiginous nevus are caused by different recessive mutations, and that the two genes reside at different sites on the same chromosome (Fig. 57.12). If an embryo is a double heterozygote, then, following DNA replication, somatic crossing over can result in two populations of cells, each homozygous for one of two recessive mutations (Happle and Steijlen, 1989).

Animal Models None.

Treatment A common strategy for longitudinal care is baseline photographic documentation followed by serial clinical observation for the development of macules or papules with atypical features or signs suggestive of cutaneous melanoma. Any such change would prompt the performance of a biopsy. The patient and family also should be educated regarding the clinical signs of melanoma. In general, speckled lentiginous nevi are much easier to follow clinically than classic congenital nevi, which are often dark brown or black, thicker throughout, and covered by dense terminal hairs. In addition, to date, there have been no reports of the development of melanoma within the dermal component of speckled lentiginous nevi as there have been for classic congenital nevi. Some authors have advocated dermatoscopy or digital epiluminescence microscopy as aids in longitudinal clinical evaluation of speckled lentiginous nevi (Johr and Binder, 2000; Johr et al., 1998). An alternative is surgical excision (Casanova et al., 1996;

A

B

C

D

E Fig. 57.12. Mechanism of non-allelic twin spotting in an embryo that is doubly heterozygous for two different recessive mutations at neighboring loci. A pair of homologous chromosomes (A) undergoes semiconservative replication (B). Crossing-over occurs, with exchange of the region carrying the two mutations (C); as a result, each chromosome is composed of two different chromatids (D). The random assortment of the chromatids during mitosis may result in two different daughter cells, each homozygous for one of the mutations (E). Recently, heterozygous germline mutations in the VG5Q gene on chromosome 5q13, which encodes a potent angiogenic factor, were identified in a subset of patients with Klippel–Trenaunay syndrome (Tian et al., 2004). As twin spotting is thought to explain the association of Klippel–Trenaunay syndrome with speckled lentiginous nevi in patients with PPV IIIb or IVb, this finding implies that genes involved in the pathogenesis of speckled lentiginous nevi may potentially be located on chromosome 5q.

Page and Windhorst, 1972; Rhodes, 1996), but because a speckled lentiginous nevus represents a “field defect,” the entire area of hyperpigmentation has to be removed in order to avoid recurrence, usually resulting in a scar of significant size. Several groups have reported use of the Q-switched ruby, 1107

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Q-switched neodymium:yttrium-aluminum-garnet (Nd:YAG), or Q-switched alexandrite laser to treat both the background hyperpigmentation and the speckles within a speckled lentiginous nevus (Grevelink et al., 1997; Moreno-Arias et al., 2001; Nehal et al., 1996; Nelson and Applebaum, 1992; Taylor and Anderson, 1993; Tse et al., 1994). Grevelink et al. (1997) reported >80% lightening in 6/6 patients (mean age 30 years; range 15–42 years) who received one to five treatments with the Q-switched ruby laser; one patient was noted to develop hyperpigmentation at the periphery of the lesion, and focal recurrences occurred after two and three years in the two patients for whom follow-up information was available. In three of the patients in this study, a comparison of test sites treated with the Q-switched ruby versus the Q-switched Nd:YAG showed 0–30% and 50–80% lightening, respectively. Specific details are available for four other patients treated with the Q-switched ruby laser (Nehal et al., 1996; Taylor and Anderson, 1993; Tse et al., 1994). Post treatment, both hypo- and hyperpigmentation were observed, as were return of the background pigmentation (n = 1), lack of response despite seven treatments (n = 1), and partial (25–60%) improvement (n = 2; one patient was also treated with the Q-switched Nd:YAG laser). In the single case reported to date, only 50% lightening was seen after 16 treatments with the Q-switched alexandrite laser (Morena-Arias et al., 2001). Lastly, 50–75% clearance was observed after four sessions with intense pulsed light in the four patients reported thus far (Gold et al., 1999; Moreno-Arias and Ferrando, 2001). In general, it appears that the background component of a speckled lentiginous nevus, like the café-au-lait macule, is particularly resistant to treatment and prone to recurrence (Hruza, 1997). From a cosmetic standpoint, although Grevelink et al. (1997) had more encouraging shortterm results than the other groups, the durability of the response remains to be seen. Moreover, from a medical standpoint, laser treatment of the nevi within a speckled lentiginous nevus is as controversial as it is for the treatment of other melanocytic nevi.

Prognosis In the past, speckled lentiginous nevi (especially those of relatively small size) were often assigned the same level of concern as a café-au-lait macule or lentigo, and routine longitudinal observation was not advised. More recently, following multiple reports of the development of cutaneous melanoma within these lesions and mounting evidence of their congenital nature, the level of concern has become more or less proportional to that given to a classic congenital nevus of the same size. It remains to be seen whether the surface area of the speckled lentiginous nevus, the number or type of nevi contained within the lesion, or the presence of atypical cytologic features in the speckles will correlate with the risk of developing cutaneous melanoma.

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COMMON BENIGN NEOPLASMS OF MELANOCYTES Orita, M., K. Obara, T. Irimajiri, and M. Kobayashi. Multiple agminate juvenile melanoma. Dermatologica 172:58–61, 1986. Page, E. V., and D. B. Windhorst. Unusual nevus (nevus spilus). Arch. Dermatol. 105:115, 1972. Paik, S.-C., J.-H. Shim, J.-D. Lee, B.-K. Cho, and C.-W. Kim. Bilateral segmental neurofibromatosis with speckled lentiginous nevus. Int. J. Dermatol. 35:360–361, 1996. Palazzo, J. P., and P. H. Duray. Congenital agminated Spitz nevi: immunoreactivity with a melanoma-associated monoclonal antibody. J. Cutan. Pathol. 15:166–170, 1988. Park Y. M., H. Kang, and B. K. Cho. Plaque-type blue nevus combined with nevus spilus and smooth muscle hyperplasia. Int. J. Dermatol. 38:775, 1999. Perkinson, N. G. Melanoma arising in a café-au-lait spot of neurofibromatosis. Am. J. Surg. 93:1018–1020, 1957. Pickering, J. G. Partial unilateral lentiginosis with associated developmental abnormalities. Guys Hospital Rep. 122:361–370, 1973. Piñol-Aguadé, J., and J. Peyri Rey. Nevus sobre nevus. Med. Cutan. Ibero Lat. Am. 7:85–93, 1973. Piqué, E. P., A. Aguilar, M. C. Fariña, M. A. Gallego, P. Escalonilla, and L. Requena. Partial unilateral lentiginosis: report of seven cases and review of the literature. Clin. Exp. Dermatol. 20:319–322, 1995. Pock, L., J. Trnka, F. Vosmik, and F. Zaruba. Systematized progradient multiple combined melanocytic and blue nevus. Am. J. Dermatopathol. 13:282–287, 1991. Port, M., J. Courniotes, and M. Podwal. Zosteriform lentiginous naevus with ipsilateral rigid cavus foot. Br. J. Dermatol. 98:693– 698, 1978. Prigent, F., A. Kahn, C. Martinet, and T. Saigot. Phacomatose pigmento-vasculaire IIIa. Ann. Dermatol. Venereol. 118:531–533, 1999. Pritchett, R. M., and P. S. Pritchett. Zosteriform speckled lentiginous nevus with giant melanosomes. Cutis 30:329–334, 1982. Prose, N. S., E. Heilman, Y. M. Felman, F. Tanzer, and J. Silber. Multiple benign juvenile melanoma. J. Am. Acad. Dermatol. 9:236–242, 1983. Quirino, A. P., F. Azevedo, and M. B. Monteiro. Nevus de Spitz multiples agminés. Ann. Dermatol. Venereol. 123:S145, 1996. Renfro, L., J. M. Grant-Kels, and S. A. Brown. Multiple agminate Spitz nevi. Pediatr. Dermatol. 6:114–117, 1989. Rhodes, A. R. Pigmented birthmarks and precursor melanocytic lesions of cutaneous melanoma identifiable in childhood. Pediatr. Clin. North Am. 30:435–463, 1983. Rhodes, A. R. Nevus spilus: A potential precursor of cutaneous melanoma worthy of aggressive surgical excision? Pediatr. Dermatol. 13:250–252, 1996. Rhodes, A. R., and M. C. Mihm Jr. Origin of cutaneous melanoma in a congenital dysplastic nevus spilus. Arch. Dermatol. 126:500– 505, 1990. Rivers, J. K., R. MacLennan, J. W. Kelly, A. E. Lewis, B. J. Tate, S. Harrison, and W. H. McCarthy. The eastern Australian childhood nevus study prevalence of atypical nevi, congenital nevus-like nevi, and other pigmented lesions. J. Am. Acad. Dermatol. 32:957–963, 1995. Roma, P., H. Sanjeev, and K. Bhusan. Generalized naevus spilus: A rare entity? J. Eur. Acad. Dermatol. 14:430, 2000. Roth, M. R., R. Martinez, and W. D. James. Segmental neurofibromatosis. Arch. Dermatol. 123:917–920, 1987. Rütten, A., and M. Goos. Nevus spilus with malignant melanoma in a patient with neurofibromatosis. Arch. Dermatol. 126:539–540, 1990. Ruth, W. K., J. D. Shelbourne, and B. V. Jegasothy. Zosteriform lentiginous nevus. Arch. Dermatol. 116:476, 1980. Saraswat, A., S. Dogra, A. Bbansali, and B. Kumar. Phakomatosis pigmentokeratotica associated with hypophosphataemic vitamin

D-resistant rickets: improvement in phosphate homeostasis after partial laser ablation. Br. J. Dermatol. 148:1074–1076, 2003. Sato, S., H. Kato, and A. Hidano. Divided nevus spilus and divided form of spotted grouped pigmented nevus. J. Cutan. Pathol. 6:507–512, 1979. Sawada, Y., M. Iwata, and Y. Mitsusashi. Nevus Pigmentovascularis. Ann. Plast. Surg. 25:142–145, 1990. Schaffer, J. V., S. J. Orlow, R. Lazova, and J. L. Bolognia. Speckled lentiginous nevus: Within the spectrum of congenital melanocytic nevi. Arch. Dermatol. 137:172–178, 2001a. Schaffer, J. V., R. Lazova, and J. L. Bolognia. Partial unilateral lentiginosis with ocular involvement. J. Am. Acad. Dermatol. 44:387–390, 2001b. Selvaag, E., P. Thune, and T. E. Larsen. Segmental neurofibromatosis presenting as a giant naevus spilus. Acta Derm. Venereol. 74:327, 1994. Sigg, C., and F. Pelloni. Oligosymptomatic form of Klippel-Trenaunay-Weber syndrome associated with giant nevus spilus. Arch. Dermatol. 125:1284–1285, 1989. Sigg, C., F. Pelloni, and U. W. Schnyder. Frequency of congenital nevi, nevi spili and café-au-lait spots and their relation to nevus count and skin complexion in 939 children. Dermatologica 180:118–123, 1990. Simoes, G. A. Speckled zosteriform lentiginous nevus. J. Am. Acad. Dermatol. 4:236–238, 1981. Skogh, M., and H. Moi. Naevus-spilus-artige hyperpigmentierungen bei psoriasis. Hautarzt 29:607, 1978. Sourriel, P., C. Beylot, and X. Carrard. Mélanome de Spitz (forme multiple agminée). Bull. Soc. Franc. Dermatol. Syphil. 76:77–79, 1969. Stein, K. M., E. Shumunes, and M. Thew. Neurofibromatosis presenting as the epidermal nevus syndrome. Arch Dermatol. 105:229–232, 1972. Steijlen, P. M., and J. P. Kuiper. Phacomatose vasculaire pigmentée. Phlébologie 43:34–35, 1990. Stern, J. B., H. M. Haupt, and C. M. Aaronson. Malignant melanoma in a speckled zosteriform lentiginous nevus. Int. J. Dermatol. 29:583–584, 1990. Stewart, D. M., J. Altman, and A. H. Mehregan. Speckled lentiginous nevus. Arch. Dermatol. 114:895–896, 1978. Suzuki, K., H. Ishizake, and H. Takahashi. [Phacomatosis pigmentovascularis IIIb associated with porokeratosis, acanthosis nigricans and endocrinopathy in brother and sister.] [Japanese] Skin Res. (Hifu) 32:65–70, 1990. Tadini, G., E. Ermacora, and G. Carminati. Unilateral speckledlentiginous naevus, contralateral verrucous epidermal naevus, and diffuse ichthyosis-like hyperkeratosis: an unusual example of twin spotting? Eur. J. Dermatol. 5:659–663, 1995. Tadini, G., L. Restano, R. Gonzales-Perez, M. A. Gonzales-Ensenat, M. A. Vincente-Villa, S. Cambiaghi, P. Marchettini, M. Mastrangelo, and R. Happle. Phacomatosis pigmentokeratotica: Report of new cases and further delineation of the syndrome. Arch. Dermatol. 134:333–337, 1998. Takahashi, M. Studies on café au lait spots in neurofibromatosis and pigmented macules of nevus spilus. Tohoku J. Exp. Med. 118:255– 273, 1976. Taylor, C. R., and R. R. Anderson. Treatment of benign pigmented epidermal lesions by Q-switched ruby laser. Int. J. Dermatol. 32:908–912, 1993. Thompson, G. W., and A. K. Diehl. Partial unilateral lentiginosis. Arch. Dermatol. 116:356, 1980. Tian, X.-L., R. Kadaba, S.-A. You, M. Liu, A. A. Timur, L. Yang, Q. Chen, P. Szafranski, S. Rao, L. Wu, D. E. Housman, P. E. DiCorleto, D. J. Driscoll, J. Borrow, and Q. Wang. Identification of an angiogenic factor that when mutated causes susceptibility to Klippel-Trenaunay syndrome. Nature 427:640–645, 2004.

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CHAPTER 57 Toda, K. Phacomatosis pigmentovascularis Ota. In: Biology and Diseases of Epidermal Pigmentation, T. B. Fitzpatrick, M. M. Wick, and K. Toda (eds). Tokyo: University of Tokyo Press, 1986. pp. 179–181. Toppe, F., and N. Haas. Naevus spilus géant avec naevus bleus. A propros d’un cast. Ann. Dermatol. Venereol. 115:703–707, 1988. Torrelo, A., and A. Zambrano. What syndrome is this: Phakomatosis pigmentokeratotica (Happle). Pediatr. Dermatol. 15:321–323, 1998. Torrelo, A., I. de Prada, A. Zambrano, and R. Happle. Extensive speckled lentiginous nevus associated with giant congenital melanocytic nevus: An unusual example of twin spotting? Eur. J. Dermatol. 13:534–536, 2003a. Torrelo, A., A. Zambrano, and R. Happle. Cutis marmorata telangiectatica congenita and extensive mongolian spots: type 5 phacomatosis pigmentovascularis. Br. J. Dermatol. 148:342–345, 2003b. Trattner, A., and A. Metzker. Partial unilateral lentiginosis. J. Am. Acad. Dermatol. 29(5 Pt 1):693–695, 1993. Trattner, A., and E. Hodak. Segmental neurofibromatosis clinically appearing as a nevus spilus. Int. J. Dermatol. 33:75, 1994. Tse, Y., V. J. Levine, S. A. McClain, and R. Ashinoff. The removal of cutaneous pigmented lesions with the Q-switched ruby laser and the Q-switched neodymium: yttrium-aluminum-garnet laser. A comparative study. J. Dermatol. Surg. Oncol. 20:795–800, 1994. Tsuruta, D., K. Fukai, M. Seto, K. Fujitani, K. Shindo, T. Hamada, and M. Ishii. Phakomatosis pigmentovascularis type IIIb associated with moyamoya disease. Pediatr. Dermatol. 16:35–38, 1999. van der Horst, J. C., and H. J. Dirksen. Zosteriform lentiginous naevus. Br. J. Dermatol. 104:104–105, 1981. Vásquez-Doval, J., M. A. Sola, F. Contreras-Mejuto, P. Redondo, J. Soto, and E. Quintanilla. Malignant melanoma developing in a speckled lentiginous nevus. Int. J. Dermatol. 34:637–638, 1995. Vignale, R. A., J. Espasandin, and H. Deneo. Melanoma extensivo superficial en nevo lentiginoso zosteriforme moteado. Med. Cut. Ibero. Lat. Am. 22:365–368, 1994. Vion, B., S. Belaïch, M. Grossin, and J. Préaux. Les aspects évolutifs du naevus sur naevus. Revue de la littérature à propos de 7 observations. Ann. Dermatol. Venereol. 112:813–819, 1985. Wagner, R. F., Jr., and W. I. Cottel. In situ malignant melanoma arising in a speckled lentiginous nevus. J. Am. Acad. Dermatol. 20:125–126, 1989. Walton, R. G., A. H. Jacobs, and A. J. Cox. Pigmented lesions in newborn infants. Br. J. Dermatol. 95:389–396, 1976. Wauschkunh, J., and B. Rohde. Systematisierte talgdüsen-, pigmentund epitheliale naevi mit neurologischer symptomatik: FeuersteinMimsches neuroektodermales syndrom. Hautarzt 22:10–13, 1971. Weinberg, J. M., P. J. Schutzer, R. M. Harris, I. A. Tangoren, S. Sood, and R. I. Rudolph. Melanoma arising in nevus spilus. Cutis 61:287–289, 1998. Welch, M. L., and W. D. James. Widespread nevus spilus. Int. J. Dermatol. 32:120–122, 1993. Woerdeman, M. J. Multiple agminate juvenile melanoma in a naevusspilus-like hyperpigmented area. Br. J. Dermatol. 110:119–120, 1984. Wollenberg, A., C. Butnaru, and T. Oppel. Phacomatosis pigmentokeratotica (Happle) in a 23-year-old man. Acta Derm. Venereol. 82:55–57, 2002. Wong, S. S. Bilateral segmental neurofibromatosis with partial unilateral lentiginosis. Br. J. Dermatol. 136:380–383, 1997. Zahorcsek, Z., A. Schmelas, and I. Schneider. Progrediente zirkumskripte lentiginose–Phakomatosis pigmentovascularis III/A. Hautarzt 39:519–523, 1988. Zvulunov, A. Segmental neurofibromatosis versus giant nevus spilus. Acta Derm. Venereol. 75:408, 1995.

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Melanocytic (Nevocellular) Nevi and Their Biology Julie V. Schaffer and Jean L. Bolognia

Definitions Melanocytic nevi (moles) are benign proliferations of a type of melanocyte known as a “nevus cell” (Magana-Garcia and Ackerman, 1990). The two major differences between ordinary melanocytes that reside in the basal layer of the epidermis and nevus cells are: (1) nevus cells cluster as nests within the lower epidermis and/or dermis, whereas epidermal melanocytes are evenly dispersed as single units; and (2) nevus cells do not have dendritic processes (with the exception of those within blue nevi). Both melanocytes and nevus cells are capable of producing the pigment melanin. Melanocytic nevi can be acquired or congenital, banal or atypical (“dysplastic”). The names applied to acquired nevi reflect the location of the nests of melanocytes; i.e., nests are at the dermal–epidermal junction in junctional nevi, in the dermis in intradermal nevi, and at both sites in compound nevi. Clinically, with progressive migration of melanocytes from the dermal–epidermal junction into the dermis, nevi become more elevated and less pigmented (Clark et al., 1984; Lund and Stobbe, 1949). Atypical nevi are benign acquired melanocytic nevi that share, usually to a lesser degree, some of the clinical features of melanoma (i.e., asymmetry, border irregularity, color variability, and diameter ≥6 mm). Considerable controversy has surrounded terms such as “dysplastic nevus,” and the 1992 National Institutes of Health Consensus Conference recommended the use of the more clinically descriptive term “atypical nevus.” They also recommended that these lesions be described histologically as “nevi with architectural disorder,” with specification of the degree of melanocytic atypia present. Of note, however, several studies have found that clinical and histologic atypia in melanocytic nevi are not highly correlated (Annessi et al., 2001; Klein and Barr, 1990). There are several other variants of melanocytic nevi, such as blue nevi and Spitz nevi, that have specific clinical, histologic, and molecular characteristics (see below). In addition to the basic clinicopathologic subtypes of melanocytic nevi, distinct patterns of nevus morphology can be observed. For example, some individuals tend to develop multiple melanocytic nevi with a particular clinical appearance, e.g., uniformly pink in color, light brown with central brown-black pigmentation, or targetoid (i.e., cockarde nevi) (Schaffer et al., 2001). This results in a predominant type of nevus, or “signature nevus.” The presence of signature nevi suggests that the nevi on a given individual are “programmed,” with nevus morphology depending on genetic as well as environmental factors. Indeed, McGregor et al. (1999) found a significantly higher degree of concordance in the color, size, symmetry, border irregularity, and edge distinctness of nevi (as assessed by quantitative computer image analysis) in monozygotic than dizygotic twins, with genetic influences esti-

COMMON BENIGN NEOPLASMS OF MELANOCYTES

mated to account for 40–80% of variation in these morphologic features.

Molecular Pathogenesis Clonality Molecular investigations have provided evidence that acquired nevi are clonal, and therefore represent true neoplasms rather than hamartomas (Hui et al., 2001; Maitra et al., 2002; Robinson et al., 1998). Polymerase chain reaction-based analysis of the human androgen receptor gene on the X chromosome demonstrated a nonrandom pattern of inactivation in 74%, 91%, and 89% of acquired junctional (26/34), compound (10/11), and intradermal (8/9) nevi from female patients, respectively (Hui et al., 2001; Robinson et al., 1998). The presence of nontumor cells within a specimen may lead to a “false” polyclonal result in a monoclonal lesion, potentially accounting for the lower proportion of junctional nevi found to be monoclonal (as it is difficult to separate these nevus cells from the surrounding epidermis). Together with other technical issues, this may also explain the inability of an earlier study using similar methodology to demonstrate clonality in nevi (Harada et al., 1997). More recently, Maitra et al. (2002) analyzed five chromosomal regions in samples obtained from banal acquired melanocytic nevi via laser capture microdissection, and found loss of heterozygosity in 75% of the nevi (9/12). Hui et al. (2001) emphasized that although the finding of monoclonality verifies the neoplastic nature of nevi, it does not necessarily imply a first step in the development of melanoma or even a precursor state, as suggested by others (Maitra et al., 2002; Robinson et al., 1998). Only one of six congenital melanocytic nevi analyzed in the aforementioned studies was found to be clonal (Hui et al., 2001; Maitra et al., 2002; Robinson et al., 1998). As discussed above, this may represent false polyclonal results due to the intimate admixture of congenital nevus cells with normal dermal elements. However, an alternative explanation, as previously proposed by Ackerman (1993), is that congenital nevi are polyclonal lesions more correctly classified as hamartomas.

Molecular and Cellular Features Several molecular and cellular features differentiate nevus cells from ordinary melanocytes. These include production of basic fibroblast growth factor, a lack of induction of apoptosis when exposed to transforming growth factor b in culture, and a higher intrinsic potential for adhesion (e.g., nesting) and migration (Alanko and Saksela, 2000; Mengeaud et al., 1996; Uead et al., 1994). The expression of E-cadherin, the major mediator of adhesion between epidermal melanocytes and keratinocytes, is relatively strong in junctional nevus cells, but decreases with depth in dermal nevus cells (with the exception of those in blue nevi) (Gontier et al., 2004; Herlyn et al., 2000; Krengel et al., 2004). This suggests that a reduction in keratinocyte control may play a role in the migration of nevus cells from the dermal–epidermal junction into the dermis. Some authors have proposed that breakdown of the basement

membrane by proteolytic enzymes such as matrix metalloproteinases represents another factor in the exodus of junctional nevus cells (Gontier et al., 2003; Krengel et al., 2002); however, both dermal nevi and invasive melanomas are surrounded by and able to synthesize basement membrane material, leading others to postulate that the basement membrane accompanies rather than acts as a barrier to melanocyte movement (Schaumburg-Lever et al., 2000). Compared with ordinary melanocytes, nevus cells at or near the dermal–epidermal junction exhibit increased expression of tyrosinase related protein 1 (TYRP1; involved in melanin biosynthesis) and gp100/Pmel-17 (a marker of melanocyte activation), detected by the MEL-5 and HMB-45 immunohistochemical stains, respectively (Clarkson et al., 2001; Meije et al., 2000). The expression of these proteins diminishes deeper in the dermis in banal nevi, congenital nevi, and most Spitz nevi, but not in blue nevi and some atypical nevi (Bergman et al., 1995; Shah et al., 1997; Skelton et al., 1991; Wood et al., 1991). Nevus cells undergo a process of “maturation” (also referred to as “atrophy”) as they descend into the dermis, associated with a decrease in the size and number of most cellular constituents, lower bcl-2 expression, increased apoptosis, and fewer proliferating cell nuclear antigen (PCNA)- or Ki-67-positive cells (Table 57.2) (Clark et al., 1984; Goovaerts and Buyssens, 1988; Li et al., 2000; MoralesDucret et al., 1995; Sprecher et al., 1999; Tokuda et al., 1994). In addition, dermal descent is often accompanied by Schwannian differentiation and increased expression of nerve growth factor receptor (Argenyi et al., 1996; Clark et al., 1984; Fullen et al., 2001; Goovaerts and Buyssens, 1988; Kanik et al., 1996; Smolle et al., 1988).

Genetic Features Analysis of somatic mutations in melanocytic nevi has provided further insight into their pathogenesis. Gain-of-function mutations in the BRAF gene, which encodes a serine/ threonine kinase in the mitogen-activated protein kinase (MAPK) signaling cascade (Fig. 57.13), have recently been identified in almost three-quarters of banal melanocytic nevi (184/256; particularly intradermal and papillomatous compound variants) and more than one third of primary cutaneous melanomas (170/452; see Table 57.2) (Cohen et al., 2004; Edwards et al., 2004; Kumar et al., 2004; Maldonado et al., 2003; Omholt et al., 2003; Pollock et al., 2003; Reifenberger et al., 2004; Sasaki et al., 2004; Sekulic et al., 2004; Shinozaki et al., 2004; Uribe et al., 2003; Yazdi et al., 2003). This implicates activation of the MAPK pathway as an important step in tumorigenesis rather than malignancy (Arbiser, 2003). Of note, a single phosphomimetic substitution (V599E) accounts for >90% of the activating BRAF mutations in melanocytic tumors, and “ultraviolet (UV) signature” (e.g., pyrimidine dimer) mutations have not been found (Kumar et al., 2004; Pollock et al., 2003; Uribe et al., 2003). BRAF mutations are seen most often in melanomas that occur on intermittently sun-exposed skin, and infrequently in those that arise on chronically sun-exposed skin, on mucous membranes, or 1113

1114

10 76 78 60 9 0 38 38

Nevi Junctional Compound Intradermal Congenital Blue Spitz Atypical Primary melanomas <10–20 <10–20 <10–20 50 0 5 <2 10–30

NRAS mutation (%)b

Rare Rare Rare 0 0 20 NA <0.2

HRAS mutation and/or 11p amplification* (%)c

Rare Rare Rare† ‡ Rare Rare <30 >95§

Chromosomal alterations other than 11p (%)d

0–25 0–25 0–25 NA NA 15–35 25–50 15–>30

Microsatellite instability (%)e

0.7 0.4 0.2 NA NA 6.9 2.6 24

MIB-1/ Ki-67+ (% of nuclei)f

95 95# 50 5‡ <5 NA >80# 60 (RGP) 30 (VGP)

KIT+ (%)g

50 50# 0 NA 100 NA NA 90

CD68+ (%)h

NA 0 0 NA NA 30 0 80

tPA+ (%)i

NA 38 养 33 养 NA 50 养 100 养 88 养 33 养

S100A6+ (%)j

0 <10** <10** NA NA 100 <10 10 (RGP) 80 (VGP)

integrin+ (%)k

b3

NA <50 75** <25** 75 75 <50 50***

p75 NGF receptor+ (%)l

*Most often, multiple copies of an isochromosome 11p are present. †Aneuploidy may be seen in fibromatous or lipomatous intradermal nevi (“ancient” nevi). ‡Atypical nodular proliferations that develop during infancy often have numerical aberrations of whole chromosomes exclusively, and >95% express KIT. §Acral melanomas have a particularly high frequency of focused amplifications and aberrations involving 5p, 11q, 12q, and 15. #Primarily in the junctional component of nevi. 养 Expression of S100A6 in Spitz nevi is strong and diffuse in both junctional and dermal components, while expression in other nevi and melanomas is typically weak and limited to the dermal component. **Primarily in the dermal component of neurotized nevi; 95% of neural nevi are p75 NGF-receptor positive. ***Up to 100% of spindle cell melanomas are p75 NGF-receptor positive. NA, not assessed; tPA, tissue-type plasminogen activator; NGF, nerve growth factor; RGP, radial growth phase; VGP, vertical growth phase. a From Cohen et al., 2004; Edwards et al., 2004; Gill et al., 2004; Kumar et al., 2004; Maldonado et al., 2003; Mihic-Probst et al., 2004; Omholt et al., 2003; Palmedo et al., 2004; Pollock et al., 2003; Reifenberger et al., 2004; Saldanha et al., 2004; Sasaki et al., 2004; Sekulic et al., 2004; Shinozaki et al., 2004; Uribe et al., 2003; Yazdi et al., 2003. b From Herlyn and Satyamoorthy, 1996; Kumar et al., 2004; Omholt et al., 2003; Papp et al., 1999, 2003; Pollock et al., 2003; Saldanha et al., 2004. c From Bastian, 2003; Bastian et al., 1999, 2000, 2003. d From Bastian, 2003; Bastian et al., 1999, 2002, 2003; Korabiowska et al., 1999. e From Birindelli et al., 2000; Hussein et al., 2001; Palmieri et al., 2002; Rubben et al., 2002; van Dijk et al., 2002. f From Li et al., 2000. g From Herron et al., 2004; Montone et al., 1997; Ohashi et al., 1996; Stefanou et al., 2004. h From Shah et al., 1997. i From Ferrier et al., 2002. j From Fullen et al., 2001; Ribé and McNutt, 2003. k From van Belle et al., 1999. l From Argenyi et al., 1996; Brocker et al., 1991; Fanburg-Smith et al., 2001; Innominato et al., 2001; Kanik et al., 1996; Thompson et al., 1989.

BRAF mutation (%)a

Type of melanocytic lesion

Table 57.2. Molecular characteristics of melanocytic nevi of various types and primary melanomas.

COMMON BENIGN NEOPLASMS OF MELANOCYTES

Fig. 57.13. The BRAF protein plays a critical role in the RAS-RAF-MAPK (mitogen-activated protein kinase) signaling pathway. The latter is a pivotal molecular cascade through which extracellular signals can be transmitted into the nucleus, to control cell proliferation or differentiation via changes in gene expression. Extracellular signals (i.e., growth factors) that activate one of two types of receptors (tyrosine kinase or G protein-coupled) can lead to activation of RAS, leading then to activation of BRAF and the remainder of the cascade. Activating mutations in the gene that encodes the BRAF protein have been detected in benign melanocytic nevi as well as melanoma. Reprinted with permission from Nature Publishing.

G-proteincoupled receptor

Growth factor

P GRB P

SOS

RAS BRAF

Receptor tyrosine kinase

MAPK signalling pathway

Growth factor

Cyclic AMP Adenylate cyclase G protein

NUCLEUS Changes in gene expression

CYTOPLASM

within the uveal tract (Edwards et al., 2004; Kilic et al., 2004; Maldonado et al., 2003). Interestingly, at least 166 Spitz nevi have been analyzed to date, and no BRAF mutations have been identified (Gill et al., 2004; Mihic-Probst et al., 2004; Palmedo et al., 2004; Saldanha et al., 2004; Yazdi et al., 2003). Activating mutations in the RAS family of protooncogenes, which encode GTPase proteins that function upstream of BRAF in the MAPK cascade, also occur in a subset of melanocytic lesions. In general, BRAF and RAS mutations are mutually exclusive, reflecting their functional equivalence in cellular transformation (Kumar et al., 2004; Omholt et al., 2003; Pollock et al., 2003). Activating mutations in NRAS have been found in approximately 50% of congenital melanocytic nevi and 10–30% of melanomas, but <2% of atypical nevi (Herlyn and Satyamoorthy, 1996; Papp et al., 1999, 2003). In contrast, mutations and/or amplifications in HRAS (located at the distal end of chromosome 11p) have been detected in approximately 20% of Spitz nevi (particularly the predominantly intradermal, desmoplastic subset), but <0.2% of melanomas (Bastian, 2003, 2004; Bastian et al., 1999, 2000, 2003; see Table 57.2). Other molecular features that characterize the various types of melanocytic nevi are presented in Table 57.2.

Natural History of Nevi Acquired melanocytic nevi begin to appear after the first 6 months of life, increase in number during childhood and adolescence, reach a peak count in the third decade, and then slowly regress with age (Cooke et al., 1985; Harrison et al., 2000; MacKie et al., 1985; Nicholls, 1973) (Figures 57.14 and 57.15; Tables 57.3 and 57.4). The classic clinical and histologic evolution of individual lesions from junctional to compound to intradermal nevi corresponds to this cycle (Brodell

Fig. 57.14. White children living in a warm, sunny climate develop more acquired melanocytic nevi than those residing in a cooler environment. Canadian Hutterite children (who receive additional protection from sun exposure by traditional clothing covering most of their bodies) have relatively few nevi, similar to Asian and Native American children. (Data compiled from Table 57.3.)

et al., 1998; Clark et al., 1984; Greene et al., 1985; Green and Swerdlow, 1989; Lund and Stobbe, 1949; Maize and Foster, 1979; Stegmaier, 1963; Stegmaier and Montgomery, 1953) (Fig. 57.16). In white adults living in temperate climates, the mean peak number of nevi is approximately 25–35 (Bataille et al., 2000; Holly et al., 1987; Mackie et al., 1985; Rampen et al., 1986; Stegmaier and Becker, 1960); by the sixth decade, the mean number of nevi in this population decreases to <5 (Lund and Stobbe, 1949; Stegmaier, 1959). Atypical nevi have a somewhat different natural history, usually beginning to appear at puberty and, unlike banal nevi, continuing to 1115

CHAPTER 57

Fig. 57.15. Acquired melanocytic nevi increase in number during childhood and adolescence, reach a peak count in the third decade, then slowly regress with age. Although lightly pigmented children and young adults living in tropical climates tend to have more nevi than those residing in temperate zones, this “latitude gradient” reverses by late adulthood, leading some to suggest a role of sun exposure in nevus involution as well as development. (Data compiled from Table 57.4 and Bataille et al., 2000.)

develop throughout life (Barnes and Nordlund, 1987; Consensus Conference Panel, 1984; Greene et al., 1985; Halpern et al., 1993; Kopf et al., 1985; Nordlund et al., 1985; Slade et al., 1995; Tucker et al., 1983). However, atypical nevi also tend to undergo a process of involution, declining in number during mid and late adulthood (Nordlund et al., 1985; Halpern et al., 1993; Tucker et al., 2002).

Pathways of Evolution Almost 50 years ago, Stegmaier (1959) described two pathways for the evolution of melanocytic nevi: (1) the formation of soft, skin-colored papules (intradermal nevi); and (2) a gradual fading away via atrophy and/or fibrosis. More recently, Tucker et al. (2002) prospectively followed 844 members of melanoma-prone families over a 25-year period, and observed that clinically atypical nevi regressed in two major patterns, identical to those identified by Stegmaier. Likewise, Siskind et al. (2002) followed the nevi on the faces and necks of a cohort of 110 adolescents over a three-year period, and noted a dynamic process of nevus turnover. Most new nevi were small and flat (i.e., junctional), and there was a general tendency for existing flat nevi to either develop a raised profile or disappear (i.e., to follow Stegmaier’s first or second pathway). Over the study period, the proportion of raised nevi increased substantially, 16% of nevi regressed completely, and there was a 56% net increase in nevus number (Siskind et al., 2002). A number of authors have provided further documentation of the involution of banal and atypical nevi via Stegmaier’s first (Cintra et al., 1994; Clark et al., 1984; Goovaerts and Buyssens, 1988) and second (Braitman, 1958; Clark et al., 1984; Halpern et al., 1993; Shelly, 1960) pathways. 1116

In 1883, Unna proposed the theory of “Abtropfung,” the “dropping off” of nevus cells from the dermal–epidermal junction into the dermis, to explain the histogenesis of compound and intradermal melanocytic nevi. The aforementioned observations of individual lesions evolving from junctional to intradermal nevi, as well as several histopathologic series showing a decrease in the proportion of nevi with a junctional component with age (e.g., from >80% in children and adolescents to <20% in adults >50 years old), support this hypothesis (Lund and Stobbe, 1949; Maize and Foster, 1979; Stegmaier, 1959; Stegmaier and Becker, 1960; Stegmaier and Montgomery, 1953; Winkelmann and Rocha, 1962). A more recent study of >3500 nevi submitted for routine histopathologic examination found a less striking decrease in the proportion of compound nevi with age, and similar frequencies of junctional nevi among different age groups (Worret and Burgdorf, 1998). Possible explanations for these results include a preferential removal of papillomatous intradermal nevi in younger patients for cosmetic reasons, biopsy of junctional lentiginous lesions in sun-damaged skin of older adults to exclude the possibility of melanoma (Kossard, 2002), and a greater tendency to biopsy small, flat, darkly pigmented nevi in adults to exclude melanoma in situ (as compared to 40–50 years ago). The hypothesis proposed by some authors that flat and raised nevi have different histogenic origins (Schmoeckel, 1997) must be reconciled with the direct observations of nevus evolution described above (Siskind et al., 2002). That said, different pathways are likely involved in the development of certain types of nevi. The particular tendency of blue nevi, which represent benign proliferations of dendritic dermal melanocytes that actively produce melanin, to develop in sites where active dermal melanocytes are still present at the time of birth (i.e., the head and neck, dorsal aspects of the distal extremities, and presacral area) gives insight into their pathogenesis. Lastly, congenital melanocytic nevi are thought to represent hamartomas that develop due to mosaicism or a “field defect” in the neural crest-derived melanocyte precursors that populate a localized area of skin (Fig. 57.17), explaining their tendency to involve adnexae, blood vessels, nerves, and deeper structures (Happle, 1995).

Effects of Anatomic Site on Natural History The development of scalp nevi during childhood represents an early indicator of a “moley” individual, often heralding the eventual development of numerous nevi. In a recent population-based study of 8–9-year-old Swedish children (n = 1069), 7% had at least one scalp nevus, the presence of which was associated with an almost twofold higher total number of nevi than children without scalp nevi (median 14 versus 8, P < 0.001) (Synnerstad et al., 2004). Tucker et al. (1983) examined 5–19-year-old children and adolescents from melanoma-prone families, and found at least one scalp nevus in 10% of the patients overall (8/81) and 42% of the subset with histologically confirmed atypical nevi (8/19). The proportion of the latter group that had scalp nevi declined with

1117

White

Australia Australia Australia Canada Germany Canada Israel Canada Australia Scotland Australia Australia Germany

English and Armstrong, 1994 Harrison et al., 1994

Kelly et al., 1994 Hancock et al., 1996

Luther et al., 1996

Enta and Kwan, 1998 Azizi et al., 2000 Gallagher et al., 2000 Harrison et al., 2000 White White White

Hutterite White White White

White Native American White 332 974 309 115 157 211 193 1812

866

1123 57

506

2552

939 1146 446 1953

N

*Number of nevi ≥2 mm in diameter. †When there is no *, refers to total number of nevi, regardless of size. M, male; F, female.

Darlington et al., 2002 Whiteman et al., 2003a Wiecker et al., 2003

White

UK

Pope et al., 1992

White White Asian White

Switzerland Canada

Sigg and Pelloni, 1989 Gallagher et al., 1990a

Primary ethnicity

Population

Study

—

—

— — — 1* 0* — 1/2* —

6 0* — — 10/9 4/4* — — — 9* 2* — 7/8* 3*

27 5* — —

—

— —

— —

—

2–3 years

0–1 years

Table 57.3. Studies examining the number of nevi in children of different ages.

— — —

1* — — —

56 11* — 1*

14/13 1/1* 3/3*

— —

4–5 years

— 19*

1* 17/14 41 —

73 18* 34* 1*

— 7/5* 2* 24/19 2/2* 5/5*

6–7 years

28 — —

40/39 16/15* 2* — — —

54* 2*

—

17/16 9/6* 3* 37/32 4/3* 9/9*

8–9 years

— —

3* — 68 —

— 3*

—

15/9* 4* 44/38 5/4* 9/8*

10–11 years

147/113 — —

188/151 — —

5* — — —

—

— 4* 46/26 — —

97* 5*

—

16/19*

28/26* 4* —

14–15 years

80* 3*

—

11/9*

22/19 17/15* 3* —

12–13 years

Mean total number of nevi† (M/F) at ages 0–17 years

235/185 — —

— — —

—

— 5*

—

—

29/27* 5* —

16–17 years

CHAPTER 57 Table 57.4. Studies examining the number of nevi in individuals of different ages. Study

Nicholls, 1973 Reddy et al., 1976 Cooke et al., 1985 Mackie et al., 1985 Rokuhara et al., 2004

Population

Australia India New Zealand UK Japan

Ethnicity

White Indian White White Asian

N

1518 2014 872 432 600

Mean total number of nevi ≥2 mm in diameter (M/F) at ages 0–70 years 0–9 years

10–19 years

20–29 years

30–39 years

40–49 years

50–59 years

60–69 years

70+ years

— 5/5 — 3/2* 3

— 5/6 — 23/18*

15/27 6/7 17/16 33/22* 7

18/12 6/6 17/16 25/11*

5/3 6/6 12/16 22/20* 4

9/10

5/7 6/6 14/9 10/6* 4

—

11/13 16/7*

— 6/5* 3

*Number of nevi ≥3 mm in diameter. UK, United Kingdom; M, male; F, female.

B A

C

age, ranging from 100% in children <10 years old to 18% in those >14 years old. The authors concluded that scalp nevi represent an early sign of the atypical nevus syndrome, allowing affected family members to be identified well before the 1118

Fig. 57.16. Fig. (A,B,C) The three major types of acquired melanocytic nevi are junctional, compound and dermal. (A) An acral junctional nevus on the heel that is dark brown in color; note the linear striations that are characteristic of nevi in this location. (B) A compound nevus that is elevated and medium brown in color; the lesion is soft to palpation. (C) A dermal nevus that is skin-colored, elevated even more so than the compound nevus and very soft to palpation.

appearance of clinically atypical nevi in the peripubertal period (Tucker et al., 1983). Despite their role as a harbinger of future moliness, scalp nevi themselves are characterized by a tendency to involute over time. A process of gradual light-

COMMON BENIGN NEOPLASMS OF MELANOCYTES

D

A

B

C

E

Fig. 57.17. Examples of congenital melanocytic nevi. (A) Minimally elevated, medium-sized congenital nevus involving the external ear. (B) Medium-sized congenital nevus of the antecubital fossa; a folding of the surface of both congenital and acquired nevi is common in intertriginous zones; (C) A large-sized congenital nevus of the back demonstrating the hypertrichosis and variation in color seen in these nevi; (D) Satellite congenital nevi in a patient with a large-sized congenital nevus; (E) A halo medium-sized congenital nevus (late stage II) that has lost its pigmentation and has nearly disappeared.

1119

CHAPTER 57

ening and regression has been observed for various types of scalp nevi, ranging from eclipse nevi (benign acquired lesions with a tan center and stellate brown rim) to medium- and large-sized congenital melanocytic nevi (Bonifazi, 2001; Schaffer et al., 2001). In contrast to the early development and subsequent involution of nevi on the scalp, nevi on the legs have been found to reach their peak density later than those in other locations (English and Armstrong, 1994). The particular tendency of women and girls to develop nevi on the legs usually does not become manifest until adolescence or early adulthood (Gallagher et al., 1990a; Synnerstad et al., 2004). Moreover, mature raised nevi are uncommonly seen on the distal extremities, including the lower legs as well the palms and soles (Clark et al., 1984; Lund and Stobbe, 1949). Potential explanations for the relatively late onset and “arrested development” of nevi on the legs include genetic programming, hormonal influences, and sun-exposure patterns (e.g., increased sunbathing by adolescent girls).

Effects of Sun Exposure on Natural History In general, sun exposure is thought to play an important role in the natural history of nevi, with influences on nevus disappearance as well as appearance (see below). White children living in Australia develop significantly more nevi by age 3 years than those from similar ethnic backgrounds living in the United Kingdom (Harrison et al., 2000). English and Armstrong (1994) found that nevus density in Australian children reached a plateau at age 9 years, with the exception of the legs, while Gallagher et al. (1990a) showed that nevus density in Canadian children (who have fewer nevi overall) continued to rise steeply until age 15 years. Accordingly, the “latitude gradient” in number of nevi has been noted to peak near the end of the first decade, then diminish with age (see Fig. 57.15) (Kelly et al., 1994; MacLennan et al., 2003). By mid adulthood the gradient reverses, with lower numbers of nevi in Australian adults >45 years old than those living in the United Kingdom (Bataille et al., 1998a). These observations suggest that sun exposure may have a role in accelerating the process of nevus induction and subsequent involution (Armstrong et al., 1986; Augustsson et al., 1992; Harth et al., 1992; Kopf et al., 1978). However, nevi in the oral cavity also begin to regress in the fifth decade (Buchner and Hansen, 1980), implying that genetic programming is also an important factor in nevus evolution.

Genetic and Environmental Factors that Influence the Formation of Nevi Acquired melanocytic nevi are extremely common. In Caucasian populations, >97% of individuals develop at least one nevus by early childhood (Dulon et al., 2002; Gallagher et al., 2000; Green et al., 1989; Harrison et al., 1999; Luther et al., 1996; Sigg and Pelloni, 1989). However, the number of nevi on a given person can range from a few to hundreds and, as discussed above, varies with age. The prevalence of clinically atypical nevi in white populations has been reported in most 1120

studies to be between 2% and 10% (Slade et al., 1995; Sober 1993), although the prevalence of at least one nevus with architectural disorder confirmed histologically can be as high as 50% (Piepkorn et al., 1989). Both environmental and genetic factors play a role in the development of melanocytic nevi (Tables 57.5 and 57.6). Sun exposure is the primary environmental influence, while hereditary components include pigmentary phenotype as well as a particular predisposition to “moliness.”

Sun Exposure Intermittent Sun Exposure Sun exposure, especially during childhood, has a major influence on the number and location of nevi. Nevi are typically concentrated on intermittently sun-exposed areas of the trunk, the lateral upper arms, and, particularly in women, the lower extremities (Augustsson et al., 1992; Autier et al., 2001; Carli et al., 2002; Crijns et al., 1993; English and Armstrong, 1994; Gallagher et al., 1990a; Harrison et al., 1999; Krüger et al., 1992; MacKie et al., 1985; MacLennan et al., 2003; Richard et al., 1994; Synnerstad et al., 2004). The density of nevi on the extremities decreases from proximal to distal sites, also suggesting a relationship to episodic rather than chronic sun exposure (Autier et al., 2001; MacLennan et al., 2003). Several studies in children and adults have demonstrated significantly higher nevus densities on intermittently exposed areas of skin than either adjacent sun-protected areas or chronically exposed areas (Augustsson et al., 1992; Gallagher et al., 1990a; Kopf et al., 1978 and 1985; Stierner et al., 1992). Nguyen et al. (1997) analyzed the distribution of nevi on the face and neck of adolescents and found the highest density in regions receiving an intermediate level of UV radiation (UVR); this differed significantly from the distribution of actinic keratoses in adults, which favored sites of maximal UVR exposure. Gallagher et al. (1990a) noted that the differences in nevus counts between intermittently and chronically exposed sites were more pronounced in younger children than in adolescents, suggesting that episodic sun exposure during early childhood might be particularly critical to nevus development. Nevi ≥5 mm in diameter are most often found on the trunk, with a predilection for the back (Autier et al., 2001; English and Armstrong, 1994; Harrison et al., 1999; MacLennan et al., 2003; Whiteman et al., 2003a). Several authors have postulated that different exposures and responses of melanocytes to sunlight account for variation in nevus size and density depending on anatomic location (Autier et al., 2001; Green, 1992; MacLennan et al., 2003). For example, a combination of intense, episodic (“traumatizing”) sun exposure and inherent, site-specific “nevus instability” may explain the development of large nevi on the back (Green et al., 1992; Richard et al., 1994). Indeed, the presence of large nevi is associated with factors that predispose to solar injury (e.g., fair skin and residence at lower latitudes) as well as an overall, oftentimes inherited propensity to “moliness” (MacLennan et al., 2003). In contrast, habitually exposed sites such as the

COMMON BENIGN NEOPLASMS OF MELANOCYTES

dorsal hands, which tend to develop fewer, smaller nevi, may be protected by tanning, a thickened stratum corneum, and perhaps intrinsic melanocyte resistance to nevogenesis. Several studies have shown a positive correlation between nevus counts and sun exposure, particularly when intense and intermittent. In children, increased numbers of nevi have been associated with multiple or severe sunburns, frequent holidays spent in warm climates, and higher levels of routine daily sun exposure (see Table 57.5). Surprisingly, a few studies have also found a correlation between sunscreen use in children and an increased number of nevi (Autier et al., 1998; Azizi et al., 2000; Darlington et al., 2002; Dulon et al., 2002; Pope et al., 1992; Luther et al., 1996); in contrast, a negative relationship between wearing clothes and nevus count has been observed (Autier et al., 1998). Sunscreens, which act as incomplete barriers to UVR, presumably allow sun exposures of longer duration that lead to a net nevogenic effect (Autier et al., 1998). However, sunscreen use can be protective against nevus development if combined with a sensible approach to sun exposure. In one randomized controlled study, school-aged children who were supplied with and instructed to use a broad-spectrum sunscreen developed significantly fewer new nevi over a 3-year period than controls (Gallagher et al., 2000). Latitude With decreasing latitude (i.e., increased ambient UVR and warmer climate/lighter clothing), higher total numbers of nevi are seen in children (Green et al., 1988; Kelly et al., 1994; Harrison et al., 1999, 2000). The prevalence of nevi on the forearms is inversely proportional to age at arrival for immigrants to Australia, but directly proportional for immigrants to Washington state (Armstrong et al., 1986; Dennis et al., 1996b; English and Armstrong, 1988). The time course of the “latitude gradient” in nevus density and its relationship to the natural history of nevi are discussed above. Young Age of Exposure Studies of adults have more often demonstrated an association between higher nevus counts and a history of intense sun exposure prior to the age of 20 years (Breitbart et al., 1997; Briollais et al., 1996; Dennis et al., 1996a; Duffy et al., 1992; Easton et al., 1991; Garbe et al., 1994b; Kennedy et al., 2003) as compared to an increased lifetime number of sunburns or tropical vacations (Augustsson et al., 1992; Fariñas-Álvarez et al., 1999). In some of these studies, chronic occupational sun exposure was found to be inversely correlated with the total number of nevi (Augustsson et al., 1992; Fariñas-Álvarez et al., 1999; Harth et al., 1992). Considering the natural history of nevi (see above) together with such observations, several authors have postulated that habitual sun exposure hastens the maturation and eventual disappearance of nevi (Armstrong et al., 1986; Augustsson et al., 1992; Kopf et al., 1978). In some series of school-aged children and adolescents, boys were noted to have significantly more nevi than girls (Coombs et al., 1992; Darlington et al., 2002; English and Armstrong,

1994; Green et al., 1989; Pope et al., 1992; Sigg and Pelloni, 1989; Synnerstad et al., 2004). Higher levels of sun exposure during outdoor play activities likely account for this difference. In addition, nevus density on the trunk and posterior neck tends to be higher in boys and men, while nevi are often concentrated on the lower extremities in girls and women (Augustsson et al., 1992; Autier et al., 2001; Fariñas-Álvarez et al., 1999; Gallagher et al., 1990a; Harrison et al., 1999; Holly et al., 1987; Karlsson et al., 2000; Kelly et al., 1989; MacLennan et al., 2003; Stierner et al., 1992; Synnerstad et al., 2004). These distribution patterns are thought to result from gender-related differences in clothing and hairstyle. However, one study of Canadian Hutterite children showed that even with protection from sun exposure by traditional clothing covering all areas of the body except the face and hands, boys tended to develop nevi on the trunk and girls on the extremities (P < 0.05) (Enta and Kwan, 1998). Whether this reflects subtle differences in sun-exposure patterns or inherent sex-related “programming” of nevus distribution remains to be determined. Direct Effects of Sun Exposure Dermoscopic and histologic changes in existing melanocytic nevi as a result of UVR exposure have also been described. Nevi excised during the summer from sun-exposed sites have been observed to have a higher proliferation fraction and greater junctional component than those removed during the winter (Armstrong et al., 1984; Fleming et al., 1991; Holman et al., 1983). UVR exposure can lead to transient dermoscopic changes in the pigmentation and border of melanocytic nevi (Hofman-Wellenhof et al., 1997, 1998; Stanganelli et al., 1996, 1997). Tronnier et al. (1995) found enhanced immunohistochemical staining with HMB-45 and a significant increase in suprabasal melanocytes one week after nevi were exposed to a single erythemal dose of UVR, indicating melanocytic activation and cellular dyshesion. Morphologic changes and enhanced melanocyte proliferative activity were found to be greater in nevi irradiated with a single erythemal dose of UV than those given repeated suberythemal doses, supporting the importance of intermittent or intense UV exposure in the development of melanocytic neoplasms (Tronnier et al., 1997).

Other Environmental Factors Other environmental triggers for the development and/or growth of melanocytic nevi are listed in Table 57.7. Cutaneous Injury Cutaneous injury from blistering disorders can lead to the formation of nevi, ranging from eruptive nevi in areas affected by toxic epidermal necrolysis to large (e.g., palm-sized), dark nevi with irregular borders at sites of previous blistering in patients with recessively inherited forms of epidermolysis bullosa (EB), particularly generalized atrophic benign junctional EB. The pathogenesis of these nevi is likely related to increased production of cytokines and growth factors (e.g., basic fibroblast growth factor) in the microenvironment of 1121

1456 327 211

939

913

446

349 2140

2552

506

866

Sigg and Pelloni, 1989

Gallagher et al., 1990a,b

Gallagher et al., 1991

Coombs et al., 1992 Pope et al., 1992

English and Armstrong, 1994

Harrison et al., 1994 and 1999

Luther et al., 1996

N

Nicholls, 1968 Rampen et al., 1986 Green et al., 1989

Study

Germany

Australia (W)

Canada (Asian or Indian) New Zealand UK (W) UK (non–W) Australia

Canada (W)

Switzerland

Australia (W) Netherlands Australia

Population

1–6 7–11

1–6

14–15 4–11 4–11 5 14

6–18

8–12 13–16 6–18

7–17 6–18 7–11

Age (y)

10/9, 4/4† 40/39, 16/15†

27/19† 28/24, 3/3† 8/8, 1/1† 40/51*, 3/3*† 96/120*, 16/22*† 41, 9†

3†

17/16 22/19 17/16†

17/12† 30/24 28

Mean no. of nevi, M/F

–‡

–

–**

–** –**

NS

–

–

NR – –

Skin type

NS

+

NS – NS

–

+‡

NR + +

+

+‡

NR

+

+

NS

NR NR +

Fair hair

NR + +

Light skin

NR

–

–

NS –

NR

NR

NR

NR NR –

Red hair

Phenotypic features

NR NR

+ +

+‡

+

NR

NR

NR

NR

+

+ (– if heavy)

NR

NR

NR NR +

+

+

NR NR NS

Freckles

No. of nevi in parents

+‡ (– if >28 days/y)

+

NR

+ NR

NR

NR

NR

NR NR NS

Habitual

Factors associated with the number of nevi

Table 57.5. Studies examining genetic and environmental factors associated with the number of nevi in children.

NS

NR

NR

NS +

NR

NR

NR

NR NR NR

Holiday

+‡

+

NR

NS +

NS

+

NR

NR NR +

Sunburns

Sun exposure

+‡

NS

NR

NR +

NR

NR

NR

NR NR NR

Sunscreen

Density: lateral arms, face, neck, back ≥2 mm: back Density: dorsal forearms, lateral arms, face, neck (boys) ≥2 mm: trunk, upper arms, thighs/legs (girls) NR

NR NR

Density: face, neck, trunk (boys); arms (girls) NS

NR NR Number: upper back, chest NR

density of nevi

Sites with greatest number and/or

332

447 974

309

3127 111

11478 1123

193

1812

1069

Enta and Kwan, 1998

Graham et al., 1999 Azizi et al., 2000

Gallagher et al., 2000

Carli et al., 2002 Darlington et al., 2002

Dulon et al., 2002 MacLennan et al., 2003; Kelly et al., 1994

Whiteman et al., 2003a

Wiecker et al., 2003

Synnerstad et al., 2004

Sweden

Germany

Australia

Germany Australia (W)

Italy Australia

Canada (W)

Canada (Hutterite) United Kingdom Israel

Europe (W)

2 7 8–9

1–3

7 7–8 12–13 6–7 9–10 13–14 12–13 16–17 5–6 6–15

5–15

6–7

3* 19* 9/7*†

5/5

32 17/14 46/26 41* 68* 11† 143/114 240/183 12/11* 65†

3/2†

6*†

–

–**

NR

–** –** (+ for leg, dorsal hand)

–** –**

NR

– +

NS

– (<5 mm)

NR

NS

NR

NR –

+

NR

– – (+ for leg)

– NR

–

NS NR

NS

NR

NS

NR

NS NS

+ NR

+ NR NR +** (– for face)

–

NS NR

NS

NR

NS

NS +

NS

NR

NR

+

+ NS

NR

NR

NR NR

+ +

NR

+ NR NR

+ NR

+ NR

NS +

NR

NR

NS

NR

NR

+

NR

NS +

NR +

+

NR +

NR

+ (<5 mm)

*Median rather than mean. †Nevi ≥2 mm in diameter. ‡Associated with an increase in the number of nevi over time. **With the exception of patients with skin type I, who had fewer nevi. M, male; F, female; W, white; B, black; NR, not recorded; NS, not significant; herit., heritability; +, positive correlation; –, negative correlation.

649

Autier et al., 1998, 2001, 2003

NR

+

NR

+ NR

NR NS

NS

NR NR

NR

+ (<5 mm)

NR

NS

NR

+ +

+ NR

+

NR NS

NR

+ (≥5 mm)

NR

NR

NR

+ NR

NR +

–

NR +

NR

+

Density: upper trunk (boys>girls); lateral arms

NR Density: lateral arms, anterior neck, back (boys), thighs (girls) ≥5 mm: back (≠ with low latitude, fair skin, freckling) Density: face and neck (boys), upper extremities (girls) ≥5 mm: trunk NR

Density: face, neck, back Number: back, shoulder

NR

Density: face, neck (boys), trunk ≥5 mm: back Number: trunk (boys), extremities (girls) NR NR

1124

327 139 121§ 615

Rampen et al., 1986 Kelly et al., 1989

UK (twins)

900

201

Karlsson et al., 2000

30–50

19–73

21–78

20–29 30–59 30–50

18–23

18–30 20–74

Adults Adults 0–80

Age (y)

15

35

11*‡ 5*‡ 67† 113† 36/35†

30/24 36† 97† 7‡

15 6 16/11‡

M/F

Mean no. of nevi,

NS

– (≥5 mm)

NS

NS

NS

–

– NR

NR – NS

Skin type

NR NS +

+ NR +

NR

NS

NS

NR

NS

NS

NS

NS

NS

NR NR NS

+ + NR

NR

Fair hair

Light skin

NS

NS

NS

NS

NS

NR

NR NS

NR NR NS

Red hair

Phenotypic features

NR

NS

NS

(36% and 84% herit. at <45 and ≥45 y) NR

NR

(82% herit.) NR

+ NR

NR

NR NR

NR NR NR

NR

NR +

NR NR NS

Freckles

Other genetic factors

+

NR

– (occupational)

– (occupational)

+ (at age <20)

NR

NR NR

NR NR NS

Habitual

Factors associated with the number of nevi

*Median rather than mean. †Nevi ≥2 mm in diameter. ‡Nevi ≥3 mm in diameter. §Subset of patients with a personal history of melanoma (nonacral). M, male; F, female; W, white; B, black; NR, not recorded; NS, not significant; herit., heritability; +, positive correlation; –, negative correlation.

Northern Sweden

Southern Sweden Spain

379 121§ 146

Italy (men only) UK (twins)

Netherlands US (W)

US (W) US (B) UK

Origin of population

Easton et al., 1991; Duffy et al., 1992 Augustsson et al., 1991b, 1992 Fariñas–Álvarez et al., 1999 Bataille et al., 2000

90

1000 251 432

Pack et al., 1952 Coleman et al., 1980 Mackie et al., 1985

Brogelli et al., 1991

N

Study

Table 57.6. Studies examining genetic and environmental factors associated with the number of nevi in adults.

NS

NR

NS

NR

+

+

NR

NR

NR

NR

+ NS

NR

NR NR

NR NR NR

Sunscreen

NR

NR

NR NS

NR NR NR

Sunburns

NR

NR

NR

NR NR

NR NR NS

Holiday

Sun exposure

Density: back (men); lateral arms, legs (women)

NS differences in heritability based on site Density: lateral arms, back (men); thighs/legs (women) Number: arms, trunk (men); legs (women) Number: heritability of sun-protected > sun-exposed sites (62% vs 42%)

Number: trunk

Number: trunk, arms NR Number: trunk (men); lateral arms and legs (women) NR Number: trunk (men)

Sites with greatest number and/or density of nevi

COMMON BENIGN NEOPLASMS OF MELANOCYTES Table 57.7. Other triggers for the development and/or growth of melanocytic nevi. Cutaneous injury Blistering processes Toxic epidermal necrolysis/Stevens–Johnson syndrome* Epidermolysis bullosa — junctional (particularly generalized atrophic benign)>recessive dystrophic> recessive simplex* Bullae secondary to sulfur mustard gas exposure* Severe sunburn* Scarring processes Lichen sclerosus‡ Systemic immunosuppression Chemotherapy, particularly for childhood hematologic malignancies*,†,‡ Allogeneic bone marrow transplantation† Solid organ transplantation, particularly renal*†

Goerz and Tsambaos, 1978; Jullien et al., 1995; Kirby and Darley, 1978; Kopf et al., 1977; Shoji et al., 1997; Soltani et al., 1979 Annicchiarico et al., 2000; Bauer et al., 2001; Bichel et al., 2001; Grubauer et al., 1989; Hinter and Wolff, 1982; Hoss et al., 1994; Lanschuetzer et al., 2003; Soltani et al., 1984; Stavrianeas et al., 2003; Voglino and Voglino, 1998 Firooz et al., 1999 Hendricks, 1981; Krakowski et al., 1981 Carlson et al., 2002b; Carlson and Mihm, 1997; El Shabrawi-Caelen et al., 2004

Baird et al., 1992; Bogenrieder et al., 2002; de Wit et al., 1990; Green et al., 1993; Happle and Koopman, 1990; Hughes et al., 1989; Jappe et al., 1996; Karrer et al., 1998; Naldi et al., 1996 Andreani et al., 2002 Alaibac et al., 2003; Barker and MacDonald, 1988; Greene et al., 1981; Grob et al., 1996; McGregor et al., 1991; Smith et al., 1993a; Szepietowski et al., 1997 Betlloch et al., 1991; Duvic et al., 1989; Grob et al., 1996; Smith et al., 1993b

Human immunodeficiency viral infection/acquired immunodeficiency syndrome* Chronic myelocytic leukemia* Infliximab therapy

Richert et al., 1996 Katsanos et al., 2003

Increased hormone levels Pregnancy*§ Growth hormone Addison disease* Thyroid hormone*

Aloi et al., 1995; Ellis, 1991; Lee et al., 2000; Onsun et al., 1999 Bourguignon et al., 1993; Pierard et al., 1996 Ibsen and Clemmensen, 1990 Redondo et al., 1998; Tofahrn and Hundeiker, 1976

Other Atopic dermatitis in children Postoperative fever* Seizures or electroencephalographic abnormalities*

Dellavalle et al., 2004 Smith et al., 1986 Capetanakis, 1975; Wallace, 1974

*Eruptive nevi have been reported. †Nevi have a predilection for the palms and soles. ‡An increased number of atypical nevi may also be seen. §Relative immunosuppression may also play a role.

epidermal regeneration, with relative preservation and subsequent stimulation of the melanocytes in the basal layer (Lanschuetzer et al., 2003; Shoji et al., 1997). Some authors have postulated that the etiology of “EB nevi” is associated with loss of adhesion of the basal layer in the setting of a complete lack of a particular type of structural protein, leading to the proliferation and migration of “free-floating” melanocytes and incipient nevus cells (Grubauer et al., 1989; Lanschuetzer et al., 2003; Riley, 1997). Interestingly, large or eruptive nevi have not been described in association with autoimmune blistering disorders. However, darkly pigmented, irregularly shaped nevi that may mimic melanoma histologically as well as clinically have been reported to develop in association with genital lichen sclerosus (Carlson et al., 2002b; Carlson and Mihm, 1997; El Shabrawi-Caelen et al., 2004). The pathogenesis of such nevi, which exhibit histologic features

and a phenotype of melanocytic activation reminiscent of recurrent nevi, may be related to the scar-like inflammatory milieu (Carlson et al., 2002a; Shabrawi-Caelen et al., 2004). Systemic Immunosuppression Systemic immunosuppression can also lead to the development of increased number of nevi, occasionally with atypical clinical and histologic features and/or eruptive onset (Barker and MacDonald, 1988; Betloch et al., 1991; Bogenrieder et al., 2002; Green et al., 1993; Greene et al., 1981; Grob et al., 1996; Karrer et al., 1998; Richert et al., 1996). This phenomenon is seen most frequently in children and young adults, and may occur following chemotherapy for childhood malignancies, in renal transplant recipients, in human immunodeficiency virus-infected individuals, and occasionally in leukemia 1125

CHAPTER 57

patients prior to the initiation of chemotherapy (see Table 57.7). An increased propensity for nevogenesis in the setting of immunosuppression implies that an intact immune system normally has inhibitory effects on the development of nevi (Alaibac et al., 2003). Interestingly, the nevi that develop following chemotherapy or renal transplantation have a particular predilection for the palms and soles (Green et al., 1993; Happle and Koopman, 1990; Hughes et al., 1989; Naldi et al., 1996; Smith et al., 1993a; Szepietowski et al., 1997). Tissue injury related to excretion of chemotherapeutic agents via eccrine glands, which are found in higher concentrations on the palms and soles, may have a role in the etiology of these nevi in the former group. In contrast, one study found that renal transplant recipients (n = 115) had significantly fewer nevi on the anterior trunk than immunocompetent controls (n = 101) (Güleç et al., 2002). Andreani et al. (2002) showed that although leukemia patients who received allogeneic bone marrow transplantation (ABMT; n = 73) overall had more nevi on the palms and legs than controls from the general population, the subset of ABMT patients with extensive chronic cutaneous graftversus-host disease (GVHD; n = 20) had a significantly lower total number of nevi. Possible explanations for the latter observation include a specific immune reaction against melanocytes, indirect destruction of nevi due to increased cutaneous inflammation, and decreased visibility of nevi in the setting of chronic cutaneous GVHD (Andreani et al., 2002; Claudy et al., 1983). Lastly, although adults with a history of severe atopic dermatitis since childhood have been reported to have fewer nevi than controls, a diagnosis of “eczema” was recently found to be associated with higher nevus counts in children (Broberg and Augustsson, 2000; Dellavalle et al., 2004). The authors of both studies postulated a possible role of an altered inflammatory response and cytokine milieu in atopic skin, possibly accelerating the process of nevus development and subsequent elimination. Hormones Increased levels of various hormones have been associated with the development and/or enlargement of melanocytic nevi. Eruptive nevi have been described in single cases of Addison disease (Ibsen and Clemmensen, 1990) and administration of excessive thyroid hormone therapy (Tofahrn and Hundeiker, 1976). Elevated levels of growth hormone (and occasionally thyroid hormone) have been associated with enlargement of pre-existing nevi (Bourguignon et al., 1993; Redondo et al., 1998). Nevi in patients undergoing growth hormone therapy demonstrate increased immunohistochemical staining with HMB-45 (detecting gp100/Pmel-17) and MIB1 (detecting Ki-67), indicating melanocytic activation and cellular proliferation, respectively (Bourguignon et al., 1993; Pierard et al., 1996). However, administration of growth hormone does not lead to higher numbers of nevi (Wyatt, 1999; Zvulunov et al., 1997a,b). The specific effects of estrogen and progesterone on nevi have not been clearly established. Although Ellis and Wheeland (1986) showed that nevi excised from women who were 1126

pregnant or taking oral contraceptives had increased numbers of estrogen- and progesterone-binding cells, other groups have failed to find evidence of nuclear estrogen receptors in either banal or atypical nevi (Lecavalier et al., 1990). While uniform darkening of nevi may occur together with the generalized hyperpigmentation that can be seen in pregnant women, a tendency for an increase in the size, number, or atypical features of nevi in association with pregnancy has not been convincingly demonstrated. Several studies (Esteve et al., 1994; Foucar et al., 1985; Sanchez et al., 1994) found that 10–30% of pregnant women (total n = 535) reported noticing enlargement or color change in their nevi during pregnancy; however, the changes were not objectively evaluated, and no significant histologic differences were seen between lesions biopsied from these women and nonpregnant controls. Pennoyer et al. (1997) monitored melanocytic nevi located on the back in 22 pregnant women, and saw no net change in the size of the nevi between the first and third trimesters. In contrast, Ellis (1991) followed 17 women with the atypical nevus syndrome, and found an increased likelihood of changing nevi and atypical histologic features during pregnancy in this specific subset of patients. Interestingly, Lee et al. (2000) observed a banal melanocytic nevus that enlarged during pregnancy, then underwent postpartum regression accompanied by apoptosis of nevus cells. Eruptive Spitz nevi in a widespread distribution (Onsun et al., 1999) or within a speckled lentiginous nevus (Aloi et al., 1995) have also been reported to develop during pregnancy. However, in general, it is important that clinicians do not allow unfounded beliefs about “normal” changes in nevi during pregnancy to deter them from performing biopsies of suspicious lesions.

Pigmentary Phenotype Individuals with lightly pigmented skin tend to have a higher number of nevi than those with darker complexions (see Tables 57.5 and 57.6). Children and adults of African, Asian, IndoPakistani, and Native American heritage have substantially lower nevus counts than their Caucasian counterparts (Coleman et al., 1980; English and Armstrong, 1994; Gallagher et al., 1991; Hancock et al., 1996; Lewis and Johnson, 1968; Pack et al., 1963; Pope et al., 1992; Rampen and de Wit, 1989; Rokuhara et al., 2004). However, the relationship of skin type with nevus density is not linear (Fig. 57.18). Individuals with skin type I (i.e., who always burn with sun exposure and are unable to tan), especially when accompanied by red hair (i.e., the “red hair phenotype”; see below), have significantly fewer nevi than those with skin type II or III (see Tables 57.5 and 57.6). Possible explanations for this apparent paradox include a tendency of individuals with skin type I to completely avoid sun exposure (which, as discussed above, represents a major factor in the development of nevi), to have nevi that lack pigmentation and are therefore more difficult to recognize clinically, and to have an inherently decreased potential for nevogenesis as well as melanogenesis. Pigmentary phenotype can also influence the distribution of nevi. For example, individuals with darkly pigmented skin have a relative predis-

COMMON BENIGN NEOPLASMS OF MELANOCYTES

on pigmentary phenotype, likely has indirect, nonlinear influences on nevogenesis. However, a recent study failed to find a significant association between MC1R variants and the presence of >50 melanocytic nevi (Matichard et al., 2004).

Factors related to the Development of Atypical Nevi

Fig. 57.18. Although individuals with lightly pigmented skin tend to have more nevi than those with darker complexions, the relationship between skin type and nevus density is not linear. Individuals with skin type I (i.e., who always burn and are unable to tan) have significantly fewer nevi than those with skin type II or III. (Data compiled from Coombs et al., 1992; Carli et al., 2002; Darlington et al., 2002; Dulan et al., 2002; English and Armstrong, 1994; Pope et al., 1992; Wiecker et al., 2003.)

position to develop nevi on the palms and soles, a phenomenon that is not related to sun exposure (Allyn et al., 1963; Coleman et al., 1980; Jaramillo-Ayerbe and Vallejo-Contreras et al., 2004; Martin et al., 1994). Freckling represents an additional phenotypic feature that has been associated with increased numbers of nevi (see Tables 57.5 and 57.6). However, other than the well-established tendency of individuals with red hair to have fewer nevi, a consistent association between hair or eye color and number of nevi has not been found (see Tables 57.5 and 57.6). Dwyer et al. (2000) demonstrated that lower cutaneous melanin density, hair eumelanin concentration, and hair eumelanin:pheomelanin ratio were stronger predictors of increased nevus density than less objective markers of phenotype. Although human pigmentation is genetically complex, to date the only gene that has been shown to play a major role in physiologic variation in hair and skin color is that encoding the melanocortin-1 receptor (MC1R). The melanocyte MC1R has a central role in the production of photoprotective, brownblack eumelanin, and a dysfunctional MC1R favors production of photosensitizing, yellow-red pheomelanin. The MC1R gene is highly polymorphic, and approximately 50% of individuals in white populations carry at least one of the more than 65 variant alleles reported to date (Rees, 2004). Homozygous or compound heterozygous loss-of-function MC1R mutations have been shown to largely account for the red hair phenotype, which approximates an autosomal recessive trait and is associated with a paucity of nevi. On the other hand, in comparisons among individuals without red hair, those who are heterozygous for MC1R variants tend to have more lightly pigmented skin, a decreased ability to tan, freckling and a lower eumelanin:pheomelanin ratio, phenotypic characteristics associated with higher numbers of nevi. Thus MC1R status, via its effects

An increased total number of nevi is the best predictor of the presence of clinically atypical nevi (Fig. 57.19) (Augustsson et al., 1991b; Autier et al., 2001; Ballone et al., 1999; Carli et al., 1997, 1998; Cooke et al., 1989; Garbe et al., 1994b; Holly et al., 1987; Titus-Ernstoff et al., 1998). For example, in a series of 121 patients with a history of cutaneous melanoma, the mean number of atypical nevi was 2 in the subset with <25 total nevi, compared to 21 in those with >100 total nevi (Holly et al., 1987). Conversely, Cooke et al. (1989) found that individuals with >2 clinically atypical nevi were 40 times more likely to have >50 total nevi than those with no atypical nevi (P < 0.002). Another population-based study reported that the mean total number of nevi was almost doubled in patients with at least one clinically atypical nevus compared to those without atypical nevi (106 versus 58; P < 0.001) (Augustsson et al., 1991b). Like banal nevi, atypical nevi are associated with fair skin and a tendency to burn with sun exposure (Augustsson et al., 1992; Ballone et al., 1999; Bataille et al., 1998a; Kopf et al., 1988; Weinstock et al., 1991). Although atypical and banal nevi share a predilection for the trunk (Kelly et al., 1989), some studies have shown that atypical moles differ in their lack of a propensity to spare sun-protected sites such as the breasts and buttocks (Augustsson et al., 1992; Stierner et al., 1992). However, other studies have demonstrated that patients with the atypical mole syndrome and/or numerous nevi were significantly more likely to have banal-appearing, but not clinically atypical, nevi on the buttocks, suggesting a critical role for sun exposure in the pathogenesis of atypical nevi (Abadir et al., 1995; Carli et al., 1998). In support of sun exposure as an important factor in the development of atypical nevi, Bataille et al. (1998a) found that the prevalence of ≥3 atypical nevi was significantly higher in Australian adults than in English adults of similar ethnic backgrounds, but observed no significant differences between these groups in the number of banal nevi. A few studies have found that atypical nevi contained significantly higher levels of pheomelanin than banal nevi, with differences beyond that accounted for by the tendency for patients with atypical nevi to have lightly pigmented skin (Pavel et al., 2004; Salopek et al., 1991; Smit et al., 1998). The additional observation of increased concentrations of reactive oxygen species in atypical nevi (Pavel et al., 2004) could be explained by exposure of photolabile pheomelanin to UVR leading directly to the generation of free radicals and oxidative stress, or by depletion of the antioxidant molecule glutathione due to high demands for cysteine during pheomelanogenesis. Preferential pheomelanin synthesis in atypical nevi, which may reflect a deranged overall pattern of melanogenesis in this type of pigmented lesion, could itself contribute to further DNA damage. In addition, a baseline propensity for pheomelanin 1127

CHAPTER 57

B

A

C

production due to germline loss-of-function MC1R mutations, which have been shown to increase the sensitivity of melanocytes to the cytotoxic effects of UVR (Scott et al., 2002), may potentially have a role in the pathogenesis of atypical nevi.

Other Genetic Factors Several studies have demonstrated a significant positive correlation between nevus counts in parents and children, or between siblings, that was independent of pigmentary phenotype and patterns of sun exposure (Graham et al., 1999; Green et al., 1989; Wiecker et al., 2003; Zhao et al., 1992). Twin studies have also provided evidence for a strong genetic influ1128

Fig. 57.19. (A, B, C) Three examples of atypical nevi. Note the larger size, more irregular outline and greater variation in color as compared to banal compound nevi.

ence on nevus number. Based on a significantly higher degree of concordance in nevus density in monozygotic than dizygotic adolescent twin pairs from the United Kingdom and Australia, it was calculated that genetic factors account for approximately 65% and 68% of variation in nevus density in these populations, respectively (Zhu et al., 1999; Wachsmuth et al., 2001). Likewise, results of adult twin studies have suggested that nevus number is 36–84% heritable, with an increase in the relative contribution of genetic as compared to environmental factors with age (Bataille et al., 2000; Easton et al., 1991). The similarity in the estimated degree of heritability of nevus number in studies from Australia and the United

COMMON BENIGN NEOPLASMS OF MELANOCYTES

Kingdom (where individuals have similar ethnic backgrounds but markedly different levels of sun exposure and mean nevus densities), together with an observation of higher heritability in sun-exposed versus sun-protected sites of the body, suggests a threshold UV level for genetic expression that is attained in sun-exposed areas even in the United Kingdom (Wachsmuth et al., 2001; Zhu et al., 1999). In contrast, Bataille et al. (2000) found that nevus counts on sun-exposed sites in adults of all ages had lower heritability than sun-protected areas, and postulated that this may be related to a critical role for sun exposure in the involution of nevi with age (see above). Transmission of the familial atypical multiple mole and melanoma syndrome (FAMMM; i.e., families with melanoma in at least two first- or second-degree relatives in association with large numbers of melanocytic nevi, some of which are clinically and histologically atypical) is thought to have an autosomal dominant pattern with incomplete penetrance (Bale et al., 1986; Greene et al., 1983; Lynch et al., 1983). However, analyses of FAMMM kindreds that included individuals with atypical nevi but no history of melanoma have had variable results, likely related in part to inconsistent definitions and subjectivity inherent in the clinical and histological assessment of atypical nevi. Bale et al. (1986) concluded that hereditary melanoma and the atypical nevus trait result from the pleiotropic effects of a single gene, while Goldgar et al. (1991) estimated that a major gene was responsible for approximately 50% of the phenotypic variability in total nevus density in kindreds with multiple cases of melanoma and/or atypical nevus syndrome. The results of other studies have suggested that inheritance of melanocytic nevi within melanoma kindreds involves more complex (e.g., polygenic) mechanisms (Briollais et al., 1996; Risch and Sherman, 1992). More recently, several studies have analyzed the specific role of the melanoma susceptibility gene CDKN2A, located on chromosome 9p21, in nevogenesis. CDKN2A encodes p16INK4a, a tumor suppressor protein that inhibits cyclindependant kinases 4 and 6 (CDK4/6), thereby preventing inactivation of the retinoblastoma (Rb) protein and arresting progression of the cell cycle at the G1 checkpoint. Of note, the p14ARF protein, which prevents degradation of p53, is also produced from CDKN2A via an alternate reading frame (Chin, 2003). High-penetrance germline mutations in CDKN2A have been found in 25–40% of melanoma-prone families (with an additional 25% of these kindreds linked to the 9p21 locus but lacking an identifiable mutation), 5–10% of unselected familial melanoma patients in population-based studies, and 0.2–2% of sporadic melanoma patients (Aitkin et al., 1999; Chin, 2003; Platz et al., 1997). Interestingly, although somatic deletion of CDKN2A can be detected in approximately half of primary melanomas, the majority of Spitz nevi express high levels of p16INK4a, with significantly higher levels in those lesions with an increased copy number of chromosome 11p or HRAS mutation than those without 11p changes (see above) (Maldonado et al., 2004). Bastian (2004) proposed that in Spitz nevi with constitutive activation of the MAPK pathway (see Fig. 57.11), p16INK4a functions as

an essential mediator of oncogene-induced melanocyte senescence, preventing progression to melanoma. Phenotypic studies in melanoma-prone families with CDKN2A mutations have demonstrated that the atypical nevus phenotype and the total number of nevi correlate significantly with mutation carrier status (Cannon-Albright et al., 1994; Goldstein et al., 2000a; Hashemi et al., 1999; Newton Bishop et al., 2000; Wachsmuth et al., 1998). However, the nevus phenotype within these families is variable, with considerable clinical overlap between carriers and noncarriers of the mutant gene. This suggests that other genetic and/or environmental factors have important effects on nevogenesis (Wachsmuth et al., 2001). The lack of detectable differences in the proliferation status of nevi from CDKN2A mutation carriers and non-carriers may also potentially be explained by a requirement for environmental stimuli (e.g., UVR exposure or hormonal milieu) or additional genetic “hits” in order to express an activated nevus phenotype; alternatively, impaired CDKN2A activity may lead to decreased cellular senescence rather than increased proliferation (Florell et al., 2002). The observation of FAMMM kindreds in which affected individuals have multiple atypical nevi but no linkage to 9p21 provides further support for the existence of additional genes with a role in the development of nevi and melanoma (Gruis et al., 1995). For example, activating germline mutations in the CDK4 protooncogene, which to date have been reported in only three melanoma kindreds, manifest with a nevus phenotype and melanoma risk similar to that seen with CDKN2A mutations (Goldstein et al., 2000b). Although there is substantial evidence that the development of nevi is under genetic control, the gene(s) that determine nevus phenotype in the general population have not yet been identified. A few population-based studies have examined the contribution of the 9p21 locus to nevogenesis. In a study of Australian adolescent twin pairs and their siblings, Zhu et al. (1999) analyzed linkage to a highly polymorphic marker in the CDKN2A region of chromosome 9p21 (D9S942), and found that a quantitative trait locus (QTL) accounted for 27% of the variation in total nevus count. Interestingly, although total heritability was higher for raised than flat nevi (69% versus 44%), the QTL effect was limited to flat nevi, and was estimated to account for 75% of genetic variation in the number of flat nevi. Longer alleles at D9S942 were also associated with significantly higher numbers of flat nevi, but accounted for only 1% of total variation. Since germline mutations in CDKN2A are extremely uncommon, the authors concluded that variants in the noncoding region of CDKN2A or a nearby gene may represent important determinants of nevus number (Zhu et al., 1999). Recently, Barrett et al. (2003) performed a similar analysis in adolescent twin pairs from the United Kingdom and found no evidence of a QTL influencing nevus density in the CDKN2A region, although they did note a weak association between longer D9S942 alleles and higher nevus density, similar to that seen by Zhu et al. Bertram et al. (2002) found no association between the Ala148Thr missense polymorphism in CDKN2A (which is present in approximately 5% of the general popula1129

CHAPTER 57

tion) and either nevus number or history of melanoma in a population-based sample. However, a significant protective effect of this allele on the number of atypical nevi was noted in a sample of individuals with a family history of melanoma or the atypical nevus phenotype (Bertram et al., 2002). A few other genes have been analyzed for a potential role in nevus and/or melanoma susceptibility. Although BRAF is often somatically mutated in nevi and melanomas (see above), several studies have failed to find an association between germline BRAF variants (either mutations or polymorphisms) and a predisposition to melanoma or FAMMM syndrome (Casula et al., 2004; Laud et al., 2003). However, Meyer et al. (2003) reported that germline single nucleotide polymorphisms in BRAF noncoding regions were associated with melanoma in German men, but not women. Recent investigations showed that epidermal growth factor (EGF) gene polymorphisms were not significantly associated with the total number of banal or atypical nevi (James et al., 2004; Randerson-Moor et al., 2004). Lastly, the relationship between MC1R status and nevus phenotype has not been specifically studied; however, MC1R variants presumably have indirect influences on nevogenesis via their effects on cutaneous pigmentation (see above), and may confer a small risk of melanoma that is independent of pigmentary phenotype (Matichard et al., 2004). An increased number of acquired melanocytic nevi can been seen as a feature of various genodermatoses. These include Turner, Noonan, Goeminne (torticollis, keloids, cryptorchidism, and renal dysplasia), Crouzon, and Mulvill-Smith (progeroid short stature with pigmented nevi) syndromes (Gines et al., 1996; Lowenstein et al., 2004). Multiple blue nevi, particularly the epithelioid variant, are a manifestation of the Carney complex/LAMB syndrome (lentigines, atrial myxomas, mucocutaneous myxomas, blue nevi). Of note, none of these disorders has been shown to be associated with an increased risk of cutaneous melanoma.

Genetic Factors in the Development of Congenital Melanocytic Nevi Large congenital melanocytic nevi typically have a mosaic pattern of distribution, which is usually patchy and without midline demarcation (type 4; Happle, 1993). In contrast, extensive speckled lentiginous nevi, which represent a subtype of congenital melanocytic nevus, often have a mosaic configuration that is block like (“checkerboard”; type 2) or that follows Blaschko lines (type 1) (Happle, 1993). Overexpression of hepatocyte growth factor, a promoter of melanocyte proliferation and motility, and/or post-zygotic mutations in its receptor, the c-Met protooncogene, may be involved in the pathogenesis of large congenital melanocytic nevi and neurocutaneous melanosis (Kos et al., 1999; Takayama et al., 2001). Analysis of gene expression profiles in congenital melanocytic nevi may help to increase our understanding of the genetic factors involved in the pathogenesis of these lesions (Dasu et al., 2004). Although the vast majority of congenital nevi are sporadic, there are at least 16 reports of familial occurrence of large congenital melanocytic nevi (Crosti and Betti, 1994; Danarti 1130

et al., 2003; Suzuki et al., 1990). Recently, the concept of paradominant inheritance has been proposed to explain the genetic basis of congenital melanocytic nevi and other conditions (e.g., Klippel–Trenaunay syndrome and organoid nevi) that are characterized by a mosaic distribution of lesions and occurrence that is primarily sporadic, but occasionally familial (Danarti et al., 2003; Happle, 2002). In the case of congenital nevi, zygotes that are homozygous for a paradominant mutation in a putative nevogenic gene would die during early embryogenesis, while individuals who are heterozygous would be phenotypically normal; this would allow the mutant gene to be transmitted without clinical expression through many generations. The trait would only become manifest as a congenital nevus when a somatic mutation during embryogenesis resulted in loss of heterozygosity, thereby forming a population of cells homozygous for the mutation. In addition, the genetic concept of twin spotting (didymosis) has been proposed to explain the combination of a nevus flammeus plus a speckled lentiginous nevus or an organoid nevus plus a speckled lentiginous nevus in phakomatosis pigmentovascularis and phakomatosis pigmentokeratotica, respectively (Happle and Steijlen, 1989; Tadini et al., 1998). The presumption is that the nevus flammeus (or the organoid nevus) and the speckled lentiginous nevus are caused by homozygous mutations in two different genes that reside at neighboring sites on the same chromosome. If an embryo is a double heterozygote, then, following DNA replication, somatic crossing over can result in two populations of cells, each homozygous for one of two mutations (Happle and Steijlen, 1989; see Fig. 57.12 in Speckled lentiginous nevus section above).

Relationship to Melanoma Phenotypic Markers of Risk Both numerous melanocytic nevi and multiple clinically atypical nevi represent phenotypic markers that are associated with a significantly increased risk for the development of cutaneous melanoma (Table 57.8). Moreover, several studies have concluded that the total number of nevi and/or the number of clinically atypical nevi are stronger melanoma risk factors than other phenotypic features (e.g., skin type, hair color, and freckling/solar lentigines) or sun exposure history (Carli et al., 1995; Bataille et al., 1996; Garbe et al., 1994a; Grob et al., 1990; Grulich et al., 1996; Halpern et al., 1991; Landi et al., 2001; Mackie et al., 1989; Matichard et al., 2004; Naldi et al., 2000; Tucker et al., 1997; Weiss et al., 1991; Youl et al., 2002). Although multiple atypical nevi and an increased total number of melanocytic nevi tend to cosegregate as phenotypic traits (see above), not all individuals with numerous nevi have clinically atypical nevi. For example, Huynh et al. (2003) described a distinct clinical phenotype characterized by numerous (>100) small, symmetric, uniformly dark brown to black melanocytic nevi with regular borders (the “cheetah” phenotype); of the six patients reported, four had a history of multiple primary melanomas. Several studies have shown that the risk of cutaneous

COMMON BENIGN NEOPLASMS OF MELANOCYTES Table 57.8. Risk of cutaneous melanoma associated with an increased number of nevi. Study

N, patients/ controls

Total number of nevi

Total number of nevi of greater importance than atypical nevi Swerdlow et al., 1986 180/197 ≥50 Holly et al., 1987 121/139 >100 Mackie et al., 1989 280/280 ≥20 Grob et al., 1990 Weiss et al., 1991 Garbe et al., 1994a Carli et al., 1995 Grulich et al., 1996 Rodenas et al., 1997 Bataille et al., 1998a Naldi et al., 2000 Youl et al., 2002 Matichard et al., 2004

207/295 204/200 513/498 106/109 244/276 116/116 183/162 Australia 542/538 201/205 108/105

≥120 >50 >100 >30 ≥100 >50 ≥100 ≥46 >100 >50

Atypical nevi of greater importance than total number of nevi Roush et al., 1988 246/134 ≥16 Augustsson et al., 1991a 121/378 ≥150 105/181 ≥25 Halpern et al., 1991 Bataille et al., 1996 426/416 ≥100 Tucker et al., 1997 716/1014 ≥100 Bataille et al., 1998 117/163 UK ≥46 Landi et al., 2001 183/179 ≥52 Bakos et al., 2002 103/206 ≥30

Relative risk* (95% CI)

Number of atypical nevi

Relative risk† (95% CI)

12.1 (2.7–53.9) 9.8 (2.5–38.6) M: 13.9 (2.7–71) F: 6.7 (2.9–15) 13.7 (4.9–38.4) 14.9(5.8–38.4)§ 7.6 (3.5–16.2) 10.1 (3.1–22.9) 7.4 (2.9–18.6)‡ 6.9 (1.6–30.3)‡§ 12.7 (4.9–33.5)‡§ 9.5 (5.4–16.8)‡§ 46.5 (11.4–190.8)‡§ 12.7 (4.8–33.3)‡

NR ≥6 ≥3 >1 ≥3 ≥5 ≥1 ≥5 NR ≥3 NR NR NR

NR 6.3 (1.9–21.5) M: 4.5 (0.8–26) F: 4.4 (1.5–13) 2.6 (1.3–5.4) NS 6.1 (2.3–16.1) 2.4 (0.5–11.4) 2.9 (0.9–9.3)‡ NR 4.6 (2.0–10.7)‡# NR NR NR

1.2 (0.7–2.0) 2.6 (1.1–6.1) 6.5 (3.0–14.1)‡§ 3.1 (P<0.01)‡ 3.4 (2.0–5.7) 16.5 (4.5–60.3)‡§ 3.4 (1.3–8.9)‡ 2.1 (0.7–6.3)‡

≥1 ≥3 ≥1 ≥4 ≥10 ≥3 ≥1 ≥1

7.7 (3.5–17.1) 5.6 (2.5–12.5) 6.4 (3.1–13.3)‡养 14.3 (P<0.001)‡ 12 (4.4–31) 51.7 (6.5–408.4)‡# 4.2 (2.4–7.4)‡# 2.7 (1.0–7.0)‡

*Adjusted for number of atypical nevi (unless otherwise indicated). †Adjusted for total number of nevi (unless otherwise indicated). ‡Odds ratio rather than relative risk. §Not adjusted for number of atypical nevi. #Not adjusted for total number of nevi. 养Odds ratio is 8.8 when not adjusted for total number of nevi.

melanoma associated with numerous banal nevi remained significant after adjusting for other known melanoma risk factors, including atypical nevi, by multiple logistic regression analysis (Augustsson et al., 1991a; Bataille et al., 1996; Carli et al., 1995; Garbe et al., 1994a; Grob et al., 1990; Grulich et al., 1996; Holly et al., 1987; Landi et al., 2001; Mackie et al., 1989; Matichard et al., 2004; Swerdlow et al., 1986; Tucker et al., 1997). These studies demonstrated that the risk of melanoma increased continuously, often almost linearly, with rising nevus counts. Conversely, atypical nevi have also been found to represent an independent melanoma risk factor when adjustments were made for the total number of melanocytic nevi (Augustsson et al., 1991a; Bakos et al., 2002; Bataille et al., 1996; Garbe et al., 1994a; Grob et al., 1990; Halpern et al., 1991; Holly et al., 1987; Mackie et al., 1989; Rousch et al., 1988; Tucker et al., 1997). The conclusions reached by the various studies have been inconsistent as to which feature, an increased total number of nevi or the presence of atypical nevi, confers a greater risk of melanoma (see Table 57.8). However, upon analysis of all epidemiologic

studies performed to date, the total number of melanocytic nevi appears to be the most important risk factor for the development of cutaneous melanoma (Bauer and Garbe, 2003).

Relationship with Genetic Factors Earlier work suggested that the lifetime risk of developing melanoma approached 100% in patients with the FAMMM syndrome (Greene et al., 1985). More recently, germline mutations in CDKN2A have been shown to confer a lifetime risk of melanoma of 50 to >95%, with increased penetrance associated with residence at a lower latitude and the presence of loss-of-function MC1R variants (representing melanomapredisposition alleles with a low penetrance but high population frequency) (Box et al., 2001; Hansen et al., 2004; van der Velden, 2001). However, as less than half of FAMMM kindreds have an identifiable CDKN2A mutation (see above), genetic testing has a low sensitivity in predicting melanoma risk, even in this highly selected group. Interestingly, among CDKN2A mutation carriers, those with the atypical nevus phenotype and/or an increased total number of nevi have both 1131

CHAPTER 57

a significantly increased risk of developing melanoma and a lower mean age at first diagnosis of melanoma (Chaudru et al., 2004; Goldstein et al., 2000a; Hashemi et al., 1999). Possible explanations include different CDKN2A mutations, environmental influences, and modifier genes that contribute to both the atypical nevus phenotype and risk of melanoma. Nonetheless, despite the overall usefulness of nevus phenotype as a marker of risk, up to a third of melanomas in multiplex melanoma families occur in individuals without clinically atypical nevi or an increased number of nevi (Bertram et al., 2002; Newton Bishop et al., 1994). Thus, although the presence of CDKN2A mutations, numerous nevi, and multiple atypical nevi represent important indicators of melanoma risk, genetic and phenotypic heterogeneity preclude the development of a precise marker of melanoma susceptibility.

Relationship with Anatomic Site Some authors have investigated the relevance of nevi located at specific anatomic sites to the overall risk of melanoma. The number of nevi on the arms or, especially in women, the anterior thighs has been shown to predict the total body nevus count (Augustsson et al., 1992; Farinas-Alvarez et al., 1999; Loria and Matos, 2001). Accordingly, several studies have found that the number of nevi on the arms represented a significant risk factor for melanoma (Beral et al., 1983; English and Armstrong, 1988; Grob et al., 1990; Green et al., 1985; Holman and Armstrong, 1984; Loria and Matos, 2001; Österlind et al., 1988). However, the numbers of nevi on the legs (particularly in women) and trunk (particularly in men) appear to represent the best local predictors of overall melanoma risk (Krüger et al., 1992; Rieger et al., 1995; Swerdlow et al., 1986; Veierod et al., 2003; Weinstock et al., 1989). An increased number of nevi on the buttocks (Bataille et al., 1998a; Grob et al., 1990; Grulich et al., 1996), dorsal feet (Bataille et al., 1996; Grulich et al., 1996), palms and/or soles (Swerdlow et al., 1986), scalp (Bataille et al., 1996, 1998a; Grulich et al., 1996; Swerdlow et al., 1986), and iris (Bataille et al., 1998a; Grulich et al., 1996) have also been associated with a significant risk of melanoma. Lastly, Vajdic et al. (2001) reported that the presence of ≥4 nevi on the back was associated with an elevated risk of ocular melanoma. Melanomas and melanocytic nevi tend to have similar patterns of distribution, with both preferentially affecting intermittently sun-exposed sites such as the upper back in men and the legs in women (Elwood and Gallagher et al., 1998; Gallagher et al., 1990a; Krüger et al., 1992; Stierner et al., 1992). A few studies showed that the number of banal nevi on the back or legs represented a significantly stronger predictor of melanoma risk for that particular anatomic location than the total body nevus count (Grulich et al., 1996; Krüger et al., 1992; Rieger et al., 1995; Rodenas et al., 1997). Rodenas et al. (1997) found that patients with superficial spreading melanoma had higher numbers of nevi at the sites of the melanomas than age- and sex-matched controls. These observations likely reflect patterns of sun exposure (e.g., episodic and intense), and perhaps site-specific differences in 1132

melanocyte “stability,” that lead to a localized propensity to develop both nevi and melanoma (Autier et al., 2001; Chen et al., 1996; Green, 1992).

Risk of Transformation Most cutaneous melanomas arise de novo from previously normal skin, with only 20–30% developing in histologic association with a melanocytic nevus (Bevona et al., 2003; Carli et al., 1999; Gruber et al., 1989; Harley and Walsh, 1996; Kaddu et al., 2002; Krüger et al., 1992; Marks et al., 1990; Massi et al., 1999; Smolle et al., 1999; Stoltz et al., 1989; Tsao et al., 2003; Urso et al., 1991). Of note, the nevi found in association with melanomas can include banal acquired and congenital variants, with <50% representing histologically atypical nevi (Marks et al., 1990; Tsao et al., 2003). Tsao et al. (2003) recently calculated a 0.01–0.03% (1:3500– 1:10 000) lifetime risk of any selected nevus transforming into a melanoma. Estimated annual transformation rates for a single nevus ranged from <0.0005% (<1:200 000) for individuals <40 years of age to 0.003% (1:33 000) for men >60 years of age, with the age-related increase in melanoma incidence and decrease in nevus number contributing to the higher rate in the latter group (Tsao et al., 2003). Tucker et al. (2002) prospectively followed 844 members of melanoma-prone families for up to 25 years, and documented that the vast majority of clinically atypical nevi either remained stable over time or regressed. These studies provide convincing evidence that both atypical and banal melanocytic nevi primarily represent risk markers rather than precursors of melanoma, and confirm that “prophylactic” surgical eradication of melanocytic nevi is not a useful means of melanoma risk reduction (Barnes and Nordlund, 1987; Bauer and Garbe, 2004).

Relationship with Melanoma Pathogenesis Analysis of characteristic patterns of melanoma development, especially correlations between the presence of risk factors such as numerous nevi and other features such as clinicopathologic subtype, anatomic site, and patient age, has provided evidence of divergent pathways in melanoma pathogenesis (Rivers, 2004). Several studies have shown that an increased number of nevi is a risk factor for the development of superficial spreading melanoma and, to a lesser extent, nodular melanoma, but not lentigo maligna melanoma or acral melanoma (Holman and Armstrong, 1984; Marks et al., 1990; Rodenas et al., 1997; Rokuhara et al., 2004; Swerdlow et al., 1986; Weiss et al., 1991). Superficial spreading melanomas, particularly when located on the trunk, are more likely than other subtypes of melanoma to arise in or be contiguous with a pre-existing melanocytic nevus (Bevona et al., 2003; Gruber et al., 1989; Skender-Kalnenas et al., 1995). In contrast, patients with melanomas of the head and neck and/or the lentigo maligna subtype are significantly less likely than those with melanomas of the trunk to have numerous melanocytic nevi, but more likely to have multiple actinic keratoses and solar lentigines (Bataille et al., 1998b; Carli and Palli, 2003; Whiteman et al., 2003b).

COMMON BENIGN NEOPLASMS OF MELANOCYTES

Grulich et al. (1996) found that 41% of melanomas in individuals <40 years of age were statistically attributable to the presence of ≥100 nevi, compared to 5% in those ≥60 years of age. The proportion of melanomas associated with a nevus histologically likewise decreases from as high as 50% in patients <30 years old to 20% in those >60 years old (Bevona et al., 2003b; Tsao et al., 2003). Carli et al. (1999) compared patients who developed melanomas with (n = 27) versus without (n = 104) histologic association with a nevus, and found that individuals in the former group were significantly more likely to have >10 banal nevi and a history of >5 sunburns, while those in the latter group were significantly more likely to have blond or red hair. Together, all these observations support the existence of at least two different pathways to cutaneous melanoma: (1) an inherently high propensity for melanocyte proliferation, as evidenced by the development of numerous nevi, which is stimulated by intermittent sun exposure; and (2) long-term, cumulative sun exposure of lightly-pigmented skin leading to melanocyte damage and subsequent malignant transformation (Whiteman et al., 2003b; Rivers, 2004). Reports of BRAF mutations in a significantly higher proportion of banal nevi and melanomas arising on intermittently sun-exposed skin than melanomas developing on sun-damaged skin provides support for this theory on a molecular level (Maldonaldo et al., 2003).

Monitoring The presence of numerous nevi and/or multiple atypical nevi, as a phenotypic marker of increased risk for the development of cutaneous melanoma, signals the need for surveillance via periodic total body skin examination. It is important to assess individual lesions in the context of previous experience with a particular patient and the complete examination (e.g., to recognize the “signature” nevus). A nevus that has different characteristics from the other nevi in a given individual (the “ugly duckling”) should be regarded with particular suspicion (Grob and Bonerandi, 1998). The authors do not, however, recommend (as others have) the removal of two nevi simply to confirm the presence of architectural disorder. Dermoscopy, total cutaneous photography, and sequential imaging may be useful adjuncts in deciding which lesions deserve further investigation and avoiding unnecessary surgery.

Animal Models Although epidemiologic studies have provided strong evidence that sun exposure plays an important role in the pathogenesis of both melanocytic nevi and melanoma, obvious ethical considerations do not permit stringent in vivo experimentation in humans. Animal models have enabled further examination of the processes of nevogenesis and malignant transformation of human melanocytes, particularly with regard to the effects of UVR. Several animal species can spontaneously develop cutaneous melanocytic nevi. However, the nevi in most of these animals (e.g., hamsters and dogs) typically arise from dermal melanocytes, analogous to human blue

nevi (Ghadially et al., 1986; Kraft and Frese, 1976; Levine, 1980; Pawlowski and Lea, 1983). An ideal animal model would develop UVR-inducible nevi and cutaneous melanomas of epidermal derivation, with histologic features and a natural history similar to human lesions, without the need for a concomitant chemical carcinogen (Menzies et al., 2004). Accessibility of the animal to genetic and immunologic manipulation represents an additional consideration (Noonan et al., 2003). Unfortunately, most existing models do not meet these criteria (Table 57.9). In an attempt to overcome biologic obstacles and further explore the molecular pathways of nevus and melanoma development, human xenograft mouse models, genetically engineered mouse models, and human skin reconstructs have been created.

Models with Nevi and Melanomas of Dermal Origin Ley et al. (1989) found that after 40–70 weeks of UVB radiation, approximately 20% of adult South American opossums (Monodelphis domestica) developed dermal melanocytic hyperplasia/nevi and/or melanoma. In a follow-up study, 80 weeks of UVA radiation resulted in an incidence of melanocytic hyperplasia similar to that observed with UVB radiation (22% and 31%, respectively) (Ley, 1997). However, dermal nevi were seen in 10% of opossums exposed to UVB for three weeks as neonates, but only 0.01% of those exposed to UVA (Robinson et al., 1994, 2000), suggesting that UVB represented the more active waveband. In the platyfish-swordtail hybrid (Xiphophorus) model, 20–40% of the fish developed melanocytic tumors after even a single exposure to UVA or UVB radiation (Setlow et al., 1989). The melanocytic lesions that were seen differed considerably from human nevi and melanomas in histology and biology, particularly with respect to their origin from melanophores (the pigment cell of lower vertebrates) rather than melanocytes. Nonetheless, despite the considerable phylogenetic distance from humans, melanoma induction by 405 nm light in these fish was found to be linked to the Diff locus, their CDKN2A ortholog (Nairn et al., 1996). The ability to induce melanomas with UV exposure alone, without the application of chemical carcinogens, represents an advantage of the opossum and platyfish-swordtail hybrid models over rodent systems. However, the solely dermal origin of the melanocytic neoplasms that arise compromises the models, making results regarding the UV action spectrum especially difficult to generalize to humans. In addition, the strong photoreactivation system found in nonplacental animals, whereby DNA photolyase repairs UVR-induced pyrimidine dimers by using the near UV-visible component of sunlight, is not present in humans. This difference further limits the ability to draw conclusions about human disease based on these models (Menzies et al., 1998).

Models with Nevi and Melanomas of Epidermal Origin Hartley albino guinea pigs and miniature pigs can develop epidermally derived pigmented melanocytic nevi and melanomas, both of which are clinically and histologically similar to their 1133

1134

Melanophore

Melanocyte

Melanocyte

Melanocyte

Melanocyte

Human melanocyte

Melanocyte

Platyfish-swordtail hybrid

Opossum

Angora goat

Hartley albino guinea pig

MeLiM miniature pig

Human skin xenograft

HGF/SF transgenic mouse

Epidermis

Epidermis

Epidermis

Epidermis

Epidermis

Dermis

Dermis

Site of origin

No

No

No

No

No

Yes

Yes

Photoreactivation

None UVB ¥ 42 wks UVB ¥ 42 wks None UVB ¥ 6–40 wks None UVB ¥ 2–4 wks UVB+A ¥ 1 at 3 days§ UVB+A ¥ 1 at 6 wks§ UVB+A ¥ 2 at 3 days, 6 wks§ UVB ¥ 3 at 3–5 days§ UVA ¥ 3 at 3–5 days§

¥1 ¥1 ¥1 ¥1 ¥1

None None

DMBA ¥1 None DMBA ¥1 bFGF bFGF bF+ET+SC bF+ET+SC None None None

DMBA DMBA DMBA DMBA DMBA None

20% 10% 0.1% Numerous solar lentigines, some with atypical melanocytes 1.5 (mean # of nevi)† 0.3 0.2 0.7 0.4 Represented 20% of lesions biopsied; AIMP

None None None None

UVB ¥ 52 wks UVA ¥ 52 wks Visible ¥ 52 wks UVB+A+visible ¥ 52 wks None None — hereditary predisposition

10–30%

None

UVB ¥ 40–80 wks (adults) UVA ¥ 80 wks (adults) UVB ¥ 3 wks (neonates) UVA ¥ 3 wks (neonates) Routine sun exposure

Neonates Adults 16% None 68% 6% 77% 4% 66% 75% 100% 100% 86% Nd 89% Nd “Nevoid” lesions (with exposure to UVB)

ND

None None

UVB ¥ 1–20 days UVA ¥ 1–20 days

Nevi/benign melanocytic hyperplasias

Other inducers

Regimen of UV induction

# = 2.4

Adults None None None None 17%‡ 45%‡ 56%‡ # = 1

55%; mean # = 1.7 None

Neonates None None 2% None None None 34% 80%; mean None 80%; mean

50% of members of the breed; superficial spreading, nodular

40–65% with higher-dose DMBA

None * None 2%

5%

20–40% 20–40%

Melanomas

Continuum from AIMP to melanoma; tendency of melanomas to regress

Progression from junctional to intradermal nevi over time

>80% of melanomas were on the dorsal ear

Spontaneous melanomas also develop

Natural history/ other features

*Developed in 40% of the subset with nevi subsequently exposed to UVB ¥ 45 weeks. †Statistically significant increase compared with all other groups. ‡In situ lesions only. §Erythemal dose was given. UV, ultraviolet; DMBA, 7,12-dimethylbenz[a]anthracene; AIMP, atypical intraepidermal melanocytic proliferation; bFGF or bF, basic fibroblast growth factor; ET, endothelin-3; SC, stem cell factor; MeLiM, melanoblastoma-bearing Libechov minipig; HGF/SF, hepatocyte growth factor/scatter factor; wks, weeks; ND, not described.

Target cell

Animal model

Table 57.9. Animal models of nevi and melanoma.

COMMON BENIGN NEOPLASMS OF MELANOCYTES

human counterparts (Menzies et al., 1998, 2004; Pawlowski et al., 1976; Vincent-Naulleau et al., 2004). In the albino guinea pig model, amelanotic melanocytes are converted into melanin-producing “nevus cells” (Pawlowski et al., 1976). The natural history of the nevi that develop is similar to that of human nevi (albeit more rapid), with evolution of individual lesions from pigmented macules (junctional nevi) to raised papules (compound and intradermal nevi) over a four to six month period (Menzies et al., 1998). Recently, Menzies et al. (2004) reported that the active waveband for melanocytic nevus induction in the guinea pig model falls within the UVB range (see Table 57.9). Interestingly, these authors noted a significant increase in the proportion of nevi with a junctional component in animals receiving the full solar spectrum (UVB + UVA + visible) compared with all other groups. Of note, induction of melanocytic proliferation in the guinea pig model requires topical application of the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator, and the relevance of this agent to human disease is unknown (Jhappan et al., 2003). At least three breeds of miniature pigs have been reported to develop hereditary melanoma: Sinclair miniature swine, the Munich miniature swine (MMS) Troll breed, and the melanoblastoma-bearing Libechov minipigs (MeLiM) (Apiou et al., 2004; Vincent-Naulleau et al., 2004). In contrast to other animal models, swine melanoma shares histologic features with human melanoma (e.g., superficial spreading and nodular subtypes). Junctional and compound nevi with a benign clinical course have also been described in Sinclair and MMS Troll swine (Vincent-Naulleau et al., 2004). However, in MeLiM the disease spectrum differs from human melanocytic neoplasia, extending continuously from intraepidermal atypical melanocytic proliferations to melanoma, with all clinically elevated lesions representing melanomas (Vincent-Naulleau et al., 2004). Another distinctive feature of both primary and metastatic miniature pig melanomas is their marked propensity to regress spontaneously (Pathak et al., 2000; Vincent-Naulleau et al., 2004). In addition, pigmented lesions in miniature pigs have no particular pattern of anatomic distribution or apparent relationship to UV exposure (Borovansky et al., 2003; Vincent-Naulleua et al., 2004). Of note, the CDKN2A gene on swine chromosome 1q25 was recently excluded as a candidate for melanoma susceptibility, although the analysis suggested linkage with the 1q25 chromosomal region (homologous to human 9p21) (Le Chaloney et al., 2003). Further attempts to identify swine melanoma susceptibility loci are ongoing (Apiou et al., 2004; Geffrotin et al., 2004).

Human Skin Xenograft Models The animal models discussed thus far have provided useful information on nevus and melanoma biology, but the findings may not pertain to the specific genetic characteristics and cutaneous environment of human nevi and melanoma. In order to overcome these limitations, Herlyn and colleagues grafted human skin onto immunodeficient mice, thus preserving the

physiologic milieu and allowing in vivo study of the role of UVR and other factors in stimulating human melanocytic neoplasia (Berking and Herlyn, 2001). In 1998, Atillasoy et al. reported the first experimental system to yield a de novo human melanoma, providing evidence of a direct causal relationship between UVB light and melanomagenesis. Full-thickness human neonatal foreskin was grafted onto immunodeficient mice (recombinase activating gene-1 knockouts), and the grafts were exposed to UVB radiation for 10 months, a single application of DMBA, or a combination of UVB and DMBA. Melanocytic hyperplasia was found in three quarters of all UVB-treated xenografts, and a nodular melanoma developed in one graft that had been treated with the combination of DMBA and UVB (see Table 57.9). Interestingly, when the same xenograft experiments were performed using adult human skin from the trunk and face, actinic keratoses rather than melanocytic lesions were observed (Berking et al., 2002). This suggested that melanocytes from adult human skin are less susceptible to UVinduced proliferation than those in neonatal skin. However, overexpression of basic fibroblast growth factor (bFGF) via adenoviral gene transfer led to melanocytic hyperplasia in adult human skin xenografts, and additional exposure to UVB radiation resulted in the development of a severely atypical lesion resembling a lentiginous melanoma (Berking et al., 2001). Recently, a combination of bFGF, stem cell factor, and endothelin-3 together with UVB exposure was found to transform 34–45% of human skin xenografts into a melanoma phenotype within four weeks. Two-thirds of the melanomas that occurred in neonatal skin grafts were invasive; although only in situ melanomas were seen in adult skin grafts, these developed both with and without UVB exposure (see Table 57.9) (Berking et al., 2004). The authors postulated that melanocytes in young skin, perhaps due to a baseline state of increased proliferation, are overall more susceptible to exogenous tumor promoters, while adult melanocytes may have pre-existing acquired mutations that render them particularly vulnerable to tyrosine kinase receptor activation in a growth factor milieu mimicking that induced by UVR.

Transgenic Mouse Models Spontaneous melanomas are extremely rare in wild-type mice, and melanoma induction typically requires an initiator such as DMBA in addition to UVR (Jhappan et al., 2003). Because murine melanocytes are found primarily in the hair follicle rather than in the interfollicular epidermis, the nevi and melanomas that develop in such animal systems arise in the dermis and have little histologic resemblance to their human counterparts (Noonan et al., 2003). A dermal origin of melanocytic neoplasms is also seen in most transgenic mice susceptible to spontaneous or UVR-induced melanomagenesis, including, for example, those expressing Tyr-RAS on an Ink4a–/– or Arf–/– background (Kannan et al. 2003) or TyrSV40E (which, as in Ink4a–/– and Arf–/– mice, respectively, 1135

CHAPTER 57 Control

Activated Ras Activated B-RafV599E + + TERT TERT + + Dominant negative p53 Dominant negative p53 and/or and Activated CDK4 Activated CDK4

Activated Ras + TERT

p16

Ras* Raf

Rb E2F CDK4* Cyclin D

P13K

MEK

Rb

Cell survival and proliferation TERT p53 Telomerase

Raf* P P MEK E2F G1 to S transition ERK Cell survival and proliferation

Akt

ERK

Telomere maintenance

Ras

Damaged cell

Cell cycle arrest and apoptosis

Fig. 57.20. Genetic alterations sufficient to induce melanocytic neoplasia in human skin reconstructs. In the context of telomerase reverse transcriptase (TERT) expression, Ras activation alone induced junctional melanocytic hyperplasia resembling an atypical nevus. Additional inactivation of either Rb (via cyclin-dependent kinase 4 [CDK4] activation) or p53 (via a dominant negative mutation) led to invasive polyclonal melanocytic neoplasia with the features of malignant melanoma. However, B-Raf activation (via the V599E mutation commonly found in both nevi and melanomas) together with TERT expression plus both Rb and p53 inactivation induced only junctional melanocytic hyperplasia. Thus, despite its stimulation of the mitogen-activated protein kinase (MAPK) signaling cascade, B-Raf lacked the oncogenic potency of Ras, which also activates the phosphatidylinositol 3-kinase (PI3K)/AKT effector pathway. *Constitutively activated protein. MEK, MAPK kinase; ERK, extracellular signal-regulated kinase. (Based on data from Chudnovsky et al., 2005.)

results in inactivation of the Rb and p53 pathways) (KleinSzanto et al., 1994). However, in albino mice overexpressing the hepatocyte growth factor/scatter factor (HGF/SF) transgene, melanocytes are also prominently situated in the dermis and in the basal layer of the epidermis, likely related to the effects of HGF/SF in promoting melanocyte survival and proliferation as well as decreasing E-cadherin expression (Noonan et al., 2003). In adult HGF/SF-transgenic mice, spontaneous melanomas develop with increasing age, and chronic suberythemal UVR results in the formation of nonmelanoma skin cancers, but not melanoma. In contrast, a single erythemal UVR dose in neonatal HGF/SF-transgenic mice (but not in adults) was found to induce the formation of nevus-like lesions and melanomas with a junctional as well as a dermal component after a relatively short latency period (Recio et al., 2002). A second erythemal exposure as young adults increased the multiplicity of the melanocytic lesions that developed. Interestingly, a subsequent study showed that UVB, but not UVA, was effective in inducing melanoma in these transgenic mice (de Fabo et al., 2004). The molecular as well as histopathologic features of the melanomas that develop in HGF/SF transgenic mice are remarkably similar to those of human melanoma, including loss of Ink4a/Arf (Recio et al., 2002). Moreover, these results 1136

provide support for epidemiologic data implicating childhood sunburn in the pathogenesis of nevi and cutaneous melanoma (Noonan et al., 2003).

Human Skin Reconstructs with Defined Genetic Elements The creation of human skin reconstructs represents a novel approach to the study of melanocyte and melanoma biology (Berking and Herlyn, 2001). Recently, Khavari and coworkers engineered human melanocytes to express different combinations of oncogenic Ras, constitutively active CDK4 (which results in Rb inactivation), dominant negative p53 mutations, and hTERT (the catalytic subunit of human telomerase), and combined them with normal human keratinocytes to regenerate human skin on immunodeficient mice (Fig. 57.20) (Chudnovsky et al., 2004). In the context of hTERT as an accelerator, Ras expression alone induced junctional melanocytic hyperplasia resembling an atypical nevus. Additional inactivation of either the Rb or p53 pathway led to invasive polyclonal melanocytic neoplasia with the features of malignant melanoma. This model of human melanocytic neoplasia thus allows direct investigation of the contributions of specific genes to nevus and melanoma development, as well as the identification of potential therapeutic targets.

COMMON BENIGN NEOPLASMS OF MELANOCYTES

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Rare Benign Neoplasms of Melanocytes Sections Nevus Aversion Phenomenon James J. Nordlund Melanotic Neuroectodermal Tumor of Infancy Julie V. Schaffer and Jean L. Bolognia Pilar Neurocristic Hamartoma Julie V. Schaffer and Jean L. Bolognia

Nevus Aversion Phenomenon

exhaustive review of the literature on congenital nevi and their treatment.

James J. Nordlund Congenital nevi can be of any size and cover from minor areas of the integument to virtually the entire body of the infant. The latter nevi have been termed garment nevi because they literally enclose the infant in a garment of pigment cells (Figs 58.1 and 58.2). Some of the congenital nevi are smaller, a few to many centimeters in longest diameter. They can be located anywhere on the integument. This author has observed many such nevi. Some are associated with a nevus flammeus and are called nevus pigmentovascularis (Chapter 52). Many of these lesions are located on the chest or on the perineum. Of interest and note, small (see Fig. 58.1) and medium (see Fig. 58.2) sized, and garment nevi (Fig. 58.3) seem to spare the areolae and nipple although they affect all the surrounding skin. Similarly the nevus seems to spare the penis (Fig. 58.4) although the surrounding tissues are massively infiltrated with nevus cells. The significance of this aversion of congenital nevi for some areas of the integument is not known. Extensive surveys have not been done to determine if this phenomenon occurs in all or only in some individuals with congenital nevi. In addition, an infant with a large sacral spot with extra sacral blue spots had a concomitant congenital nevus over the sacrum. The two lesions seemed to have an aversion for each other and halos are present around the congenital nevi where the sacral spots are not visible (Fig. 58.5). No histologic studies have been done in these lesions. The adult woman in Fig. 58.2 illustrates that this aversion of congenital nevus cells in at least some individuals is a life-long phenomenon. The biological basis for the phenomenon is not known but presumably is not chance. No other cases illustrating this phenomenon were found in an extensive but not 1148

Melanotic Neuroectodermal Tumor of Infancy Julie V. Schaffer and Jean L. Bolognia

Historical Background In a study of adamantinomas, Krompecher (1918) described a pigment-producing tumor the size of a hazelnut in the maxilla of a 2-month-old infant. He believed that this tumor represented a melanocarcinoma that had arisen from debris in the peridental region (Zone, 1970). Borello and Gorlin (1966) suggested that a more appropriate term for this tumor was “melanotic neuroectodermal tumor of infancy” (MNTI). In the interim, however, the tumor has had a number of different names.

Synonyms Over the past 85 years, numerous synonyms for MNTI have appeared in the literature (Borello and Gorlin, 1966; Nozicka and Spacek, 1978), including congenital melanocarcinoma (Krompecher, 1918), melanotic epithelial odontome (Mummery and Pitts, 1926), melanoma (Soderberg and Padgett, 1941), retinal anlage tumor (Allen et al., 1969; Halpert and Patzer, 1947; Martin and Foote, 1951; Reyes et al., 1964; Zone, 1970), melanotic adamantinoma (Ashley, 1964; Jones and Williams, 1960; Wass, 1948), pigmented retinoblastoma (Clarke and Parsons, 1951), pigmented adamantinoma (Battle et al., 1952), pigmented congenital epulis (Henry and Bodian, 1960; MacDonald and White, 1954; Mitchell and Read, 1960; Willis, 1958), retinoblastic teratoma (Eaton and Ferguson, 1956), melanotic progonoma (Alipchenko et al., 1976; Blanc et al., 1958; de Cholnokoy, 1970; Dilu et al., 1998; Kasumova et al., 1978; Medenis et

RARE BENIGN NEOPLASMS OF MELANOCYTES

Fig. 58.1. A small congenital nevus on a 14-year-old boy. The nevus cells seem to avoid the areolae (see also Plate 58.1, pp. 494–495).

Fig. 58.3. A garment nevus. Careful inspection will show that the nevus seems to avoid both areolae (see Plate 58.3, pp. 494–495).

Fig. 58.2. A medium-sized congenital nevus on the breast of a pregnant 27-year-old woman. The cells seem to avoid the areolae (see Plate 58.2, pp. 494–495).

Fig. 58.4. A garment nevus that spares the glans and shaft of the penis (see also Plate 58.4, pp. 494–495).

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et al., 2003). The most common sites of origin are the maxilla (60%), skull (12%), brain (7%), epididymis/paratesticular area (7%), and mandible (6%); (Table 58.1). Overall, at least 90% of the cases occurred in children under the age of 1 year, and no sex predilection was found (Cutler et al., 1981; Mosby et al., 1992). However, only 30% of the cases originating in the central nervous system (CNS; n = 26) occurred before the age of 1 year, with a median age at diagnosis of 3.5 years; in this subset of patients, the male : female ratio was 3 : 1 (Gorhan et al., 2001; Rickert et al., 1998).

Clinical Description

Fig. 58.5. Congenital nevus over a sacral spot. There are clear halos around the congenital nevus (see also Plate 58.5, pp. 494–495).

al., 1962; Misugi et al., 1965; Neustein, 1967; Porter and Cummings, 1963; Rickert et al., 1998; Stowens, 1957; Vajtai et al., 2000), pigmented ameloblastoma (Kerr and Weiss, 1963), melanotic ameloblastic odontoma (Duckworth and Seward, 1965); melanotic tumor of the infantile jaw (Mårtensson et al., 1965), melanotic ameloblastoma (Adeloye et al., 1977; Pontius et al., 1965; Williams, 1967), melanocytoma (Stokke, 1968), pigmented tumor of the jaw of infants (Hayward et al., 1969), pigmented teratoma (Bolande, 1974), retinal choristoma (Bolande, 1974), melanotic anlage tumor (Bolande, 1974), atypical melanoblastoma (Bolande, 1974), and melanogenic neuroectodermal tumor (Freitag et al., 1997).

Epidemiology/Clinical Findings To date, approximately 400 cases of MNTI have been reported in the literature (Abdin et al., 1982; Adad et al., 2004; Alipchenko et al., 1976; Al-Marzooq et al., 2003; Atanackovic et al., 1986; Barrett et al., 2002; Bouckaert and Raubenheimer, 1998; Caird et al., 2000; Carnevale and Mortelliti, 2001; Dashti et al., 1999; de Cholnokoy, 1970; Deminiere et al., 2002; de Souza et al., 1999; de Vita et al., 1983; Dilu et al., 1998; Dourov et al., 1987; Eckardt et al., 2001; el-Saggan et al., 1998; Franchi et al., 2002; Freitag et al., 1997; Galera-Ruiz et al., 1999; Gomez et al., 1996; Gorhan et al., 2001; Gupta et al., 2002; Hall et al., 1979, Haider et al., 2003; Handley et al., 1988; Hoeffel et al., 1998; Hoshina et al., 2000; Howell and Cohen, 1996; Kacker et al., 1993; Kalra et al., 2001; Kasumova et al., 1978; Kaya et al., 2000; Khoddami et al., 1998; Kim et al., 1996; Kirtane et al., 1984; Klaeren et al., 1999; Klingenberg et al., 1998; Kobayashi et al., 1996; Kraus et al., 2002; Mast et al., 1999; Mello et al., 2000; Morera Martinez et al., 1984; Nishio et al., 1999; Patankar et al., 1998; Puchalski et al., 2000; Paueksakon et al., 2002; Raila et al., 1997; Ramon et al., 1979; Rege et al., 1999; Rickert et al., 1998; Ricketts et al., 1985; Rustemeyer et al., 2001; Shaia et al., 2002; Sidhu et al., 1981; Toda et al., 1998; Vajtai et al., 2000; Woessmann

1150

The tumor usually presents as a firm, fairly well-circumscribed mass (Atkinson et al., 1989). Its surface may be lobulated or smooth (Irving et al., 1993; Wass, 1948), and occasionally it contains areas that are cystic and soft (Mårtensson et al., 1965). In addition, the mass may be adherent to deeper structures (Ashley, 1964; Borello and Gorlin, 1966). A history of rapid growth is characteristic of MNTI (Kapadia et al., 1993; Pierre-Kahn et al., 1992). Although these lesions usually range from 1 cm to 4 cm in diameter (Morin et al., 1992; Pettinato et al., 1991), they may attain a size as large as 18 cm, referred to as a “gigantiform variant” (Bouckaert et al., 1998). It usually presents as a single lesion; however, there have been reports of a multicentric distribution (Jones and Williams, 1960; Pontius et al., 1965; Steinberg et al., 1988). Because the majority of these tumors appear during infancy and the most common location is the maxilla, a classic presentation is difficulty in feeding due to an intraoral mass (Mosby et al., 1992; Patel and Balar, 1991). On oral examination, a mass arising from the maxilla, or less often the mandible, is seen; the overlying mucosa may be normal in appearance (Allen et al., 1969; Irving et al., 1993; Wass, 1948) or it can be gray to blue-black in color (Demas et al., 1992; Eckardt et al., 2001; Jones and Williams, 1960; Kim et al., 1996; Koudstaal et al., 1968; Puchalski et al., 2000; Shokry et al., 1986; Soderberg and Padgett, 1941; Steinberg et al., 1988). Occasionally, the swelling has a deep red to red-blue hue (Anil et al., 1993; Borello and Gorlin, 1966; Kerr and Weiss, 1963), and displaced teeth may be attached to its surface (Eckardt et al., 2001; Handley and Peters, 1988; Hayward et al., 1969). When the intraoral lesions are large, they can cause disfigurement of the face, most commonly distortion of the upper lip (Henry and Bodian, 1960; Irving et al., 1993; Mosby et al., 1992; Steinberg et al., 1988). In addition, tumors originating in the maxilla may extend upward to invade the maxillary sinus and orbit, occasionally involving the optic nerve (el-Saggan et al., 1998; Mello et al., 2000; Shaia et al., 2002; Woessman et al., 2003). Less frequently, a firm, noninflammatory subcutaneous swelling is noted on the face or scalp that is a reflection of a tumor that has arisen in the underlying bone (Ashley, 1964; Clarke and Parsons, 1951; Yu et al., 1992). Usually, the overlying skin is normal and nonadherent (Atkinson et al., 1989; Haider et al., 2003; Jones et al., 1990; Koudstaal et al., 1968;

RARE BENIGN NEOPLASMS OF MELANOCYTES Table 58.1. Anatomic location of MNTIs reported in the literature (n = 400). Location

Through 1979a

1980–89b

1990–99c

2000–04d

Total (%e)

Maxilla Skull Brain Epididymis/paratesticular/testes Mandible Soft tissuesg Orbit Dura Ovary Mediastinum Maxillary sinus Oropharynx Uterus Scapula Femur Metatarsal Forehead Retina Unknowni

99e,f 19 14 6 9 2 1

37 7 8 7 5 2 1 1

58 13 3 11 7 1

19 3 1 1 2 1 2

213 (60) 42 (12) 26 (7) 25 (7) 23 (6) 6 (2) 4 (1) 4 (1) 4 (1) 2 2 1 1 1 1 1 1 1 42

3

3h 2 1 1 1f 1f

1 1

1 1 1 1 24

8

10

a

From de Cholnoky (1970), Alipchenko et al. (1976), Kasumova et al. (1978), Hall et al. (1979), Ramon et al. (1979), Cutler et al. (1981), Carpenter et al. (1985), and Diamond et al. (1992). b From Sidhu et al. (1981), Abdin et al. (1982), de Vita et al. (1983), Kirtane et al. (1984), Morera et al. (1984), Atanackovic et al. (1986), Diamond et al. (1992), and Mosby et al. (1992). c From Judd et al. (1990), Claros et al. (1990), Chossegros et al. (1990), Rao et al. (1990), Jones et al. (1990), Sondule and Bhowate (1990), Pettinato et al. (1991), Yacobi (1991), Milrich et al. (1991), Siracusano et al (1991), Cheung et al. (1991), Aryya et al. (1991), Patel and Balar (1991), Machado de Sousa et al. (1992), Diamond et al. (1992), Yu et al. (1992), Slootweg (1992), Mosby et al. (1992), Demas et al. (1992), Morin et al. (1992), Greschnoik et al. (1992), Anagnostake et al. (1992), Pierre-Kahn et al. (1992), deChiara et al. (1992), Kapadia et al. (1993), Irving et al. (1993), Anil et al. (1993), Kacker et al. (1993), Shah et al. (1994), Kanugo and Chandi (1994), Jurincic-Winkler et al. (1994), Hoshino et al. (1994), Dammann et al. (1995), Nitta et al. (1995), Raziq et al. (1995), Calabrese et al. (1995), George et al. (1995), Gomez et al. (1996), Howell and Cohen (1996), Kim et al. (1996), Koybayashi et al. (1996), Moriuchi et al. (1996), Freitag et al. (1997), Raila et al. (1997), Bouckaert and Raubenheimer (1998), Dilu et al. (1998), el-Saggan et al. (1998), Hoeffel et al. (1998), Khoddami et al. (1998), Klingenberg et al. (1998), Kurabayashi et al. (1998), Patankar et al. (1998), Richert et al. (1998), Toda et al. (1998), Dashti et al. (1999), de Souza et al. (1999), Galera-Ruiz et al. (1999), Klaeren et al. (1999), Mast et al. (1999), Nishio et al. (1999), and Rege et al. (1999). d Caird et al. (2000), Hoshina et al. (2000), Kaya et al. (2000), Mello et al. (2000), Puchalski et al. (2000), Vajtai et al. (2000), Carnevale and Mortelliti (2001), Eckardt et al. (2001), Gorhan et al. (2001), Kalra et al. (2001), Rustemeyer et al. (2001), Barrett et al. (2002), Deminiere et al. (2002), Franchi et al. (2002), Gupta et al. (2002), Paueksakon et al. (2002), Shaia et al. (2002), Al-Marzooq et al. (2003), Haider et al. (2003), Woessmann et al. (2003), and Adad et al. (2004). e Denominator is total number of cases for which the anatomic location is known. f Excluded by Borello and Gorlin (1966), including one case from the maxilla; Kerr and Weiss (1963) questioned if the uterine tumor was a malignant teratoma. g Anatomic sites included the face (2 cases), shoulder/upper arm (2 cases), and thigh (2 cases). h Two diagnoses were questioned by Carpenter et al. (1985). i Some original articles were not available for review.

Yu et al., 1992), but a few patients have had associated cutaneous findings, in particular a dusky color or prominent dilated blood vessels (Adeloye et al., 1977; Clarke and Parsons, 1951; Patankar et al., 1998). In one case, recurrent tumor was heralded by facial swelling and a bluish discoloration (Dehner et al., 1979). There are also scattered reports of MNTI originating in the soft tissues of the cheek (Jimenez et al., 1981; Pettinato et al., 1991), shoulder (Blanc et al.,

1958), upper arm (Al-Marzooq et al., 2003), and thigh (Scheck et al., 1989; Vlachos and Papadimitriou, 1969). The overlying skin may be normal in appearance (Scheck et al., 1989; Vlachos and Papadimitriou, 1969), slightly bluish in color (Blanc et al., 1958), or tense with congested vessels (Jimenez et al., 1981) (Fig. 58.6). At the time of surgical excision, the presence of pigment within the tumor suggests the diagnosis of MNTI. Although

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Fig. 58.6. A large, soft tumor mass of the right cheek in a 6-monthold child with MNTI and fetal hydantoin syndrome. The overlying skin was tense with prominent conjested vessels. Note the epicanthal folds, flat nasal bridge, and mild hypertelorism. (From Jimenez et al. Melanotic neuroectodermal tumor of infancy and fetal hydantoin syndrome. Am. J. Pediatr. Hematol. Oncol. 3:9–15, 1981, with permission of the publisher.)

the reasons for the recurrence rate of at least 15% (see Prognosis below). The form of MNTI that arises in the CNS has a particularly aggressive course (Hahn et al., 1976; Rickert et al., 1998; Yu et al., 1992). Patients may present with focal neurologic deficits (Mirich et al., 1991) and increased head circumference due to hydrocephalus as well as symptoms secondary to increased intracranial pressure, such as headaches, nausea, and vomiting (Hahn et al., 1976). The 26 cases of primary CNS MNTI reported to date were all located in or near the midline, most often involving the cerebellum (n = 16; particularly the vermis) or the pineal region (n = 6) (Gorhan et al., 2001; Rickert et al., 1998). Approximately a third of the tumors had spread to additional sites within the CNS, including the meninges and spinal cord, by the time of surgery; three-quarters of the patients died as a result of the tumor, with a mean postoperative survival time of 9 months (Rickert et al., 1998). Approximately 3% of MNTIs that arise at sites other than the CNS (e.g., maxilla, mandible, femur, and epididymis/ paratesticular area) exhibit malignant behavior. Metastases to regional lymph nodes, soft tissue, and visceral organs, such as the liver, bone, and adrenal glands, has been observed (Cutler et al., 1981; De Chiara et al., 1992; Dehner et al., 1979; Johnson et al., 1982; Lindahl, 1970; Mosby et al., 1992; Navas-Palacios, 1980; Pettinato et al., 1991). Metastases were composed of neuroblastlike cells, but the histologic features of the primary tumors were similar to those of nonmetastatic tumors [(Carpenter et al., 1985); see Histology below].

Associated Disorders There is one report of MNTI in a child with fetal hydantoin syndrome (Jimenez et al., 1981). The authors point to the association of neuroblastomas and ganglioneuroblastomas with fetal hydantoin syndrome as evidence of a possible oncogenic effect for hydantoin compounds.

Histology Fig. 58.7. In this gross specimen of MNTI, the cut surface exhibits irregular black pigmentation in a white fibrous stroma. Note the greater amount of melanin in the center of the tumor compared to its periphery. (From Jimenez et al. Melanotic neuroectodermal tumor of infancy and fetal hydantoin syndrome. Am. J. Pediatr. Hematol. Oncol. 3:9–15, 1981, with permission of the publisher.)

its outer surface may be white and fibrous, sectioning of the tumor reveals multiple pigmented lobules (Williams, 1967); the pigmentation can range from blue to gray to black (Carpenter et al., 1985; Pettinato et al., 1991) (Fig. 58.7). Surgical excision is easier when the tumor is surrounded by a capsule (Wass, 1948) and there has been compression rather than infiltration of adjacent structures. However, there are cases where “tongues” of tumor invade the surrounding bone (Koudstaal et al., 1968; Mummery and Pitts, 1926), and incomplete removal of these islands of tumor may be one of 1152

In addition to fibroblasts, two characteristic cell types have been described: (1) small, lymphocytelike cells that are round to oval in shape with a darkly staining nucleus and a small rim of cytoplasm (Carpenter et al., 1985); these cells can be densely packed or loosely arranged, are often surrounded by a fibrillary stroma, and have been referred to as neuroblastlike (Borello and Gorlin, 1966); and (2) larger cells that are cuboidal or polygonal in shape with an abundant cytoplasm which often contains melanin (positive Fontana-Masson stain); these cells are found primarily in the center of the tumor, are arranged around clefts forming glandular and alveolar structures, and have been described as epithelioid (Carpenter et al., 1985). On fine-needle aspiration smears, the two major types of cells can be recognized and the diagnosis of MNTI confirmed (Adad et al., 2004; Al-Marzooq et al., 2003; Rao et al., 1990; Rege et al., 1999; Toda et al., 1998). By electron microscopy, there are also three different cell types (Carpenter et al., 1985; Neustein, 1967; Nikai et al., 1977; Scheck et al., 1989): (1) undifferentiated cells, some of which

RARE BENIGN NEOPLASMS OF MELANOCYTES

contain a few electron-dense neurosecretory granules as well as microtubules within neurite-like cytoplasmic processes, features that reflect a neuroblastic origin; (2) larger, melanin-containing cells with premelanosomes and melanosomes that also contain intermediate filaments (8–10 nm) as evidence of their epithelial origin; some authors have observed desmosomes between these cells (Carpenter et al., 1985; Claman et al., 1991; Hayward et al., 1969), though others have not seen such structures (Cutler et al., 1981); and (3) stromal fibroblasts. According to Nikai et al. (1977), the large melanin-producing cells have characteristics of a neuroepithelium, whereas the small cells that lack pigment are similar to immature neuroblasts. In addition to these three types of cells, some tumors have exhibited bone formation within their stroma (Slootweg, 1992; Williams, 1967) and areas of myoid differentiation (Anagnostaki et al., 1992; Rao et al., 1990). The MNTIs that arise from the maxilla and mandible can contain displaced deciduous teeth (Kim et al., 1996; Barretteta et al., 2002; Williams, 1967). Although mitoses and necrosis are typically rare to absent (Kapadia et al., 1993), an aggressive MNTI with a higher mitotic rate has been described (Barrett et al., 2002), and there is one report of spindle cells exhibiting mitotic activity admixed with the classic cell types (Slootweg, 1992). The tumor can infiltrate adjacent tissues, such as skeletal muscle and bone (Barrett et al., 2002; Pettinato et al., 1991; Pierre-Kahn et al., 1992). Immunohistochemical studies have shown positive staining for neuron-specific enolase in both major cell types (Barrett et al., 2002; Calabrese et al., 1995; De Chiara et al., 1992; Hoshino et al., 1994; Machado de Sousa et al., 1992; Pettinato et al., 1991; Slootweg, 1992). The large melanincontaining cells typically stain positively for cytokeratin, vimentin, and HMB-45 (Anagnostaki et al., 1992; Barrett et al., 2002; Calabrese et al., 1995; Kapadia et al., 1993); however, staining for S-100 is usually negative (Machado de Sousa et al., 1992; Pettinato et al., 1991). For example, in 3 series of patients where 29 tumors were examined, only 4 stained positively for S-100 (Barrett et al., 2002; Kapadia et al., 1993; Pettinato et al., 1991). In these series, expression of the following proteins was noted in small and/or large cells: synaptophysin (16/29), Leu 7 (13/21), glial fibrillary acidic protein (6/21), chromogranin (3/29), and epithelial membrane antigen (7/19; large cells only) (Barrett et al., 2002; Kapadia et al., 1993; Pettinato et al., 1991). Lastly, Barrett et al. (2002) found that PGP 9.5, a neuroendocrine marker, was often present (6/8; primarily in large cells) and NB84, a marker of childhood neuroblastoma, was absent in the eight cases studied; the single aggressive tumor that had higher mitotic activity expressed CD99, a marker of peripheral neuroectodermal tumors, as well as Ki-67. One explanation for the variable pattern of staining is the immaturity of the tumor cells (Machado de Sousa et al., 1992). In malignant MNTI, the metastases have a monomorphic histologic appearance and are composed of neuroblastlike cells (Dehner et al., 1979; Johnson et al., 1982; Shokry et al., 1986). However, when the primary tumors were examined, they did not differ significantly from MNTIs that did not

metastasize (Carpenter et al., 1985); i.e., there was no suggestion of malignancy. Rarely, foci of pseudoglandular structures composed of melanin-containing cells have been observed within lymph nodes (De Chiara et al., 1992; Dehner et al., 1979). Interestingly, in one case where neoadjuvant chemotherapy was successful in reducing tumor volume (see Treatment, below), comparison of the subsequent excisional specimen to the initial biopsy revealed a marked reduction in the neuroblastlike cell component but persistence of the pigmented cell component (Mello et al., 2000).

Laboratory Findings and Investigations In plain radiographs of involved bones, cystic and lytic lesions often are seen (Allen et al., 1969; Batsakis, 1987; Dehner et al., 1979) and adjacent bone may be sclerotic (Atkinson et al., 1989; Khoddami et al., 1998; Nishio et al., 1999). If the MNTI involves the maxilla or mandible, displaced deciduous teeth can be seen within the tumor, resulting in “floating” teeth (Demas et al., 1992; Hoshina et al., 2000; Koudstaal et al., 1968; Williams, 1967). Computed tomography (CT) also is used to define the extent of involvement and bony infiltration (Cheung et al., 1991; Irving et al., 1993). It was anticipated that, because MNTI are melanincontaining tumors, by magnetic resonance (MR) scanning they would be hyperintense relative to brain on T1-weighted images and relatively hypointense on T2-weighted images (Batsakis, 1987; George et al., 1995). This characteristic pattern has been reported by some authors, especially in the central portion of the tumor, where the greatest concentration of melanin is seen (George et al., 1995; Mirich et al., 1991). However, there have been reports of the tumor being isointense on T2-weighted images (Atkinson et al., 1989), in particular the peripheral portion (George et al., 1995), as well as tumors that were hypo- and isointense on both T1- and T2weighted images (Hoshino et al., 1994; Nishio et al., 1999; Toda et al., 1998; Klingenberg et al., 1998) or slightly hyperintense on T2-weighted images (Mirich et al., 1991). The prediction that one would see increased signal on T1 images was based on the paramagnetic effect of melanin (George et al., 1995). Melanin can act as a free-radical trap that chelates paramagnetic metal ions. The presence of unpaired electrons results in enhanced proton relaxation and increased T1 signal (Atkinson et al., 1989). The T2 image is related to magnetic field inhomogeneity; the presence of melanin leads to a rapid T2 decay and low signal intensity (George et al., 1995). The inconsistency of the findings by MR imaging has been explained by variability in the histology and amount of melanin (George et al., 1995). Borello and Gorlin (1966) first reported an elevated level of vanilmandelic acid (3-methoxy, 4-hydroxymandelic acid; VMA) in the urine of a patient with MNTI. When the tumor was removed, the levels returned to normal. Because increased urinary VMA has been described in other tumors of neural crest origin, including retinoblastoma, ganglioneuroblastoma, neuroblastoma, ganglioneuroma, and pheochromocytoma, this finding was used to support the theory that MNTI was 1153

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of neural crest origin (Borello and Gorlin, 1966). Brekke and Gorlin (1975) confirmed this observation when they found increased levels of urinary VMA in the immediate postoperative period in a patient with MNTI, which subsequently returned to normal. However, in a review of the literature from 1980 to 1990 (Mosby et al., 1992), only one of nine patients tested had increased urinary VMA; in 22 patients evaluated since 1990 the urinary VMA was elevated in five (Anil et al., 1993; Chossegros et al., 1995; Claros et al., 1990; Dammann et al., 1995; De Chiara et al., 1992; el-Saggan et al., 1998; Haider et al., 2003; Hoshina et al., 2000; Hoshino et al., 1994; Howell et al., 1996; Irving et al., 1993; Kaya et al., 2000; Kim et al., 1996; Klingenberg et al., 1998; Mirich et al., 1991; Nishio et al., 1999; Pierre-Kahn et al., 1992; Rao et al., 1990). There are also a few reports of elevated serum levels of a-fetoprotein in patients with MNTI (Dourov et al., 1987; Klingenberg et al., 1998; Ricketts and Majmudarr, 1985). Nitta et al. (1995) suggested that expression of mRNA for melanotransferrin within the tumor also supported the theory that MNTIs are derived from neural crest cells. In addition, cell culture studies have shown that the neuroblastic cells have tyrosinase activity, and following exposure to dibutyryl cyclic adenosine 3¢,5¢-monophosphate (cAMP), increases in pigmentation and dendricity were seen in the melanotic cells (Dehner et al., 1979), effects similar to those observed with melanocytes. de Souza et al. (1999) found that only the large, melanin-containing cells of MNTIs expressed the MDM-2 protein (a protooncogene product that inhibits the regulatory function of p53), proliferating cell nuclear antigen, cyclin D1, and cyclin A; they postulated that these cells rather than the small neuroblastlike cells represent the proliferative component of the tumor. In support of this contention, a predominance of melanin-containing cells in cultures from MNTI has been reported (Claman et al., 1991), and a cell line established from a MNTI (ONS-99) was found to have melanotic differentiation, with positivity for HMB45 as well as neuron-specific enolase and formation of desmosomes (Moriuchi et al., 1996).

Criteria for Diagnosis On gross examination, the diagnosis of MNTI is suggested by the presence of melanin in the tumor (see Fig. 58.7). Histologically, the two major cell types described above should be present to confirm the diagnosis of MNTI.

Differential Diagnosis Clinically, intraoral lesions may be confused with benign odontogenic lesions, such as an eruption cyst or congenital epulis as well as malignancies, such as osteogenic sarcoma (Cheung et al., 1991). The rare pigmented intraosseous odontogenic carcinoma can mimic MNTI both clinically and histologically, but differs in the presence of squamous differentiation and the absence of neuroblastic elements (Ijiri et al., 2001). Histologically, MNTI must also be distinguished from other neural and neuroectodermal tumors, in particular neuroblastoma, olfactory neuroblastoma, ganglioneuroblastoma, and peripheral neuroepithelioma (Kapadia et al., 1993; 1154

Scheck et al., 1989). Although the lack of rosette or pseudorosette formation as well as solid sheets of primitive cells helps to exclude neuroblastoma and peripheral neuroepithelioma (Scheck et al., 1989), melanin-containing cells have been observed in some cases of olfactory neuroblastoma, ganglioneuroblastoma, and melanotic paraganglioma. In addition, MNTI may be confused with rhabdomyosarcoma (Diamond et al., 1992; Kapadia et al., 1993; Rao et al., 1990; Scheck et al., 1989). The differential diagnosis of primary CNS MNTI includes meningioma, neurofibroma, ependymoma, and medulloblastoma (Borello and Gorlin, 1966; Navas-Palacios, 1980; Yu et al., 1992). Although some authors have preferred the terms “pigmented papillary medulloblastoma,” “medulloblastomalike tumor,” and “melanotic or pigmented medulloblastoma,” Hahn et al. (1976) have argued in favor of the name MNTI because of the similarities in histologic features between the CNS and the extra-CNS tumors, in addition to their sites of origin. For example, there are reports of these tumors arising in the pineal gland or entirely within the third ventricle, unlikely sites of origin for medulloblastoma.

Pathogenesis A number of theories regarding the origin of this tumor have been advanced (Borello and Gorlin, 1966), including an early proposal that it represented a congenital melanoma (Krompecher, 1918; Soderberg and Padgett, 1941). However, the benign histologic and biologic behavior of the vast majority of these tumors is against this theory. A second proposal was that MNTI was of odontogenic origin because of its proximity histologically to proliferating odontogenic epithelium (Battle et al., 1952; Jones and Williams, 1960; Kerr and Weiss, 1963; Mitchell and Read, 1960; Mummery and Pitts, 1926; Pontius et al., 1965; Wass, 1948; Williams, 1967; Willis, 1958). This belief was reflected in several of the previous names for MNTI, including epithelial odontome, adamantinoma, ameloblastoma, and pigmented congenital epulis (epulis = tumor on the gum). Although the majority of these tumors arise in the maxilla or mandible (sites of tooth development), in extramaxillary locations this odontogenic epithelium has not been observed (Borello and Gorlin, 1966). The retinal anlage theory of ectopic foci of retinal cells in the maxilla arose as a result of the histologic similarities between the tumor cells and the cells in the primitive optic cup and retina (Clarke and Parsons, 1951; Halpert and Patzer, 1947; Martin and Foote, 1951). This idea received little support due to several observations: the retina is well organized by the time the maxilla is beginning to form and none of the patients with MNTI have had associated eye abnormalities (Borello and Gorlin, 1966; Stowens, 1957). The name “melanotic progonoma” (pro = before, gonos = germ) arose from the theory of atavism and the belief that these tumors arose from sensory neuroectoderm that was related to a transiently existing embryonic structure, in this case the vomeronasal organs of Jacobson (Medenis et al., 1962; Stowens, 1957). Neither this theory nor the proposals that

RARE BENIGN NEOPLASMS OF MELANOCYTES

MNTI is a form of teratoma (Eaton and Ferguson, 1956; Soderberg and Padgett, 1941) or is related to the fetal pineal gland (Dooling et al., 1977) are accepted currently. Today, there is a general consensus that MNTIs are of neural crest origin (Borello and Gorlin, 1966; Dehner et al., 1979; Misugi et al., 1965; Neustein, 1967; Nikai et al., 1977). This proposal is based on several observations, including ultrastructural findings, histochemical studies, and biochemical assays (e.g., tyrosinase activity). As discussed under Histology above, the pigmented cells contain premelanosomes and melanosomes and the neuroblastic cells contain neurosecretory granules (Carpenter et al., 1985); by immunohistochemistry, there is positive staining for neuron-specific enolase in both major cell types (Calabrese et al., 1995; De Chiara et al., 1992; Hoshino et al., 1994; Mosby et al., 1992). However, although the MNTI shares some features with other small cell neuroectodermal tumors of childhood (e.g., neuroblastoma, peripheral primitive neuroectodermal tumor, and desmoplastic small round cell tumor), Khoddami et al. (1998) did not find any of the molecular genetic changes that characterize the latter (e.g., MYCN gene amplification, deletions in chromosome 1p, and translocations involving chromosomes 11 and 22) in three classic cases of MNTI.

Treatment Complete surgical excision or enucleation is the recommended treatment for MNTI. The operating microscope may be helpful in ensuring complete removal (Carnevale and Mortelliti, 2001). If complete excision is not possible, local excision followed by curettage of the involved bone can be performed (Irving et al., 1993). Occasionally, radiation therapy or chemotherapy (see below) has been given prior to surgical removal (Borello and Gorlin, 1966). Recurrences also can be treated successfully with re-excision (Chossegros et al., 1995; Franchi et al., 2002; Jones and Williams, 1960; MacDonald and White, 1954; Shaia et al., 2002). Some authors have suggested that if gross mutilation would be required to remove the entire tumor, then it should be debulked and the patient allowed to destroy residual tumor cells (Judd et al., 1990). In patients where the entire tumor cannot be removed, radiation therapy or chemotherapy (see below) has been given following surgery (Atkinson et al., 1989; Mosby et al., 1992; Shaia et al., 2002). In malignant, extensive facial, or primary CNS MNTI, radiation therapy and chemotherapy have been administered (Dehner et al., 1979; Hahn et al., 1976; Navas-Palacios, 1980; Pierre-Kahn et al., 1992; Shokry et al., 1986). The chemotherapeutic agents that have been tried include vincristine, prednisone, lomustine, cyclophosphamide, methotrexate, doxorubicin, etoposide, procarbazine, hydroxyurea, cytosine arabinoside, and cisplatin (Cohen et al., 1988; De Chiara et al., 1992; Dehner et al., 1979; Johnson et al., 1982). In two children, preoperative chemotherapy with vincristine or doxorubicin plus cyclophosphamide failed to decrease the size of the tumor (Jimenez et al., 1981; Pierre-Kahn et al., 1992). However, in an infant with an initially unresectable facial tumor, neoadjuvant combination chemotherapy with vin-

cristine, cyclophosphamide, etoposide, doxorubicin and cisplatin resulted in a significant reduction in tumor volume that allowed complete excision (Mello et al., 2000). Furthermore, two infants with massive facial MNTIs were successfully treated with aggressive combination chemotherapy (vincristine/vinblastine, ifosfamide/cyclophosphamide, etoposide, doxorubicin, and dactinomycin) alone; in both cases, the tumors underwent shrinkage and calcification, and have not recurred over follow-up periods of two and six years (Klaeren et al., 1999; Woessmann et al., 2003). In other patients with aggressive and/or recurrent disease who have done well, chemotherapy was administered as adjuvant therapy (De Chiara et al., 1992; Johnson et al., 1983; Shaia et al., 2002). Lastly, Brekke and Gorlin (1975) have suggested an early examination by a speech therapist in patients with maxillary or mandibular tumors, followed by speech therapy.

Prognosis There are two clinical features of MNTI that are troublesome: rapid growth and a propensity for recurrence (Borello and Gorlin, 1966; Brekke and Gorlin, 1975). The recurrence rate usually quoted is 10–15% (Cutler et al., 1981), although a review of cases from 1980 to 1989 (Mosby et al., 1992) revealed a recurrence rate of at least 30% (excluding malignant cases). The recurrence rate is approximately 15% for cases reported since 1990 (Franchi et al., 2002; Kapadia et al., 1993; Pettinato et al., 1991; Shaia et al., 2002). To date, “benign” recurrences of MNTI have been limited to tumors that arose in the head and neck region, including the maxilla, mandible, skull, brain, and soft tissue (Brekke and Gorlin, 1975; Chossegros et al., 1990; Franchi et al., 2002; Hoshino et al., 1994; Kapadia et al., 1993; Mosby et al., 1992; Pettinato et al., 1991; Shaia et al., 2002). A bimodal curve has been described for these recurrences: one group recurs within 30 days and the second group from 6 weeks to 2 years after the original tumor has been removed (Brekke and Gorlin, 1975). When MNTIs arise in the neurocranium but are relatively small or located extraarachnoidally, they can be excised completely (Yu et al., 1992). In a review of 27 cases of MNTI of the calvarium, 19 did well but six died in the postoperative period (Jones et al., 1990). One explanation for the postoperative mortality was adherence of the midline lesions to the sagittal sinus and extensive intraoperative bleeding (Cinalli, 1995; Pierre-Kahn et al., 1992). However, a second theory was a rapid decrease in circulating levels of catecholamines (Hoshino et al., 1994). The poor prognosis of primary CNS MNTI is a result of several factors, including inability to remove the entire tumor, inaccessible sites, presence of metastases to other areas of the CNS at the time of surgery, and inability of radiation therapy to improve survival (Yu et al., 1992). Malignant MNTIs that arise outside the CNS also have a poor prognosis, with the majority of patients dying of disease within 3 years (Pettinato et al., 1991). Because the histologic features of the primary tumor do not offer a clue to the subsequent biologic behavior, the hope is 1155

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that in the future DNA ploidy as determined by flow cytometry or N-myc amplification may provide prognostic information, as in the case of neuroblastomas (Scheck et al., 1989). To date, however, DNA ploidy has not been able to predict benign recurrences of MNTI (Kapadia et al., 1993; Pettinato et al., 1991).

References Abdin, H.A., S.R. Prabhu, and L. Jose. Melanotic neuroectodermal tumor of the mandible in a Sudanese infant. Int. J. Oral Surg. 11:383–387, 1982. Adad, S.J., S.W. Pinheiro, E.O. Marinho, M.A. Reis, A.M. Rua, and D.B.R. Rodrigues. Melanotic neuroectodermal tumor of infancy. Diagn. Cytopathol. 30:67–69, 2004. Alipchenko, L.A., G.V. Ardatova, and T.F. Rostovtseva. Melanoticheskaia progonoma cherepa. Vopr. Onkol. 22:95–98, 1976. Al-Marzooq, Y.M., M.H. Al-Bagshi, R. Chopra, and H. Hashish. Melanotic neuroectodermal tumor of infancy in the soft tissues of the arm: Fine needle aspiration biopsy and histologic correlation— A case report. Diagn. Cytopathol. 29:352–355, 2003. Atanackovic, M., M. Isvaneski, I. Sjerobabin, and M. Vukadinovic. Melanomski neuroektodermalni tumor gornje vidice u dece. Srp. Arh. Celok. Lek. 114:867–873, 1986. Barrett, A.W., M. Morgan, A.D. Ramsay, P.M. Farthing, L. Newman, and P.M. Speight. A clinicopathologic and immunohistochemical analysis of melanotic neuroectodermal tumor of infancy. Oral Surg. Oral Med. Oral Pathol. Radiol. Endod. 93:688–698, 2002. Bouckaert, M.M., and E.J. Raubenheimer. Gigantiform melanotic neuroectodermal tumor of infancy. Oral Surg. Oral Med. Oral Pathol. Radiol. Endod. 86:569–572, 1998. Caird, J., M. McDermott, and M. Farrell. 5 month old boy with occipital bone mass. Brain Pathol. 10:317–319, 2000. Carnevale, G.G., and A.J. Mortelliti. The operating microscope in the management of melanotic neuroectodermal tumor of infancy. Am. J. Otolaryngol. 22:76–79, 2001. Dashti, S.R., M.L. Cohen, and A.R. Cohen. Role of radical surgery for intracranial melanotic neuroectodermal tumor of infancy: Case report. Neurosurgery 45:175–178, 1999. de Cholnokoy, T. Melanotic progonoma (retinoblastoma) of maxilla. Case report. Plast. Reconstr. Surg. 46:600–603, 1970. Deminiere, C., M.T. Akele-Akpo, J. Rivel, and P. Vergnes. Tumeur épididymaire chez un nourrisson de six mois. Ann. Pathol. 22:52–55, 2002. de Souza, P.E.A., F. Merly, D.M.F. Maia, W.H. Castro, R.S. Gomez, and B.H.M. Gerais. Cell cycle-associated proteins in melanotic neuroectodermal tumor of infancy. Oral Surg. Oral Med. Oral Pathol. Radiol. Endod. 88:466–468, 1999. de Vita, C., G. Corradino, N. Mansi, and C. Marinelli. Il tumore melanotico neuroectodermico dell’infanzia: Osservazione di un caso a locallizzazione mascellare. Acta Otorhinolaryngol. Ital. 3:555–565, 1983. Dilu, N.J., A. Bobe, and M.R. Sokolo. Progonome melanotique. A propos d’une volumineuse tumeur du nourrisson. Rev. Stomatol. Chir. Maxillofac. 99:103–105, 1998. Dourov, N., R. Mayer, F. de Martelaere, S. Godart, W. Gepts, and R. Maurus. Melanotic neuroectodermal tumor of infancy with high serum levels of alpha-fetoprotein. J. Oral Pathol. 16:251–255, 1987. Eckardt, A., G. Swennen, and T. Teltzrow. Melanotic neuroectodermal tumor of infancy involving the mandible: 7-year follow-up after hemimandibulectomy and costochondral graft reconstruction. J. Craniofac. Surg. 12:350–354, 2001. el-Saggan, A., G. Bang, and J. Olofsson. Melanotic neuroectodermal tumour of infancy arising in the maxilla. J. Larngol. Otol. 112:61–64, 1998.

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Franchi, G., F. Sleilati, V. Soupre, S. Boudjemaa, P. Josset, P.A. Diner, and M.P. Vazquez. Melanotic neuroectodermal tumour of infancy involving the orbit and maxilla: Surgical management and followup strategy. Br. J. Plast. Surg. 55:526–529, 2002. Freitag, S.K., R. Eagle, J.A. Shields, J.S. Duker, and R.L. Font. Melanogenic neuroectodermal tumor of the retina (primary malignant melanoma of the retina). Arch. Ophthalmol. 115:1581–1584, 1997. Galera-Ruiz, H., D. Gomez-Angel, F.J. Vazquez-Ramirez, J.C. Sanguino-Fabre, C.I. Salazar-Fernandez, and J. Gonzalez-Hachero. Fine needle aspiration in the pre-operative diagnosis of melanotic neuroectodermal tumour of infancy. J. Laryngol. Otol. 113:581–584, 1999. Gomez, R.S., E. Carvalho Silva, and W.H. Castro. Melanotic neuroectodermal tumor of infancy: Report of a case. J. Clin. Pediatr. Dent. 20:253–255, 1996. Gorhan, C., G. Soto-Ares, M.-M. Ruchaux, S. Blond, and J.-P. Pruvo. Melanotic neuroectodermal tumour of the pineal region. Neuroradiology 43:944–947, 2001. Gupta, A., A. Trehan, R.K. Marwaha, R.K. Sharma, and R. Nijhawan. Melanotic neuroectodermal tumour in an infant. Indian J. Pediatr. 69:725–726, 2002. Hall, W.C., D.M. O’Day, and A.D. Glick. Melanotic neuroectodermal tumor of infancy: An ophthalmic appearance. Arch. Ophthalmol. 97:922–925, 1979. Haider, N., M. McDermott, and R.J. Fitzgerald. Melanotic neuroectodermal tumour of infancy arising in skull. Med. Pediatr. Oncol. 41:495–496, 2003. Handley, G.H., and G.E. Peters. Melanotic neuroectodermal tumor of infancy. South. Med. J. 81:1170–1172, 1988. Hoeffel, C., J.M. Vignaud, A. Clement, C. Chelle, and J.C. Hoeffel. Melanotic neuroectodermal tumor of infancy. Klin. Pädiatr. 210:99–101, 1998. Hoshima, Y., Y. Hamamoto, I. Suzuki, T. Nakajima, H. Ida-Yonemochi, and T. Saku. Melanotic neuroectodermal tumor of infancy in the mandible: Report of a case. Oral Surg. Oral Med. Oral Pathol. Radiol. Endod. 89:595–599, 2000. Howell, R.E., and M.M. Cohen Jr. Pathological case of the month: Melanotic neuroectodermol tumor of infancy. Arch. Pediatr. Adolesc. Med. 150:1103–1104, 1996. Ijiri, R., K. Onuma, M. Ikeda, K. Kato, Y. Toyoda, Y. Ngashima, Y. Ito, Y. Abiko, and Y. Tanaka. Pigmented intraosseous odontogenic carcinoma of the maxilla: A pediatric case report and differential diagnosis. Hum. Pathol. 32:880–884, 2001. Kacker, A., S. Bahadur, and M. Singh. Melanotic neuro-ectodermal tumour of infancy arising from the squamous and occipital bone. J. Laryngol. Otol. 107:843–844, 1993. Kalra, N., D. Srivstava, A. Goswami, A. Narang, and U. Rani. Melanotic neuroectodermal tumour of infancy: a case report. J. Indian Soc. Pedod. Prev. Dent. 19:134–136, 2001. Kasumova, S.I., V.I. Komarov, and L.A. Alipchenko. Sluchai melanoticheskoi progonomy svoda cherepa. Vopr. Neirokhir. 2:46–49, 1978. Kaya, S., O.F. Ünal, S. Sarac, and G. Gedikog˘lu. Melanotic neuroectodermal tumor of infancy: Report of two cases and review of literature. Int. J. Pediatr. Otorhinolaryngol. 52:169–172, 2000. Khoddami, M., J. Squire, M. Zielenska, and P. Thorner. Melanotic neuroectodermal tumor of infancy: A molecular genetic study. Pediatr. Devel. Pathol. 1:295–299, 1998. Kim, Y.G., J.H. Oh, S.C. Lee, and D.M. Ryu. Melanotic neuroectodermal tumor of infancy. J. Oral Maxillofac. Surg. 54:517–520, 1996. Kirtane, M.V., S.B. Ogale, S.N. Merchant, and S.Y. Sane. Melanotic neuroectodermal tumour of the maxilla (a case report). J. Postgrad. Med. 30:250–252, 1984. Klaeren, R., V. Aumann, K. Radig, and U. Mittler. Pigmentierter neuroektodermaler tumor des kindesalters. Monatsschr. Kinderheilkd. 147:562–566, 1999.

RARE BENIGN NEOPLASMS OF MELANOCYTES Klingenberg, C., M. Kearney, R. Hennig, R. Sagsveen, B. Nilsen, and T. Flaegstad. Melanotic neuroectodermal tumor of infancy located in the skull. Med. Pediatr. Oncol. 31:555–557, 1998. Kobayashi, T, K. Kunimi, T. Imao, M. Ohkawa, K. Komatsu, Y. Mizukami, and M. Namiki. Melanotic neuroectodermal tumor of infancy in the epididymis: Case report and literature review. Urol. Int. 57:262–265, 1996. Kraus, J.A., J. Felsberg, J.C. Tonn, G. Reifenberger, and T. Peitsch. Molecular genetic analysis of the TP53, PTEN, CDKN2A, EGFR, CDK4 and MDM2 tumour-associated genes in supratentorial primitive neuroectodermal tumours and glioblastomas of childhood. Neuropathol. Appl. Neurobiol. 28:325–333, 2002. Kurabayashi, T., M. Ida, H. Yoshimasu, N. Yoshino, and T. Sasaki. Computed tomography in the diagnosis of maxillofacial mass lesions in younger children. Dentomaxillofac. Radiol. 27:334–340, 1998. Mast, B.A., S.B. Kapadia, E. Yunis, and M. Bentz. Subtotal maxillectomy for melanotic neuroectodermal tumor of infancy. Plast. Reconstr. Surg. 103:1961–1963, 1999. Mello, R.J., A.K. Vidal, H.M. Fittipaldi Jr., L.T. Montenegro, L.M. Calheiros, and G.I. Rocha. Melanotic neuroectodermal tumor of infancy: Clinicopathologic study of a case, with emphasis on chemotherapeutic effects. Int. J. Surg. Pathol. 8:247–251, 2000. Morera Martinez, H., J. Bonet Marco, and H. Morera Faet. Tumor neuroectodermico melanotico de la infancia. Presentacion de un caso en maxilar y revision de la literatura. Rev. Esp. Estomatol. 32:421–428, 1984. Moriuchi, S., K. Shimizu, Y. Miyao, and T. Hayakawa. A novel PNET cell line with melanotic differentiation. Anticanc. Res. 16:779–784, 1996. Nishio, S., T. Morioka, N. Murakami, M. Fukui, T. Inamitsu, and S. Ishihari. Melanotic neuroectodermal tumour of infancy at the anterior fontenelle. Neuroradiology 41:202–204, 1999. Patankar, T., S. Prasad, A. Goel, J. Perumpillichira, and A.P. Desai. Malignant melanotic neuroectodermal tumour of infancy affecting the occipital squama. J. Postgrad. Med. 44:73–75, 1998. Paueksakon, P., J.R. Parker, X. Fan, G. Miles, H. Ruiz, C. Wushensky, and M.D. Johnson. Melanotic neuroectodermal tumor of infancy discovered after head trauma. Pediatr. Neurosurg. 36:33–36, 2002. Puchalski, R., U.K. Shah, D. Carpentieri, R. McLaughlin, and S.D. Handler. Melanotic neuroectodermal tumor of infancy (MNTI) of the hard palate: presentation and management. Int. J. Pediatr. Otorhinolaryngol. 53:163–168, 2000. Raila, F.A., A.D. Parent, B.A. Ward, and J.D. Fratkin. Threedimensional reconstruction of melanotic neuroectodermal tumor of infancy involving the occipital bone and dura. J. Neuroimaging 7:123–126, 1997. Ramon, Y., M. Oberman, I. Horowitz, and A. Freedman. Melanotic neuroectodermal tumor of infancy (pigmented melanoameloblastoma). Int. J. Oral Surg. 8:312–317, 1979. Rege, J.D., T. Shet, H.V. Sawant, and L.P. Naik. Cytologic diagnosis of a melanotic neuroectodermal tumor of infancy occurring in the cranial bones. Diagn. Cytopathol. 21:280–283, 1999. Rickert, C.H., S. Probst-Cousin, S. Blasius, and F. Gullotta. Melanotic progonoma of the brain: A case report and review. Child. Nerv. Syst. 14:389–393, 1998. Ricketts, R.R., and B. Majmudarr. Epididymal melanotic neuroectodermal tumor of infancy. Hum. Pathol. 16:416–420, 1985. Rustemeyer, J., V. Thieme, S. Loeschke, A. Bremerich, and F.K. Kossling. Der melanotische neuroektodermale tumor des sauglingsalters. Klin. Pädiatr. 213:69–73, 2001. Shaia, W.T., L.J. DiNardo, T.E. Underhill, and C.E. Cesca. Recurrent melanotic neuroectodermal of infancy. Am. J. Otolaryngol. 23:249–252, 2002. Sidhu, S.S., H. Parkash, and M. Jetley. Melanotic neuroectodermal tumor of the maxilla. J. Dent. 9:299–301, 1981.

Toda, T., A.M. Sadi, M. Kiyuna, H. Egawa, T. Tamamoto, and Z. Toyoda. Pigmented neuroectodermal tumor of infancy in the epididymis. A case report. Acta Cytol. 42:775–780, 1998. Vajtai, I., J. Suták, and Z. Varga. Melanotic progonoma as a component of ovarian teratoma. Histopathology 36:279–289, 2000. Woessmann, W., M. Neugebauer, R. Gossen, R. Blütters-Sawatzki, and A. Reiter. Successful chemotherapy for melanotic neuroectodermal tumor of infancy in a baby. Med. Pediatr. Oncol. 40:198–204, 2003.

Pilar Neurocristic Hamartoma Julie V. Schaffer and Jean L. Bolognia

Historical Background In 1982, Tuthill et al. were the first to describe the entity “pilar neurocristic hamartoma.” The name reflected a differentiation of the tumor towards melanocytic nevus cells as well as Schwann cells, both derivatives of the neural crest. “Pilar” was included in the name because of the prominent perifollicular arrangement of the cells. The authors considered this hamartoma to be distinct from either patch- or plaque-type blue nevus. When they reviewed previous reports of plaque-type blue nevi (Tuthill et al., 1982), they were unable to identify a case with the same clinical and histologic findings. Pearson et al. (1996) later proposed the broader term “neurocristic hamartoma” for pigmented skin lesions composed of a complex proliferation of spindled and dendritic blue nevus cells, other melanocytic nevus cells, and Schwann cells. These authors considered the pilar neurocristic hamartoma to represent a folliculocentric subtype of neurocristic hamartoma.

Synonyms Neurocristic cutaneous hamartoma is another name for neurocristic hamartoma (Mezebish et al., 1998; Smith et al., 1998), but there are no specific synonyms for pilar neurocristic hamartoma. Some clinicians may consider these entities to represent variants of plaque-type blue nevus (Grichnik et al., 2003). For example, Misago (2000) noted that some cases described as cellular or common blue nevi with peripheral nerve sheath differentiation had histologic features identical to those of a neurocristic hamartoma. In addition, Smith et al. (2001) suggested that a CD34-positive variant of cellular blue nevus falls within the spectrum of neurocristic hamartomas. Lastly, Betti et al. (1997) reported a variant of speckled lentiginous nevus consisting of an “agminate and plaque-type blue nevus combined with lentigo, associated with follicular cyst and eccrine changes” that had features of a pilar neurocristic hamartoma. It is important not to confuse the aforementioned entities with proliferative neurocristic hamartomas, malformations that usually develop within giant congenital melanocytic nevi (CMN) on the trunk (Bastian et al., 2002; Clark et al., 1990). Proliferative neurocristic hamartomas can present either as a large, somewhat polypoid mass with a broad-based attachment or as multiple nodules; the former frequently ulcerates and may involve a significant part of the underlying CMN 1157

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(Clark et al., 1990). Proliferative neurocristic hamartomas are usually present at birth and may increase markedly in size during the first few weeks of life. Histologically, a nondescript mesenchyme is observed, sometimes admixed with a variety of more differentiated tissues such as cartilage and skeletal muscle.

Epidemiology To date, only 13 patients with a neurocristic hamartoma have been described (Bevona et al., 2003; Crowson et al., 1996; Kikuchi et al., 1983; Pathy et al., 1993; Pearson et al., 1996; Smith et al., 1998; Resnik et al., 1994; Tuthill et al., 1982; Table 58.2). The mean age of onset in these individuals was 14 years (range from birth to 49 years), and 5/13 lesions (38%) were congenital. This entity has no apparent sex predilection.

Clinical Description Over half of the neurocristic hamartomas reported to date have been located on the scalp (7/13); the upper trunk (4/13) and hip or buttock (2/13) represent other sites of predilection (see Table 58.2). The lesions can range from 1 cm to >10 cm in diameter (see Table 58.2), and often have an irregular shape. One large congenital neurocristic hamartoma located unilaterally on the chest and back was noted to follow the distribution of the long thoracic nerve (Pearson et al., 1996). In the initial case of pilar neurocristic hamartoma, there was a large cluster (9.5 ¥ 10 cm) of blue, black, and brown macules and papules on the upper chest (Fig. 58.8) (Tuthill et al., 1982). The lesions had been present for ten years, but the area of involvement had recently enlarged. Prominent peripilar distribution was described, with hairs emerging from many of the papules (Fig. 58.9). However, a clinically obvious perifollicular arrangement of macules and papules has not been noted in subsequent reports of pilar neurocristic hamartoma (Kikuchi et al., 1983, Pathy et al., 1993, Crowson et al., 1996). The spectrum of clinical findings in other cases of neurocristic hamartoma has ranged from one or more bluish nodules (Pearson et al., 1996; Resnik et al., 1994) to a blue plaque with a “pebbly” or multinodular surface (Bevona et al., 2003; Pathy et al., 1993; Pearson et al., 1996; Smith et al., 1998) to a patch of blue pigmentation with or without overlying blue-black macules and/or papules (Kikuchi et al., 1983; Pearson et al., 1996) to multiple blue papules within a localized area (Pearson et al., 1996). In several cases, additional superimposed (Bevona et al., 2003; Mezebish et al., 1998; Pearson et al., 1996; Smith et al., 1998) or satellite (Bevona et al., 2003; Pathy et al., 1993; Resnik et al., 1994) whitish-blue to black papules or nodules appeared over time, sometimes heralding malignant degeneration (Pathy et al., 1993; Pearson et al., 1996; see below). One patient with a neurocristic hamartoma involving the parietal scalp developed subacute swelling of the parotid gland due to infiltration by benign melanocytes (Bevona et al., 2003), while invasion of the parotid gland by malignant melanoma cells was seen in two other cases (Pathy et al., 1993; Pearson et al., 1996). 1158

In a few instances, neurocristic hamartomas have had clinical manifestations related to aberrant epithelial structures. For example, Mezebish et al. (1998) reported a neurocristic hamartoma that presented as an 8 cm area of congenital, nonscarring alopecia on the temporoparietal scalp with subtle increased pigmentation at the periphery. Betti et al. (1997) described a speckled lentiginous nevus composed in part of a plaque-type blue nevus with several superimposed gray-brown papules that discharged a serous fluid; histologic examination revealed both features of a pilar neurocristic hamartoma (see below) and a proliferation of eccrine glands and ducts in the papillary dermis.

Associated Disorders Pilar neurocristic hamartomas have been seen in association with dermal melanocytosis (Kikuchi et al., 1983) as well as blue nevi (Bevona et al., 2003; Pathy et al., 1993). Clinically and histologically, there is significant overlap with both patchand plaque-type blue nevi (Jimenez et al., 1994; Mevorah et al., 1977; Pittman and Fisher, 1976; Tsoïtis et al., 1983). As noted above, plaque-type blue nevi with features of a pilar neurocristic hamartoma can develop within speckled lentiginous nevi (Betti et al., 1997). Of concern is the potential for development of cutaneous melanoma within neurocristic hamartomas (Pathy et al., 1993; Pearson et al., 1996). Malignant transformation occurred in more than half of the lesions described in the literature to date (7/13; see Table 58.2). Although this proportion is likely inflated due to reporting bias, it suggests that patients with a neurocristic hamartoma may have a higher risk of melanoma than those with the closely related plaque-type blue nevus. For example, at least 30 of the latter lesions have been described in the literature, with most located on the trunk (approximately half of cases) and distal extremities (a quarter of cases). However, thus far, melanoma has been reported to develop in only two plaque-type blue nevi, both of which were located on the scalp (one coexisting with a nevus of Ota) (Hartmann et al., 1989; Woltzke et al., 1995). Of note, an elevated risk for lesions on the scalp is not surprising, as it represents the site of the majority of malignant blue nevi as well as five of the seven melanomas reported to arise within neurocristic hamartomas. Pearson et al. (1996) coined the term “cutaneous malignant melanotic neurocristic tumor” (CMMNT) for the subtype of melanoma developing within a neurocristic hamartoma. Like malignant blue nevi and a subset of melanomas that originate within large CMN, these tumors arise in the dermis or subcutaneous tissues rather than at the dermal–epidermal junction. However, distinctive features in the cases of CMMNT reported thus far have included circumscribed, multinodular lesions composed of small and monotonous cells, relatively slow growth, multiple local recurrences, and late hematogenous metastases (most often pulmonary); 3/7 patients had died of disease by the time of reporting, with a mean survival of five years after diagnosis (Pathy et al., 1993; Pearson et al., 1996). CMMNTs occurred at a mean age of 38 years (range

Plaque-type BN and NH §

Bevona et al., 2003

F M M F

M

32 Birth 49 Birth

Birth

Early childhood

M

F

F

Birth

Childhood

M M

F

M

M

F

Sex

5 Birth

42

Childhood

30

8

Age at onset (y)

Thigh

Forehead, parietal scalp

Buttock

Posterior auricular scalp Upper chest and back† Scalp Temporo-parietal scalp

Upper back Posterior auricular scalp Temple, scalp, ear

Hip

Parietal scalp

Upper back

Upper chest

Location

6

>10

5

5 8

>10

3

>5

>6 8

1.3

5

11

10

Size (cm)

Blue plaque, overlying blue-white nodules, satellite papule# Blue plaque with gray-brown papules; background tan patch

Gray-blue plaque, superimposed nodule

Pigmented lesion Area of alopecia with subtle pigmentation, 5 superimposed nodules

Bluish patch

Bluish plaque

Pigmented nodule with satellite papules Pigmented lesion Bluish nodules, macules, and papules Pigmented papules

Black macules w/in a blue patch Blue-black plaque with satellite nodules

Rows of peripilar blue, black, and brown papules

Clinical features

+

+

NR

+ +

NR

NR

+

NR NR

+

+

+

+

NR

+

NR

NR +

+

NR

NR

NR NR

NR

NR

NR

NR

NR

+

NR

NR +

+

+

NR

NR NR

NR

NR

NR

NR

Melanocytes surrounding: Adnexae* Vessels Nerves

NR

+

+ + (also enlarged, tortuous nerves and tactoid bodies) + (also tactoid bodies)

NR

NR

NR + (also tactoid bodies) NR

NR

NR

NR

+

Schwannian differentiation

NR

Mucinous with CD34+ spindle cells, dilated vessels, mast cells NR

NR Mucinous with CD34+ spindle cells, floretlike giant cells, dilated vessels, mast cells

NR

NR

NR

NR NR

NR

Fibrous; melanophages

NR

Collagenous; scattered melanophages

Stromal features

Grouped hyperplastic eccrine glands and ducts in the papillary dermis; ruptured epidermoid cyst

Moderate regular acanthosis; no hair follicles NR

NR Elongated, wide rete ridges; alopecia with rare dystrophic follicles

NR

Focal lentiginous epidermal hyperplasia; Aberrant basaloid follicular structures in the dermis NR

Basaloid proliferation in the dermis NR NR

NR

Mild epidermal hyperplasia with hyperkeratosis, horn cysts, and basilar hyperpigmentation NR

Epithelial features

—

—

—

+ (50) —

+ (67)

+ (35)

+ (25)

+ (11) + (12)

—

+ (65)

—

—

Melanoma** (age in y)

*Perifollicular and perieccrine distribution. †Following the distribution of the long thoracic nerve. ‡Excluded case 3 from Pearson et al. (features suggestive of a neurotized congenital melanocytic nevus) and case 1 from Smith et al. (no melanocytic component). #Clinically apparent melanocytic infiltration of underlying muscle, nearby nerves, the parotid gland, and regional lymph nodes (the latter limited to the capsule). §Variant of speckled lentiginous nevus consisting of an “agminate and plaque-type blue nevus combined with lentigo, associated with follicular cyst and eccrine changes” that was noted to have features of a PNH; because PNH was not the overall diagnosis, this case was not included in tabulations of the clinical and histologic features of PNH. **Melanoma variant termed “cutaneous malignant melanotic neurocristic tumor.” PNH, pilar neurocristic hamartoma; DM, dermal melanocytosis; BN, blue nevus; NH, neurocristic hamartoma; NCH, neurocristic cutaneous hamartoma; NR, not reported.

Betti et al., 1997

NCH

Smith et al., 1998‡; Mebish et al., 1998

Crowson et al., 1996/ Resnik et al., 1994 Pearson et al., 1996‡

NH

PNH and DM BN with features of PNH PNH

Kikuchi et al., 1983

Pathy et al., 1993

PNH

Tutill et al., 1982

Diagnosis

Table 58.2. Clinical and histologic features of neurocristic hamartomas.

CHAPTER 58

Fig. 58.8. Pilar neurocristic hamartoma with clustering of blue, black, and brown macules and papules.

Fig. 58.9. Close-up of the papules in Fig. 58.8, showing their rowlike perifollicular arrangement. (From Tuthill, R. J., W. H. Clark, Jr., and A. Levine. Pilar neurocristic hamartoma: its relationship to blue nevus and equine melanotic disease. Arch. Dermatol. 118:592–596, 1982, with permission from the publisher.)

11–67 years; mean = 35 years for the subset of lesions that were congenital) (Pathy et al., 1993; Pearson et al., 1996). Although malignant blue nevi develop in a similar age range and, as noted above, share a predilection for the scalp, they tend to have increased cytologic atypia and more aggressive behavior (Connelly and Smith, 1991; Goldenhersh et al., 1988). Lastly, in contrast with CMMNTs, more than half of melanomas originating in large CMN develop before the age of 5 years (Marghoob et al., 1996).

epithelioid melanocytes containing variable amounts of fine brown pigment. These melanocytes were diffusely positive with HMB-45 immunohistochemical staining (Mezebish et al., 1998; Smith et al., 1998). In 8/13 cases, 4 of which were referred to as pilar neurocristic hamartomas (see Table 58.2), a prominent perifollicular and/or perieccrine distribution of such melanocytes was noted (Bevona et al., 2003; Crowson et al., 1996; Kikuchi et al., 1983; Pathy et al., 1993; Pearson et al., 1996; Smith et al., 1998; Tuthill et al., 1982); the melanocytes were described as “streaming along” the connective tissue sheath of the hair follicle (Kikuchi et al., 1983), “wrapped around” hair follicles and eccrine glands (Pathy et al., 1993), “investing” adnexal structures in whorls and fascicles (Pearson et al., 1996), and “invad[ing]” the adventitial dermis of the hair follicle (Bevona et al., 2003). Tuthill et al. (1982) argued that the combination of a striking accumulation of melanocytes around adnexal structures and only a few scattered pigmented spindle cells in the dermis between the hair follicles distinguished the pilar neurocristic hamartoma from patch- and plaque-type blue nevi (see below). However, most lesions subsequently reported as pilar neurocristic hamartoma or neurocristic hamartoma also had fascicles, cords, and/or sheets of spindled or epithelioid melanocytes elsewhere in the dermis, in some cases surrounding blood vessels and/or nerves and extending into the subcutaneous fat, fascia, and even muscle (Bevona et al., 2003; Pearson et al., 1996; Smith et al., 1998). One patient had benign pigmented spindle cells within the capsule of regional lymph nodes (Bevona et al., 2003). Neuroid structures showing schwannian differentiation were seen in 6/13 cases of neurocristic hamartoma (Bevona et al., 2003; Pearson et al., 1996; Smith et al., 1998; Tuthill et al., 1982; see Table 58.2). Several cases demonstrated abnormal epithelial elements such as basaloid or eccrine proliferations (Crowson et al., 1996; Betti et al., 1997), aberrant basaloid follicular structures (Pearson et al., 1996), and dystrophic follicles (Smith et al., 1998). These observations, together with a diffusely CD34-positive lesional stroma (Mezebish et al., 1998), suggest that neurocristic derivatives in the dermis may have a “mesenchymal” role in influencing the induction of appendageal epithelial development.

Laboratory Findings There are no laboratory findings that are specific for neurocristic hamartoma, except perhaps the histologic finding of pigmented dermal melanocytes, particularly in a periadnexal distribution, in combination with evidence of schwannian differentiation.

Histology Cases reported as neurocristic hamartoma have shown considerable histologic heterogeneity, with different proportions, distributions, and types of melanocytic, schwannian, stromal, and epithelial components (Bevona et al., 2003; see Table 58.2). Nonetheless, all of the lesions demonstrated to some degree a dermal proliferation of dendritic spindled and/or 1160

Differential Diagnosis The major considerations in the differential diagnosis are patch- and plaque-type blue nevi. Patch-type blue nevi present as blue-gray patches that histologically demonstrate a more prominent proliferation of pigmented dendritic dermal melanocytes than Mongolian spots. In plaque-type blue nevi, the

RARE BENIGN NEOPLASMS OF MELANOCYTES

lesions are elevated and are often composed of a cluster of papules and nodules on a blue-gray background (Busam et al., 2000, Pittman and Fisher, 1976). As noted above, these entities have a predilection for the trunk rather than the scalp. Like pilar neurocristic hamartomas, some plaque-type blue nevi demonstrate a periappendageal accumulation of pigmented spindle cells (Tsoïtis et al., 1983; Velez et al., 1993; Wen, 1997), although this is typically in the setting of diffuse infiltration of the interfollicular reticular dermis. In a study of biopsy specimens submitted for routine histologic examination, Misago (2000) found that 63% of cellular blue nevi (5/8) and 9% of common blue nevi (10/112) showed evidence of peripheral nerve sheath differentiation. Some of the lesions were histologically indistinguishable from a neurocristic hamartoma, but clinical information was not available. Smith et al. (2001) reported four cases of congenital cellular blue nevi with diffuse expression of CD34. They proposed that these lesions, which measured 3–6 cm in diameter and were found on the head, neck, and upper back, arose from primitive cells derived from the neural crest and fell within the spectrum of neurocristic hamartomas. Other neoplasms consisting of a combination of cell types derived from the neural crest have been described. For example, Karcioglu et al. (1977) reported an “ectomesenchymoma” showing ganglionic, schwannian, melanocytic, and rhabdomyoblastic differentiation that presented as a tumor on the face of an infant. Misago et al. (1999) observed a slowly enlarging blue-black plaque on the upper arm of a middleaged woman; histologically, the lesion demonstrated both dermal melanocytosis and the features of an immature nerve sheath myxoma.

Pathogenesis and Animal Models A neurocristic hamartoma is thought to result from the aberrant development of pluripotent neural crest cells after their migration to the skin (Mezebish et al., 1998; Pearson et al., 1996). Neural crest cells normally give rise to melanocytes, neurosustentacular cells (e.g., Schwann cells) and, particularly in cephalic areas, neuromesenchymal cells (Reed, 1983). The latter can serve a fibrogenic function, and may also influence the development of epithelial structures such as hair follicles and eccrine glands. Neurocristic hamartomas would fall within a spectrum defined by blue nevi at one end and schwannomas at the other. “Equine melanotic disease” is a disorder characterized by a progressive growth of nodules composed of melanin-laden cells in the dermis of the skin and other tissues. It is seen in horses that turn dappled gray then white as they age (Levene, 1971; Tuthill et al., 1982). Histologically, collections of pigmented spindle cells are observed around hair follicles and sweat glands. Because of the overlap in gross and histologic features, Tuthill et al. (1982) proposed that pilar neurocristic hamartoma was related to equine melanotic disease. However, in contrast with the pilar neurocristic hamartoma, the latter often progresses to involve lymph nodes and viscera and occasionally disseminates rapidly.

Treatment A conservative approach would be to treat neurocristic hamartomas in a manner similar to that advocated for CMN and cellular blue nevi. Any clinically significant change in the lesion would necessitate performance of a biopsy to exclude the possible development of a cutaneous melanoma. Surgical excision could be considered for reasons of cosmesis or for lesions thought to be at increased risk of malignancy and/or too difficult to follow clinically, e.g., those on the scalp.

Prognosis The development of a melanoma in more than half of the 13 reported patients with neurocristic hamartomas is concerning. Most of these individuals had lesions on the scalp, a site where the closely related cellular blue nevus is known to have an especially high risk for transformation to a malignant blue nevus (Goldenhersh et al., 1988). Longitudinal observation, perhaps aided by photography, and education of the patient regarding the clinical signs of malignant degeneration are indicated. If feasible, considering the size of the lesion, patients with a neurocristic hamartoma on the scalp should be given the option of surgical excision.

References Bastian, B. C., J. Xiong, I. J. Frieden, M. L. Williams, P. Chou, K. Busam, D. Pinkel, and P. E. LeBoit. Genetic changes in neoplasms arising in congenital melanocytic nevi: Differences between nodular proliferations and melanomas. Am. J. Pathol. 161:1163–1169, 2002. Betti, R., E. Inselvini, M. Palvarini, and C. Crosti. Agminate and plaque-type blue nevus combined with lentigo, associated with follicular cyst and eccrine changes: A variant of speckled lentiginous nevus. Dermatology 195:387–390, 1997. Bevona, C., Z. Tannous, and H. Tsao. Dermal melanocytic proliferation with features of a plaque-type blue nevus and neurocristic hamartoma. J. Am. Acad. Dermatol. 49:924–929, 2003. Busam, K. J., J. M. Woodruff, R. A. Erlandson, and M. S. Brady. Large plaque-type blue nevus with subcutaneous cellular nodules. Am. J. Surg. Pathol. 24:92–99, 2000. Clark, W. H., Jr., D. E. Elder, and D. Guerry. Dysplastic nevi and malignant melanoma. In: Pathology of the Skin, E. R. Farmer, and A. F. Hood (eds). Norwalk: Appleton and Lange, 1990, pp. 684–756. Connelly, J., and J. L. Smith Jr. Malignant blue nevus. Cancer 67:2653–2657, 1991. Crowson, A. N., C. M. Magro, and W. H. Clark, Jr.. Pilar neurocristic hamartoma. J. Am. Acad. Dermatol. 34:715, 1996. Goldenhersh, M. A., R. C. Savin, R. L. Barnhill, and K. S. Stenn. Malignant blue nevus. J. Am. Acad. Dermatol. 19:712–722, 1988. Grichnik, J. M., A. R. Rhodes, and A. J. Sober. Benign neoplasias and hyperplasias of melanocytes. In: Fitzpatrick’s Dermatology in General Medicine, 6th ed., I. M Freedberg, A. Z. Eisen, K. Wolff, K. F. Austen, L. A. Goldsmith, and S. I. Katz (eds). New York: McGraw-Hill, Inc., 2003, pp. 897–899. Hartmann, L. C., F. Oliver, R. K. Winkelmann, T. V. Colby, T. M. Sundt, and B. P. O’Neill. Blue nevus and nevus of Ota associated with dural melanoma. Cancer 64:182–186, 1989. Jimenez, E., P. Valle, and P. Villegas. Unusual acquired dermal melanocytosis. J. Am. Acad. Dermatol. 30:277–278, 1994.

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CHAPTER 58 Karcioglu, Z., A. Someren, and S. J. Mathes. Ectomesenchymoma: A malignant tumor of migratory neural crest (ectomesenchyme) remnants showing ganglionic, schwannian, melanocytic and rhabdomyoblastic differentiation. Cancer 39:2486–2496, 1977. Kikuchi, I., S. Inoue, I. Taketomi, and T. Ono. Two cases of nevus fuscocaeruleus with pain, including a case of pilar neurocristic hamartoma. J. Dermatol. 10:275–280, 1983. Levene, A. Equine melanotic disease. Tumori 57:133–168, 1971. Marghoob, A. A., S. P. Schoenbach, A. W. Kopf, S. J. Orlow, R. Nossa, and R. S. Bart. Large congenital melanocytic nevi and the risk for the development of malignant melanoma: a prospective study. Arch. Dermatol. 132:170, 1996. Mevorah, B., E. Frenk, and J. Delacretaz. Dermal melanocytosis: Report of an unusual case. Dermatologica 154:107–114, 1977. Mezebish, D., K. Smith, J. Williams, P. Menon, J. Crittenden, and H. Skelton. Neurocristic cutaneous hamartoma: A distinctive dermal melanocytosis with an unknown malignant potential. Mod. Pathol. 11:573–578, 1998. Misago, N. The relationship between melanocytes and peripheral nerve sheath cells (part II): Blue nevus with peripheral nerve sheath differentiation. Am. J. Dermatopathol. 22:230–236, 2000. Misago, N., Y. Narisawa, T. Inoue, and N. Yonemitsu. Unusually differentiating immature nerve sheath myxoma in association with dermal melanocytosis. Am. J. Dermatopathol. 21:55–62, 1999. Pathy, A. L., T. N. Helm, D. Elston, W. F. Bergfeld, and R. J. Tuthill. Malignant melanoma arising in a blue nevus with features of pilar neurocristic hamartoma. J. Cutan. Pathol. 20:459–464, 1993. Pearson, J. P., S. Weiss, and J. T. Headington. Cutaneous malignant melanotic neurocristic tumors arising in neurocristic hamartomas:

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A melanocytic tumor morphologically and biologically distinct from common melanoma. Am. J. Surg. Pathol. 20:665–677, 1996. Pittman, J. L., and B. K. Fisher. Plaque-type blue nevus. Arch. Dermatol. 112:1127–1128, 1976. Reed, R. J. Neuromesenchyme: The concept of a neurocristic effector cell for dermal mesenchyme. Am. J. Dermatopathol. 5:385–395, 1983. Resnik, K. S., G. R. Kantor, G. H. Telang, and N. R. Howe. Ichthyosis. Basal cell carcinoma. Granuloma annulare. Self-assessment examination of the American Academy of Dermatology. J. Am. Acad. Dermatol. 30:153–156, 1994. Smith, K. J., D. Mezebish, J. Williams, M. L. Elgart, and H. G. Skelton. The spectrum of neurocristic cutaneous hamartoma: clinicopathologic and immunohistochemical study of three cases. Ann. Diagn. Pathol. 2:213–223, 1998. Smith, K. J., M. Germain, J. Williams, and H. G. Skelton. CD34positive cellular blue nevi. J. Cutan. Pathol. 28:145–150, 2001. Tsoïtis, G., C. Kanitakis, and E. Kapetis. Naevus blue multinodulaire en plaque, superficiel et neuroïde. Ann. Dermatol. Venereol. 110:231–235, 1983. Tuthill, R. J., W. H. Clark Jr., and A. Levene. Pilar neurocristic hamartoma: its relationship to blue nevus and equine melanotic disease. Arch. Dermatol. 118:592–596, 1982. Velez, A., E. del-Rio, C. Martin-de-Hijas, V. Furio, E. Sanchez Yus. Agminated blue nevi: Case report and review of the literature. Dermatology 186:144–148, 1993. Wen, S. Y. Plaque-type blue nevus: Review and an unusual case. Acta Derm. Venereol. (Stockh.) 77:458–459, 1997. Wlotzke, U., U. Hohenleutner, R. Hein, R.-M. Szeimies, and M. Landthaler. Maligner infiltrierender blauer nävus vom plaque-typ: Fallbericht und übersicht. Hautarzt 45:860–864, 1995.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

8

Treatment of Pigmentary Disorders

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

59

Topical Treatment of Pigmentary Disorders Rebat M. Halder and James J. Nordlund

Hyperpigmentation Successful treatment of hyperpigmentation depends on the identification of the mechanism for the abnormal color (see Chapter 28 for discussion of pathophysiology). Many disorders are caused by deposition of excessive quantities of melanin in the epidermis. Some of these conditions are amenable to lightening with applications of topical agents that interfere with production of melanin by the melanocyte. Melasma responds rather well to topical therapies. Café-au-lait macules do not respond as easily but with higher concentrations of lightening agents, can be made to fade. Other disorders are manifestations of deposition of melanin within the dermis. The melanin can be located between collagen bundles or within macrophages called dermal melanosis (see Chapter 28). Or melanocytes replete with melanin can be located in the dermis, a phenomenon called dermal melanocytosis. Melanin within the dermis cannot be treated by applications of topical agents. All of the available topical agents such as hydroquinone function either by blocking production of melanin or enhancing its desquamation. Thus no condition characterized by the presence of melanin within the dermis can be treated topically. Dermal melanin can be treated successfully only with lasers (Chapter 64). Epidermis that is abnormally thick such as is seen in ichthyosis nigricans (Chapters 27 and 28), seborrheic keratoses, or lentigines (Chapters 27 and 28) does not respond to topical treatment with lightening agents that block synthesis of melanin. Some of these conditions might respond to retinoids that will return the structure of the epidermis back to a normal condition. Still other conditions are caused by deposition of extraneous materials in the dermis. Hemosiderin, medications, heavy metals or tattoo dyes (Chapter 54) all are examples of foreign substances located in the dermis that cause hyperpigmentation. None of these are treated with lightening agents and some may require laser therapy (Chapter 64). The chemical agents described below are useful for those conditions caused by excessive production of melanin in the epidermis such as melasma, solar lentigines, postinflammatory hyperpigmentation, and for similar conditions. Congenital forms of hyperpigmentation such as café-au-lait spots do not respond readily to topical or systemic therapy. They must be treated with lasers although the probability of success is mod-

erate. It is critical in prescribing topical agents to understand how they alter the color of skin.

Hydroquinone Hydroquinone is the best known and studied lightening agent. Often it is labeled a bleach, a misnomer that suggests it produces its effects on the skin by modification of the melanin polymer. Rather it functions as an inhibitor of melanin synthesis (Arndt and Fitzpatrick, 1965; Fitzpatrick et al., 1966). More recent studies suggest that it might block formation of melanin by alteration of cellular metabolism (Penney et al., 1984; Smith et al., 1988). In cell cultures hydroquinone inhibits the synthesis of DNA and RNA. The inhibition seems to be dependent on the presence and activity of tyrosinase rather than melanin content of the cell (Penney et al., 1984; Smith et al., 1988). Thus hydroquinone is not a useful agent for altering the color of melanin already deposited within the epidermis or dermis. It can be used to retard or stop production of new melanin in many conditions such as melasma (Grimes, 1995) or postinflammatory hyperpigmentation. Hydroquinone is available in the United States in 2%, 3%, and 4% concentrations in a variety of diluents. The 4% seems to be more effective than the 2%. The agents are applied sparingly twice daily (Figs 59.1 and 59.2). Hydroquinone by itself is a weak therapeutic agent. It generally must be applied for prolonged periods of time for an effect to be seen, usually three to six months (Engasser and Maibach, 1981) (Fig. 59.3). Hydroquinone has been used in combination with other medications such as steroids and retinoids (see below). Its efficacy seems enhanced by combining it with these other preparations. Hydroquinone has been used successfully for many forms of epidermal hyperpigmentation. The classic condition is melasma. This condition may be caused by both epidermal and dermal melanosis. Only the epidermal component responds to applications of hydroquinone. A 3% hydroalcoholic solution of hydroquinone was used to treat 46 women with melasma. The investigators noted that 40 women (87%) improved significantly (Sanchez and Vazquez, 1982). Commercially available combinations such as TriLuma® that contains hydroquinone, tretinoin, and fluocinolone acetonide have been effective in lightening conditions such as melasma. Hydroquinone has been used to prevent hyperpigmentation following procedures such as laser treatments or surgery (Fulton, 1991; Gilchrest and Goldwyn, 1981; Ho et al., 1995). 1165

CHAPTER 59

Fig. 59.1. Boy with hyperpigmented skin in a graft used to treat a thermal burn (see also Plate 59.1, pp. 494–495).

Fig. 59.3. An African American woman with hypopigmented skin from daily applications of hydroquinone. Her normal color is visible as dark streaks on her cheeks and chin (see also Plate 59.3, pp. 494–495). Fig. 59.2. Pigmentation has disappeared following daily applications of 4% hydroquinone for nine months (see also Plate 59.2, pp. 494–495).

It seems to work best when used in combination with other topical agents (see section on Combination therapy below). Hydroquinone is remarkably safe although minor toxicity has been described. Some have suggested that it causes depigmentation by destruction of the melanocyte (Kersey and Stevenson, 1981; Markey et al., 1989). However the capacity of hydroquinone to destroy melanocytes and cause depigmentation is debated. It is well documented that derivatives of hydroquinone with aliphatic or aromatic side chains do cause depigmentation [for review of this controversy see Cummings and Nordlund (1995)]. It has been noted that hydroquinone and its chemical derivatives must not be confused or considered simple variations of each other (BentleyPhillips and Bayles, 1975). The major side effect of hydroquinone is localized ochronosis associated with formation of colloid milium (Camarasa and Serra-Baldrich, 1994; Diven et al., 1990; Findlay and de Beer,

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1980; Findlay et al., 1975; Hardwick et al., 1989; Hoshaw et al., 1985; Hull and Procter, 1990; Jordaan and Mulligan, 1990; Jordaan and Van Niekerk, 1991; Lawrence et al., 1988; Tidman et al., 1986; Weiss et al., 1990) (Fig. 58.4). Ochronosis seems to occur in those individuals who use a hydroquinone preparation for excessively prolonged periods of time, usually one or more years and who are exposed to intense sunlight. Hydroquinone also has been reported to alter the color of nails (Coulson, 1993; Mann and Harman, 1983).

Azelaic Acid Azelaic acid is a dicarboxylic acid with nine carbons. It has been proposed that it can lighten skin by inhibiting the activity of the tyrosinase enzyme necessary for melanin production by interruption of the oxidative systems of the melanocyte (Nazzaro-Porro, 1987). This agent first was introduced as a therapy for lentigo maligna, a precursor of melanoma (Breathnach et al., 1989a,b; Fitton and Goa, 1991; Nazzaro-Porro et al., 1989; Rodriguez Prieto et al., 1993). Because it does not destroy the malignancy which can spread and cause the demise of the patient, azelaic acid is no longer used for the treatment

TOPICAL TREATMENT OF PIGMENTARY DISORDERS

caused by physical or photochemical agents, and lentigo maligna melanoma and other disorders characterized by abnormal proliferation of melanocytes. Its mechanism of action is to inhibit DNA synthesis and mitochondrial enzymes, thereby inducing direct cytotoxic effects toward the melanocyte (Smith et al., 1988). Topical azelaic acid has no depigmentation effect on normally pigmented skin, freckles, senile lentigines, and nevi. This specificity may be attributed to its selective effects on abnormal melanocytes. Azelaic acid can be used for postinflammatory hyperpigmentation in acne (Rodriguez Prieto et al., 1993). Free radicals are believed to contribute to hyperpigmentation, and azelaic acid acts by reducing free radical production (Weinstein et al., 1991). Azelaic acid 20% is currently available in the United States and is only indicated for the treatment of acne, although it has off-label use for hyperpigmentation. In the treatment of melasma, a 24-week study in South America found that a 20% concentration of azelaic acid was equivalent to 2% hydroquinone (Weiss et al., 1988). In the Philippines, a study found that 20% concentration of azelaic acid was better than 2% hydroquinone (Snellman et al., 2004).

Topical Steroids

Fig. 59.4. Localized ochronosis from prolonged and excessive use of hydroquinone (see also Plate 59.4, pp. 494–495).

of lentigo maligna. More recently it has been used for treatment of conditions like melasma. It is available in a 20% concentration for application twice daily. Its success in treating melasma is controversial. Some investigators have found it useful (Balina and Graupe, 1991; Piquero Martin et al., 1988; Rigoni et al., 1989; Verallo-Rowell et al., 1989) but others have reported little success with this agent (Duteil and Ortonne, 1992; Mayer-da Silva et al., 1987; Pathak et al., 1985). It is not clear why the results are discrepant. One report suggests that azelaic acid is useful for treatment of acropigmentation of Kitamura (Kameyama et al., 1992). Azelaic acid cream is applied once or twice daily to the hyperpigmented skin. The treatment must be continued for many months. Often this agent is used in combination with one or several of the other lightening agents including hydroquinone, tretinoin, and/or steroids. Its use originated from the findings that Pityrosporum spp. can oxidize unsaturated fatty acids to dicarboxylic acids, which competitively inhibit tyrosinase. Azelaic acid was initially developed as a topical drug with therapeutic effects for the treatment of acne. Because of its effect on tyrosinase, however, it has also been used to treat melasma, lentigo maligna, and other disorders of hyperpigmentation (Ho et al., 1995; Smith et al., 1988) (28,66). Azelaic acid has been reported to be effective for hypermelanosis

Topical steroids have been used for treatment of various hyperpigmentary disorders. The mechanism by which they produce a lightening of the skin is not known. However, it is well established that injection of steroids into the skin causes hypopigmentation (Fig. 59.5). The loss of color is transient, but indicates steroids might be useful in treating pigmentary disorders. It has been suggested that they might have a direct effect on the synthesis of melanin. Melanocytes are known to respond to a variety of chemical mediators like prostaglandins and leukotrienes. Steroids might alter melanocyte function by inhibition of prostaglandin or cytokine production by various cells of the epidermis. Steroids have been combined with tretinoin and hydroquinone in one of the most effective of all therapies for hyperpigmentation (Kligman and Willis, 1975). Topical steroids can be used alone as a modality for epidermal hyperpigmentation. It is hypothesized that corticosteroids suppress the biosynthetic and secretory functions of melanocytes, thus suppressing melanin production without causing destruction of the melanocyte (Kligman and Willis, 1975). High potency steroids can inhibit melanin formation but cause significant side effects, such as epidermal thinning, telangiectasia, acne, form eruption, and increased thickness of vellus hair. Generally midpotency steroids are prescribed such as triamcinolone acetonide 0.1%, desonide 0.05%, or similar steroid. Steroids are applied once or twice daily until the skin returns to normal color, a process that often takes many months or a year.

Topical Retinoids The use of tretinoin was first suggested as a combination therapy (Kligman and Willis, 1975) in which tretinoin was

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Fig. 59.6. Melasma in an Asian woman (see also Plate 59.6, pp. 494–495).

Fig. 59.5. Marked hypopigmentation of the epidermis caused by injections of steroids into the skin. The hypopigmentation follows the route of lymphatics. The hypopigmentation is reversible (see also Plate 59.5, pp. 494–495).

considered to function to enhance penetration of hydroquinone. Later tretinoin was recognized to have effects on the pigmentary system of its own. It has been used successfully as a topical treatment for solar lentigines and photodamage (Bulengo-Ransby et al., 1993; Griffiths et al., 1993, 1994; Humphreys et al., 1996; Kimbrough-Green et al., 1994; Leyden et al., 1989; Lowe et al., 1994; Olsen et al., 1992; Ortonne, 1992; Pathak et al., 1986; Rafal et al., 1992; Tadaki et al., 1993; Weinstein et al., 1991; Weiss et al., 1988). Tretinoin can be used for treatment of any form of epidermal hyperpigmentation. It might function in several ways including enhancing desquamation of preformed melanin and inhibition of tyrosinase thereby inhibiting melanin synthesis. Retinoids applied topically or taken orally also might have beneficial effects on those forms of hyperpigmentation caused by abnormal keratinization such as is found in various forms of ichthyosis or acanthosis nigricans. This agent is applied once or twice daily until the skin returns to its normal color. Because it is irritating for many individuals, the dose must be adjusted to prevent inflammation. Inflammation might cause an increase in the hyperpigmentation, especially in those with dark skin. Results of recent studies suggest that tretinoin is useful for the treatment of melasma (Griffiths et al., 1993) (Figs 59.6 and 59.7). A group of 38 women participated in the study. 1168

Fig. 59.7. The melasma has responded to applications of hydroquinone and tretinoin (see also Plate 59.7, pp. 494–495).

Nineteen were in the tretinoin treated group. Of these, 13 reported significant improvement compared to one in the control group. However, many reported irritation from the tretinoin. In another study of melasma in black patients, the investigator found that tretinoin alone produced significant improvement in a third of subjects compared with 10% in the control group. Irritation including desquamation and erythema was noted in 88% of subjects (Kimbrough-Green et al., 1994). Not all studies have confirmed the usefulness of tretinoin for melasma (Tadaki et al., 1993). Tretinoin has been used successfully for treatment of postinflammatory hyperpigmentation caused by acne, dermatitis, and abrasions (Bulengo-Ransby et al., 1993). Although tretinoin can be effective as monotherapy for hyperpigmentation and melasma, it requires 20- to 40-week treatment periods. Darker-skinned people who develop dermatitis from tretinoin may develop postinflammatory hyperpigmentation secondary to the dermatitis. In a randomized clinical trial, the efficacy of adapalene 0.1% was found to be comparable with that of tretinoin 0.05% cream in the treatment of melasma (mainly epidermal

TOPICAL TREATMENT OF PIGMENTARY DISORDERS

type). The results showed fewer side effects and greater acceptability among patients using adapalene (Dogra et al., 2002).

Kojic Acid Kojic acid (5-hydroxy-2(hydroxymethyl)-4-pyrone) is a naturally occurring hydrophilic fungal derivative, evolved from certain species of Acetobacter, Aspergillus, and Penicillium, and used in the treatment of hyperpigmentation disorders (Weiss et al., 1990). It acts by inhibiting the production of free tyrosinase with efficacy similar to hydroquinone (Cabanes et al., 1994). In Japan, kojic acid has been increasingly used in skin care products. This is because, until recently, topically applied kojic acid at 1% concentration had not exhibited any sensitizing activity (Wong and Jimbow, 1991). More recent long-term Japanese studies, however, have shown that kojic acid has the potential for causing contact dermatitis and erythema (Wong and Jimbow, 1991).

N-Acetyl-5-cysteaminylphenol This compound is a thioether that recently has been described as a useful agent for the treatment of epidermal hyperpigmentation (Ito et al., 1987; Jimbow, 1991; Pankovich et al., 1990; Wong and Jimbow, 1991). It is not yet available in North America. Results of studies on black mice suggest that the chemical is capable of completely inhibiting melanin production within the hair follicle (Wong and Jimbow, 1991). NAcetyl-5-cysteaminylphenol acts to decrease intracellular glutathione by stimulating pheomelanin rather than eumelanin. The agent has been used to treat women with melasma (Jimbow, 1991). Of the 12 women treated, one showed complete resolution of the condition. In addition eight showed marked improvement and the others moderate improvement. There were no clinical failures. Improvement of melanoderma in patients with melasma was evident after two to four weeks of daily application of N-acetyl-4-cysteaminylphenol. The investigators observed that the number of melanosomes and quantity of melanin in the keratinocytes decreased significantly following therapy (Jimbow, 1991).

tated the epidermal penetration of the hydroquinone. The tretinoin-induced irritation was reduced by the corticosteroid. The first triple combination topical therapy approved by the US Food and Drug Administration for melasma is a modified formulation comprising fluocinolone acetonide, hydroquinone 4%, and tretinoin 0.05% (TriLuma®). In studies of patients with melasma, 78% had complete or near clearing after eight weeks of therapy. Similar results and favorable safety profile were seen in a 12-month study.

Arbutin Arbutin, which is the b-D-glupyranoside derivative of hydroquinone, is a naturally occurring plant-derived compound that has been used for postinflammatory hyperpigmentation (Boissy et al., 2005; Halder and Richard, 2004; Hori et al., 2004). It is effective in the treatment of disorders of hyperpigmentation characterized by hyperactive melanocytes (Boissy et al., 2005; Halder and Richard, 2004; Hori et al., 2004). The action of arbutin is dependent on its concentration. Higher concentrations are more efficacious than lower concentrations, but they may also result in a paradoxical hyperpigmentation (Boissy et al., 2005; Halder and Richard, 2004; Hori et al., 2004). In comparative in vitro studies of various compounds used to improve the appearance of disorders of hyperpigmentation, arbutin was found to be less toxic than hydroquinone. A dose-dependent reduction in tyrosinase activity and melanin content in melanocytes was also demonstrated.

Licorice Extract Licorice extract is not yet available in North America, but has been used in other parts of the world, particularly in Egypt. Its mechanism of action is similar to that of kojic acid. The main component of the hydrophobic fraction of licorice extract is glabridin. Studies investigating the inhibitory effects of glabridin on melanogenesis and inflammation have shown that it inhibits tyrosinase activity of these cells. No effect on DNA synthesis was detectable.

Mequinol Combination Therapy The most effective preparation for the treatment of epidermal hyperpigmentation is a combination of hydroquinone, a steroid, and tretinoin (Kligman and Willis, 1975). The combination strongly inhibits the production of melanin without destruction of the melanocytes (Kligman and Willis, 1975). It is likely that the three agents each work in different ways. A common preparation is 4% hydroquinone, tretinoin 0.05%, and triamcinolone acetonide 0.1% (or desonide 0.05%) each applied separately (Gilchrest and Goldwyn, 1981; Ho et al., 1995; Pathak et al., 1986). Each medication is applied twice daily if tolerated. Even with triple therapy, it is necessary to continue treatment for many months. The first published study of combination therapy used tretinoin 0.1%, hydroquinone 5%, and dexamethasone 0.1% for postinflammatory hyperpigmentation (Kligman and Willis, 1975). Tretinoin was shown to reduce the atrophy of the corticosteroid and facili-

The chemical 4-hydroxyanisole in combination with tretinoin 0.01% has been shown to inhibit production of melanin (Colby et al., 2003; Kasraee et al., 2003). Recently it has been introduced as a chemical agent (Solagé®) for the treatment of solar lentigines (Fleischer et al., 2000). Patients report significant lightening of solar lentigines after several months of treatment (Fleischer et al., 2000; Colby et al., 2003). The effects of the combination retinoid and mequinol was superior to either agent used individually.

Hypopigmentation Hypopigmentation is defined as a paucity of melanin within the epidermis (Chapter 28). Depigmentation is defined as complete absence of melanin. Depigmentation usually is due to absence of melanocytes such as is found in vitiligo or piebald1169

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ism. Occasionally the blockage of the melanin pathway is so severe that the skin appears depigmented despite the presence of melanocytes. This phenomenon is seen in the tyrosinase related forms of oculocutaneous albinism (Chapter 31). There are few agents to repigment skin that has a paucity or absence of melanin or melanocytes. The agents are limited to topical steroids useful for acquired hypomelanoses and various forms of phototherapy. Phototherapy includes exposure to broadband ultraviolet (UV) B which has a spectrum of 290–320 nm or narrowband UV 309–311 nm (see Chapter 59). Narrowband UV has been introduced in recent years for treatment of a variety of dermatological conditions (Baron and Stevens 2003; Grundmann-Kollmann et al., 2004; Mori et al., 2004; Ohe et al., 2004; Saricaoglu et al., 2003; Schiffner et al., 2002; Snellman et al., 2004). These spectra are most effective for causing tanning. UVB is used with variable success to enhance melanin formation in skin that is hypopigmented such as pityriasis alba (Chapter 37). It has little effect on depigmentation. Hypopigmentation can be caused by several mechanisms. Some conditions are acquired and are due to suppression of melanin formation like pityriasis alba. It is possible that the inhibition of melanin formation is related to inflammation within the epidermis. These conditions respond to topical steroids. The choice of steroid seems critical. Potent steroids suppress inflammation but also might directly block the melanization process (see Fig. 59.5). Thus intermediate potency steroids like desonide seem optimal for treatment of pityriasis alba. The congenital variants like a nevus depigmentosus (Chapter 32) or genetic forms like the ash leaf (Chapter 32), or albinism spot are due to an intrinsic defect within the melanocyte and are not responsive to any therapy. Other forms of hypopigmentation are caused by absence of melanocytes. Such conditions include vitiligo and piebaldism. PUVA or psoralens and exposure to ultraviolet A (320–400 nm) and narrow band UVB (309–311 nm) (see Chapter 61) are excellent stimuli for enhancing melanin formation and for proliferation of melanocytes within hair follicles. The efficacy of PUVA or narrow band UVB in conditions characterized by absence of interfollicular melanocytes depends on the availability of a reservoir of melanocytes in hair follicles to migrate into the depigmented skin for repigmentation to occur (see Chapters 30 and 60). Piebald skin lacks melanocytes within the interfollicular epidermis but also within the follicles. Patients with piebaldism do not respond to PUVA or other forms or therapy although pigmented patches at times spontaneously gain pigmentation. Surgical transplant of melanocytes is the most effective way to repigment piebald skin. Currently the logistics and expense of transplantation make this approach impractical. In contrast vitiliginous skin can respond to PUVA or applications of topical therapy (Chapter 30). PUVA also has been used to treat conditions like the hypomelanoses. Patients with some hypomelanoses have a very mottled appearance to their skin. PUVA for individuals

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with pityriasis alba or similar conditions usually does not resolve the problem. The normal skin darkens more than the abnormal skin. The contrast can be even more severe after therapy than before. Some hypomelanoses are not caused by abnormalities of the pigment system. The normal color of the skin is determined by other factors such as the vascular supply or the quantity of collagen in the dermis. The nevus anemicus appears hypopigmented but is a vascular anomaly that cannot respond to therapies described in this section. Similarly collagen deposition can give a whitish appearance to the skin. The collagen causes the epidermis to appear white. Examination of such skin with Wood’s light confirms that melanin is present and that the abnormality is dermal. A biopsy is rarely necessary but can confirm the diagnosis. Such abnormalities do not respond to therapies described in this section.

Depigmentation and Use of Monobenzone Monobenzone is the only agent available in the United States and in some European countries for the treatment of vitiligo that is too extensive to repigment (Chapter 30). Monobenzone is a chemical that can destroy epidermal melanocytes and leave the epidermis depigmented. For that reason it is never used for treating hyperpigmented conditions like melasma or postinflammatory hyperpigmentation (Figs 59.8–59.10). The goal of treatment for these latter conditions is inhibition of the synthesis of melanin so that the color of the skin returns to normal. Although monobenzone is a potent inhibitor of melanin synthesis, its capacity to destroy melanocytes makes it useless except for completing the loss of pigment in those with vitiligo. The best is for an individual to have their own normal skin color. The worst is having two colors. The second best is losing

Fig. 59.8. An African American woman incorrectly treated with monobenzone for melasma. She has depigmentation of the face that is not readily repigmented (see also Plate 59.8, pp. 494–495).

TOPICAL TREATMENT OF PIGMENTARY DISORDERS

Fig. 59.9. Depigmentation of the hand by improper use of monobenzone.

Fig. 59.11. Woman with vitiligo too extensive to repigment (see also Plate 59.9, pp. 494–495).

Fig. 59.10. Depigmentation of the forearm from improper use of monobenzone. The photo illustrates the confetti depigmentation from use of this agent.

all one’s color so that the skin color is uniform although depigmented. Thus the optimal therapy for those with vitiligo is repigmentation of the skin. However that is not always possible. For those with vitiligo that is unresponsive to repigmentation or too extensive for repigmentation, monobenzone can produce a uniform depigmented state that is cosmetically very attractive (Figs 59.11 and 59.12) (Mosher et al., 1977). The individual has a very young appearance because the usual spots such as keratoses and lentigines associated with aging do not mar depigmented skin. Monobenzone is available as a 20% cream. It can be purchased from pharmaceutical companies. It is more readily obtained at this time from pharmacists who formulate the preparation. The patient applies the cream on a small spot once daily for a week to ensure that he or she has no allergy to the chemical, the most common side effect (Nordlund et al., 1985). If there is no inflammation, the cream is applied to the pigmented skin twice daily. Loss of pigment occurs slowly over

months to a year or longer. The pigment fades imperceptibly. Usually the person applies the monobenzone to the hands, arms, neck, and face first. When these areas are depigmented the person can then depigment the legs and thighs. Later the trunk can also be treated although most individuals choose to stop when the exposed skin is depigmented. The resulting skin has an attractive, healthy pink color and is not white. At times the commercially available monobenzone is ineffective. It can be used under occlusion or the concentration can be increased to 30%. These maneuvers can be successful at times in depigmentation in a patient whose skin is resistant. Occasionally a patient who has successfully undergone depigmentation suddenly regains much or most of his or her pigmentation back, generally following a sunburn. This is very alarming and requires the patient to start depigmentation again. The repigmentation might be so extensive that the person might choose to try repigmentation. For patients in whom itching and burning occur, a 5% cream is used initially. The concentration is increased every 2–3 months until the 20% preparation is tolerated. Many allergic individuals cannot tolerate any concentration of monobenzone (Chapter 30). They must stop the treatment and there is no substitute medication. Once depigmentation is

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Fig. 59.12. Woman in Figure 59.11 properly treated with monobenzone to remove remaining pigment. Her appearance is excellent (see also Plate 59.10, pp. 494–495).

complete, no maintenance is needed except an occasional retreatment may be required for focal areas of repigmentation that may appear particularly after sun exposure. The individual using monobenzone should not allow the cream to touch others. They should avoid direct contact for an hour after applying. Many individuals choose to apply the cream after children go to school and early in the evening to avoid contact with the spouse.

References Arndt, K., and T. Fitzpatrick. Topical use of hydroquinone as a depigmentating agent. J. Am. Med. Assoc. 194:965–967, 1965. Balina, L. M., and K. Graupe. The treatment of melasma. 20% azelaic acid versus 4% hydroquinone cream. Int. J. Dermatol. 30:893–895, 1991. Baron, E. D., and S. R. Stevens. Phototherapy for cutaneous T-cell lymphoma. Dermatol. Ther. 16:303–310, 2003. Bentley-Phillips, B., and M. A. Bayles. Cutaneous reactions to topical application of hydroquinone. Results of a 6-year investigation. S. Afr. Med. J. 49:1391–1395, 1975. Boissy, R. E., M. Visscher, and M. A. DeLong. Deoxyarbutin: a novel reversible tyrosinase inhibitor with effective in vivo skin lightening potency. Exp. Dermatol. 14:601–608.

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Breathnach, A. C., M. Nazzaro-Porro, S. Passi, and G. Zina. Azelaic acid therapy in disorders of pigmentation [review]. Clin. Dermatol. 7:106–119, 1989a. Breathnach, A. S., E. J. Robins, M. Nazzaro-Porro, S. Passi, and M. Picardo. Hyperpigmentary disorders–mechanisms of action. Effect of azelaic acid on melanoma and other tumoral cells in culture [review]. Acta Derm. Venereol. Suppl. (Stockh.) 143:62–66, 1989b. Bulengo-Ransby, S. M., C. E. Griffiths, C. K. Kimbrough-Green, L. J. Finkel, T. A. Hamilton, C. N. Ellis, and J. J. Voorhees. Topical tretinoin (retinoic acid) therapy for hyperpigmented lesions caused by inflammation of the skin in black patients. N. Engl. J. Med. 328:1438–1443, 1993. Cabanes, J., S. Chazarra, and F. Garcia-Carmona. Kojic acid, a cosmetic skin whitening agent, is a slow-binding inhibitor of catecholase activity of tyrosinase. J. Pharm. Pharmacol. 46:982–985, 1994. Camarasa, J. G., and E. Serra-Baldrich. Exogenous ochronosis with allergic contact dermatitis from hydroquinone. Contact Dermatitis 31:57–58, 1994. Colby, S. I., E. H. Schwartzel, F. J. Huber, A. Highton, D. J. Altman, W. W. Epinette, and E. Lyon. A promising new treatment for solar lentigines. J. Drugs Dermatol. 2:147–152, 2003. Coulson, I. H. “Fade out” photochromonychia. Clin. Exp. Dermatol. 18:87–88, 1993. Cummings, M. P., and J. J. Nordlund. Chemical leukoderma: fact or fancy. Am. J. Contact Dermatitis 6:122–127, 1995. Diven, D. G., E. B. Smith, R. A. Pupo, and M. Lee. Hydroquinoneinduced localized exogenous ochronosis treated with dermabrasion and CO2 laser. J. Dermatol. Surg. Oncol. 16:1018–1022, 1990. Dogra, S., A. J. Kanwar, and D. Parsad. Adaplene in the treatment of melasma: a preliminary report. J. Dermatol. 29:539–540. Duteil, L., and J. P. Ortonne. Colorimetric assessment of the effects of azelaic acid on light-induced skin pigmentation. Photodermatol. Photoimmunol. Photomed. 9:67–71, 1992. Engasser, P., and H. Maibach. Cosmetics and dermatology: Bleaching creams. J. Am. Acad. Dermatol. 5:143–147, 1981. Findlay, G. H., and H. A. de Beer. Chronic hydroquinone poisoning of the skin from skin-lightening cosmetics. A South African epidemic of ochronosis of the face in dark-skinned individuals. S. Afr. Med. J. 57:187–190, 1980. Findlay, G., J. Morrison, and J. Simson. Exogenous ochronosis and pigmented colloid milium from hydroquinone bleaching creams. Br. J. Dermatol. 93:613–622, 1975. Fitton, A., and K. L. Goa. Azelaic acid. A review of its pharmacological properties and therapeutic efficacy in acne and hyperpigmentary skin disorders. Drugs 41:780–798, 1991. Fitzpatrick, T. B., K. A. Arndt, A. M. El Mofty, and M. A. Pathak. Hydroquinone and psoralens in the therapy of hypermelanosis and vitiligo. Arch. Dermatol. 93:589–600, 1966. Fleischer, A. B., Jr., E. H. Schwartzel, S. I. Colby, and D. J. Altman. The combination of 2% 4-hydroxyanisole (Mequinol) and 0.01% tretinoin is effective in improving the appearance of solar lentigines and related hyperpigmented lesions in two double-blind multicenter clinical studies. J. Am. Acad. Dermatol. 42:459–467, 2000. Fulton, J. E., Jr. The prevention and management of postdermabrasion complications. J. Dermatol. Surg. Oncol. 17:431–437, 1991. Gilchrest, B. A., and R. M. Goldwyn. Topical chemotherapy of pigment abnormalities in surgical patients. Plast. Reconstr. Surg. 67:435–439, 1981. Griffiths, C. E., L. J. Finkel, C. M. Ditre, T. A. Hamilton, C. N. Ellis, and J. J. Voorhees. Topical tretinoin (retinoic acid) improves melasma. A vehicle-controlled, clinical trial. Br. J. Dermatol. 129:415–421, 1993. Griffiths, C. E., M. T. Goldfarb, L. J. Finkel, V. Roulia, M. Bonawitz, T. A. Hamilton, C. N. Ellis, and J. J. Voorhees. Topical tretinoin (retinoic acid) treatment of hyperpigmented lesions associated with

TOPICAL TREATMENT OF PIGMENTARY DISORDERS photoaging in Chinese and Japanese patients: a vehicle-controlled trial. J. Am. Acad. Dermatol. 30:76–84, 1994. Grimes, P. E. Melasma: Etiologic and therapeutic considerations. Arch. Dermatol. 131:1453–1457, 1995. Grundmann-Kollmann, M., R. Ludwig, T. M. Zollner, F. Ochsendorf, D. Thaci, W. H. Boehncke, J. Krutmann, R. Kaufmann, and M. Podda. Narrowband UVB and cream psoralen-UVA combination therapy for plaque-type psoriasis. J. Am. Acad. Dermatol. 50:734–739, 2004. Halder, R. M. and G. M. Richards. Topical agents used in the management of hyperpigmentation. Skin Ther. Lett. 9:1–3. Hardwick, N., L. van Gelder, and C. van der Merwe. Exogenous ochronosis: an epidemiologic study. Br. J. Dermatol. 120:229–238, 1989. Ho, C., Q. Nguyen, N. J. Lowe, M. E. Griffin, and G. Lask. Laser resurfacing in pigmented skin. Dermatol. Surg. 21:1035–1037, 1995. Hori, I., K. Nihei, and I. Kubo. Structural mechanism for depigmenting mechanism of arbutin. Phytother. Res. 18:475–479. Hoshaw, R. A., K. G. Zimmerman, and A. Menter. Ochronosislike pigmentation from hydroquinone bleaching creams in American blacks. Arch. Dermatol. 121:105–108, 1985. Hull, P. R., and P. R. Procter. The melanocyte: an essential link in hydroquinone-induced ochronosis. J. Am. Acad. Dermatol. 22:529–531, 1990. Humphreys, T. R., V. Werth, L. Dzubow, and A. Kligman. Treatment of photodamaged skin with trichloroacetic acid and topical tretinoin. J. Am. Acad. Dermatol. 34:638–644, 1996. Ito, Y., K. Jimbow, and S. Ito. Depigmentation of black guinea pig skin by topical application of cysteaminylphenol, cysteinylphenol, and related compounds. J. Invest. Dermatol. 88:77–82, 1987. Jimbow, K. N-acetyl-4-S-cysteaminylphenol as a new type of depigmenting agent for the melanoderma of patients with melasma. Arch. Dermatol. 127:1528–1534, 1991. Jordaan, H. F., and R. P. Mulligan. Actinic granuloma-like change in exogenous ochronosis: case report. J. Cutan. Pathol. 17:236–240, 1990. Jordaan, H. F., and D. J. Van Niekerk. Transepidermal elimination in exogenous ochronosis. A report of two cases. Am. J. Dermatopathol. 13:418–424, 1991. Kameyama, K., M. Morita, K. Sugaya, S. Nishiyama, and V. J. Hearing. Treatment of reticulate acropigmentation of Kitamura with azelaic acid: an immunohistochemical and electron microscopic study. J. Am. Acad. Dermatol. 26:817–820, 1992. Kasraee, B., F. Handjani, and F. S. Aslani. Enhancement of the depigmenting effect of hydroquinone and 4-hydroxyanisole by alltrans-retinoic acid (tretinoin): the impairment of glutathionedependent cytoprotection? Dermatology 206:289–291, 2004. Kersey, P., and C. J. Stevenson. Vitiligo and occupational exposure to hydroquinone from servicing self-photographing machines. Contact Dermatitis 7:285–287, 1981. Kimbrough-Green, C. K., C. E. Griffiths, L. J. Finkel, T. A. Hamilton, S. M. Bulengo-Ransby, C. N. Ellis, and J. J. Voorhees. Topical retinoic acid (tretinoin) for melasma in black patients. A vehiclecontrolled clinical trial. Arch. Dermatol. 130:727–733, 1994. Kligman, A. M., and I. Willis. A new formula for depigmenting human skin. Arch. Dermatol. 111:40–48, 1975. Lawrence, N., C. Bilgard, R. Reed, and W. Perret. Exogenous ochronosis in the United States. J. Am. Acad. Dermatol. 18:1207–1211, 1988. Leyden, J. J., G. L. Grove, M. J. Grove, E. G. Thorne, and L. Lufrano. Treatment of photodamaged facial skin with topical tretinoin. J. Am. Acad. Dermatol. 21:638–644, 1989. Lowe, P. M., J. Woods, A. Lewis, A. Davies, and A. J. Cooper. Topical tretinoin improves the appearance of photo damaged skin. Australas. J. Dermatol. 35:1–9, 1994. Mann, R., and R. Harman. Nail staining due to hydroquinone skinlightening creams. Br. J. Dermatol. 108:363–365, 1983.

Markey, A. C., A. K. Black, and R. J. Rycroft. Confetti-like depigmentation from hydroquinone. Contact Dermatitis 20:148–149, 1989. Mayer-da Silva, A., H. Gollnick, E. Imcke, and C. E. Orfanos. Azelaic acid vs. placebo: effects on normal human keratinocytes and melanocytes. Electron microscopic evaluation after long-term application in vivo. Acta Derm. Venereol. 67:116–122, 1987. Mori, M., P. Campolmi, L. Mavilia, R. Rossi, P. Cappugi, and N. Pimpinelli. Monochromatic excimer light (308 nm) in patch-stage IA mycosis fungoides. J. Am. Acad. Dermatol. 50:943–945, 2004. Mosher, D. B., J. A. Parrish, and T. B. Fitzpatrick. Monobenzyl ether of hydroquinone: A retrospective study of treatment of 18 vitiligo patients and a review of the literature. Br. J. Dermatol. 97:669–679, 1977. Nazzaro-Porro, M. Azelaic acid. J. Am. Acad. Dermatol. 17:1033– 1041, 1987. Nazzaro-Porro, M., S. Passi, G. Zina, and A. S. Breathnach. Ten years experience of treating lentigo maligna with topical azelaic acid. Acta. Derm. Venereol. 143(Suppl):4957, 1989. Nordlund, J. J., B. Forget, J. Kirkwood, and A. B. Lerner. Dermatitis produced by applications of monobenzone in patients with active vitiligo. Arch. Dermatol. 121:1141–1145, 1985. Ohe, S., K. Danno, H. Sasaki, T. Isei, H. Okamoto, and T. Horio. Treatment of acquired perforating dermatosis with narrowband ultraviolet B. J. Am. Acad. Dermatol. 50:892–894, 2004. Olsen, E. A., H. I. Katz, N. Levine, J. Shupack, M. M. Billys, S. Prawer, J. Gold, M. Stiller, L. Lufrano, and E. G. Thorne. Tretinoin emollient cream: a new therapy for photodamaged skin. J. Am. Acad. Dermatol. 26:215–224, 1992. Ortonne, J. P. Retinoic acid and pigment cells: A review of in-vitro and in-vivo studies. Br. J. Dermatol. 127:43–47, 1992. Pankovich, J. M., K. Jimbow, and S. Ito. 4-S-cysteaminylphenol and its analogues as substrates for tyrosinase and monoamine oxidase. Pigment Cell Res. 3:146–149, 1990. Pathak, M. A., E. R. Ciganer, M. Wick, A. J. Soger, W. A. Farinelli, and T. B. Fitzpatrick. An evaluation of the effectiveness of azelaic acid as a depigmenting and chemotherapeutic agent. J. Invest. Dermatol. 85:222–228, 1985. Pathak, M. A., T. B. Fitzpatrick, and E. W. Kraus. Usefulness of retinoic acid in the treatment of melasma. J. Am. Acad. Dermatol. 15:894–899, 1986. Penney, K. B., C. J. Smith, and J. C. Allen. Depigmenting action of hydroquinone depends on disruption of fundamental cell processes. J. Invest. Dermatol. 82:308–310, 1984. Piquero Martin, J., J. Rothe de Arocha, and D. Beniamini Loker. [Double-blind clinical study of the treatment of melasma with azelaic acid versus hydroquinone] [Spanish]. Med. Cutan. IberoLat-Am. 16:511–514, 1988. Rafal, E. S., C. E. Griffiths, C. M. Ditre, L. J. Finkel, T. A. Hamilton, C. N. Ellis, and J. J. Voorhees. Topical tretinoin (retinoic acid) treatment for liver spots associated with photodamage. N. Engl. J. Med. 326:368–374, 1992. Rigoni, C., P. Toffolo, R. Serri, and R. Caputo. [Use of a cream based on 20% azelaic acid in the treatment of melasma] [Italian]. G. Ital. Dermatol. Venereol. 124:I-VI, 1989. Rodriguez Prieto, M. A., P. Manchado Lopez, I. Ruiz Gonzalez, and D. Suarez. Treatment of lentigo maligna with azelaic acid. Int. J. Dermatol. 32:363–364, 1993. Sanchez, J. L., and M. Vazquez. A hydroquinone solution in the treatment of melasma. Int. J. Dermatol. 21:55–58, 1982. Saricaoglu, H., S. K. Karadogan, E. B. Baskan, and S. Tunali. Narrowband UVB therapy in the treatment of lichen planus. Photodermatol. Photoimmunol. Photomed. 19:265–267, 2003. Schiffner, R., J. Schiffner-Rohe, M. Gerstenhauer, M. Landthaler, F. Hofstadter, and W. Stolz. Dead Sea treatment – principle for outpatient use in atopic dermatitis: safety and efficacy of synchronous

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CHAPTER 59 balneophototherapy using narrowband UVB and bathing in Dead Sea salt solution. Eur. J. Dermatol. 12:543–548, 2002. Smith, C. J., K. B. O’Hare, and J. C. Allen. Selective cytotoxicity of hydroquinone for melanocyte-derived cells is mediated by tyrosinase activity but independent of melanin content. Pigment Cell Res. 1:386–389, 1988. Snellman, E., T. Klimenko, and T. Rantanen. Randomized half-side comparison of narrowband UVB and trimethylpsoralen bath plus UVA treatments for psoriasis. Acta Derm. Venereol. 84:132–137, 2004. Tadaki, T., M. Watanabe, K. Kumasaka, Y. Tanita, T. Kato, H. Tagami, I. Horii, T. Yokoi, Y. Nakayama, and A. M. Kligman. The effect of topical tretinoin on the photodamaged skin of the Japanese. Tohoku J. Exp. Med. 169:131–139, 1993. Tidman, M. J., J. J. Horton, and D. M. MacDonald. Hydroquinoneinduced ochronosis: light and electronmicroscopic features. Clin. Exp. Dermatol. 11:224–228, 1986. Verallo-Rowell, V. M., V. Verallo, K. Graupe, L. Villafuerte, and M.

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Garcia-Lopez. Double-blind comparison of azelaic acid and hydroquinone in the treatment of melasma. Acta Derm. Venereol. 143(Suppl):58–61, 1989. Weinstein, G. D., T. P. Nigra, P. E. Pocha, R. C. Savan, A. Allan, K. Benik, E. Jeffes, L. Lufrano, and E. G. Thorne. Topical tretinoin for treatment of photodamaged skin: a multicenter study. Arch. Dermatol. 127:659–665, 1991. Weiss, J. S., C. N. Ellis, J. T. Headington, T. Tincoff, T. A. Hamilton, and J. J. Voorhees. Topical tretinoin improves photoaged skin. A double-blind vehicle-controlled study [published errata appear in J. Am. Med. Assoc. 259:3274, 1988 and 260:926, 1988]. J. Am. Med. Assoc. 259:527–532, 1988. Weiss, R. M., E. del Fabbro, and P. Kolisang. Cosmetic ochronosis caused by bleaching creams containing 2% hydroquinone [letter]. S. Afr. Med. J. 77:373, 1990. Wong, M., and K. Jimbow. Selective cytotoxicity of N-acetyl-4-S-cysteaminylphenol on follicular melanocytes of black mice. Br. J. Dermatol. 124:56–61, 1991.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Chemophototherapy of Pigmentary Disorders Rebat M. Halder and James J. Nordlund

Psoralens and PUVA

Oral Psoralen Therapy

Plant extracts combined with natural sunlight have been used for thousands of years to successfully treat hypopigmentation and vitiligo in Egypt and India (Danielssen, 1966; Ortonne et al., 1983). The plants are Ammi majus in Egypt and Psoralea corylifolia in India (Singh et al., 1974). Several crystalline compounds, including ammodin (8-methoxypsoralen, 8MOP) and bergapten (5-methoxypsoralen, 5-MOP) were isolated from the powder of Ammi majus in 1947 (el-Mofty, 1948). Other psoralens such as trioxsalen (4,5¢,8 trimethylpsoralen, TMP) were developed later. Currently psoralen photochemotherapy is the most widely used and most effective therapy available for repigmentation in vitiligo and other disorders of hypopigmentation although it is not a panacea for any of these conditions. In the United States, photochemotherapy can be administered either systemically or topically. Oral 5-MOP is being used in other countries (Beretti et al., 1989; Hann et al., 1991). The combination of Psoralen and UltraViolet A has been given the acronym PUVA. Psoralens are furocoumarins consisting of a double-ringed coumarin moiety to which a furan group is attached (Fitzpatrick et al., 1966; Parrish et al., 1976; Pathak et al., 1984). The furan ring is joined at its 3¢2¢ bond to the 6,7 bond of the coumarin in a linear fashion. Psoralens are photoactive and have no known effects on their own (Kao and Yu, 1992). For psoralens to be active they must be combined with ultraviolet A (UVA). Psoralens absorb photons forming short-lived, high-energy compounds. The exact mechanism by which photosensitivity occurs following PUVA therapy is not exactly known. However, two distinct independent photoreactions take place when psoralen-treated skin is exposed to UVA light. The type I photoreaction is oxygen independent and forms monofunctional and bifunctional bonds called adducts between the psoralen and DNA. Type II reactions are oxygen dependent and form similar adducts. Psoralen reactions are predominately type I. Those psoralens that form bifunctional adducts generally cause photosensitization. Psoralens are metabolized in the liver where there is saturable, first pass metabolism which reduces the amount of active drug reaching the systemic circulation (Pathak et al., 1984). Metabolism of 8-MOP and TMP have been identified but data on their activity are sparse. 8-MOP induces drugmetabolizing enzymes in the liver as well as cytochrome P450, effects not caused by TMP.

Currently, oral 8-MOP is available in the United States in a liquid formulation contained inside a soft gelatin capsule (Oxsoralen Ultra‘). This preparation is much better absorbed than the previous commercial form that was a crystalline powder inside a hard gelatin capsule. TMP is available in the United States as a tablet (Trisoralen‘). Because of better absorption, the liquid formulation of 8-MOP produces an earlier peak in blood and tissues and is much more photosensitizing than the crystalline powder formulation. Psoralens are absorbed in the small intestine and absorption is decreased by high-fat meals. TMP is highly insoluble in water, less than 8MOP. TMP is poorly absorbed in the small intestine. Measurements of serum levels of psoralen indicate that there is great variability in absorption of TMP by an individual from day to day and from person to person (Chakrabarti et al., 1982). Because of the poor absorption of TMP, it is a poor photosensitizer in oral tablet formulation. TMP has a higher binding affinity to serum proteins than 8-MOP. The binding causes lower tissue concentrations of TMP in the skin. As a general principle TMP is not the agent of choice for oral PUVA (Bleehen, 1972; Kligman and Goldstein, 1973). PUVA therapy for vitiligo is a protracted, time consuming process to which the patient should be committed (el-Mofty et al., 1994). The response rate is moderate. PUVA treatment for other hypopigmentary disorders such as progressive macular hypopigmentation does not require prolonged times. Relative contraindications to PUVA include some ocular disorders, liver dysfunction, young age, small areas of depigmentation, prior history of skin cancers, and prior exposure to cutaneous carcinogens (Nordlund et al., 1996). Absolute contraindications include pregnancy, aphakia, liver failure, photosensitive disorders like lupus erythematosus, depigmentation confined to glabrous skin, and depigmentation confined to skin with white hairs (see also Chapter 30) (Nordlund et al., 1996). In general children are not good candidates for systemic psoralen therapy. Age under 12 years is considered a relative contraindication for oral PUVA therapy (Halder et al., 1987). However, exceptions are made for children with very extensive involvement for which topical PUVA or topical steroid therapy is impractical and for whom the vitiligo is debilitating (Figs 60.1 and 60.2). For children under age 12 years, TMP is sometimes substituted for 8-MOP because TMP is a 1175

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Fig. 60.1. Child with extensive vitiligo before therapy with oral PUVA.

Fig. 60.2. Child in Figure 60.1 after treatment with oral PUVA showing excellent repigmentation.

weaker photosensitizer and the margin of safety is greater, lessening the risk of phototoxicity. TMP metabolism is not as dependent on drug-metabolizing enzymes in the liver as is 8-MOP. These enzymes are not fully developed in young children. In adults and some children with vitiligo, 8-MOP, especially in the liquid form (Oxsoralen-Ultra‘), is the most effective drug for PUVA therapy. There are two ways to administer PUVA. The first recommends higher doses of Oxsoralen and lower doses of UVA. The second uses lower doses of psoralen and higher doses of UVA. It is not known whether the two are biologically and biochemically identical but the clinical outcomes of the two forms of therapy seem to be equivalent. For high-dose psoralen therapy, the recommended dosage of 8-MOP (as Oxsoralen-Ultra) is 0.4–0.5 mg/kg ingested 1.5 hours prior to exposure to UVA light. Initial UVA exposure is based on skin type. For skin types I–III, an initial dose of 0.5 J/cm2 of UVA is given whereas for skin types IV–VI, 1.0 J/cm2 is administered. At subsequent treatments increments of 0.25–0.5 J/cm2 are added until persistent, moderate, asymptomatic erythema is maintained between treatments. At that stage, no further increments in UVA dosage are given. Treat-

ments are given two to three times weekly but never on consecutive days. A maximum treatment exposure usually ranges between 1.0 J/cm2 and 4.0 J/cm2. The dose is individualized for each patient and determined by the appearance of erythema. For the second type PUVA called low-dose psoralen therapy, the patient takes 10 mg Oxsoralen-Ultra and is exposed to 4 J UVA. The dose of UVA is increased 1–2 J per treatment until erythema is noted. If the dose of UVA is over 16 J, the patient is given 20 mg of Oxsoralen-Ultra. The dose of UVA is started at 4 J and increased until erythema is noted. The final dose of UVA is usually large, around 14–20 J. The risk of serious burns from inadvertent exposure to UVA in sunlight is minimal in the second form of PUVA utilizing low doses of Oxsoralen (Figs 60.3 and 60.4). Regardless of the method utilized, the course of PUVA should be continued for at least three to four months before the therapy is considered unsuccessful. If repigmentation is not observed in four months, the PUVA probably should be discontinued and tried again at a later time. If repigmentation does occur manifested by perifollicular pigmentation, it should be continued until no further progress is noted. Sunlight must be used with great caution when using 8-MOP (heliotherapy)

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Fig. 60.3. A young man before treatment with PUVA (see also Plate 60.1, pp. 494–495).

because the quantity of UVA reaching the earth’s surface varies widely from hour to hour and day to day. Physicians properly trained can give heliotherapy with 8-MOP but must titrate the exposure to sunlight carefully to avoid burns. In those children selected for oral PUVA therapy, TMP is given in a dosage of 0.6 mg/kg ingested two hours prior to UVA exposure. UVA exposure is determined just as for 8MOP by titrating the amount of UVA to produce erythema. For adults who choose heliotherapy, TMP can be substituted for 8-MOP. It is poorly absorbed and less photosensitizing. The results of treatment seem to be less satisfactory than those with 8-MOP. TMP is taken at a dosage of 0.6–0.8 mg/kg two hours prior to sunlight exposure. Exposure should begin with five minutes of mid-day summer sun. Subsequent exposures are increased in increments of five minutes with each treatment until moderate asymptomatic erythema is achieved. Usually the maximum exposure will be 30–45 minutes. Treatments are done two to three times per week but not on consecutive days. There are important precautions to be taken with oral PUVA therapy, especially if the individual is taking larger doses of 8-MOP. These precautions include the avoidance of

Fig. 60.4. The young man in Figure 60.3 approximately 25 months after treatment with low-dose psoralen PUVA. The response is excellent. Note that glabrous skin on the hands and umbilicus did not respond (see also Plate 60.2, pp. 494–495).

unnecessary exposure to sunlight from the time of ingestion of the medication until sunset of the same day to prevent the risk of developing a burn. Patients should wear UVA blocking, wraparound sunglasses for 12–24 hours after ingestion of psoralen to protect the eyes from psoralen-induced photodamage. Studies have not found an increase in incidence of premature cataracts in vitiligo patients treated with PUVA therapy. Human eyelid skin blocks completely the penetration of UVA. Following oral PUVA therapy, patients must apply a broad spectrum sunscreen to sun-exposed areas of treated skin. Although the incidence of squamous cell carcinoma of the skin has been found to be increased in patients with psoriasis treated with PUVA, skin cancers have not been observed in patients with vitiligo (see Chapter 30 for discussion of skin cancer and vitiligo). Acute side effects of oral PUVA therapy include nausea from the psoralen. This is particularly a problem with higher doses of psoralen. Patients receiving psoralens are subject to severe, blistering burns that are very painful and long-lasting. Many patients note fatigue after each treatment, pruritus, and 1177

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Fig. 60.5. Vitiligo before treatment with topical PUVA.

Fig. 60.6. The same skin as in Figure 59.5 during treatment with topical PUVA (see also Plate 60.3, pp. 494–495).

headaches. Generally these are tolerable. Because of the immediate and long-term toxicity of PUVA, the prescribing physician should be familiar with this form of treatment.

Topical PUVA Therapy Topical PUVA therapy for vitiligo is indicated in adults with less than 20% skin surface involvement and for children of any age (Grimes et al., 1982). It can also be used by patients in whom oral PUVA therapy is contraindicated. Topical PUVA seems to avoid some of the potential side effects associated with oral PUVA therapy but has other toxicities that limit its usefulness (Figs 60.5–60.7). Topical PUVA therapy is difficult to use because of the extreme photosensitivity associated with application of psoralens to the skin (Kanof, 1955; Kelly and Pinkus, 1955). Severe blistering burns are common in patients treated with topical PUVA. However, topical PUVA therapy for vitiligo is safe and effective if used judiciously by those properly trained (Fulton et al., 1969; Parrish et al., 1971). Adverse reactions

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Fig. 60.7. The patient in Figures 60.5 and 60.6 after successful treatment with topical PUVA.

PHOTOTHERAPY OF PIGMENTARY DISORDERS

are minimized by careful monitoring of the dose of UVA irradiation. Thus, topical PUVA therapy for vitiligo should never be used with natural sunlight (heliotherapy). Topical psoralens are not usually dispensed to patients to avoid inadvertent misuse of these potent agents. Various studies have determined that 0.1% or 0.01% concentration of 8-MOP is optimal for topical PUVA therapy for vitiligo (Grimes et al., 1982; Halder, 1991). One percent Oxsoralen (8-MOP) is diluted to 0.1% or 0.01% concentration in one of the following vehicles: alcohol, propylene glycol, Aquaphor, or acid mantle cream. The formulated cream is applied thinly and sparingly with a cotton-tipped swab within the margins of a vitiliginous patch by the dermatologist, nurse, or medical assistant. Patients who are reliable can be taught how to apply properly the medication in the physician’s office. Because it dries quickly, the alcohol or propylene glycol vehicle is preferred for areas that will be covered with clothing soon after application. Acid mantle cream or Aquaphor is preferred on the face to prevent the Oxsoralen from dripping and causing undesired hyperpigmentation. Normal skin surrounding a depigmented patch is not treated. After an interval of 30 minutes the painted areas are exposed to UVA. Maximal penetration of the psoralen into the epidermis occurs within this time (Arora and Willis, 1976). The initial dose of UVA depends on the skin type. The dose is 0.12 J/cm2 in those with types I–III skin and 0.25 J/cm2 for those with darker skin (Nordlund et al., 1996). The dose is increased by that amount at every other treatment until a mild erythema is produced. Adverse reactions to topical PUVA are common and include excessive erythema and edema, blistering, or extreme pruritus. Treatments must be discontinued until the reactions subside (usually one to two weeks). At reinstitution of therapy, UVA doses are decreased to half the previous value. Blistering may be due to using a preparation of Oxsoralen that is too concentrated, exposure to too much UVA, failure to wash the areas after the treatment or exposure to sunlight following treatment. Blistering can be treated by carefully incising the blister roof, draining the fluid, but leaving the blister roof intact and applying a sterile dressing. Other phototoxic side effects of topical PUVA therapy include itching and hyperkeratosis of treated skin. A mild topical steroid cream, such as desonide, can be used for the itching on facial areas. Triamcinolone acetonide can be used on other areas. Urea or lactic acid containing creams or lotions can soften hyperkeratotic skin. Since topical Oxsoralen is not absorbed through the skin in appreciable amounts (Coleman et al., 1988), risks of premature cataract formation or liver toxicity are thought to be nil. However, there have been isolated reports of elevated liver transaminases with topical PUVA (Park et al., 1994). Topical PUVA is also an acceptable treatment for children too young for oral PUVA. Children as young as 2 years of age have been successfully treated with topical PUVA. The child must be able to stay in the PUVA box for a sufficient time to develop an erythema. Topical or oral

PUVA therapy should never be used in combination due to the much increased risk of severe phototoxic reactions.

Results of PUVA Therapy There are many reports on the results of treatment of vitiligo with topical and oral PUVA therapy. With both forms of therapy there is some degree of repigmentation in 60–80% of patients (Grimes, 1993). Cosmetically acceptable repigmentation ranges from 11% to 60% depending on the criteria used but results of most studies indicate that 40–60% of individuals respond to a course of PUVA (see Figs 60.1–60.7). Complete repigmentation occurs in 20–25% of patients treated. Some investigators suggest that those who fail the first course of therapy be given a second course six months later. A higher percentage regain some pigment, possibly as high as 70%. PUVA therapy, either topical or oral, is not a panacea for the treatment of vitiligo or other pigmentary disorders but is the most effective treatment currently available. Why some patients respond to PUVA therapy and others do not is unknown. The permanence of repigmentation in vitiligo has been reported to be high (Kenney, 1971) but it has been stated that loss of new pigmentation will occur unless the area has completely repigmented. This suggestion has not been substantiated by careful studies. There are factors that influence the response to treatment. Psoralen dosage, either topical or oral, in excessive amounts producing severe erythema or blistering does not give better results. It may decrease the rate of repigmentation. Increased frequency of treatment does not increase response rate but may increase the risk of phototoxic reactions. A good response correlates best with duration of continuous treatment. The course must be at least three to four months. If pigment is developing in the vitiliginous skin, the therapy should be continued until no further progress is noted. Age of the patient is unrelated to response although some have suggested that the younger the patient, the earlier and greater the amount of repigmentation (Grimes, 1993). However this suggestion is still controversial. The site of the lesion is an important factor in determining response to therapy. In most studies, the face and neck respond the best with a 60–70% response with full repigmentation (Grimes, 1993). Areas that characteristically respond poorly are glabrous skin such as the lips, parts of the hands and fingers, feet, toes, ankles, palms, soles, and nipples (see Figs 60.3 and 60.4) (see Chapter 30). This poor response is due to lack of hair follicles that serve as a reservoir of melanocytes. White (not gray) hair itself in an area of vitiligo is also associated with a poor response to therapy (see Figs 60.8 and 60.9). The white color of the hair is an indicator that the reservoir has been destroyed by the vitiliginous process. The extent of vitiligo and duration of disease are not related to the response to PUVA therapy. The activity of the disease can be

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Fig. 60.8. White hair in a patch of vitiligo. This skin has lost its reservoir of melanocytes and cannot respond to medical therapies but can be treated with surgical therapies described in Chapter 63.

considered a bad prognostic sign for repigmentation if the disease is spreading rapidly. PUVA does not halt the progression of depigmentation, particularly if the disease is spreading rapidly. Slowly spreading vitiligo can respond to PUVA. It has been suggested that patients with darker skin types, i.e., IV–VI, obtain a better response to PUVA therapy (Grimes et al., 1983). The reason for this is unknown but may be related to a residual pool of melanocytes that may aid in repigmentation. It also might be related to the greater need for patients with dark skin to regain color. Although skin cancer has been reported to be higher in incidence in psoriatic patients treated with PUVA, this has not been the case in vitiligo (Halder et al., 1995; Harrist et al., 1984). The mechanism by which PUVA stimulates proliferation and migration of melanocytes is not known. In normal skin PUVA causes an increase in the number of melanocytes and stimulates melanogenesis (Becker, 1967). Light microscopy (dopa reaction) and scanning electron microscopy have shown proliferation of melanocytes in hair follicles and perifollicular areas in repigmenting vitiligo (Ortonne et al., 1979, 1980). Proliferation of melanocytes within hair follicles has been documented by [3H]-thymidine labeling. The melanocyte reservoir probably resides in the middle and/or lower parts of the outer root sheaths of the hair follicles (Cui et al., 1991; Horikawa et al., 1996). The melanocytes migrate upward along the surface of the outer root sheet to the epidermis where they spread radially. Repigmentation also occurs at the margins of a lesion. It is not known if the mechanism for this type of repigmentation is migration of melanocytes from a marginal follicular reservoir or from uninvolved interfollicular epidermis. It is not known if PUVA stimulates melanocyte proliferation and migration directly or indirectly. Results of recent studies suggest that immune cytokines and inflammatory mediators like leukotriene C4 (LTC4) and transforming growth factor a (TGFa) are potent mitogens for melanocytes (Morelli et al., 1989, 1992). 1180

Fig. 60.9. Depigmented skin in person with vitiligo responding to PUVA. Note that the skin with pigmented terminal hairs responds well, that with white terminal hairs did not respond. The case shows the importance of the reservoir of melanocytes in the follicles (see also Plate 60.4, pp. 494–495).

Khellin and UVA Light (KUVA) Khellin (4,9-dimethoxy-7-methyl-5H-furo[3,2-g][1]-benzophyran-5-one) is a furanochrome isolated from the seeds of the plant Ammi visnaga and has a nucleus that is a structural isomer of the psoralen nucleus (Abdel-Fattah et al., 1982). In the 1940s and 1950s, khellin was used for the treatment of angina pectoris and asthma because of its relaxing effect on smooth muscle via ganglionic blockade. Promising results from limited clinical trials using khellin and natural sunlight to treat vitiligo were first reported in 1982. Daily treatments consisting of 100 mg of khellin administered orally 45 minutes prior to exposure to mid-day sunlight for 15 minutes for four months resulted in 40% of the patients regaining at least 50% repigmentation. Serious side effects were not observed. In another study, patients were exposed three times per week to artificial UVA 2.5 hours after ingesting 100 mg of khellin (Ortel et al., 1988). Each patient received a constant UVA dose per treatment at 10 J/cm2 for skin types II and III and 15 J/cm2 for skin type IV. At least 70% repigmentation was achieved in 41% of the patients who had received 100–200 treatments. This success rate was noted to be comparable to the rate obtained with PUVA using 8-MOP or TMP. Limited success was also reported using a topical 2% solution of khellin although the number of patients was too small to be significant (Orecchia and Perfetti, 1992). Glabrous skin without a melanocyte reservoir did not respond to khellin. Phototoxic erythema was not observed with either topical or oral khellin even at high doses of irradiation. The lack of severe phototoxicity makes khellin a useful drug for home treatments or heliotherapy. Khellin has other short-term side effects. Systemically administered khellin produces nausea, hypotension, or loss of appetite in 30% of patients. These effects usually subsided after two weeks of treatments. In 25% of the patients, signif-

PHOTOTHERAPY OF PIGMENTARY DISORDERS

icant increases in liver transaminases require cessation of treatment (Ortel et al., 1988). These abnormalities spontaneously revert to normal. A marked but reversible increase in liver transaminases was reported in one patient after five weeks of topical applications of khellin (as a 2% solution) to one extremity (Duschet et al., 1989). One case of pseudoallergic reaction to orally administered khellin was reported consisting of a generalized urticarial reaction (Jung and Fingerhut, 1988). Although no long-term clinical side effects were noted after 12–15 months of therapy, the potential for such effects exists since khellin can induce formation of monofunctional adducts and cross-links with DNA and exhibits photogenotoxic properties. Compared with the psoralens, however, khellin produces photoadducts less efficiently than psoralens (Cassuto et al., 1977; Morliere et al., 1988). Like the psoralens, khellin induces an increased tolerance to natural sunlight in vitiliginous skin after a few months, but unlike the psoralens, it does not enhance the tanning properties of UVA in normal skin. Khellin is not approved for use in the United States by the Food and Drug Administration.

Phenylalanine and UVA (Phe-UVA) L-Phenylalanine (l-phe), an essential amino acid normally found in dietary protein, has been reported to induce repigmentation of vitiligo when given orally followed by UVA exposure. Repigmentation was reported in more than 90% of patients treated for six to eight months on a twice weekly regimen. Each individual received an oral dose of l-phe, 50 mg/kg, 30–45 minutes prior to UVA exposure (Cormane et al., 1985). In 26.3%, there was “dense” repigmentation and in 46% there was 50–90% repigmentation (Schulpis et al., 1989). When l-phe at 50 mg/kg was combined with sunlight, a follicular pattern of repigmentation was reported in 81% of cases (Kuiters et al., 1986). Oral phe-UVA was compared with combined oral and topical phe-UVA using a 10% cream of lphe applied to affected areas 20 minutes before UVA exposure (Antoniou et al., 1989). Although blood levels of l-phe were comparable in both groups, 90% of the patients treated with the combined oral and topical mode had greater than 75% repigmentation compared with 45% of the patients on oral treatment alone. The total dose of UVA required was also lower with the combined treatment mode. More recently the protocol was revised to daily intake of l-phe with UVA exposure two to three times per week. Side effects were not encountered in any of the above studies while an increased tolerance of vitiliginous skin to sunlight was observed. The repigmented areas remained stable after one year. Contraindications to phe-UVA include phenylketonuria, impaired liver and kidney function, premalignant and malignant skin lesions, pregnancy, breastfeeding, a history of using arsenates, a history of exposure to ionizing radiation, topical medications such as tar, light-induced dermatoses, autoimmune diseases, significant actinic damage, and cardiovascular disorders.

Although the mechanism by which phe-UVA causes repigmentation is not known, it is probably not related to increased tyrosine formation because no repigmentation was obtained when oral tyrosine was combined with UVA (Cormane et al., 1985). There are many speculations. One proposed mechanism is based on the finding that l-phe reduces an antibody response that might diminish an autoimmune-mediated destruction of melanocytes while allowing UVA or sunlight to stimulate proliferation of melanocytes (Ryan and Carver, 1964). Other suggested mechanisms include the stimulatory effect of UVA and l-phe or one of its metabolites on melanocyte activity and melanosome formation, and a shift in Langerhans cell subpopulations that results in an altered immune response (Cormane et al., 1985).

References Abdel-Fattah, A., M. N. Aboul-Enein, G. M. Wassel, and B. S. El-Menshawi. An approach to the treatment of vitiligo by khellin. Dermatologica 165:136–140, 1982. Antoniou, C., H. Schulpis, T. Michas, A. Katsambas, N. Frajis, S. Tsagaraki, and J. Stratigos. Vitiligo therapy with oral and topical phenylalanine with UVA exposure. Int. J. Dermatol. 28:545–547, 1989. Arora, S. K., and I. Willis. Factors influencing methoxsalen phototoxicity in vitiliginous skin. Arch. Dermatol. 112:327–332, 1976. Becker, S. Psoralen phototherapeutic agents. J. Am. Med. Assoc. 202:120–122, 1967. Beretti, B., D. Grupper, B. Bermejo, A. Borenstein, Y. Charpentier, D. Edelson, A. Thioly-Bensoussan, and R. Triller. PUVA 5-MOP + phenylalanine in the treatment of vitiligo. Study of 125 patients: preliminary results. In: Psoralens: Past, Present and Future of Photochemoprotection and Other Biological Activities. T. B. Fitzpatrick, P. Forlot, M. Pathak, and F. Urbach (eds). Paris: John Libbey Eurotext, 1989, pp. 103–108. Bleehen, S. S. Treatment of vitiligo with oral 4,5¢,8-trimethylpsoralen (Trisoralen). Br. J. Dermatol. 86:54–60, 1972. Cassuto, E., N. Gross, E. Bardwell, and P. Howard-Flanders. Genetic effects of photoadducts and photocross-links in the DNA of phage lambda exposed to 360 nm light and trimethylpsoralen or khellin. Biochim. Biophys. Acta 475:589–600, 1977. Chakrabarti, S. G., P. E. Grimes, H. R. Minus, J. A. Kenney Jr., and T. K. Pradhan. Determination of trimethylpsoralen in blood, ophthalmic fluids and skin. J. Invest. Dermatol. 79:374–377, 1982. Coleman, W. R., N. J. Lowe, M. David, and R. M. Halder. Palmoplantar psoriasis: Experience with 8-methoxypsoralen soaks plus ultraviolet A with the use of a high-output metal halide device. J. Am. Acad. Dermatol. 20:1078–1082, 1988. Cormane, R. H., A. H. Siddiqui, W. Westerhof, and R. B. Schutgens. Phenylalanine and UVA light for the treatment of vitiligo. Arch. Dermatol. Res. 277:126–130, 1985. Cui, J., L. Shan, and G. Wang. Role of hair follicles in the repigmentation of vitiligo. J. Invest. Dermatol. 97:410–416, 1991. Danielssen, B. 1966. Quoted in Goldman, L., R. S. Moraites, and K. W. Kitzmiller. White spots in biblical times. Arch. Dermatol. 93:744–753, 1966. Duschet, P., T. Schwartz, M. Pusch, and F. Gschnait. Marked increase of liver transaminases after khellin and UVA therapy. J. Am. Acad. Dermatol. 21:592–594, 1989. el-Mofty, A. M. A preliminary clinical report on the treatment of leucodermia with Ammi majus Linn. J. Egypt. Med. Assoc. 31:651, 1948. el-Mofty, A. M., H. el Sawalhy, and M. el Mofty. Clinical study of a new preparation of 8-methoxypsoralen in photochemotherapy. Int. J. Dermatol. 33:588–592, 1994.

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CHAPTER 60 Fitzpatrick, T. B., K. A. Arndt, A. M. El Mofty, and M. A. Pathak. Hydroquinone and psoralens in the therapy of hypermelanosis and vitiligo. Arch. Dermatol. 93:589–600, 1966. Fulton, J. E., Jr., J. Leyden, and C. Papa. Treatment of vitiligo with topical methoxsalen and blacklite. Arch. Dermatol. 100:224–229, 1969. Grimes, P. E. Vitiligo. An overview of therapeutic approaches. Dermatol. Clin. 11:325–338, 1993. Grimes, P. E., H. R. Minus, S. G. Chakrabarti, J. Enterline, R. Halder, J. E. Gough, and J. A. Kenney Jr. Determination of optimal topical photochemotherapy for vitiligo. J. Am. Acad. Dermatol. 7:771– 778, 1982. Grimes, P. E., H. R. Minus, R. M. Halder, J. Enterline, and J. A. Kenny. Vitiligo: Racial variations in the response to topical photochemotherapy [abstract]. J. Invest. Dermatol. 80:367, 1983. Halder, R. M. Topical PUVA therapy for vitiligo. Dermatol. Nurs. 3:178–180,198, 1991. Halder, R. M., P. E. Grimes, C. A. Cowan, J. A. Enterline, S. G. Chakrabarti, and J. A. Kenney Jr. Childhood vitiligo. J. Am. Acad. Dermatol. 16:948–954, 1987. Halder, R. M., E. F. Battle, and E. M. Smith. Cutaneous malignancies in patients treated with psoralen photochemotherapy (PUVA) for vitiligo [letter]. Arch. Dermatol. 131:734–735, 1995. Hann, S. K., M. Y. Cho, S. Im, and Y. K. Park. Treatment of vitiligo with oral 5-methoxypsoralen. J. Dermatol. 18:324–329, 1991. Harrist, T. J., M. A. Pathak, D. B. Mosher, and T. B. Fitzpatrick. Chronic cutaneous effects of long-term psoralen and ultraviolet radiation therapy in patients with vitiligo. Natl. Cancer Inst. Monogr. 66:191–196, 1984. Horikawa, T., D. A. Norris, T. W. Johnson, T. Zekman, N. Dunscomb, S. D. Bennion, R. L. Jackson, and J. G. Morelli. DOPA-negative melanocytes in the outer root sheath of human hair follicles express premelanosomal antigens but not a melanosomal antigen or the melanosome-associated glycoproteins tyrosinase, TRP-1 and TRP2. J. Invest. Dermatol. 106:28–35, 1996. Jung, E. G., and W. Fingerhut. Pseudoallergic reaction from Khellin in photochemotherapy of vitiligo: a case report. Photodermatology 5:235–236, 1988. Kanof, N. B. Melanin formation in vitiliginous skin under the influence of external applications of 8-methoxypsoralen. J. Invest. Dermatol. 24:5, 1955. Kao, C. H., and H. S. Yu. Comparison of the effect of 8-methoxypsoralen (8-MOP) plus UVA (PUVA) on human melanocytes in vitiligo vulgaris and in vitro. J. Invest. Dermatol. 98:734–740, 1992. Kelly, E. W., Jr., and H. Pinkus. Local application of 8-methoxypsoralen in vitiligo. J. Invest. Dermatol. 25:453, 1955. Kenney, J. A., Jr. Vitiligo treated by psoralens: A long-term follow-up study of the permanency of repigmentation. Arch. Dermatol. 103:475, 1971. Kligman, A. M., and F. P. Goldstein. Ineffectiveness of trioxsalen as an oral photosensitzer. Arch. Dermatol. 107:413–414, 1973.

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Kuiters, G. R., J. M. Hup, A. H. Siddiqui, and R. H. Cormane. Oral phenylalanine loading and sunlight as source of UVA irradiation in vitiligo on the Caribbean island of Curacao NA. J. Trop. Med. Hygiene 89:149–155, 1986. Morelli, J. G., J. J. Yohn, M. B. Lyons, R. C. Murphy, and D. A. Norris. Leukotrienes C4 and D4 as potent mitogens for cultured human melanocytes. J. Invest. Dermatol. 93:719–722, 1989. Morelli, J. G., J. Kincannon, J. J. Yohn, T. Zekman, W. L. Weston, and D. A. Norris. Leukotriene C4 and TGF-alpha are simulators of human melanocyte migration in vitro. J. Invest. Dermatol. 98:290–295, 1992. Morliere, P., H. Honigsmann, D. Averbeck, M. Dardalhon, G. Huppe, B. Ortel, R. Santus, and L. Dubertret. Phototherapeutic, photobiologic, and photosensitizing properties of khellin. J. Invest. Dermatol. 90:720–724, 1988. Nordlund, J. J., P. E. Grimes, R. M. Halder, and H. R. Minus. Guidelines of care for vitiligo. J. Am. Acad. Dermatol. 35:620–626, 1996. Orecchia, G., and L. Perfetti. Photochemotherapy with topical khellin and sunlight in vitiligo. Dermatology 184:120–123, 1992. Ortel, B., A. Tanew, and H. Honigsmann. Treatment of vitiligo with khellin and ultraviolet A. J. Am. Acad. Dermatol. 18:693–701, 1988. Ortonne, J. P., D. M. MacDonald, A. Micoud, and J. Thivolet. PUVAinduced repigmentation of vitiligo: a histochemical (split-DOPA) and ultrastructural study. Br. J. Dermatol. 101:1–12, 1979. Ortonne, J. P., D. Schmitt, and J. Thivolet. PUVA-induced repigmentation of vitiligo: scanning electron microscopy of hair follicles. J. Invest. Dermatol. 74:40–42, 1980. Ortonne, J.-P., D. B. Mosher, and T. B. Fitzpatrick. Topics in Dermatology: Vitiligo and Other Hypomelanoses of Hair and Skin. New York: Plenum Medical Book Company, 1983, pp. 129–461. Park, Y. M., T. Y. Kim, H. O. Kim, and C. W. Kim. Reproducible elevation of liver transaminases by topical 8-methoxypsoralen. Photodermatol. Photoimmunol. Photomed. 10:261–263, 1994. Parrish, J. A., M. A. Pathak, and T. B. Fitzpatrick. Prevention of unintentional overexposure in topical psoralen treatment of vitiligo. Arch. Dermatol. 104:281–283, 1971. Parrish, J. A., T. B. Fitzpatrick, C. Shea, and M. A. Pathak. Photochemotherapy of vitiligo. Use of orally administered psoralens and a high-intensity long-wave ultraviolet light system. Arch. Dermatol. 112:1531–1534, 1976. Pathak, M. A., D. B. Mosher, and T. B. Fitzpatrick. Safety and therapeutic effectiveness of 8-methoxypsoralen, 4,5,8-trimethylpsoralen and psoralen in vitiligo. Natl. Cancer Inst. Monogr. 66:165–173, 1984. Ryan, W. L., and M. J. Carver. Inhibition of antibody synthesis by L-phenylalanine. Science 143:479–480, 1964. Schulpis, C. H., C. Antoniou, T. Michas, and J. Strarigos. Phenylalanine plus ultraviolet light: Preliminary report of a promising treatment for childhood vitiligo. Pediatr. Dermatol. 6:332–335, 1989. Singh, G., Z. Ansari, and R. N. Dwivedi. Letter: Vitiligo in ancient Indian medicine. Arch. Dermatol. 109:913, 1974.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

61

UVB Therapy for Pigmentary Disorders Thierry Passeron and Jean-Paul Ortonne

Introduction Ultraviolet (UV) A and B phototherapies are both widely used in dermatology. UVA radiation includes electromagnetic waves with wavelengths between 320 nm and 400 nm. It is used with systemic or topical psoralens, which selectively absorbs the radiation [psoralen and UVA (PUVA) therapy]. The main action of PUVA on biologic systems is the inhibition of DNA synthesis due to photoadducts formed between psoralen and pyrimidine bases in the nucleic acid. Although for a long time PUVA was considered to be the phototherapy of choice, its potential side effects — including cutaneous cancers (Garland et al., 2003; Lindelof et al., 1999; Stern, 2001; Stern et al., 1997) — mean that many authors recommend the use of UVB therapy instead.

Sources of UVB UVB wavelengths are between 290 nm and 320 nm. Unlike UVA, no prior photosensitization is required for efficacy. Initially broadband UVB therapy (290–320 nm) was based on the use of copper vapor lamps. Later, studies with monochromatic irradiations showed that the spectrum of action of phototherapy in psoriasis was optimal when UVB wavelengths of between 300 nm and 313 nm were used (Parrish, 1981). So, the concept of selective UVB phototherapy was developed, leading to narrowband UVB (NB-UVB) therapy (around 311 nm), which is widely used in dermatology today. Data now confirm that NB-UVB is more efficient than broadband UVB, with a better risk:benefit ratio (Tjioe et al., 2003); however, it is reserved for skin disorders that affect more than 20% of the body surface area. In the past few years, some devices have been developed that selectively deliver UVB to smaller surface areas, allowing surrounding healthy skin to be preserved (Leone et al., 2003; Menchini et al., 2003; Tanghetti and Gillis, 2003a). At the same time, excimer lasers have begun to be used for pigmentary disorders (Esposito et al., 2004; Ostovari et al., 2004; Spencer et al., 2002; Taneja et al., 2003). This type of laser represents the latest advance in the field of selective phototherapy. It emits a wavelength of 308 nm and capitalizes on the physical properties of lasers: monochromatic light; possibility of delivering high fluencies; and selectivity of the treatment. Because it represents an interesting new approach and to date few prospective studies have been performed with this type of laser (especially in pigmentary disorders), studies with long-term follow-up are needed.

Moreover, the high cost of this device has limited its use to a few dermatological centers.

Photobiological Effects of UVB Radiation The mechanisms by which UVB radiation exerts its activity on skin are still not well known. Immunomodulatory effects are the best understood. Animal models have shown that low doses of UVB are able to induce local immunosuppression and high doses can induce systemic immunosuppression (Miyauchi and Horio, 1995; Miyauchi-Hashimoto and Horio, 1996; Toews et al., 1980). UVB radiation acts directly on the antigen-presenting cells by depleting Langerhans cells from the epidermis and impairing antigen-presenting functions (Toews et al., 1980). UVB also works on keratinocytes; after UVB irradiation, the keratinocytes produce many cytokines, such as interleukin (IL)-1, IL10, tumor necrosis factor a (TNF-a), prostaglandin E2 or a-melanocytic stimulating hormone (aMSH). The injection of recombinant TNF-a intracutaneously at the site of sensitization suppresses the induction of contact hypersensitivity, whereas antibodies to TNF-a reverse UVBinduced immunosuppression (Yoshikawa and Streilein, 1990). Pre-treatment of Langerhans cells with IL-10 impairs their ability to present antigen to T helper (Th)1 clones, while antigen-presenting capacity to Th2 clones was not altered (Enk et al., 1993). a-MSH and prostaglandin E2 may also be involved in UV-induced immunosuppression (Luger et al., 1999; Shreedhar et al., 1998). One of the most important photobiological effects of UVB is the induction of apoptosis of T lymphocytes. T cells seem to be affected by UV radiation to a greater extent than do B lymphocytes. The mechanism of action is still not well known, but direct lesions on lymphocytic DNA and expression of Fas ligand on the surface of the keratinocytes could both be involved (Gutierrez-Steil et al., 1998). The exact photobiological effects of the 308 nm excimer laser radiation on skin have not so far been investigated. It is probable that they are similar to those produced by NB-UVB phototherapy. However, it has been shown that the apoptotic dose 50 (apoptosis in 50% of the T cells) is 95 mJ/cm2 with the 308 nm excimer laser and 320 mJ/cm2 with NB-UVB light (Novak et al., 2002). Irradiating at 200 mJ/cm2 with the 308 nm excimer laser induces apoptosis in 59–65% of T cells. This efficiency gain could allow the cumulative dose necessary to obtain clinical results to be reduced, thus limiting the side effects of the phototherapy. However, the need for long-term follow-up is obvious, and the risk that the 308 nm excimer 1183

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laser might have long-term mutagenic effects cannot be excluded. Finally, decreased numbers of natural killer cells (Toda et al., 1986), decrease in the expression of adhesion molecules on vascular endothelial cells (Yamawaki et al., 1996), and suppression of the degranulation and secretion of histamine by mast cells (Danno et al., 1986) have also been reported, and may contribute to the immunomodulatory effects of UVB phototherapy. Immunomodulating effects explain the efficacy of UVB therapy on inflammatory disorders such as psoriasis and atopic dermatitis. Apoptosis of T cell infiltrate is also very useful in pityriasis lichenoides and mycosis fungoides. These photobiological effects probably also play an important role in the treatment of autoimmune pigmentary disorders such as vitiligo. Indeed, recent work on the autoimmune origin of vitiligo underlines the importance of the immunomodulatory effect of UVB in the treatment of vitiliginous lesions (Ongenae et al., 2003). The stimulation of migration and proliferation of melanocytes from the hair follicle niche is necessary and essential to obtain repigmentation — including in vitiligo. This stimulation is so far not fully understood, although many studies have demonstrated the direct and indirect action of UVB in melanogenesis. DNA damage and the formation of thymidine dimers directly induced by UVB irradiation of melanocytes have been shown to enhance pigmentation (Eller and Gilchrest, 2000). The increase of a-MSH receptors (Chakraborty et al., 1999) and the formation of nitric oxide (Romero-Graillet et al., 1997), also induced by UVB irradiation, also promote melanogenesis. However, the indirect action of the cytokines secreted by the keratinocytes is probably the major component in this stimulation (Hirobe et al., 2002). This indirect action of UVB could explain its increased efficiency in inducing repigmentation by comparison with UVA, even though UVA radiation can penetrate deeper into the dermis and directly stimulate the melanocytes located in the hair follicle niche.

Vitiligo There is increasing evidence that UVB therapy is superior to UVA in treating vitiligo. Many studies have demonstrated the efficiency of PUVA therapy in this indication; however, there are specific contraindications associated with it and a higher risk of side effects, including skin carcinomas (Garland et al., 2003; Lindelof et al., 1999; Stern, 2001; Stern et al., 1997). It is important to note that long-term follow-up of UVB therapy is much more limited than UVA, but, by means of a dose–response model, it has been calculated that long-term NB-UVB therapy may carry substantially less risk for skin cancer than PUVA therapy (Slaper et al., 1986). A metaanalysis of the literature concludes that UVB therapy is the most effective and safe therapy for generalized vitiligo (Njoo et al., 1998). However, comparison of all of the studies must

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be undertaken carefully, as great variability is apparent in the patient population (particularly localization, age, and skin type) and in the duration of treatment.

Broadband UVB Only a few studies have evaluated the use of broadband UVB therapy in the treatment of vitiligo. In the first, 8 of the 14 patients (57.1%) showed repigmentation of at least 75% at the end of 12 months of treatment (Koster and Wiskemann, 1990). However, these results were essentially obtained in Fitzpatrick skin types IV–VI. These interesting results are not confirmed by a recent intraindividual comparative study that showed 22% of patients treated by NB-UVB achieved a repigmentation of at least 75% after 12 months of treatment against none with broadband UVB (Anke Hartmann, 2004). The latter study involved only ten patients, and the localization of the treated lesions was different between the two groups (upper part of the body for NB-UVB and lower part for broadband).

Narrowband UVB First results obtained with NB-UVB were also very promising. In a retrospective study, five of the seven treated patients achieved more than 75% repigmentation following a mean of 19 treatments (Scherschun et al., 2001). In an open trial involving 51 children with generalized vitiligo, 53% of the patients showed at least 75% repigmentation after 1 year of twice-weekly NB-UVB treatment (Njoo et al., 2000). On the other hand, in another study only two of nine patients achieved 75% repigmentation after 12 months of treatment (Anke Hartmann, 2004). In a larger study, 5 of 60 patients (8%) achieved 75% repigmentation after three months of treatment and 32 of the 51 other patients (63%) showed at least 75% repigmentation after 12 months of treatment (Westerhof and Nieuweboer-Krobotova, 1997). In the last few years, new devices for delivering UVB light have been developed to treat localized vitiligo. An open study with NB-UVB micro-phototherapy has shown 510 of 734 patients (69.5%) achieving more than 75% of repigmentation after 12 months of treatment (Menchini et al., 2003). No sideeffects were reported. The device produces a focused beam of NB-UVB, which allows selective treatment of the vitiligo lesions. However, these promising results have to be confirmed in a control prospective study. Another approach was developed using a 308 nm monochromatic excimer lamp. In a pilot study, 12 of 37 patients (32.4%) showed more than 75% repigmentation after three months of treatment and 18 patients (48.6%) after six months (Leone et al., 2003). Side effects were limited to transient erythema. These results are encouraging and seem to be similar to those obtained with 308 nm excimer lasers. The use of a lamp instead of a laser decreases the cost of the device and allows larger surfaces to be treated; however, the device does not use a coherent light and selective treatment with sparing of healthy surrounding skin is not possible.

UVB THERAPY FOR PIGMENTARY DISORDERS

308 nm Excimer Laser The efficiency of the 308 nm excimer laser in the treatment of vitiligo was first reported by Baltas et al. (2001). Since then, many studies have shown the potential of this device in the treatment of vitiligo (Baltas et al., 2002; Esposito et al., 2004; Ostovari et al., 2004; Spencer et al., 2002; Taneja et al., 2003). Only low fluencies are used (starting at between 50 mJ/cm2 and 200 mJ/cm2). Sessions were carried out two or three times a week for one to six months depending on the series. The number of lesions which showed repigmentation at the end of the treatment was excellent (79–100%). However, except in one study, the percentage of treated lesions achieving at least 75% repigmentation was about 30%. Among the factors which influence the therapeutic response, localization of the lesions plays a key role. In their study, Taneja et al. (2003) obtained at least 75% repigmentation in all lesions situated on the face versus none on the hands or the feet. In our series, we found significant statistically inferior results for “UV resistant” areas (extremities and bony protuberances) by comparison with the rest of the body (Ostovari et al., 2004). The stability of the repigmentation with time has so far been difficult to evaluate, as follow-up of the patients is poor or nil; however, one recent series showed an absence of depigmentation of the treated lesions after one year (Esposito et al., 2004). Finally, patients’ tolerance of the treatment is usually good; side effects are limited to erythema and rare bullous lesions. Thus, the 308 nm excimer laser appears to be an efficient and well-tolerated treatment for localized vitiligo.

Combination Therapies Interest in combination treatments was first demonstrated with the combination of UVA and topical steroids. In a prospective, randomized, controlled, left–right comparison study, it was shown that the combination of UVA and fluticasone propionate was much more effective than UVA or topical steroid alone (Westerhof et al., 1999). To the best of our knowledge, the combination of UVB therapy with topical steroids has not yet been evaluated, although some series have studied the association of UVB and other synergistic drugs. Oxidative stress has been shown to be involved in the pathogenesis of vitiligo. Pseudocatalase has the ability to remove hydrogen peroxide and so could be interesting in the treatment of vitiligo. The combination of topical pseudocatalase with UVB showed promising results in a pilot study (complete repigmentation on the face and the dorsum of the hands in 90% of patients) (Schallreuter et al., 1995). Unfortunately, these results were not confirmed in a recent study (Patel et al., 2002). The occurrence of repigmentation of vitiligo in patients treated with calcipotriol (a vitamin D3 analog) for psoriasis has suggested that it might be efficacious in treating vitiligo. The use of calcipotriol with sun or PUVA therapy has provided some interesting rates of repigmentation. However, the results are controversial (Baysal et al., 2003; Ermis et al., 2001; Parsad et al., 1998). To date, only one study with calcipotriol and UVB has been reported. The results confirmed

the efficiency of NB-UVB and its superiority over broadband UVB for treating vitiligo, but also showed that the combination of calcipotriol with UVB had no enhancing effect on repigmentation (Anke Hartmann, 2004). Tacrolimus ointment has recently shown some interesting results in the treatment of vitiligo (Lepe et al., 2003). However, the best results were achieved in sun-exposed areas. Two recent studies have evaluated if the combination of a 308 nm excimer laser and topical tacrolimus could be synergistic. These compared the efficiency of a 308 nm excimer combined with tacrolimus ointment with excimer laser monotherapy (Passeron et al., 2004) or excimer laser monotherapy associated with placebo ointment (Kawalek et al., 2004). In both cases, a total of 24 sessions were completed, and tacrolimus ointment 0.1% was applied twice a day. The results were similar and showed that the combined treatment was more efficient than laser used alone. Tolerance was good and side effects were limited to constant erythema, tingling, and rare bullous lesions. These encouraging results are corroborated by two other reports associating UVB light and topical tacrolimus (Castanedo-Cazares et al., 2003; Tanghetti and Gillis, 2003b). However, the increased risk of skin cancers — promoted by the association of two immunosuppressive treatments — cannot be excluded. In the meanwhile, until long-term follow-up is available, this association should be reserved to control studies.

Other Hypopigmented Disorders Postsurgical Leukoderma Although no prospective studies have been conducted as yet, two cases of post-resurfacing leukoderma have been successfully treated with a 308 nm excimer laser (Friedman and Geronemus, 2001). An improvement of at least 75% was obtained in eight sessions in the first case and in ten sessions in the second one, without any depigmentation after one month follow-up. In one other case of persistent laser-induced hypopigmentation after tattoo removal, repigmentation was achieved in 40 sessions using a 308 nm excimer laser (Gundogan et al., 2004). In all cases, no adverse events were noted and the selectivity of the treatment prevented perilesional hyperpigmentation.

Hypopigmented Mycosis Fungoides UVB phototherapy is one of the best treatments available for cutaneous T lymphomas. Hypopigmentation can occur in mycosis fungoides and in Sézary syndrome. The use of NBUVB therapy has been shown to both improve the lymphocytic infiltrate and hypopigmentation (Akaraphanth et al., 2000; Gathers et al., 2002).

Mature Hypopigmented Striae An open prospective study on 75 patients evaluated the repigmentation of mature hypopigmented striae with a 308 nm

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excimer laser (Goldberg et al., 2003). Increased pigmentation was observed after eight sessions, leading to esthetic improvements for 80% of patients. The absence of a control group and the weakness of the evaluation methods moderate these encouraging results, especially as maintenance sessions will probably be necessary to sustain the results. However, this technique remains one of the few possible therapeutic approaches for this frequent esthetical problem.

References Akaraphanth, R., M. C. Douglass, and H. W. Lim. Hypopigmented mycosis fungoides: treatment and a 6(1/2)-year follow-up of 9 patients. J. Am. Acad. Dermatol. 42(1 Pt 1):33–39, 2000. Anke Hartmann, C. L., H. Hamm, E.-B. Bröcker, and U. B. Hofmann. Narrow-band UVB311 nm vs. broad-band UVB therapy in combination with topical calcipotriol vs. placebo in vitiligo. Int. J. Dermatol. 44:736–742, 2005. Baltas, E., Z. Csoma, F. Ignacz, A. Dobozy, and L. Kemeny. Treatment of vitiligo with the 308-nm xenon chloride excimer laser. Arch. Dermatol. 138:1619–1620, 2002. Baltas, E., P. Nagy, B. Bonis, Z. Novak, F. Ignacz, G. Szabo, Z. Bor, A. Dobozy, and L. Kemeny. Repigmentation of localized vitiligo with the xenon chloride laser. Br. J. Dermatol. 144:1266–1267, 2001. Baysal, V., M. Yildirim, A. Erel, and D. Kesici. Is the combination of calcipotriol and PUVA effective in vitiligo? J. Eur. Acad. Dermatol. Venereol. 17:299–302, 2003. Castanedo-Cazares, J. P., V. Lepe, and B. Moncada. Repigmentation of chronic vitiligo lesions by following tacrolimus plus ultravioletB-narrow-band. Photodermatol. Photoimmunol. Photomed. 19:35–36, 2003. Chakraborty, A. K., Y. Funasaka, A. Slominski, J. Bolognia, S. Sodi, M. Ichihashi, and J. M. Pawelek. UV light and MSH receptors. Ann. N. Y. Acad. Sci. 885:100–116, 1999. Danno, K., K. Toda, and T. Horio. Ultraviolet-B radiation suppresses mast cell degranulation induced by compound 48/80. J. Invest. Dermatol. 87:775–778, 1986. Eller, M. S., and B. A. Gilchrest. Tanning as part of the eukaryotic SOS response. Pigment Cell Res. 13(Suppl 8):94–97, 2000. Enk, A. H., V. L. Angeloni, M. C. Udey, and S. I. Katz. Inhibition of Langerhans cell antigen-presenting function by IL-10. A role for IL-10 in induction of tolerance. J. Immunol. 151:2390–2398, 1993. Ermis, O., E. Alpsoy, L. Cetin, and E. Yilmaz. Is the efficacy of psoralen plus ultraviolet A therapy for vitiligo enhanced by concurrent topical calcipotriol? A placebo-controlled double-blind study. Br. J. Dermatol. 145:472–475, 2001. Esposito, M., R. Soda, A. Costanzo, and S. Chimenti. Treatment of vitiligo with the 308 nm excimer laser. Clin. Exp. Dermatol. 29:133–137, 2004. Friedman, P. M., and R. G. Geronemus. Use of the 308-nm excimer laser for postresurfacing leukoderma. Arch. Dermatol. 137:824– 825, 2001. Garland, C. F., F. C. Garland, and E. D. Gorham. Epidemiologic evidence for different roles of ultraviolet A and B radiation in melanoma mortality rates. Ann. Epidemiol. 13:395–404, 2003. Gathers, R. C., L. Scherschun, F. Malick, D. P. Fivenson, and H. W. Lim. Narrowband UVB phototherapy for early-stage mycosis fungoides. J. Am. Acad. Dermatol. 47:191–197, 2002. Goldberg, D. J., D. Sarradet, and M. Hussain. 308-nm Excimer laser treatment of mature hypopigmented striae. Dermatol. Surg. 29:596–598; discussion 598–599, 2003. Gundogan, C., B. Greve, I. Hausser, and C. Raulin. [Repigmentation of persistent laser-induced hypopigmentation with an excimer laser following tattoo removal]. Hautarzt 55:549–552, 2004.

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Gutierrez-Steil, C., T. Wrone-Smith, X. Sun, J. G. Krueger, T. Coven, and B. J. Nickoloff. Sunlight-induced basal cell carcinoma tumor cells and ultraviolet-B-irradiated psoriatic plaques express Fas ligand (CD95L). J. Clin. Invest. 101:33–39, 1998. Hirobe, T., R. Furuya, S. Akiu, O. Ifuku, and M. Fukuda. Keratinocytes control the proliferation and differentiation of cultured epidermal melanocytes from ultraviolet radiation Binduced pigmented spots in the dorsal skin of hairless mice. Pigment Cell Res. 15:391–399, 2002. Kawalek, A. Z., J. M. Spencer, and R. G. Phelps. Combined excimer laser and topical tacrolimus for the treatment of vitiligo: a pilot study. Dermatol. Surg. 30(2 Pt 1):130–135, 2004. Koster, W., and A. Wiskemann. [Phototherapy with UV-B in vitiligo]. Z. Hautkr. 65:1022–1024, 1029, 1990. Leone, G., P. Iacovelli, A. Paro Vidolin, and M. Picardo. Monochromatic excimer light 308 nm in the treatment of vitiligo: a pilot study. J. Eur. Acad. Dermatol. Venereol. 17:531–537, 2003. Lepe, V., B. Moncada, J. P. Castanedo-Cazares, M. B. Torres-Alvares, C. A. Ortiz, and A. B. Torres-Rubalcava. A double-blind randomized trial of 0.1% tacrolimus vs 0.05% clobetasol for the treatment of childhood vitiligo. Arch. Dermatol. 139:581–585, 2003. Lindelof, B., B. Sigurgeirsson, E. Tegner, O. Larko, A. Johannesson, B. Berne, B. Ljunggren, T. Andersson, L. Molin, E. NylanderLundqvist, and L. Emtestam. PUVA and cancer risk: the Swedish follow-up study. Br. J. Dermatol. 141:108–112, 1999. Luger, T. A., T. Schwarz, H. Kalden, T. Scholzen, A. Schwarz, and T. Brzoska. Role of epidermal cell-derived alpha-melanocyte stimulating hormone in ultraviolet light mediated local immunosuppression. Ann. N. Y. Acad. Sci. 885:209–216, 1999. Menchini, G., E. Tsoureli-Nikita, and J. Hercogova. Narrow-band UV-B micro-phototherapy: a new treatment for vitiligo. J. Eur. Acad. Dermatol. Venereol. 17:171–177, 2003. Miyauchi, H., and T. Horio. Ultraviolet B-induced local immunosuppression of contact hypersensitivity is modulated by ultraviolet irradiation and hapten application. J. Invest. Dermatol. 104:364–369, 1995. Miyauchi-Hashimoto, H., and T. Horio. Suppressive effect of ultraviolet B radiation on contact sensitization in mice. II. Systemic immunosuppression is modulated by ultraviolet irradiation and hapten application. Photodermatol. Photoimmunol. Photomed. 12:137–144, 1996. Njoo, M. D., P. I. Spuls, J. D. Bos, W. Westerhof, and P. M. Bossuyt. Nonsurgical repigmentation therapies in vitiligo. Meta-analysis of the literature. Arch. Dermatol. 134:1532–1540, 1998. Njoo, M. D., J. D. Bos, and W. Westerhof. Treatment of generalized vitiligo in children with narrow-band (TL-01) UVB radiation therapy. J. Am. Acad. Dermatol. 42(2 Pt 1):245–253, 2000. Novak, Z., B. Bonis, E. Baltas, I. Ocsovszki, F. Ignacz, A. Dobozy, and L. Kemeny. Xenon chloride ultraviolet B laser is more effective in treating psoriasis and in inducing T cell apoptosis than narrowband ultraviolet B. J. Photochem. Photobiol. B 67:32–38, 2002. Ongenae, K., N. Van Geel, and J. M. Naeyaert. Evidence for an autoimmune pathogenesis of vitiligo. Pigment Cell Res. 16:90–100, 2003. Ostovari, N., T. Passeron, W. Zakaria, E. Fontas, J. C. Larouy, J. F. Blot, J. P. Lacour, and J. P. Ortonne. Treatment of vitiligo by 308nm excimer laser: an evaluation of variables affecting treatment response. Lasers Surg. Med. 35:152–156, 2004. Parrish, J. A.. Phototherapy and photochemotherapy of skin diseases. J. Invest. Dermatol. 77:167–171, 1981. Parsad, D., R. Saini, and N. Verma. Combination of PUVAsol and topical calcipotriol in vitiligo. Dermatology 197:167–170, 1998. Passeron, T., N. Ostovari, W. Zakaria, E. Fontas, J. C. Larrouy, J. P. Lacour, and J. P. Ortonne. Topical tacrolimus and the 308-nm excimer laser: a synergistic combination for the treatment of vitiligo. Arch. Dermatol. 140:1065–1069, 2004.

UVB THERAPY FOR PIGMENTARY DISORDERS Patel, D. C., A. V. Evans, and J. L. Hawk. Topical pseudocatalase mousse and narrowband UVB phototherapy is not effective for vitiligo: an open, single-centre study. Clin. Exp. Dermatol. 27:641–644, 2002. Romero-Graillet, C., E. Aberdam, M. Clement, J. P. Ortonne, and R. Ballotti. Nitric oxide produced by ultraviolet-irradiated keratinocytes stimulates melanogenesis. J. Clin. Invest. 99:635–642, 1997. Schallreuter, K. U., J. M. Wood, K. R. Lemke, and C. Levenig. Treatment of vitiligo with a topical application of pseudocatalase and calcium in combination with short-term UVB exposure: a case study on 33 patients. Dermatology 190:223–229, 1995. Scherschun, L., J. J. Kim, and H. W. Lim. Narrow-band ultraviolet B is a useful and well-tolerated treatment for vitiligo. J. Am. Acad. Dermatol. 44:999–1003, 2001. Shreedhar, V., T. Giese, V. W. Sung, and S. E. Ullrich. A cytokine cascade including prostaglandin E2, IL-4, and IL-10 is responsible for UV-induced systemic immune suppression. J. Immunol. 160:3783–3789, 1998. Slaper, H., A. A. Schothorst, and J. C. van der Luen. Risk evaluation of UVB therapy for psoriasis: comparison of calculated risk for UVB therapy and observed risk in PUVA-treated patients. Photodermatology 3:271–283, 1986. Spencer, J. M., R. Nossa, and J. Ajmeri. Treatment of vitiligo with the 308-nm excimer laser: a pilot study. J. Am. Acad. Dermatol. 46:727–731, 2002. Stern, R. S. The risk of melanoma in association with long-term exposure to PUVA. J. Am. Acad. Dermatol. 44:755–761, 2001. Stern, R. S., K. T. Nichols, and L. H. Vakeva. Malignant melanoma in patients treated for psoriasis with methoxsalen (psoralen) and ultraviolet A radiation (PUVA). The PUVA Follow-Up Study. N. Engl. J. Med. 336:1041–1045, 1997. Taneja, A., M. Trehan, and C. R. Taylor. 308-nm excimer laser for the treatment of localized vitiligo. Int. J. Dermatol. 42:658–662, 2003.

Tanghetti, E., and P. R. Gillis. Photometric and clinical assessment of localized UVB phototherapy systems for the high-dosage treatment of stable plaque psoriasis. J. Cosmet. Laser Ther. 5:101–106, 2003a. Tanghetti, E. A., and P. R. Gillis. Clinical evaluation of B Clear and Protopic treatment for vitiligo. Lasers Surg. Med. 32(S15): 37, 2003b. Tjioe, M., T. Smits, P. C. van de Kerkhof, and M. J. Gerritsen. The differential effect of broad band vs narrow band UVB with respect to photodamage and cutaneous inflammation. Exp. Dermatol. 12:729–733, 2003. Toda, K., Y. Miyachi, N. Nesumi, J. Konishi, and S. Imamura. UVB/PUVA-induced suppression of human natural killer activity is reduced by superoxide dismutase and/or interleukin 2 in vitro. J. Invest. Dermatol. 86:519–522, 1986. Toews, G. B., P. R. Bergstresser, and J. W. Streilein. Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J. Immunol. 124:445–453, 1980. Westerhof, W., and L. Nieuweboer-Krobotova. Treatment of vitiligo with UV-B radiation vs topical psoralen plus UV-A. Arch. Dermatol. 133:1525–1528, 1997. Westerhof, W., L. Nieuweboer-Krobotova, P. G. Mulder, and E. J. Glazenburg. Left-right comparison study of the combination of fluticasone propionate and UV-A vs. either fluticasone propionate or UV-A alone for the long-term treatment of vitiligo. Arch. Dermatol. 135:1061–1066, 1999. Yamawaki, M., S. Futamura, and T. Horio. UVB radiation suppresses the TNF-alpha-induced expression of E-selectin and ICAM-1 on cultured human umbilical vein endothelial cells. J. Dermatol. Sci. 13:11–17, 1996. Yoshikawa, T., and J. W. Streilein. Tumor necrosis factor-alpha and ultraviolet B light have similar effects on contact hypersensitivity in mice. Reg. Immunol. 3:139–144, 1990.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Sunscreens and Cosmetics James J. Nordlund and Rebat M. Halder

Sunscreens The cutaneous pigmentary system is markedly affected by electromagnetic radiation. There are two spectra of ultraviolet light that stimulate production of pigmentation in the skin, the process called tanning. They are ultraviolet A (UVA) defined as the spectrum from 320 nm to 400 nm and ultraviolet B (UVB) defined as the spectrum from 290 nm to 320 nm. Ultraviolet C (UVC) is the spectrum from 290 nm to 250 nm. UVC is emitted by the sun but is absorbed by the ozone layer and none reaches the surface of the earth. In contrast large amounts of UVB strike the earth. There is about 1000-fold more UVA in sunlight than UVB. Suntan is caused by two mechanisms: immediate pigment darkening and the production of melanin (Agin et al., 1985; Porges et al., 1988). Immediate pigment darkening is thought to be due mainly to oxidation of the melanin polymer (Beitner and Wennersten, 1985; Honigsmann et al., 1986) within the skin although rearrangement of melanosomes within keratinocytes might also be involved (Lavker and Kaidbey, 1982). It occurs within minutes after exposure to ultraviolet light and reverses within 24 hours after exposure is stopped (Porges et al., 1988). In contrast delayed tanning dependent on melanin synthesis takes several days but persists for prolonged periods of time, up to many weeks (Porges et al., 1988). UVA is especially efficient in causing immediate pigment darkening (Irwin et al., 1993; Kaidbey and Barnes, 1991; Lavker and Kaidbey, 1982; Porges et al., 1988) but can cause melanin synthesis as well. UVB is responsible for delayed tanning that starts about 72 hours after exposure to sunlight. It is caused by enhanced synthesis of melanin and transfer of melanosomes into the surrounding keratinocytes. The mechanisms responsible for stimulating melanin formation are complex. Because sunlight can profoundly affect melanin production and skin color, it is used to treat skin conditions of hypopigmentation like vitiligo and pityriasis alba. It is also important to prevent sunlight from penetrating to skin that is hyperpigmented, such as melasma (Balina and Graupe, 1991; Pathak et al., 1986; Vazquez and Sanchez, 1983; Verallo-Rowell et al., 1989). The best way to stop sunlight from penetrating the skin is by application of sunscreens. The potency of sunscreen or its ability to prevent ultraviolet light from penetrating the epidermis can be measured (Lowe and Frielander, 1995). The potency is called Sun Protective Factor or SPF. An SPF of 2 means that it takes twice as long for a sunburn to develop in the treated skin than in untreated skin. That is, the sunscreen absorbed about half of 1188

the incident light. An SPF of 4 means that it takes four times more exposure to sun to develop a burn in treated skin than in unprotected skin. About a quarter of the incident light penetrates the epidermis. Sunscreens have potencies of 50 and higher. An SPF of 25 absorbs all but 4% of the sunlight. A sunscreen with an SPF of 50 absorbs all but 2% of the ultraviolet light. Practically the difference is not important. Thus a sunscreen with an SPF of 20–25 is considered to have maximum practical protection. The SPF is a measure only of absorption of UVB. Most UVA penetrates through the sunscreen although a moderate amount of the shorter UVA in the 320–340 nm spectrum is absorbed (Cole, 1994; Diffey and Farr, 1991; Gange et al., 1986; Leenutaphong, 1992; Lowe et al., 1987; Roelandts, 1991). There is one chemical called parasol available for protection against UVA (Anonymous, 1989, 1993; Leenutaphong, 1992; Lowe and Frielander, 1995). There are few commercial products containing UVB screens and parasol for complete protection against the effects of UVA and UVB (Anonymous, 1993). Despite this, it is important for those being treated with conditions of hyperpigmentation such as melasma to use sunscreens to block some of the tanning effects of sunlight. Those with depigmenting conditions such as vitiligo, piebaldism, and albinism must use sunscreen to prevent burning. These individuals have lost the sun-protective effects of the pigmentary system. Their skin requires some other form of protection against the burning effects of sunlight. Clothing can be an excellent sunscreen but is not practical during the hot summer months. Sunscreen can act as a substitute for protective clothes. Treatment of individuals with hyperpigmentation such as melasma (Balina and Graupe, 1991; Pathak et al., 1986; Vazquez and Sanchez, 1983; Verallo-Rowell et al., 1989) requires the use of sunscreens. Postinflammatory hyperpigmentation on exposed skin also is benefited by applications of sunscreens (Bekhor, 1995; Ho et al., 1995; Leenutaphong, 1992; Pathak et al., 1986).

Other Therapies for Pigmentary Disorders Cosmetics In darker-skinned individuals cover-up cosmetics (Dermablend, Covermark) can hide the vitiligo area with remarkable color matching and are often used between treatments with other modalities (Figs 62.1 and 62.2). Because they can be rubbed off easily, they are best used on areas that are subject to minimal contact such as the face. Camouflage stains

SUNSCREENS AND COSMETICS

Fig. 62.1. A woman with extensive vitiligo.

Fig. 62.2. Woman in Figure 62.1 after application of cosmetics. Her appearance is excellent.

(Vitadye, Dyoderm), on the other hand, have the advantage of being long-lasting but usually do not offer exact color matching and may interfere with treatment using UV light. They are best used for limited areas that are subject to constant friction. Many women wear cosmetics daily to cover pigmentary blemishes. Many cosmetics contain sunscreens. These combination products seem especially useful to protect the skin from pigmentary changes.

Cole, C. Multicenter evaluation of sunscreen UVA protectiveness with the protection factor test method. J. Am. Acad. Dermatol. 30:729–736, 1994. Diffey, B. L., and P. M. Farr. Sunscreen protection against UVB, UVA and blue light: an in vivo and in vitro comparison [published erratum appears in Br. J. Dermatol. 125:609, 1991]. Br. J. Dermatol. 124:258–263, 1991. Gange, R. W., A. Soparkar, E. Matzinger, S. H. Dromgoole, J. Sefton, and R. DeGryse. Efficacy of a sunscreen containing butyl methoxydibenzoylmethane against ultraviolet A radiation in photosensitized subjects. J. Am. Acad. Dermatol. 15:494–499, 1986. Ho, C., Q. Nguyen, N. J. Lowe, M. E. Griffin, and G. Lask. Laser resurfacing in pigmented skin. Dermatol. Surg. 21:1035–1037, 1995. Honigsmann, H., G. Schuler, W. Aberer, N. Romani, and K. Wolff. Immediate pigment darkening phenomenon. A reevaluation of its mechanisms. J. Invest. Dermatol. 87:648–652, 1986. Irwin, C., A. Barnes, D. Veres, and K. Kaidbey. An ultraviolet radiation action spectrum for immediate pigment darkening. Photochem. Photobiol. 57:504–507, 1993. Kaidbey, K. H., and A. Barnes. Determination of UVA protection factors by means of immediate pigment darkening in normal skin. J. Am. Acad. Dermatol. 25:262–266, 1991. Lavker, R. M., and K. H. Kaidbey. Redistribution of melanosomal complexes within keratinocytes following UV-A irradiation: a possible mechanism for cutaneous darkening in man. Arch. Dermatol. Res. 272:215–228, 1982. Leenutaphong, V. Evaluating the UVA protection of commercially available sunscreens. J. Med. Assoc. Thai. 75:619–624, 1992.

References Agin, P. P., D. L. Desrochers, and R. M. Sayre. The relationship of immediate pigment darkening to minimal erythemal dose, skin type, and eye color. Photodermatology 2:288–294, 1985. Anonymous. Photoplex — a broad spectrum sunscreen. Med. Lett. Drugs Ther. 31:59–60, 1989. Anonymous. Shade UVAGuard — a second broad-spectrum sunscreen. Med. Lett. Drugs Ther. 35:53–54, 1993. Balina, L. M., and K. Graupe. The treatment of melasma. 20% azelaic acid versus 4% hydroquinone cream. Int. J. Dermatol. 30:893–895, 1991. Beitner, H., and G. Wennersten. A qualitative and quantitative transmission electronmicroscopic study of the immediate pigment darkening reaction. Photodermatology 2:273–278, 1985. Bekhor, P. S. The role of pulsed laser in the management of cosmetically significant pigmented lesions [Review]. Australas. J. Dermatol. 36:221–223, 1995.

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CHAPTER 62 Lowe, N. J., and J. Frielander. Prevention of photodamage with sunprotection and sunscreens. In: Photodamage, B. A. Gilchrest (ed.). Cambridge, MA: Blackwell Science, Inc., 1995, p. 295. Lowe, N. J., S. H. Dromgoole, J. Sefton, T. Bourget, and D. Weingarten. Indoor and outdoor efficacy testing of a broad-spectrum sunscreen against ultraviolet A radiation in psoralen-sensitized subjects. J. Am. Acad. Dermatol. 17:224–230, 1987. Pathak, M. A., T. B. Fitzpatrick, and E. W. Kraus. Usefulness of retinoic acid in the treatment of melasma. J. Am. Acad. Dermatol. 15:894–899, 1986. Porges, S. B., K. H. Kaidbey, and G. L. Grove. Quantification of visible

1190

light-induced melanogenesis in human skin. Photodermatology 5:197–200, 1988. Roelandts, R. Which components in broad-spectrum sunscreens are most necessary for adequate UVA protection? J. Am. Acad. Dermatol. 25:999–1004, 1991. Vazquez, M., and J. L. Sanchez. The efficacy of a broad-spectrum sunscreen in the treatment of melasma. Cutis 32:92–96, 1983. Verallo-Rowell, V. M., V. Verallo, K. Graupe, L. Villafuerte, and M. Garcia-Lopez. Double-blind comparison of azelaic acid and hydroquinone in the treatment of melasma. Acta Derm. Venereol. 143(Suppl):58–61, 1989.

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

63

Surgical Treatments of Pigmentary Disorders Rebat M. Halder and James J. Nordlund

Repopulation of Melanocytes by Grafting The possibility of treating vitiligo or other forms of depigmentation with “exchange grafts” using the patient’s own clinically normal skin was first recognized in 1952 (Spencer and Tolmach, 1952). Since then, various adaptations of this basic concept have evolved into rather promising additions to the armamentarium for the treatment of depigmentation. Successful repigmentation using these modalities is dependent on proper case selection of patients, especially those with vitiligo. Most importantly, only patients in whom the vitiligo is stable (no progression within four to six months) should be considered as good candidates for transplantation. In this respect, patients with segmental vitiligo are ideal candidates because this type of vitiligo, once established, tends not to progress. Many of these individuals have white hairs within the patch, an indication that the reservoir of melanocytes is destroyed (see Chapter X). Following these selection criteria usually yields relatively high success rates.

Autologous Epidermal Grafting This technique was described in a report published in 1964. It was found that viable epidermis could be separated from dermis in vivo by induction of suction blister by applying negative pressure at 200 mm Hg to the skin for three to four hours (Kiistala and Mustakallio, 1964). Dermal epidermal separation occurs through the level of the lamina lucida. Successful repigmentation of vitiliginous skin was achieved when the resulting melanocyte-bearing blister top was used as a graft, dermal side down, and placed onto recipient area of depigmentation previously denuded by blistering via suction, applications of liquid nitrogen, or by topical UVA (Falabella, 1989; Kiistala and Mustakallio, 1964; Koga, 1988; Suvanprakorn et al., 1985). Pigmentation usually develops in three to six months. There may be areas of achromic fissures between grafts in the recipient areas. Koebnerization at donor sites has been reported in conjunction with depigmentation of transplanted grafts in patients with unstable vitiligo (Hatchome et al., 1990). This technique has been used to treat leukoderma following burns, vitiligo, and piebaldism. Results of treatment of segmented vitiligo are better than of treatment of generalized vitiligo (Koga, 1988). However, most physicians do not have the mechanical apparatus needed for the production of the

blisters at the donor site. Although this technique is time consuming and requires special equipment, its attractive features include the lack of scarring in both donor and recipient sites and the regeneration of new pigmented skin at donor sites. The same sites can be used as a source of melanocytes every few months.

Autologous Thin Thiersch Grafting The application of these thin split-thickness grafts in the treatment of vitiligo was first introduced in 1964 (Behl, 1964). Strips of these grafts obtained using the scalpel or dermatome are placed onto recipient sites prepared in a similar manner or by dermabrasion. Achromic areas ranging in size from 6 cm2 to 100 cm2 were treated. Patients were satisfied with the results in more than 85% of cases although objective evaluations were not done. The same method was reported as successful in over 2500 patients treated for vitiligo over a 20-year period (Behl, 1984, 1985; Behl and Bhatia, 1973). Larger areas were grafted with an average of 180–300 cm2 done at one time. Post-burn leukoderma was treated in 32 patients by using 0.1–0.2 mm thin split-thickness grafts obtained by hand dermatome (Taki et al., 1985). All patients responded with 95–100% repigmentation. Recently the technique of thin splitthickness grafts has been modified for the treatment of vitiligo with the harvesting of grafts by mechanical dermatome with excellent results (Kahn and Cohen, 1995; Kahn et al., 1993). This technique has also been used to successfully treat vitiligo of the lip (Chitale, 1991). The advantage of this technique is it allows the grafting of large areas in a relatively short time. However, this must be weighed against the need for general anesthesia and the risk of hypertrophic scarring of both donor and recipient sites (Falabella, 1989).

Suction Blister Grafts Separation of viable epidermis from dermis can be accomplished via the production of suction blisters at 200 mm Hg negative pressure in three to four hours (Kiistala, 1968; Kiistala and Mustakallio, 1964). This finding was adapted to the treatment of achromic areas of skin where pigmented epidermis is harvested by this technique and is used to cover achromic areas that have been prepared by denuding them with liquid nitrogen blisters (Falabella, 1971, 1984). Melanocytes are contained within the top of 2–2.5 cm suction blisters. The tops of these blisters are removed with iris scissors and are directly applied to the denuded area of achromic skin. These are placed in a mosaic type of pattern. Healing 1191

CHAPTER 63

Fig. 63.1. Minigrafts placed in a depigmented patch on the ankle of a young man have successfully repigmented the area.

occurs in seven days in both donor and grafted sites. Grafted areas are covered with Telfa dressing and elastic bandages. Pigmentation usually develops in three to six months. There may be areas of achromic fissures between grafts in the recipient areas. This technique has been used to treat leukoderma following burns, vitiligo, and piebaldism. Results of treatment of segmented vitiligo are better than of nonsegmented vitiligo (Koga, 1988). An advantage of suction blister grafts is that scarring is minimal, as the dermis is left intact in both donor and recipient sites. However, most physicians do not have the mechanical apparatus needed for the production of the blisters at the donor site.

Autologous Minigrafts This technique was initially reported in one patient for the treatment of depigmentation secondary to a thermal burn. The investigators used a 2 mm punch to obtain grafts from normally pigmented skin that were transplanted into the achromic patch (Orentreich and Selmanowitz, 1972). The technique was subsequently reported to be successful in repigmenting leukodermic areas of the scalp in a patient with scarring alopecia secondary to discoid lupus erythematosus (Lobuono and Shatin, 1976) (Fig. 63.1). This technique was later found to be of benefit in two patients with vitiligo who also received PUVA therapy (Bonafe et al., 1983). This technique has been refined over the years (Falabella, 1978, 1983, 1984, 1986, 1987, 1988, 1989) and presently can be used for stable cases of leukoderma including segmental or localized vitiligo (Fig. 63.1). Post-burn achromia, piebaldism, post-dermabrasion achromia, and depigmentation from monobenzyl ether of hydroquinone can be treated by this technique (Selmanowitz, 1979; Selmanowitz et al., 1977). This technique places islands of normally pigmented skin within achromic or depigmented areas from burns or vitiligo (Falabella, 1989). The islands of pigment enlarge up to 25 times the original surface area of a 1 mm or 2 mm punch graft. A 1192

gradual process of pigment migration takes place during a period of three to six months (Falabella, 1989). The actual technique involves the harvesting of 1.2–2.0 mm punch grafts from the pigmented donor site, usually an area on the lower back below the waistline (Falabella, 1988). They can be harvested virtually next to each other. A 2 ¥ 2 cm area can yield up to 100 minigrafts. The grafts are placed 3–4 mm from each other in the recipient site where the site is prepared by making defects with the same size punch. The grafts are embedded by manual compression with saline soaked gauze and are held in place by Steri strips (cover strips) which are removed in seven days. PUVA therapy given subsequent to autologous minigrafting can hasten the repigmentation process (Skouge et al., 1992). Complications of this technique include cobblestoning, infection at the donor or recipient site, and koebnerization of the donor site. The success of autologous minigrafting is high, if the patient is selected carefully.

Autologous Mini Punch Grafts This technique is analogous to hair transplantation in that skin punches are used. The concept of donor dominance, however, is not applicable to vitiligo since viable transplanted grafts can lose their pigment even in cases where vitiligo appears stable. In 1972, 2 mm punch grafts of normally pigmented skin were used to repigment leukodermic skin (Orentreich and Selmanowitz, 1972). Grafts of 10 ¥ 3 mm with PUVA have been used by others to successfully repigment vitiliginous skin (Bonafe et al., 1983). The technique was refined by using 1.2–1.25 mm full thickness punch grafts placed 4–5 mm apart into comparable-sized recipient sites (Falabella, 1986, 1988). This graft size was found to minimize both the “cobblestoning” (trap door) effect and the cosmetic damage to the donor site that is especially prominent with larger-sized grafts, while containing enough pigment source to stimulate the spotty perifollicular repigmentation that is typically seen with spontaneous repigmentation or repigmentation with PUVA. The extent of maximal pigment spread is approximately four to five times the diameter of the graft and is reached after about four to six months without adjunctive therapy with PUVA (Figs 63.2–63.8). Resumption of the preoperative course of PUVA two weeks postoperatively can shorten this period to two to three months. Histologic studies have shown that the pigmentation that develops around either epidermal or punch grafts consists of normal melanin content and functional melanocytes (Falabella, 1988; Suvanprakorn et al., 1985).

Transplantation of Cultured Autologous Melanocytes The technique of transplanting melanocyte-containing cultures of cells has the theoretical advantage of potentially treating large areas using cells harvested from a small piece of donor skin by expanding the culture in vitro. Its major disadvantage lies in the complexities and cost of the culture systems and equipment required to achieve the culture. Thus

SURGICAL TREATMENTS OF PIGMENTARY DISORDERS

Fig. 63.2. Vitiligo on the forehead before grafting with 1 mm punches (see also Plate 63.1, pp. 494–495).

Fig. 63.3. The skin in Figure 63.2 immediately after the placement of grafts (see also Plate 63.2, pp. 494–495).

Fig. 63.5. The cheek of a woman before grafting with 1 mm punches.

Transplantation of Autologous Pure Melanocyte Culture

Fig. 63.4. The skin in Figures 63.2 and 63.3 after spread of the pigmentation showing excellent response (see also Plate 63.3, pp. 494–495).

this technique is impractical for most dermatologists. Also of concern is the uncertainty about what, if any, effects the chemicals used in culturing the cells have on the melanocytes and subsequently on the patient.

Melanocytes have been observed to survive and proliferate in a medium containing phorbol esters (Eisinger and Marko, 1982). Later, methods were developed to successfully grow melanocytes from individuals with vitiligo (Lerner, 1988; Lerner et al., 1987; Medrano and Nordlund, 1990). These techniques permitted the expansion of a small number of melanocytes into a sufficient number of cells to repigment large areas of depigmented skin. Cultured melanocytes are injected into blisters raised on achromic skin either by suction or by using a combination of liquid nitrogen and a heated oscillating disk. Although the number of cases treated in this way is small, it appears that successful repigmentation does occur. It has been noted that for success the vitiligo should be stable and the melanocyte suspension must contain about 5 ¥ 105 cells/1 cm blister. There is no pigment spread beyond the transplanted area and the color match was perfect. Ultrastructural studies showed that the transplanted melanocytes had localized to the basal layer of the epidermis and were producing and transferring pigment as in the adjacent “normal” 1193

CHAPTER 63

Fig. 63.6. The cheek of the woman in Figure 63.5 immediately after placement of 1 mm punch grafts.

Fig. 63.7. The skin in Figures 63.5 and 63.6 after partial repigmentation.

Fig. 63.8. The skin in Figures 63.5–63.7 after complete repigmentation.

Fig. 63.9. The hand of a patient with vitiligo before treatment with autologous melanocytes grown in culture.

skin (Lerner et al., 1990) (Figs 62.9–62.12). Subsequent to the initial reports of the success of melanocyte transplants for vitiligo, others have had similar successful results (Falabella et al., 1989, 1992; Gauthier and Surleve-Bazeille, 1992; Lontz

et al., 1994; Olsson and Juhlin, 1993; Olsson et al., 1994; Plott et al., 1989; Zachariae et al., 1993). This technique was initially limited by the fact that tissue plasminogen activator (TPA), the essential ingredient for

1194

SURGICAL TREATMENTS OF PIGMENTARY DISORDERS

grow well in the presence of TPA. However, the more recent reports have used growth modifiers other than TPA.

Transplantation of Autologous Melanocyte and Keratinocyte Cocultures

Fig. 63.10. The hand of the patient in Figure 63.9 after transplantation of cultured melanocytes. (Courtesy of Dr. Aaron Lerner.)

Melanocytes can be cultured in the presence of keratinocytes. Such cultures can be used to repigment areas of skin depigmented by diseases or injuries. One report emphasized that melanocytes proliferate despite the lack of “growth enhancers, feeder layers, or hormones” like phorbol esters in the culture medium (Boyce and Ham, 1983). After confluence and differentiation of the culture in about 21 days, the sheet of cells is detached from the culture flask, transferred to a layer of petrolatum gauze, and grafted onto recipient sites previously denuded with liquid nitrogen. With subsequent pigment spread, satisfactory repigmentation and color match have been achieved. Although only primary cultures have been used for grafting, each of these cultures yielded a sheet of cells that was estimated to be ten times the size of the original donor specimen. It is reasonable to assume that with subculturing, further expansion can be obtained. Another study used the culture medium MCDB-153 which was found to support the clonal growth of melanocytes and keratinocytes while inhibiting the growth of fibroblasts (Brysk et al., 1989; Plott et al., 1989). With this culture technique, 50-fold expansion of cells could be achieved in 10–14 days with a melanocyte to keratinocyte ratio of 1:10. The cells were plated on collagen-coated membranes and, once the culture reached 50–70% confluence in about two weeks, the entire composition was then grafted onto dermabraded vitiliginous skin. Repigmentation occurred in three of four cases reported and covered 40–90% of each vitiliginous area with perfect color match and subsequent spreading of pigment beyond the graft margins. The presence of viable melanocytes in the grafted skin was confirmed in both methods by biopsy.

Other Surgical Treatments Micropigmentation

Fig. 63.11. The hand of a patient with vitiligo (see also Plate 63.4, pp. 494–495).

expansion of pure melanocyte cultures, is also a potent tumor promoter and therefore poses long-term risks (Brysk et al., 1989). Furthermore, it was noted that melanocytes from vitiligo patients, unlike those from normal people, did not

The practice of tattooing goes back to prehistoric times. In 1987, the first application of the process in the treatment of vitiligo using nonallergenic iron oxide pigments was reported (Halder et al., 1989). This was an adaptation of the technique of permanent eyeliner tattooing (Patipa, 1987; Patipa et al., 1986). This form of treatment is recommended for limited areas that are not only refractory to other treatments but are also deemed by the patient to be especially cosmetically bothersome. These areas usually include exposed areas on the face such as the lips (Figs 63.13 and 63.14), hands (Figs 63.15 and 63.16), and forearms, but occasionally include more private areas such as the nipples. The results are permanent although some fading will occur with time, requiring an occasional touch-up in one to two years. Patients receiving this treatment nonetheless are very pleased with both their appearance and the convenience 1195

CHAPTER 63

Fig. 63.14. The lips in Figure 63.13 treated with micropigmentation (tattooing). The stark area of depigmentation is no longer visible. The color match is good (see also Plate 63.7, pp. 494–495).

Fig. 63.12. The hand of the patient in Figure 63.11 after successful grafting of cultured melanocytes (see also Plate 63.5, pp. 494–495). (Courtesy of Dr. Aaron Lerner.)

Fig. 63.13. Depigmentation on the lips of an African American patient with vitiligo (see also Plate 63.6, pp. 494–495).

of being able to go about their daily activities without the hassle associated with the use of cover-up cosmetics. The best results in terms of color match and pigment retention are obtained when micropigmentation is performed on soft, thin pigmented skin such as that of the lips and nipples. 1196

Fig. 63.15. The hand of an African American with depigmentation from vitiligo.

Fig. 63.16. The hand in Figure 63.15 after micropigmentation (tattooing) showing an excellent cosmetic response.

Although the potential for koebnerization is real, it has not been reported. The most common complication of tattooing of the lips is herpes simplex. This infection can be prevented by prophylactic treatment with acyclovir.

SURGICAL TREATMENTS OF PIGMENTARY DISORDERS

References Behl, P. N. Treatment of vitiligo with homologous thin Thiersch’s skin grafts. Curr. Med. Pract. 8:218, 1964. Behl, P. N. Repigmentation of leukoderma. J. Dermatol. Surg. Oncol. 10:669–670, 1984. Behl, P. N. Repigmentation of segmental vitiligo by autologous minigrafting. J. Am. Acad. Dermatol. 12:118–119, 1985. Behl, P. N., and R. K. Bhatia. Treatment of vitiligo with autologous thin Thiersch’s grafts. Int. J. Dermatol. 12:329–331, 1973. Bonafe, J. L., J. Lassere, J. P. Chavoin, J. P. Baro, and R. Jeune. Pigmentation induced in vitiligo by normal skin grafts and PUVA stimulation: a preliminary study. Dermatologica 166:113–116, 1983. Boyce, S. T., and R. G. Ham. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J. Invest. Dermatol. 81:33s– 40s, 1983. Brysk, M. M., R. C. Newton, S. Rajaraman, T. Plott, E. Barlow, T. Bell, P. Penn, and E. B. Smith. Repigmentation of vitiliginous skin by cultured cells. Pigment Cell Res. 2:202–207, 1989. Chitale, V. R. Overgrafting for leukoderma of the lower lip: a new application of an already established method. Ann. Plast. Surg. 26:289–290, 1991. Eisinger, M., and O. Marko. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc. Natl. Acad. Sci. U. S. A. 79:2018–2022, 1982. Falabella, R. Epidermal grafting: An original technique and its application in achromic and granulating areas. Arch. Dermatol. 104:592–600, 1971. Falabella, R. Repigmentation of leukoderma by minigrafts of normally pigmented, autologous skin. J. Dermatol. Surg. Oncol. 4:916–919, 1978. Falabella, R. Repigmentation of segmental vitiligo by autologous minigrafting. J. Am. Acad. Dermatol. 9:514–521, 1983. Falabella, R. Repigmentation of leukoderma by autologous epidermal grafting. J. Dermatol. Surg. Oncol. 10:136–144, 1984. Falabella, R. Repigmentation of stable leukoderma by autologous minigrafting. J. Dermatol. Surg. Oncol. 12:172–179, 1986. Falabella, R. Postdermabrasion leukoderma. J. Dermatol. Surg. Oncol. 13:44–48, 1987. Falabella, R. Treatment of localized vitiligo by autologous minigrafting. Arch. Dermatol. 124:1649–1655, 1988. Falabella, R. Grafting and transplantation of melanocytes for repigmenting vitiligo and other types of leukoderma. Int. J. Dermatol. 28:363–369, 1989. Falabella, R., C. Escobar, and I. Borrero. Transplantation of in vitrocultured epidermis bearing melanocytes for repigmenting vitiligo. J. Am. Acad. Dermatol. 21:257–264, 1989. Falabella, R., C. Escobar, and I. Borrero. Treatment of refractory and stable vitiligo by transplantation of in vitro cultured epidermal autografts bearing melanocytes. J. Am. Acad. Dermatol. 26(2 Pt 1):230–236, 1992. Gauthier, Y., and J. E. Surleve-Bazeille. Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions. J. Am. Acad. Dermatol. 26(2 Pt 1):191–194, 1992. Halder, R. M., H. N. Pham, J. Y. Breadon, and B. A. Johnson. Micropigmentation for the treatment of vitiligo. J. Dermatol. Surg. Oncol. 15:1092–1098, 1989. Hatchome, N., T. Kato, and H. Tagami. Therapeutic success of epidermal grafting in generalized vitiligo is limited by the Koebner phenomenon. J. Am. Acad. Dermatol. 22:87–91, 1990. Kahn, A. M., and M. J. Cohen. Vitiligo: treatment by dermabrasion and epithelial sheet grafting. J. Am. Acad. Dermatol. 33:646–648, 1995. Kahn, A. M., M. J. Cohen, L. Kaplan, and A. Highton. Vitiligo: treat-

ment by dermabrasion and epithelial sheet grafting — a preliminary report. J. Am. Acad. Dermatol. 28:773–774, 1993. Kiistala, U. Suction blister device for separation of viable epidermis from dermis. J. Invest. Dermatol. 50:129–137, 1968. Kiistala, U., and K. K. Mustakallio. In vivo separation of epidermis by production of suction blisters. Lancet 1:144, 1964. Koga, M. Epidermal grafting using the tops of suction blisters in the treatment of vitiligo. Arch. Dermatol. 124:1656–1658, 1988. Lerner, A. B. Repopulation of pigment cells in patients with vitiligo. Arch. Dermatol. 124:1701–1702, 1988. Lerner, A. B., R. Halaban, S. N. Klaus, and G. E. Moellmann. Transplantation of human melanocytes. J. Invest. Dermatol. 89:219–224, 1987. Lerner, A. B., R. Halaban, and D. Leffell. Melanocytes in culture from patients with disorders of hypopigmentation [abstract]. In: Proceedings of the XIVth International Pigment Cell Conference, Y. Mishima (ed.). Kobe, Japan: International Pigment Cell Society, 1990, p. 100. Lobuono, P., and H. Shatin. Transplantation of hair bulbs and melanocytes into leukodermic scars. J. Dermatol. Surg. Oncol. 2:53–55, 1976. Lontz, W., M. J. Olsson, G. Moellmann, and A. B. Lerner. Pigment cell transplantation for treatment of vitiligo: a progress report. J. Am. Acad. Dermatol. 30:591–597, 1994. Medrano, E. E., and J. J. Nordlund. Successful culture of adult human melanocytes from normal and vitiligo donors. J. Invest. Dermatol. 95:441–445, 1990. Olsson, M. J., and L. Juhlin. Repigmentation of vitiligo by transplantation of cultured autologous melanocytes. Acta Derm. Venereol. 73:49–51, 1993. Olsson, M. J., G. Moellmann, A. B. Lerner, and L. Juhlin. Vitiligo: repigmentation with cultured melanocytes after cryostorage. Acta Derm. Venereol. 74:226–228, 1994. Orentreich, N., and V. J. Selmanowitz. Autograft repigmentation of leukoderma. Arch. Dermatol. 105:734–736, 1972. Patipa, M. Eyelid tattooing. Dermatol. Clin. 5:335, 1987. Patipa, M., F. A. Jakobiew, and W. Krebs. Light and electron microscopic findings with permanent eyeliner. Ophthalmologica 93:1361, 1986. Plott, R. T., M. M. Brysk, R. Newton, S. S. Raimer, and S. Rajaraman. A surgical treatment for vitiligo: Transplantation of autologous cultured epithelial grafts. J. Dermatol. Surg. Oncol. 15:1161– 1166, 1989. Selmanowitz, V. J. Pigmentary correction of piebaldism by autografts. II. Pathomechanism and pigment spread in piebaldism. Cutis 24:66–71, 1979. Selmanowitz, V. J., A. D. Rabinowitz, N. Orentriech, and E. Wenk. Pigmentary correction of piebaldism by autografts. J. Dermatol. Surg. Oncol. 3:615–622, 1977. Skouge, J. W., W. L. Morison, R. V. Diwan, and S. Rotter. Autografting and PUVA. A combination therapy for vitiligo. J. Dermatol. Surg. Oncol. 18:357–360, 1992. Spencer, G. A., and J. A. Tolmach. Exchange grafts in vitiligo. J. Invest. Dermatol. 19:1, 1952. Suvanprakorn, P., S. Dee-Ananlap, C. Pongsomboon, and S. N. Klaus. Melanocyte autologous grafting for the treatment of leukoderma. J. Am. Acad. Dermatol. 13:968–974, 1985. Taki, T., S. Kozuka, Y. Izawa, T. Usuda, M. Hiramatsu, T. Matsuda, K. Yokoo, Y. Fukaya, M. Tsubone, and J. Aoka. Surgical treatment of skin depigmentation caused by burn injuries. J. Dermatol. Surg. Oncol. 11:1218–1221, 1985. Zachariae, H., C. Zachariae, B. Deleuran, and P. Kristensen. Autotransplantation in vitiligo: Treatment with epidermal grafts and cultured melanocytes. Acta Derm. Venereol. 73:46–48, 1993.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

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Laser Treatment of Pigmentary Disorders Rebat M. Halder, Lori M. Hobbs, and James J. Nordlund

Development of lasers adapted for medical and dermatological uses has been remarkable during the past decade. The field of dermatology has benefited greatly. The treatment of many pigmentary disorders had been difficult or not feasible in the past but lasers have provided a successful treatment modality for some of these conditions. The use of lasers for the treatment of pigmentary disorders is based on the theory of selective photothermolysis (Anderson et al., 1993; Murphy et al., 1983). This theory proposes that the specific spectrum of light emitted by a particular laser is selectively absorbed by a cell or tissue type. That cell or tissue type is selectively damaged or destroyed by the heat energy of the laser utilized. Several lasers emit monochromatic light that is preferentially absorbed by melanin. Specific destruction of melanocytes or dispersion of melanin by these light beams is the probable mechanism for their successful use. Melanin has a very broad absorption spectrum that includes ultraviolet, visible, and near infrared wavelengths (Spicer and Goldberg, 1996). The curve of absorption is highest in the ultraviolet spectrum and lowest in the infrared (Spicer and Goldberg, 1996). The lasers currently used for the treatment of pigmented lesions include the pigmented lesion dye laser, copper vapor laser, Q-switched ruby laser, Q-switched alexandrite laser, Q-switched neodymium:yttrium aluminum garnet (Nd:YAG) laser, continuous wave Nd:YAG laser, frequency doubled Q-switched Nd:YAG laser, and the carbon dioxide laser. The carbon dioxide laser emits continuous radiation with a wavelength of 10 600 nm. Low fluence irradiation (3.0– 4.4 J cm2) is optimal for treating pigmented lesions. The beam causes nonselective thermal damage to the entire epidermis and the melanocytes within. It has been used successfully to treat solar lentigines (Bailin et al., 1980; Dover et al., 1988; Groot et al., 1986). In one series 121 lesions were treated. Six weeks later 12 lentigines had cleared completely, 81 lightened substantially, and 28 were unchanged. There were atrophic changes in two lesions. There are other lasers discussed below that can more effectively treat solar lentigines. Tattoos were formerly treated with the carbon dioxide laser (Spicer and Goldberg, 1996). The infrared beam of the carbon dioxide laser causes coagulation necrosis extending into the reticular dermis. This laser causes changes in the texture of the epidermis and sometimes obvious scarring. Other lasers are now used to treat tattoos. The argon laser (488–514 nm) emits a blue-green light. It is a continuous wave laser and penetrates 1–2 mm in the skin. 1198

It produces heat from high energy fluences required to penetrate melanin in the epidermis (Neumann et al., 1992). Nonspecific destruction of epidermis including the resident melanocytes and papillary dermis can occur. With this laser skin texture changes and scarring are common and are permanent. Hypopigmentation may develop following treatments with this laser. Many benign pigmented lesions have been treated with the argon laser including lentigines, ephelides, café-au-lait spots, blue nevi, nevus of Ota, epidermal nevi, pigmented seborrheic keratosis, melasma, and postinflammatory hyperpigmentation (Brauner et al., 1991; McBurney, 1993; Ohshiro et al., 1980; Trelles et al., 1992). The effectiveness of the argon laser in the treatment of these lesions is variable. Published reports are for the most part anecdotal. Café-au-lait macules and Becker nevi respond poorly (Dover et al., 1990). Recent studies show that freezing of solar lentigines with liquid nitrogen was superior to that of the carbon dioxide or argon laser (Stern et al., 1994). Lentigo maligna has been treated with the argon laser but has recurred (Arndt, 1984, 1986). The pigmented lesion dye laser (PLDL) (500–520 nm) produces a green light. The optimal chromophore is melanin at 510 nm. Light from the PLDL penetrated only to a depth of 0.25–0.5 mm, thus it is best suited for superficial pigment. Benign epidermal pigmented lesions that respond are solar lentigines, ephelides, café-au-lait macules, some seborrheic keratoses, dermatosis papulosa nigra, Becker nevi, some cases of epidermal melasma, and superficial postinflammatory hyperpigmentation (Alster, 1993; Fitzpatrick et al., 1993a; Grekin et al., 1993; Yasuda et al., 1991). Café-au-lait macules do not always clear completely following treatment with this laser (Alster, 1995a). It is not effective in the treatment of deep dermal pigmented lesions such as nevus of Ota, dermal melasma, and dermal melanosis following inflammatory processes (Fitzpatrick et al., 1993a; Grekin et al., 1993). The Q-switched ruby laser (694 nm) emits a red light and causes selective destruction to pigment-containing cells by damaging melanized melanosomes (Hruza et al., 1991; Polla et al., 1987). It is melanin and not hemoglobin that preferentially absorbs 694 nm light. Blue-black and green tattoo pigments also absorb this wavelength well (Dover et al., 1989; Kilmer and Anderson, 1993). The pulse duration of the Q-switched ruby laser is 20–50 ns which is shorter than the estimated 50–100 ns thermal relaxation time of melanosomes (Sherwood et al., 1989) resulting in selective photothermolysis of these organelles (Dawber and Wilkinson,

LASER TREATMENT OF PIGMENTARY DISORDERS

1986). The depth of penetration of light from the Q-switched ruby laser is deep, about 2–3 mm. Lesions that have been treated successfully with this laser include solar lentigines, ephelides, café-au-lait macules, nevus of Ota, melasma and postinflammatory hyperpigmentation, penile lentigines and labial lentigines, Becker nevi, and seborrheic keratoses (Goldberg and Nychay, 1995; Grevelink et al., 1992; Levins and Anderson, 1995; Nelson and Applebaum, 1992; Ono et al., 1993). The rate of response is variable. Solar lentigines and ephelides respond well to one treatment. Café-au-lait macules respond less well and require more than one treatment (Dover et al., 1993). Often it is impossible to completely and permanently clear café-au-lait macules and recurrence of pigmentation is common within 6–12 months (Goldberg, 1993a,b; Grossman et al., 1995). They may become hyperpigmented before clearing and hypopigmentation or mottled pigmentation may occur. The response of Becker nevi is similar. Nevus of Ota responds to treatment by the Q-switched ruby laser. In one series 57% of patients achieved a good response after one treatment (Geronemus, 1992a,b; Taylor et al., 1994; Tse et al., 1994). Biopsy confirmed that dendritic melanocytes were destroyed to a depth of 2.6 mm (Tse et al., 1994). Melasma has a variable response to the Q-switched ruby laser. Most studies report poor results with recurrences common soon after treatment (Goldberg, 1993a,b). Those patients who have the epidermal variant of melasma and who have lighter complexions may respond better as well as those who have shown partial response to previous hydroquinone therapy (Dover et al., 1993). Sunscreens must be used after Qswitched laser therapy for melasma. Postinflammatory hyperpigmentation can occur. Labial and periorbital lentigines in Peutz–Jeghers syndrome have been successfully treated with the Q-switched ruby laser (Ashinoff and Geronemus, 1992; DePadova-Elder and Milgraum, 1994; Ohshiro et al., 1980). Caution must be taken when treating pigmented lesions in darker skin types with the Q-switched ruby laser. Because of increased amounts of melanin present, higher amounts of laser energy are absorbed within the epidermis (Taylor and Anderson, 1994). This may lead to erosions, hyperpigmentation, and hypopigmentation as well as texture changes such as atrophy or depression. The red light of the Q-switched ruby laser is absorbed well by blue-black and green tattoo ink. Tattoos containing these dyes are effectively removed (Achauer et al., 1994; Lowe et al., 1994; Reid et al., 1990, 1993; Scheibner et al., 1990; Taylor et al., 1990, 1991). Other colors are removed less efficiently. Amateur tattoos require fewer treatment sessions than do professional tattoos (Kilmer and Anderson, 1993). Because the 694 nm wavelength of the Q-switched ruby laser is absorbed well by melanin, damage to melanocytes can occur leading to hypopigmentation and hyperpigmentation and rarely even to depigmentation (Dover et al., 1993; Kilmer et al., 1993b). Otherwise, scarring is minimal in the treatment of tattoos by the Q-switched ruby laser. Q-switched ruby laser at exposure doses below the threshold for melanosomal disruption has not been found efficacious in treating vitiligo

Fig. 64.1. A tattoo before laser ablation (see also Plate 64.1, pp. 494–495).

despite instances of hyperpigmentation observed when applied in the treatment of other skin conditions (Renfro and Geronemus, 1992). The Q-switched Nd:YAG laser has a wavelength of 1064 nm and can effectively remove black tattoo ink (Figs 63.1 and 63.2) (Anderson and Dover, 1989; Kilmer and Anderson, 1993; Kilmer et al., 1993b; Levine and Geronemus, 1993). Other colors are removed less efficiently. Because of the long wavelength emitted, the disruption to melanincontaining cells is minimal, so that pigmentary changes are less than with the Q-switched ruby laser (Dover et al., 1993). Temporary texture changes can occur but scarring is rare. A frequency-doubling crystal can be added to the Qswitched Nd:YAG laser which halves the wavelength from 1064 nm to 532 nm (Kilmer et al., 1994). This wavelength is visible green light that is absorbed well by red tattoo ink. Thus, the frequency-doubled Nd:YAG laser can better treat red tattoos than the Q-switched ruby laser or the 1064 nm ND:YAG laser (Kilmer et al., 1993a). However, since this wavelength is also absorbed well by melanin, side effects of hypopigmentation, hyperpigmentation and blistering similar to the Q-switched ruby laser can occur. Also since it does not penetrate into the dermis as deeply as the Q-switched ruby laser, deeper dermal pigmented lesions cannot be treated. The Q-switched Nd:YAG set at 532 nm is very effective at removing lentigines with no scarring (Figs 63.3 and 63.4) (Kilmer 1199

CHAPTER 64

Fig. 64.3. Solar lentigines before laser ablation.

Fig. 64.2. The tattoo in Figure 64.1 treated with a Nd:YAG laser set at 1064 nm (see also Plate 64.2, pp. 494–495). Note the laser removal of black pigmentation in Fig. 64.1. However, the green pigment is not removed by Nd:YAG laser.

et al., 1994). Café-au-lait macules exhibit a variable response (Figs 63.5 and 63.6). The copper vapor laser emits green light at 511 nm and yellow light at 578 nm. A filter provides choice of wavelength (Thibault and Wlodarczyk, 1992). The 511 nm wavelength is used for pigmented lesions. Lentigines and ephelides respond well. Scarring and pigmentary sequelae are uncommon (Neumann et al., 1993). However, other treatment modalities can be used to treat these lesions as well (Dinehart et al., 1993; McMeekin, 1992). Café-au-lait macules have a variable response. The copper vapor laser has been used in treating post-sclerotherapy hyperpigmentation with good results even though the pigment is hemosiderin and not melanin (Thibault and Wlodarczyk, 1992). The Q-switched alexandrite laser (755 nm) has been used successfully in the treatment of blue and black tattoos without long-term pigmentary changes (Alster, 1995b; Fitzpatrick and Goldman, 1994; Fitzpatrick et al., 1992, 1993b; Stafford and Tan, 1995; Stafford et al., 1995). In particular, bright-colored 1200

Fig. 64.4. Hands in Figure 64.4 three months after treatment with a Nd:YAG laser set at 532 nm.

Fig. 64.5 Café-au-lait spot before laser ablation (see also Plate 64.3, pp. 494–495).

ink such as in teal-colored tattoos are amenable to treatment by Q-switched alexandrite laser. The longer wavelength of the Q-switched alexandrite laser penetrates deeper into the skin without disrupting epidermal melanocytes. Such an advantage

LASER TREATMENT OF PIGMENTARY DISORDERS

References

Fig. 64.6 Café-au-lait spot in Figure 64.5 after four treatments with a Nd:YAG laser set at 532 nm. The hyperpigmentation has been removed (see also Plate 64.4, pp. 494–495).

has also enhanced the treatment of deeper dermal melanocytes of the nevus of Ota by the Q-switched alexandrite laser (Alster and Williams, 1995) with very good results without scarring or pigmentary changes. The broadband light source, the intense pulse light (IPL), has been used to treat a variety of pigmented and vascular lesions, as well as unwanted hair and rhytids. The IPL emits a polychromatic light between 515 nm and 1200 nm. Depending upon the intended target and the photo type of the patient, cut-off filters are employed to allow the proper spectrum of light to selectively target the intended chromophore. For example, higher cut-off filters are used to reduce the absorption of melanin and provide a better safety margin when treating darker photo types. The pulse duration of the IPL systems is in the millisecond domain. Single, double, and triple pulses can be utilized. Multiple pulses are often necessary to divide the energy of high fluences. The delay time between pulses varies between 1 millisecond and 300 milliseconds. The delay time follows the principle of thermokinetic selectivity and allows smaller nonselective targets to be protected by cooling down between pulses, and selective larger targets to retain heat, resulting in precise thermal damage. A variety of pigmented disorders can be treated with the IPL (Raulin, 2003). These disorders range from lentigines, nevi of Ota and Ito, café-au-lait macules, postinflammatory hyperpigmentation, melasma, and even traumatic tattoos. Multiple treatments with the IPL are generally required to achieve the desired clinical outcome. The cutaneous response after treatment with the IPL is generally mild to transient erythema and edema lasting for a few hours. When used properly, scarring and permanent dyspigmentation are rare (Greve and Raulin, 2002). Darker photo types and persons who are tanned may experience more adverse events such as transient purpura, crusting, and dyspigmentation. With the use of the IPL, it is crucial to warn patients that unwanted hair loss can occur at the treatment site.

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and J. A. Parrish. Organelle-specific injury to melanin-containing cells in human skin by pulsed laser irradiation. Lab. Invest. 49:680– 685, 1983. Nelson, J. S., and J. Applebaum. Treatment of superficial cutaneous pigmented lesions by melanin-specific selective photothermolysis using the Q-switched ruby laser. Ann. Plast. Surg. 29:231–237, 1992. Neumann, R. A., R. M. Knobler, H. Leonhartsberger, and W. Gebhart. Comparative histochemistry of port-wine stains after copper vapor laser (578) nm and argon laser treatment. J. Invest. Dermatol. 99:160–167, 1992. Neumann, R. A., H. Leonhartsberger, K. Bohler-Sommeregger, R. Knobler, E. M. Kokoschka, and H. Honigsmann. Results and tissue healing after copper-vapour laser (at 578 nm) treatment of port wine stains and facial telangiectasias. Br. J. Dermatol. 128:306– 312, 1993. Ohshiro, T., Y. Maruyama, N. Nakajima, and M. Mima. Treatment of pigmentation of the lips and oral mucosa in Peutz-Jeghers syndrome using ruby and argon lasers. Br. J. Plast. Surg. 33:346–349, 1980. Ono, I., H. Gunji, M. Sato, et al. Treatment of pigmented seborrheic keratosis by ruby laser irradiation. Eur. J. Dermatol. 3:206, 1993. Polla, L. L., R. J. Margolis, J. S. Dover, D. Whitaker, G. F. Murphy, S. L. Jacques, and R. R. Anderson. Melanosomes are a primary target of Q-switched ruby laser irradiation in guinea pig skin. J. Invest. Dermatol. 89:281–286, 1987. Reid, W. H., I. D. Miller, M. J. Murphy, J. P. Paul, and J. H. Evans. Q-switched ruby laser treatment of tattoos: a 9-year experience. Br. J. Plast. Surg. 43:663–669, 1990. Raulin C., B. Greve, and H. Grema. IPL technology: a review. Lasers Surg. Med. 32:78–87, 2003. Reid, W. H., P. J. McLeod, and A. Ritchie. Q-switched ruby laser treatment of black tattoos. J. Dermatol. Surg. Oncol. 19:330, 1993. Renfro, L., and R. G. Geronemus. Lack of efficacy of the Q-switched ruby laser in the treatment of vitiligo [letter]. Arch. Dermatol. 128:277–278, 1992. Scheibner, A., G. Kenny, W. White, and R. G. Wheeland. A superior method of tattoo removal using the Q-switched ruby laser. J. Dermatol. Surg. Oncol. 16:1091–1098, 1990. Sherwood, K. A., S. Murray, A. K. Kurban, and O. T. Tan. Effect of wavelength on cutaneous pigment using pulsed irradiation. J. Invest. Dermatol. 92:717–720, 1989. Spicer, M. S., and D. J. Goldberg. Continuing medical education: lasers in dermatology. J. Am. Acad. Dermatol. 34:1–25, 1996. Stafford, T. J., and O. T. Tan. 510-nm pulsed dye laser and alexandrite crystal laser for the treatment of pigmented lesions and tattoos. [Review]. Clin. Dermatol. 13:69–73, 1995. Stafford, T. J., R. Lizek, J. Boll, and O. T. Tan. Removal of colored tattoos with the Q-switched alexandrite laser. Plast. Reconstr. Surg. 95:313–320, 1995. Stern, R. S., J. S. Dover, J. A. Levin, and K. A. Arndt. Laser therapy versus cryotherapy of lentigines: a comparative trial. J. Am. Acad. Dermatol. 30:985–987, 1994. Taylor, C. R., and R. R. Anderson. Ineffective treatment of refractory melasma and post inflammatory hyperpigmentation by Q-switched ruby laser. J. Dermatol. Surg. Oncol. 20:592–597, 1994. Taylor, C., R. Gange, J. Dover, T. Flotte, E. Gonzalez, N. Michaud, and R. Anderson. Treatment of tattoos by Q-switched ruby laser. Arch. Dermatol. 126:893–899, 1990. Taylor, C. R., R. R. Anderson, R. W. Gange, N. A. Michaud, and T. J. Flotte. Light and electron microscopic analysis of tattoos treated by Q-switched ruby laser. J. Invest. Dermatol. 97:131–136, 1991. Taylor, C. R., T. J. Flotte, W. Gange, and R. R. Anderson. Treatment of nevus of Ota by Q-switched ruby laser. J. Am. Acad. Dermatol. 30:743–751, 1994. Thibault, P., and J. Wlodarczyk. Postsclerotherapy hyperpigmenta-

LASER TREATMENT OF PIGMENTARY DISORDERS tion. The role of serum ferritin levels and the effectiveness of treatment with the copper vapor laser. J. Dermatol. Surg. Oncol. 18:47–52, 1992. Trelles, M. A., W. Verkruysse, J. W. Pickering, M. Velez, J. Sanchez, and P. Sala. Monoline argon laser (514 nm) treatment of benign pigmented lesions with long pulse lengths. J. Photochem. Photobiol. B 16:357–365, 1992.

Tse, Y., V. J. Levine, S. A. McClain, and R. Ashinoff. The removal of cutaneous pigmented lesions with the Q-switched ruby laser and the Q-switched neodymium: yttrium-aluminum-garnet laser. A comparative study. J. Dermatol. Surg. Oncol. 20:795–800, 1994. Yasuda, Y., O. T. Tan, A. K. Kurban, et al. Electron microscopic study on black pig skin irradiated with pulsed dye laser (504 nm). Proc. SPIE: Lasers Derm. Tissue Welding 1422:19, 1991.

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The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

Index

Notes Page numbers in bold refer to tables and those in italics refer to figures. Where genes/proteins from both human and other species are mentioned the gene will be listed under the human gene/protein unless otherwise stated. ABCD1 gene, adrenoleukodystrophy, 772 ABCD syndrome, 146 Abtropfung, 1116 acanthosis nigricans, 907–914 animal models, 912 associated disorders, 910, 910 benign, 909 clinical findings, 908, 908–910, 909 diagnosis/differential diagnosis, 910–911 endocrinopathy-related, 909 epidemiology, 908 familial, 909 historical background, 907–908 laboratory findings/investigations, 910 malignant, 908, 909, 911 obesity-related, 908, 909, 909 pathology/pathogenesis, 911 treatment/prognosis, 912 types, 909–910 N-acetyl-4-S-cystaminylphenol (4-SCAP), 675 achalasis, Rozycki syndrome, 551 achromatous pityriasis faciei, see pityriasis alba achromias, classification, 501 achromic nevus, see nevus depigmentosus acidity, see pH ACK2 (anti-KIT) antibody, 429, 429–430, 430 acoustic neuromas, neurofibromatosis type 2, 813 acquired bilateral nevus of Ota-like macules (ABNOM), 1017–1018, 1018 acquired generalized dermal melanocytosis, 1016 acquired hepatic porphyria, see porphyria cutanea tarda acquired idiopathic benign lentiginosis, see Laugier–Hunziker syndrome acquired leukoderma, see vitiligo vulgaris (vitiligo) acquired leukopathia, see vitiligo vulgaris (vitiligo) acquired linear dermal melanocytosis, 1016 acquired porphyria, see porphyria cutanea tarda acral acanthotic anomaly, 909 acral lentiginous melanoma, 473 Down syndrome, 658 nail, 1063, 1065 acrochordons (skin tags), acanthosis nigricans, 909 acro-dermato-ungual-lacrimal-tooth syndrome (ADULT syndrome), 902 acrogeria, 792, 884–886, 885 acrokeratotic poikiloderma of Weary, see hereditary acrokeratotic poikiloderma acromegaly Carney complex/myxoma syndrome, 855 hyperpigmentation, 1072 acromelanosis albopunctata, see reticulated acropigmentation of Dohi acromelanosis progressiva (acropigmentation), 914, 914–915 acropigmentation of Dohi, 789, 797, 914 acropigmentation of Kitamura, 789, 797 acropigmentation symmetrica of Dohi, see reticulated acropigmentation of Dohi ACTH, see adrenocorticotropic hormone (ACTH) actin filaments, intracellular transport, 37, 38, 38, 171–172

actinic lentigo, see lentigo senilis et actinicus actinic purpura, hemosiderosis, 990 adaptor complex-3 (AP-3), 615 Addison disease adrenoleukodystrophy, 771, 772 associated disorders, 970–971 clinical features, 970, 970, 971 differential diagnosis, 972 epidemiology, 970 histology, 971 hyperpigmentation, 667 laboratory abnormalities, 971–972 melanocytic nevi development, 1126 mucosal hyperpigmentation, 1072 pathogenesis, 971 pigmentary changes, 969–972 treatment, 972 adhesive tape-induced skin discoloration, 1045 adiposogenital syndrome (Fröhlich syndrome), 839 adolescents, skin color, 511–512 adrenal insufficiency, vitiligo vulgaris, 566 adrenergic receptors, color change and, 36–37 adrenocorticotropic hormone (ACTH), 194, 196, 414–415 Addison disease, 971 adrenoleukodystrophy, 771 carcinoid tumors, 920 induction by UVR, 413 adrenoleukodystrophy (ALD), 771–774 adrenomyeloneuropathy (AMN), 771, 772 adriamycin (doxorubicin)-induced skin discoloration, 1046 adult progeria, see Werner syndrome ADULT syndrome (acro-dermato-unguallacrimal-tooth syndrome), 902 AEC syndrome (ankyloblepharon, ectodermal dysplasia), 902 aganglionic megacolon, 145, 146 age/aging disorders associated with, 884–897, see also specific disorders melanocyte senescence, 464–471 melanoma incidence, 473 pigmentation changes, 511, 511–513 premature, ataxia telangiectasia, 621 age spot, see lentigo senilis et actinicus agminated lentigines (AL), 863, 863–867 animal models, 867 associated disorders, 863 clinical description, 865 differential diagnosis, 865, 1106 epidemiology, 865 histopathology, 865 historical background, 864–865, 866 pathogenesis, 865, 867 treatment, 865 agminated Spitz nevi, 1106 agouti protein, 196–198, 199, 399–401 allelic variants, 395 biochemical action, 197 cAMP signaling and, 395 eumelanin vs. pheomelanin biosynthesis, 196–197, 197, 395, 416 evolutionary conservation, 400–401 homologs, 395

human (agouti signaling protein, ASP), 198, 400–401, 416, 514 inverse agonist, see alpha-melanocyte stimulating hormone (a-MSH) isoforms, 399–400 melanocortin receptor binding, 399, 404, 416 MIF relationship, 43 spatiotemporal gene expression, 399–400, 400 AIDS, see HIV infection/AIDS AIM-1, see membrane-associated transport protein (MATP) AK amyloid, 926 albinism, see also ocular albinism (OA); oculocutaneous albinism (OCA); piebaldism/piebald trait auditory abnormalities, 100, 602 clinical features, 571 definition, 600 historical aspects, 8, 599–600 nonmammalian vertebrates, 48, 118 nystagmus, 602 partial (mosaic), see piebaldism/piebald trait visual system abnormalities, 92–93, 571, 600, 600–602 foveal hyperplasia, 601–602 iris pigmentation, 600 optic nerve development, 601–602 retinal pigmentation, 601–602 Albinism Database, 605 albinism–deafness syndrome (Tietz syndrome), 245, 546, 546–547, 630–631 albinoid disorders, 613–621 Albright’s syndrome (disease), see McCune–Albright syndrome alcohol ingestion, porphyria cutanea tarda, 981 alcoholism, centrofacial lentiginosis pathogenesis, 840–841 Alezzandrini syndrome, 559, 725–726, 726, 738 alkaptonuria, see ochronosis allergic contact dermatitis, plants, 949 allergic shiners, 879 allergy testing, progressive macular hypomelanosis, 749 allogeneic bone marrow transplantation (ABMT), melanocytic nevi formation, 1126 all trans retinoic acid (ATRA), see tretinoin (all trans retinoic acid; ATRA) aloesin, 675 alopecia, see also specific types Cronkhite–Canada syndrome, 984, 984 evolutionary significance, 71, 72 thumb deformity and, 904 alopecia areata, 371, 754–756 clinical description, 754, 755 diagnostic criteria, 755 differential diagnosis, 755 Down syndrome, 658 epidemiology, 754 histology, 754–755 historical background, 754 pathogenesis, 755 prognosis, 755–756 sudden whitening of hair, 766 Vogt–Koyanagi–Harada syndrome, 736, 754 alopecia mucinosa, see follicular mucinosis

1205

INDEX alpha-melanocyte stimulating hormone (a-MSH), 414–415 Addison disease, 1072 AIDS patients, 943 amphibian cell differentiation, 117–118 antagonists, 36 binding sites, 762 induction by UVR, 413 iridophore effects, 30, 30–31 melanocortin receptor binding, 399, 404, 415 inverse agonist, see agouti protein as melanocyte mitogen, 448 melasma, 1021 MITF regulation, 246 normal pigmentation role, 404 alveolar rhabdomyosarcoma, pax3 (PAX3) mutations, 144 amalgam tattoo, 1080, 1080–1081 amelanosis, definition, 527 ametantrone-induced skin discoloration, 1045 3-amino-3-hydroxyphenylethylamine (3-AHPEA), 301, 301 4-amino-3-hydroxyphenylethylamine (4-AHPEA), 301, 301, 314 aminoglycoside antibiotics, ototoxicity, 376 3-aminohydroxyphenylalanine (3-AHP), 297, 297, 299–300, 300 4-aminohydroxyphenylalanine (4-AHP), 282, 283, 296–297, 297, 300, 300–301 amiodarone-induced skin discoloration, 1038, 1038–1039, 1039 Ammi majus, 1175 amphibians, see also specific types cellular associations in color change, 40–42 chromatophore development, 115–119 differentiation, 117–118 morphogenesis, 115–117, 116 mutants, 118–119 chromatophores, 108 egg as, 24, 24–25, 25 MSH effects on xanthophores, 31 amyloid, 926 amyloid K, 926 amyloidosis, cutaneous, 924–928 associated disorders, 926 clinical findings, 924–926, 925 diagnosis/differential diagnosis, 926 familial, 926 pathology/pathogenesis, 926 treatment/prognosis, 926–927 anal canal, melanocyte numbers, normal, 1069 analgesics, fixed drug eruption, 1028 anal melanoma, 1085 anal skin, melanocyte numbers, normal, 1069 ancient Greeks, 5 ancient Romans, 5 androgens, melanocyte regulation, 416–417 anemia canities, premature, 659, 666 Fanconi, 776, 776–778 hypopigmentation, 768 iron deficiency, 666 pallor, 530 pernicious, see pernicious anemia Angelman syndrome, 235, 620, 629 anhidrotic ectodermal dysplasia and immunodeficiency (EDA-ID), 877 aniline dyes, skin discoloration, 1048 ankyloblepharon, ectodermal dysplasia (AEC syndrome), 902 anonychia with flexural pigmentation, 789, 797, 873, 874, 902 antibiotics, see also specific drugs confluent and reticulated papillomatosis, 923 fixed drug eruption, 1028 ototoxicity, 376 antigen in melanoma-1 (AIM-1), see membraneassociated transport protein (MATP) antimalarials, hyperpigmentation-induction, 940, 1042, 1042, 1081 anti-melanocyte antibodies, vitiligo vulgaris, 572 antimicrobials, fixed drug eruption, 1028 anti-mitochondrial antibody (AMA) titers, primary biliary cirrhosis, 994 antimonials, post-kala-azar dermatosis, 697 antioxidants depigmentation, 673 melanin as, 72, 311, 331–334, 333, 334, 345 vitiligo vulgaris, 577

1206

antipyretics, fixed drug eruption, 1028 antithyroid antibodies, vitiligo vulgaris, 566 AP2 transcription factor/binding sites, melanoma genetics, 481–482 AP3 (adaptor protein-3), 159–160, 160, 160 AP3B1 gene, Hermansky–Pudlak syndrome type 2, 615 APAF1 mutations, melanoma, 472, 479 apoptosis suppression, melanoma, 472, 481 apudomas, 919 arbutin, 675, 1169 ARF protein, melanoma and, 475, 476 argon laser, 1198 argyria, 525, 1030–1032, 1031 arsenic-induced skin discoloration, 1034, 1034, 1035 arterial dissection, lentiginosis, 868 arthropathy, hereditary hemochromatosis, 987 Asboe–Hansen disease, see incontinentia pigmenti (IP) ascorbate (ascorbic acid), 331, 677 ashen mouse mutant, 173, 174–175 ash-leaf spots, 515, 531, 651, 653, 654, 654 ashy dermatosis of Ramirez, see erythema dyschromicum perstans Aspergillus, pityriasis alba, 700 aspirin, vitiligo vulgaris, 577 ataxia telangiectasia, 621–622 atomic force microscopy, melanins, 312–313, 313–314 atomic weapons, depigmentation due to, 684 atopic dermatitis hyperpigmentation, 911 hypopigmentation, 702–703, 703 ATPases, 37, 183, 233, 237 atrophic degenerative pigmentary dermatitis, see poikiloderma of Civatte atrophic scleroderma d’emblée, see atrophoderma of Pasini et Pierini atrophie brilliante, see confluent and reticulated papillomatosis (CRP) atrophoderma of Pasini et Pierini, 963, 963–965, 964 atrophoscleroderma superficialis circumscripta, see atrophoderma of Pasini et Pierini attractin, 401–403 atypical melanoblastoma, see melanotic ectodermal tumor of infancy atypical melanocytic hyperplasia, 1066, 1066 atypical mole syndrome (AMS), familial melanoma and, 474, 474 atypical nevi, 1112 blue nevus, Carney complex/myxoma syndrome, 857 development, 1127 auditory system albinism, 100, 602 development, 99 melanocytes in, see otic melanocytes toxic lesions, 376 Vogt–Koyanagi–Harada syndrome, 735 autocrine factors human melanocyte regulation, 413–417 melanomas, 490 autocytotoxic hypothesis, vitiligo vulgaris, 574 autoimmunity idiopathic guttate hypomelanosis, 728 vitiligo vulgaris, 565, 572–574 T-cell response, 573 autologous epidermal grafting, 1191 autologous melanocyte and keratinocyte coculture transplantation, 1195 autologous minigrafts, 1192, 1192, 1193, 1194 autologous pure melanocyte culture transplantation, 1193–1195, 1194, 1195, 1196 autologous thin Thiersch grafting, 1191 autonomic nervous system, depigmented skin, 564 auto-oxidation, melanin, 317, 328–329 autosomal recessive ocular albinism (AROA), 605 avian species, see birds axanthic mutants, 118 axillary freckles, 532, 811 azelaic acid (AZA) hyperpigmentation disorders, 1166–1167 mechanism of action, 676, 1167 melasma, 1167

postinflammatory hyperpigmentation, 1167 reticulated acropigmentation of Kitamura, 804 tinea versicolor, 694 azidothymidine (AZT) mucosal discoloration, 1081 skin discoloration, 1045, 1045 azo dyes, skin discoloration, 1048 B7-1, melanoma regression and, 718 B7-2, melanoma regression and, 718 babides, 686 background adaptation, 25–26, 29, 32–33 adaptive mechanisms, 32–33 high vs. low albido environments, 33 morphologic color change, 27–28 physiologic color change, 27 balanitis xerotica obliterans, 1086, 1086 baldness, see alopecia Bannayan–Riley–Ruvalcaba syndrome (BRR), 867–868, 1084 animal models, 868 genital macules, 1084 historical background, 867–868 Barrere, Pierre, 7 Barton, Benjamin, 9 basal cell nevus syndrome (Gorlin Goltz syndrome), 655 basic fibroblast growth factor (bFGF), see fibroblast growth factor-2 (FGF-2) basic helix–loop–helix transcription factors (bHLH), 243, 248 Bateman purpura, 990 Baynham, William, 7 BCL-2 gene/protein deficiency, melanocyte apoptosis, 528 melanoma association, 481 BCNU (carmustine) mycosis fungoides, 976 skin discoloration, 1045 Becker melanosis, see Becker nevus Becker nevus, 915, 915–917 bejel, 688 benign fibrous histiocytoma (dermatofibroma), 433–435 benzathine penicillin, 687, 688 benzothiazines, late pheomelanogenesis, 292, 292–293, 293 Berkshire neck, see poikiloderma of Civatte Berlin syndrome, 780–781 Berloque dermatitis, see phytophotodermatitis 6-beta-alanyl-2-carboxy-4-hydroxybenzothiazide (BTCA), 297, 297 betamethasone, vitiligo vulgaris, 577 big endothelins (big ETs), 422, 427 “bihumoral theory,” 13 bile, as skin pigment, 7 bindhi, chemical depigmentation, 562, 562 biomarkers melanoma, 293–294 replicative senescence, 464 biopterin deficiency, 634 biphasic amyloidosis, 924 bird-headed dwarfism (Seckel syndrome), 657–658 birds eye color, 98, 98–99 melanocyte differentiation, 109, 121–122 melanocyte morphogenesis, 119, 119–121 dynamics, 119–120 mechanism of migration, 120 mutants, 122 birthmarks, see nevi 1,3-bis(chloroethyl)-1-nitrosourea (BCNU), 976, 1045 N,N-bis-acetanilidedimethylamine (QX-572), 378, 378–379 bisisolylmaleimide, 673 bismuthia, 1034 bismuth-induced skin discoloration, 525, 1033–1034 black skin, see also ethnicity; skin types (human) color, early theories, 5–8 inherited patterned lentiginosis, 869 white skin vs., 8 Wood’s light examination, 532 “black tongue,” 998 Blaschko lines, 636–637, 637

INDEX bleaching agents melanin modification, 357–358 melasma, 1022, 1022 bleeding diathesis, Hermansky–Pudlak syndrome, 614 bleomycin-induced skin discoloration, 1045, 1045–1046 blinding filariasis, see onchocerciasis blistering disorders, melanocytic nevi formation, 1121, 1125 BLOC-2, 615 Bloch–Siemens syndrome, see incontinentia pigmenti (IP) Bloch–Sulzberger syndrome, see incontinentia pigmenti (IP) BLOC proteins, 160, 160, 160–161, 615 Bloom syndrome, 806 blue coloration human dermal melanocytes, 69–70 iridophores, 19, 20, 21 “plerodeles blue,” 22, 32 blue-gray macule of infancy, see Mongolian spot blue macules mycosis fungoides, 975 progressive systemic sclerosis, 1018 blue nevi Carney complex/myxoma syndrome, 856, 857 oral mucosa, 1078, 1078, 1079 patch-type, 1160 plaque-type, 1160–1161 speckled lentiginous nevus, 1105 Blumenbach, Johann Friedrich, 7 bone marrow, dyskeratosis congenita, 898 bone morphogenetic proteins (BMPs) avian melanocyte specification, 121 mammalian pigmentary system development, 83 Böök syndrome (PHC syndrome), 661 borderline borderline leprosy, 690 borderline tuberculoid leprosy, 690 boron neutron capture therapy (BNCT), 382–383 Borrelia burgdorferi, morphea, 969 bouba, 686–687 Bourneville disease, see tuberous sclerosis complex (TSC) bowel disorders, hypopigmentation, 665–666 Bowman-Birk protease inhibitor, 678 Boyle, Robert, 6 BRAF gene/protein melanocytic nevi, 1113, 1115 melanoma and, 453, 472, 492 familial, 477 sporadic, 479–480 nevogenesis, 1130 BRCA2 gene/protein, familial melanoma and, 477 breast, Carney complex/myxoma syndrome, 855, 856–857, 858 Breschet, G.H., 8 Breslow index anal melanoma, 1085 ungual melanoma, 1066–1067 BRN2 (N-Oct3; POU3F2) gene, 248 Bronze–Schilder disease, 771–774 Brown, Sir Thomas, 6 buba, 686–687 Buscke heat melanosis, see erythema ab igne busulfan, skin discoloration and, 940, 1046 N-butyldeoxynojirimicin, 673 Byrd, William, 9 C2-ceramide, 673 C57BK/6 MivitMivit mouse model, vitiligo vulgaris, 576, 576 E-cadherin, melanocytic nevi, 1113 N-cadherin, melanoma and, 482 Caenorhabditis elegans, Bannayan–Riley–Ruvalcaba syndrome model, 868 café-au-lait spots (macules: CALMs), 515, 917–919 agminated lentigines, 864 clinical findings, 531–532, 917, 917–918, 918 diagnostic criteria, 918 differential diagnosis, 918 Down syndrome and, 658 epidemiology, 917 familial multiple, 809

genetic epidermal syndromes, 809–823 historical background, 917 laser treatment, 1198, 1200, 1200, 1201 LEOPARD syndrome, 844 McCune–Albright syndrome, 522, 817, 817, 917–918 neurofibromatosis type 1, 435, 810–811, 811, 813 paracrine interactions, 435–437, 436, 440, 441, 441 pathogenesis, 522, 522, 918 pathology, 918 piebaldism, 543 prognosis, 918 Q-switched ruby laser, 1199 Silver–Russell syndrome, 821 solitary, 917 treatment, 918 tuberous sclerosis complex, 654 café noir spots, LEOPARD syndrome, 844 calcipitriol acanthosis nigricans, 912 UVB phototherapy and vitiligo vulgaris, 1185 calcium D-pantetheine-S-sulfonate (PaSSO3Ca), 673 Caldwell, Charles, 8, 9 calphostine, 673 camouflage, melanin and, 75–76 camouflage stains, 1188–1189 cancer, see malignancy; specific cancers Candida infections nail pigmentation, 1061 vitiligo vulgaris, 565 canities definition, 527 premature (premature graying of hair), 659–660 associated disorders, 760 definition, 761 Down syndrome, 658 Fisch syndrome, 659 iron deficiency anemia, 666 mandibulofacial dysostosis, 660 myotonic dystrophy, 661 pernicious anemia, 666 prolidase deficiency, 662 senile, 512, 512, 527, 760–764 animal models, 762 associated disorders, 760 clinical features, 760 diagnosis/differential diagnosis, 761–762 epidemiology, 760 histology, 760–761 historical background, 760 immune tolerance hypothesis, 762 laboratory findings, 761 pathogenesis, 762 prognosis, 763 repigmentation, 762–763 treatment, 762–763 canthaxanthin-induced skin discoloration, 1038 Cantu syndrome, 781 capillaritis, 1029–1030, 1030 capillary hemangiomas, 1069–1070 carate, 687–688 carbolic acid-induced skin discoloration, 1041 carbon baby (universal acquired melanosis), 906 carbon dioxide laser, 1198 7-carboxyl porphyrins, porphyria cutanea tarda, 982 carcinogenesis drug-binding to melanin(s), 373, 374, 383 psoralens and ultraviolet light (PUVA), 582 carcinoid syndrome (carcinoidosis), 919–921 clinical findings, 919, 919–920 pellagra, 998 carcinoid tumors, 920 cardiac anomalies, LEOPARD syndrome, 844–845 cardiac myxomas Carney complex/myxoma syndrome, 854, 854, 856, 858 treatment, 859 cardiac rhabdomyomas, tuberous sclerosis complex, 653 cardiocutaneous syndrome, see LEOPARD syndrome Carleton–Biggs syndrome, 1017

carmustine (BCNU) mycosis fungoides, 976 skin discoloration, 1045 Carney complex/myxoma syndrome associated disorders, 856–857 clinical description/presentation, 846, 856–857 clinical findings, 853–856, 854, 855 diagnosis, 853–856 differential diagnosis, 859 epidemiology, 853 histology, 857–858 histopathology, 1073 historical background, 851, 851–853 laboratory investigations, 858–859 mucosal pigmentation, 1072–1073 pathogenesis, 859 prognosis, 860 treatment, 859–860 carotenemia, 530, 1036–1038, 1038 b-carotene, skin discoloration, 1036–1038, 1038 carotenoids, 19, 22 amphibian egg, 24–25 deficiency, 25 skin discoloration, 1036–1038, 1038 Casal’s necklace, 996 castrated males, skin coloration, 667 cataract, 96, 375 catecholamines, color change (nonmammalian) role, 36–37 cathecols, 675 cativa, 687–688 cats, pigment type switching, 404–405 CD4+ cells melanocytic neoplasia associated hypomelanosis, 716–717 melanoma regression, 718 vitiligo autoimmunity, 573 CD8+ cytotoxic T cells (CTL) melanocytic neoplasia associated hypomelanosis, 716 melanoma-associated depigmentation, 719 melanoma regression, 718 vitiligo autoimmunity, 573 CD8+ T cells, fixed drug eruption, 1029 CDK4 mutations, melanoma, 472, 476 CDKN2A gene/protein atypical mole syndrome, 474 gene products (p16/ARF), 474–475, 475 mutation in familial melanoma, 472, 474–476 nevogenesis, 1129–1130 “celery burns,” see phytophotodermatitis celiac disease, 666 cell adhesion molecules (CAMs) melanoma and, 481–482 pigmentation pattern formation, 45, 47, 112–113 cell–cell interactions, amphibian morphogenesis, 116–117 cell cycle regulation, see also specific molecules melanocyte senescence, 465 melanoma, 453–454 cell death fish pattern formation, 112 melanin-induced, 72 cellular immunity halo nevi, 709–710 tinea versicolor, hypopigmentation pathogenesis, 693–694 Celtic peoples epidermal melanocytes and, 66 melanin content, 74 red hair color, 69 central nervous system (CNS), see also neuromelanin defects hypomelanosis of Ito, 638 Vogt–Koyanagi–Harada syndrome, 735 xeroderma pigmentosum, 890 drug-induced toxicity, 376–378 melanocortin signaling mutants, 402 centrofacial lentiginosis, 837–842 age of onset, 840 animal models, 841 associated disorders, 839 Carney complex/myxoma syndrome vs., 859 clinical description, 838–839, 846 diagnostic criteria, 840 differential diagnosis, 840 epidemiology, 839–840

1207

INDEX historical aspects, 837–838 inheritance, 839 laboratory findings/investigations, 840 neuropsychiatric disorders, 839 pathogenesis, 840–841 alcoholism, 840–841 mechanistic theories, 841 syphilis-induced mutation hypothesis, 840 prevalence, 839 sexual distribution, 839 cerebrovascular anomalies, agminated lentigines, 865 ceroid-lipofuscin, Hermansky–Pudlak syndrome, 615 chaperone proteins, 159–162, 160, 160, 232 Chediak–Higashi syndrome (CHS), 530, 614, 616, 616–617 chaperone protein mutations, 159, 161–162 molecular pathogenesis, 617 “cheetah” phenotype, 1130 chemical melanocytotoxins, 562 chemokines, in pigmentary disorders, 421–444, see also specific molecules chemophototherapy, see photochemotherapy chemotherapy, see also specific agents/treatments drug-binding to melanins, 379, 380–383, 381, 383–384 melanocytic nevi and, 1125–1126 melanoma (see melanoma) mycosis fungoides, 977–978 photochemotherapy (see photochemotherapy) skin discoloration induction, 1044, 1044–1045, 1045, 1045–1047 children, skin color, 511–512 CHK2 gene/protein, familial melanoma and, 477 chloasma, 513, 1021 chloroquine porphyria cutanea tarda, 982 skin discoloration, 982 toxicology of melanin binding, 375 eye, 375–376 ototoxicity, 376 skin, 374 chlorpromazine CNS toxicity and parkinsonism, 377 ocular bodies, 375, 1044 skin discoloration and, 1044 choroid anatomy/structure, 93, 93, 94, 96, 96 development, 91–92 melanocytes, see uveal melanocytes variation in, 96 Christ–Siemens–Touraine syndrome (hypohidrotic ectodermal dysplasia), 901, 902 “chromatic abnormalities,” terminology, 499–503 chromatics, 501, 501 chromatophore(s), 108, see also color change (nonmammalian); specific types adaptive mechanisms, 33–35 amphibian egg as, 24, 24–25, 25 characterization, 14–27 definition/terminology, 11, 12, 14, 14 embryology/development, 11, 12, 42, 51, 52, 76–77 amphibians, 115–119 fish, 109–115 reptiles, 119 extracutaneous, 17, 51–52, 91 functions, 25–28 hormone actions on, 12–13, 32–35, see also individual hormones mechanisms of, 35–37 innervation, 26–27 patterns/patterning, 42–48, 45, 52–53 photosensitive, 35, 52 responses, 27–28 adaptive mechanisms, 33–35 integration, 26 morphologic color change, 27–28 physiologic color change, 27 signaling mechanisms, 26 unknown type, 24 chromatophoromas, 50 chromoheteropia, see heterochromia irides chromometer/chromatometry, 533–534 chromophores human skin color and, 505

1208

melanotic, see melanin(s) nonmelanotic, 501, 505 terminology, 501 UV absorption, 343–344 chronic cyclitis, see heterochromia irides chronic nonsuppurative destructive cholangitis, see primary biliary cirrhosis (PBC) chrysiasis, 525, 1032, 1032 CHS1 gene, Chediak–Higashi syndrome, 616 ciclosporin-induced skin discoloration, 1047 ciliary bodies, 96, 96–97 ciliary epithelium, 97 circumscribed hypermelanosis, see café-au-lait spots (macules: CALMs) circumscribed melanotic macule, see café-au-lait spots (macules: CALMs) circumscribed scleroderma, see morphea cisplatin, skin discoloration, 1030, 1046 Clark levels oral melanoma, 1080 ungual melanoma, 1066 cleft lip-Mongolian spot, 1005 cliff sign, 963 clofazamine hyperpigmentation induction, 943, 1042–1043, 1043 leprosy, 692 clonality, melanocytic nevi, 1113 clonidine, carcinoid syndrome, 921 Clouston syndrome (hidrotic ectodermal dysplasia), 902–903, 903 “Club Med dermatitis,” see phytophotodermatitis clump cells of Kogenei, 97 CMM4 mutations, melanoma, 472, 476 coat, see hair/coat cochlea, melanocytes, 99, 100, 100–101 COL3A1 gene mutations, acrogeria, 885 colitis, Hermansky–Pudlak syndrome, 615 color change (nonmammalian) adaptation to darkness, 33, 33–35, 34 background adaptation, 25–26, 29, 32–33, 33 cellular associations, 40–42, 41 control of, 12–13, 28–35, see also specific hormones adaptive mechanisms, 33–35 mechanisms of hormone action, 35–37 melanophore stimulation, 30–31 pituitary role, 12–13, 28–29 morphologic, 27–28, 28, 29, 31 physiologic, 12, 16, 16, 27 pigment granule translocation, 13, 37–40 subcellular associations, 41–42 compound nevi Carney complex/myxoma syndrome, 857 melanocytic, 1118 computed tomography (CT), Carney complex/myxoma syndrome, 858 confluent and reticulated papillomatosis (CRP), 922–924 clinical features, 910, 922, 922 diagnosis, 923 endocrine dysfunction, 923 epidemiology, 922 pathology/pathogenesis, 922–923 treatment, 923 congenital bilateral dermal melanocytosis, 1015 congenital generalized dermal melanocytosis, 1015 congenital histiocytosis, hemosiderosis, 990 congenital lentiginosis, see LEOPARD syndrome congenital melanocarcinoma, see melanotic ectodermal tumor of infancy congenital nevus, 515 depigmentation halos, 707, 707, 708 congenital poikiloderma with blisters of Kindler–Salamon, see Kindler syndrome congenital poikiloderma with bullous lesions “Weary type,” see hereditary acrokeratotic poikiloderma congenital segmental dermal melanocytosis, 1015 congenital universal hypomelanosis, 624 conservatism, mammalian pigmentary system evolution, 76–78 constitutive skin color (CSC), 65, 507 histological basis, 65–67 UV response, 410–412 contact leukoderma, genital, 1087

contact sensitivity, Riehl’s melanosis, 962 copper ions binding agents, 361, 361–362 melanin binding, 326, 326 Menkes’ kinky hair syndrome, 632 tyrosinase active site, 217–218, 265, 356, 361–362, 365, 365 copper vapor laser, 1200 cortical microadrenomatosis, see primary pigmented nodular adrenal disease corticosteroids injected, hypopigmentation-induction, 1167, 1168 melasma, 678 mycosis fungoides, 976 systemic, vitiligo vulgaris, 580–582 topical combination therapy, 1169 hyperpigmentation disorders, 1167, 1169 hypopigmentation treatment, 1170 lichen sclerosus, 1087 UVB phototherapy and, vitiligo vulgaris, 1185 vitiligo vulgaris, 579–580 cosmetics, 1188–1189 camouflage stains, 1188–1189 cover-up, 1188, 1189 melasma-induction, 1021–1022 Riehl’s melanosis, 962 co-trimoxazole, fixed drug eruption, 1027 coumarins, 675 counseling, vitiligo vulgaris, 583–584 craniofacial–deafness–hand syndrome, 143–144, 544–545 “craw-craw,” 694 “crazy paving stone” dermatitis, kwashiorkor, 664 creole dyschromia, see progressive macular hypomelanosis Crohn’s disease, 995 Cronkhite–Canada syndrome, 983–986 associated disorders, 984 clinical features, 984, 984 epidemiology, 983–984 histology, 984–985 pathophysiology, 985 Cross syndrome, see oculocerebral syndrome with hypopigmentation Crow sign, LEOPARD syndrome, 844 CRP of Gougerot and Carteaud, see confluent and reticulated papillomatosis (CRP) Cruickshank, William, 7 curettage, dermatosis papulosa nigra, 929 Cushing syndrome, 667 Carney complex/myxoma syndrome, 855, 857 pigmented adrenocortical nodular dysplasia, 852–853, 859 treatment, 859 cutaneous hepatic porphyria, see porphyria cutanea tarda cutaneous malignant melanotic neurocristic tumor (CMMNT), 1158, 1160 cutaneous myxomas, Carney complex/myxoma syndrome, 855, 856, 856, 857–858 cutaneous papillomatosis, see confluent and reticulated papillomatosis (CRP) cute, 687–688 cutis trunci variata, see progressive macular hypomelanosis cyanophores, 14, 24 MSH effects, 31 cyanosomes, 24 cyclic AMP (cAMP) signaling adrenergic receptors and, 37 dendrite formation and, 171 melanocortin-1 receptor (Mc1R), 395 agouti protein and, 395 MSH action and, 35–36, 85, 246, 359, 395 tyrosinase and, 403 melanosome translocation, 39 cyclin-dependent kinases (CDKs), see also specific CDKs in aging melanocytes, 465 in melanoma, 454, 472, 474–476 cyclobutane pyrimidine dimers, 344, 347–348, 349 L-cyclodopa (leukodopachrome), 263, 263–264, 284 cyclophosphamide, skin discoloration, 940, 1046

INDEX CYP2D gene/protein, familial melanoma and, 477 cyproheptadine acanthosis nigricans, 912 carcinoid syndrome, 921 cystathionine b synthase deficiency, 529 cystathionine synthase, 626 cysteinyldopamine-melanin, 301, 301–302 cysteinyldopas, 282, 284, 286, 286, 291–292, 292, 354 melanoma markers, 293–294 pigmented spindle cell nevi, 1095 protective roles, 295 thiol source, 397 cystinosis, 370 cytochrome P450, familial melanoma and, 477 cytokines, see also specific molecules human melanocyte regulation, 412, 413 melanoma and, 491–492 in pigmentary disorders, 421–444 cytoskeleton keratinocyte intracellular transport, 182–186 maintenance of melanin cap, 182, 185–186, 186 melanocyte intracellular transport, 13, 37–40, 38, 171–173 cytotoxicity melanin and, 72, see also drug-binding to melanin(s) phototoxicity, 73–74, 334, 334 precursors, 294–295, 295, 366–367 melanocyte sensitivity to, 354, 371 neurotoxicity, 376–378 protective mechanisms, 367, 509–510 dactinomycin-induced skin discoloration, 1046 danthron, skin discoloration, 1048 dapsone, leprosy, 692 Darier sign, 955 Darier–White disease, 647–649, 648 dark dot disease, see Dowling–Degos disease darkness, adaptation to, 33, 33–35, 34, 35 pineal gland role, 33–34 daunorubicin-induced skin discoloration, 1046 Davy, Humphrey, 7 deafness, see hearing loss delayed tanning (DT), 343 Delleman–Oorthuys syndrome (oculocerebrocutaneous syndrome), 630 dendrite formation (melanocytes), 171–173, 173, 176, 176–177, 356 dental defects, hypomelanosis of Ito, 638 dento-oculo-cutaneous syndrome, 903 depigmentation, see also hypomelanosis acquired, 370, 551–598, see also depigmenting agents chemically-induced, 562 drug-induced, 370–371, 582–583, 715, 715–716 monobenzone-induced, 1170, 1170–1172, 1171, 1172 occupational, 669–670 Rozycki syndrome, 551 toxicology, 369–371 Vogt–Koyanagi–Harada syndrome, 735–736 yaws, 686–687 animal, 576–577 confetti-like, 567, 567 confusing terms, 500 congenital, 370 definition, 527 generalized, 369 halos, 707, 707, 708 human skin color evolution, 73–74 lupus erythematosus, 703 melanin modification, 358 melanoma, 566–569, 567, 568 morphological/functional defects, 510 onchocerciasis, 695, 695 pinta, 687 Scarpa triangle, onchocerciasis, 695 segmental, 370, 371, see also piebaldism/piebald trait vitiligo, see vitiligo vulgaris (vitiligo) vitiligo-like, atopic dermatitis, 702, 703 depigmenting agents, 562 bleaching agents, 357–358, 1022 classification, 672 drugs, 370–371, 582–583, 715, 715–716

mechanism of action, 670–671, 672 melanogenic enzyme regulators, 671–677 occupational, 669–670 dermal chromatophore unit, 16–17, 22, 40–41, 41 dermal hyperpigmentation, Mongolian spots, 63, 69 dermal hypoplasia, focal, 645, 646 “dermal light sense,” 13 dermal melanocytes, 63 blue color, 69–70 humans, 69–70 Mongolian spot, 63, 69, 358, 1005 nonhuman mammals, 70 dermal melanocytic hamartoma (persistent Mongolian spot), 1005 dermal melanocytic nevus, 1118 dermal melanocytosis, 523, 524 acquired, 1016–1017 classification, 1016 pathogenesis, 1017 congenital, 1015–1016 clinical findings, 1015 histology, 1015–1016 definition, 1165 dermal melanophores, 11, 14, 16, 16–17, see also specific types extracutaneous, 17 functions, 40–41 mosaic, 42 MSH sensitivity, 17, 28, 29 phyllomedusine melanosomes, 17, 17, 18 structure, 17 dermal melanosis, 522, 523–524 dermal neurofibromas, 812 dermatitis allergic contact, 949 atopic hyperpigmentation, 911 hypopigmentation, 702–703, 703 atrophic degenerative pigmentary, see poikiloderma of Civatte paederus, 949 phototoxic, see phytophotodermatitis pigmented contact, see Riehl’s melanosis dermatitis bullosa striata pretensis, see phytophotodermatitis dermatofibroma, 433–435, 434, 440, 441, 441 dermatomal vitiligo, 500–501 dermatopathia pigmentosa reticularis (DPR), 784–786, 789, 797 clinical findings, 784–785 diagnosis/differential diagnosis, 785, 785–786 genetics, 784 histology, 785 pathogenesis, 786 dermatophyte infection, pigmenting pityriasis alba, 700 dermatosis papulosa nigra, 928, 928–929 dermatospectrometer, 534 dermis, see also entries beginning dermal development, 124 human pigmentation, 69–70 melanoma cell invasion, 490 dermoscopy, 533, 1063 De Sanctis–Cacchione syndrome, 890 desferrioxamine, hemosiderosis, 991 desipramine-induced skin discoloration, 1044 development, see embryology (pigment cells) DHICA oxidase, see tyrosinase-related protein-1 (TYRP-1) diabetes mellitus acanthosis nigricans, 908 hereditary hemochromatosis, 987 vitiligo vulgaris, 564, 566 dibromomannitol-induced skin discoloration, 1046 didymosis (twin spotting), 1015, 1107, 1107, 1130 diethylcarbamazine, onchocerciasis, 696 diffuse lentigo, see LEOPARD syndrome digital malformations, Pierre Robin syndrome, 661 dihydrolipoic acid, 673 2,2¢-dihydroxy-5-5¢-dipropyl-biphenyl (DDB), 677 dihydroxyacetone, skin discoloration, 1048 dihydroxybenzene, see hydroquinone (dihydroxybenzene; HQ)

5,6-dihydroxyindole (DHI), 264, 282, 283, 284 DHICA ratio in late eumelanogenesis, 290–291 melanoma marker, 293 protective role, 295 5,6-dihydroxyindole-2-carboxylic acid (DHICA), 264, 269, 282, 283, 284 DHI ratio in late eumelanogenesis, 290–291 melanoma marker, 293 protective role, 295 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase, see tyrosinase-related protein-1 (TYRP-1) dihydroxyphenylalanine reaction, 534 3,4-dihydroxyphenylalanine (DOPA) staining, see dopa histochemistry N, N¢-dilinoleylcystamine, 676 dilute mouse mutant, 173–174 2,4-dinitrophenol, skin discoloration, 1048 dinitrosalicylic acid, skin discoloration, 1048 dioxin-induced skin discoloration, 1047 discoid lupus erythematosus, pigmentary disturbances, 703, 703 disseminated hypopigmented keratoses, 745–746, 746 pathogenesis, 745–746 DKC1 gene, dyskeratosis congenita, 899 DNA aging/senescence, 468 diagnosis, KIT gene mutations, 142 photodamage, 185, 342, 347–348 action spectra, 344, 344 melanin-induced cell death and, 72 melanogenesis induction, 348–349 ploidy analysis, lentigo simplex, 826 repair, 342 melanogenesis induction, 348–349 skin types and, 411, 412 DNTNBP1 gene, Hermansky–Pudlak syndrome type 7, 616 dogs mycosis fungoides, 976 pigment type switching, 405 dominant-negative mutations, KIT gene, 142 L-dopa formation, 365 oxidation, 262, 263, 266 assay, 272, 274, 274–275 skin discoloration, 1047 L-dopachrome, 263, 264, 274, 284 dopachrome conversion factor (DCF), see tyrosinase-related protein-2 (TYRP-2) dopachrome oxidoreductase, see tyrosinaserelated protein-2 (TYRP-2) dopachrome tautomerase (Dct), see tyrosinaserelated protein-2 (TYRP-2) dopa histochemistry, 155, 534 leprosy, 690–691 pityriasis alba, 700 senile canities, 760–761 dopa-melanin, 289–290, 313 physical properties, 311–341 dopamine-melanins, 301, 301–302 L-dopaquinone, 263, 263, 282, 284, 284 conjugation with 2-thiouracil, 368–369, 369 inhibitory effect, 671 intrinsic reactivity, 285, 285, 354 melanogenesis control by, 285, 285–287 redox exchange, 365–366 thiols and, 367 dorsal spinal line, 881 Dowling–Degos disease, 882–883 clinical features, 803, 882, 882 differential diagnosis, 882–883, 883, 910 histology, 882 historical background, 882 treatment, 883 vulvar, 1084 Down syndrome, 658–659 doxorubicin (adriamycin)-induced skin discoloration, 1046 Drosophila melanogaster, “granule group” mutants, 162 drug-binding to melanin(s), 356, 356, 372, 509–510, see also specific drugs mechanisms of binding, 371–372 pharmacological uses, 378–383 applied toxicity (chemotherapy), 380–383, 381, 383–384

1209

INDEX biological monitoring, 379–380 photochemotherapy, 379 tinnitus treatment, 378, 378–379 toxicity/toxicology, 373–374, 383, see also drug-induced pigmentation changes carcinogenesis, 373, 374, 383 chloroquine, 374, 374, 375–376 CNS, 376–378, 377 eye, 375–376 inner ear, 376 kinetics, 372–373 skin, 374–375 thioureylenes, 368–369, 369 drug-induced phototoxic reactions, 949 drug-induced pigmentation changes, 1026–1054, see also fixed drug eruption; specific drugs depigmentation, 370–371, 582–583 immunotherapy, 715, 715–716 monobenzone, 1170, 1170–1172, 1171, 1172 hyperpigmentation, 940 mycosis fungoides treatment, 977 nails, 1081 oral mucosa, 1081, 1081 “dual filament transport model,” 39 ductal adenomas (breast), Carney complex/myxoma syndrome, 856–857 dye-induced skin discoloration, 1048 dynactin, melanosome trafficking in keratinocytes, 183 dyneins, 185 keratinocyte intracellular transport, 182–186, 184 melanin cap formation, 185–186, 186 melanocyte intracellular transport, 38, 172–173, 174, 183 structure, 182–183 dysacousia, Vogt–Koyanagi–Harada syndrome, 559 dysbindin, 616 dyschromatosis hereditaria, see dyschromatosis universalis hereditaria dyschromatosis universalis hereditaria, 786–788 associated disorders, 786–787 clinical features, 785, 786, 787, 788 pathology, 787 dyschromic and atrophic scleroderma, see atrophoderma of Pasini et Pierini dyskeratosis congenita, 777, 785–786, 898–901 associated findings, 898–899 clinical findings, 898, 899 histology, 899 pathogenesis, 899 prognosis, 899–900 dyskerin, dyskeratosis congenita, 899 dysplastic nevi HIV, 944 melanocytic, 1112 melanoma and, 473, 473, 482 nevus spilus, 1099 dysraphia, centrofacial lentiginosis, 838–839 dystopia canthorum, Waardenburg syndrome, 544 dystrophia bullosa hereditaria, see Mendes da Costa syndrome dystrophia myotonica, 660–661 dystrophie papillaire et pigmentaire, see acanthosis nigricans ear, see also auditory system drug toxicity, 376 melanocytes, see otic melanocytes pigment cells, vitiligo vulgaris, 560 Ebstein’s disease, 1104 echocardiographic screening, Carney complex/myxoma syndrome, 858 ectodermal dysplasia, ectrodactyly, cleft lip and palate (EEC syndrome), 901–902 ectodermal dysplasias, 901–905 abnormal thumbs, 904 classification, 901 hidrotic (Clouston syndrome), 902–903, 903 hypohidrotic, 901, 902 tricho-odonto-onychial type, 903 X-linked anhidrotic, 901, 902 ectomesenchymoma, 1161 ectopic ACTH syndrome, 938–939, 939 eczema, nevus counts, 1126, see also dermatitis

1210

edema, skin, 768 EDN3 gene mutation, Waardenburg syndrome, 544 EDNRB (Ednrb) gene/protein melanocyte morphogenesis, 123–124 mutations, 544, 545 EEC syndrome (ectodermal dysplasia, ectrodactyly, cleft lip and palate), 901–902 EGF motif, tyrosinase gene family, 217 egg, amphibian as chromatophore, 24, 24–25, 25 egr-1, melanoma growth role, 452 electrical properties, melanins, 320–322 electrocardiography (ECG; EKG) abnormalities, LEOPARD syndrome, 844–845, 846 leads, skin discoloration, 1045 electroencephalography (EEG), tuberous sclerosis complex, 655 electron microscopy (EM), 534–535 familial progressive hyperpigmentation, 775 hypermelanocytic punctata et guttata hypomelanosis, 747, 747 Kindler syndrome, 783 leprosy, 691 melanins, 312–313 melanotic ectodermal tumor of infancy, 1152–1153 oculocerebral syndrome with hypopigmentation, 627–628 pityriasis alba, 700 sarcoidosis, 752 senile canities, 761, 761, 762 speckled lentiginous nevus, 1105 tinea versicolor, 693 ultrastructural, 535 Westerhof syndrome, 742–744 electron paramagnetic resonance (EPR) spectroscopy, melanins, 311, 322–325, 323, 328 electron–proton coupling, melanins, 319 electron resonance (ER) spectroscopy, melanins, 299 electrophoresis, enzyme activity assays, 276 Elejalde syndrome, 175 elemental melanin analysis, 298–299, 315 ellagic acid (EA), 676 embryology (pigment cells), 76–77, 108–139, see also melanoblast(s); neural crest; specific genera/species comparative biology, 127 gene regulation, 243–248 growth factors and signal transduction regulating, 445–463, see also specific molecules/pathways historical background, 108–139, 445 mammalian, 78–86, 122–126, 489, 541 genetic disorders, 527–528, 541 identification of biological growth factors, 445–448 murine coat development, 78–82 murine morphogenesis, 123–124 ocular melanocytes, 91–93, 95 otic melanocytes, 99 nonmammalian, 11, 12, 42 amphibians, 115–119 birds, 119, 119–122 organelles, 16, 52 pattern formation, see pattern formation (nonmammalian) reptiles, 119 teleost fish, 109–115 organellogenesis, 16, 52, 155–170, see also melanosome biogenesis pattern formation, 108 stem cells, 489 emtricitabine (FTC), hyperpigmentationinduction, 943 en coup d’sabre, 968 endemic syphilis, 688 endocochlear potential (EP), otic melanocyte role, 100–101 endocrine system, see also hormones; specific components disorders/dysfunction Carney complex/myxoma syndrome, 855 centrofacial lentiginosis, 839 hypomelanosis, 667–668 LEOPARD syndrome, 845

melanonychia, 1061 NAME syndrome, 854 POEMS syndrome, 952 vitiligo and, 564–566 human melanocyte regulation, 411 endoplasmic reticulum (ER), melanosome biogenesis, 155–157 endothelin(s), 422, see also individual types avian melanocyte morphogenesis, 121 fish chromatophore development, 114 mammalian pigmentary system development, 83 as melanocyte mitogens, 446, 491 receptors, 422 B receptor, see endothelin-B (ETB) receptor gene mutations, 145, 146 melanoma and, 453 Waardenburg’s syndrome and, 145, 146 endothelin-1 (ET-1), 421 café-au-lait spots and, 435 fibroblast–melanocyte interactions, 435 inflammatory cytokine induction, 425, 426–427 keratinocyte–melanocyte interactions, 422–427 lentigo senilis, 424–425, 425 seborrheic keratosis, 425–427, 426 UVB melanosis, 422–424, 423, 424, 425 melanocyte regulation, 412–413, 414, 446, 670 endothelin-3 (ET-3), melanocyte regulation, 446, 491 endothelin-B (ETB) receptor, 414, 422 keratinocyte–melanocyte interactions, 422–427 lentigo senilis, 424–425, 425 seborrheic keratosis, 425–427 UVB melanosis, 422–424, 423, 424, 425 melanocyte differentiation/proliferation role, 446 melanoma and, 453 Waardenburg syndrome, 527, 545 endothelin-converting enzyme (ECE), 422, 427 environment adaptation to background, see background adaptation darkness, 33, 33–35, 34, 35 hair/coat color and, 75–76 enzyme assays, 261, 270–276, 272–273, see also specific methods L-dopa oxidation, 272, 274, 274–275 historical background, 270–271 melanin formation, 273, 276 tyrosine hydroxylase, 271, 272 TYRP-2 activity, 273, 275–276 eotaxin, incontinentia pigmenti, 877 ephelides, 515, 515, 929–931 associated conditions, 929–930 axillary, 532, 811 Carney complex/myxoma syndrome, 857 clinical findings, 929, 929–930 differential diagnosis, 930 histopathology, 1074 intertriginous, 810, 811 lentigines vs., 824 lip vermilion, 1073–1074 melanocytic nevi association, 1127 melanoma association, 929–930 NAME syndrome, 853–854, 854 pathology, 930 ephelis ab igne, see erythema ab igne ephelis ignealis, see erythema ab igne ephrins, avian melanocyte morphogenesis, 120 epidermal cysts, normal color, 515–516 epidermal growth factor 2 (EGF2), familial melanoma and, 477 epidermal hypermelanoses, see also individual diseases/disorders acquired, 907–978 congenital, 898–906 melanocytic, 521, 522 melanotic, 521–522, 522 epidermal hyperplasia, pigmented spindle cell nevi, 1095 epidermal melanin unit, 16, 17, 41, 66, 312 epidermal melanocytes evolution, 77–78 extracutaneous vs., 102 human, 65, 65–67 neural crest origin, 122

INDEX epidermal melanophores, 11, 14, 15, 15–16, 41 morphologic color change, 27–28 epidermal nevi, clinical features, 911 epidermal pigmentation, lentigine associated, 824–872 epidermal syndromes (genetic), see also individual disease/disorders aging disorders, 884–897 café-au-lait macules, see café-au-lait spots (macules: CALMs) generalized hyperpigmentation, 771–779 localized hyperpigmentation, 873–883 reticulated hyperpigmentation, 780–808 epidermis, see also entries beginning epidermal development, 124 human, 65, 65, 185, 342, 505, 505, 506, 507 synthesis of pigmentary regulatory factors, 412–413 epidermolysis bullosa (EB) melanocytic nevi formation, 1121, 1125 Mendes da Costa variant, see Mendes da Costa syndrome with mottled pigmentation, 788–790 clinical findings, 788–789, 789, 797 diagnosis/differential diagnosis, 789, 789–790 genetics, 788 histology, 789 pathogenesis, 790 epidermolysis bullosa simplex (EBS), Mendes da Costa variant, see Mendes da Costa syndrome epigenetics, melanocyte aging/senescence, 468 epiloia, see tuberous sclerosis complex (TSC) epiluminescence microscopy, see dermoscopy epinephrine, color change and, 36–37 epithelioid blue nevus (EBN), 1073 eponychium, 1057, 1059 equine melanotic disease, 1161 erbb3 gene/protein, fish chromatophore differentiation, 114 ERK2, MITF-M phosphorylation, 246 erythema minimal dose causing, 344 skin type and, 346–347 erythema ab igne, 931–933, 932 erythema a colore, see erythema ab igne erythema caloricum, see erythema ab igne erythema dyschromicum perstans, 879, 933–935 clinical findings, 933–934, 934 differential diagnosis, 934 epidemiology, 933 HIV, 943 pathology/pathogenesis, 934 erythema streptogenes, see pityriasis alba erythromelanosis follicularis faciei et coli, 935–937, 936, 937 erythrophores, 14, 14, 19–24 migration speed, 39 erythrophoroma, goldfish, 48, 49, 49 differentiation, 49–50, 51 erythrose péribuccale pigmentaire of Brocq, 937–938, 938 erythrosis pigmenta faciei, 937–938, 938 erythrosis pigmentosa peribuccalis, 937–938, 938 esculetin, 675 esculin, 675 essential cutaneo-mucous hyperpigmentation, see Laugier–Hunziker syndrome estrogen melanocyte regulation, 416–417 melanocytic nevi development, 1126 melanogenesis regulation, 195 ethiops, 8 ethnicity, see also skin types (human) eye color, 95, 507 Golger’s rule, 504–505 hair color, 507 MC1R variations, 401 oral mucosa pigmentation, 1072, 1072 skin color and, 63, 65, 66, 66–67, 342, 411, 505–508 chemistry of, 499–500 etretinate confluent and reticulated papillomatosis, 923 Darier–White disease, 649 xeroderma pigmentosum, 892

eumelanin(s), 159, 192, 282, 355, 395 biosynthesis, 193, 263, 263–264, 276–277, 282, 284, 284–285, 342, 365, 397 agouti protein signaling and, 196–197, 197, 395, 416 control, 285, 285–286, 315 early stages, 290, 291 late stages, 290–291, 292 P protein and, 232 brown oculocutaneous albinism, 606 carboxyl content, 296 classification, 287–289, 288 degradation, 295, 295–296, 296, 300 permanganate vs. peroxide oxidation, 302, 302 quantitative analysis, 299–300 ethnic variation in, 506–507 eumelanin/pheomelanin switch (see pigment type switching) functional groups, 315 human hair, 69 human skin, 64–65 hypomelanosis of Ito, 640 isolation, 289 pheomelanin ratio, 350 pheomelanins vs., 298, 298–299, 397 photogeneration of free radicals, 330 photoprotection, 412 structure, 287–289, 288 synthetic, 289–290 eumelanosomes, 159, 312 evolution mammalian pigmentary system, 63–78, 91 hair/coat color, 67–69 human skin color, 64–65, 72–74, 504 melanin adaptations, 63, 72–74 nonhuman primates, 71, 74 radicalism and conservatism in, 76–78 of mammals from reptiles, 63–64, 76–77 MATP gene, 237 melanin and, 357 pigmentary organelles, 41 P protein, 235 tyrosinase gene family, 215 evolutionary conservation agouti protein, 400–401 tyrosinase, 249, 361 excimer laser, 1183 postsurgical leukoderma, 1185 vitiligo vulgaris, 580, 1185 “exclamation mark” hairs, alopecia areata, 755 exocytosis, melanosome transfer to keratinocytes, 177 Experiments and Considerations Touching Colours, 6 extracellular matrix (ECM), amphibian chromatophore morphogenesis, 114–115 extracellular signal-related kinase (ERK) signaling, see mitogen-activated protein kinase (MAPK) signaling cascade extracutaneous melanocytes, 91–107, see also specific types/locations auditory (see otic melanocytes) epidermal vs., 102 historical background, 91 internal organs, 101–102, 102 nonmammalian, 17, 52 ocular (see ocular melanocytes) eye(s), see also choroid; entries beginning oculo-/ ocular; retina; visual system anatomy, 96 chloroquine effects, 375–376 color Drosophila “granule group” mutants, 162 ethnic variation, 507 mammalian, 98 development, 91–93 melanocytes (see ocular melanocytes) protection, oral PUVA, 582, 1177 structure, 93, 93–94, 94, 95 toxicology of melanin-affinic compound, 373 vitiligo vulgaris, 558–560, 559, 560 eyebrows, vitiligo vulgaris, 560 eyelashes, vitiligo vulgaris, 560, 561 eye–skin syndrome, 374 eyewear, protective in PUVA, 582, 1177

FACES syndrome, 1104 facial angiofibromas, tuberous sclerosis complex, 653, 653 facial dermal dysplasia, focal, 904 facial malformations, LEOPARD syndrome, 844 facultative skin color (FSC), 343, 507 histological basis, 65–67 UV response, 410–412 familial atypical multiple mole and melanoma syndrome (FAMMM), 1129, 1131–1132 familial mandibuloacral dysplasia, 790–792, 792 familial melanoma, 472, 474–477 atypical mole syndrome, 474, 474 low-penetrance genes, 477 susceptibility/high-penetrance genes, 474–476 familial multiple café-au-lait spots (neurofibromatosis 6), 809, 813 familial obstructive cardiomyopathy, see LEOPARD syndrome familial progressive hyperpigmentation, 774–776, 906, 914 histology, 775 familial universal or diffuse melanosis, see familial progressive hyperpigmentation Fanconi anemia, 776, 776–778 fatty acids melanogenesis regulation, 200 very long chain (VLCFAs) in adrenoleukodystrophy, 771 Felty syndrome, 939–941, 940 female(s), see also pregnancy facial melanosis, see Riehl’s melanosis human skin color, 513 Fenton reaction, 334, 334, 357–358 ferroportin 1 (IREG1; MTP1) gene, hemochromatosis, 988 fetal hydantoin syndrome, 1152 b-fibrilloses, see amyloidosis, cutaneous fibroblast(s) choroidal, 93, 94 dermatofibroma, 433–435, 434 melanocyte interactions, 433–437 melanocyte mitogen production, 447 melanoma progression, 490, 491 fibroblast growth factor-2 (FGF-2) café-au-lait spots, 435–436 fibroblast–melanocyte interactions, 435–436 human melanocyte regulation, 412, 413–414 mammalian melanocyte requirement, 445–448 melanomas and, 451–453, 490 growth promotion, 451–452 mechanism of action, 452–453 overexpression in tumorigenesis, 491 transcriptional control, 452–453 fibroblast growth factor receptors (FGFRs), 446 in melanoma growth, 451–452 mutations, 447–448 fibroblast growth factors (FGFs) basic (FGF2), see fibroblast growth factor-2 (FGF2) hair follicle growth and, 86 human melanocyte regulation, 412, 413–414 fibrolamellar hepatoma, Carney complex/myxoma syndrome, 857 filopodia, melanocytes, 176, 176–177, 177 Fisch syndrome, 659 fish, see teleost fish Fitzpatrick skin type classification, 506–507, 507 fixed drug eruption, 1026–1029 clinical findings, 1026–1027, 1027 cross sensitivity, 1028 epidemiology, 1026 histology/histopathology, 1027–1028, 1073, 1074 historical background, 1026 mucosal hyperpigmentation, 1073, 1074 nonpigmenting, 1027 pathogenesis, 1028, 1028–1029 polysensitivity, 1028 sensitization, 1027 treatment, 1029 flag sign, kwashiorkor, 664, 665 “flaky paint/enamel paint” rash, kwashiorkor, 664, 665 “floating teeth,” melanotic ectodermal tumor of infancy, 1153 fluconazole, tinea versicolor, 694

1211

INDEX fluorometric assay/analysis melanins, 319, 320 tyrosinase activity, 272, 274 5-fluorouracil-induced skin discoloration, 1046 flushing, carcinoid syndrome, 919–920 focal dermal hypoplasia, 645, 646 folate/folic acid deficiency, 666, 666 hyperpigmentation in HIV, 943 follicular melanocytes, 63, 67–69 bulb, 1059 hair follicle growth regulators and, 86 humans, 68–69 mice, 67–68, 68 regulation of differentiated cells in mice, 85 follicular mucinosis, 702 clinical features, 702, 702 hypopigmentation, 702 mucosis fungoides and, 974 treatment, 702 Fontana–Masson stains, 534 forensic toxicology, 372–373, 379–380 foveal hyperplasia, albinism, 601–602 FOX01A–PAX3 fusions, 144 FoxD3, avian melanocyte specification, 122 framboesia tropica, 686–687 framboesides, 686 frameshift mutations, KIT gene, 142 Franceschetti–Jadassohn syndrome, see Naegeli–Franceschetti–Jadassohn syndrome freckles, see ephelides free fatty acids, melanogenesis regulation, 200 free radicals in melanins, 311, 322–325, 323 inducible vs. intrinsic, 324 photoformation, 329–330, 330 stability, 323 scavengers, 677–678 (see also antioxidants) melanin as, 72, 311, 331–334, 333, 345 freezing, depigmentation, 684 friction amyloidosis, 925 Fröhlich syndrome (adiposogenital syndrome), 839 Fuch’s heterochromic uveitis, 96 fungi non-melanocytic melanins, 301–302 tyrosinase gene family, 214–215 furocoumarins, see psoralens Fusarium solani, melanonychia, 1061 Futcher lines, see pigmentary demarcation lines “gale filarienne,” 694 gamma-glutamyl transpeptidase (Gpt), 403 gammopathy, associated hyperpigmentation, 965 garment nevi, 1148, 1149 Garrod, Sir Archibald, 599 gastrointestinal disorders dyskeratosis congenita and, 898 hypermelanosis and, 979–1002 KIT mutations in, 141, 143 gastrointestinal stromal tumors (GIST), KIT mutations, 141, 143 Gaucher cells, 778 Gaucher disease, 778–779 Gaultier, M., 8 gender, human skin color, 513 generalized bullous fixed eruption, 1027 generalized morphea, 967, 968 generalized nevoid pigmentation, see familial progressive hyperpigmentation gene repression, melanomas, 452–453 genetics, see also individual diseases/disorders hypomelanoses, see hypomelanosis mammalian eye color, 98 mammalian hair pigmentation, 68, 69 mammalian pigmentary system development, 68, 82–85 melanoma (see under melanoma) zebrafish patterning, 47, 113, 115 genitalia abnormalities, LEOPARD syndrome, 845 extramammary Paget disease, 1087 hyperpigmentation, 1081–1085 benign, 1081–1084 malignant, 1084–1085 hypopigmentation, 1085–1087 melanocyte numbers, normal, 1069 nevi, 1083, 1084 pigmented carcinomas, 1085

1212

premature infants, 511 vitiligo vulgaris, 555, 555 gentisic acid (MG), 676 geographic variation in pigmentation, 73–74, 75 glabridin, 678–679, 1169 glaucoma, ocular pigmentation, 96 glomeruloid angiomata, POEMS syndrome, 953, 953 glucocerebrosidase deficiency, Gaucher disease, 778 glucose tolerance tests, porphyria cutanea tarda, 981 glutathione, 676 glutathione-S-transferases, familial melanoma and, 477 GM1 type 1 gangliosidosis, Mongolian spot, 1004 GNAS1 gene, McCune–Albright syndrome, 818 gnetol, 676 goldfish erythrophoroma, 48, 49, 49 differentiation, 49–50, 51 gold-induced skin discoloration, 525, 940, 1032, 1032 gold sodium thiosulfate, chrysiasis, 1032 Golger’s rule, 504–505 Golgi body, role in melanosome biogenesis, 156–157 Goltz’s syndrome, see focal dermal hypoplasia Gordon, John, 8 Gorlin Goltz syndrome (basal cell nevus syndrome), 655 Gottron’s syndrome, see acrogeria goundou, 686 gp100 (Silver/Pmel17), see Pmel-17 gene/protein “granule group” mutants, 162 granulocyte–macrophage colony-stimulating factor (GM-CSF), 421, 435 granulomas, sarcoidosis, 752 gray hair, see canities Griscelli–Prunieras syndrome, 530 Griscelli syndrome (GS), 614, 617–618, 618 melanosome biogenesis and, 159, 162 melanosome trafficking and, 174, 175 molecular pathogenesis, 618 types, 618 Grover disease (transient acantholytic dyskeratosis), 649 growth factors, see also specific growth factors amphibian neural crest cells and, 117 induction by UVR, 412–413 mammalian melanogenesis regulation, 199 mammalian pigmentary system development, 83 melanocyte proliferation/differentiation and, 445–463, 446 identification of, 445–448 melanoma cells, 450–454, 451 autocrine, 490 FGF2 role, 451–452, 490, 491 melanosis from, 1025 paracrine, 491–492 nonmitogenic, 448–450 growth hormone, melanocytic nevi development, 1126 growth-related oncogene a (GROa), 421 keratinocyte–melanocyte interactions, 437–440 melanoma, 490 receptor, 437 Riehl’s melanosis, 437–440, 439 growth retardation, LEOPARD syndrome, 845 guanine, iridophores, 18 guanophores, 12 guidance cues, avian melanoblasts, 120 gumma, 688 gunpowder tattoos, 1035, 1037 guttate leukoderma Darier–White disease, 648 differential diagnosis, 647 hypomelanosis with punctate keratosis of the palms and soles, 646, 647 guttate parapsoriasis (acute pityriasis lichenoides), 703 H63D gene, hemochromatosis, 988 Haber syndrome, 882 HAIR-AN syndrome, 908 hair bulb melanocytes albinism test, 600, 602

alopecia areata, 755 loss, vitiligo vulgaris, 555, 560, 560–561 hair/coat, 63, 67 color, see hair color development, 78–86 embryonic, 78–82 wild-type mice, 78 drug affinity/binding, 372–373, 379–380 evolutionary origin, 77 growth cycle, 67, 80, 80–81 aging/senescence and, 465 growth regulators, 92 hair types, 81 human, see human hair structure, 67, 78, 79 hair color, 63 development embryonic establishment in mice, 78–82 melanoblast migration, 82 wild-type mice, 78 epidermal melanocytes and, 66, 66–67 evolutionary basis environmental factors, 75–76 follicular melanocytes, 67–69 juvenile mammals, 63, 76 primates, 71–72 gray/graying, see canities human, see human hair color white, see leukotrichia (white hair) hair follicles, 67, 79 embryonic development, 81 growth regulators, 92 melanocyte reservoir, vitiligo vulgaris, 560, 577–579, 578 hair shaft diameter, kwashiorkor, 664–665 halides, fixed drug eruption, 1028 Hallerman–Streiff syndrome, 622, 623, 623 halo effect, kwashiorkor, 664 halo nevi, 705–710 clinical features, 706, 706–707, 707, 708 definition, 705 disappearance, 706–707 epidemiology, 705–706 historical aspects, 705 melanoma association, 707–708 multiple, 707, 707 pathogenesis/pathology, 709–710 regression, 709 treatment, 710 vitiligo vulgaris, 558, 558, 708–709 Vogt–Koyanagi–Harada syndrome, 735–736 halo nevus antigens, 709 hamartin, 652 “hanging groin,” onchocerciasis, 695 harderian gland, melanocytes, 101, 102 Harlan, Richard, 8 Hartnup disease, pellagra, 998 Hay–Wells syndrome, 902 hearing loss age-related changes, 468 albinism, 602 Fanconi anemia, 776 mandibulofacial dysostosis, 660 otic melanocytes and, 101 sensorineural LEOPARD syndrome, 844 Waardenburg syndrome, 544 heavy metal excretion, melanin role, 357 heliotherapy, 1176–1177 hemangiomas, 1069–1070 hematoxylin and eosin, 534 hemochromatosis, 986–992 classification, 991 hereditary, 986–989 animal models, 988 associated disorders, 987 clinical features, 987 diagnosis, 988 differential diagnosis, 988 epidemiology, 986–987 historical background, 986 laboratory findings, 988 pathogenesis, 988 treatment, 988–989 histology, 987–988 neonatal, 987 oral mucosa, 1074 types, 987, 988

INDEX hemodialysis patients, lentigo senilis et actinicus, 834–835 hemoglobin, skin color role, 64, 70–71 hemosiderin deposits, 1070, 1071 hemosiderosis, 524, 986, 989–992 animal models, 991 associated disorders, 989–990 classification, 991 clinical features, 989–990, 990 definition, 989 histology, 990–991 oral hyperpigmentation, 1070 pathogenesis, 991 trauma, 990, 990 treatment, 991 hemosiderotic fibrohistocytic lipomatous lesion, 990 Hemostix test, subungual hematoma, 1061 henna, skin discoloration, 1048 hepatic porphyria, see porphyria cutanea tarda hepatitis C infection, porphyria cutanea tarda, 980–981 hepatocyte growth factor (HGF) fibroblast–melanocyte interactions, 433–437 café-au-lait spots, 435–437, 436, 437 dermatofibroma, 433–435 hair follicle growth and, 92 as melanocyte mitogen, 446, 447 nevi and melanoma, 490, 1134 hereditary acrokeratotic poikiloderma, 792–795 clinical features, 783, 785, 792–793, 793 diagnosis/differential diagnosis, 793–794, 794 differential diagnosis, 790 histology, 793 hereditary porphyria, see porphyria cutanea tarda hereditary sclerosing poikiloderma, 795–796 clinical features, 783, 785, 795, 795 diagnosis/differential diagnosis, 795–796, 796 histology, 795 Hermansky–Pudlak syndrome (HPS), 613–616, 614 chaperone protein mutations, 159–160 genetics, 528 histology, 614 molecular pathogenesis, 615 oculocutaneous albinism, 613, 614 phenotypes, 613–615, 614 types, 615–616 herpes zoster hypopigmentation, 697, 697 Vogt–Koyanagi–Harada syndrome, 738 heterochromia, see heterochromia irides heterochromia irides, 756–760 albinism–deafness syndrome, 547 animal models, 758 associated disorders, 757–758 clinical description, 756–757 diagnosis/differential diagnosis, 758 epidemiology, 756 histology, 758 historical background, 756 laboratory findings/investigations, 758 pathogenesis, 758–759 Waardenburg syndrome and, 544 heterochromic cyclitis, see heterochromia irides HFE (hemochromatosis) gene hemochromatosis, 1074 hereditary hemochromatosis, 988 porphyria cutanea tarda, 980, 982 high albido environments, light perception in, 33 high-performance liquid chromatography (HPLC), 535 Hippocrates, 551 Hirschsprung disease, 146–147 piebaldism, 543 Waardenburg syndrome type 4, 545 histidinemia, 623–625 histiocytoma (dermatofibroma), 433–435 HIV infection/AIDS hyperpigmentation associated, 941–946, 942 clinical features, 941, 941–943, 942 etiology, 943 histology, 941–943 Kaposi sarcoma, 1070–1072 labial melanotic macules, 1074 melanoma, 944 vitiligo vulgaris, 572, 573 homeotherms, 12

homocystinuria, 370, 625, 625–626 Hori’s macules, see acquired bilateral nevus of Ota-like macules (ABNOMs) hormones, see also individual hormones mammalian melanocyte regulation, 416–417 melanocytic nevi development, 1126 pigmentary system development, 83 pigmenting (human skin), 513–514 nonmammalian amphibian neural crest cells and, 117 “bihumoral theory,” 13 color change, 11, 12–13, 28–35 mechanisms of, 35–37 melanogenesis regulation, 193–195, 194 pigmentation patterns and, 48, 52–53 sex hormones, see sex hormones Horner syndrome clinical features, 756–757 heterochromia irides, 756–760 ocular pigmentation, 96 horses, pigmentary lesions, 569 HPLC enzyme assay, 272, 274, 275–276 melanin analysis, 298, 299–300, 302 HPS1 gene, Hermansky–Pudlak syndrome type 1, 615 HPS3 gene, Hermansky–Pudlak syndrome type 3, 615 HPS4 gene/protein, Hermansky–Pudlak syndrome type 4, 615–616 HPS5 gene, Hermansky–Pudlak syndrome type 5, 616 HPS6 gene, Hermansky–Pudlak syndrome type 6, 616 HRAS mutations, melanocytic nevi, 1115 human brain, melanocytes, 101 human chimerism, 637 human evolution hair graying/balding and, 72 melanin and adaptation, 72–73 skin color, 64–65, 72–74, 504 human eye color, 98 ethnic variation, 507 human hair, 71–72 Addison disease, 970 age-related changes, 512 ethnic variation, 507 follicles, 79 melanocytes, 68–69 halo nevi, 706 hypertrichosis, 71–72, 980, 980 kwashiorkor, 664–665 Menkes’ kinky hair syndrome, 631–632 structure, 68 vitiligo vulgaris and, 554, 556–557, 560, 560–561, 561 human hair color age-related changes, 512, 512, 659 ethnic variation, 507 genetic hypomelanoses, 657–663 gray/graying (see canities) red (Celtic) phenotype, 66, 69, 415 epidermal melanocytes and, 66 kwashiorkor and, 665 MC1R mutation, 1127 white, see leukotrichia (white hair) human immunodeficiency virus (HIV), see HIV infection/AIDS human leukocyte antigen (HLA) halo nevi, 709 Vogt–Koyanagi–Harada syndrome, 738 human skin color (see human skin color) melanocytic nevi models, 1135, 1136, 1136 Menkes’ kinky hair syndrome, 631 solar radiation effects, 343, 410–412, 411 structure, 65, 65 types, see skin types (human) human skin color, 499, 504–520, see also melanin(s); melanocyte(s) age effects, 511, 511–513 adults, 512 childhood/adolescence, 511–512 premature infants, 511 chemistry, 499–500 constitutive (CSC), 65–67, 410–412, 507 contributing factors, 500 demarcation lines, 514, 514

dermal melanocytes, 69–70 development melanocyte differentiation, 126 melanocyte morphogenesis, 124–125 epidermal melanin unit, 66 epidermal melanocytes, 65, 65–67, 66 evolution of, 64–65, 504 depigmentation, 73–74 melanin and adaptation, 72–73 melanin content, 73–74 facultative (FSC), 65–67, 343, 410–412, 507 glutathione levels and, 363 hemoglobin role, 64, 70–71 histological basis, 65–67 melanin content, 342, 410, 506–507 melanin distribution and, 66, 505, 505 normal, 521, 522 physics of, 500 regulation, 410–420, 411 epidermally-synthesized factors, 412–413 paracrine/autocrine, 413–417 response to UV radiation, 410–412 sex hormones and, 513, 513–514 unusual, 530 variations, 63, 66–67, 342, 499, 504–505, see also ethnicity; skin types (human) ethnicity, 505–508 genetic basis, 237 gray-blue, 530 yellow, 530 yellow-orange, 530 vitamin D synthesis and, 72, 73, 509 Hunter syndrome, Mongolian spot, 1004 Hunter, William, 7 Hurler syndrome, Mongolian spot, 1004 Hutchinson–Gilford syndrome, see progeria Hutchinson sign Laugier–Hunziker syndrome, 1076 melanonychia, 1062, 1062 hydantoin-induced skin discoloration, 1047 hydroquinone (dihydroxybenzene; HQ), 674, 1165–1166 application, 1165 combination therapy, 1169 hyperpigmentation disorders, 1165–1166, 1166 mechanism of action, 1165 melasma treatment, 1022, 1022 ochronosis-induction, 1040, 1041, 1041, 1166, 1167 side effects, 1166 hydroquinone monobenzyl ether (MBEH), 674 6-hydroxy-3,4-dihydrocumarins, 677–678 a-hydroxy acids, 678 4-hydroxyanisole (para-hydroxymethoxybenzene), 369, 675 hydroxychloroquine-induced skin discoloration, 1042, 1042 5-hydroxyindoleacetic acid (5-HIAA), carcinoid syndrome, 920 4-hydroxyphenyl a-D-glucopyranoside (aarbutin), 674 8-hydroxyquinoline (and analogs), 361, 361–362 hydroxyurea, skin discoloration, 943, 1047 hyperchromias, 501, 501 mixed dermal/epidermal, 1020–1025 hyperkeratoses kwashiorkor, 664 warty, 805, 806 hypermelanocytic punctata et guttata hypomelanosis (HPGH), 746–748, 747, 747 hypermelanosis blue (ceruloderma), 521, 522, 522–523, 523 brown, 521 definition, 521 dermal acquired, 1016–1018 congenital, 1003–1019 epidermal (see epidermal hypermelanoses) gastrointestinal disorders and, 979–1002 mixed dermal/epidermal, 521, 1020–1025 universal, 531 hypermelanotic macule, see café-au-lait spots (macules: CALMs) hyperpigmentation, see also specific disorders adrenoleukodystrophy, 772 cytokine involvement, 421–444 drug-induced, 523, 524–526

1213

INDEX familial progressive, 774–776 Fanconi anemia, 776 focal dermal hyperplasia, 645 genital, 1081–1085 kwashiorkor, 664 leprosy, 691 mycosis fungoides, 974–975, 975 oral mucosa, 1069–1081 periorbital, 879, 879–880 piebaldism, 542–543 premature infants, 511 topical treatment, 1165–1169 vulvar, 1081 hyperthyroidism, vitiligo vulgaris, 565–566, 566 hypertrichosis, 71–72 porphyria cutanea tarda, 980, 980 hypoadrenalism, see Addison disease hypochromias, 501, 501 hypocorticism, see Addison disease hypogonadism, pigmentation, 667 hypomelanosis, see also specific types chemical/pharmacologic, 669–685 congenital universal, 624 definition, 527 endemic syphilis, 688 endocrine disorders, 667–668 extracutaneous pigmentation loss, 754–766 genetic acquired depigmentation, 551–598 congenital white spotting, 541–550 defective melanosome biogenesis/transport, 613–621 generalized hypopigmentation, 599–635 hair hypopigmentation, 657–663 immune deficiency, 617–618 localized hypopigmentation, 636–656 infectious, 686–698 inflammatory, 699–704 leprosy, 691 melanocytic neoplasia associated, 705–724 melanocytopenic, 527, 527 acquired, 529 melanopenic, 527, 528 metabolic, 664–667 mosaicism associated, 571–572 nutritional, 664–667 physical agents, 683–685 postinflammatory, 701–704 hypomelanosis of Ito (HI), 636–645 cutaneous symptoms, 637, 638, 638 differential diagnosis, 637–638, 651 exclusion criteria, 637–638 extracutaneous anomalies, 638–639 heterogeneity cytogenic evidence, 639, 640–641 phenotypic evidence, 639, 639 histology, 639–640 historical background, 636 inclusion criteria, 636–637 inheritance, 640, 642 melanin composition, 640 P protein and, 235 terminology, 636 hypomelanosis with punctate keratosis of the palms and soles, 646, 646–647 hypomelanotic macules, idiopathic, 655 hyponychium, 1057, 1059 hypophysis background adaptation, 32–33 morphologic color change, 27 hypopigmentation, see also specific disorders albinism–deafness syndrome, 547 Angelman syndrome, 619–620, 620 atopic dermatitis, 702–703, 703 bowel disorders, 665–666 causes, 1170 Chediak–Higashi syndrome, 613, 616, 616–617 focal dermal hyperplasia, 645, 646 follicular mucinosis, 702, 702 genital, 1085–1087 Griscelli syndrome, 617 Hermansky–Pudlak syndrome, 616 herpes zoster, 697, 697 HIV, 944 kwashiorkor, 664 leprosy, 690, 690, 691 lichen sclerosis et atrophicus, 732 mucous membranes, 1085–1087

1214

mycosis fungoides, 973, 973–974, 974, 975, 976, 977 nutritional causes, 664–666 oculocerebral syndrome with, 627 post-kala-azar dermatosis, 696 P protein and, 235 Prader–Willi syndrome, 620, 620 as therapeutic effect, 670 topical treatment, 1169–1170 Waardenburg syndrome, 545 without hypomelanosis, 767–768 hypopigmented macules, 515 hypopituitarism, pigmentation, 667 hypothalamus, melanogenesis regulation, 194–195 ichthyoses, histology, 966–967 ichthyosis nigrans, keratoses and epidermal hyperplasia, 965–967, 966, 967 idiopathic guttate hypomelanosis (IGH), 726–729 clinical description, 531, 727, 728, 730, 747 diagnostic criteria, 728 differential diagnosis, 728 epidemiology, 727 histochemistry, 727–728 histology, 727–728, 747 historical background, 726–727 pathogenesis, 728 repigmentation, 729 treatment, 728–729 idiopathic lenticular mucocutaneous pigmentation, see Laugier–Hunziker syndrome idiopathic multiple large-macule hypomelanosis, see progressive macular hypomelanosis idiopathic neuraxitis, see Vogt–Koyanagi–Harada syndrome id reaction, yaws, 686 image analysis, 534 imipramine-induced skin discoloration, 1044 imiquimod, xeroderma pigmentosum, 892 immediate pigment darkening (IPD), 343, 358 immune system dyskeratosis congenita and, 898 melanocytes and, 510 UVR effects, 349–350 immunocytochemistry, 534 immunoglobulin(s), fixed drug eruption, 1029 immunoglobulin G (IgG), vitiligo vulgaris, 572 immunohistochemistry acanthosis nigricans, 911 melanotic ectodermal tumor of infancy, 1152–1153 immunomodulators, vitiligo vulgaris, 579–580 immunosuppression melanocytic nevi formation, 1125, 1125–1126 UVR induced, 349–350 impetigo, chronic, see pityriasis alba incontinentia pigmenti (IP) clinical features, 637, 873–875, 874, 875 diagnostic criteria, 876, 877 differential diagnosis, 637, 876 epidemiology, 873 extracutaneous manifestations, 875, 876 genetics, 873 histology, 875–876 historical background, 873 laboratory findings, 876 pathogenesis, 876–877 prognosis, 878 treatment, 877–878 incontinentia pigmenti achromians, see hypomelanosis of Ito (HI) indeterminate leprosy, 690 infants blue-gray macules, see Mongolian spot hemangiomas, 1069–1070 melanotic ectodermal tumor, see melanotic ectodermal tumor of infancy (MNTI) premature, skin color, 511 infections, see also specific infections/organisms hypomelanosis, 686–698 nail, 942, 942 inflammation, see also cytokines; specific mediators of melanocytes and, 74, 412, 413, 510 pigmentation, 670 inflammatory bowel disease, pigmentation, 995

inflammatory mediators, human melanocyte regulation, 412, 413 infrared absorption spectroscopy, melanins, 299, 315 infrared (IR) photography, 533 injury, see trauma INK4A-ARF, see CDKN2A gene/protein “ink-spot” lentigo, see reticulated black solar lentigo “ink-spot” macules, NAME syndrome, 854 innervation, chromatophore(s), 26–27 insulin-like growth factor-1 (IGF-1), melanoma, 490, 491 insulin-like growth factor-1 receptor (IGF-1R), melanoma and, 453 insulin receptor mutations, acanthosis nigricans, 911 intense pulse light (IPL), 1201 intercellular adhesion molecule-1 (ICAM-1), fixed drug eruption, 1029 interferon-g (IFN-g), depigmented skin, 564 interleukin-1a (IL-1a) ET-1 induction, 425, 426–427 GROa induction, 440 induction by UVR, 412, 423 interleukin-1b (IL-1b), induction by UVR, 412, 413 interleukin-1b (IL-1b), POEMS syndrome, 953 interleukin-2 (IL-2), depigmentation induction, 568 interleukin-6 (IL-6), POEMS syndrome, 953 interleukin-8 (IL-8), melanoma, 490 interleukin-8 (IL-8) receptor, 437 “intermedin,” see melanocyte-stimulating hormone (MSH) internal organs, pigmentation, 101–102, 102, see also extracutaneous melanocytes; specific locations nonmammalian, 17, 52 intertriginous freckling, neurofibromatosis type 1, 810, 811 intestinal polyposis type II, see Peutz–Jeghers syndrome intestinal polyposis with hyperpigmentation, see Cronkhite–Canada syndrome intestinal polyposis with nail loss, see Cronkhite–Canada syndrome intracellular pigment transport, see also melanosome trafficking higher vertebrates keratinocytes, 181–186 melanocytes, 65, 171–176 melanogenesis regulation, 199–200 lower vertebrates, 13, 26, 37–40, 38 “dual filament transport model,” 39 speed, 38–39, 39 invertebrates melanogenesis, 193–195 non-melanocytic melanins, 301–302 tyrosinase gene family, 214–215 iodothiouracil, 380, 381, 381 ionizing radiation, depigmentation and, 684 irideterochromia, see heterochromia irides iridomelanophoroma, 48 iridophores, 14, 14, 17, 17, 17–19 adrenergic receptors, 36–37 apparent color and optical properties, 18, 19, 20, 21, 42, 43 avian eye color, 98, 98 leukophores vs., 19 morphologic color change, 27, 31 MSH stimulation, 30, 30–31 physiologic color change, 31 platelet morphology, 18–19, 19, 20, 21 iridosarcoma, 48, 50 iris/iris pigmentation albinism, 600 oculocutaneous albinism type 1, 604 anatomy/structure, 96, 97–98 clump cells of Kogenei, 97 Griscelli syndrome, 617 Lisch nodules in neurofibromatosis, 811, 812, 820 mammalian eye color, 98 melanocyte variation in, 97 nonmammalian eye color, 98, 98–99 pathology, 96 tuberous sclerosis complex, 654 vitiligo vulgaris, 558, 559, 560

INDEX iris pigment epithelium (IPE), 97–98 iritis, vitiligo vulgaris, 559, 560 iron deficiency, anemia, 666 melanin binding, 326–327 overload, porphyria cutanea tarda, 980 skin discoloration and, 1029–1030, 1030 tattoos, 1030 ischemia, depigmentation due to, 371 isolated mucocutaneous melanotic pigmentation (IMMP), 868–869 isomorphic response sunburn, 562–563 vitiligo vulgaris, 562–563, 563 isotretinoin, confluent and reticulated papillomatosis, 923 Ito syndrome, 636 itraconazole, tinea versicolor, 694 ivermectin, onchocerciasis, 696 jaundice carotenemia vs., 1037 melanosis from melanoma vs., 1024 skin color, 530 Jefferson, Thomas, 9 jentigo, 825, 864, 1106 Johanson–Blizzard syndrome, 903 Josselyn, John, 8 junctional nevus, 1112, 1118 Carney complex/myxoma syndrome, 857 juvenile polyposis, 1001 kang cancers, 932 Kangri cancers, 932 Kant, Immanuel, 7–8 Kaposi sarcoma (KS), 1070–1072 histopathology, 1071, 1071 oral mucosa, 1070–1072, 1071 treatment, 1071–1072 kappa-chain deficiency, 631 kathon CG, 960 keratinocyte(s) human, 65, 65 melanin caps, 181–182, 182, 185–186, 186 melanin-induced cell death, 72 melanocyte interactions/regulation, 171, 172, 412–413 mitogen production, 446–447, 447 paracrine factors, 421–444, 422 melanosome acquisition, 65–66, 175–178, 312, 356 melanosome processing, 181–190, see also melanosome trafficking intracellular transport, 181–186, 183, 184 lysosomal processing, 186–187 migration through epidermis, 181 pityriasis alba, 700 keratoses, arsenical, 1034, 1035 keratosis follicularis, see Darier–White disease keratosis follicularis (Darier–White disease), 647–649, 648 ketoconazole, tinea versicolor, 694 khellin, 582, 1180 khellin and UVA light (KUVA), 1180–1181 KIND-1 gene, 783 Kindler syndrome, 781–784 clinical findings, 781–782, 782, 783, 785 differential diagnosis, 783, 783 histology, 782–783 inheritance, 781 pathogenesis, 783 kindlin-1, 783 kinesins, intracellular transport, 38, 172 kinky hair disease, see Menkes’ kinky hair syndrome KIT (Kit) gene/protein functions, 140 human protein, 141 ligand, see stem cell factor (SCF) melanocyte–keratinocyte interactions, 427–433 lentigo senilis, 430, 430–431, 431 UVB melanosis, 423, 424, 427–430, 429 vitiligo vulgaris, 431–433, 432, 433 melanoma and, 453, 481 mutations, 140–143, 141 piebaldism, 543–544 pdgfra gene association, 141 pigmentary system development, 83, 111, 114, 123–124, 446

signal transduction pathway, 140–141 urticaria pigmentosum, 958 Kit ligand (KL), see stem cell factor (SCF) c-Kit tyrosine receptor kinase (c-kitTRK), 83 Klein–Waardenburg syndrome (Waardenburg syndrome type III), 143–144, 544–545 Koebner phenomenon, see isomorphic response kojic acid, 675–676, 1169 kojyl-APPA, 676 Kramer syndrome, see oculocerebral syndrome with hypopigmentation KU-MEL-1, melanoma-associated depigmentation, 719 KUVA therapy, 1180–1181 kwashiorkor, 664–665, 665, 665 labial melanotic macules (lentigines), 1074–1075, 1075 LAMB syndrome, 852, 854, 854, see also Carney complex/myxoma syndrome laminopathies, 791 lamins, 791, 887, 888 Langerhans cell, depigmented skin, 569–570 lareotide, carcinoid syndrome, 921 laser treatment, 1198–1204, see also individual lasers café-au-lait spots, 1198, 1199, 1200, 1200, 1201 ephelides, 930 lentigines, 1200, 1200 lentigo senilis et actinicus, 834 melasma, 1199 nevus of Ota, 1009, 1010, 1199 speckled lentiginous nevus, 1108 tattoos, 1035, 1198, 1199, 1199, 1200 Latanoprost, ocular pigmentation, 96 Laugier disease, see Laugier–Hunziker syndrome Laugier–Hunziker syndrome, 869, 1001, 1075–1076, 1076 lead gingival pigmentation, 1033, 1081 skin discoloration, 1033, 1033 leaden mouse mutant, 173, 175 “lead line,” gingival, 1081 Le Cat, Claude, 8 Leishmania infections, post-kala-azar dermatosis, 696 lens, drug-induced toxicity, 375 lentigines, see also lentiginosis agminated, see agminated lentigines (AL) Carney complex/myxoma syndrome, 855, 856, 857 centrofacial lentiginosis, 838 clinical findings, 825 ephelides vs., 824 generalized (see LEOPARD syndrome) genital, 825, 826 isolated generalized, 869 labial (see labial melanotic macules) laser treatment, 1199, 1200 mucosal, 825 patterned, 827 penile, 825, 826, 1082–1083, 1083 psoriasis, 870 PUVA and, 830, 830, 832, 833, 834 sunbed, 832–833 vulvar, 825, 1081–1082 lentigines, electrocardiographic abnormalities, ocular hypertension, pulmonary stenosis, abnormalities of genitalia, retardation of growth, and deafness, sensorineural type, see LEOPARD syndrome lentiginocardiomyopathic syndrome, see LEOPARD syndrome lentiginosis, 824 arterial dissection, 868 cardiac pre-excitation, 868 centrofacial, see centrofacial lentiginosis eruptive, 869 genital, 825 inherited patterned, in black people, 869 systemic abnormality unknown, 869–870 white, 869–870 lentiginosis profusa, see LEOPARD syndrome lentiginous mosaicism, see agminated lentigines (AL) lentigo benign melanonychia vs., 1064–1065, 1065

maligna, 473, 834, 1166 multiple, see lentigines senilis, see lentigo senilis et actinicus simplex, see lentigo simplex solar, 830–831, 832 lentigo-nevus, 1064 lentigo senilis et actinicus, 829–837 animal models, 833–834 clinical description, 830–832, 831, 832 differential diagnosis, 833 epidemiology, 830 histology, 832–833 historical background, 829 paracrine interactions, 440, 441, 441 ET-1/ETB receptors, 424–425, 425, 426 mSCF/KIT interactions, 430, 430–431, 431 pathogenesis, 833 prevention, 834 prognosis, 834–835 treatment, 834 variants, 830 lentigo simplex, 824–829 animal models, 827 clinical findings, 825, 825–826 course/prognosis, 827 differential diagnosis, 826–827 epidemiology, 824–825 histology/histochemistry, 826 historical background, 824 nail bed, 825 pathogenesis, 827 treatment, 827 lentigo solaris, see lentigo senilis et actinicus “leopard skin,” onchocerciasis, 695 LEOPARD syndrome, 842–851 animal models, 847 associated disorders, 844–845 Carney complex/myxoma syndrome vs., 859 clinical description, 843, 843–844, 844, 1077 diagnosis/differential diagnosis, 846 epidemiology, 843 histology/histochemistry, 845–846, 1077 historical background, 842–843 laboratory findings, 846 pathogenesis, 846–847 treatment, 847 lepromatous leprosy (LL), 689, 690 lepromin skin test, 691 leprosy, 689–692 classification, 689–690 diagnosis, 691 epidemiology, 689 histology, 690–691 historical aspects, 9, 689 hyperpigmentation, 691 hypomelanosis pathogenesis, 691 hypopigmentation, 690, 691 macular hypopigmentation, 571 treatment, 691–692 leptomeninges, melanocytes, 101 l’erthrose pigmentée peri-buccale, see erythrose péribuccale pigmentaire of Brocq leucine zipper, 243 leukemias, ataxia telangiectasia, 621 leukoderma, see also specific types acquired, see vitiligo vulgaris (vitiligo) chemical, 574 classification, 501 piebaldism, 541–543, 542 terminology, 500, 501, 501 leukoderma acquisitum centrifugum, 705, 708, 710 leukoderma punctata, 729–731 animal models, 731 clinical description, 729–730, 730, 747 diagnosis/differential diagnosis, 730–731 histology, 730, 747 pathogenesis, 731 spontaneous partial repigmentation, 731 treatment, 731 leukoderma syphiliticum, 688–689, 689, 1087 leukodermia lenticular disseminada, see idiopathic guttate hypomelanosis leukodopachrome (L-cyclodopa), 263, 263–264 leukopathia, acquired, see vitiligo vulgaris (vitiligo) leukopathia guttata et reticularis symmetrica, see idiopathic guttate hypomelanosis

1215

INDEX leukopathia punctata et reticularis symmetrica, see reticulated acropigmentation of Dohi leukopathie symétrique progressive des extremities, see idiopathic guttate hypomelanosis leukophores, 14, 18–19, 98 leukopigmentary nevus, see halo nevi leukosome, 19 leukotrichia (white hair), 659 sudden whitening, 371, 764–766 associated disorders, 765 clinical features, 764, 765 diagnosis/differential diagnosis, 765 histology, 765 historical background, 764 pathogenesis, 765–766 prognosis/treatment, 766 tuberous sclerosis complex, 654, 654 leukotrienes, human melanocyte regulation, 413 levodopa-induced skin discoloration, 1047 Leydig cell tumor, Carney complex/myxoma syndrome, 858 lichen amyloidosis, 924 lichen aureus, 1030, 1030 lichen nitidus, 731 lichen pigmentosum, see erythema dyschromicum perstans lichen planus, 523, 523 lichen planus pigmentosus, see erythema dyschromicum perstans lichen sclerosis et atrophicus, 731–732, 732, 968 lichen sclerosus (LS), 1086–1087 lichen simplex chronicus, 966, 967 licorice extract, 1169 lidocaine, 378 Li–Fraumeni syndrome, melanoma and, 477 light cells (otic melanocytes), 99–100 light microscopy, 534–535 acanthosis nigricans, 911 chrysiasis, 1032 stains, 534 light responses, RPE melanocytes, 94 light scattering, melanins, 318 lilac rings, morphea, 968, 968 linea nigra, 513, 513 linear morphea, 967 linoleic acid, 677, 678 lipodystrophy, familial mandibuloacral dysplasia, 791 lipophores, 19 b-lipotropin, melasma, 1021 lipoxygenase derivatives, induction by UVR, 413 liquid nitrogen, lentigo senilis et actinicus, 834 liquiritin, 678 Lisch nodules, 810, 811, 812, 820 little leopard, see LEOPARD syndrome Littre, Alexis, 7 livedo reticularis e calore, see erythema ab igne liver fibrosis, hereditary hemochromatosis, 987 liver spot, see lentigo senilis et actinicus LMNA gene, familial mandibuloacral dysplasia, 790 Lorenzo oil, adrenoleukodystrophy, 773 loss-of-function mutations, KIT gene, 142 loss of heterozygosity, melanoma, 480 low albido environments, light perception in, 33 lupus erythematosus, 571, 703, 703 lupus pernio, see sarcoidosis L value, 533 lycopene-induced skin discoloration, 1038 lycopenemia, 1038 lymphokine secretion, defective, melanoma regression, 717 lymphomas ataxia telangiectasia, 621 cutaneous, see mycosis fungoides vitiligo vulgaris, 572 lymphomatoid papulosis, 974 lysosome(s) melanosomes and, 66, 233 biogenesis, 159 processing in keratinocytes, 186–187 RPE melanocytes and, 95 lysosome-associated membrane protein (LAMP-1), 200, 222 lysosome-related organelles (LRO) disorders, 613–621

1216

lysosomes, see lysosome(s) melanosomes, see melanosome(s) LYST gene/protein, 617 Chediak–Higashi syndrome, 617 melanosome biosynthesis, 161–162 macrocheilia, port wine stain, 1070 macromelanocytes, melanoma-associated depigmentation, 714 macromelanosomes, neurofibromatosis type 1, 814 macular amyloidosis, 924 macular blue nevus (aberrant Mongolian spot), 1003 macular degeneration, ocular pigmentation, 96 macular nevus unilateralis, see agminated lentigines (AL) magnesium-1-ascorbate-3-phosphate (MAP), 677 magnetic resonance imaging (MRI) albinism, 601 iron tattoos, 1030 melanotic ectodermal tumor of infancy, 1153 magnetoencephalography, albinism, 601 magot chinosis, 1043, 1043 mahogonoid (Mgrn1), 401–403 major histocompatibility complex (MHC) fixed drug eruption, 1026 melanocytic neoplasia associated hypomelanosis, 716–717 Malassezia furfur, see Pityrosporum orbiculare (ovale) mal de la rosa, see pellagra mal del pinto, 687–688 males, human skin color, 513 malignancy, see also specific cancers/tumors ataxia telangiectasia and, 621 ectopic ACTH syndrome, 938–939 KIT mutations, 142–143 neurofibromatosis type 1 (NF1), 812 nonmammalian vertebrates, 48–50, 49, 51 xeroderma pigmentosum associated, 890, 890, 891 malnutrition, hypopigmentation in, 665–666 Malpighi, Marcello, 6 mammals environmental interactions, 75–76 evolution from reptiles, 63–64, 76–77 eye color, 98 pigmentary system, 63–84 melanin, see melanin pigmentary system (mammalian) mandibuloacral dysplasia (MAD syndrome), 796 mandibulofacial dysostosis (Treacher Collins syndrome), 660 mandrill (Mandrillus sphinx), skin color, 70–71 manganese ions melanin binding, 325–326 neurotoxicity, 378 MAPK, see mitogen-activated protein kinase (MAPK) signaling cascade Marcy, Samuel, 8 MART-1, melanoma regression, 717 mask of pregnancy (chloasma), 513, 1006 mast cell growth factor (MCGF), see stem cell factor (SCF) mast cells café-au-lait spots (macules: CALMs), 437 dermatofibroma, 435 mastocytoma, 141, 143, 955 mastocytosis, 141, 143, 434–435, see also urticaria pigmentosum MATP, see membrane-associated transport protein (MATP) Matricaria chamomilla extract, 678 M-box element, tyrosinase gene family, 218–219, 220–221 MC1R, see melanocortin-1 receptor (MC1R) MCAM expression, melanoma and, 481–482 McCune–Albright syndrome, 817–819 clinical findings, 817, 817–818, 1072 clinical manifestations, 743 pathogenesis, 522, 818 ME20 (Silver/Pmel17), see Pmel-17 gene/protein mechlorethamine mycosis fungoides, 976 skin discoloration, 1047 meladinine, tinea versicolor, 694

melaginina, vitiligo vulgaris, 582 Melan-A/MART-1 protein, 163 melanin(s), 159, 192–193, 193, 282–310, 355, see also melanin pigmentary system (mammalian); melanosome(s); specific types assay, 273, 276, 282, 535 auto-oxidation, 317, 328–329 characterization techniques, 535 chemical analysis, 298–303 chemical properties, 293, 298, 298–299 classification, 287–289, 288 cytotoxicity (see cytotoxicity) degradation, 282, 295, 295–298, 299–300, 302, 303–304, 314, 356 detection of nascent, 368–369 disorders, see also pigmentary anomalies/disorders; specific disorders circumscribed, 531 clinical history, 530–531 diagnosis, 530–535 physical examination, 531–532 removal abnormalities, 530 dispersion agents, 678 drug affinity/binding (see drug-binding to melanin) forensic toxicology, 372–373, 379–380 formation (see melanogenesis) functions, 345, 356–357, 504–505, 508–511 adaptive in mammals, 63, 91, 345 camouflage, 75–76, 357 controversy, 509 human evolution and, 72–74 photoprotection, 72–74, 295, 345–346, 410, 412, 508–509, see also melanin photobiology toxin removal, 509–510 historical aspects, 261–262, 282–283, 311–312 isolation/preparation, 289–290 location, 355 melanoma diagnosis, 293–294, 354 metabolites, 282, 293–295, see also specific metabolites modification, 357–358 molecular weights, 290 morphologic color change, 27–28 ocular, 93, 95, 601, see also retinal pigment epithelium (RPE) other biopolymers vs., 283, 283 otic, 99, 101, 357, see also auditory system photobiology, see melanin photobiology physical properties, 298, 298–299, 311–341, 355, 356 antioxidant, 72, 311, 331–334, 333, 334, 345 band model, 322 cation binding, 311, 324, 325–327, 334, 371–372 electrical conduction, 320–321 energy transfer, 319 free radical centers, 311, 322–325, 329–330, 330 functional groups, 311, 315 optical, 311, 316–320, 317, 319, 346, 522–523 photoreactivity, 329, 329–331, 330, 332 redox reactions, 311, 324, 327–329 P protein interaction, 232 “primary particles,” 312 skin color role, 66, 70–71, 410, 505–507 structures, 287–289, 288, 312–316, 354 supranuclear caps, 181–182, 182, 185–186, 186 synthetic, 289–290, 313 toxicological aspects, 354–355, 371–383 transport, 356, see also intracellular pigment transport; melanosome trafficking melanin-affinic compounds, see drug-binding to melanin(s) melanin-concentrating hormone (MCH), 11, 31–32, 194 melanogenesis regulation, 194–195 “melanin loading,” tinea versicolor, 693 melanin macroglobules (giant melanosomes), familial progressive hyperpigmentation, 775

INDEX melanin photobiology, 329, 329–331, 330, 332, 342–353, see also DNA, photodamage; ultraviolet (UV) radiation DNA damage/repair induced melanogenesis, 348–349 effect of solar UVR on human skin, 343 erythema, 346–347 events initiating melanogenesis, 344, 344 optical properties of melanin, 311, 316–320, 317, 320, 346 photobleaching, 311, 320, 330, 331 photoprotection, 72–74, 295, 345–346, 508–509 effectiveness, 72 eumelanin vs. pheomelanin, 412 SPF, 508 phototoxicity/photosensitization, 73–74, 334, 350 skin chromophores and, 343–344 skin type and, 344–345 melanin pigmentary system (mammalian), see also hair color; skin color; specific components dermal melanocytes, 69–70 development, 78–86, 109, 122–126, 541, see also embryology (pigment cells); melanoblast(s); neural crest differentiation, 125–126 embryonic establishment, 78–82 hair coat of wild-type mice, 78 identification of growth factors, 445–448 morphogenesis, 122–125, 123–125 regulation, 82–85 environmental factors, 75–76 ethnic skin color and, 505–506 evolution of, see evolution follicular melanocytes, 67–69 functional adaptation, 63, 72–74 historical background, 63–64 melanin formation, see melanogenesis melanization inhibitors, 199 ocular, 95–96 melanization-inhibiting factor (MIF), 43–44, 44, 45, 52, 117–118 melanization-stimulating factor (MSF), 44–45 melanoacanthoma (melanoacanthosis), 946–948 cutaneous type, 946, 946 differential diagnosis, 947 mucosal type, 946, 946, 1077, 1077, 1078 pathology/pathogenesis, 946, 946–947, 947 melanoblast(s), 358–359, 489, see also neural crest differentiation, 108–139, 359 amphibians, 117–118 birds, 121–122 fish, 113–115 mammalian, 125–126 reptiles, 119 disorders of, 140–154, 541, see also specific disorders gene regulation, 243–248 MITF, see MITF (mitf) gene/protein growth factors and signal transduction in, 445–463 migration, see melanoblast migration neural crest origin, 108, 359, 541 melanoblast migration, 82, 91–92, 108–139, 359 amphibians, 115–117, 116 birds, 119, 119–121 dorsolateral/lateral, 109 fish, 111–113 mammalian, 82, 122–126 humans, 124–125 mice, 123–124 ocular melanocytes, 91–92 otic melanocytes, 99 reptiles, 119 ventromedial/medial, 109 melanoblastosis cutis linearis, see incontinentia pigmenti (IP) melanocortin-1 (MCR-1), see alpha-melanocyte stimulating hormone (a-MSH) melanocortin-1 receptor (MC1R), 395, 415, 448 accessory proteins, 401–403 basal activity, 404

cyclic AMP signaling, 395 familial melanoma and, 477 gene, 399 allelic variants, 395, 401, 415 human, 415 MC1R mutation, red hair phenotype, 1127 homologs, 395 ligands, see agouti protein; alpha-melanocyte stimulating hormone (a-MSH) molecular biology, 399 melanocyte(s) aging/senescence, 464–471, 469, 511, 511–513, 512 epigenetics, 468 MITF loss, 468 molecular biology/biochemistry, 465–468, 466 mosaicism, 465, 467, 467 signal transduction, 466, 466–467 in vivo studies, 464–465 avian, 98, 98–99, 119, 119–122, see also birds benign neoplasms, 1093–1147, 1148–1162 bipolar, nevus of Ota, 1008, 1008 cell culture, 445–446 autologous transplantation and, 1192–1193 piebaldism, 543 cell death, see also cytotoxicity melanin-induced, 72 protective mechanisms, 367 susceptibility, 354, 371 dendrite formation, 171–173, 173, 176, 356, see also melanosome trafficking development, see embryology (pigment cells) extracutaneous, see extracutaneous melanocytes factors affecting, 191 fibroblast interactions, 433–437 filopodia, 176, 176–177, 177 grafting, 1191–1195 vitiligo vulgaris, 578–579, 583, 583 halo nevi, 709 human, 64–67 dermal, 63, 69–70 epidermal, 65, 65–67, 66 ethnic skin color and, 506 eye color, 98, 507 functions, 508–511 hair color, 507 lifespan, 465–466, 468, 469 morphogenesis, 124–125 regulation of, 410–420, 411 signal transduction in, 449, 450–451 in idiopathic guttate hypomelanosis, 727 immune role, 510 inflammation and, 74 “internal clock,” 762 keratinocyte contacts/interactions, see keratinocyte(s) leprosy and, 690–691 lower vertebrate, see melanophores melanin, see melanin(s) mitogens, 445–463, 446, see also specific growth factors/pathways FGF2 as, 445–448 historical background, 445 identification of, 445–448 a-MSH as, 448 signal transduction, 449, 450–454 transformation and, 491 mouse development, 123–124 differentiation, 125–126 lifespan, 466, 469 nail, 1057–1068, see also nail melanin unit nevus cells vs., 1112 nonhuman primates, 74 nonmitogenic growth factors, 448–450 ocular, see ocular melanocytes organelles, see melanosome(s) otic, see otic melanocytes photolysis, 509 pinta, 688 postnatal disappearance, 528 regulation of differentiated, 85 senile canities, 762

transcriptional regulation, 242–260, see also transcriptional control; specific genes/proteins transformed phenotype, 489–494, see also melanoma vitamin D synthesis, 72, 73, 343, 346, 509 melanocyte differentiation antigen, melanoma regression, 717 melanocyte-directed enzyme-activated prodrug therapy (MDEPT), 369 melanocyte-stimulating hormone (MSH), 11, 28–30, 414–415 Addison disease, 667 a-MSH, see alpha-melanocyte stimulating hormone (aMSH) dendrite formation and, 171 historical aspects, 12–13 human skin color, 513–514 as melanocyte mitogen, 446 melanophore stimulation, 17, 28, 30, 30–31 melanosome trafficking, 30, 175–176, 177 morphologic color change, 27 mutations, mouse coat color, 529 pattern formation and, 48, 48, 52–53 receptors, 36, 52, 85 regulation of differentiated melanocytes in mice, 85 signaling pathway, 35–36, 85, 246, 359, 395 melanocytic nevi, 514, 1112–1147 acquired, 1112, 1115, 1118 environmental factors, 1120–1130, 1122–1123, 1124, 1125 genetic factors, 1120–1130, 1122–1123, 1124 age-related changes, 512–513 children, 1115, 1116, 1117, 1122–1123 clinical features, 911 clonality, 1113 congenital, 1130 cutaneous injury, 1121, 1125, 1125 definitions, 1112–1113 experimental models animals used, 1133, 1134 human skin constructs, 1136, 1136 human skin xenografts, 1135 melanoma association, 1133–1136, 1136 models with dermal origin, 1133 models with epidermal origin, 1133, 1135 transgenic mice, 1135–1136 “field defect theory,” 1116 histogenic origins, 1116 “latitude gradient,” 1116, 1120 melanoma association, 1130–1136, see also melanoma anatomic site relationship, 1132 genetic factors, 1131–1132 models, 1133–1136, 1134, 1136 monitoring, 1133 pathogenesis, 1132–1133 phenotypic risk markers, 1130–1131, 1131 transformation risk, 1132 molecular pathogenesis, 1113–1115, 1114 morphological patterns, 1112 nail, 1063, 1065 natural history, 1115, 1115–1120, 1116, 1117, 1118 oral mucosa, 1077–1078, 1078, 1079, 1120 pathways of evolution, 1116 pigmentary phenotype, 1122–1123, 1124, 1126–1127, 1127 scalp, 1116–1120 sun exposure, 1120–1121 age at exposure, 1121 direct effects, 1121 intermittent, 1120–1121, 1122–1123 latitude, 1121 natural history, 1120 twin studies, 1128–1129 melanocytoma, Mongolian spot, 1004 melanoderma, adrenoleukodystrophy, 772 melanodermatitis toxica (tar melanosis), 1048 melanodermic leukodystrophy, 771–774 melanogenesis, 191–212, 231, 342, 354, 355–356 aging/senescent melanocytes, 467, 467–468 assay, see enzyme assays avian melanocyte specification, 121 biochemical control, 366

1217

INDEX biosynthetic pathways, 192, 193, 213, 231, 282, 284, see also eumelanin; pheomelanin; specific components chemistry, 284–289 dopaquinone regulation, 285, 285–287 enzymology, 261–281 mammalian, 262–264, 263 MITF regulation, 246 phase I, 364, 364–366 switching between, see pigment type switching tyrosinase and related proteins, see tyrosinase gene family cellular vs. subcellular regulation, 192 chromophores, 344 depigmenting drugs and, 670–671 diagnostic utilization of, 368–371 DNA damage/repair induced, 348–349 enzyme regulators, 671–677 factors affecting, 191 historical background, 191–192, 282–283 induction of, 359 inflammation-induced response inhibitors, 678–679 inhibition, 670–671 thiazolidines, 367 tyrosinase glycosylation, 673 invertebrates, 193–195 lower vertebrates, 191, 193–195 hormonal control, 193–195, 194 zebrafish mutants, 47 mammalian, 191, 195–200, 262–264, 284–285, 354 growth factors, 199 hormonal control, 195–198 inhibitory factors, 199 melanosome biosynthesis/transport, 199–200 pH and, 200 proteasome function, 200 substrate availability, 198, 367 UV radiation, 198 melanogenic paracrine network, 421, 422 melanosome transporter proteins and, 230–241 mixed, 282, 287, 287, 350, 350 overview, 192–193 photosensitization and, 350 physiological control, 359–360 recent advances, 290–293 therapeutic utilization of, 368–371 thiol effects, 363–364 toxic intermediates, 366–367 protection from, 367 toxicological aspects, 354, 358–371 melanogenic complex, 222, 261, 269, 277 melanogenic neuroectodermal tumor, see melanotic ectodermal tumor of infancy melanogenic paracrine network, 421, 422 melanoid mutants, 118 melanoma, 489–494 age-related incidence, 473 anal, 1085 animal pigs, 1135 platyfish–swordtail hybrid, 454–455, 1133 biological properties, 490 biomarkers, 293–294 chemotherapy boron neutron capture therapy (BNCT), 382–383 melanin precursor cytotoxicity and, 294–295 resistance, 481, 483, 489 targeting, 354, 380–383, 381, 383–384 therapy-induced depigmentation, 715, 715–716 classification, 473 definition, 472 depigmentation and, see melanoma-associated depigmentation dermal invasion, 490 ephelides association, 929–930 etiology/risk factors, 473–474 familial, 472, 474–477 genetics, 472–488, 1114, see also specific genes/proteins AP2 and, 481–482 APAF1 mutations, 472, 479 apoptosis suppression, 472, 481

1218

BRAF mutations, 472, 477, 479–480, 492 CDK4 mutations, 472, 476 CDKN2A mutations, 472, 474–476, 475 CMM4 mutations, 472, 476 familial melanoma, 474–477 high-penetrance genes, 474–476 loss of heterozygosity, 480 low-penetrance genes, 477 p53 gene/pathway, 477, 478–479 protein tyrosine kinases, 479 PTEN mutations, 472, 480–481 RB gene/pathway, 475, 477, 478 sporadic melanoma, 478–480 telomerase, 482 XP genes and, 476, 833, 891 growth factors/signal transduction in, 450–454, 451, 478–480, 480, 1134 autocrine, 490 cell cycle regulation, 453–454, 474–476, 475 cell surface receptors, 453 FGF2, 451–453, 490, 491 MAPK signaling in, 453–454, 472, 479–480, 480, 492 metastatic cells, 450 paracrine, 491–492 transformation by, 491 halo nevi association, 707–708 historical background, 472 HIV infection and, 944 incidence, 473 Li–Fraumeni syndrome and, 477 melanin-affinic compounds and diagnosis by, 354 induction by, 374–375 targeting by, 380–383, 381, 383–384 melanosis from, 1023–1025, 1024 metastases chemotherapy, 383–384 melanosis, 1023 nail, 1062, 1063 regression, 710 molecular characteristics, 1114 nails, 1066–1067 nevus of Ota, 1008 nonmammalian, 454–455, 1133 oral, 1079, 1079–1080, 1080 penile, 1085 photosensitization, 350 regression, 710–713 clinical features, 711, 711 complete spontaneous, 710 differential diagnosis, 712 epidemiology, 710–711 extent, 711–712 mechanisms, 717–718 pathology, 711, 711–712 prognosis, 712, 712–713 sentinel lymph node involvement, 712 transcriptional control in, 452–453 tumor progression, 473, 473, 489, 490 genetic model, 482–483 “tumor stem cells,” 489–490 TYRP2 expression in, 222–223 UVB and, 473, 491 uveal, 96 viability assessment, 382 vulvar, 1084–1085 xeroderma pigmentosum and, 476, 833, 891 melanoma-associated depigmentation, 566–569, 567, 568, 713–715 “booster function,” 715 cellular mechanism, 719 clinical features, 713, 714 epidemiology, 713 humoral mechanisms, 718–719 mechanisms, 716–719 pathology, 713–714 significance/prognosis, 715 T-cell responses, 544 vitiligo vs., 713–714 melanoma in situ, 1061, 1062, 1066 melanonychia, 1059–1066 benign, 1064–1066 lentigo vs., 1064–1065, 1065 differential diagnosis, 1060–1062, 1061 drug-induced, 1060 fungal, 1061 linear, 1061

longitudinal, 1059–1060, 1060 dermoscopic examination, 1062–1063 spontaneous regression, 1067, 1067 surgical intervention, 1063–1064 melanin/nonmelanin pigment differentiation, 1061 nail plate melanin topography, 1060 non-drug-induced, 1061 striata longitudinalis, 825–826 toxic-induced, 1060 transverse, 1060 melanophages, incontinentia pigmenti, 875–876 melano-phagosome/phagolysosomes distribution in keratinocytes, 181–186 lysosomal processing, 186–187 morphologies, 187 melanophilin, 618 Griscelli syndrome, 162, 174 melanosome biogenesis, 162 melanosome trafficking, 175, 175 melanophores, 14, 14, 15, 15–25, 20, 21, see also color change (nonmammalian); specific types adrenergic receptors, 36–37 avian eye color, 98 darkness adaptation, 34, 35 dermal, see dermal melanophores epidermal, see epidermal melanophores extracutaneous, 17, 52 follicular, 63, 67–69 historical background, 12 melanogenesis regulation, 193–195 pattern formation, see pattern formation (nonmammalian) stimulation of, 30–31 unknown, 24 melanosis, see also specific types paracrine interactions, 440, 441, 441 endothelins in UVB melanosis, 422–424, 423, 424, 425 mSCF/KIT interactions in UVB melanosis, 423, 424, 427–430, 428, 429, 430 Riehl’s (PAN-induced), 437–440, 439, 440 transient neonatal pustular, 905–906, 906 universal acquired, 906 melanosis diffusa congenita, see familial progressive hyperpigmentation melanosis neviformis Becker, see Becker nevus melanosis perioralis et peribuccalis, see erythrose péribuccale pigmentaire of Brocq melanosis universalis hereditaria, see familial progressive hyperpigmentation melanosome(s), see also keratinocyte(s); melanin(s) aging/senescence, 465, 467 architecture, 201 biogenesis, see melanosome biogenesis body distribution variations, 67 darkness adaptation, 34 degradation, 66 abnormalities, 530 ethnic/racial variations, 66, 66–67, 342, 411 intracellular transport, see melanosome trafficking keratinocyte processing of, 181–190 key proteins, 236 LEOPARD syndrome, 846 melanin composition, 312–313 melanization abnormalities, 529 microenvironment, 162–163, 233 nevus of Ota, 1008–1009 ocular choroidal, 93 retention of, 95–96 otic, 99–100 phyllomedusine frogs, 17, 17, 18 porphyria cutanea tarda, 981 stabilization pathway abnormalities, 528–529 transporter proteins, 230–241, 236 historical background, 230–231 MATP, see membrane-associated transport protein (MATP) P protein, see P protein tuberous sclerosis complex, 654 ultrastructure, 312–313 UV-damaged, 72

INDEX melanosome biogenesis, 155–170, 164, 367 “bipartite theory,” 155 disorders of, 159–162, 160, 528–529, 613–621 DOPA histochemistry, 155, 156 melanogenesis regulation and, 199–200 specialization, 162–163 stage I (premelanosomes), 155–157, 156, 157, 164, 231 chaperoning of TYRPs into, 159–162, 160, 232, see also chaperone proteins development and protein trafficking into, 157–159, 158 ER vs. Golgi origin, 155–157 stage II, 155, 156, 164, 231 stage III, 155, 156, 164, 231 melanosome complex formation, aging melanocytes, 467 melanosome trafficking in keratinocytes, 181–186, 183 direction, 185 microtubules, 182, 184–185 molecular motors, 182–186, 184 in melanocytes, 65, 171–176, 183 actin filaments, 37, 38, 38, 171–172 Griscelli syndrome and, 174, 175 melanogenesis regulation, 199–200 melanophilin, 175, 175 microtubules, 37, 38, 171–173 molecular motors, 38–40, 172–173, 174 mouse mutants, 173–176, 174 myosin 5A, 173–174, 175 Rab27A, 174, 175 Rac protein, 171 Rho protein, 171 speed of migration, 38–39, 39 in melanophores, 37–40, 40 transfer abnormalities, 529–530 transfer to keratinocytes, 65–66, 175–178, 312, 356 exocytosis, 177 filopodia and, 176, 176–177, 177 inhibition, 678 molecular mediators, 175–178 MSH role, 175–176, 177 phagocytosis, 178, 181 UVR role, 175–176, 177 melanotic adamantinoma, see melanotic ectodermal tumor of infancy melanotic ameloblastic odontoma, see melanotic ectodermal tumor of infancy melanotic ameloblastoma, see melanotic ectodermal tumor of infancy melanotic anlage tumor, see melanotic ectodermal tumor of infancy melanotic ectodermal tumor of infancy (MNTI), 1148–1157 anatomic locations, 1150, 1151 associated disorders, 1152 bone formation, 1152 central nervous system, 1152, 1155 clinical features, 1150–1152, 1152 diagnosis/differential diagnosis, 1154 epidemiology, 1150 gigantiform variant, 1150 histology, 1152–1153 historical background, 1148 laboratory findings/investigations, 1153–1154 malignant, 1153, 1155 metastases, 1153 neuroblast-like cells, 1152 pathogenesis, 1154–1155 prognosis, 1155–1156 recurrence, 1155 retinal anlage theory, 1154 treatment, 1155 melanotic epithelial odontome, see melanotic ectodermal tumor of infancy melanotic progonoma, see melanotic ectodermal tumor of infancy melanotic tumor of the infantile jaw, see melanotic ectodermal tumor of infancy melanotropin, melanogenesis regulation, 194, 194, 196 melasma, 1020–1023 clinical findings, 1020, 1021 differential diagnosis, 1020–1021 genetics, 1021 histopathology, 1020

pathogenesis, 1021–1022 treatment, 1022, 1022, 1165, 1167, 1168, 1168 hydroquinone, 1165 laser, 1199 types, 1020 melatonin, 11, 13, 194, 514 adaptation to darkness, 33–34 melanogenesis regulation, 195 membrane-associated transport protein (MATP), 163, 230, 231, 235–236, 261, 270 evolution of gene, 237 human skin color variation and, 237 mouse mutants, 235–236 OCA type 4 and, 230, 237, 270, 607 structure and function, 236, 236–237 transporter homologies, 236 memory T-suppressor/cytotoxic cells, fixed drug eruption, 1029 Mendes da Costa syndrome, 789, 796–798, 797 meninges, 101, 559 Menkes’ kinky hair syndrome, 529, 631–633 animal models, 632 clinical features, 631–632 diagnosis/differential diagnosis, 632 epidemiology, 631 histology, 632 investigations, 632 pathogenesis, 632 treatment/prognosis, 633 menopausal solar dermatitis, see poikiloderma of Civatte mequinol, solar lentigines, 1169 Mercurialis, 552 mercury-induced skin discoloration, 525, 1032–1033 Merkel cell, depigmented skin, 570 mesenchymal tissues, melanocytes, 102 metabolic dysfunction, centrofacial lentiginosis, 839 metageria, 792, 886 clinical findings, 886 differential diagnosis, 885, 886 metallothioneins, 678 metals, see also specific metals fixed drug eruption, 1028 melanin binding, 311, 324, 325–327, 371–372 antioxidant properties and, 334 oral mucosa discoloration, 1081 skin discoloration, 523, 524–526, 1029–1030 tyrosinase-related protein-2, binding, 268–269 MET (Met) gene/protein, 446, 447 ligand, see hepatocyte growth factor (HGF) melanocyte–fibroblast interactions, 433–437 melanoma and, 453 methacycline-induced skin discoloration, 1040 methimazole, 677 methoin-induced skin discoloration, 1047 methotrexate, hyperpigmentation-induction, 940 8-methoxypsoralens (8-MOP), 1175 tinea versicolor, 694 vitiligo vulgaris, 580, 581 5-methoxypsoralens (5-MOP), vitiligo vulgaris, 582 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) parkinsonism, 377 methylene blue (MTB), 380 methysergide, carcinoid syndrome, 921 Mgrn1 (mahoganoid), 401–403 mice, see also specific mutants Bannayan–Riley–Ruvalcaba syndrome model, 868 coat development, 78–82 coat pigmentation, 67–68, 68, 143, 230–231, 396, 396 mutants, 396 wild-type phenotype, 78, 85 developmental regulation of pigmentation, 82–85 eye color, 231–232 informative mitf alleles, 244–245 melanocyte(s) differentiation, 125–126 lifespan, 466, 469 migration/morphogenesis, 82, 123–124 melanogenesis regulation, 196–197, 395–409 melanosome trafficking, 173–176, 174 mutants, 84, 123, 126

transgenic, melanocytic nevi models, 1135–1136 miconazole, 675 microphthalmia-associated transcription factor, see MITF (mitf) gene/protein micropigmentation (tattooing), 1195–1196, 1196 vitiligo vulgaris, 583 microtrabeculae, 182 microtubule-mediated transport in keratinocytes, 182–186 in melanocytes, 37, 38, 171–173 polarity, 184–185 milk line nevi, 1083 minimal deviation melanoma, 1097 minimal erythema dose (MED), 344 minocycline confluent and reticulated papillomatosis, 923 skin discoloration, 526, 940, 1039–1040, 1040 missense mutations KIT gene, 142 P protein in OCA, 234 tyrosinase in OCA, 223 MITF (mitf) gene/protein, 243–246, 671, 673 aging/senescent melanocytes, 468 fish chromatophore differentiation, 113–114 gene structure, 243, 243–244, 244 promoter, 242, 245–246, 247 informative alleles, 244–245 isoforms, 242, 244, 244 mammalian melanocyte specification, 125–126, 245 mutations Tietz syndrome, 245 Waardenburg syndrome type 2A, 126, 144–145, 245, 544 post-translational regulation, 246, 248 RPE melanocyte development, 92, 252–253 SOX10 and, 247 transcriptional control, 245–246, 247 tyrosinase gene family expression and, 218–219, 220–221, 252 mitogen-activated protein kinase (MAPK) signaling cascade, 449, 450–451 melanocyte aging/senescence, 466, 466–467 melanocytic nevi pathogenesis, 1113, 1115 melanoma and, 453–454, 472, 479–480, 480, 492 mixed melanogenesis, 282, 287, 287, 350, 350 MLPH gene, 175, 618 MMAC1 gene, see PTEN gene/protein molecular motors keratinocyte transport, 182–186 melanocyte transport, 38–40, 40, 172–173, 174 moles, see melanocytic nevi Mongolian spot, 63, 69, 358, 1003–1006 aberrant (macular blue nevus), 1003 adult onset, 1005 associated disorders, 1003–1005 clinical findings, 1003, 1004, 1005 differential diagnosis, 1005 histopathology, 1005 persistent (dermal melanocytic hamartoma), 1005 premature infants, 511 regression, 1003, 1005 monkeys juvenile coat colors, 76 skin color, 74 monobenzone allergy, 582–583, 1171 application, 1171 contraindications, 1170, 1170, 1171 depigmentation, 555, 555, 562, 562, 1170–1172 repigmentation, post-treatment, 1171 vitiligo vulgaris treatment, 582–583, 1171, 1171, 1172 monoclonal antibodies, 534 monocyte-chemoattractive protein-1 (MCP-1), melanoma and, 491 morbus Besnier–Boeck–Schaumann, see sarcoidosis morphea, 967–969 associated findings, 968 clinical findings, 967, 967–968, 968, 969 epidemiology, 967

1219

INDEX histology, 968 pathogenesis, 969 morphea plana atrophica, see atrophoderma of Pasini et Pierini morphologic color change, 27–28, 28, 29, 31 Mortimer’s malady, see sarcoidosis mosaicism aging/senescent melanocyte(s), 465, 467, 467 hypomelanosis of Ito, 636–645 mosaic speckled lentiginous nevus, see speckled lentiginous nevus Moss, Henry, 9 mother yaw, 686 motor proteins, intracellular transport, 38–40, 40, 172–173, 174 mouse, see mice Moynahan’s syndrome, see LEOPARD syndrome MPTP parkinsonism, 377 Mucha–Habermann disease (chronic pityriasis lichenoides), 703 mucous membranes melanocyte numbers, normal, 1069 melanocytic nevi, 1120 normal color, 515–516 pigmentary abnormalities/discolorations, 946, 946, 1069–1089 vitiligo vulgaris, 557–558 multifocal partial unilateral lentiginosis, see agminated lentigines (AL) multiple endocrine neoplasia (MEN) Carney complex/myxoma syndrome vs., 859 MEN type 2a (Sipple syndrome), 926 multiple lentigines syndrome, see LEOPARD syndrome murine models, see mice mycelium, 1061 Mycobacterium lepra, 689, 691 mycosis fungoides, 972–978 animal models, 976 associated disorders, 974–975 clinical features, 973–974 diagnosis/differential diagnosis, 976 disseminated, 973 epidemiology, 973 histology, 975 historical background, 972–973 hyperpigmentation, 974, 975 hypopigmentation, 973, 973–974, 974, 975, 976, 977 macular, 571 UVB phototherapy, 1185 investigations, 975–976 patch stage, 973, 975 pathogenesis, 976 plaque phase, 973 prognosis, 977 treatment, 976–977, 1185 myelin figure, leukoderma punctata, 730 myeloid leukemia, KIT mutations, 141, 143 myosin(s) intracellular transport, 38, 174, 175 myosin 5A, 618 Griscelli syndrome, 162, 176 melanosome trafficking, 173–174, 175 myotonia atrophica, 660–661 myotonia dystrophica (dystrophy), 660–661 MYOVA gene, Griscelli syndrome type 1, 617 myoxin, 529–530 myxoid leiomyomas, Carney complex/myxoma syndrome, 857 myxoid mammary fibroadenomas, 855, 856, 858, 859 myxoma syndrome, see Carney complex/myxoma syndrome Naegeli–Franceschetti–Jadassohn syndrome, 789, 798–799 clinical features, 785, 797, 798, 904 diagnosis/differential diagnosis, 798–799 Naegeli syndrome, see Naegeli–Franceschetti–Jadassohn syndrome naevus fusocaeruleus ophthalmomaxillaris, see nevus of Ota naevus sur naevus, see speckled lentiginous nevus nail(s) absence (anonychia), 789, 797, 873, 874, 902 anatomy, 1057, 1058 bacteria-induced pigmentation, 1060–1061

1220

black (see melanonychia) exogenous pigmentation, 1060 hyperpigmentation HIV infection and, 942, 942 lead-induced, 1033 zidovudine-induced, 943 invasive melanoma, 1066–1067 melanocyte system, 1057–1059, see also nail melanin unit disorders, 1059–1068 nail apparatus (unit), 1057, 1058 nevi, 1065, 1065–1066 nail bed, lentigo simplex, 825 nail matrix, 1057, 1059 biopsy, 1064 removal, 1064 nail melanin unit, 1057–1059 epithelium types, 1057 melanocyte bicompartmentalization, 1057–1058, 1058, 1059 melanocyte distribution, 1057 intraepithelial, 1058, 1059 skin color type, 1058 nail plate, surgery, 1063–1064 NAME syndrome, see also Carney complex/myxoma syndrome clinical features, 852, 852 clinical findings, 853–884, 854 diagnostic criteria, 853–854 historical background, 852 naproxen, fixed drug eruption, 1027 narrow-band spectrophotometers, 533 narrow-band UVB (NBUVB) therapy, 1170, 1183 delivery devices, 1184 vitiligo vulgaris, 580, 1184 National Organization for Albinism and Hypopigmentation (NOAH), 630 natural killer cells (NK), melanocytic neoplasia associated hypomelanosis, 716 natural selection, human skin color, 504 necklace of Venus, 571, 688 neonatal progeroid syndrome of Wiedemann–Rautenstrauch, 822, 886 nerve growth factor (NGF), melanocytes and, 448–450 neural crest, 11, 12, 108, 449, see also melanoblast(s); melanoblast migration depigmentation syndromes, 527 dispersion from, 359 multipotent cells, 109 reptiles, 119 neural hypothesis, vitiligo vulgaris, 575 neural tube, 12, 111, 116 neurocristopathies, 541 neurodegeneration, melanocortin signaling mutants, 402 neuroendocrine melanoderma, extracutaneous, 938–939, 939 neurofibromas, neurofibromatosis type 1, 811, 812, 812 neurofibromatosis, 809–816 animal models, 815 associated disorders, 813 clinical findings, 810, 810–813, 811, 812 diagnosis/differential diagnosis, 814 epidemiology, 810 genetics, 435, 436, 810 histology, 813–814 historical background, 809–810 laboratory investigations, 814 pathogenesis, 814 prenatal diagnosis, 815 treatment, 815 type 1 (NF1) associated tumors, 812 café-au-lait spots, 435–437, 522, 810–811, 811 Carney complex/myxoma syndrome vs., 859 clinical findings, 810, 810–812, 811, 812 diagnosis, 814 gene, 435, 436 histology, 813–814 neurofibromas, 811, 812, 812 with Noonan syndrome, 813, 816–817 piebaldism association, 543 treatment, 815 type 2 (NF2; central, bilateral acoustic), 812–813, 814

type 3 (NF3), 813 type 4 (NF4), 813 type 5 (NF5), see segmental neurofibromatosis type 6 (NF6), 809, 813 type 7 (NF7; late-onset), 813 neurofibromatosis not otherwise specified (NFNOS), 813 neurofibromin, 436, 522, 814 neuroleptics, CNS toxicity and parkinsonism, 376–377 neuromelanin, 301, 312, 314, 373 parkinsonism and, 376–378 nevi, 69, see also specific types age-related changes, 512–513 dysplastic, 473, 473, 482, 944, 1099 genital, 1083, 1084 melanoma association, 473, 473–474, 482 nevocellular nevi, see melanocytic nevi nevoid nail area melanosis, 1067 nevus anemicus, 515, 651, 767–768 associated disorders, 767 clinical features, 767, 768 nevus aversion phenomenon, 1148, 1149, 1150 nevus cells, 1112–1113 “dropping off,” 1116 maturation, 1115–1116 normal melanocyte vs., 1112 nevus depigmentosus, 571, 649–652 associated findings, 650 clinical findings, 649–650, 650, 655 clinical manifestations, 743 diagnosis/differential diagnosis, 650–651 epidemiology, 649 histology, 650 historical background, 649 prognosis, 651 treatment, 651 nevus flammeus, see port wine stain nevus incipiens, 1105 nevus ischemicus, see nevus anemicus nevus of Ito, 1012, 1012–1013 nevus of Ota, 69, 70, 1006–1012 associated disorders, 1009–1010 classification, 1007, 1009 clinical findings, 1006–1008, 1007 complications, 1008 epidemiology, 1006 histology, 1008, 1008–1009 melanoma association, 1008 ocular, 1007, 1007 pathogenesis, 1010 treatment, 1009, 1010 nevus oligemicus, 767 nevus on nevus, see speckled lentiginous nevus nevus pigmentosus systematicus, see incontinentia pigmenti (IP) nevus pigmentovascularis, see phakomatosis pigmentovascularis nevus pilaris, see Becker nevus nevus sobre nevus, see speckled lentiginous nevus nevus spilus, see speckled lentiginous nevus nevus spilus en nappe, see speckled lentiginous nevus nevus spilus zoniforme, see speckled lentiginous nevus nevus tardif de Becker, see Becker nevus nf1 gene, neurofibromatosis type 1, 435, 436, 814 nf2 gene, neurofibromatosis type 2, 813 NFJ syndrome, see Naegeli–Franceschetti–Jadassohn syndrome niacinamide, 678 niacin (nicotinic acid) deficiency, pellagra, 997–998 nicotinamide deficiency, pellagra, 997–998 nitrazepam, skin discoloration, 1048 nitric acid, skin discoloration, 1048 nitrogen mustard-induced skin discoloration, 1044 NOAH (National Organization for Albinism and Hypopigmentation), 630 nobiletin, 676 N-Oct3 (BRN2; POU3F2), 248 nodular (tumefactive) amyloidosis, 925 nodular melanoma, 473 noise-related hearing loss, otic melanocytes and, 101

INDEX nonmammalian pigment cells, 11–62, see also specific types anomalies, 11, 48–50 color change, see color change (nonmammalian) current concepts, 14–51 embryology, 11, 12, 42 growth factors and signal transduction, 454–455 historical background, 11–13 lower vertebrate, see chromatophore(s) melanomas, 454–455 patterns/patterning, 42–48, 45, 52–53 perspectives, 51–53 terminology, 11, 12, 14, 14 nonmelanocytic melanins, 301, 301–302 nonpigmented epithelial cells (NPE), 97 nonsense mutations, KIT gene, 142 Noonan syndrome, 813, 816–817, 846 norepinephrine, color change and, 36–37 norfloxacin-induced skin discoloration, 1047 notalgia paresthetica, amyloidosis, 926 NRAS gene/protein, 480, 1115 nuclear factor kB essential modulator (NEMO), incontinentia pigmenti, 876–877 nuclear magnetic resonance spectroscopy (NMR), melanins, 299, 315–316 nude mouse xenografts, neurofibromatosis type 2, 815 nummular and confluent papillomatosis, see confluent and reticulated papillomatosis (CRP) nystagmus, 602, 627 obesity, acanthosis nigricans, 908, 909, 909 OCA2 gene, 163 occupational pigmentation changes, 669–670, 1030–1031 ochronosis, 526, 1040–1042, 1041 octreotide acanthosis nigricans, 912 carcinoid syndrome, 921 ocular abnormalities, see visual system abnormalities/defects ocular albinism (OA) autosomal recessive, 605 classification, 603 definition, 600 hearing loss, 602 melanosome biogenesis and, 159 type 1 (OA1) gene, 159, 253 X-linked, 529 ocular melanocytes, see also retinal pigment epithelium (RPE) melanocytes; uveal melanocytes development, 91–93 morphology/structures, 93–102 pathological conditions, 96 ocular telangiectasia, 621 oculocerebral hypopigmentation syndrome of Preus, see oculocerebral syndrome with hypopigmentation oculocerebral syndrome with hypopigmentation (OCSH), 626–630 clinical description, 627, 628–629 differential diagnosis, 628–629 electron microscopy, 627–628 epidemiology, 626 hair shaft examination, 628 histology, 627–628 historical background, 626 pathogenesis, 630 treatment/prognosis, 630 oculocerebrocutaneous syndrome (Delleman–Oorthuys syndrome), 630 oculocutaneous albinism (OCA), 222, 234, 602–613 autosomal dominant, 607–608 brown, 606, 607 classification, 602–603, 603 definition, 600 Hermansky–Pudlak syndrome, 613, 614 incidence, 234 prevalence, 603, 603 type 1 (OCA1), 223, 603–605 clinical features, 603–604 molecular pathogenesis, 605 temperature-sensitive, 605 type 1 A (OCA1A), 604, 604

type 1 B (OCA1B), 604, 604 tyrosinase mutations, 222, 223, 603, 605 type 2 (OCA2), 163, 230, 234–235, 270, 605–606 African-American/African individuals, 606, 606 Angelman syndrome, 620 Caucasian individuals, 605–606 molecular pathogenesis, 606 P protein mutations, 230, 234–235, 270, 606 Prader–Willi syndrome, 620 type 3 (OCA3; red; ROCA), 214, 222, 223, 606–607 molecular pathogenesis, 529, 607 ocular features, 607 type 4 (OCA4), 230, 237, 270, 607 MATP mutations, 230, 237, 270, 607 oculocutaneous syndrome, see Vogt–Koyanagi–Harada syndrome Onchocerca volvulus, 694 onchocerciasis, 694–696, 695 onchodermatitis, 694 oncocercomas, 695 oncogenes, KIT as, 142–143 ONC-Xmrk receptor tyrosine kinase, 454–455 ondansetron, carcinoid syndrome, 921 ophthalmologic abnormalities, see visual system abnormalities/defects optical properties iridophores, 18, 19, 20, 21, 43 blue coloration, 19 of melanins, 311, 316–320, 346, 522–523 fluorescence, 319, 320 human skin melanin, 317, 318 photodynamics, 319–320 synthetic melanin, 317, 317–318 optic nerve, albinism, 601–602 optic whiteners, Riehl’s melanosis pathogenesis, 962 oral contraceptives melanocytic nevi development, 1126 melasma-induction, 1021 oral mucosa hyperpigmentation, 1069–1081 drug-induced, 1081, 1081 endogenous chromophores, 1069–1072 exogenous chromophores, 1080–1081 heavy metal-induced, 1081 melanin/melanocytes, 1072–1080 melanocyte numbers, normal, 1069 melanocytic nevi, 1077–1078, 1078, 1079 normal color, 515–516 physiologic (racial) pigmentation, 1072, 1072 organellogenesis, 16, 52, 155–170, see also melanosome biogenesis ortho-quinones cytotoxicity, 294–295, 295, 366–367 intrinsic reactivity, 285, 285, 354 osteoma cutis, tetracycline-induced, 1040 otic melanocytes, 99–101, 100 aging/senescence, 468 development, 99 drug-induced toxicity, 376 functions, 100–101, 357 locations, 99 tinnitus treatment, 378–379 ototoxic drugs, 376 OTX2 gene/protein, 253 ovarian tumors, Carney complex/myxoma syndrome, 857, 859 oxoprenolol-induced skin discoloration, 1047 Oxsoralen UltraTM, 1175 oxygen consumption during melanin photoexcitation, 330, 332 quenching by melanin, 332, 333, 333 redox reactions, 328 oxyresveratrol, 676 p14 (ARF) gene/protein, melanoma and, 475, 476 p16 (INK4A) gene/protein familial melanoma role, 474–476 normal functions, 475, 475–476 sporadic melanoma role, 478 p53 gene/protein familial melanoma role, 477

role in melanogenesis, 349 sporadic melanoma role, 478–479 p63 gene, ectodermal dysplasias, 901 paederus dermatitis, 949 Paget disease, extramammary, 1087 pagetoid melanocytosis, pigmented spindle cell nevi, 1094 pale ear (ep) locus, 615 pallidin, 528 pallid locus mutations, 528 palms, vitiligo vulgaris, 553–554, 554 pancytopenia, Fanconi anemia, 776 pangeria, see Werner syndrome panhypopituitarism, congenital, 667 papillomatose pigmentee confluente et reticulee innominee, see confluent and reticulated papillomatosis (CRP) papillomatose pigmentee innominee, see confluent and reticulated papillomatosis (CRP) paracrine factors melanocyte regulation, 411, 413–417 melanoma formation, 491–492 in pigmentary disorders, 421–444, 422, 441, 441, see also specific disorders pigmentary system development, 83 para-hydroxylated monophenols, 675 parakeratose brilliante, see confluent and reticulated papillomatosis (CRP) parakeratose pigmentogene peribuccale, 937 parangi, 686–687 parapsoriasis, large plaque, 974 paraquat, parkinsonism and, 377, 377–378 parasol, 1188 parkinsonism, 376–378, 377 partial albinism, see piebaldism/piebald trait partial albinism with immunodeficiency syndrome (PAID), 618 partial unilateral lentiginosis (PUL), see agminated lentigines (AL) paru, 686–687 Patch mutant, 126 pattern formation (nonmammalian), 42–48, 45, 52–53 anomalies, 11, 45, 46, 48–50 cell death and, 112 chromophore interactions, 112–113 endogenous integument factors, 42–45, 44, 45 hormonal influences, 48, 52–53 zebrafish, 45–47, 47, 110, 110–111 Pautrier microabscesses, 975 PAX3 (pax3) gene/protein, 126, 246–247 avian melanocyte specification, 122 mutations, 143–144 Waardenburg syndrome, 544 protein product function, 144 SOX10 and, 144 PAX6 (pax6) gene/protein, 253 Peale, Charles, 9 pearl (pe) locus, 615 Pechlin, Johann, 6 pecrolimus, vitiligo vulgaris, 580 pefloxacin-induced skin discoloration, 1047 pellagra, 995–999 animal models, 998 associated disorders, 995–997 clinical features, 911, 996, 996–997 differential diagnosis, 997 epidemiology, 995 glove/gauntlet of, 995–996 histology, 997 laboratory findings, 997 medication-induced, 998 pathogenesis, 997–998 treatment, 998 pemphigus vegetans, 911 penicillin, leukoderma syphiliticum, 689 penile lentigines, 825, 826 atypical penile lentigo, 1082–1083, 1083 penile melanoma, 1085 perinevoid leukoderma, see halo nevi perinevoid vitiligo, see halo nevi periorbital hyperpigmentation, 879, 879–880 periorificial lentiginosis, see Peutz–Jeghers syndrome (PJS) peripheral demyelinating neuropathy, central demyelinating leukodystrophy, Waardenburg syndrome, Hirschsprung disease (PCWH), 545–546

1221

INDEX periungual fibroma, tuberous sclerosis complex, 653, 653 periungual pigmentation, surgery, 1063 pernicious anemia hyperpigmentation, HIV, 943 premature canities, 659, 666 vitiligo vulgaris, 566 peroxidase inhibitors, 677 Peutz–Jeghers syndrome (PJS), 999–1002 cancer predisposition, 1000 clinical findings, 531, 846, 999, 999–1000, 1000 oral mucosa, 1076, 1076–1077 diagnosis/differential diagnosis, 1001 differential diagnosis Carney complex/myxoma syndrome vs., 859 genetics, 999 histopathology, 1000–1001, 1076 treatment, 1001, 1076–1077 Peutz–Touraine–Jeghers syndrome, see Peutz–Jeghers syndrome (PJS) pH effect on melanin binding of metal ions, 325, 371 melanogenesis regulation by, 200 melanosome microenvironment, 162–163, 233 phagocytosis melanosome transfer to keratinocytes, 178, 181, 356 RPE melanocytes, 94–95 phakomatosis pigmentokeratotica, 1104 phakomatosis pigmentovascularis (PPV), 1013–1015 classification, 1013 clinical findings, 1013, 1014, 1103 differential diagnosis, 1013 lentigines, 868 pharmacological nevus, 530 pharyngeal arch abnormalities, mandibulofacial dysostosis, 660 PHC syndrome (Böök syndrome), 661 phenacetin-induced skin discoloration, 1047 phenazopyridine, skin discoloration, 1048 phenolic compound-induced skin discoloration, 1040–1042 phenol oxidases, 261–262 phenolphthalein, fixed drug eruption, 1073 phenothiazines CNS toxicity and parkinsonism, 376–377 skin discoloration due to, 374, 1043, 1043–1044 phenyazonaphthol (PAN) allergy-induced melanosis, 437–440, 438, 439 phenylalanine, 582, 1181 phenylalanine and UVA (Phe-UVA), 1181 phenylketonuria (PKU), 370, 629–630, 633–635, 634 phenylthiourea, tyrosinase inhibition, 362 phenytoin-induced skin discoloration, 1047 pheochromocytoma, 920 pheomelanin(s), 159, 192–193, 282, 355, 395 atypical nevi, 1126 biosynthesis, 193, 263, 263, 276, 282, 284, 284–285, 342, 365, 395, 397 agouti protein signaling and, 196–197, 197, 395, 416 control, 285, 286–287 cysteinyldopa formation, 286, 286 gaps in understanding, 403 late stages, 291–292, 292, 293 P protein and, 232 classification, 287–289, 288 degradation, 296–298, 297, 300 4-AHP as specific marker, 300–301 permanganate vs. peroxide oxidation, 302, 302 quantitative analysis, 299–300 ethnic variation in, 506–507 eumelanin/pheomelanin switch, see pigment type switching eumelanin ratio, 350 eumelanins vs., 298, 298–299, 397 human hair, 68–69 human skin, 65 isolation, 289 photodestruction, 331 photoprotection, 412 solubility, 303, 396 structures, 287–289, 288

1222

synthesis, 85 synthetic, 290 trichochromes, 288, 288, 288–289 wild-type mouse hair, 85 pheomelanosomes, 85, 159, 312 phlebotomy hereditary hemochromatosis, 988–989 porphyria cutanea tarda, 982 phlogiston, 7–8 photo-acoustic spectroscopy, melanins, 318–319 photobiology of melanins, see melanin photobiology photobleaching, melanins, 311, 320, 330, 331 photochemotherapy, 379, 1175–1182 hypopigmentation treatment, 1170 KUVA therapy, 1180–1181 phenylalanine and UVA (Phe-UVA), 1181 PUVA, see psoralens and ultraviolet light A (PUVA) photolysis, melanocyte(s), 509 photoprotection, melanin and, 72–74, 295, 345–346, 412 photoreactivity melanins, 329, 329–331, 330, 332 type I and II reactions, 1175 photoreception, 35, 52 photoreceptor degradation, RPE melanocytes, 94–95 photosensitivity, xeroderma pigmentosum, 889, 889 photosensitization, melanogenesis-related, 350 phototherapy, see photochemotherapy phototoxic dermatitis, see phytophotodermatitis phototoxicity depigmentation, 684 melanin and, 73–74, 334, 334, 350 phototypes, melano-phagolysosome processing, 186 phylloid hypomelanosis, 642, 642–643 phyllomedusine frogs eggs as chromatophores, 24, 24–25, 25 melanosomes, 17, 17, 18 physiologic color change, 12, 27 dermal melanophores, 16, 16 speed, 38–39, 39 physiologic (racial) pigmentation, see ethnicity phytophotodermatitis, 948–951 clinical findings, 532, 948, 948–949 diagnosis/differential diagnosis, 949 dose-dependent reaction, 950 epidemiology, 948 pathology/pathogenesis, 950–951 residual hyperpigmentation, 949 treatment, 951 pian, 686–687 pianides, 686 picric acid, skin discoloration, 1048 piebaldism/piebald trait, 125, 140–143, 427, 446, 541–544 associated conditions, 142–143, 543 clinical features, 541–543, 542, 570–571, 655, 738, 743 genetic diagnosis, 142 genetics, 543–544 histology, 738 homozygous, 543 incidence, 543–544 KIT mutations, 140–142, 141, 142 management, 543, 1170 phenotype, 140 pigmentary anomalies, 542–543 piebald mutant mice, 123–124 Pierre Robin syndrome, 661–662 PIG3.V gene, vitiligo vulgaris, 574 pigmentary anomalies/disorders, 500, see also specific disorders abnormal darkening, 521–526, see also hypermelanosis; hyperpigmentation epidermal thickening, 524, 526 exogenous/endogenous pigment-related, 524–526 exogenous/endogenous related disorders, 523 hemoglobin-related disorders, 523, 524 melanin-related disorders, 521–524 abnormal lightening, 526–530, see also hypomelanosis; hypopigmentation hemoglobin-related disorders, 530 melanin-related disorders, 21–524

chimeras, 45, 46 classification, 501, 501–502, 502 confusing terms, 500–501 definitions, 502 mechanisms, 521–537 melanoblast development and, 140–154 melanosome biogenesis and, 159–162, 160 nonmammalian, 11, 45, 48–50 zebrafish, 47 paracrine interactions, 421–444 pteridine biosynthetic mutants, 21, 25 surgical treatment of, 1191–1197 terminology, 499–503 tyrosinase gene family mutations, 214, 222–223 pigmentary demarcation lines, 514, 880, 880–882 classification, 514, 880, 880–881 histology, 881 pathogenesis, 881 pregnancy, 515 pigmentary dysplasia, see hypomelanosis of Ito (HI) pigmentary glaucoma, 96 pigmentary incontinence, 522, 523–524 pigmentary mosaicism, see hypomelanosis of Ito (HI) pigmentary organelles, 14, see also melanosome(s); specific types evolution, 41 intracellular transport, see intracellular pigment transport organellogenesis, 16, 52, 155–170 subcellular associations, 41–42 unusual, 17, 17, 18, 52 pigmentary spots, normal, 514–515 pigmentation cutaneous, 1026 regulation in humans, 410–420 science of, 5–10 early anatomic discoveries, 5–6 experiments of nature, 8–9 modern research, beginnings of, 10 terminology, 499–503 pigment cells, 11, 108, see also specific types embryology, see embryology evolution, see evolution extracutaneous, see extracutaneous melanocytes mammalian, see melanocyte(s) nonmammalian, see nonmammalian pigment cells “pigment dilution,” 526 pigmented adamantinoma, see melanotic ectodermal tumor of infancy pigmented ameloblastoma, see melanotic ectodermal tumor of infancy pigmented congenital epulis, see melanotic ectodermal tumor of infancy pigmented contact dermatitis, see Riehl’s melanosis pigmented erythroderma, HIV, 944 pigmented hairy epidermal nevus, see Becker nevus pigmented lesion dye laser (PLDL), 1198 pigmented peribuccal erythema of Brocq, see erythrose péribuccale pigmentaire of Brocq pigmented purpura, 524 pigmented retinoblastoma, see melanotic ectodermal tumor of infancy pigmented spindle cell nevi, 1093–1098 clinical features, 1093–1094 diagnosis/differential diagnosis, 1096–1097 epidemiology, 1093 growth pattern, 1094, 1095 histology, 1094, 1094–1095, 1095 historical background, 1093 laboratory findings/investigations, 1095–1096 Spitz nevus vs., 1096 treatment/prognosis, 1097 pigmented spindle cell nevus (tumor) of Reed, see pigmented spindle cell nevi pigmented teratoma, see melanotic ectodermal tumor of infancy pigmented tumor of the jaw of infants, see melanotic ectodermal tumor of infancy

INDEX pigment granule translocation, 356, see also melanosome trafficking color change (nonmammalian), 13, 37–40 pigmenting hormones, 513–514 pigmenting pityriasis alba, 699–700 pigmentophages, 766 pigments, see specific pigments pigment type switching, 395–409, 396, 415–416 accessory proteins, 401–403 agouti protein (see agouti protein) cats, 404–405 dogs, 405 genetic variation and, 395, 401, 404–405 historical background, 395–397 melanocortin signaling, 399, 403 tyrosinase activity vs. cysteine levels in, 397–399, 398, 403 pilar neurocristic hamartoma, 1157–1162 animal models, 1161 associated disorders, 1158, 1160 clinical description, 1158, 1159, 1160 differential diagnosis, 1160–1161 epidemiology, 1158 histology, 1159, 1160 laboratory findings, 1160 malignant transformations, 1158, 1160, 1161 pathogenesis, 1161 treatment/prognosis, 1161 pilomatricomas, myotonic dystrophy, 661 PIMSF, 438, 440, 440 pineal gland, adaptation to darkness, 33–34 pink-eyed dilution (p) gene, see P protein pinta, 687–688 pintids, 686 pituitary, see also specific hormones control of color change (nonmammalian), 12–13, 28–30, 32–33 melanogenesis regulation, lower vertebrates, 194 tumors, Carney complex/myxoma syndrome, 857, 858 pityriasis alba, 699–701 clinical description, 699–700, 700, 750 diagnosis/differential diagnosis, 700–701 vitiligo vulgaris vs., 571 epidemiology, 699 histology, 700, 750 history, 699 laboratory examination, 750 pathogenesis, 700–701 pigmenting, 699–700 treatment, 701 pityriasis alba faciei, see pityriasis alba pityriasis corporis, see pityriasis alba pityriasis impetigo furfuracea, see pityriasis alba pityriasis lichenoides, 703–704 achromic, 703–704 acute (guttate parapsoriasis), 703 chronic (Mucha–Habermann disease), 703 hypopigmentation, 703–704, 704 pityriasis sicca faciei, see pityriasis alba pityriasis simplex faciei, see pityriasis alba pityriasis streptogenes, see pityriasis alba pityriasis versicolor, 750 Pityrosporum orbiculare (ovale), 571, 692 Pityrosporum, progressive macular hypomelanosis, 749 PKA holoenzyme, Carney complex/myxoma syndrome, 853 plaque morphea, 967 platelet-derived growth factor receptor-a (Pdgfra), 126, 141, see also KIT (Kit) gene/protein platelets (reflecting), 18–19, 19, 20, 42 avian eye color, 98, 98 malignancy, 50 platinum-induced skin discoloration, 1030 platyfish-swordtail hybrid, melanoma, 454–455, 1133 “plerodeles blue,” 22, 32 plexiform neurofibromas, 812, 812 plumbism, 1033, 1033, 1081 Pmel-17 gene/protein, 155–157, 252, 261, 269–270, 529 “pocket proteins,” in melanoma, 454 POEMS syndrome, 951–954 clinical features, 952, 952–953 epidemiology, 951–952 pathogenesis, 953

poikiloderma, 782, 783, see also specific types poikiloderma atrophicans, see Rothmund–Thomson syndrome poikiloderma congenitale, see Rothmund–Thomson syndrome poikiloderma of Civatte, 500, 959, 959–961 poikiloderma vasculare atrophicans, 973, 974 poikilotherms, 12 dermal melanophores(cytes), 17 pigmentation patterns, 42–48 point mutations, KIT gene, 142 poliosis, 527, 654 polychlorinated biphenyl-induced skin discoloration, 1047 polyglandular disease, vitiligo vulgaris, 564–565 polyneuropathy, organomegaly, endocrinopathy, M protein and skin changes, see POEMS syndrome polyostotic fibrous dysplasia, 817 polyps Cronkhite–Canada syndrome, 984 Peutz–Jeghers syndrome, 1000, 1001 porphyria cutanea tarda (PCT), 979–983 animal models, 982 associated disorders, 980–981 clinical findings, 979–980, 980 diagnosis/differential diagnosis, 981 epidemiology, 979 histology, 981 laboratory findings, 981 pathogenesis, 981–982 treatment, 982 type I (sporadic), 979, 981 type II (familial), 979, 981–982 port wine stain, 506, 1070 histopathology, 1070 intraoral, 1070 nevus anemicus association, 767 phakomatosis pigmentovascularis, 1013 postinflammatory hyperpigmentation, acne, azelaic acid treatment, 1167 postinflammatory hypopigmentation pityriasis lichenoides, 703–704, 704 psoriasis, 701, 701–702 post-kala-azar dermatosis, 696–697 postzygotic mutations, hypomelanosis of Ito, 640, 642 POU3F2 (BRN2; N-Oct3), 248 P protein, 163, 230, 270 evolution, 235 human skin color variation and, 237 mouse gene (pink-eyed dilution (p) gene), 231–232 mutations, 230, 234–235 oculocutaneous albinism, 528, 606 Prader–Willi syndrome, 629 structure and function, 232–233, 236 transporter homologies, 233 Prader–Willi syndrome (PWS), 620, 620–621 genetic mutation, 629 P protein and, 235 prednisone, vitiligo vulgaris, 577 pregnancy acanthosis nigricans, 908 human skin color and, 513, 513 melanocytic nevi development, 1126 pigmentary demarcation lines, 515, 881 premature aging, ataxia telangiectasia, 621 premature infants, skin color, 511 premelanosomes, 155–157, 159–162, see also melanosome biogenesis prenatal diagnosis KIT gene mutations, 142 neurofibromatosis, 815 primary autoimmune cholangitis, see primary biliary cirrhosis (PBC) primary biliary cirrhosis (PBC), 992–995 animal models, 994 associated disorders, 992 clinical features, 992, 993 diagnosis/differential diagnosis, 993 epidemiology, 992 histology, 992–993 laboratory findings, 993 pathogenesis, 994 prognosis, 994–995 treatment, 994 primary biliary hepatitis (PBH), see primary biliary cirrhosis (PBC)

primary cortical nodular dysplasia, 857, 858 primary pigmented nodular adrenal disease, 857, 858 primates (nonhuman) hair color, 71–72, 74 graying patterns, 71, 71 juvenile coat colors, 76 melanin pigmentation of skin, 74 tylotrich follicles, 70 Pringle disease, see tuberous sclerosis complex (TSC) PRKKAR1A gene mutation, Carney complex/myxoma syndrome, 853, 1073 progeria, 792, 886–888 adult, see Werner syndrome clinical findings, 886–887, 887 differential diagnosis, 885, 887 histology, 887 laboratory investigations, 887 pathogenesis, 887–888 progeroid syndromes, with hyperpigmentation, 792 progesterone melanocytic nevi development, 1126 melanogenesis regulation, 195 progressive cardiomyopathic lentiginosis syndrome, see LEOPARD syndrome progressive idiopathic atrophoderma, see atrophoderma of Pasini et Pierini progressive macular hypomelanosis, 748–751 clinical features, 748–749, 749 diagnosis/differential diagnosis, 749–750, 750 histology, 749 historical background, 748 laboratory findings/investigations, 749 pathogenesis, 750 prognosis/treatment, 750 progressive systemic sclerosis, blue macules, 1018 prolidase deficiency, 662–663 proliferative neurocristic hamartoma, 1157–1158 promontory sign, 1071 proopiomelanocortin (POMC) congenital deficiency, 404 melanogenesis regulation, 399, 414–415 lower vertebrates, 194 mammals, 195–196 Propionibacterium acnes, progressive macular hypomelanosis, 750 prostaglandins human melanocyte regulation, 413 ocular pigmentation, 96 protease-activated receptor 2 (PAR-2), 678 proteasomes, melanogenesis regulation, 200 proteinase-activated receptor-2 (PAR-2) activation, 178 induction by UVR, 413 protein folding, 218, 233 protein interactions, tyrosinase-related proteins, 222 protein kinase C (PKC), melanocyte proliferation role, 446, 450–451 protein malnutrition, kwashiorkor, 664–665 protein sorting, tyrosinase-related proteins, 158, 158–159, 199, 222 protein tyrosine kinases (PTKs), melanoma and, 479 proton pumps, 163, 200, 233, 236 psammomatous melanotic schwannoma, 857, 858, 860, 1073 pseudoatrophic macule, 812 pseudocatalase, 580, 1185 pseudo-Hutchinson sign, 1062, 1062, 1063, 1064 “pseudomelanoma change,” lichen sclerosus, 1087 Psoralea, 1175 psoralens, 379, 950, 1175 hypermelanosis due to, 950, 950 phototherapy, see psoralens and ultraviolet light A (PUVA) phytophotodermatitis, 948 structure, 1175 systemic, vitiligo vulgaris, 580–582 topical, vitiligo vulgaris, 580 psoralens and ultraviolet light A (PUVA), 379 carcinogenicity, 582 children, 1175–1176, 1176, 1177 hypopigmentation treatment, 1170

1223

INDEX lentigines, 830, 830, 832, 833, 834 mechanism of action, 379, 1180 melanoma-induction, 582 mycosis fungoides, 976–977 oral, 1175–1178 contraindications, 1175 high-dose, 1176 low-dose, 1176, 1177 precautions, 1177 side effects, 1177–1178 vitiligo, 1175 psoralens, 1175 repigmentation permanence, 1179 results, 1176, 1177, 1179–1180, 1180 hair color, 1179–1180, 1180 lesion site, 1179–1180 topical, 1178–1179 adverse reactions, 1178–1179 application, 1179 children, 1179 initial dose, 1179 photosensitivity, 1178 vitiligo, 1178, 1178 vitiligo vulgaris, 580–582, 581, 1175 dosage, 581 psoriasis, 701, 701–702, 870 psychotropic drug-induced skin discoloration, 1043–1044 PTEN gene/protein Bannayan–Riley–Ruvalcaba syndrome, 867–868 Cowden syndrome, 867 melanoma and, 472, 480–481 pteridine pigments, 19, 21 autonomy, 21–22 biosynthesis, 21, 23 mutants, 21, 25 pterinosomes, 19, 22, 24 pterorhodin, phyllomedusine frogs, 17, 17, 18 PTPN11 gene, LEOPARD syndrome, 843 pulmonary fibrosis, Hermansky–Pudlak syndrome, 615 pulmonary hemosiderosis, 990 pulmonary valvular dysplasia, LEOPARD syndrome, 845 pulsed tunable dye laser (PDL), poikiloderma of Civatte, 960 punch biopsy, nail, 1064 purines, iridophores, 18 putrefaction, technique, 6 PUVASOL, leukoderma punctata, 729, 731 PUVA therapy, see psoralens and ultraviolet light A (PUVA) pyridine-2,3,4,6-tetracarboxylic acid, 297, 297 pyridoxine, homocystinuria, 626 pyrimidine dimers, 344, 347–348 pyrrole-2,3-dicarboxylic acid (PDCA), 282, 296, 296 pyrrole-2,3,5-tricarboxylic acid (PTCA), 282, 283, 296, 296, 299, 300, 535 Q-switched alexandrite laser, 1200–1201 speckled lentiginous nevus, 1108 Q-switched lasers, 1198–1201, see also specific types speckled lentiginous nevus, 1108 Q-switched Nd:YAG laser, 1199–1200 speckled lentiginous nevus, 1108 tattoo removal, 1199, 1199, 1200 Q-switched ruby laser, 1198–1199 café-au-lait macules, 1199 dark skin types, 1199 nevus of Ota, 1199 speckled lentiginous nevus, 1108 tattoo, 1199 quantum mechanics, melanins, 315, 318 quinacrine-induced skin discoloration, 1042 quinine ototoxicity, 376 skin discoloration, 1042 quinone detoxification pathway, 367 QX-572, 378, 378–379 Rab27A gene/protein, 618 Griscelli syndrome, 162, 174, 618 melanosome biosynthesis, 162 melanosome trafficking, 174–175, 175 Rab GTPases melano-phagolysosome processing, 187

1224

melanosome biogenesis, 162 melanosome trafficking, 174–175 melanosome transfer to keratinocytes, 177 Rac GTPases, melanosome trafficking, 171 racial (physiologic) pigmentation, see ethnicity radial growth phase (RGP) melanoma, 473, 473, 482, 489 dermal invasion, 490 radiation lentigo, 830, 833 radicalism in mammalian pigmentary system evolution, 76–78 radiodermatitis, chronic, depigmentation, 684 radiodermatitis, depigmentation, 684 radiometric assays, 272, 273 radionuclide labeling, 380 RAF mutations, melanoma, 479–480, 492 Raper–Mason pathway, 262–277, 263, 284, 284–285, 342, see also melanogenesis; specific enzymes RAS mutations melanocytic nevi, 1115 melanoma, 453, 479–480, 492 Rayleigh (Tyndall) scattering, 1026 RB (retinoblastoma) gene/protein functions, 475, 475 in melanoma, 454, 475, 477, 478 reactive oxygen species (ROS), see free radicals Recklinghausen disease, clinical manifestations, 743 recombinant interferon-a, carcinoid syndrome, 921 RECQL2 gene, Werner syndrome, 896 RECQL4 gene, Rothmund–Thomson syndrome, 806 red hair phenotype, 69, 415 epidermal melanocytes and, 66 kwashiorkor and, 665 MC1R mutation, 1127 melanocytic nevi association, 1127 redox reactions/agents, 677–678 L-dopaquinone, 365–366 melanins, 311, 324, 327–329 Reed, Alexander, 6 Reed’s nevus, see pigmented spindle cell nevi Reed’s pigmented spindle cell nevus, see pigmented spindle cell nevi reflectance mode confocal microscopy (RCM), 533 reflective spectrophotometry, 533–534 refractosome, 19 renal disease, tuberous sclerosis complex, 656 renal hemosiderosis, 990–991 replicative senescence, 464, 467, 467–468 reptiles, see also specific types chromatophores, 108 iridosarcoma, 50 mammal evolution from, 63–64, 76–77 neural crest and chromatophore development, 119 resorcinol-induced skin discoloration, 1041 resveratrol, 676 rete mucosum, 6–8 RET gene mutations, 146–147, 448 reticulated acropigmentation of Dohi, 799–802 associated disorders, 800 clinical findings, 799–800, 800 diagnosis/differential diagnosis, 800–801, 801 pathology/pathogenesis, 800, 801 reticulated acropigmentation of Kitamura, 802–804, 914 clinical findings, 802, 802–804 diagnosis/differential diagnosis, 803 differential diagnosis, 803 genetics, 802 histology, 803 pathogenesis, 804 reticulated black solar lentigo, 830, 831, 833 reticulated pigmented anomaly of the flexures, see Dowling–Degos disease retina, see also entries beginning retino-/retinal anatomy, 93 drug-induced toxicity, 375–376 pink-eyed dilution (p) gene and, 231–232, 232 retinal anlage tumor, see melanotic ectodermal tumor of infancy retinal choristoma, see melanotic ectodermal tumor of infancy retinal pigment epithelium (RPE), 242, 558 age-related changes, 468, 513

albinism and, 92–93, 601–602 anatomy/structure, 93, 93–94, 95, 96, 97 chloroquine and, 375, 375–376 development, 91–92, 242, 252 melanocytes, see retinal pigment epithelium (RPE) melanocytes melanosomes, 312 optical properties, 320 in premature infants, 511 transcription factors, 252–253 retinal pigment epithelium (RPE) melanocytes, 91 aging/senescence, 468, 513 anatomy/morphology, 93–94 variation in, 97–98 development, 91–93 differentiation, 92 functions, 94–95 retinoblastic teratoma, see melanotic ectodermal tumor of infancy retinoic acid, 199, 367, 678 retinoids confluent and reticulated papillomatosis, 923 topical, hyperpigmentation disorders, 1167–1169 xeroderma pigmentosum, 892 retrotransposons, hypomelanosis of Ito, 642 rheumatoid arthritis, 939–941, 1032, 1032 RhoB gene, 83 Rho GTPases, 83 melanosome trafficking role, 171, 172 rickets, phakomatosis pigmentokeratotica, 1104 Riehl’s melanosis, 438, 961–963 clinical findings, 961, 961 differential diagnosis, 962 histopathology, 961–962 HIV, 944 paracrine interactions, 437–440, 439, 440, 441, 441 pathogenesis, 962 rifabutin, hyperpigmentation-induction, 943 rifampicin in leprosy, 692 skin discoloration due to, 1047 Riley–Smith syndrome, see Bannayan–Riley–Ruvalcaba syndrome (BRR) Riolan, Jean, 5 river blindness, see onchocerciasis Rothmund–Thomson syndrome, 804–808 clinical features, 783, 785, 805, 805–806 diagnosis/differential diagnosis, 806–807, 807 histology, 806 laboratory investigations, 806 management/treatment, 807 pathogenesis, 807 Rozycki syndrome, 551 RPE1 (Silver/Pmel17), 155–157 ruby eye (ru) locus, 616 Rush, Benjamin, 9 Ruvalcaba–Myhre–Smith syndrome, see Bannayan–Riley–Ruvalcaba syndrome (BRR) Ruvalcaba–Zonana–Smith syndrome, see Bannayan–Riley–Ruvalcaba syndrome (BRR) Ruvalcaba–Zonana syndrome, see Bannayan–Riley–Ruvalcaba syndrome (BRR) sacral spot of infancy, see Mongolian spot salamanders chromatophore development, 115–119 color change, 14 light perception in high vs. low albedo environments, 33 neural tube, 116 sandy (sdy) locus, 616 sarcoidosis, 751–753 associated disorders, 752 clinical description, 751–752, 752 diagnosis/differential diagnosis, 753 epidemiology, 751 histology, 752 laboratory findings/investigations, 752 macular hypopigmentation, 571 pathogenesis, 753 prognosis/treatment, 753

INDEX scalp melanocytic nevi, 1116–1120 vitiligo vulgaris, 554, 555 scatter factor, see hepatocyte growth factor (HGF) schwannomas, Carney complex/myxoma syndrome, 855–856, 857, 858, 860 sclera, anatomy, 93 scleroderma, localized, see morphea scleromyxedema, associated hyperpigmentation, 965 sclerosing hemangioma (dermatofibroma), 433–435 sclerotherapy, hyperpigmentation, 1029 Scytalidium dimidiatum, 1061 seasonal variation in pigmentation, 75 seborrheic keratosis, 708, 965, 966, 967 clinical features, 910–911 histology, 967 paracrine interactions, 440, 441, 441 ET-1/ETB receptors, 425–427, 426 Seckel syndrome (bird-headed dwarfism), 657–658 sectorial neurofibromatosis, see segmental neurofibromatosis segmental lentiginosis, see agminated lentigines (AL) segmental morphea, 967 segmental neurofibromatosis, 819–820 agminated lentigines, 867 clinical features, 532, 813, 819, 819 epidemiology, 819 histology, 819 pathogenesis, 820 prognosis/treatment, 820 speckled lentiginous nevus, 1104 segmental nevus spilus, see speckled lentiginous nevus segmental vitiligo, 500–501 segmented heterochromia, iron deficiency anemia, 666 sella turcica, centrofacial lentiginosis, 839 semiconductors, melanin as, 320–322 semimetal-induced skin discoloration, 1034–1035 senescence, see also age/aging associated heterochromatic foci, 468 melanocytes, 464–471, 475 senile purpura, 990 sensorineural hearing loss LEOPARD syndrome, 844 Waardenburg syndrome, 544 serotonin, carcinoid syndrome, 920 Sertoli cell tumors, Carney complex/myxoma syndrome, 858 sex hormones color change (nonmammalian) and, 32 melanocyte regulation, 416–417 melanogenesis regulation, 195 skin color and, 71, 513, 513–514 “threshold theory” and, 82 Sézary syndrome, 973 shagreen patch (connective tissue nevus), 653, 653 Shah-Waardenburg syndrome, see Waardenburg syndrome type 4 (WS4) “Shiro bito,” 551 siderophages, oral mucosa, 1070 siderosis, secondary, 988 Siemens–Bloch pigmented dermatosis, see incontinentia pigmenti (IP) Siemens syndrome, 790 Siemerling–Creutzfeldt disease, 771–774 signaling mechanisms, see also specific pathways chromatophores, 26 melanocytes aging, 466, 466–467 development, 83, 450–451 proliferation and differentiation, 445–463, 449 melanoma cells, 450–454, 451, 474–476, 475, 478–480, 480, 492 FGF2 role, 451–453 signature nevus, 1112 Silver gene, see Pmel-17 gene/protein silver-induced skin discoloration, 1030, 1030–1032, 1031 silver nitrate stains, 534

Silver–Russell syndrome, 820–822 Sinclair swine model, vitiligo vulgaris, 576 Sipple syndrome, amyloidosis and, 926 skeletal abnormalities Becker nevus, 916 dyskeratosis congenita, 898 hypomelanosis of Ito, 638–639 LEOPARD syndrome, 844 Menkes’ kinky hair syndrome, 631–632 Rothmund–Thomson syndrome, 805, 805 skin aging/senescence, 465 chromophores, 343–344 color, see skin color depigmented inflammatory response, 564 physiologic functioning, 564 development melanoblast migration, 82 structural/functional organization, 82 drug-binding to melanin(s), 374–375 evolutionary role, 76 human, see human skin intercellular communication, 342 melanogenic paracrine network, 421, 422 turnover, acceleration, 678 skin cancer, see also specific types immunosuppression and, 349 pigmentation relationship, 345 UVR relationship, 342, 345, 349 skin color, 499, see also melanin; melanin pigmentary system (mammalian) constitutive (CSC), 65–67, 410–412, 507 contributing factors, 500 development, melanoblast migration, 82 facultative (FSC), 65–67, 507 hemoglobin role, 64, 70–71 histological basis, 65–67 human, see human skin color image analysis, 533 melanin role, 70–71, 342 nonhuman primates, 74 mandrill (Mandrillus sphinx), 70–71 physics of, 500 skin myxomas, Carney complex, 855 skin types (human), 506–507, 507 DNA repair and, 411, 412 melanin photobiology and, 344–345, 345, 508–509 physical properties, 509–510 tanning response and, 410, 412 variation in eumelanin:pheomelanin ratios, 412, 506–507 SKT11 gene, Peutz–Jeghers syndrome, 1076 slaty locus, see tyrosinase-related protein-2 (TYRP-2) SLEV1 gene, vitiligo vulgaris, 574 Slit proteins, avian melanocyte morphogenesis, 120–121 SLUG (SNAI2) gene mutation mammalian pigmentary system development, 83 piebaldism and, 142, 543–544 Waardenburg syndrome type 2D, 145, 544 Smith, Samuel Stanhope, 7, 9 smoker’s melanosis, 1079 smooth muscle hamartomas (congenital Becker nevus), 915, 916 Smyth chicken model, vitiligo vulgaris, 577 snake, iridosarcoma, 50 SNAP proteins, melanosome biosynthesis, 162 SNARE proteins melano-phagolysosome processing, 187 melanosome biosynthesis, 160–161, 162 melanosome transfer to keratinocytes, 177 sodium–hydrogen exchangers, 163, 236 sodium nitrate, skin discoloration, 1048 sodium stibogluconate, post-kala-azar dermatosis, 697 sodium thiosulfate, tinea versicolor, 694 solar lentigo, 830–831, 832 solar radiation, see ultraviolet (UV) radiation soles, vitiligo vulgaris, 553–554, 554 somatostatin, carcinoid syndrome, 921 SOS response, 349 SOX10 (Sox10) gene/protein, 247 avian melanocyte specification, 122 fish chromatophore differentiation, 113, 114 MITF and, 247

PAX3 and, 144 Waardenburg syndrome type 3 (WS3), 145 Waardenburg syndrome type 4 (WS4), 146, 247, 545–546 soybean trypsin inhibitor (STI), 678 speckled lentiginous nevus, 515, 869, 870, 1098–1112 associated disorders, 1101–1104 background hyperpigmentation, 1100, 1101, 1102 blue nevi, 1105 clinical features, 1101, 1102 as congenital melanocytic nevus subtype, 1099–1100, 1100 diagnosis/differential diagnosis, 1105–1106, 1106 epidemiology, 1099–1100 eyelids, 1101 histology, 1104–1105 historical background, 1098–1099 hybrid lesions, 1100 laboratory findings/investigations, 1105 lines of Blaschko, 1100, 1102 melanoma development, 1101–1102, 1103, 1105 misdiagnosis, 1100, 1102 pathogenesis, 1106–1107 prognosis, 1108 shape, 1100 terminal hair, 1100, 1101 treatment, 1107–1108 speckled lentiginous nevus syndrome, 1104 speckled nevus (spilus), see speckled lentiginous nevus speckled zosteriform lentiginous nevus, see speckled lentiginous nevus spectrophotometry enzyme assay, 271, 272, 273, 274, 275 melanin analysis, 298, 302–303, 315 sphingosine, 673 spitzenpigment, see acromelanosis progressiva Spitz nevus, pigmented spindle cell nevi vs., 1096 splotch mutant, see PAX3 (pax3) gene/protein sporadic porphyria, see porphyria cutanea tarda spotted grouped pigmented nevus, 1106, 1106 spotting heart disease, see LEOPARD syndrome spotty nevus, see speckled lentiginous nevus squamous cell carcinoma in situ (SCCIS), 1085 Staphylococcus aureus, pityriasis alba, 700 status dysraphicus, centrofacial lentiginosis, 838–839 steel factor (SLF), see stem cell factor (SCF) steely hair disease, see Menkes’ kinky hair syndrome Steinert–Curschmann disease (myotonic dystrophy), 660–661 stem cell factor (SCF) fibroblast–melanocyte interactions, 433–437 café-au-lait macules, 435–437 dermatofibroma, 433–435, 434 mastocytosis, 434–435 keratinocyte–melanocyte interactions, 427–433 lentigo senilis, 430, 430–431, 431 in UVB melanosis, 423, 424, 427–430, 428, 429, 430 vitiligo vulgaris, 431–433, 432, 433 mammalian melanocyte development, 83–85, 124, 427 as melanocyte mitogen, 446, 447, 491 membrane-bound (mSCF), 421, 427–433 receptor (see KIT (Kit) gene/protein) soluble (sSCF), 421, 433–437 urticaria pigmentosum, 958 stem cells, melanoma, 489–490 steroid hormones, see also corticosteroids; sex hormones; specific steroids color change and, 32 melanocyte regulation, 416–417 strawberry nevi, 1069–1070 stress, vitiligo vulgaris, 563 stria, hypopigmented, UVB phototherapy, 1185–1186 stria vascularis, 99, 560 strimmer rash (string trimmer dermatitis), see phytophotodermatitis stromal-derived factor 1a (sdf1a), fish chromatophore morphogenesis, 111–112

1225

INDEX styphnate hexanitrodiphenylamine, skin discoloration, 1048 subcutaneous neurofibromas, 812 subungual hematoma, 1061 subungual tumors, incontinentia pigmenti, 875 suction blister grafts, 1191–1192 sunbed lentigines, 832–833 sunburn, see also erythema amyloid deposits, 925–926 freckles, 831 isomorphic response, 562–563 sun, emission spectra, 343–344, see also ultraviolet (UV) radiation sun exposure atypical nevi, 1127 halo nevi, 705–706 lentigo induction, see lentigo senilis et actinicus pityriasis alba, 700 sun protection factor (SPF), 508, 1188 sunscreens, 508, 1188–1190 melanocytic nevi, 1121 oral PUVA, 1177 potency, 1188 suntans, see tanning superficial spreading melanoma (SSM), 473 superoxide radicals, 330–331, 332–333, 345 suprarenal insufficiency, see Addison disease surgical treatment of pigmentary disorders, 1191–1197, see also specific techniques Sutton nevus, see halo nevi Swiss syndrome, see Carney complex/myxoma syndrome swordtails, melanoma, 454–455, 1133 sympathetic ophthalmia, 738 symptomatic cutaneous porphyria, see porphyria cutanea tarda symptomatic porphyria, see porphyria cutanea tarda synapsids, 76–77 syndrome myxoma, see Carney complex/myxoma syndrome syphilis (secondary), 689 leukoderma, 688–689, 689, 1083 vitiligo vulgaris vs., 571 T4 endonuclease, xeroderma pigmentosum, 892 tacrolimus UVB phototherapy and, vitiligo vulgaris, 1185 vitiligo vulgaris, 580 tanning, 1188, see also facultative skin color (FSC) delayed, 1188 immediate pigment darkening, 1188 variation in ability to, 504, 507–508, 508 targetoid hemosiderotic hemangioma, 990 tar-induced skin discoloration, 1048 tar melanosis (melanodermatitis toxica), 1048 tattoos, 1035–1036, 1036, 1037 ablation, 1035–1036 amalgam, 1080, 1080–1081 colors, 1035 granulomatous reactions, 1035, 1037 laser treatment, 1198, 1199, 1199, 1200 traumatic, 1035 Tay syndrome, 868 T cells halo nevi pathogenesis, 710 vitiligo pathogenesis, 544 teeth, minocycline-induced discoloration, 1039, 1040 telangiectasia macularis eruptiva perstans, 956 teleost fish, see also individual species chromatophores, 15, 19, 108, see also specific types development, 109–115 differentiation, 113–115 light sensitivity, 35 morphogenesis, 111–113 mutants, 47, 113, 114, 115 pigment retention, 110 color pattern formation, 45–47, 47 erythrophoroma, 48, 49, 49 differentiation, 49–50, 51 tellurium toxicity, 1034–1035 telomerase, melanoma genetics, 482

1226

telomeres lengthening, in melanoma, 482 replicative senescence, 464 temperature-sensitive oculocutaneous albinism (OCA), 605 temporary threshold shift (TTS), albinism, 602 TEP1 gene, see PTEN gene/protein TERC gene, dyskeratosis congenita, 899 terminology, 499–503 4-tertiary butylphenol, 675 testicular tumors, 855, 857, 858, 859 treatment, 860 testosterone, 667 tetracosactide (ACTH), hyperpigmentationinduction, 943 tetracycline(s), skin discoloration and, 1039–1040 tetrahydrobiopterin, vitiligo vulgaris, 575 tetryl, skin discoloration, 1048 T helper cells (Th) melanocytic neoplasia-associated hypomelanosis, 716 melanoma-associated depigmentation, 719 melanoma regression, 717 thermal burns, depigmentation, 683, 683–684 thermoregulation, mammalian evolution, 77 thiazole-2,4,5-tricarboxylic acid (TTCA), 282, 297, 297 thiazole-4,5-dicarboxylic acid (TDCA), 297, 297 thiazolidines, melanogenesis inhibition, 367 thioctic acid (a-lipoic acid), 678 thiol compounds, 363–364, 397 thioredoxin, vitiligo vulgaris, 575 thioridazine, retinal toxicology, 375 2-thiouracil, 381 dopaquinone conjugation, 368–369, 369 melanoma targeting, 380, 382 thioureylenes, 381 incorporation into melanin, 368–369 melanoma targeting, 380–383, 381 “threshold theory,” 82 thumb deformity, and alopecia, 904 thymine dimers, melanogenesis induction, 348–349 thymomas, vitiligo vulgaris, 572 thyroid amphibian morphogenesis role, 117 color change (nonmammalian) and, 32 dysfunction, vitiligo vulgaris, 565–566, 566 tumors, Carney complex/myxoma syndrome, 857 thyroxine amphibian morphogenesis, 117 color changes and, 32 Tietz syndrome (albinism–deafness syndrome), 245, 546, 546–547, 630–631 tinea capitis, pigmenting pityriasis alba, 700 tinea versicolor, 692–694 diagnosis/differential diagnosis, 571, 694 electron microscopy studies, 693 epidemiology, 692–693 histology, 693 hypopigmentation, 692, 693 pathogenesis, 693–694 treatment, 694 tinnitus, management, 378, 378–379 tissue plasminogen activator (tPA), melanocyte transplantation, 1194–1195 toasted skin syndrome, see erythema ab igne a-toc, 677 a-tocopherol ferulate (a-Toc-F), 677 toenail plate, orange-brown staining, iron induced, 1030 toxicology, 354–394 melanin, 371–383 melanogenesis, 358–371 TP53 gene, see p53 gene/protein transcriptional control, see also specific genes/proteins historical background, 242–243 melanomas, 452–453 pigment cell development, 243–248 tyrosinase gene family, 218–219, 220, 248–253

transcription factors mammalian pigmentary system development, 83, 450 MITF (see MITF (mitf) gene/protein) tyrosinase gene family control, 218–219 transferrin saturation testing, hereditary hemochromatosis, 988 transgenic mic, melanocytic nevi models, 1135–1136 transient acantholytic dyskeratosis (Grover disease), 649 transient neonatal pustular melanosis, 905–906, 906 transient receptor potential 7 (trmp7), 111 transporter proteins, 230–241, see also specific proteins trauma depigmentation, 684 hemosiderosis, 990, 990 idiopathic guttate hypomelanosis, 728 melanocytic nevi, 1121, 1125, 1125 melanonychia, 1061 oral hyperpigmentation, 1070 tattooing, 1035 thermal burns, depigmentation, 683, 683–684 Treacher Collins syndrome (mandibulofacial dysostosis), 660 Treponema carateum, 686 Treponema endemicum, 688 Treponema pallidum, 686 Treponema pertenue, 686 treponematoses, 686–689, see also individual diseases/disorders tretinoin (all trans retinoic acid; ATRA) combination therapy, 1169 hyperpigmentation disorders, 1167–1169 inflammation–induction, 1168 mechanism of action, 671, 673 melasma, 1168, 1168 melasma treatment, 1022 postinflammatory hyperpigmentation, 1168–1169 side effects, 1168–1169 trichochromes, 288, 288, 288–289 degradation, 297 isolation, 289 melanosis from melanoma, 1024 tricho-oculo-derma-vertebral syndrome, 903 tricho-odonto-onycho-dermal (TOOD) syndrome, 904 tricho-odonto-onychodysplasia, 903 tricho-onycho-hypohidrotic ectodermal dysplasia, 904 Trichophyton rubrum, melanonychia, 1061 Trichophyton sousanense, nail pigmentation, 1061 trichopoliodystrophy, see Menkes’ kinky hair syndrome tricho-rhino-phalangeal syndrome, 903 trichrome vitiligo, 553, 554, 554 tricyclic antidepressant-induced skin discoloration, 1044 triiodothyronine, color changes and, 32 TriLuma®, melasma treatment, 1022, 1169 2,5,6-trimethyl-para-hydroxy-methoxybenzene, 675 4,5¢,8 trimethylpsoralen (TMP), 1175 trimethylpsoralens, 1175 vitiligo vulgaris, 580 trinitrotoluene, skin discoloration, 1048 “tripe palms,” 909 triploidy, hypomelanosis of Ito, 639 triquin, fixed-drug reactions, 934 trisomy 21 (Down syndrome), 658–659 TrisoralenTM, 1175 tristimulus colorimeters, 533 Troisser–Hanot–Chauffard syndrome, see hemochromatosis, hereditary TYRPs (tyrosinase-related proteins), see tyrosinase gene family; specific proteins TYRPS1 gene, tricho-rhino-phalangeal syndrome, 903 L-tryptophan, 264 TSC1 gene, tuberous sclerosis complex, 652 TSC2 gene, tuberous sclerosis complex, 652

INDEX tuberculoid leprosy, 689, 690 tuberin, 652 tuberous sclerosis complex (TSC), 652–656 ash leaf macule (hypomelanotic macule), 531, 651, 653, 654, 654 clinical manifestations, 652–653, 653, 743 cutaneous, 653–654, 747 diagnosis/differential diagnosis, 653, 655 epidemiology, 652 genetics, 652 historical background, 652 laboratory findings/investigations, 655 macules, 638 pathogenesis, 655 pathology, 654–655 pigmentary disorders, 653–654 prognosis/treatment, 655–656 Tu melanoma locus, 454 tumor-infiltrating lymphocytes (TIL), melanoma regression, 717 tumor necrosis factor-a (TNF-a) endothelin induction, 426–427 human melanocyte regulation, 413 induction by UVR, 412 tumors, see malignancy “tumor stem cells,” 489–490 twin spotting (didymosis), 1015, 1107, 1107, 1130 twin studies, melanocytic nevi, 1128–1129 tylotrich follicles, 70, 82 Tyndall (Rayleigh) scattering, 1026 typus maculatus of Mendes da Costa, see Mendes da Costa syndrome tyrosinase, 157, 191–192, 199, 217–219, 242, 249–250, 261, 265–267, 354, 355, 360 acidity and, 200 biochemical control of, 366 in control of melanogenesis, 287, 397, 403 enzyme reactions, 217–218, 261, 262, 262–263, 364, 364–368 assay, 271, 272, 274–275 cytotoxicity and, 366–367, 368 L-dopa oxidation, 262, 263, 266, 272, 274, 274–275 mechanism of action, 265–266, 266, 365, 365 rates, 364 tyrosine hydroxylation, 262, 263, 265, 271, 272, 274, 274 evolutionary conservation, 249, 361 gene structure, 218, 249, 250 historical background, 213–214, 262, 282 inhibition, 673–677 competitive/noncompetitive, 674–676 copper-binding agents, 361, 361–362 shift to pheomelanin synthesis, 676 thiols, 363–364 melanoma marker, 294 mutations, 223–224 oculocutaneous albinism, 605 post-transcription control, 362–363, 676–677 proteasomal regulation, 200 senile canities, 761 structure, 360 active site, 217–218, 261, 265, 356, 361–362 carboxyl sites, 361 glycosylation, 362–363, 673 protein folding/maturation, 218, 233, 363 substrate availability and, 198, 367 substrate specificity, 360–361 targeting to melanosome, 230 transcriptional control, 218–219, 246, 249–250, 671, 673 vitiligo vulgaris and, 573 tyrosinase gene family, 213–229, 242, 248–249, 267, see also specific genes/proteins evolution, 215 historical background, 213–214 invertebrates, 214–215 lower vertebrates, 215 pH and, 162–163 pigmentation disorders and, 214, 222–224 protein interactions, 222, 261, 269, 277 protein sorting/trafficking, 157–159, 158, 164, 199, 222, 232 sequence similarity, 215, 215–217, 216 structural similarity, 217, 217 structure, 157

transcriptional regulation, 248–253 common elements, 221–222 tyrosinase pseudogene, 215 tyrosinase pseudogene (TYRL), 215 tyrosinase-related protein-1 (TYRP-1), 219–220, 242, 250–251, 261, 267 amino acid sequence, 216 avian melanocyte specification, 122 enzyme reaction, 219, 267 evolution, 215 gene structure, 215, 215, 219–220, 250–251, 267 historical background, 214 late eumelanogenesis, 291, 292 melanocytic nevi, 1113 mutations, 220 OCA type 3 and, 214, 222, 606–607 nail melanocytes, 1057 pheomelanogenesis and, 197 protein sorting, 158, 158–159 protein structure, 250, 251 synthesis, 157–158 transcriptional control, 220, 221, 246, 251 tyrosinase-related protein-2 (TYRP-2), 220–221, 242, 251–252, 261, 267–269, 360, 529 activity inhibition, 676 amino acid sequence, 216 homology to tyrosinase, 268 enzyme reaction, 220, 267, 282, 284 assay, 273, 275–276 mechanism of action, 268, 268 evolution, 215 expression in melanoma, 222–223 gene structure, 215, 215, 220, 251 historical background, 214 late eumelanogenesis, 291 mammalian pigmentary system development, 84, 85 metal ion binding sites, 268–269 mutations, oculocutaneous albinism type 3, 529 nail melanocytes, 1057 pheomelanogenesis and, 197 protein sorting, 158, 159 RPE development, 253 synthesis, 157–158 transcriptional control, 220–221, 251–252 tyrosinase-related proteins (TYRPs), see tyrosinase gene family; specific proteins L-tyrosine, 264 deficiency, kwashiorkor, 665 hydroxylation, 262, 263, 264, 265, 354 assay, 271, 272, 274 Tyson, Edward, 8 ulcerative colitis, 666, 995 ultraviolet A (UVA), sunscreen penetration, 1188 ultraviolet B (UVB) immunomodulatory effects, 1183–1184 melanoma risk, 473 melanosis, paracrine interactions in, 440, 441, 441 ET-1/ETB receptor interactions, 422–424, 423, 424, 425 mSCF/KIT interactions, 423, 424, 427–430, 428, 429, 430 photobiological effects, 1183–1184 phototherapy, 1183–1187 broadband, 1183 combination therapies, 1185 hypopigmented mycosis fungoides, 1185 mycosis fungoides, 976–977 narrowband, see narrowband UVB (NB-UVB) therapy postsurgical leukoderma, 1185 stria, hypopigmented, 1185–1186 UVB sources, 1183 vitiligo vulgaris, 1184–1185 solar lentigo, 833 T lymphocyte apoptosis, 1183 ultraviolet B (UVB), delayed tanning, 1188 ultraviolet C (UVC), 1188 ultraviolet (UV) photography, 533 ultraviolet (UV) radiation, see also facultative skin color (FSC); melanin photobiology; tanning; specific wavelengths chromophore absorption, 343–344

dendrite formation and, 171 DNA damage, 185 human skin and, 65, 343, 410–412, 411 idiopathic guttate hypomelanosis, 727, 728 immunosuppression due to, 349–350 induction of epidermally synthesized factors, 412– 413 mammalian melanogenesis and, 198 melanin adaptation to, 72–73 melanoma risk, 473 melanosome transfer to keratinocytes, 175–176, 177 photosensitization, 350 psoriasis treatment, 702 Riehl’s melanosis pathogenesis, 962 skin cancer, 342, 345, 349, 473 skin type, 344–345, 345, 410 tinea versicolor, hypopigmentation pathogenesis, 693 underwhite protein, see membrane-associated transport protein (MATP) unilateral lentigines, see agminated lentigines (AL) universal acquired melanosis (carbon baby), 774–776, 906, 915 upstream regulatory factor 1 (USF1), 248 urocanic acid (UCA), 350 uroporphyrinogen decarboxylase (URO-D) deficiency, 981–982 urticaria pigmentosum, 954–959 adult onset, 956 animal models, 958 of childhood, 955, 955–956 classification, 955 clinical manifestations, 955, 955–956, 957 diagnosis/differential diagnosis, 958 histology, 956, 957 historical background, 954–955 laboratory findings, 956–958 pathogenesis, 958 treatment, 958 uterine tumors, Carney complex/myxoma syndrome, 857 UVB, see ultraviolet B (UVB) uveal melanocytes, 91, 558 age-related changes, 513 anatomy/morphology, 93 variation in, 96–97 development, 91–93 differentiation, 92 ethnic variations, 95 melanoma, 96 uveal tract, 96, 96–97 melanocytes, see uveal melanocytes melanoma, 96 uveitis Fuch’s heterochromic, 96 Vogt–Koyanagi–Harada syndrome, 559, 559 uveoencephalitis, see Vogt–Koyanagi–Harada syndrome uveomeningoencephalitis (syndrome), see Vogt–Koyanagi–Harada syndrome UV light, see ultraviolet (UV) radiation vaccination-induced autoimmune “vitiligo,” 716 vacuolar proton pump, 163, 200 vagabond disease, see vagabond leukomelanoderma vagabond leukomelanoderma, 732–734 clinical description, 532, 733, 733, 733 histology, 733, 733–734 historical background, 732–733 pathogenesis, 734 Vaghbhata, 551–552 vaginal mucosa, normal color, 515–516 VAMP proteins, melano-phagolysosome processing, 187 vanilmandelic acid (VMA), 1153–1154 varicose dyschromia, hemosiderosis, 990 variegated translocation mosaicism, 896 vascular endothelial growth factor (VEGF), 953 vascularity, skin color and, 70–71 vasoconstriction, skin color, 530 Vauzeme, Roussel de, 8 venous lakes, lips, 1070, 1070, 1071 Versalius, 5 vertebrates, see also specific groups/species avian species, see birds

1227

INDEX embryology, see embryology evolution, see evolution intracellular transport, see intracellular pigment transport lower, 11 melanocyte(s), see melanophores melanogenesis, 47, 191, 193–195, 194 tyrosinase gene family, 215 mammals, see mammals nonmammalian albinism, 48, 118 color change, see color change (nonmammalian) malignancy, 48–50, 49, 51 pigment cells, see pigment cells vertical growth phase (VGP) melanoma, 473, 473, 482–483, 489, 490 very long chain fatty acids (VLCFA), adrenoleukodystrophy, 771, 772 Virchow cells, leprosy, 690 “visage mauve,” 1043, 1043 visual acuity, 601, 614 visual evoked potentials (VEPs) albinism, 601 Chediak–Higashi syndrome, 616 Prader–Willi syndrome, 620 visual pigments, photosensitive chromatophores, 35, 52 visual system, see also entries beginning oculo-/ ocular; eye(s); retina defects, see visual system abnormalities/defects development, 91–93 melanocytes, see ocular melanocytes visual system abnormalities/defects, see also specific disorders albinism, see under albinism Alezzandrini syndrome, 725 centrofacial lentiginosis, 839 hypomelanosis of Ito, 638 incontinentia pigmenti, 875 LEOPARD syndrome, 844 melanoma-associated depigmentation, 713 onchocerciasis, 695 Rothmund–Thomson syndrome, 805 Vogt–Koyanagi–Harada syndrome, 735 xeroderma pigmentosum, 890 VIT 1 gene, vitiligo vulgaris, 574 vitamin A supplements, Darier–White disease, 649 vitamin B3 deficiency, 997–998 vitamin B12 deficiency, see pernicious anemia vitamin D (and metabolites) melanocyte regulation, 416 synthesis, 72, 73, 343, 346, 509 vitiliginous amyloidosis, 925 vitiligo vulgaris (vitiligo), 370, 551–584 age of onset, 553 animal models, 576–577 associated conditions, 564–569, 565, 667 Crohn’s disease, 995 Down syndrome, 658 endocrine, 564–566 halo nevi, 558, 558, 708–709 HIV-associated, 944 lupus-associated, 703 psoriasis, 701 skin cancer, 563, 563–564 thyroid dysfunction, 565–566, 566 bilateral, symmetrical (generalized), 553, 553–556, 554 clinical features, 552–553, 730–731, see also alopecia areata cutaneous, 531, 553–557, 655, 738 hearing loss, 101, 560 mucosal depigmentation, 557–558, 558 ocular, 558–560, 559, 560, 757, 757–758 polyglandular dysfunction, 564–565 confusing terms, 500–501 counseling, 583–584 definition, 552 dermatoglyphics, 557 diagnosis/differential diagnosis, 570–572 epidemiology, 552 genetics, 552 genitalia, 555, 555, 1085–1086 hair bulb melanocyte loss, 555, 560, 560–561, see also alopecia areata heterochromia irides, 757–758

1228

histology/histopathology, 563, 569, 569–570, 738, 1086 historical aspects, 9, 551–552 inflammation/inflammatory response, 564, 570 inner ear pigment cells, 560 laboratory findings, 570 lesions hyperpigmented borders, 554, 554 inflammatory, 555 physiologic functioning, 564 medical therapies, 579–582 morphological/functional defects, 510 paracrine interactions, 431–433, 432, 433, 440, 441, 441 pathogenesis, 370, 431, 572–576 autocytotoxic hypothesis, 574 autoimmune hypothesis, 572–574 genetic hypothesis, 574–575 neural hypothesis, 575 pattern of onset, 555 phenolic compounds and, 368 photography, 579 precipitating factors, 562–563 psychologic impact, 561–562 sex differences, 553 spontaneous repigmentation, 556, 556 stress-induced, 563 treatment, 577–584, 1086 age and, 579 chemical depigmentation, 582–583, 1171, 1171, 1172 limitations, 578–579 melanocyte reservoir, 577–579, 578 micropigmentation, 583 photochemotherapy, 579–582 surgical, 583, 583, 1191–1196 UVB phototherapy, 580, 1184–1185 unilateral, asymmetrical (segmented), 553, 556, 556–557, 557, 579, 738 Vogt–Koyanagi–Harada syndrome (VKHS), 734–741 alopecia areata, 736, 754 animal models, 739 associated disorders, 736 clinical features, 559, 559, 560, 735–736, 736, 738 cutaneous, 737 ocular, 735, 736–737, 737 depigmentation, 735–736 diagnosis/differential diagnosis, 737–738 Alezzandrini syndrome vs., 726 dysacousia, 560 epidemiology, 735 histology, 736–737, 738 historical background, 734–735 laboratory findings/investigations, 737 pathogenesis, 738–739 prognosis, 739 “specific antigens,” 739 treatment, 739 Voight lines, see pigmentary demarcation lines vulva hyperpigmentation, 1081 lentigines, 825, 1081–1082 melanoma, 1084–1085 melanosis, 1082, 1082 nevi, 1083, 1084 Waardenburg–Hirschsprung disease (HSCR2), 546 Waardenburg–Shah syndrome (Shah–Waardenburg syndrome), see Waardenburg syndrome type 4 Waardenburg syndrome (WS), 357, 358, 544–546, 738, see also specific types clinical features, 544 genetics, 544 heterochromia irides, 757, 757 historical background, 544 incidence, 544 subtypes, 544–546 Waardenburg syndrome type 1 (WS1), 143–144, 544–545 Waardenburg syndrome type 2 (WS2), 144–145 clinical features, 527, 545 mutation, 527, 630 type 2A (WS2A), 126, 144–145, 245

type 2B (WS2A), 144 type 2C (WS2A), 144 type 2D (WS2A), 145 Waardenburg syndrome type 3 (WS3), 145, 544–545 Waardenburg syndrome type 4 (WS4), 146, 247, 545, 545–547 Wangiella dermatidis, melanonychia, 1061 warty hyperkeratoses, 805, 806 Watson syndrome, 813, 823 Weary–Kindler syndrome, see hereditary acrokeratotic poikiloderma Werner syndrome, 661, 806, 894–897 associated disorders, 895 clinical findings, 792, 894–895, 895 differential diagnosis, 885, 896 genetics, 894 histology, 895 laboratory investigations, 895 pathogenesis, 896 treatment, 896 Westerhof syndrome, 741–744 clinical description, 741–742, 742, 742 diagnosis/differential diagnosis, 743, 744 histopathology, 742, 742–744 white forelock, piebaldism, 570, 655 white hair, see leukotrichia (white hair) white halo, Mongolian spot, 1003 white mutants, 118–119 white pinta, 687 white skin, black skin vs., 8, see also ethnicity; skin types (human) Wnt protein signaling avian melanocyte specification, 121–122 fish chromatophore differentiation, 114 mammalian melanocyte specification, 83, 125, 448 MITF regulation and, 245–246 Wood’s light examination, 531, 532–533 black skin, 532 melasma, 1020 nevus anemicus, 767 nevus depigmentosus, 651 pityriasis alba, 699 tuberous sclerosis complex, 655 vitiligo vulgaris, 553, 554 Woolf syndrome, see albinism–deafness syndrome Woronoff ring, 701, 768 psoriasis, 701 Wright, Sewell, 395–396 WRN, Werner syndrome, 896 xanthelasma, 992 xanthomas, 992 xanthophores, 14, 17, 19–24, 20, 40–41 avian eye color, 98 MSH effects, 31 zebrafish patterning, 47 fate specification, 114–115 X-chromosome inactivation, 357 xenograft, melanocytic nevi models, 1135 Xenopus laevis color change, 29 darkness adaptation, 33, 33–34, 34, 35 morphologic, 27, 28 dermal melanophores, dispersed vs. aggregated, 16 melanophore line, regulation, 455 xeroderma pigmentosum, 889–894 animal models, 892 associated malignancy, 891 clinical findings, 889, 889–891, 890 diagnosis/differential diagnosis, 891 epidemiology, 889 histology, 891 historical background, 889 laboratory findings, 891 lentigines, 831, 890, 891 malignancy association, 890, 890 pathogenesis, 892 pigmentary features, 891 subtypes, 889, 890–891, 892 therapy, 892–893 Xiphophorine Gordon–Kosswig Melanoma System, 48 X-linked albinism–deafness syndrome, 147

INDEX X-ray depigmentation, 684 X-ray diffraction studies, melanin, 313, 314 yaws, 686–687 Yemenite deaf–blind hypopigmentation syndrome, 146 zebra, coat coloration, 75–76 zebrafish chromatophore development, 109–115

differentiation, 113–115 morphogenesis, 111–113 color pattern formation, 45–47, 47 hormonal influences, 48 larval vs. adult, 110, 110–111 molecular genetics, 47 mutants, 47, 111, 113, 114, 115 zidovudine (AZT), hyperpigmentation–induction, 943

Ziehl–Nielsen method, leprosy diagnosis, 691 Ziprkowski–Margolis syndrome, see albinism–deafness syndrome zosteriform lentiginous nevus, see speckled lentiginous nevus zosteriform speckled lentiginosis, see agminated lentigines (AL) zosteriform speckled lentiginous nevus, see speckled lentiginous nevus

1229

The Pigmentary System: Physiology and Pathophysiology, Second Edition Edited by James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, Richard A. King, William S. Oetting, Jean-Paul Ortonne Copyright © 2006 Blackwell Publishing Ltd

Plate 2.2 (Fig. 2.2)

Plate 2.1 (Fig. 2.1)

Plate 2.4 (Fig. 2.9)

Plate 2.3 (Fig. 2.3)

Plates 2.1–22.2: please refer to text for legends.

Plate 2.5 (Fig. 2.11)

Plate 2.6 (Fig. 2.15)

Plate 2.7 (Fig. 2.16)

Plate 2.8 (Fig. 2.20)

Plate 2.9 (Fig. 2.21)

Plate 2.10 (Fig. 2.22)

Plate 2.11 (Fig. 2.23)

Plate 2.12 (Fig. 2.42)

Plate 2.13 (Fig. 2.44)

Plate 2.14 (Fig. 2.45)

Plate 2.15 (Fig. 2.46)

Plate 2.16 (Fig. 2.47)

Plate 2.17 (Fig. 2.48)

Plate 2.18 (Fig. 2.49)

A

C

Plate 2.19 (Fig. 2.50)

B

Plate 2.20 (Fig. 2.53)

Plate 2.21 (Fig. 2.56)

S

Sinus

C RPE

R Plate 4.1 (Fig. 4.1)

a

b

P

Early larva

Adult

mel

xan irid

mel

mel irid

mel Plate 5.1 (Fig. 5.1)

wild-type

picasso (erbb3)

puma

panther (csf1r)

sparse (kit)

panther (csf1r); sparse (kit)

rose (ednrb1)

panther (csf1r); rose (ednrb1)

sparse (kit); rose (ednrb1)

nacre (mitfa)

leopard (cx40)

jaguar (kir7.1)

Plate 5.2 (Fig. 5.2)

Endoplasmic reticulum Golgi

Tyrosinase

Silver/Pmell7

M ul

t

iv

esi

c ula r / s o r ti ng

bo

o som

el

an

de

os o

me

C o a te

nd

e / p re m

dy

Plate 8.2A (Fig. 8.3A)

Melanoso

e stag me –

II

Clathrins -

Adaptins Molecules for recognition, docking and fusion -

Plate 7.1 (Fig. 7.5)

Plate 8.2B (Fig. 8.3B)

Basement membrane 1 µm

Plate 8.1 (Fig. 8.1)

Plate 8.2C (Fig. 8.3C)

A Tyrosine

Cysteine

Tyrosinase r3

r1

Dopaquinone

Cysteinyldopa r4

Cysteinyldopaquinone

Cyclodopa

r2

Tyrosinase

Dopachrome

Dopa

Eumelanin

Pheomelanin

Level of tyrosinae activity

B Eumelanin

Pheomelanin

Non-mutant (Tyr +)

Tyr ch/Tyr ch Tyr: Ay/a Agouti:

Tyr ch/Tyr ch a/a

+/+ Ay/a

Chinchilla (Tyr ch)

a/a Ay/a Level of Agouti expression Level of Mc1r signaling Plate 19.1 (Fig. 19.2)

light-bellied agouti (AW)

agouti (A)

black-and-tan (at )

Hair cycle: Ventral:

1A

1A¢

Ventralspecific

(100 kb)

1B

1C

ATG

(18 kb)

2

3

4

TGA

Hair cyclespecific

Region-specific promoters

Protein-coding exons

Plate 19.2 (Fig. 19.3)

A

UVB

100mm

Lesion B Non UVB 100mm

Nonlesion

Plate 21.1 (Fig. 21.3) Plate 21.2 (Fig. 21.8)

3 days after 2MED UVB irradiation

Non-specific IgG

Anti-SCF

Non UVB

100mm

Non-specific IgG

Anti-SCF

UVB

100mm

Plate 21.3 (Fig. 21.9)

Plate 21.4 (Fig. 21.11)

UVB irradiation (288 mJ/cm2)

0

1 Day 6

ACK2 injection (5 mg/50 ml)

2

3

Measurement

4

A

non UVB

5

6

7

8

B

C

UVB + ACK2

UVB + IgG

9

10 (day)

Plate 21.5 (Fig. 21.12)

A

A

Lesion B B

Nonlesion Plate 21.6 (Fig. 21.14) Plate 21.7 (Fig. 21.15)

A

B

Nonlesion

Plate 21.8 (Fig. 21.18)

PAN 19 Days

Lesion 16 Days

28 Days

DNCB

28 Days

Plate 21.9 (Fig. 21.21)

A

Plate 22.1 (Fig. 22.1)

19 Days

16 Days

13 Days

7 Days

Cont

1 Day

7 Days

13 Days

1 Day

Cont

B

Growth Factor

ligand GPCR

RTK

K

PTEN

Src

Shc

Ras

PI3

AKT PI3K

Grb2 SOS1 GrB1

STAT RAF PLCγ

PI3K

IKK

BAD GSK3 NFκB β-catenin

MEK

PKC+ Ca+

MAPK

p70S6K

RSK

β-catenin Tcf/Lef CREB MITF

DAG/Ca

RSK STAT NFκB

+

Ras/RAF Rap1/BRAF

Protein MAPK synthesis

RSK

MAPK

PDK1 PKC

Gq/PLCγ

RSK

MAPK

SRF c-Fos, CBP Elk-1 TCF

Plate 26.1 (Figs. 26.1, 50.51) Poikiloderma of Civatte. Differentiation Anti-apoptosis

Proliferation

Plate 22.2 (Fig. 22.2)

Plate 27.1 Greenish discoloration caused by venous engorgement of the areola of a pubescent girl.

Plate 24.1 (Fig. 24.2) Nevocellular nevi.

Plate 27.2 (Fig. 27.4) Nevus flammeus.

Plate 27.3 Nevas anemicus. The hypopigmentation is caused by decreased blood flow.

Plate 27.5 Microvesicular dermatitis affecting exclusively the pigmented skin of a patient with vitiligo.

Plate 27.4 A heavily pigmented Peruvian woman with photodamage (solar elastosis) from intense sun exposure at high altitude.

Plate 27.6 Penile and scrotal hyperpigmentation in a normal neonate. The lower panel shows a dizygotic twin with albinism.

Plate 27.7 Vulvar hyperpigmentation in a newborn infant.

Plate 27.8 (Fig. 27.8) White hair.

Plate 27.11 Type C hypopigmented demarcation line emanating from the nipple.

Plate 27.9 (Fig. 27.9) Linea nigra.

Plate 27.10 Futcher’s type A demarcation line.

Plate 27.12 (Fig. 27.11) Freckles.

Plate 28.1 (Fig. 28.7) Lichen planus.

Plate 28.2 (Fig. 28.8) Hyperpigmented lichen planus. Plate 28.5 Marked hyperpigmentation of the palms of a patient with adrenal insufficiency (normal hands are shown for comparison).

Plate 28.3 (Fig. 28.9) Hemosiderin deposition.

Plate 29.1 (Fig. 29.1) Piebaldism: father and daughter.

Plate 28.4 (Fig. 28.13) Alkaptonuria.

Plate 29.3 (Fig. 29.3) Piebaldism.

Plate 29.2 (Fig. 29.2) Piebaldism.

Plate 29.5 (Fig. 29.5) Waardenburg syndrome type IV.

Plate 29.4 (Fig. 29.4) Waardenburg syndrome type I.

Plate 30.4 (Fig. 30.5) Trichrome vitiligo.

Plate 30.1 (Fig. 30.2) Bilateral vitiligo vulgaris.

Plate 30.5 (Fig. 30.6) Vitiligo on palms.

Plate 30.2 (Fig. 30.3) Segmental vitiligo.

Plate 30.6 (Fig. 30.8) Hyperpigmented borders.

Plate 30.3 (Fig. 30.4) Vitiligo vulgaris.

Plate 30.7 (Fig. 30.10) Vitiligo: raised erythematous borders.

Plate 30.8 (Fig. 30.11) Monobenzone dermatitis.

Plate 30.10 (Fig. 30.16) Segmental vitiligo.

Plate 30.9 (Fig. 30.14) Spontaneous repigmentation.

Plate 30.11 (Fig. 30.18) Depigmentation of gums.

Plate 30.12A (Fig. 30.15) A,B. Segmental vitiligo of chin extending to mouth.

Plate 30.12B (Fig. 30.19)

Plate 30.13 (Fig. 30.21) Vogt–Koyanagi–Harada syndrome.

Plate 30.14 (Fig. 30.22) Uveitis in patient in Plate 30.13.

Plate 30.15 (Fig. 30.23) Iritis in vitiligo patient.

Plate 30.17 (Fig. 30.29) Bindhi depigmentation.

Plate 30.16 (Fig. 30.27) White hairs in vitiligo.

Plate 30.18 (Fig. 30.31) Squamous carcinoma in vitiligo.

Plate 30.19A (Fig. 30.33A) A. Depigmentation in patient with melanoma.

Plate 30.19B (Fig. 30.33B) B. Progressive pigment loss.

Plate 30.20 (Fig. 30.34) Depigmentation with melanoma.

Plate 30.21A (Fig. 30.35A) A,B. Depigmentation of hair associated with metastatic melanoma.

Plate 30.21B (Fig. 30.35B)

Plate 30.22 (Fig. 30.40) Repigmenting vitiligo.

Plate 30.23 (Fig. 30.42) No repigmentation with white hair.

Plate 31.1 (Fig. 31.1) Oculocutaneous albinism type 1A.

Plate 31.2 (Fig. 31.2) Oculocutaneous albinism type 1B.

Plate 31.3 (Fig. 31.3) Oculocutaneous albinism type 2.

Plate 31.4 (Fig. 31.4) Brown oculocutaneous albinism type 2.

Plate 31.6 (Fig. 31.7) Griscelli Syndrome.

Plate 31.5 (Fig. 31.6) Chediak–Higashi syndrome.

Plate 31.7 (Fig. 31.8) Angelman syndrome.

Plate 31.8 (Fig. 31.10) Hellerman–Streiff syndrome.

Plate 31.9 (Fig. 31.12) Homocystinuria.

Plate 31.10 (Fig. 31.13) Oculocerebral syndrome.

Plate 31.11 (Fig. 31.14) Phenylketonuria.

Plate 31.12 (Fig. 31.15) Inborn error of biopterin synthesis.

Plate 31.13 (Fig. 31.16) Brittle hair of patient in Plate 31.12.

Plate 32.2 (Fig. 32.4) Hypomelanosis of Ito.

Plate 32.1 (Fig. 32.2) Hypomelanosis of Ito.

Plate 32.3 (Fig. 32.5) Asymmetric pigmentation.

Plate 32.5 (Fig. 32.9) Focal dermal hypoplasia.

Plate 32.4 (Fig. 32.6) Sweat test of patient in Plate 32.2.

Plate 32.6 (Fig. 32.13) Nevus depigmentosus.

Plate 32.7 (Fig. 32.14) Nevus depigmentosus.

Plate 32.8 (Fig. 32.16) Congenital hypomelanotic nevus.

Plate 32.11 (Fig. 32.19) Periungual fibromata.

Plate 32.9 (Fig. 32.17) Acquired nevus depigmentosus.

Plate 32.12 (Fig. 32.20) Shagreen patch.

Plate 32.10 (Fig. 32.18) Angiofibromata in tuberous sclerosis.

Plate 32.13 (Fig. 32.21) Ash leaf spot.

Plate 32.14 (Fig. 32.22) Leukotrichia of tuberous sclerosis.

Plate 34.2 African child with Kwashiorkor showing hypopigmented skin and light hair. The mother is normally pigmented.

Plate 34.1 (Fig. 34.1) Kwashiorkor.

Plate 34.3 (Fig. 34.2) Hyperpigmentation of folic acid deficiency.

Plate 34.4 Hyperpigmentation of the gums of a patient with folic acid deficiency (courtesy Dr. Fred Miller).

Plate 35.1 (Fig. 35.2) Depigmentation from thermal burn and follicular repigmentation.

Plate 35.2A (Fig. 35.3A) A,B. Pigmentary changes induced by Xray burns.

Plate 34.5 Melasma-like hyperpigmentation associated with folic acid deficiency.

Plate 35.2B (Fig. 35.3B)

Plate 36.1 (Fig. 36.2) Hypopigmentation of leprosy.

Plate 36.3A (Fig. 36.4A) A,B. Tinea versicolor.

Plate 36.2 (Fig. 36.3) Hyper- and hypopigmentation of leprosy. Plate 36.3B (Fig. 36.4B)

Plate 37.1 Typical pityriasis alba on the cheeks of a young boy.

Plate 36.4 (Fig. 36.5) Onchocerciasis. Plate 37.2 (Fig. 37.1) Pityriasis alba.

Plate 36.5 (Fig. 36.6) Post herpes zoster hypopigmentation.

Plate 37.5 (Fig. 37.5) Alopecia mucinosa.

Plate 37.3 (Fig. 37.3) Psoriasis. Plate 37.6 (Fig. 37.6) Atopic dermatitis.

Plate 37.4 (Fig. 37.4) Woronoff ring.

Plate 37.7 Depigmentation on the dorsum of the hands of a patient with scoid lupus erythematosus.

Plate 37.8 (Fig. 37.7) Discoid lupus erythematosus.

Plate 37.9 (Fig. 37.8) Discoid lupus erythematosus.

Plate 37.10 (Fig. 37.9) Pityriasis lichenoides acuta.

Plate 38.1 (Fig. 38.2) Halo nevus.

Plate 38.3 (Fig. 38.4) Halo seborrheic keratoses.

Plate 38.2 (Fig. 38.3) Multiple halo nevi.

Plate 38.4 (Fig. 38.5) Halo congenital nevus.

Plate 38.5 (Fig. 38.6) Depigmented congenital nevus.

Plate 39.1 (Fig. 39.2) Guttate hypomelanosis.

Plate 38.6 (Fig. 38.7) Depigmented congenital nevus.

Plate 39.2 (Fig. 39.6) Lichen sclerosis et atrophicus.

Plate 38.7 (Fig. 38.8) Depigmentation in a melanoma.

Plate 39.3 (Fig. 39.7) Lichen sclerosis et atrophicus.

Plate 40.2 (Fig. 40.4) Progressive macular hypomelanosis.

Plate 40.1 (Fig. 40.2) Hypermelanocytic punctata et guttata hypomelanosis.

Plate 40.3 (Fig. 40.5B) Sarcoidosis.

Plate 41.1 (Fig. 41.1) Regrowing white hair in alopecia areata.

Plate 41.4 (Fig. 41.4) Heterochromia irides.

Plate 41.2 (Fig. 41.2) Heterochromia irides.

Plate 41.3 (Fig. 41.3) Heterochromia irides.

Plate 42.1 (Fig. 42.1) Nevus anemicus.

Plate 43.1 (Fig. 43.1) Hyperpigmentation in Fanconi anemia.

Plate 44.3 (Fig. 44.3) Dyschromatosis universalis hereditaria.

Plate 44.1 (Fig. 44.1) Kindler syndrome.

Plate 44.2 (Fig. 44.2) Kindler syndrome.

Plate 44.4 (Fig. 44.4) Dyschromatosis universalis hereditaria.

Plate 44.5 (Fig. 44.5) Dyschromatosis universalis hereditaria.

Plate 44.7 (Fig. 44.7) Epidermolysis bullosa and mottled hyperpigmentation.

Plate 44.8 (Fig. 44.8) Hereditary acrokeratotic poikiloderma.

Plate 44.6 (Fig. 44.6) Dyschromatosis universalis hereditaria.

Plate 44.9 (Fig. 44.9) Hereditary acrokeratotic poikiloderma.

Plate 44.10 (Fig. 44.10) Hereditary sclerosing poikiloderma.

Plate 44.11 (Fig. 44.11) Hereditary sclerosing poikiloderma.

Plate 44.12 (Fig. 44.12) Acropigmentation of Dohi.

Plate 44.13 (Fig. 44.13) Acropigmentation of Dohi.

Plate 44.14 (Fig. 44.14) Acropigmentation of Dohi.

Plate 44.15 (Fig. 44.15) Acropigmentation of Kitamura.

Plate 44.16 (Fig. 44.16) Acropigmentation of Kitamura.

Plate 44.17 (Fig. 44.17) Acropigmentation of Kitamura.

Plate 44.19 (Fig. 44.19) Rothmund–Thomson syndrome.

Plate 44.18 (Fig. 44.18) Rothmund–Thomson syndrome.

Plate 45.1 (Fig. 45.1) Café-au-lait macules in neurofibromatosis (NF) 1.

Plate 45.2 (Fig. 45.2) Café-au-lait macules in NF1.

Plate 45.5 (Fig. 45.5) Lisch nodules on iris.

Plate 45.3 (Fig. 45.3) Café-au-lait macules in NF1. Plate 45.6 (Fig. 45.6) Plexiform neurofibroma.

Plate 45.4 (Fig. 45.4) Axillary freckling in NF1. Plate 45.7 (Fig. 45.7) McCune–Albright syndrome.

Plate 45.8 (Fig. 45.8) McCune–Albright syndrome.

Plate 45.9 (Fig. 45.9) Segmental neurofibromatosis.

Plate 46.1 (Fig. 46.1) Lentigo simplex.

Plate 46.2 (Fig. 46.2B) PUVA lentigines.

Plate 46.3 (Fig. 46.3) Actinic lentigines.

Plate 46.5 (Fig. 46.5) Eruptive lentigines.

Plate 46.6 (Fig. 46.6) Multiple lentigines (LEOPARD) syndrome.

Plate 46.4 (Fig. 46.4) Actinic lentigines.

Plate 46.7 (Fig. 46.7) LEOPARD syndrome.

Plate 46.10 (Fig. 46.10) Lentigines in Carney complex.

Plate 46.8 (Fig. 46.8) LEOPARD syndrome.

Plate 46.11 (Fig. 46.11) Cardiac myxoma.

Plate 46.9 (Fig. 46.9) LEOPARD syndrome.

Plate 46.14 (Fig. 46.14) Labial lentigines.

Plate 46.12 (Fig. 46.12) Labial lentigines.

Plate 46.15 (Fig. 46.15) Cutaneous myxomata.

Plate 46.13 (Fig. 46.13) Periorbital lentigines.

Plate 46.16 (Fig. 46.16) Zosteriform speckled lentiginosis.

Plate 46.17 (Fig. 46.17) Nevus spilus. Plate 47.2 (Fig. 47.2) Incontinentia pigmenti stage 3.

Plate 47.1 (Fig. 47.1) Incontinentia pigmenti stage 1.

Plate 47.3 (Fig. 47.3) Incontinentia pigmenti stage 4.

Plate 47.4 (Fig. 47.4) Periorbital hyperpigmentation.

Plate 47.7 (Fig. 47.7) Demarcation line type A.

Plate 47.5 (Fig. 47.5) Periorbital hyperpigmentation.

Plate 47.8 (Fig. 47.8) Dowling–Degos disease.

Plate 47.6 (Fig. 47.6) Demarcation line type A.

Plate 47.9 (Fig. 47.9) Dowling–Degos disease.

Plate 48.4 (Fig. 48.8) Werner syndrome.

Plate 48.1 (Fig. 48.1) Progeria.

Plate 48.2 (Fig. 48.2) Xeroderma pigmentosum. Plate 49.1 (Fig. 49.1) Dyskeratosis congenita.

Plate 48.3 (Fig. 48.5) Xeroderma pigmentosum.

Plate 50.1 (Fig. 50.1) Acanthosis nigricans.

Plate 49.2 (Fig. 49.2) Dyskeratosis congenita.

Plate 50.2 (Fig. 50.6) Becker’s nevus.

Plate 49.3 (Fig. 49.4) Clouston syndrome.

Plate 50.3 (Fig. 50.10) Carcinoid syndrome.

Plate 50.4 (Fig. 50.11) Confluent and reticulated papillomatosis.

Plate 50.7 (Fig. 50.17) Macular amyloid.

Plate 50.5 (Fig. 50.13) Confluent and reticulated papillomatosis.

Plate 50.8 (Fig. 50.21) Erythema ab igne.

Plate 50.9 (Fig. 50.22) Erythema dyschromicum perstans.

Plate 50.6 (Fig. 50.14) Macular amyloid.

Plate 50.10 (Fig. 50.24) Erythromelanosis faciei.

Plate 50.11 (Fig. 50.28) Felty syndrome.

Plate 50.12 (Fig. 50.33) Blue nails with HIV.

Plate 50.13 (Fig. 50.34) Melanoacanthoma.

Plate 50.14 (Figs. 28.20, 50.39) Psoralen hyperpigmentation.

Plate 50.17 (Fig. 50.43) Angiomata of POEMS syndrome.

Plate 50.15 (Fig. 50.40) POEMS syndrome.

Figure 50.16 (Fig. 50.41). Hyperpigmentation of POEMS syndrome.

Plate 50.18 (Fig. 50.44) Urticaria pigmentosum.

Plate 50.19 (Fig. 50.45) Diffuse mastocytosis.

Plate 50.20 (Fig. 50.46) Diffuse mastocytosis.

Plate 50.21 (Fig. 50.47) Hyperpigmentation from diffuse mastocytosis.

Plate 50.22 (Fig. 50.53) Atrophoderma of Pasini and Pierini.

Plate 50.23 (Fig. 50.54) Atrophoderma of Pasini and Pierini.

Plate 50.25 (Fig. 50.57) Hyperpigmentation with ichthyosis.

Plate 50.26A (Fig. 50.69A) Addisonian pigmentation before therapy.

Plate 50.24 (Fig. 50.56) Ichthyosis.

Plate 50.26B (Fig. 50.69B) Addisonian pigmentation after therapy.

Plate 50.27 (Fig. 50.70) Addisonian oral hyperpigmentation.

Plate 51.1 (Fig. 51.3) Porphyria cutanea tarda.

Plate 51.2 (Fig. 51.5) Cronkhite–Canada syndrome.

Figure 50.28 (Fig. 50.73) Reticulated hyperpigmentation in mycosis fungoides.

Plate 51.3 (Fig. 51.7) Biliary cirrhosis.

Plate 51.4 (Fig. 51.8) Pellagra.

Plate 51.5 (Fig. 51.9) Pellagra.

Plate 51.6 (Fig. 51.10) Pellagra.

Plate 51.7 (Fig. 51.11) Peutz–Jeghers syndrome.

Plate 51.8 (Fig. 51.12) Peutz–Jeghers syndrome.

Plate 52.2 (Fig. 52.3) Extrasacral blue spots.

Plate 52.1 (Fig. 52.2) Mongolian sacral spot.

Plate 52.3A (Fig. 52.7A) A. Nevus of Ota.

Plate 52.3B (Fig. 52.7B) B. Nevus of Ota laser treated.

Plate 52.5 (Fig. 52.10) Phakomatosis pigmentovascularis.

Plate 52.4 (Fig. 52.9) Phakomatosis pigmentovascularis.

Plate 52.6 (Fig. 52.11) Phakomatosis pigmentovascularis. Plate 52.8 (Fig. 52.14) Acquired facial blue macules.

Plate 52.7 (Fig. 52.13) Phakomatosis pigmentovascularis.

Plate 53.1A (Fig. 53.2A) A. Melasma before therapy.

Plate 53.1B (Fig. 53.2B) B. Melasma after therapy.

Plate 54.1 (Fig. 54.1) Fixed drug eruption.

Plate 53.2 (Fig 53.3) Diffuse melanomatosis.

Plate 54.2 (Fig 54.2) Fixed drug eruption.

Plate 54.3 (Fig. 54.5) Lichen aureus.

Plate 54.5 (Fig. 54.7) Argyria.

Plate 54.4 (Figs 28.10, 54.6) Argyria.

Plate 54.6 (Figs 28.11, 54.8) Blue nails from argyria.

Plate 54.9 (Figs 28.12, 54.12) Gingival lead line.

Plate 54.7 (Figs 28.11, 54.10) Chrysiasis.

Plate 54.10 (Fig. 54.16) Tattoo.

Plate 54.8 (Fig. 54.11) Chrysiasis and normal hand.

Plate 54.11 (Fig. 54.17) Tattoo.

Plate 54.14 (Fig. 54.22) Amiodarone pigmentation.

Plate 54.12 (Fig. 54.18) Tattoo.

Plate 54.13 (Fig. 54.21) Carotenemia.

Plate 54.15 (Fig. 54.23) Amiodarone pigmentation.

Plate 54.16 (Fig. 54.24) Minocycline pigmentation.

Plate 54.19 (Fig. 54.29) Atrabine discoloration.

Plate 54.17 (Fig. 54.25) Minocycline pigmentation. Plate 54.20 (Fig. 54.30) Chloroquine-induced light hair.

Plate 54.18 (Fig. 54.26) Minocycline discoloration of teeth.

Plate 54.21 (Fig. 54.31) Clofazimine pigmentation and normal hand.

Plate 54.24 (Fig. 54.34) Nitrogen mustard hyperpigmentation.

Plate 54.22 (Fig. 54.32) Clofazimine pigmentation.

Plate 54.25 (Fig. 54.35) Bleomycin-induced hyperpigmentation.

Plate 54.23 (Fig. 54.33) Thorizine-induced discoloration.

Plate 54.26 (Fig. 54.36) Bleomycin hyperpigmentation.

Sagittal section through finger nail

Proximal matrix Proximal Nail Fold Eponychium Cuticle Lunula Nest of pigment producing cells Nail plate

Distal matrix

Nail

bed

Hyponychium Distal groove

A

B

Plate 55.1 (Fig. 55.1) Schematic drawing of nail. Plate 55.4 (Fig. 55.7) Longitudinal melanonychia: proximal widening.

Plate 55.2 (Fig. 55.5) Longitudinal melanonychia (single band). Plate 55.5 (Fig. 55.8) Hutchinson sign.

Plate 55.3 (Fig. 55.6) Longitudinal melanonychia (multiple bands).

Plate 55.6 (Fig. 55.9) Pseudo-Hutchinson sign.

Plate 55.8 (Fig. 55.11) Benign nevus.

Plate 55.7 (Fig. 55.10) Longitudinal melanonychia from metastatic melanoma.

Plate 55.9 (Fig. 55.12) Acrolentiginous melanoma.

Plate 56.1 (Fig. 56.3) Oral Kaposi sarcoma.

Plate 56.5 (Fig. 56.18) Blue nevus. Plate 56.2 (Fig. 56.8) Labial melanotic macule.

Plate 56.6 (Fig. 56.21) Gingival melanoma. Plate 56.3 (Fig. 56.10) Laugier–Hunziker syndrome.

Plate 56.4 (Fig. 56.12) Peutz–Jeghers syndrome.

Plate 56.7 (Fig. 56.24) Amalgam tattoo.

Plate 58.1 (Fig. 58.1) Congenital nevus sparing the areola. Plate 56.8 (Fig. 56.26) Vulvar melanosis.

Plate 56.9 (Fig. 56.29) Penile lentigo.

Plate 58.2 (Fig. 58.2) Congenital nevus sparing the areola.

Plate 58.5 (Fig. 58.5) Halos around sacral spots.

Plate 58.3 (Fig. 58.3) Congenital nevus sparing the areola.

Plate 59.1 (Fig. 59.1) Post-graft hyperpigmentation before treatment.

Plate 58.4 (Fig. 58.4) Congenital nevus sparing the genitalia.

Plate 59.2 (Fig. 59.2) Post-graft hyperpigmentation after treatment.

Plate 59.3 (Fig. 59.3) Hypopigmentation from hydroquinone.

Plate 59.4 (Figs 28.14, 54.27, 59.4) Ochronosis from hydroquinone.

Plate 59.6 (Fig. 59.6) Melasma before treatment.

Plate 59.5 (Fig. 59.5) Steroid-induced hypopigmentation.

Plate 59.7 (Fig. 59.7) Melasma after treatment.

Plate 59.8 (Fig. 59.8) Monobenzone-induced depigmentation.

Plate 59.9 (Fig. 59.11) Vitiligo before monobenzone therapy.

Plate 59.10 (Fig. 59.12) Vitiligo after monobenzone therapy.

Plate 60.1 (Figs. 30.43A, 60.3) Vitiligo before PUVA.

Plate 60.2 (Figs. 30.43B, 60.4) Vitiligo after PUVA.

Plate 60.4 (Fig. 60.9) White hair and failure of PUVA.

Plate 60.3 (Fig. 60.6) PUVA repigmentation.

Plate 63.1 (Fig. 63.2) Vitiligo before grafting.

Plate 63.2 (Fig. 63.3) Vitiligo after grafting.

Plate 63.3 (Fig. 63.4) Repigmentation from grafts.

Plate 63.4 (Fig. 63.11) Vitiligo before grafting.

Plate 63.5 (Fig. 63.12) Vitiligo after grafting.

Plate 63.6 (Fig. 63.13) Vitiligo before micropigmentation.

Plate 63.7 (Fig. 63.14) Vitiligo after micropigmentation.

Plate 64.1 (Fig. 64.1) Tattoo before laser removal.

Plate 64.2 (Fig. 64.2) Tattoo after laser removal.

Plate 64.3 (Fig. 64.5) Café-au-lait spot before laser therapy.

Plate 64.4 (Fig. 64.6) Café-au-lait spot after laser therapy.

A

D B

C

E

F

H

G

I

A: These two individuals illustrate the wide range of skin and hair color. [Marianne Greenwood (right) graciously shared the photographs . (from her book Varför Grater Puman?) that compose this frontispiece.] B: Classical Celtic women with blue eyes and red hair. C: Typical Scandinavian with blue eyes. D: Native American. E: A Peruvian girl. F: Himalayan woman and child. G: A Venezuelan woman. H: Two teenagers from New Guinea. I: Man from the New Hebrides Islands. See Chapter 27 for further information.