PEDIATRIC BONE Biology and Diseases
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PEDIATRIC BONE Biology and Diseases Editor-in-Chief
FRANCIS H. GLORIEUX Genetics Unit, Shriners Hospital McGill University Montreal, Quebec, Canada
Associate Editors II
JOHN M. PETTIFOR
HARALD lUPPNER
Mineral Metabolism Research Unit, Department of Pediatrics Chris Hani Baragwanath Hospital Soweto, Johannesburg, South Africa
Endocrine Unit, Department of Medicine and Pediatrics Massachusetts General Hospital and Harvard Medical Center Boston, Massachusetts
ACADEMIC PRESS A Harcourt Science and Technology Company
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Table of Contents
Contributors xi Foreword xv Preface xvii
1. Structure of Growth Plate and Bone Matrix WILLIAM G. COLE
Introduction 1 Type I Collagen 1 Type V Collagen 6 Type II Collagen 8 Type IX Collagen 10 Type XI Collagen 11 Type X Collagen 14 Aggrecan 16 Cartilage Link Protein 1 16 Small, Leucine-Rich, Interstitial Proteoglycans Perlecan 20 Matrilins 21 Thrombospondins 22 Osteonectin 23 Osteocalcin 24 Matrix Gla Protein 25 Bone Sialoprotein 25 Bone Acidic Glycoprotein-75 26 Dentin Matrix Acidic Phosphoprotein-1 26 Osteopontin 26 References 27
Osteoprogenitor Cells and Regulation of Osteoblast Differentiation and Activity 50 Regulation of Osteoblast Differentiation and Activity 54 Osteocytes 58 Morphological Features of Osteoclasts 59 Mechanisms of Osteoclastic Bone Resorption 60 Origin of Osteoclasts 61 Regulation of Ostoclast Activity and Differentiation 61 Osteoclast Size: Multinucleation and Function 63 Stem Cell, Osteoblast, and Osteoclast Changes in Disease 63 Tissue Engineering and Stem Cell Therapy for Skeletal Diseases 64 References 65
17 3. P r e n a t a l B o n e D e v e l o p m e n t : O n t o g e n y and Regulation BENOITST-JACQUESANDJILLA. HELMS Introduction 77 Skeletogenesis 78 Skeletal Organization and Embryonic Origin of Bones 82 Molecular Regulation of Bone Formation 96 References 104
4. Postnatal B o n e Growth: Growth Plate 2. B o n e Cell Biology: O s t e o b l a s t s , O s t e o c y t e s , and O s t e o c l a s t s JANEE.AUBINANDJOHANN. M. HEERSCHE Introduction 43 Ontogeny of Osteoblasts and Control of Osteoblast Development 44
Biology, Modeling, and Remodeling GERARD KARSENTYAND HENRY M. KRONENBERG
Endochondral and Intramembranous Bone Formation 119 Growth Hormone and Insulin-Like Growth Factor- 1 121
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Fibroblast Growth Factors 122 Thyroid Hormone 124 Estrogen and Androgen 124 Osteoblasts and Bone G r o w t h 124 Endocrine Regulation of Bone Formation References 130
References
9. P e a k B o n e M a s s a n d Its R e g u l a t i o n JEAN-PHILIPPEBONJOUR,THIERRYCHEVALLEY,SERGEFERRARI, AND RENF.RIZZOLI
126
5. P a r a t h y r o i d H o r m o n e a n d C a l c i u m Homeostasis GORDON J. STEWLER
Cellular and Extracellular Calcium Homeostasis Parathyroids and Secretion and Metabolism of Parathyroid Hormone 139 Assay of PTH 147 Parathyroid Hormone Action 148 References 160
135
6. P h o s p h a t e H o m e o s t a s i s R e g u l a t o r y Mechanisms JOSEPH CAVERZASIO, HEINI MURER, AND HARRIET S. TENENHOUSE
Introduction 173 Physiological Aspects 173 Cellular and Molecular Aspects 178 Pathophysiological Aspects 181 References 187
Definition and Importance of Peak Bone Mass 235 Characteristics of Peak Bone Mass Acquisition 235 Calcium-Phosphate Metabolism During Growth 236 Determinants of Bone Mass Gain 238 Conclusions 242 References 243
10. P r e g n a n c y a n d L a c t a t i o n ANN PRENTICE
Introduction 249 Mineral Fluxes from Mother to Offspring Pregnancy 250 Lactation 255 Summary 264 References 264
249
1 1. Fetal M i n e r a l H o m e o s t a s i s CHRISTOPHER S. KOVACS
7. Vitamin D Biology RENEST-ARNAUDANDMARIEB. DEMAY Metabolic Activation of Vitamin D 193 Mechanism of Action 198 Role of Vitamin D in Calcium Homeostasis Summary and Perspectives 208 References 209
201
8. O t h e r Factors C o n t r o l l i n g B o n e G r o w t h a n d D e v e l o p m e n t " C a l c i t o n i n , CGRP, O s t e o s t a t i n , Amylin, a n d A d r e n o m e d u l l i n JILLIANCORNISHANDTHOMASJOHNMARTIN Introduction 217 Calcitonin 217 Calcitonin Gene-Related Peptide 220 Parathyroid Hormone-Related Protein Amylin 225 Adrenomedullin 228
229
Fetal Adaptive Goals 271 Placental Calcium Transport 280 Placental Transport of Magnesium and Phosphate 288 Fetal Parathyroids 288 Calcium-Sensing Receptor 289 Thymus 290 Fetal Kidneys and Amniotic Fluid 290 Fetal Skeleton 291 Maternal Skeleton 292 Fetal Response to Maternal Hyperparathyroidism 293 Fetal Response to Maternal Hypoparathyroidism 293 Integrated Fetal Calcium Homeostasis 293 References 296
12. N o n i n v a s i v e T e c h n i q u e s for B o n e M a s s Measurement STEFANO MORA, LAURA BACHRACH, AND VINCENTE GILSANZ
223
Indications for Bone Mass Measurements 303 Challenges to Interpreting Bone Mass Measurements in Childhood and Adolescence 303 Noninvasive Measurement Techniques 304
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Data Interpretation References 319
316
1 3. A s s e s s m e n t of M a t u r a t i o n " B o n e A g e a n d Pubertal Assessment NO[L CAMERON
Background 325 Initial Considerations 325 Methods of Assessment 329 Reliability 333 Comparability of the Atlas and Bone-Specific Methods 333 Secondary Sexual Development 334 References 337
14. B i o c h e m i c a l M a r k e r s o f B o n e Metabolism ECKHARDSCHONAUAND FRANKRAUCH
Introduction 339 Markers of Bone Formation 341 Markers of Bone Resorption 342 Bone Markers During Normal Development 344 Puberty 346 Bone and Collagen Markers in Metabolic Bone Diseases 346 Clinical and Research Value of Biochemical Markers of Bone Metabolism 352 References 353
15. B o n e H i s t o m o r p h o m e t r y FRANK RAUCH
Introduction 359 Methodology 359 Pediatric Bone Histomorphometry in Health and Disease 366 Indications for Bone Biopsy and Histomorphometry in Pediatric Bone Diseases 372 References 373 16. A D i a g n o s t i c A p p r o a c h t o S k e l e t a l Dysplasias SHEILA UNGER, ANDREA SUPERTI-FURGA, AND DAVID L. RIMOIN
Introduction 375 Background 375
VII
History and Physical Examination 376 Diagnostic Imaging 389 Biochemical Investigations 393 Cartilage Histology 395 Molecular Basis 396 Prenatal Detection of Suspected Skeletal Dysplasia 397 Conclusion 398 References 398
1 7. T h e S p e c t r u m of P e d i a t r i c Osteoporosis LEANNE M. WARD AND FRANCIS H. GLORIEUX
Abstract 401 Introduction 401 Definition and Diagnosis of Osteopenia/Osteoporosis in Pediatric Patients 401 The Role of the Mechanostat in the Pathogenesis of Pediatric Osteoporosis 403 The Scope of the Problem 405 Approach to Prevention and Intervention 426 Differentiating Child Abuse from Bone Fragility Conditions 428 Summary and Future Directions 430 References 431
18. O s t e o g e n e s i s I m p e r f e c t a HORACIAO PLOTKIN, DRAGAN PRIMORAC, AND DAVID ROWE
Introduction 443 Classification 444 Types of OI 445 Differential Diagnosis 449 General Clinical Findings 450 Pathophysiology 453 Therapy 457 References 463
19. S c l e r o s i n g B o n y D y s p l a s i a L. LYNDONKEY,JR.,AND WILLIAML. RIES
Introduction 473 Defects in Osteoclastic Bone Resorption 473 Skeletal Abnormalities Related to Overproduction of Bone: Involvement of Transforming Growth Factors 478 Summary 480 References 481
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20. Parathyroid Disorders MURAT BASTEPE, HARALD JUPPNER, AND RAJESH V THAKKER
Introduction 485 PTH Gene Structure and Function 485 Hypocalcemic Disorders 488 Hypercalcemic Diseases 496 Conclusion 501 References 502
2 1 . Fibrous Dysplasia PAOLO BIANCO, PAMELA GEHRON ROBEY, AND SHLOMO WIENTROUB
Introduction 509 Clinical Features 510 Molecular Genetics 516 Determinants of Phenotypic Variability 518 Pathology 520 Pathogenesis 526 Management and Treatment 530 References 533
22. Nutritional Rickets JOHN M. PETTIFOR
Introduction 541 Definition of Rickets 541 Classification of Rickets 542 Nutritional Rickets 542 References 560
23.Metabolic Bone Disease of Prematurity NICK BISHOP AND MARY FEWTRELL
Introduction 567 In Utero Mineral Accretion and Bone Growth 567 Physiological Changes in Mineral Homeostasis at Birth 568 Metabolic Bone Disease 569 Skeletal Health in Preterm Infants 572 Suggested Guidelines for the Prevention of Metabolic Bone Disease in Preterm Infants 578 Conclusions 579 References 579
24. Rickets Due to Hereditary Abnormalities of Vitamin D Synthesis or Action ANTHONYA. PORTALE AND WALTER L. MILLER
Introduction
583
Biosynthesis of Vitamin D 583 Vitamin D Biosynthetic Enzymes 584 Vitamin D 25-Hydroxylase and 24-Hydroxylase 584 Vitamin D la-Hydroxylase 584 Rickets Due to Abnormalities of Vitamin D Metabolism 585 Rickets Due to Abnormalities of Vitamin D Action 592 References 598
25. Familial Hypophosphatemia and Related Disorders INGRID A. HOLM, MICHAEL J. ECONS. AND THOMAS 0. CARPENTER
Introduction 603 Clinical Description of Disease Entities 603 Treatment 613 Family/Genetic Studies 616 Molecules, Pathophysiology, and Lessons from Animal Models 617 Current Problems and Unresolved Questions 623 References 624
26. Rickets Due to Renal Tubular
Abnormalities RUSSELL W. CHESNEY AND DEBORAH I? IONES
Introduction 633 Fanconi Syndrome 633 Renal Magnesium Wasting 640 Hypercalciuria 642 Renal Tubular Acidosis 644 Conclusion 647 References 647
27. Hypophosphatasia DAVID E. C. COLE
Biology of Alkaline Phosphatase 651 Clinical Hypophosphatasia 661 References 672
28. Renal Osteodystrophy: Pathogenesis, Diagnosis, and Treatment BEATRIZ D. KUIZON AND ISIDRO B. SALUSKY
Introduction 679 The Spectrum of Renal Osteodystrophy 679 Pathogenesis of Renal Osteodystrophy 682
Table of Contents Clinical Manifestations 685 Biochemical Determinations 686 Histologic Manifestations 687 Radiographic Features of Renal Osteodystrophy 689 Long-Term Consequences 690 Treatment 691 References 695
2 9 . B o n e T u m o r s in C h i l d r e n MARCH. ISLERAND ROBERTE. TURCOTTE Introduction 703 General Principles of Treatement 703 Diagnosis 704 Classification and Nomenclature 706
ix
Back Pain and Spinal Neoplasms in Children 707 Fibrous Tumors of Bone 709 Cartilage-Forming Tumors 713 Bone-Forming Tumors 722 Hematopoietic 730 Vascular 731 Neurogenic 733 Adipose 734 Mixed 734 Notochord 735 Tumor-Like 735 Conclusion 740 References 740
Index
745
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Contributors
Russell W. Chesney (633) University of Tennessee Health Science Center, Deparment of Pediatrics, Memphis, Tennessee 38103
Numbers in parentheses indicate the pages on which the authors' contribution begin.
Jane E. Aubin (43) Department of Medical Genetics and Microbiology, Faculty of Medicine, Toronto, Ontario M5S 1A8, Canada
Thierry Chevalley (235) Department of Internal Medicine, Division of Bone Diseases, World Health Organization Collaboration Center for Osteoporosis and Bone Diseases, University Hospital, CH 1211 Geneva, Switzerland
Laura Baehraeh (303) Division of Endocrinology, Stanford University School of Medicine, Stanford, California 94305
David E. C. Cole (651) Departments of Laboratory Medicine and Pathobiology, Medicine, and Genetics, University of Toronto, Toronto, Ontario MSG 1X8, Canada
Murat Bastepe (485) Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston Massachusetts 02115
William G. Cole (1) Division of Orthopaedics and Research Institute, The Hospital for Sick Children, University of Toronto, Toronto, Ontario MSG 1X8, Canada
Paolo Bianeo (509) Department of Experimental Medicine and Pathology, La Sapienza University School of Medicine, 00161 Rome, Italy Nick Bishop (567) Academic Unit of Child Health, University of Sheffield, Sheffield Children's Hospital, Western Bank, Sheffield S10 2TH, United Kingdom
Jillian Cornish (217) Department of Medicine, University of Auckland, Auckland 1020, New Zealand Marie B. Demay (193) Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston Massachusetts 02114
Jean-Philippe Bonjour (235) Department of Internal Medicine, Division of Bone Diseases, World Health Organization Collaborating Center for Osteoporosis and Bone Diseases, University Hospital, CH 1211 Geneva, Switzerland
Michael J. Econs (603) Indiana University School of Medicine, Indianapolis, Indiana 46202 Serge Ferrari (235) Department of Internal Medicine, Division of Bone Diseases, Worlth Health Organization Collaboration Center for Osteoporosis and Bone Diseases, University Hospital, CH 1211 Geneva, Switzerland
Noel Cameron (325) Human Biology Research Centre, Department of Human Sciences, Loughborough University, Loughborough, Leicestershire LEll 3TU, United Kingdom Thomas O. Carpenter (603) Yale University School of Medicine, New Haven, Conneticut 06510
Mary Fewtrell (567) MRC Childhood Nutrition Research Center, Institute of Child Health, London WC 1N 1EH, United Kingdom
Joseph Caverzasio (173) Division of Bone Diseases, Department of Internal Medicine, University Hospital of Geneva, CH-1211 Geneva, Switzerland
Vicente Gilsanz (303) Department of Radiology, Children's Hospital Los Angeles, University of Southern
xi
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Contributors
California School of Medicine, Los Angeles, California 90027 Francis H. Glorieux (401) Departments of Surgery, Pediatrics and Human Genetics, McGill University and the Shriners Hospital for Children, Montreal, Quebec, Canada H3A 2T5
Jill A. Helms (77) Department of Orthopedic Surgery, University of California San Francisco, San Francisco, California 94143-0514 Johan N. M. Herschel (43) Dental Research Institute, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada lngrid A. Holm (603) Harvard Medical School, Boston Massachusetts 02115
Mare H. Isler (703) University of Montreal, Department of Orthopedic Oncology, Hopital Maisonneuve Rosemont and Hopital Sainte Justine, Montreal, Quebec, Canada H 1T 2M4 Deborah P. Jones (633) University of Tennessee Health Science Center, Deparment of Pediatrics, and Children's Foundation Research Center at Le Bonheur Children's Medical Center, Memphis, Tennessee 38103 Harald Jiippner (485) Endocrine Unit, Department of Medicine and Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02115 G6rard Karsenty (119) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston Texas 77030
L. Lyndon Key, Jr. (473) Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425 Christopher S. Kovacs (271) Faculty of Medicine, Endocrinology, Memorial University of Newfoundland, Health Sciences Centre, St. John's Newfoundland A1B 3V6, Canada Henry M. Kronenberg (119) Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 Beatriz D. Kuizon (679) Department of Pediatrics, UCLA School of Medicine, Los Angeles, California 90095 Thomas John Martin (217) St. Vincent's Institute of Medical Research, Melbourne, Victoria, Australia Walter L. Miller (583) Department of Pediatrics, University of California San Francisco, San Francisco, California 94143
Stefano Mora (303) Laboratory of Pediatric Endocrinology, Scientific Institute H San Raffaele, 20132 Milan, Italy Heini Murer (173) Institute of Physiology, University of Zurich, CH-8057 Zurich, Switzerland John M. Pettifor (541) MRC Mineral Metabolism Research Unit, Department of Pediatrics, University of the Witwatersrand and Chris Hani Baragwanath Hospital, Johannesburg, South Africa Horaeio Plotkin (443) Inherited Metabolic Diseases Section, Department of Pediatrics, University of Nebraska Medical Center and Children's Hospital, Omaha, Nebraska 98198 Anthony A. Portale (583) Department of Pediatrics, University of California San Francisco, San Francisco, California 94143 Ann Prentice (249) Medical Research Council Human Nutrition Research, Elsie Widdowson Laboratory, Cambridge CB 1 9NL, United Kingdom Dragan Primorae (443) Laboratory of Clinical and Forensic Genetics, Split University Hospital and School of Medicine, Split, Croatia Frank Raueh (339, 359) Genetics Unit, Shriners Hospital for Children, Montreal, Quebec H3G 1A6, Canada
William L. Ries (473) Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425 David L. Rimoin (375) Medical Genetics Birth Defects Center, Cedars-Sinai Health System and Department of Pediatrics and Medicine, UCLA School of Medicine, Los Angeles, California 90048 Ren6 Rizzoli (235) Department of Internal Medicine, Division of Bone Diseases, Worlth Health Organization Collaboration Center for Osteoporosis and Bone Diseases, University Hospital, CH 1211 Geneva, Switzerland Pamela Gehron Robey (509) Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892 David Rowe (443) Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Conneticut 06030
lsidro B. Salusky (679) Department of Pediatrics, UCLA School of Medicine, Los Angeles, California 90095 Eckllard Sch6nau (339) Children's Hospital and Health Center, University of K61n, 50924 K61n, Germany
Contributors
xiii
Ren~ St-Arnaud (193) Genetics Unit, Shriners Hospital for Children, Montreal, Quebec H3G 1A6 Canada and Departments of Surgery and Human Genetics, McGill University, Montreal, Quebec H3A 2T5 Canada
Rajesh V. Thakker (485) Molecular Endocrinology Group, Nuttfield Department of Medicine, John Radcliffe Hospital Headington, Oxford OX3 9DU, United Kingdom
Benoit St-Jacques (77) Department of Human Genetics,
Robert E. Turcotte (703) University of Montreal and
McGill University and Genetics Unit, Shriners Hospital for Children, Montreal, Quebec H3G 1A6, Canada
McGill University, Department of Orthopedic Oncology, Hopital Maisonneuve Rosemont, Montreal, Quebec, Canada H 1T 2M4
Gordon J. Strewler (135) Walter Bradford Cannon Soci-
Sheila Unger (375) Division of Clinical and Metabolic
ety, Harvard Medical School, Boston, Massachusetts 02115
Genetics, Hospital for Sick Children, University of Toronto, Toronto, Canada M5G 1 X8
Andrea Superti-Furga (375) University of Lausanne, Div-
Leanne M. Ward (401) Department of Pediatrics, Div-
ision of Molecular Pediatrics, Centre Hospitalier Universitaire Vaudois, CH- 1011 Lausanne, Switzerland
ision of Endocrinology and Metabolism, University of Ottawa and the Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada K1H 8L1
Harriet S. Tenenhouse (173) Departments of Pediatrics
and Human Genetics, McGill University, Montreal Children's Hospital Research Institute, Montreal, Quebec H3Z 2Z3 Canada
Shlomo Wientroub (509) Department of Pediatric Orthopedic Surgery, Dana Children's Hospital, Tel Aviv Medical School, 64239 Tel Aviv, Isreal
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Foreword
Harold Harrison's contributions to our understanding of pediatric bone disease (4). Chapters in the book, each one written by an authoritative member of the corresponding "academy," address particular topics, such as the cellular architecture of bone itself, how it develops, and how it remodels. The vast complexity of calcium and phosphate ion metabolism, and the complexity of their hormonal controls and of the corresponding nutritional dimensions are subjects of other chapters. There are chapters that describe how to measure the features of bone, and there are tables of mutations that point to online databases and the locus specific alleles that are causes of certain bone diseases. A series of twelve chapters address diseases of bone development (skeletal dysplasias), of bone density (osteoporosis), of tensile strength (osteogenetics imperfecta) and remodeling (osteopetrosis). Aberrations of hormonal control and ion homeostasis, are discussed. Acquired diseases of bone (e.g. tumors and renal osteodystrophy) are not forgotten. More important still, the patient with the problem is not forgotten. Between the covers of this book, there is a great deal of information from which the scientist and the clinician can acquire knowledge. More important the reader will gain wisdom about the precious skeleton of youthful Homo sapiens.
The human skeleton is a product of biological evolution; it accommodates an upright bipedal organism. It is still imperfect for the job it must do; there are speculations on how the human skeleton might be improved (1). Meantime, Homo sapiens has what it has. The healthy human skeleton and the organic matrices of which it is made are mysterious and wonderful structures. In the fetus, they are pliable and miniature, they accommodate both fetal growth and development and also the trauma of passage through the maternal pelvis. The infant's skeleton must then grow enormously in volume and span, remodeling itself all the while until adult dimensions are achieved. At the same time, it must be resisting the continuous action of gravity, while maintaining its integrity under physical trauma, on average for 8 decades! A truly mysterious and marvelous structure. This book, which reflects the vision of its outstanding editors, addresses the biology of bone, and the diseases that interfere with its structure and function. Industrialized societies of the northern hemisphere witnessed childhood rickets in epidemic proportions during the 19 th and early 20 th century. Rickets was an important pediatric disease. Then came the discovery of the anti-rachitic factor (vitamin D); awareness of the origins and importance of vitamin D, combined with improvements in living conditions, led to the virtual disappearance of nutritional infantile rickets, leaving the much rarer intrinsic biological forms of rickets to be recognized--the inherited forms of calcium or phosphate dishomeostasis. The first such report appeared in 1937 (2). It heralded the search for, and eventually the molecular definition of, heritable forms of rickets, and of metabolic and organic bone diseases in general in human societies (3). In the past half century, there has been an impressive growth of knowledge about cellular, ionic, organic, and hormonal aspects of bone metabolism, and of the diseases that interfere with the processes of development, growth, remodeling, and mineralization of bone. This book is a record of that understanding at the present time. It is the first of its kind, to my knowledge, to follow an earlier book honoring Helen and
(1)
Olshansky, S. J., Carnes, B. A., Butler, R. N. If humans were built to last. Sci. Am. 284:50-55, 2001.
(2)
Albright, F., Butler, A. M., Bloomberg, E. Rickets resistant to vitamin D therapy. Am. J. Dis. Child 54:529-547, 1937
(3) Scriver, C. R., Tenenhouse, H. S. On the heritability of rickets, a common disease (Mendel, Mammals and Phosphate). Johns Hopkins Med. J. 149:179-187, 1981. (4)
DeLuca H. F., Anast C. S. Pediatric Diseases Related to Calcium. Elsevier. New York. 1980. Charles R. Scriver, M D C M Montreal Children's Hospital, Canada
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Preface
The description and investigation of bone diseases both in adults and in children have been, for a long time, part of the realms of Endocrinology, Nephrology, Rheumatology and also Orthopedics and Medical Genetics. In the past three decades, as new knowledge and technology rapidly developed, bone biology and its related diseases progressively became a well identified domain in medicine. Thus was published a series of important books in adult medicine dealing with the following: metabolic bone disease, diseases of connective tissue, osteoporosis (a field in itself), and bone biology. In some of these textbooks, chapters dealing with pediatric bone disease have been included. However, not since the book by Maroteaux (1) was published in 1974 in French, which contains a unique collection of radiographs, and not since the Proceedings of a Symposium held in 1980 to celebrate the careers of Harold and Helen Harrison (2), has there been a book attempting to integrate basic knowledge and clinical facts as they concern pediatric bone disease. When we started drawing up the outline for this book, we decided that its usefulness would be greatly enhanced by first providing detailed descriptions of the basic concepts underlying the development, structure, and homeostatic control of bone tissue. We also wanted to provide descriptions of the tools and methodologies now validated for the precise evaluation of most of the disturbances of bone and mineral metabolism in children. Thus the book opens with chapters on the structure of the growth plate and the bone matrix, bone cell biology, mineral ion homeostasis and prenatal and postnatal bone growth. This is followed by the descriptions of how to assess bone maturation by measuring biochemical bone markers in blood and urine, and of the unique information provided by bone histomorphometry. The next part comprises fourteen chapters discussing specific disorders, or groups of disorders, including basic and clinical aspects.
We hope that the book will be useful both to researchers interested in learning about the clinical expression of the biological processes they study at the bench, and to the practitioners who wish to better understand the molecular and genetic intricacies of the conditions they evaluate and treat in the clinical setting. We wish to express our gratitude to Tari Paschall and Jasna Markovac at Academic Press for their understanding and support. We also thank all of the contributors for their willingness to be a part of this large effort. Finally we hope that the summation of knowledge and observations under one single heading will give substance to the concept of "pediatric osteology," which has become an entity of its own and may need to be recognized as a speciality in itself. As such, it may become an important and exciting career opportunity for some of our younger colleagues. (1) Maroteaux, P. (1974). Les maladies osseuses de l'Enfant. Flammarion M6decine-Sciences, Paris. (2) DeLuca, H. F., and Anast, C. E. (Eds.) (1980). Pediatric Diseases Related to Calcium. Elsevier, North Holland, New York, NY. Francis H. Glorieux Harald W. Jueppner John M. Pettifor
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1 Structure of Growth Plate and Bone Matrix WILLIAM G. COLE Division of Orthopaedics and the Research Institute, The Hospital for Sick Children, and University of Toronto, Toronto, Ontario, Canada
INTRODUCTION This chapter focuses on the main macromolecules of the extracellular matrices of the growth plates and bone. These diverse macromolecules often perform critical biomechanical, physiological, and biochemical functions. The hyaline cartilage growth plates are highly organized structures responsible for the elongation of bones by endochondral ossification. The cellular events are described in detail in subsequent chapters. Hydrated hyaline cartilages contain approximately 65-80% water, 10-20% collagen, 4-7% aggrecan, and <5% other proteoglycans, collagens, glycoproteins, and lipids [1]. The highly hydrophilic nature of aggrecan and other proteoglycans, and less so collagen, accounts for the high level of hydration of cartilage. Approximately 20-30% by weight of bone is organic, 10% is water, and the remainder is mineral [2]. The organic matrix contains approximately 90% collagen and 10% noncollagenous proteins, proteoglycans, and lipids. Plasma proteins are also represented in the organic matrix. Many of the plasma proteins are present within the blood vessels of bone but have little or no affinity for bone, whereas others such as OLz-HS glycoprotein have a higher affinity for the mineralized bone matrix. A description of the primary structure, synthesis, and assembly of the major macromolecules of hyaline cartilage growth plates and bone follows. Some of the macromolecules are specific for one or the other of these tissues and many are found in both. The bone collagens are described first, followed by the cartilage collagens, cartilage aggregating and nonaggregating proteoglycans, cartilage glycoproteins, and a group of noncollagenous proteins of bone. Some macromolecules are not
PediatricBone
discussed, including the microfibrillar proteins, such as type VI collagen and the fibrillins, as well as fibronectin, type XII collagen, and the lipoproteins.Details concerning the latter macromolecules as well as other macromolecules of the extracellular matrix and further details about the described macromolecules can be found online at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/), National Library of Medicine, National Institutes of Health.
TYPE i COLLAGEN Type I collagen, the most abundant protein of bone, is also present in periosteum, perichondrium, ligaments, tendons, annulus fibrosus, menisci, dermis, sclera, dentin, fascia, and the adventitial layers of viscera [3,4]. It is one of the fibrillar collagens. The characteristic feature of the fibrillar collagens is that they contain a long, continuous triple helix that assembles into highly organized collagen fibrils [5]. The fibrils have high tensile strength, which is critical for the function of bone and other tissues. Each molecule of type I collagen is composed of two OLl(I) chains and one oL2(I) chain [5]. A small number of molecules contain three OLl(I) chains. The importance of type I collagen in normal development is highlighted by the phenotypes that result from the homozygous or heterozygous loss of the OLl(I) chains. Homozygous loss in the Mov 13 mouse, in which the OLl(I) gene is inactivated by insertional mutagenesis, results in a prenatal lethal phenotype because of the lack of type I collagen in the tissues [6]. Heterozygous loss of one aa(I) allele, either in the Mov 13 mouse or in humans, yields the osteogenesis imperfecta type IA phenotype [7]. Copyright 2003, Elsevier Science (USA). All rights reserved.
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William G. Cole
The Otl(I) and ot2(I) chains are encoded by the COL1A1 and COL1A2 genes, respectively. The COL1A1 gene is located on chromosome 17q21.3-q22 and the COL1A2 gene on chromosome 7q21.3-q22 [8,9]. Both genes have a similar structure, but because exons 33 and 34 are fused in COL1A1, the COL1A1 gene contains 51 exons, whereas the COL1A2 gene contain 52 exons [10,11]. The differences of 18 kb for COL1A1 and 38 kb for COL1A2 are due to differences in the sizes of their introns. For the pre-pro-oL2(I) chain, the signal peptide is encoded by part of exon 1; the N-propeptide is encoded by part of exon 1, exons 2-5, and part of the junctional exon 6; the N-telopeptide is encoded by part of exon 6; the triple helix is encoded by part of exon 6, exons 7-48, and part of the junctional exon 49; the C-telopeptide is encoded by part of exon 49; the C-propeptide is encoded by part of exon 49 and exons 50-52 [5,10]. The same arrangement exists for the pre-pro-oLl(I) chain, except that the exon numbering is reduced by one beyond exons 33 and 34, which are fused in COL1A1 [5]. The exons encoding the main triple helix of the two chains have similar sizes [9]. Each exon encodes complete Gly-X-Y triplets, where X and Y are often proline so that they commence with a codon for Gly and end with a codon for Y. All exons encoding the triple-helical domain are 45,54,99,108, or 162, base pairs (bps). It is likely that 54 bp was the ancestral exon size. The 108- and 162-bp exons arise from loss on introns. The 45- and 99-bp exons result from recombinations between two 54-bp exons [12]. The pre-pro-otl(I) chain contains 1464 amino acid residues [5,10]. It contains a signal peptide of 22 residues, a N-propeptide of 139 residues, a N-nonhelical telopeptide of 17 residues, a main triple helix of 1014 residues, a C-nonhelical telopeptide of 26 residues, and a C-propeptide of 246 residues. The mature tissue form of the OLl(I) chain contains 1057 residues, including the main triple helix and the N- and C-telopeptides. The N-propeptide begins with a globular domain of 86 residues, including a von Willebrand type C repeat and 10 cysteine residues. It is followed by a triple-helical domain of 48 amino acids and a short globular domain. The procollagen N-proteinase cleavage site is at the Pro161-Gln162 bond, numbered from the start of the signal peptide. Allysine cross-linking sites are located at residues 170 and 1208. There are two hydroxylysines that can be glycosylated at residues 265 and 1108. The mammalian collagenase cleavage site is at the Gly 953Ile 954 bond. Proline residue 1164 may be 3-hydroxylated. There are two potential R G D cell attachment sites at residues 745-747 and 1093-1095. Procollagen C-proteinase cleaves at Alal21S-Asp 1219. The globular C-propeptide contains 4 cysteine residues at positions 1259,1265,1282, and 1291 that are involved in interchain
disulfide bonding [13]. It also contains cysteine residues that are involved in intrachain disulfide bonding between residues 1299 and1462 and between residues 1370 and 1415. There is also a putative Asn 1365 site for attachment of an N-linked oligosaccharide. The pre-pro-oL2(I) chain is shorter than the prepro-oLl(I)chain, although the main triple-helical domains are the same size [11,14]. The pre-pro-ot2(I) chain has a signal peptide of 22 amino acid residues, an N-propeptide of 57 residues, an N-nonhelical telopeptide of 11 residues, a main helical domain of 1014 residues, a C-terminal nonhelical telopeptide of 15 residues, and a globular C-propeptide of 247 residues. The very short globular domain of the N-propeptide contains only 2 cysteine residues. It is followed by a triple-helical domain of 42 residues and a second short globular domain. Procollagen N-proteinase cleaves at the Asn Y9-Glns~ bond. Lysine residues 177 and 1023 are potential sites for hydroxylation and glycosylation. There are potential cell attachment sites at R G D sequences at positions 777-779,822-824, and 1005-1007. The procollagen C-proteinase cleavage site is at the Ala 1119_Asp1120 bond. Three sites for interchain disulfide bonds are at residues 1163, 1186, and 1195. Intrachain disulfide bonds may form between cysteine residues at positions 1203 and 1364 and between cysteine residues at positions 1272 and 1317. There is a potential N-linked oligosaccharide attachment site at A s n 1267. The structures of the C-propeptide of human type III procollagen and the C-telopeptides of type I collagen have been described [15,16]. The structures of the C-propeptides of types I and III procollagen are likely homologous. In contrast to the elongated structure of the C-terminal triple-helical region and the C-telopeptide the C-propeptide has a low-resolution structure composed of three large lobes and one small lobe [16]. This structure is readily interpretable in terms of the subunit composition and known positions of interand intrachain disulfide bonds. Among the eight cysteines found in the C-propeptide domains of type III procollagen, cysteines 1-4 are involved in interchain disulfide bonding, whereas cysteines 5-8 form intrachain disulfide bonds [17]. Consequently, it is likely that the three large lobes correspond to the intrachain disulfide bonded region of each of the three polypeptide chains, whereas the small lobe corresponds to the junction region containing the interchain disulfide bonds and linking to the rest of the procollagen chain. Such an arrangement would place the chain-recognition region at the core of the structure, well positioned to determine the specificity of chain-chain interactions [16]. The C-telopeptide of type I collagen had a hairpin conformation, with the C terminus folded back onto the triple helix [15].
1. Structure of Growth Plate and Bone Matrix
After being transcribed, the pre-mRNA for the prepro-oLl(I) and pre-pro-cx2(I) chains undergoes exon splicing, capping, and the addition of a polyA tail, which gives rise to mature mRNAs. The mRNAs are translated in polysomes bound to the rough endoplasmic reticulum [18]. Formation of the triple-helical heterotrimeric type I procollagen molecule requires the synthesis of the constituent pro-oLl(I) and pro-oL2(I) chains from their distinct mRNAs. Transfer to the Golgi apparatus and movement into the secretory pathway require completion of both posttranslational modifications and triple helix formation. The [pro-oLl(I)]2[pro-oL2(I)] heterotrimer is the major product, whereas stable [proOtl(I)]3 homotrimer is formed in only small amounts. A [pro-otl(I)][pro-ot2(I)]2 heterotrimer or a [pro-oL2(I)]3 homotrimer have not been detected in cell cultures or tissues. Efficient type I procollagen heterotrimeric assembly appears to require that the elongating nascent pro-~l(I) and pro-ot2(I) chains be inserted into the same compartments of the rough endoplasmic reticulum and that a mechanism for chain selection be operational within the compartment. Coordinated expression of the COL1A1 and COL1A2 genes is one way of ensuring that the appropriate 2:1 ratios of the protein chains are available for the assembly of heterotrimers [19-21]. However, the ratios of the pro-oLl(I):pro-oL2(I) mRNAs vary widely, even though the resultant heterotrimers maintain the protein chain ratio of 2:1 [22]. Consequently, chain selection probably plays an important role in molecular assembly [23]. In support of the latter proposal, distinct chain recognition sequences of 15 amino acid residues have been identified within the C-propeptides of the type I procollagen chains [24]. The interactions between the C-propeptides that lead to registration of nascent pro-oLl(I) chains occur while the chains are still associated with polysomes [25-27]. Nascent collagen chains are translated on complex polyribosomal aggregates associated with more than one strand of mRNA. Consequently, it is likely that the organization of the m R N A translocons is a critical factor in molecular assembly [18]. Elongation pauses are also a prominent feature of synthesis of pro-oLl(I) and pro-oL2(I) chains [28]. There also appears to be an interaction between the translation complexes for the synthesis of the two chain types [23]. These interactions may serve two purposes: to bring the translation complexes to the same regions of the endoplasmic reticular membrane to ensure the colocalization of the nascent chains for interaction and to regulate or coordinate the rates of synthesis of the two pro-oL chains. In many systems producing type I collagen, the pro-e~l(I):pro-a2(I) ratio is greater than 2:1 [29]. In such situations, it is likely that all of the pro-~2(I) m R N A is engaged in heterotrimer synthesis,
3
whereas the excess pro-~xl(I) chains may either produce [pro-o~l(I)]3 trimer or be degraded [30]. The signal peptides are removed as the nascent chains enter the rough endoplasmic reticulum. Both procollagen chains undergo hydroxylation and glycosylation of specific residues as a prerequisite for the formation of a stable triple-helical heterotrimer. Many of these enzymatic modifications occur as cotranslational events. Approximately 100 proline residues in the Y position of Gly-X-Y repeats undergo 4-hydroxylation by prolyl 4-hydroxylase. A few proline residues in the X position undergo 3-hydroxylation by prolyl 3-hydroxylase. A variable number of lysine residues, also in the Y position, may undergo lysyl hydroxylation by lysyl hydroxylase. Hydroxylation of proline residues to hydroxyproline is critical for the formation of a stable triple helix. The hydroxylases require the procollagen chains to be in a nascent state and various cofactors need to be present, such as ferrous ions, molecular oxygen, oL-ketoglutarate, and ascorbate [31]. Prolyly 4-hydroxylase, also called procollagen proline 2 oxoglutarate 4-dioxygenase, is a tetramer consisting of two oL and two [3 subunits with a molecular weightof 240 kDa. The [3 subunit is also known as protein disulfide isomerase. The gene for the [3 subunit, P4HB, is located on chromosome 17q25 [32]. The gene is expressed ubiquitously. There are two ot subunits whose genes are named P4HA1 and P4HA2 [33]. The P4HA1 gene is located on chromosome 10q21.3-23.1 [34], and the P4HA2 gene is located on chromosome 5q31 [33]. The mRNAs encoded by P4HA1 and P4HA2 are expressed ubiquitously, although the ratios of the mRNAs vary between tissues. There are twoe~(I) subunit m R N A isoforms that result from mutually exclusive alternative splicing of exons 9 or 10 of P4HA1 [35]. Tetramers containing either two oL(I)/two [3 subunits or two oL1(II)/ two [3 subunits have similar enzyme activities [33]. Coexpression of recombinant P4HA1, P4HA2, and P4HB in insect cells did not show any tetramers that contained both P4HA1 and P4HA2 protein chains [36]. There appear to be at least four lysyl hydroxylases. The enzyme is also called procollagen-lysine 2 oxoglutarate 5-dioxygenease. Lysyl hydroxylase 1, a 85-kDa membrane-bound homodimeric protein, is localized to the cisternae of the rough endoplasmic reticulum [37]. It hydroxylates specific lysine residues in X-Lys-Gly sequences. Its gene, PLOD, is located on chromosome l p36.3-p36.2 [37]. It is expressed in the skeleton and in many other tissues. Lysyl hydroxylase 2 is encoded by PLOD2, which is located on chromosome 3q23-q24 [38]. It also forms homodimers. It is highly expressed in nonskeletal tissues [39]. Lysyl hydroxylase 3 is encoded by PLOD3, which is located on chromosome 7q36 [40,41]. It is also expressed mostly in nonskeletal tissues. There is
4
William G. Cole
also a putative telopeptide lysyl hydroxylase that is specific for bone. The gene called either B R K S or T L H 1 is predicted to be located on chromosome 17p12 [42]. When lysyl residues become hydroxylated, they may serve as a substrate for a glycosyltransferase and for a galactosyltransferase, which add glucose and galactose, respectively, to the hydroxyl group. These modifications also require the chains to be in the nascent state. Increased levels of glycosylation tend to reduce the size of the collagen fibrils presumably due to interference with packing of the molecules. A mannose-rich oligosaccharide may also attach to an asparagine residue in the C-propetide. Chaperones play an important role in the assembly of procollagens in the rough endoplasmic reticulum. Protein disulfide isomerase, which has both enzymatic and chaperone functions, interacts transiently with procollagen chains early in the procollagen assembly pathway [43,44]. Release of prolyl 4-hydroxylase, including its [3 subunit protein disulfide isomerase, from the triplehelical domain coincides with the assembly of thermally stable triple-0helical molecules. However, if triple helix formation is prevented, prolyl 4-hydroxylase remains associated with the triple-helical domain, suggesting a role for the enzyme in preventing aggregation of this domain. Protein disulfide isomerase is also able to independently associate with the C-propeptide of monomeric procollagen chains prior to trimer formation, indicating a role for this isomerease in coordinating the assembly of heterotrimeric molecules [43]. It is also able to catalyze disulfide bond formation within and between the C-propeptides. Hsp47 is a heat shock protein that resides in the endoplasmic reticulum [45]. It interacts transiently with procollagen during its folding, assembly, and transport from the endoplasmic reticulum of mammalian cells. It has been suggested to carry out a diverse range of functions, such as acting as a molecular chaperone facilitating the folding and assembly of procollagen molecules, retaining unfolded molecules within the endoplasmic reticulum, and assisting the transport of correctly folded molecules from the endoplamic reticulum to the Golgi apparatus. The association of Hsp47 with procollagen coincides with the formation of a collagen triple helix. The importance of Hsp47 in normal type I collagen biosynthesis is highlighted by the phenotype of mice lacking the heat shock protein [45]. The homozygous mice did not live longer than 11.5 days, and their tissues were deficient in collagen fibrils. The findings indicated that type I collagen was unable to form a rigid triplehelical structure without the assistance of Hsp47 and that Hsp47 was essential for normal development. The C-propeptides undergo registration and stabilization by the formation of interchain and intrachain disulfide bonds in the rough endoplasmic reticulum.
The formation of the disulfide bonds is catalyzed by the enzyme protein disulfide isomerase, which is also the [3 subunit ofprolyl 4-hydroxylase. This process is critical for the correct alignment of the main triple-helical domain because the helix winds up from the C terminus. Triple helix formation is initiated in the rough endoplasmic reticulum immediately after the synthesis of the pro-oL chains and after the formation of the interchain disulfide bonds within the C-propeptide [46,47]. It is likely that formation of the triple helix is a posttranslational event because the production of triple-helical molecules requires approximately 8 or 9 min after completion of the synthesis of the pro-oL chains. The helix propagates from a single C-terminal nucleation site toward the N terminus and is interrupted by the random occurrence of peptide bonds in the cis configuration. The C-propeptide and C-telopeptide do not appear to play a role in nucleation of triple helix formation [48]. However, a minimum of two hydroxyproline-containing Gly-X-Y triplets at the C-terminal end of the triple helix are required for nucleation to occur [48]. Direct nuclear magnetic resonance measurements of chick calvarial collagen showed that approximately 16% of the X-Pro and 8% of X-Hyp bonds were cis in the unfolded collagen [49]. Studies by Sarkar et al. and many others have shown that the rate-limiting step in the zipper-like propagation of the helix is the process of cis-trans isomerization [46,47,49,50]. Peptidyl prolyl cis-trans isomerase catalyzes the cis-trans isomerization of X-Pro peptide bonds in collagen [51]. Protein disulfide isomerase is also present in the rough endoplasmic reticulum. However, protein disulfide isomerase does not appear to act as a cis-trans isomerase [51]. Full hydroxylation of proline residues in the Y position of Gly-X-Y triplets also enhances the rate of propagation of the triple helix from the site of nucleation to the N terminus [52]. Biophysical studies using model collagen peptides have also shown that the folding of Gly-X-Y peptides is best described as an all-or-none third-order reaction [53-55]. The formation of the triple helix occurs in the rough endoplasmic reticulum. The type I procollagen molecules move to the Golgi apparatus, where oligosaccharides may be added to a C-propeptide asparagine residue. The molecules are secreted from the cell, during which or soon after the Nand C-propeptides are rapidly cleaved. The N-propeptide is specifically cleaved by procollagen 1 N-endoproteinase. The enzyme is encoded by the A D A M T S 2 gene, which is an abbreviation for a disintegrin-like and metalloproteinase with thrombospondin type I motif [56]. The enzyme exists in a long and a short form as a result of alternative splicing [57]. The C-propeptide is cleaved by procollagen C-endoproteinase, which is the same as bone morphogenetic protein-1 (BMP-1) [58]. The gene is located on
1. Structure of Growth Plate and Bone Matrix
chromosome 8q21 [59]. Procollagen C-endoproteinase is a secreted, neutral zinc metalloproteinase. The Drosophila equivalent gene is called tolloid (TLD). There are two isoforms of the human enzyme as a result of alternative splicing [60]. The long form appears to be an inactive proenzyme that can be activated by removal of the prodomain. There are four mammalian BMP-1/TLD-like proteases [61]. One of them, tolloid-like-1 (TLL1), is also an astracin-like metalloproteinase. Its gene, TLL1, is located on chromosome 4q32-q33 [61]. The activity of procollagen C-endoproteinase is enhanced by procollagen C-endopeptidase enhancer, which is a glycoprotein that binds to the C-propeptide and enhances the activity of the C-proteinase enzyme [62]. Its gene, PCOLCE, is located on chromosome 7q21.3-q22 approximately 6 Mb from the COL1A2 gene that encodes pro-oL2(I) chains of type I procollagen [63]. A second procollagen C-endopeptidase enhancer has been isolated. Its gene, PCOLCE2, is located on chromosome 3q21-q24 [64]. A number of functions have been proposed for the released ~l(I) N-propeptide, including prevention of premature intracellular molecular association, facilitation of transcellular transport and secretion, regulation of extracellular fibrillogenesis, and feedback regulation of procollagen synthesis [65]. However, there is little evidence to support these proposals. For example, deletion of exon 2 of collal in mice, which deleted the 65-amino acid cysteine-rich globular domain of the N-propeptide of pro-e~a(I) chains, did not produce any demonstrable anomalies in type I collagen biosynthesis, collagen cross-linking, or collagen fibrillogenesis [65]. Following removal of the N- and C-propeptides, the type I collagen molecules can self-assemble, cross-link, undergo further growth, and pack into thick collagen fibers. The nucleation steps that initiate the formation of collagen fibrils may commence within crypts on cell surfaces [66,67]. Various biophysical studies as well as rotary shadowing electron microscopy have shown that type I collagen monomers are rod-like structures with a length of approximately 300nm and a diameter of approximately 1.4nm. The overall helical symmetry is lefthanded, with 10 residues in three turns and a pitch of approximately 3 nm. The three helical chains are further coiled about a central axis to form a right-handed helix with a repeat distance of approximately 10nm [68, 69]. The high content of glycine and its occurrence in every third residue of the triple-helical domain give rise to a polymer of tripeptide units with the formula (-Gly-X-Y-)n. Glycine is the smallest amino acid; as such, it is the only amino acid that can pack tightly at the center of the triple-stranded collagen fibril monomers. The side chains of amino acids in the remaining - X - and -Y-positions protrude from the chain, and this arrange-
5
ment allows a variety of amino acid residues to be accommodated in the molecule. The high amino acid content, particularly the high 4-hydroxyproline content, has a stabilizing effect on the triple-helical structure. In type I collagen, the triple-helical configuration occurs throughout 95% of the rod-like monomer. The N- and C-telopeptides do not contain glycine residues at every third position. The long triple-helical domain not only provides the molecule with the stability required for its biomechanical functions but also makes it resistant to enzymatic cleavage apart from specific peptide bonds that can be cleaved by mammalian collagenases. The collagen monomers are able to undergo spontaneous self-assembly into fibrils. The fibrils are crossstriated as a result of the assembly of molecules in a parallel array but with a stagger of approximately onequarter of their length. The periodicity of the crossstriated fibril is a result of each monomer having five highly charged regions at approximately 67-nm intervals. The repeat period, called a D period, is approximately 67 nm in length and contains 234 amino acids. The overall length of the collagen fibril monomer is 4.4 D units, which also corresponds to 300 nm. Because of the nonintegral length of the monomers, overlapping by D divides the fibril into overlap zones that include the N and C termini of the molecules and gap zones that do not. The quarter-stagger arrangement of the collagen molecules provides the appropriate substrate conformation for the action of lysyl oxidase. The enzyme, which is a copper-dependent amine oxidase, requires molecular oxygen for activity. It acts on specific lysine and hydroxylysine residues to produce the corresponding aldehydes that are required for the formation of covalent collagen cross-linkages. The enzyme, which is also called proteinlysine 6 oxidase, is encoded by LOX, which is located on chromosome 5q23.3-q31.2 [70]. Alternative splicing produces three m R N A isoforms [70]. There are also two lysyl oxidase-like loci. LOXL1 is located on chromosome 15q22 and LOXL2 on chromosome 8p21-p21.2 [71,72]. The lysine aldehyde pathway occurs primarily in adult dermis, cornea, and sclera, whereas the hydroxylysine aldehyde pathway predominates in bone, ligaments, tendons, and embyronic dermis. The first step in both pathways is the oxidative deamination of the e-amino group in telopeptide lysine and hydroxylysine residues to form their corresponding aldehydes, called allysine and hydroxyallysine, respectively. In the lysine aldehyde pathway, two allysines may condense to form the aldol condensation product, which forms intramolecular bonds. Aldimine cross-links are formed when allysine in the telopeptides reacts with lysine or hydroxylysine residues in adjacent helices to provide covalent intermolecular cross-linkages. In the hydroxylsine aldehyde pathway, hydroxyallysine can condense with an
6
William G. Cole
hydroxylysine residue to form a reducible cross-link that can undergo an Amadori rearrangement to form hydroxylysino-5-oxo-norleucine. The hydroxylyinsederived aldimine cross-links can also occur as galactosyl or glucosylgalactosyl derivatives. In the hydroxylysine aldehyde pathway, the major mature cross-link is based on trivalent 3-hydroxypyrididinium residues [73]. It includes hydroxylysyl-pyridinoline, derived from three hydroxylysine residues, and lysyl-pyridinoline, derived from two hydroxylysine residues and one lysine residue. These two cross-links are naturally fluorescent and can be assayed directly in tissue hydrosylates as well as in blood and urine. The formation of collagen fibrils has been studied extensively. Of particular interest are in vitro studies that use intact procollagen as well as procollagen lacking either the N-propeptide (pC) or C-propeptide (pN) with fibril formation initiated by the addition of specific N- and C-proteases [74,75]. This approach leads to the formation of fibril-like structures with the characteristic collagen D-periodic banding pattern but with a distinctive, bipolar needle-like morphology that is different from that of fibrils isolated from native tissue [67,75]. These fibril structures show a single polarity reversal where the orientation of the collagen is reversed with amino termini at both ends of the fibrils [75]. Newly formed fibrils with characteristic D periodicity have been isolated from various embyronic tissues [76]. These fibrils also frequently show a single polarity reversal [77]. Fibrils may increase in size by the fusion of small (1-10 ~m) segments, and the lateral association of long or short fibrils may lead to thicker fibrils [76,78]. The growth of type I collagen fibrils appears to be partly regulated by other collagen molecules that are included within the heterotypic fibers [79]. For example, other collagens in the heterotypic type I collagen fibrils of dermis include types III, V, XII, and possibly XIV collagen [80]. In bone, the other collagens are type V and type V/XI hybrid molecules [81]. It is likely in these various types of heterotypic type I collagen fibrils that the N-propeptides that remain attached to the collagens, other than type I collagen, play a role in regulating the growth of the fibrils [80]. A number of other molecules, including the small leucine-rich proteoglycans such as decorin, fibromodulin, lumican, as well as hyaluronan, also appear to regulate the growth of fibrils [82]. The molecular packing of collagen fibrils has been determined mainly in tissues such as tendon. X-ray diffraction studies indicate the presence of threedimensional crytallinity admixed with liquid-like lateral order [83,84]. The lateral unit cell, which contains five molecules in cross section, gives rise to row lines with a maximum spacing of 3.8 nm. Electron microscopy of a transverse section of tendon fibrils reveals a similar
periodicity (~4 nm) orientated radially with respect to the fibril center. A feature of the model is that molecules are tilted obliquely in a plane orientated 30 ~ to the fibril surface [84], which results in the helicoidal organization of collagen fibrils. An additional feature of the model is that the fibril surface is coated in molecular ends, which has important consequences for fibril growth. For example, the persistence of the N-propeptides of types III, V, or XI collagen might prevent their incorporation into the center of the fibril, thereby forcing all N termini to the surface of the fibril, with prevention of further accretion and limiting fibril diameter [83]. An alternative molecular packing model is the fivestranded Smith microfibril [84]. The microfibril, with a diameter of approximately 4 nm, is the minimum filamentous structure that possesses an axial D repeat. Although their existence is still debated, evidence indicates that they do exist [83]. Three-dimensional image reconstructions of 25-nm diameter collagen fibrils show evidence of a 4-nm repeat in transverse section, which might correspond to ordered arrays of microfibrils, particularly at the level of gap-overlap junctions. Bone and other connective tissues have distinctive collagen fiber sizes and distinctive suprafibrillar architectures, as seen by polarized light microscopy [85]. In woven bone, the collagen fibrils are randomly distributed. Lamellar bone contains collagen fibrils that are arranged in parallel layers or sheets running in different directions. Bone osteons have a lamellar structure in which the lamellae are arranged in concentric cylinders. TYPE V COLLAGEN Type V collagen was first identified in human placenta and dermis, but later studies showed that it is widely expressed in type I collagen-containing tissues including bone [86,87]. The type V collagen molecules exist as heterotrimers Otl(V)2ot2(V) or Otl(V)otz(V)ot3(V ) and as homotrimers ~1(V)3 [88,89]. An apparently distinct cx4(V) chain is synthesized by Schwann cells [90]. Type V collagen chains also form heterotypic molecules with type XI collagen chains. For example, the highly homologous OLl(V) and oLI(XI) chains may yield an oL1(V)oLI(XI)oL2(V) trimer in bone and cartilage [81]. The following description of type V collagen is limited to the Otl(V ) and ot2(V ) chains because the oL3(V)and 0/.4(7 ) genes are not expressed in bone or cartilage, although the e~3(V) gene is expressed in ligament attachments to bone [91]. The gene for the oLI(V) chain, COL5A1, is located on chromosome 9q34.2-q34.3 [92]. The gene has 66 exons, more than the number of exons in types I and II collagen. Exon 1 encodes the signal peptide of 36 amino acid residues and one base of the N-propeptide. Exons 2-14
1. Structure of Growth Plate and Bone Matrix
encode the remainder of the N-propeptide, with exon 14 being a junctional exon that encodes the end of the N-propeptide and the beginning of the triple-helical domain. The N-propeptide contains 505 amino acid residues and the N-telopeptide contains 17 residues. The pro-eta(V) N-propeptide is similar in size and domain structure to the N-propeptides of the pro-etl(XI) and pro-etz(XI) chains [93-95]. The N-propeptides of the latter three chains all contain a very large globular domain, immediately downstream of the signal peptide, that is much larger than and has no apparent homology to the cysteine-rich globular domains of the types I-III collagen. The globular domains of the pro-etl(V), proetl(XI), and pro-etz(XI) chains are bisected by a cluster of two cysteines into a basic N-terminal subdomain and a C-terminal subdomain rich in acidic residues and tyrosines [93-96]. The latter region contains 27 tyrosine residues and 73% of the total number of tyrosine residues in the pre-pro-~l(V) chain. Approximately 40% of the N-propeptide tyrosine residues are sulfated [97]. The N-terminal subdomain is analogous to the thrombospondin 1 motif and is identical to the proline- and arginine-rich peptide (PARP) motif [98]. The derived three-dimensional structure of PARP suggests a conserved nine [3-stranded structure [99,100]. The PARP motif is also present in the N-propeptides of the proetl(XI) and pro-etz(XI) chains but not in the pro-etz(V) chains. The globular domain of the N-propeptide of the pro-etl(V) chain is followed by an interrupted triple-helical domain of 25 Gly-X-Y repeats and then another short noncollagenous region prior to the main triple-helical domain. Pro-etl(V), pro-etl(XI), and proetz(XI) sequences share similarities in all subdomains of their N-propeptides, with the exception of the acidic globular variable region [94]. There is no evidence of alternative splicing in the N-propeptide of pro-etl(V) chains, although it occurs in the pro-etl(XI) and proetz(XI) N-propeptides [93]. There are two hypothetical N-proteinase cleavage sites (Ala-Gln) at positions 541-542 and 546-547. The main triple-helical domain of etl(V) chains is similar to those of types I-III collagens in that each nonjunctional exon begins with a complete codon for glycine and ends with a complete codon for a residue in the Y position of Gly-X-Y triplets [92,101]. The codons are commonly 45 or 54 bps. The main triple-helical domain is encoded by exons 14-62 [92]. These exons encode a main triple-helical domain of 1014 amino acid residues. Lysine residues at positions 642 and 1482, which are important for intermolecular cross-linkages, are preserved. The helical domain lacks the Gly-Ile/Leu mammalian cleavage site at position 775-776 of types I-III collagens [102]. The absence of this sequence explains the lack of cleavage of the otl(V) chains by mammalian collagenase.
7
Carboxyl terminal of the main triple helix is the C-telopeptide of 33 amino acid residues and the C-propeptide of 233 residues. They are encoded by exons 62-66. The putative C-proteinase cleavage site is located at position 1605-1606 (Ala-Asp). The C-propeptide has a high homology with the same region of pro-eta(XI) chains. Seven cysteine residues and their vicinities are conserved. The pro-et chains that can form homotrimers or both homo- and heterotrimers of pro-et chains have 8 cysteine residues, but the pro-et chains that form heterotrimers only have 7 cysteine residues [12]. The C-propeptide of pro-etl(V) chains has 8 cysteine residues, whereas the C-propeptide of the pro-etl(XI) chains has 7 cysteine residues [103]. The pro-etl(V) chain contains numerous sites for potential attachment of N-linked oligosaccharides. It has a heparin binding site at residues 897-929 [104]. Heparin binding sites are also present in a l(XI) and etz(XI) chains but not etz(g) chains [105]. Binding of chondroitin sulfate E to type V collagen may facilitate cell binding and matrix assembly [106]. There are RGD cell attachment sites at positions 645-647 and 663-665. The gene for the etz(V) chain, COL5A2, resides on chromosome 2q14-q32 [107,108]. The gene, which spans approximately 67 kb, is located in a tail-to-tail orientation with the COL3A1 gene. The intergenic distance is approximately 22 kb. The two genes contain 51 exons and share almost identical structures. The pro-etz(V) N-propeptide is encoded by 4 complete exons and partially by the junctional exon. It encodes a pre-pro-etz(V) chain of 1496 amino acid residues [109,110], and it includes a signal peptide of 26 residues. The N-propeptide of 167 residues includes an amino-terminal globular subdomain of 82 residues, in which there is a von Willibrand type C-like repeat. This subdomain contains a central cluster of 10 cysteine residues flanked on both sides by short, hydrophilic sequences. It is similar to the equivalent subdomain of the N-propeptide of pro-etl(I) chains but dissimilar to the equivalent subdomain of the pro-etl(V) chain [101,111]. The N-propeptide also includes an interrupted helical subdomain of 78 residues and a nonhelical subdomain of 7 residues. The N-proteinase cleavage site is at the Asn-Gln bond at position 193-194 of the full-length chain. The N-telopeptide contains 15 residues, including the lysine cross-linking site at residue 175. The main triple-helical domain contains 1017 residues and is followed by a C-telopeptide of 26 residues and a C-propeptide of 246 residues. The C-proteinase cleavage site is located at the Gly-Asp bond at position 1250-1251. There are seven R G D sequences that are potential cell binding sites. There are several sites for the attachment of N-linked oligosaccarides. Also, there are 3 cysteine residues, at positions 1293, 1299, and 1325, for intermolecular
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William G. Cole
cross-linking and sites for intrachain disulfide cross-links at positions 1333-1494 and 1402-1447. In contrast to type I procollagen processing, in which the N- and C-propeptides are rapidly cleaved following secretion, type V procollagen molecules retain their N-propeptides. A variety of type V collagen molecules retaining all or parts of the N-propeptides have been extracted from tissues and from tissue culture medium [99,112,113]. Rotary shadowing confirmed the retention of the N-propeptides on some of the type V collagen molecules extracted from tissues [114]. Nonetheless, within pro-e~l(V)zpro-c~z(V) heterotrimers, some proOtl(V) N-propeptides and pro-oLz(V) C-propeptides are enzymatically removed by bone morphogenetic proteinl-like enzymes. PrO-al(V) C-propeptides are processed by furin-like proprotein convertases in vivo [115]. When type V collagen epitopes are unmasked in tissues, this collagen is found to colocalize with type I collagen [116]. The exact spatial relationship between these collagens in the type I collagen fibrils is unclear. However, copolymeric assembly is likely because crosslinkages between types I and V collagens have been isolated from bone [117]. It is also likely that type V collagen regulates the formation of the type I collagen fibrils [118]. Type V collagen molecules are capable of forming homotypic fibrils in vitro with or without an apparent 67-nm cross-striation pattern [119]. Increasing the quantity of type V collagen relative to type I collagen decreased the final fibril diameter [120]. It has been proposed that the retained N-propeptides of type V collagen molecules protrude from the surface of the type I collagen fibrils, where they regulate the growth of the fibrils [79,120]. Confirmation of the importance of the aminoterminal extension of the e~z(V) chain was provided by the phenotype of mice in which the col5a2 gene was engineered to lack exon 6, which normally encodes the N-telopeptide of Cxz(V) chains. The mice showed abnormal type I collagen fibrillogenesis in the dermis [121]. Although type V collagen, with the exception of its N-propeptides, is buried with the type I collagen fibrils, its main triple-helical domain is known to bind to thrombospondin, heparin, heparan sulfate, decorin, and biglycan [104,122-125]. Type V collagen contains seven R G D sequences on the OL2(V) and two on the OLI(V) chains that may enable type V collagen to attach to various cell types. These interactions may involve the OLl[31and az[31integrins [126].
TYPE II COLLAGEN Type II collagen is the main fibrillar collagen of growth plate cartilage and other cartilages. It is also
found in the vitreous humor, intervertebral disks, and inner ear [127]. Its importance is clearly evident from the severe lethal chondrodysplastic phenotype in mice that lack type II collagen [128,129]. There is gross disorganization of the cartilages with abnormal architecture of the chondrocytes and extracellular matrix. There is ectopic expression of types I and III collagens in the cartilage. The skeleton demonstrates membranous and periosteal bone but no endochondral ossification [128]. Type II collagen exists in two molecular forms. The first form is a homotrimeric type II collagen, [OLl(II)]3, which is largely found as the main component of the thickest collagen fibrils of cartilage. The second form is a component of heterotrimeric type XI collagen with a chain composition [txl(XI)oL2(XI)ota(XI)], in which the Ota(XI) chains are an overhydroxylated form of OLl(II) chain [130]. Type XI collagen is described separately. Type II collagen is encoded by the COL2A1 gene on chromosome 12ql 3.11-q 13.2 [127,131]. The gene structure and cDNA sequence show that COL2A1 and COL1A1 are homologous genes, even thought their expression patterns and molecular chain compositions are different [132,133]. Many aspects of type II collagen biosynthesis are the same as those described for type I collagen. Consequently, the following account of type II collagen biosynthesis focuses on specific differences between the biosynthesis of the two collagens. The COL2A1 gene contains 53 exons and encodes two alternatively spliced mRNAs and protein isoforms, referred to as type IIA and type IIB collagens [131,134,135]. Type IIA collagen is the long form, which includes a 69-amino acid cysteine-rich region of the N-propeptide that is encoded by exon 2 [134]. Type IIB collagen lacks the exon 2-encoded sequence [133]. The full-length or type IIA form of the protein has 1487 amino acid residues. It includes a signal peptide of 25 amino acid residues, an N-propeptide of 156 residues, an N-telopeptide of 19 residues, a main triple-helical domain of 1014 residues, a C-telopeptide of 27 residues, and a C-propeptide of 246 residues [133,134]. There are allysine cross-linking precursor residues at positions 190 and 1231, which are within the N- and C-telopeptides, respectively. The N-proteinase cleavage site to remove the N-propeptide is at the Ala-Gln bond 181-182. Potential sites for the hydroxylation and glycosylation of lysine residues are present at positions 287 and 1130. The mammalian collagenase cleavage site is at the Gly-Ile bond at position 834-835. Proline 1186 can be 3-hydroxylated. The C-proteinase cleavage site is at the Ala-Asp bond 1241-1242. Interchain disulfide bonds utilize the cysteine residues at positions 1283, 1289,1306, and 1315. Intrachain disulfide bonds occur between cysteine pairs 1323-1485 and 1393-1438. There is a putative Asn 1388 N-linked oligosaccharide attachment
1. Structure of Growth Plate and Bone Matrix
site. The short type IIB form of the protein contains 1418 amino acid residues due to the loss of the 69 residues encoded by exon 2 [133,134]. The type IIB isoform, lacking exon 2-encoded sequences, is the adult form of the protein that is found as the predominant isoform in the growth plates, other cartilages, vitreous humor, and the inner ear. The type IIA isoform, containing the exon 2-encoded sequence of 69 amino acid residues and rich in cysteine residues, appears to play an important role in the normal development of skeletal and nonskeletal tissues. Many studies have shown that the type IIA isoform is expressed in prechondrocytes, whereas the type IIB form is expressed by chondrocytes. During elongation of the growth plate, mature chondrocytes express the type IIB isoform and then differentiate into hypertrophic chondrocytes and initiate expression of type X collagen. In contrast, notochordal remnants and the inner annulus fibrosus of the vertebral column continue to express the type IIA isoform. However, processing of the N-propeptide of the type IIA isoform appears to be developmentally regulated in the latter tissues. In early fetal development, the N-propeptide remains attached to the main protein chain, whereas in later fetal development the N-propeptide is cleaved. In other prechondrogenic tissues, however, the N-propeptide appears to remain attached to the main type II collagen chain. In solid phase assays, transforming growth factor-j31 (TGF-[31) and BMP-2 bound to the type IIA isoform but not to the type IIB isoform. Antibodies to BMP-2 also immunoprecipitated the type IIA isoform from tissue culture medium. Consequently, the role of the cysteine-rich domain of the N-propeptide in development may, at least in part, be due to its ability to regulate the availability of specific growth factors. The removal of the N- and C-propeptides of types I and II collagens occurs by similar mechanisms. Cleavage of the N-propeptides involves the procollagen N-proteinase ADAMTS2, and cleavage of the C-propeptides involved the procollagen C-endopeptidase BMP-1 and.the procollagen C-endopeptidase enhancer [57,58,62]. Other studies indicate, however, that many other cleavage enzymes and other cleavage sites may be involved in the processing of type II procollagen. Studies of the processing of the N-propeptides of fibrillar procollagens in cattle lacking ADAMTS2 activity showed that the N-propeptides of type I procollagen in the skin were not processed, whereas the N-propeptides of type II procollagen in cartilage were processed normally [136]. The possible existence of other ADAMTS enzymes in cartilage, as an explanation for the latter findings, was investigated in Swarm rat chondrosarcoma RCS-LTC cells, which fail to process the N-propeptides of type II procollagen. Stable transfections of the cells with bovine A D A M T S 2 or human A D A M T S 3 cDNAs partially rescued the
9
processing defect. Normal human skin and skin fibroblasts showed 30-fold higher mRNA levels of ADAMTS2 than A D A M T S 3 . In contrast, A D A M T S 3 mRNA levels were 5-fold higher than A D A M T S 2 mRNA levels in normal human cartilage. Taken together, these findings explain the lack of N-propeptide processing of type I procollagen in skin and the normal processing of type II procollagen in cartilage in animals lacking ADAMTS2 activity. Because ADAMTS3 is normally more abundant that ADAMTS2 in cartilage, it is likely that ADAMTS3 is the physiologically more important enzyme [136]. Some matrix metalloproteinases (MMPs) are also able to cleave at various sites in the N-propeptides and N-telopeptides of type II procollagen [137]. Recombinant trimeric type IIA N-propeptides were exposed to MMP- 1-3, 7-9, - 13, and - 14 [138]. MMP-1, -2, and -8 did not show cleavage, whereas MMP-3, -7, -9,-13, and -14 cleaved at the procollagen N-proteinase cleavage site and in the N-telopeptide. All of the latter enzymes cleaved in domains common to both type IIA and type IIB procollagen, whereas MMP-7 a n d - 1 3 also cleaved in the type II-specific minor helix and MMP-7 cleaved in the type IIA-specific cysteine-rich domain. MMP-7 was also able to remove the N-propeptides from collagen fibrils in the extracellular matrix of fetal cartilage. These findings indicate that some of the MMPs may play an in vivo role in the processing of the N-propeptides of type II procollagen. MMP-3,-9, and -14 are expressed during the early stages of cartilage development. MMP-9 and -13 appear to play a role in the cartilage- to-bone transition in the growth plate. MMP-14 is also expressed in hypertrophic chondrocytes. Cleavage of the N-propeptides and N-telopeptides is likely to contribute to the normal processing and turnover of type II procollagen. In addition, release of the N-propeptides from the type IIA isoform is likely to provide growth factors such as TGF-[31 and BMP-2. The C-propeptide is found in high concentrations in all cartilages including the growth plate [139]. It was initially isolated as a calcium-binding protein from developing growth plate cartilage, particularly from the lower hypertrophic zone. Amino acid sequencing showed that the protein, which was called chondrocalcin, was the C-propeptide of type II procollagen [140]. The main triple-helical domain of type II collagen is susceptible to cleavage by mammalian collagenases at the Gly-Ile bond at position 834-835. The released fragments are readily degraded to small peptides [141]. This finding is in contrast to the resistance of native types IX and XI collagens, isolated from hyaline cartilage, to digestion with mammalian collagenase [141,142]. This observation also applies to the oL3(XI) chain of type XI collagen, which is an overglycosylated form of oLI(II) chain.
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William G. Cole
Following removal of the N- and C-propeptides, the OLl(II) chains self-assemble, like type I collagen chains, into a quarter-staggered array. Lysyl oxidase-derived cross-linkages form to stabilize the intramolecular and intermolecular interactions. The native molecular packing of type II collagen differs from that of type I collagen [143]. Low-angle X-ray diffraction studies showed that in wet, native type II collagen, the molecules are spaced farther apart than in type I collagen under the same conditions. The type II collagen fibrils are more hydrated than type I collagen fibrils. The additional hydration may enable cartilage to better dissipate compressive loads. The higher content of glycosylated hydroxylysine residues in the type II collagen is likely the main factor modulating the fibrillar hydration. There is considerable variation in the diameter of the collagen fibrils in growth plate and hyaline cartilages. They vary from fine fibrils of approximately 25 nm in fetal cartilage and in parts of the growth plate to fibrils of approximately 30-200 nm in other parts of the growth plate and in articular cartilages [144]. The thin fibrils contain types II, IX, and XI collagens, whereas the large fibrils contain mainly types II and IX collagens. It is likely that the amount of type XI collagens as well as the small leucine-repeat proteoglycans influences the diameters of the heterotypic type II collagen fibrils [144]. Type II collagen fibrils are strengthened by covalent intermolecular bonds provided by 3-hydroxypyridinium cross-linking residues [145]. Purification and sequencing of the cross-linkages identified two hydroxypyridinium cross-linking sites within the triple-helical region of the type II collagen molecule. They were placed symmetrically at opposite ends at residues 87 and 930 of the al(II) chains, where telopeptide aldehydes are known to react to form the initial "head-to-tail" intermolecular bonds. The cross-linkages between type II collagen and other types of collagen in the heterotypic type II collagen fibrils of cartilage are described later.
TYPE IX COLLAGEN Type IX collagen, which belongs to a group of fibrilassociated collagens with interrupted helices (FACIT), is a component of hyaline cartilages, intervertebral disks, vitreous humor, and the inner ear [146]. In these locations it is associated with types II and XI collagens. The type IX collagen molecule is a heterotrimer consisting of three genetically distinct chains, ~I(IX)-c~3(IX), and possesses three collagenous domains, numbered COL 1-COL3 from the C terminus, flanked by four noncollagenous domains, NC1-NC4 [147]. Type IX collagen
is also a proteoglycan because of a glycosaminoglycan side chain covalently attached to the NC3 domain of the et2(IX) chain [148]. The Ul(IX) chain is encoded by the COL9A1 gene located on chromosome 6q12-q14 [149-151]. Three FACIT collagen genes, COL9A1, COL12A1, and COL19A1, are located close together on chromosome 6q12-q13 [152]. The COL9A1 gene is approximately 90kb and contains 38 exons [151]. It encodes two alternatively spliced m R N A and protein isoforms referred to as a long form of 931 amino acid residues and a short form of 688 residues [153]. The long form or isoform 1 contains a signal peptide of 23 amino acid residues, an NC4 domain of 245 residues, a COL3 domain of 137 residues, an NC3 domain of 12 residues, a COL2 domain of 339 residues, an NC2 domain of 30 residues, a COL1 domain of 115 residues, and an NC1 domain of 30 residues. The overall size of the mature chain is 908 residues. There are 4 cysteine residues in the NC4 domain at positions 44,198,242, and 252. There are also 2 cysteine residues in the NC3 domain at positions 411 and 415. There is a putative N-linked oligosaccharide attachment site at A s n 171 in the NC4 domain. The NC4 domain has a calculated isoelectric point of 9.7. The short form of Otl(IX) or isoform 2 contains an NC4 domain, including the signal peptide, of 25 amino acid residues in comparison with the equivalent region of the long isoform that contains 268 residues [153]. The remainder of the amino acid sequences of the long and short isoforms are the same. The long transcript contains the 5' region encoded by exons 1-7 spliced to exon 8. The short transcript contains the 5' region encoded by an alternative exon 1" located in the intron between exons 6 and 7 and spliced to exon 8 [151,153]. The Otz(IX ) chain is encoded by the COL9A2 gene located on chromosome lp33-p32.2 [151,154,155]. The gene is approximately 15 kb and contains 32 exons [151]. There is one transcript that encodes a full-length protein of 689 amino acid residues and a mature protein of 666 residues [154]. The protein contains a signal peptide of 23 amino acid residues, an NC4 domain of 3 residues, a COL3 domain of 137 residues, an NC3 domain of 10 residues, a COL2 domain of 339 residues, an NC2 domain of 31 residues, a COL1 domain of 115 residues, and an NC1 domain of 25 residues. The glycosaminoglycan attached to Ser 169 is in the sequence Gly-Ser-Ala-Asp within the NC3 domain. Direct analyses of type IX collagen extracted from cartilages have shown that up to 70% of the molecules contain a chondroitin sulfate glycosaminoglycan side chain attached to Ser 169 [156]. There are 4 cysteine residues--2 at residues 174 and 178 of the NC3 domain and 2 at residues 664 and 669 of the NC1 domain.
1. Structure of Growth Plate and Bone Matrix
The c~3(IX) chain is encoded by COL9A3, which is located on chromosome 20q13.3 [157,158]. The gene is approximately 23 kb and contains 32 exons [157]. It encodes a single protein chain containing 684 amino acid residues [159]. The full-length ot3(IX) chain contains a signal peptide of 25 amino acid residues and a mature protein chain of 657 residues. The mature chain contains an NC4 domain of 3 amino acid residues, a COL3 domain of 137 residues, an NC3 domain of 15 residues, a COL2 domain of 339 residues, an NC2 domain of 31 residues, a COL1 domain of 112 residues, and an NC1 domain of 22 residues [159]. The domain and exon organization of COL9A3 is almost identical to that of COL9A2. However, exon 2 of the COL9A3 gene codes for one Gly-X-Y triplet less than exon 2 of COL9A2. The difference is compensated for by an insertion of 9bps coding for an additional triplet in exon 4 of COL9A3. As a result, the number of Gly-X-Y triplets in the COL3 domain is the same in both protein chains and consequently the triple helix is in register. However, the COL1 domain of the ot3(IX) chain is one triplet shorter than the corresponding domain in oLI(IX) and Otz(IX) chains. It is likely that the NC2 domain can accommodate this difference so that the register is maintained correctly for the formation of the triple helix involving the COL2 domains. Although there are many possible chain combinations for the formation of type IX collagen, the molecules extracted from the medium of cultured cells and from tissues appear to be limited to disulfide-bonded molecules containing o~I(IX)oLz(IX)oL3(IX) [146,147]. Studies of the molecular assembly of type IX collagen using recombinant chains confirm that otl(IX)oLz(IX)ot3(IX) is the favored heterotrimer. However, the chains are also able to form disulfide-bonded heterotrimers of oLI(IX) and oL3(IX ) chains as well as OLl(IX)3 , OLz(IX)3, and oL3(IX)3 homotrimers [160]. The information required for chain selection and assembly is present in the NC1 domain and the adjoining region of the COL1 domain of the type IX collagen chains. When this region of type IX collagen was prepared from tissues reduced and denatured, three types of disulfide-bonded molecules formed: Otl(IX)3, Otz(IX)3, and OLI(IX)oL2(IX)oL3(IX)[161]. Rotary shadowing electron microscopy has shown that the type IX collagen molecule is approximately 190 nm long with a globular N-terminal end representing the large NC4 domain of the C~l(IX) chain [146]. The molecule also shows a characteristic kink located at the NC3 domain. Disulfide bonds and the glycosaminoglycan side chain are also located at the NC3 domain, and additional disulfide bonds are located at the junction of the NC1 and COL1 domains. Although the heterotypic type II collagen fibrils in cartilages vary considerably in diameter, fibrils of all diameters show type IX collagen
11
distributed along their outer surfaces in a D-periodic pattern [146]. The amount of type IX collagen on individual type II collagen fibrils varies considerably and does not appear to correlate with the diameter of the fibrils. This latter finding is consistent with the observation that purified type IX collagen did not inhibit formation of large fibril-like structures formed by type II collagen in reconstitution experiments in vitro [146]. Therefore, it appears unlikely that type IX collagen, in contrast to type XI collagen, directly controls the lateral growth of the heterotypic type II collagen fibrils. The latter proposal is also consistent with findings in cell lines and mice lacking oLI(IX) chains of type IX collagen. In both instances, type IX collagen molecules did not form, although the COL9A2 and COL9A3 genes were transcribed [162-164]. In mice lacking oLI(IX) chains and type IX collagen molecules, it appeared that type IX collagen was not essential for skeletal morphogenesis or proper fibril formation [163]. However, it is necessary for the functional and mechanical stability of the weight-bearing articular surfaces, presumably reflecting the need for interactions between type IX collagen and other extracellular matrix components. The mice appeared normal at birth but developed premature osteoarthritis [163]. Type IX collagen is extensively cross-linked in cartilage [165]. Most of the cross-linking residues in mature adult cartilage are trivalent pyridinolines. The finding of cross-linkages between type IX and type II collagen molecules is in keeping with electron microscopic images of type IX collagen molecules decorating the surface of type II collagen fibrils [146]. In addition, the divalent reducible cross-links, dihydroxylysinonorleucine and hydroxylysinonorleucine, are also found in type IX collagen. Each of the type IX collagen chains contains a site of cross-linking at the N-terminus of the COL2 triple helix to which the OLl(II) N-telopeptide can bond. The oL3(IX) COL2 domain also has an attachment site for the OLl(II) C-telopeptide. The distance between the C~l(II) N-telopeptide and the C~l(II) C-telopeptide interaction sites, 137 residues, is equal to the length of the gap region (0.6 D) in a type II collagen fibril. These findings imply an antiparallel type II-to-type IX cross-linking relationship [165]. With this arrangement, it is likely that the glycosaminoglycan side chain protrudes into the gap region and that the short COL3 domain and the basic globular NC4 domain project from the surface of the fibril [166].
TYPE X! COLLAGEN Type XI collagen is a component ofheterotypic type II collagen fibrils in cartilage, and the type V/XI hybrid
12.
William G. Cole
collagen molecule is a component of heterotypic type I collagen fibrils in bone and many nonskeletal tissues [167,168]. In cartilage, it assembles with types II and IX collagens to produce an extensive network of thin, heterotypic collagen fibrils 15-25 nm in diameter [169,170]. In noncartilaginous tissues, the component chains, particularly the pro-oLa(XI) chains, take part in the formation of hybrid type V/XI molecules that coassemble with type I collagen to produce the collagen fibrils of bone and other nonskeletal tissues [171,172]. Type XI collagen is a heterotrimeric molecule consisting of c~I(XI)-oL3(XI) chains [173]. The oL3(XI) chain is an overglycosylated form of type II collagen, whereas the oLI(XI) and ~2(XI) chains are the products of distinct genes. All three chains are important in the formation of the type XI collagen molecule because the absence of any one of them results in spondyloepiphyseal dysplastic phenotypes of varying severities. Lack of the e~l(XI) chain in the cho/cho mouse results in a lethal phenotype with disorganization of the cellular and extracellular matrix architecture of the cartilages [174]. The composition of type XI collagen in these mice is not known. Consequently, it is unclear whether the oLI(V) chain substitutes for the oLI(XI) chain in type XI collagen molecules. A milder chondrodysplastic phenotype was observed in mice that lacked oL2(XI) chains. This phenotype appeared to be due to the ability of the oLI(XI) chains to partially rescue the phenotype by forming stable triple-helical molecules in cartilage, although the chain composition of such molecules is unclear [175]. Mice lacking the OLl(II) chain also have a lethal chondrodysplastic phenotype [128]. The type XI collagen molecules, containing only the oLI(XI) and Otz(XI) chains, were unstable [129]. The oLI(XI) gene, C O L l l A 1 , is located on chromosome lp21 [176]. The C O L l l A 1 gene contains 69 exons and encodes a procollagen chain that resembles the proe~l(V) chain. There are three major human pro-oLl(XI) isoforms. The longest isoform, isoform 2, contains 1818 amino acid residues [103]. It includes a signal peptide of 36 residues, an N-propeptide of 487 residues, an N-telopeptide of 16 residues, a main triple-helical domain of 1014 residues, a C-telopeptide of 23 residues, and a C-propeptide of 241 residues. The N-propeptide of the pro-~l(XI) chain contains a proline- and arginine-rich protein (PARP) domain followed by a variable region, a constant region, and a minor triple helix. Alternative splicing within the variable region in humans involves exon 6, which exists as exon 6A encoding 39 amino acid residues and as exon 6B encoding 51 residues [177]. The exon 6A-encoded sequence is highly acidic, with an estimated isoelectric point of 3.2 [177]. In contrast, exon 6B encodes a highly basic amino acid sequence, with an estimated isoelectric point of 10.6
[177]. The three isoforms are isoform 1, which uses exon 6A, isoform 2, which uses exon 6B, and isoform 3, which uses neither exon 6A nor exon 6B. It is unclear whether other isoforms exist in humans, and little information is available about the temporal and spatial expression of them. At least in chick chondrocytes, the different splice variants of pro-oLl(XI) collagen chains undergo uniform amino-terminal processing [178]. More information is available concerning the alternative splicing of the C O L l l A 1 transcript in chick, mouse, and rat tissues and cultured cells [179-182]. In mice, exons 6A, 6B, 7, and 8 of the colllal gene encode the variable region of the amino-terminal domain [183]. A complex pattern of alternative splicing occurs that is both tissue dependent and developmentally regulated. Expression of colllal is predominantly associated with cartilage, in which it plays a critical role in skeletal development. At least five splice forms (6B-7-8, 6A-7-8,7-8,6B-7, and 7) are found in cartilage. Splice forms containing exons 6B or 8 have distinct distributions in the long bone during development, whereas in noncartilage tissues splice form 6A-7-8 is typically expressed. To study the process in more detail, a minigene containing exons 5-11 was transfected into chondrocytic (RCS) and nonchondrocytic (A204) cell lines that endogenously express oLI(XI) chains as well as into 293 cells that do not express oLI(XI) chains. Alternative splicing in RCS and A204 cells reflected the appropriate cartilage and noncartilage patterns, whereas 293 cells produced only the 6A-7-8 isoform. This finding suggested that 6A-7-8 is the default splicing pathway and that cell-or tissue-specific trans-acting factors are required to obtain the pattern of the alternative splicing of Otl(XI) premRNA observed in chondrocytes. Deletional analysis revealed cis-acting regions important for regulating splicing. The presence of the intact exon 7 was required to generate the full complex chondrocytic pattern of splicing. Deletional mapping of exon 6B revealed sequences required for expression of exon 6B in RCS cells, and these may correspond to purine-rich (ESE) and AC-rich (ACE) exonic splicing enhancers. Immunohistochemical studies have also provided new insights into the temporal and spatial expression of the oLI(XI) isoforms in rats [181]. Antibodies to the basic peptide p6b (encoded by exon 6b) and the acidic peptide p8 (encoded by exon 8) localized these epitopes to the surface of the collagen fibrils and were used to determine the pattern of isoform expression during the development of the rat humerus [181]. Expression of the p6b isoform was restricted to the periphery of the cartilage underlying the perichondrium of the diaphysis, a pattern that appears de novo on Embryonic Day (E) 14. p8 isoforms appeared to be associated with early stages of chondrocyte differentiation and were detected
1. Structure of Growth Plate and Bone Matrix
throughout prechondrogenic mesenchyme and immature cartilage. After El6, p8 isoforms gradually disappeared from the diaphysis and then from the epiphysis preceding chondrocyte hypertrophy, but they were highly evident at the periarticular joint surface, where ongoing chondrogenesis accompanies the formation of articular cartilage. The spatially restricted and differentiationspecific distribution of Otl(XI) isoforms suggests that the isoforms perform distinct functions in skeletal development that may go beyond their role in collagen fibrillogenesis. The C O L l l A 2 gene that encodes the pro-oLz(XI) collagen chain resides on chromosome 6p21.3 [184]. It spans approximately 30.5 kbs and contains 66 exons [185,186]. The retinoid X receptor [3 gene is located 1.1 kb upstream of C O L l l A 2 [186]. The overall structure of the C O L l l A 2 gene is similar to that of the C O L l l A 1 gene, including a similar amino-terminal fragment as well as alternative splicing, which involves exons 6-8 [94,177,187]. The signal sequence is encoded by exon 1, the PARP subdomain by exons 2-5, the variable region by exons 5-9, the short triple-helical domain by exons 9-12, and the noncollagenous domain by exons 12-14. The N-telopeptide is encoded by exons 13 and 14. The junctional exon 14 also contains sequences for the beginning of the main triple-helical domain. Studies in mice have shown three Otz(XI) isoforms. Isoform 1 is a full-length mRNA, isoform 2 lacks exons 6 and 8 sequences, and isoform 3 lacks exons 6-8 sequences [95]. However, the number of isoforms involving exon 7 may be greater because of the additional splicing sequences observed in intron 6 [187]. A potential splice acceptor site 450bps and a potential splice donor site 227bps upstream of the published splice acceptor site of exon 7 were identified in humans. A branch point consensus sequence known to be essential for the formation of lariat intermediates in m R N A splicing exists 32-42 nucleotides 3' of these potential splice sites. Usage of these signals would result in three exon 7 splice variants [187]: exons 7A (249 bps) and 7B (227 bps) in addition to the known 63-bp exon 7, designated exon 7C. The main triple helix is encoded by 48 exons and parts of the junctional exons 14 and 63. As with the other fibrillar collagens, the codons encoding the helical domain, apart from the junctional exons, start with a complete codon for Gly and end with a complete codon for the Y residue of Gly-X-Y triplets. However, exon sizes and codon usage differed from the typical arrangements observed in types 1-III collagens. The amino acid sequences and the charge distributions within the triplehelical domains of the oL2(XI), otl(XI), and oL1(V) chains were similar. However, there was relatively little conservation of these characteristics with the oL3(XI)/ otl(II) chain.
13
The C-propeptide of the pro-oL2(XI) chain is approximately 30 amino acids shorter than the C-propeptides for pro-otl(I), pro-oLl(II), pro-oLl(V), pro-cxz(V), and prooL1(XI) because of internal deletions. As with other collagens, type XI collagen molecules assemble from the C-propeptides of their component chains and subsequently undergo cross-linking. Purified type XI collagen from fetal bovine cartilage contained divalent, borohydride-reducible structures, whereas pyridinoline residues were essentially absent [130]. The cross-linking patterns of N-telopeptides to the C-terminal region of the triple-helical domain of type XI collagen were consistent with a head- to-tail interaction of molecules staggered by 4 D periods (D = 67 nm). In addition, oLl(II) C-telopeptide was linked to the aminoterminal site of the oL~(XI) triple helix. However, type XI collagen molecules are primarily cross-linked to each other in cartilage, implying that a homopolymer is initially formed. Links to type II collagen are consistent with eventual cofibrillar assembly. Analysis of cartilage extracts showed that all three chains, OLl(XI)-ot3(XI), had at least in part retained their N-propeptides in cartilage matrix and that the oL3(XI) chain was the IIB splicing variant product of the COL2A1 gene. Of particular note was the finding that the N-telopeptide cross-linking site in both oLI(XI) and otz(XI) is located amino terminal to the putative N-propeptidase cleavage site. This structural feature provides a potential mechanism for the proteolytic depolymerization of type XI collagen by proteases that can cleave between the cross-link and the triple helix. Transglutamination has also been proposed as another mechanism for crosslinking of type XI and type V/XI hybrid molecules [171,188]. Type XI collagen appears to nucleate self-assembly and limit the lateral growth of heterotypic type II collagen fibrils [189]. In general, growth plates and fetal epiphyseal cartilages contain fibrils with diameters less than approximately 25 nm, whereas the permanent cartilage of adult tissues contains fibrils of approximately 30)-200 nm [144]. Human juvenile and newborn growth plates have thin fibrils in the hypertrophic zone, thick fibrils in the resting zone or permanent cartilage, and a mixture of thin and thick fibrils in the proliferative zone. Immunoelectron microscopy with anti-peptide antibodies to the C-telopeptide and to the amino-terminal non-triple-helical domains of oLI(XI) chains showed that both epitopes of type XI collagen were readily accessible to antibodies at the fibrillar surface and that type XI collagen was associated predominantly with fibrils <25 nm in diameter. Type XI collagen was not found in thick fibrils even after disruption with chaotropic agents. In contrast, types II and IX collagens were associated with fibrils of all sizes. These findings, which imply that
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William G. Cole
higher concentrations of type XI collagen are associated with smaller diameter heterotypic type II collagen fibrils, are consistent with the abnormally thick collagen fibrils and the abnormal growth plate architecture in the cho/ cho mouse, which lacks oLI(XI) chains [174]. Rotary shadowing imaging of type XI collagen molecules shows that the conformation and size of the various subdomains of the N-propeptides, particularly the Otl(XI) chains, would require that the N-propeptide protrude from the surface of the fibril rather than being accommodated within the gap zone of the fibril [189]. This spatial arrangement would place the N-propeptide in an appropriate position to interact with other matrix components as well as other collagen fibrils [190,191]. This proposal is also consistent the location of the type XI collagen molecules in heterotypic type II collagen fibrils as determined by immunoelectron microscopy studies and from the localization of covalent crosslinkages between types XI and II collagens [130,144].
TYPE X COLLAGEN Type X collagen is a short-chain fibrillar-type coliagen [192)-194] specifically produced by hypertrophic chondrocytes of the growth plate [192]. Each type X collagen molecule is a homotrimer consisting of three oLI(X) chains. The gene COLIOA1 is located at chromosome 6q21-q22 [195)-197]. It contains three exons, of which exons 2 and 3 encode the protein chain [195,198]. The 169-bp exon 2 encodes 15bps of the 5' untranslated sequence, 54 bps of the signal peptide, and 100 bps of the N-terminal noncollagenous domain 2 (NC2). The 3' nucleotide of exon 2 encodes the first nucleotide of the codon for the 34th amino acid residue of the NC2 domain. The 2940-bp exon 3 starts with the remaining 14 bps of the NC2 domain. It is followed by 1389 bps encoding the triple-helical domain (COL1), 483 bps encoding the complete NC1 domain, and 1054 bps of 3' untranslated sequence up to and including a potential polyadenylation signal. Consequently, the full-length protein contains 680 amino acid residues, including a signal sequence of 18 residues, an NC2 domain of 38 residues, a COL1 domain of 463 residues, and an NC1 domain of 161 residues. In contrast to the long-fibrillar types I-III collagens, in which the COL1 domains only contain repeating Gly-X-Y triplets, the COL1 domain of type X collagen contains eight interruptions in the triplet-helical repeats [195]. In five instances, the X or the Y amino acid residue is absent, and in three instances the Gly residue is replaced by another residue. Two of the latter interruptions encode mammalian collagenase cleavage sites. The
sequence at residues 151-156 is GISVPG and the sequence at residues 479-484 is GIATKG. The latter sequences correspond to the mammalian collagenase consensus sequence Gly-Ile-Y-X-Y-Gly. There are also several examples of Gly-X-Y triplets containing Gly in the X or Y positions. The first 12 amino acid residues of the NC1 domain share 67% homology with bovine oLI(X) chains, whereas 143 of the remaining 149 amino acid residues are 94% identical [195,199]. The locations of the 13 tyrosine residues, the putative N-linked oligosaccharide attachment site, and the single cysteine residue are all conserved [200]. Many studies of growth plates and isolated growth plate cells show that hypertrophic chondrocytes of the growth plate specifically express the COLIOA1 gene and produce type X collagen [201-204]. The NC1 domain contains the necessary signals to direct the assembly of the homotrimeric molecule [205,206]. This process occurs in the rough endoplasmic reticulum and is aided by chaperonins such as protein disulfide isomerase [207]. The resulting homotrimers are extremely stable because of the high content of hydrophobic residues [208]. The NC1 domain of type X collagen chains is homologous to the NC1 domain of type VIII collagen and to a C lq-like domain [209]. The crystal structure of the C-terminal domain of ACRP30, a serum protein, showed that its C lq-like domain is related to the tumor necrosis factor family both in subunit fold and in oligomeric assembly [210]. The crystal structure of the NC1 domain of human type X collagen has been resolved at 2.0 [211]. The NC 1 trimer has the shape of a squat truncated cone. The polypeptide chain termini of the subunits emerge at the broad base of the NC1 trimer, whereas the apex is constructed from tight loops organized around an intricate arrangement of Ca 2+ ions. The NC1 trimer is formed by the very tight association of three subunits. The intersubunit contacts are almost entirely hydrophobic near the base of the NC1 trimer and become progressively more hydrophilic toward the top of the trimer, forming a pronounced solvent-filled central channel. Approximately 7-11 calcium ions are present per NC1 trimer. The molecular surface of the trimer contains three strips, each containing eight partially exposed aromatic residues, that run across the shallow grooves between subunits. One of these strips has been proposed to be a hydrophobic surface patch involved in the higher order association of type X collagen trimers. There are three appropriately placed patches per NC1 trimer that would enable a polygonal network to form in two or three dimensions. The affinity for Ca 2+ ions and the observed CaZ+-regulated association of NC1 trimers provide a possible mechanism for the aggregation of type X collagen during endochondral ossification [212].
1. Structure of Growth Plate and Bone Matrix
Type X collagen produced by long-term cultured chick hypertrophic chondrocytes associated rapidly into multimeric clusters via their NC1 domains forming structures with a central nodule of NC1 domains and the triple-helical domains radiating outwards [213]. Prolonged incubation resulted in the formation of a regular polygonal lattice by lateral association of the juxtaposed triple-helical domains from adjacent multimeric clusters. These findings suggest that the NC1 domain remains attached to the c~l(X) chain in the extracellular matrix. The aggregates may also be stabilized by disulfide links that involve the single cysteine residue in the NC1 domain [195]. This proposal is in keeping with a report of human type X collagen with a molecular weight of 60 kDa when extracted from cartilage under reducing conditions and as a high-molecular-weight oligomer under nonreducing conditions [214]. The aggregates may also be stabilized by hydroxylysyl- and lysyl-derived cross-linkages [213,215]. Type X collagen extracted from chick growth plate cartilage contained a high concentration of lysylpyridinoline, a nonreducible collagen cross-link, whereas type II collagen from the same source contained hydroxylysylpyridinoline, a cross-link that predominates in cartilage other than the hypertrophic zone of the growth plate [216]. Immunoelectron microscopy of type X collagen in chick growth plate cartilage showed that it exists in two supramolecular forms [217]. In one form, type X collagen is associated with fine filaments <5 nm in diameter within matted structures that are predominately in the pericellular matrix of hypertrophic chondrocytes. It is likely that the nonstriated filamentous mats represent the regular polygonal lattice structures that form in longterm cultures of chick hypertrophic chondrocytes [213]. In a second form, farther away from the hypertrophic chondrocyte, type X collagen is associated with fibrils 10-20 nm in diameter that also contain type II collagen. The type X collagen associated with the striated fibrils most likely represents a secondary association of the molecule with preexisting heterotypic collagen fibrils containing types II, IX, and XI collagens [218,219]. The proteolytic cleavage of type X collagen likely commences with cleavage of the chains at their two mammalian collagenase cleavage sites [195,220]. In the chick, 59-kDa native type X collagen is cleaved at the two sites, yielding a final product of 32 kDa. The 32-kDa fragment has a mean temperature of 43~ due to a very high amino acid content. Consequently, the interstitial collagenase cleavage products are likely to be helical at physiological temperatures. However, the 32-kDa fragments can be readily degraded by cathepsin B at 37~ and at acid pH by osteoclast lysates [221]. One proposal is that during in vivo endochondral bone formation, type X collagen is first cleaved at neutral pH by interstitial
15
collagenase secreted by resorbing cartilage-derived cells. The resulting 32-kDa fragment is further degraded by osteoclast-derived cathepsin B supplied by the invading bone [221]. Other enzymes such as stromelysin-1, which is a neutral tissue metalloproteinase that can be extracted from cartilage, may also be involved in the degradation of type X collagen [137]. It cleaves type X collagen from the growth plate in its triple-helical domain, with a reduction in its chain size by approximately 10 kDa [137]. The proposed functions of type X collagen are based on its restricted expression within the hypertrophic zone of the growth plate. They include supporting the forming bone during the degradation of the cartilage matrix, facilitating the removal of the heterotypic type II collagen fibrils, participating in the mineralization process, and influencing the vascular invasion of the cartilage matrix [217,222]. However, the initial report of mice lacking type X collagen described normal long bone growth and development [223]. In the second study, mice lacking type X collagen had a mild skeletal phenotype resembling Schmid metaphyseal chondrodysplasia [224]. There were mild deformities of the femoral necks, reduced thickness of the resting zone of the growth plates, altered bone content, and atypical distribution of matrix components such as matrix vesicles and proteoglycans. In the third study, a variable skeletohematopoietic phenotype, from mild to lethal, was observed in mice lacking type X collagen [225]. The latter phenotypes were similar to the phenotypes of mice bearing a dominant interference mutation of type X collagen [226]. Perinatal lethality occurred in approximately 11% of null mutants, in which the growth plates were compressed, there was less trabecular bone, and the bone marrow showed hematopoietic aplasia and erythrocytefilled vascular sinusoids [225]. Lymphatic organs, which were reduced to approximately 80% of the size of control organs, displayed altered architecture and lymphocyte content. The thymus showed a paucity of cortical CD3+/CD4+/CD8 + lymphocytes, consistent with the marrow's inability to replenish maturing T cells. In the spleen, an unaltered T cell distribution was coupled with diffuse staining for IgD+/B220 + B cells, whose reduction was prominent in the poorly organized lymphatic nodules. Disorderly arrays of splenic macrophages surrounding periarteriolar lymphatic sheaths and depletion of the red pulp were also observed. Subtle growth plate compressions and hematopoietic changes were also seen in all the null mice that were apparently normal. From these diverse findings, it appears that hypertrophic cartilage and endochondral skeletogenesis may contribute to the marrow microenvironment required for differentiation of the hemopoietic and lymphoid systems. The molecular mechanisms involved in such interactions are unknown.
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William G. Cole
AGGRECAN The abundant, large cartilage-derived aggrecan belongs to the family of aggregating chondroitin sulfate proteoglycans [227]. This family of hyaluronatebinding proteoglycans also includes versican and two smaller proteoglycans, neurocan and brevican, that are expressed in neural tissue [228-230]. Versican is largely expressed in nonskeletal tissues. It is transiently expressed in the condensing mesenchyme of the developing limb bud as well as in low levels by chondrocytes of the intervertebral disk [231,232]. Consequently, this description of aggregating chondroitin sulfate proteoglycans is limited to aggrecan, which is largely specific for cartilage. Aggrecan and the other hyaluronate-binding proteoglycans form a network of proteoglycan aggregates that fill the space between collagen fibrils in the matrix, with the stability of the network maintained by the cooperative interaction of link protein [233]. In contrast, the leucine-rich repeat proteoglycans are characterized by their ability to interact with the collagen fibrils [234,235]. The human gene A GC1 is located on chromosome 15q26.1 and contains 19 exons spread over more than 50 kbs of genomic DNA [236,237]. Alternative splicing generates a long and a short isoform [238]. The long mRNA isoform encodes 2415 amino acids, whereas the short isoform encodes 2316 amino acids [239]. The protein includes a signal peptide of 19 amino acid residues. The mature protein adopts a conformation possessing three globular domains, G1-G3 [239]. The G1 region is at the N terminus and is separated by a relatively short interglobular domain from the G2 region. The G1 region is homologous to the complete link protein sequence and thus consists of three disulfide-bonded loop structures designated A, B, and Bt motifs, with B and Bt related sequences of approximately 100 amino acids [240]. The G2 region is similar to the G1 region, possessing the B and B' domains but lacking the A domain. The G3 region, at the C terminus, has a complex structure. It contains two additional disulfide-containing domains, a calcium-binding epidermal growth factor (EGF)-like motif, a C-type lectin motif, and a complement regulatory protein motif [238,240]. Alternative splicing generates several isoforms. In juvenile human cartilage, approximately 95% of the transcripts lack the EGF-like and the complement regulatory protein motifs [239]. There is also a full-length transcript and one that lacks the EGF-like motif but retains the complement regulatory protein motifs. The functional significance of the isoforms is unknown. The glycosaminoglycan attachment region lies between the G2 and G3 regions, beginning with a
proline-rich domain adjacent to G2 [239]. Next is a keratan sulfate-rich region from amino acid residues 772-837. It contains 12 consecutive repeating units of 7 conserved residues (E-E-P-Y-T-P-S) for the attachment of keratan sulfate. It is followed by the chondroitin sulfate attachment domain 1 (CS-1) of 649 amino acid residues. The CS-1 domain contains 77 Ser-Gly pairs for the attachment of chondroitin sulfate chains [241,242]. The CS-1 domain is followed by the CS-2 domain of 665 amino acid residues. It contains 69 Ser-Gly pairs. In addition to the many keratan sulfate and chondroitin sulfate chains, there are also many O-linked and N-linked oligosaccharides. There are also complex changes in the extent of the posttranslational modifications with age, and there complex events are involved in the specific degradation of aggrecan. Hyaluronan is a glycosaminoglycan consisting of several thousand repeating disaccharide units [243]. Five of its repeating disaccharide units are necessary for the binding of an individual aggrecan core protein with its associated link protein. The link protein and the hyaluronate binding region of aggrecan bind to hyaluronan with the same specificity and with a similar binding strength. Consequently, many proteoglycan molecules can bind to a single hyaluronan molecule. Ultrastructural studies have shown that proteoglycan aggregates contain a large number of proteoglycan monomers very closely spaced along the hyaluronan central filament. The proteoglycan molecules and link protein appear to bind to the hyaluronan in the extracellular matrix. The proteoglycans have a high fixed-charge density so that they swell if not constrained by collagen fibrils [243].
CARTILAGE LINK PROTEIN 1 Cartilage link protein 1 stabilizes the binding of the hyaluronan-binding regions of aggrecan core protein to hyaluronan in growth plate and articular cartilages. The protein is widely distributed in connective tissues but the latter function is limited to cartilages. The gene for cartilage link protein 1, CRTL1, is located on chromosome 5q 14.3 [244,245]. The full-length protein contains 354 amino acid residues. It includes a signal peptide of 15 residues and a mature protein of 339 residues. It has a modular structure consisting of one immunoglobulin V-type domain from residues 54-141 and two hyaluronan binding sites from residues 157-254 and 258-351. The importance of cartilage link protein 1 in normal cartilage physiology is demonstrated by the severe chondrodysplasia that occurs in mice lacking this protein [246].
1. Structure of Growth Plate and Bone Matrix
SMALL, LEUCINE-RICH, INTERSTITIAL PROTEOGLYCANS The small, leucine-rich, interstitial proteoglycans (SLRPs) are a large family of extracellular matrix glycoproteins/proteoglycans that share a leucine-rich repeating structural motif [247]. Many of the family members bind to various collagens and to growth factors such as TGF-[311248]. The gene family can be divided into subfamilies based on similarities in amino acid sequences and gene organization. The type I subfamily includes decorin and biglycan, which contains an N-terminal domain substituted with chondroitin or dermatan sulfate chains. These proteoglycans show 57% protein sequence identity and are encoded by genes composed of eight exons with exon/intron junctions in conserved positions [249,250]. Fibromodulin and lumican constitute the type II subfamily and exhibit 48% protein sequence identity. Their genes are composed of three exons with conserved exon/intron junctions [251,252]. Other members of the latter family include the proline- and arginine-rich and leucine-rich repeat protein (PRELP) and osteomodulin [253,254]. The class III SLRP expressed in cartilage is epiphycan, also called PG-Lb [255]. Chondroadherin represents its own subfamily, with a different gene organization and a different amino acid composition [256]. The leucine-rich repeating extracellular glycoproteins/ proteoglycans have core proteins of 32-42 kDa [257]. The protein chains can be divided into N-terminal, central, and C-terminal domains. The N-terminal domains are least conserved, but in all members of the family they contain 4 Cys residues, which form intrachain disulfide bonds [258]. The glycosaminoglycan chains in decorin and biglycan are O-glycosidically linked to Ser residues in the N-terminal region, providing polyanionic properties to the proteoglycans. In contrast, the N-terminal domains of fibromodulin and lumican carry clusters of negatively charged Tyr sulfate residues [257]. The different leucine-rich repeat proteoglycans and glycoproteins have similar C-terminal domains, which contain approximately 50 amino acid residues. This domain contains 2 Cys residues involved in an intrachain disulfide bond, leading to the formation of a 34- to 41-residue loop. The common central domain constitutes approximately 60-80% of the total protein. In most of the members of the family, it contains 10 or 11 repeats of a 20- to 25-residue-long leucine-rich motif, with Asn and Leu residues in conserved positions. Each leucine-rich repeat contains the motif LXXLXLXXNXL, with each motif being separated by 9-18 amino acids. Up to 30 adjacent leucine-rich repeats have been described in some leucine-rich repeat proteins. The leucine residues in the motif may be replaced by alanine, valine, isoleucine,
17
phenylalanine, tyrosine, or methionine. The asparagine residue at position 9 may be replaced by cysteine or threonine [256]. There are consensus site Asn residues in the central repeat domain for substitution with carbohydrates. For example, the latter Asn sites are partially substituted with keratan sulfate in fibromodulin and lumican [252]. The three-dimensional structure of one member of the family, ribonuclease inhibitor, showed that the leucinerich repeats form a horseshoe-shaped coil of parallel, alternating c~helices and 13sheets stabilized by interchain hydrogen bonds [259]. Results of structural studies of decorin and biglycan are in accordance with the latter X-ray crystallographic findings [260]. Decorin, fibromodulin, and lumican bind to fibrillar collagens in vitro, leading to delayed fibril formation and the formation of thinner fibrils [261]. These changes are likely due to binding of the leucine-rich repeat glycoproteins/proteoglycans to the surface of the axially growing fibril, which inhibits the incorporation of additional triple-helical collagen monomers [75]. Binding of the leucine-rich repeat proteoglycan to collagen alters the surface properties of the fibrils and may affect the interactions between individual collagen fibrils as well as between the fibrils and the matrix. Competitive binding and displacement of proteoglycans may regulate the growth of collagen fibrils during skeletal development [262]. Decorin and fibromodulin bind to distinct and apparently separate sites in the gap region of the D period of the collagen fibril in vivo [263,264]. Decorin has been shown to bind to a small region of the C terminus of the triple-helical domain of etl(I) chains, close to one of the intermolecular cross-linking sites [265]. This region corresponds to the Cl band of the collagen fibril D period. Proteoglycan core proteins of decorin, biglycan, and fibromodulin, prepared as fusion proteins, each bound TGF-1311266]. There was negligible binding to several other growth factors. Intact decorin, biglycan, and fibromodulin, isolated from bovine tissues, competed with the fusion proteins for TGF-f31binding. Affinity measurements suggest a two-site binding model with KDvalues ranging from 1 to 20 nM for the high-affinity binding site and from 50 to 200nM for the low-affinity binding site. Stoichiometry indicated that the high-affinity binding site was present in 1 of 10 proteoglycan core molecules and that each molecule contained a lowaffinity binding site. Tissue-derived biglycan and decorin were less effective competitors for TGF-f3 binding than fibromodulin or the nonglycosylated fusion proteins. Removal of the chondroitin/dermatan sulfate chains of decorin and biglycan (fibromodulin is a keratan sulfate proteoglycan) increased the activities of decorin and
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William G. Cole
biglycan, suggesting that the glycosaminoglycan chains may hinder the interaction of the core proteins with TGF-[3. The fusion proteins competed for the binding of radiolabeled TGF-[3 to Mv 1 Lu cells and endothelial cells. Affinity labeling showed that the binding of TGF-[3 to betaglycan and the type-I receptors in Mv 1 Lu cells and to endoglin in endothelial cells was reduced, but the binding to the type II receptors was unaffected. TGF-[32 and-3also bound to all three fusion proteins. Latent recombinant TGF-[31precursor bound slightly to fibromodulin and not at all to decorin and biglycan. These findings indicate that the three decorin-type proteoglycans each bind TGF-[3 isoforms and that slight differences exist in their binding properties. They, and possibly other members of this family of proteoglycans, may regulate TGF-[3 activities by sequestering TGF-[3 in the extracellular matrix. Changes have been reported in the abundance of decorin, biglycan, fibromodulin, and lumican during maturation of the bovine fetal growth plate [267]. Decorin protein and m R N A were readily detectable in both the reserve and proliferating zones of the growth plate but not in the zones of maturation and hypertrophy. In contrast, fibromodulin and biglycan protein and m R N A could be detected throughout the growth plate. although their abundance was decreased in the proliferative and hypertrophic zones. Lumican protein and m R N A were not detected in the growth plate even though its m R N A was detectable in isolated growth plate cells [252]. Representative members of the subfamilies of small, leucine-rich interstitial proteoglycans are described next. Decorin a n d Biglycan The decorin gene, DCN, and the biglycan gene, BGN, are located on chromosomes 12q13.2 and Xq28, respectively [250,268-270]. Both genes have a similar 8exon gene structure. The decorin gene is approximately 45 kbs, whereas the biglycan gene is approximately 8 kbs [249]. Exon 1 of decorin, which encodes the 5' untranslated region, exists as exon l a and exon 1b, which undergo alternative splicing [250]. The decorin transcript can also undergo additional alternative splicing to yield five isoforms, A-E. Isoform A, which contains the full amino acid coding sequence, is the product of transcript variants A1 from exon la and A2 from exon 2b. Isoform B lacks the exons 3 and 4 sequence, isoform C lacks the exons 3-5 sequence, isoform D lacks the exons 4-7 sequence, and isoform E lacks the exons 3-7 sequence. There are several polyadenylation sites, of which two account for the 1.6- and 1.9-kb mRNAs isolated from various connective tissues and cultured cells [250]. The biglycan m R N A sizes, also related to the use of
different polyadenylation sites, are 2.1 and 2.6kbs [249]. The BGN gene is subject to X inactivation because there is no homologous gene on the Y chromosome. However, the pseudoautosomal expression of BGN is probably due to the regulation of the BGN gene by a gene or genes that escape X inactivation [271]. Preprodecorin contains 359 amino acid residues [272]. It includes a signal peptide of 16 residues, an N-propeptide of 14 residues, and a mature chain of 329 residues. Intrachain disulfide bonds are present between Cys 54 and Cys 67 as well as between Cys 313 and Cys 346. Twelve leucine-rich repeats are present in the central domain between residues 73 and 359. The O-linked chondroitin sulfate or dermatan sulfate attachment site is at residue 34. Residues 211,262, and 303 provide potential N-linked oligosaccharide attachment sites. The core protein of 359 amino acid residues has a predicted molecular weightof 39 kDa, whereas the protein extracted from tissues has a molecular weight of approximately 130 kDa, of which the chondroitin sulfate chain contributes approximately 40 kDa [273]. Preprobiglycan contains 368 amino acid residues [249,269,270]. It contains a signal peptide of 16 residues, a propeptide of 21 residues, and a mature protein of 331 residues. A region of proteoglycan N-terminal homology is present from residues 57 to 81 of the full-length protein. Ten leucine-rich repeats are located between residues 91 and 315. Residues 316-368 contain a domain with proteoglycan C-terminal homology. Residues 42,47,180, and 198 are potential chondroitin sulfate or dermatan sulfate attachment sites. Biglycan contains two substituted sites, whereas decorin contains only one. Asn residues 270 and 311 are possible sites for N-linked oligosaccharides. The secreted core protein has a predicted molecular weight of 38 kDa, whereas the intact tissue protein has a molecular weight of approximately 270 kDa, with the two chondroitin sulfate chains contributing 40kDa [274]. BMP-1 cleaves probiglycan at a single site, removing the propeptide and producing a biglycan molecule with an N terminus identical to that of the mature form found in tissues [275]. The BMP-1related proteases, mammalian Tolloid and mammalian Tolloid-like 1 (mTLL-1), have low levels of probiglycancleaving activity. Wild-type mouse embryo fibroblasts produce only fully processed biglycan, whereas the fibroblasts derived from embryos homozygous null for the Bmpl gene, which encodes both BMP-1 and mammalian Tolloid, produce predominantly unprocessed probiglycan, and fibroblasts homozygous null for both the Bmpl gene and the m TLL-1 gene produce only unprocessed probiglycan. Consequently, all detectable probiglycanprocessing activity in the mouse embyronic fibroblasts is accounted for by the products of these two genes. Cartilage contains both proteoglycan and nonproteogly-
1. Structure of Growth Plate and Bone Matrix
can forms of biglycan, with the latter form increasing with age [276]. The importance of decorin and biglycan in the formation of the connective tissues is demonstrated by the anomalies observed in mice that fail to express these genes. In decorin-deficient mice, the mice have fragile skin due to coarse and irregular collagen fibrils, confirming the importance of decorin in normal collagen fibrillogenesis [277]. Biglycan-deficient mice show reduced longitudinal growth and decreased bone mass, indicating an important role for biglycan in bone health [278]. Decorin and biglycan are representative of a subfamily of proteins that also includes epiphycan and asporin [279,280]. The latter small leucine-rich dermatan sulfate-containing proteoglycans have been isolated from cartilage. Fibromodulin a n d Related Small P r o t e o g l y c a n s Representative members of the fibromodulin subfamily also include lumican, PRELP, and osteomodulin, also known as osteoadherin. They are found in most connective tissues but in greatest abundance in cartilage, tendon, and ligament [281]. Fibromodulin was first isolated from bovine articular cartilage and subsequently cloned and sequenced from bovine tracheal cartilage [281,282]. The fibromodulin gene, FMOD, is approximately 8.5 kbs and is located on chromosome lq32 [283]. The gene contains three exons with conserved exon/intron junctions. Exon 1 encodes the 5' untranslated region, exon 2 contains 983 bps and encodes the major part of the translated region, and exon 3 encodes the last 50 nucleotides of the translated region as well as the 3' untranslated region [251]. The fibromodulin m R N A is approximately 3 kbs [283]. The highest expression of the gene is in hyaline cartilages, tendon, and ligament, whereas lower levels of expression occur in most other connective tissues [281]. Fibromodulin is a 59-kDa protein [281]. The fulllength protein contains 376 amino acid residues, including a signal peptide of 18 amino acid residues and a mature protein of 358 amino acid residues [251,283]. The mature protein is composed of a central region containing leucine-rich repeats with up to four keratan sulfate chains flanked by disulfide-bonded terminal domains. The amino-terminal domain contains 10 tryosine residues that are partially sulfated [251]. The keratan sulfate chains were attached by N-glycosidic linkages from N-acetylglucosamine to asparagine residues in the central domain of the molecule [284]. In contrast to other SLRPs, fibromodulin does not contain glycosaminoglycan chains in the amino-terminal domain and does not contain chondroitin/dermatan sulfate in any domain.
19
Mice lacking fibromodulin showed altered tendon structure due to the high proportion of thin, irregular collagen fibrils [257]. These relatively mild changes confirmed the important role of fibromodulin in regulating collagen fibrillogenesis in vivo. The lumican gene, L UM, is located on chromosome 12q21.3-q22 [252,285]. It encodes 338 amino acid residues, including a signal peptide of 18 residues and a mature protein of 320 residues. Residues 37-53 of the full-length protein encompass a Cys-rich domain followed by a central domain of 12 leucine-rich repeat motifs from residues 59-338. An intrachain disulfide bond can form b e t w e e n Cys 295 a n d Cys 328. The residues at positions 88,127,160, and 252 are potential N-linked keratan sulfate attachment sites. The protein is present in the cornea as well as cartilage from all age groups, but it is more abundant in adult cartilage. In adult cartilage, the protein is a glycoprotein that lacks keratan sulfate, whereas in juveniles it is a proteoglycan containing keratan sulfate chains [252]. The important role of lumican in normal collagen fibrillogenesis is confirmed by the finding in lumican knockout mice of skin fragility and corneal opacities due to abnormal collagen fibrillogenesis [286]. The gene for PRELP is located on chromosome lq32 [287]. The gene of 16 kbs contains three exons. The protein is encoded by three mRNAs of 1.7,4.6, and 6.7 kbs. The differences in the size of the mRNAs appear to be due to variations in the length of their 3' untranslated regions. It encodes a 382-amino acid residue protein that includes a signal peptide of 20 residues and a mature PRELP protein of 362 residues [254,287]. The central domain containing 10 leucine-rich repeat motifs is flanked by Cys-rich regions. Potential N-linked oligosaccharide attachment sites are located at residues 124,289, and 320. However, substitution by N-linked keratan sulfate is less than for fibromodulin and lumican. The mRNAs are expressed at a high level in juvenile and adult cartilage, at a low level in neonatal cartilage, and are not detectable in fetal cartilage. The PRELP mRNAs and protein differ in their expression from those of decorin, biglycan, fibromodulin, and lumican. PRELP mRNAs were not detected in trabecular bone, skin, kidney, liver, spleen, or thymus [254]. The molecular weight of the core PRELP protein is 42 kDa, whereas that of PRELP extracted from cartilage is 55 kDa [288]. The gene for osteomodulin (also known as osteoadherin), OMD, is located on chromosome 9q22.2. The full-length human protein contains 421 amino acid residues. The primary structure of bovine osteomodulin was obtained by nucleotide sequencing of a cDNA clone from a primary bovine osteoblast expression library [289,290]. The entire translated primary sequence corresponds to a 49,116-Da protein with a calculated
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William G. Cole
isoelectric point for the mature protein of 5.2. The dominating feature is a central region consisting of 11 leucinerich repeats ranging in length from 20 to 30 residues. The full, primary sequence contains four putative sites for tyrosine sulfation, three of which are at the N-terminal end of the molecule. There are six potential sites for N-linked glycosylation, some of which are substituted with keratan sulfate chains. Osteomodulin shows highest sequence identity (42%) to bovine keratocan and 37 or 38% identity to bovine fibromodulin, lumican, and human PRELP. Unique to osteomodulin is the presence of a large and very acidic C-terminal domain. The distribution of cysteine residues resembles that of other leucine-rich repeat proteins except for two centrally located cysteines. Northern blot analysis of RNA samples from various bovine tissues showed a 4.5-kilobase pair message for osteomodulin to be expressed in bone only. Osteomodulin m R N A was detected by in situ hybridization in mature osteoblasts located superficially on trabecular bone. Osteomodulin binds to hydroxyapatite and cells. Cell binding is mediated by integrin OLv[33[289,290].
Epiphycan Epiphycan is a dermatan sulfate proteoglycan also known as PG-Lb as well as dermatan sulfate proteoglycan 3. It can be distinguished from decorin and biglycan of epiphyseal cartilage, from which it derives its name [255]. The gene, DSPG3, which is located on chromosome 12q21, contains seven exons [255]. The full-length protein contains 322 amino acid residues. Residues 121-140 contain a leucine-rich repeat N-terminal domain. Two serine residues exhibit the glycosaminoglycan attachment consensus similar to those in aggrecan, decorin, and biglycan. In the mouse, the gene is expressed in cartilage, ligaments, and the placenta. The gene is expressed in a precise temporal and spatial manner in the growth plate. It is expressed by growth plate chondrocytes after type II collagen appears but is not expressed by hypertrophic chondrocytes. However, despite the limited pattern of chondrocyte expression, the protein is widely distrubuted through the resting, proliferative, and hypertrophic zones of the growth plate [291]. Chondroadherin Chondroadherin represents its own subfamily of SLRPs, with a different gene organization and a different amino acid composition [253]. It was initially isolated from bovine cartilage as a 36-kDa protein that was involved in chondrocyte-matrix interactions [292,293]. The human gene, CHAD, is located on chromosome 17q21.33 [256]. It has three exons. Exon 1 encodes the
signal peptide, the N-terminal cysteine-rich region, and the first 9 leucine-rich repeats. Exon 2 encodes the last 2 leucine-rich repeats and part of the C-terminal cysteinerich region. Exon 3 encodes the remainder of the C-terminal cysteine-rich region. There is a single m R N A species of 1.9 kbs. The cDNA contains a 5' untranslated region of 149 bps, a coding region of 1080 bps including the stop codon, and a 3' untranslated region of 561 bps terminating in a poly(A) tail. The full-length protein contains 359 amino acid residues, including a signal peptide of 21 residues and a mature protein of 339 residues. The mature sequence contains 11 leucinerich repeats of 22- to 25-amino acid residues flanked by cysteine-rich regions. The protein is enriched in cartilage and bone [293].
PERLECAN Perlecan is a heparan sulfate proteoglycan and a major component of basement membranes and cartilage [294,295]. The gene, HSPG2, is located on chromosome lp36.1-p35 [296-298]. It encodes a full-length protein of 3205 amino acids [299,300]. The full-length protein contains a signal peptide of 21 amino acid residues and a mature protein of 4373 residues [300]. The mature protein core, without the signal peptide of 21 amino acids, has a molecular weight of 466,564. This large protein is composed of multiple modules homologous to the receptor of low-density lipoprotein, laminin, neural cell adhesion molecules, and epidermal growth factor. Domain I, near the amino terminus, appears to be unique for the proteoglycan since it shares no significant homology with any other proteins. It contains three Ser-Gly-Asp sequences that could act as attachment sites for heparan sulfate glycosaminoglycans [301]. Domain II is highly homologous to the low-density lipoprotein receptor and contains 4 repeats with perfect conservation of all six consecutive cysteines. Next is domain III, which shares homology to the short arm of laminin A chain and contains 4 cysteinerich regions intercalated among three globular domains. Domain IV, the largest module with more than 2000 residues, contains 21 repeats of the immunoglobulin type as found in neural cell adhesion molecule. Near the beginning of this domain, there is a stretch of 29 hydrophobic amino acids that could allow the molecule to interact with the plasma membrane. Domain V, similar to the C-terminal globular G domain of laminin A and to the related protein merosin, contains three globular regions and 4 EGF-like repeats. In situ hybridization and immunoenzymatic studies show a close association of this gene product with a
1. Structure of Growth Plate and Bone Matrix
variety of cells involved in the assembly of basement membranes, in addition to being localized within the stromal elements of various connective tissues. Perlecan interacts with extracellular matrix proteins, growth factors, and receptors, and it influences cellular signaling [302-304]. Perlecan expression occurs during organogenesis of the kidney, lung, liver, spleen, gastrointestinal tract, and cartilage. The levels of perlecan are low in precartilaginous tissues but high in mature cartilage [295]. In vito studies show that perlecan supports chondrocyte differentiation [295,305]. The importance of perlecan in normal cartilage development is demonstrated by the abnormal structure and function of cartilage in mice lacking this proteoglycan [306,307]. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and a reduced amount of glycosaminoglycans in the cartilage. The columnar structure of the growth plate is severely disrupted.
MATRILINS The matrilins are a family of four oligomeric, multidomain, extracellular matrix proteins [308]. The prototype member of the family is matrilin-1, which was initially referred to as cartilage matrix protein [309,310]. The four monomers are named matrilin-l-matrilin-4 [3081. The human M A T N 1 gene, encoding matrilin-1, resides on chromosome lp35. It is 12kbs and contains 8 exons [311]. The m R N A of approximately 1.5kbs is predicted to yield a monomer of 496 amino acids, including a signal peptide of 22 residues, two von Willebrand factor A (vWFA) domains separated by a single EGFlike motif, and a C-terminal coiled-coil oligomerization domain. The mature protein is predicted to have an unmodified molecular weightof 51,344. The higher molecular weight of 52 kDa observed for matrilin-1 from cartilage is likely due to the presence of N-glycosidically linked oligosaccharides [310]. Matrilin-2 is encoded by the M A TN2 gene located on chromosome 8q22 [312]. The m R N A is approximately 2.9 kbs. The cDNA sequence predicts a protein monomer of 956 amino acid residues, including a signal peptide of 23 residues. The two vWFA domains are separated by 10 EGF-like motifs. A sequence that is unique to matrilin-2 is followed by the C-terminal coiled-coil oligomerization domain. Alternative splicing can generate a short form of the protein in which 19 amino acid residues are deleted from the unique sequence domain. The matrilin-3 gene, located on chromosome 2p24-p23, yields a processed m R N A of approximately 1.5 kb [313-315]. The protein monomer is predicted to
21
contain 486 amino acids, including a signal peptide of 28 residues. Adjoining the signal peptide is a short positively charged region followed by a single vWFA domain, four EGF-like motifs, and a C-terminal coiled-coil oligomerization domain. The matrilin-4 gene is located on chromosome 20ql 3.1-13.2 [316,317]. It yields three alternatively spliced m R N A species. The long form encodes a monomer of 582 amino acids, including a signal peptide of 56 residues. The modular structure of the long isoform includes two vWFA domains separated by three EGFlike motifs and followed by the C-terminal coiled-coil domain. The two shorter forms, resulting from alternative splicing, contain either one or two EGF-like motifs. The members of the matrilin family share a similar modular structure consisting of one or two vWFA domains, a varying number of EGF-like motifs, and a C-terminal coiled-coil oligomerization domain [308]. The matrilins can self-assemble into supramolecular structures that form a filamentous network that may stabilize the extracellular matrix [318-321]. At least for matrilin1 and matrilin-3, the filamentous networks can be associated with or be independent of collagen fibrils [322,323]. Matrilin-1 appears to have a periodic association with collagen fibrils and to play a role in collagen fibrillogenesis [324,325]. In the normal zone of maturation, between the zones of proliferation and hypertrophy of the mouse growth plate, matrilin-1 has a periodicity of 59.3 nm along the heterotypic type II collagen fibrils [326]. In mice lacking matrilin-1, the zone of maturation contains abnormal collagen fibrils, in keeping with the importance of this glycoprotein in normal type II collagen fibrillogenesis [326]. Matilin-1 can also interact covalently with aggrecan core protein [309,327], and it may interact with the Otl~ 1 integrin receptor [327]. These various interactions indicate that at least this member of the matrilin family has the ability to link the major collagen and proteoglycan components of the extracellular matrix to specific integrin receptors and their associated cell signaling pathways. Matrilin-2 and matrilin-4 are present in many tissues, whereas matrilin-1 and matrilin-3 are more restricted to skeletal tissues [309,312-317,319-321,328]. In epiphyseal cartilage of growing long bones, matrilin-1 and matrilin3 are present in all cartilage regions, in contrast to matrilin-2, which is expressed in the proliferative and the upper hypertrophic zones [320,321,329]. Similarly, matrilin-4 is detected all over the epiphyseal cartilage, with the weakest expression in the hypertrophic zone. During joint development matrilin-2 and matrilin-4 are present at the developing joint surface, whereas in articular cartilage of 6-week-old mice, all matrilins are only weakly expressed. Matrilin-1 has a more widespread distribution during embyronic development [330].
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William G. Cole
Electron microscopy of matrilin-1 oligomers from cartilage revealed the presence of a bouquet-like structure consisting of three ellipsoid subunits connected at their C-terminal coiled-coil domains [331]. Although the oligomers are stabilized by disulfide bonds between the coiled-coil domains, formation of the oligomers is not dependent on formation of disulfide bonds. In contrast, oligomerization appears to be dependent on a heptad repeat of seven residues denoted a-g, with a 3,4-hydrophobic repeat of mostly apolar residues at positions a and d [332]. Studies using isolated coiled-coil domains showed .that matrilin-1, matrilin-2, and matrilin-4 formed disulfide-linked, three-stranded, parallel homotrimers, whereas the domain from matrilin-3 produced a disulfide-linked, four-stranded, parallel coiled-coil structure [308]. The preference of matrilin-3 to form tetramers rather than trimers may be due to the fact that matrilin-3 contains a single isoleucine residue at a d position of the third heptad repeat. Nine different oligomers were identified when all possible combinations of two or three different matrilin monomers were mixed in various molar ratios. With the exception of matrilin-3, which interacted only with matrilin-1, heteromer formation was observed for all other chain combinations. For all of these complexes, disulfide-linked heterotrimeric structures were found, except for matrilin-1 and matrilin-3, which folded into disulfide-linked tetramers. The concentration of the individual peptides determined the stoichiometry of the complexes. Consequently, it is likely that the relative levels of expression of the matrilin genes are the main determinant of the types of matrilin oligomers in the extracellular matrix. The molecular heterogeneity is likely to be even greater because of the alternatively spliced isoforms of matrilin-2 and matrilin-4.
THROMBOSPONDINS The five members of the thrombospondin family are designated thrombospondin-l-thrombospondin-5 [333]. Thrombospondin-5 is also known as cartilage oligomeric matrix protein (COMP). The thrombospondins are multimeric, multidomain glycoproteins that function at cell surfaces and in the extracellular matrix. The latter functions are also referred to as matricellar functions. The thrombospondin-1 gene, THBS1, is located on chromosome 15q15 [334-336]. The full-length protein monomer contains 1170 amino acids, including a signal peptide of 31 amino acids. The thrombospondin-2 gene, THBS2, is located on chromosome 6q27 [337,338]. The full-length protein contains 1172 amino acids, including a signal peptide of 18 amino acid residues. The throm-
bospondin-3 gene, THBS3, on chromosome l q21 encodes a protein monomer of 956 amino acids, including a signal peptide of 36 amino acid residues [339,340]. The thrombospondin-4 gene, THB4, on chromosome 5q13 encodes a full-length protein monomer of 961 amino acid residues, including a signal peptide of 21 residues [341,342]. Finally, the thrombospondin-5 gene, COMP, located on chromosome 19pl 3.1 encodes a full-length monomer of 757 amino acids, including a signal peptide of 20 amino acid residues [343]. Each thrombospondin is expressed in multiple tissues, particularly during fetal life. Similarly, most tissues express multiple members of the thrombospondin family [333]. Consequently, most of the thrombospondins are expressed in bone and cartilage during embryogenesis [343-346]. COMP is the most abundant form in postnatal growth plates [345,347]. Thrombospondin-2 and COMP are also present in postnatal bone and bone marrow stromal cells [347]. The thrombospondins are divided into subfamily A and subfamily B [333]. Thrombospondin-1 and thrombospondin-2 monomers, the members of subfamily A, share a complex modular structure and can assemble into disulfide-linked trimers. The modules from the amino terminus include the N-terminal domain; the oligomerization sequence; the procollagen homology region; three type 1, three type 2, and seven type 3 repeating units; and a globular C-terminal domain. Thrombospondins-3-5 are in subfamily B. They have unique N-terminal regions and lack the procollagen homology domain and type 1 repeats, but they contain four copies of the type 2 repeat and are assembled as pentamers. The globular amino- and carboxyl-terminal domains are connected to the remainder of the monomers by thin, flexible stalk regions [348]. The conformations of the stalk regions and the termini are determined, at least in part, by the calcium ion concentration [349]. The oligomerization motifs in all the thrombospondins are likely to be in a coiled-coil conformation, giving rise in subfamily A to trimers and in subfamily B to pentamers [350]. The procollagen homology region of thrombospondin1 also folds into a stable, compact, and disulfide-bonded monomer [350]. Isolated type 3 repeats of COMP produce a 14.2-nm rod-like structure [351]. The C-terminal domain and the adjoining type 3 repeats may fold together, in the presence of calcium, to form a C-terminal globular structure [351]. Thrombospondin-1 and thrombospondin-2 can form homotrimers and heterotrimers [352]. Thrombospondin trimers and pentamers are stabilized by the formation of interchain disulfide bonds between their corresponding oligomerization domains [353]. Pentamerization of COMP involves the formation of interchain disulfide
1. Structureof Growth Plate and Bone Matrix bonds between cysteine residues 68 and 71 [354]. Intrachain disulfide bonds are also present in the type 3 repeats [355]. There are also specific sites for the addition of N- and O-linked sugars and for the C-mannosylation of tryptophan [356,357]. In the extracellular matrix, the thrombospondins bind to other macromolecules as well as to cell surface receptors. Many of the interactions involve specific domains within the monomers. Some of the interactions are sequence specific, but many are also dependent on the ionic environment and the conformation of the monomers. Each thrombospondin-1 monomer can bind approximately 35 calcium ions, whereas subfamily B thrombospondins, such as COMP, are expected to bind almost double the number of calcium ions [358]. Major changes in conformation of the type 3 repeats and the molecule follow the binding of calcium ions. Binding sites are also present for heparin and heparan sulfate proteoglycan, decorin, various integrins, as well as a wide range of proteases, cytokines, and growth factors [357,359]. The binding sites and the consequences of the interactions have been most thoroughly studied for thrombospondin-1 and thrombospondin-2. For example, the activities of thrombin, plasmin, neutrophil cathepsin G, elastase, urokinase plasminogen activator, plasminogen activator inhibitor, and MMP-2 are modified following binding to the latter thrombospondins [360,361]. The small latent TGF-[31complex with the latencyassociated peptide binds to the WSIIWSPW motif in the second type 1 repeat of thrombospondin-1 [362]. An intermolecular activation effect of the KRFK motif in the first type 1 repeat of thrombospondin-1 releases mature, active TGF-[31. Thrombospondin-2, which lacks the KRFK motif, can bind the latent TGF-[31complex but cannot activate it [362]. Thrombospondin-4 and COMP bind to types I and II collagens [363]. COMP also binds type IX collagen and MMP-19 and-20, whereas thrombospondin-4 also binds to laminin, fibronectin, and matrilin-2 [364-366]. Cell surface interactions with thrombospondin-1 have been studied in detail, whereas little information is available concerning the other thrombospondins [333]. The amino-terminal domain, type 1 repeats, type 3 repeats, and the C-terminal domain all interact with the cell surface. Each region appears to act via different cell surface receptors. The interactions are calcium dependent and appear to be important for the maintenance of cell adhesion, spreading, migration, and shape. Cell attachment activity also appears to be important for thrombospondins-2 and-4 and COMP [344,367]. The C-terminal domains of thrombospondin-4 and COMP bind types I, II, and IX collagens in a zincion-dependent manner [363-365]. The binding of the
23
latter thrombospondins is to the amino and carboxyl propeptides and two triple-helical sites of these collagens. The pentameric structure of thrombospondin-4 and COMP favors the use of multiple sites of interaction, which are likely to stabilize the extracellular matrix. A candidate COMP binding site with types II and IX collagens resides between amino acid residues 579 and 595 of the C-terminal domain [364]. Further insights into the functions of thrombospondin-l, thrombospondin-2, and COMP have been obtained from murine gene knockout studies. Mice lacking thrombospondin-1 show decreased embryonic viability, early onset pneumonia, increased circulating monocytes, and reduced TGF-[31activation in the inflamed lungs and pancreas [368]. Mice lacking thrombospondin-2 develop fragile skin, lax tendons, increased thickness and density of the long-bone cortices, increased vascular density, prolonged bleeding time, and accelerated skin wound healing [369]. The changes in the skin indicate that thrombospondin-2 normally plays an important role in collagen fibrillogenesis. Abnormal fibroblast interactions with the extracellular matrix and increased production of active MMP-2 were also identified in cell cultures from thrombospondin-2-deficient mice. In contrast, mice deficient in COMP did not show any anomalies [370].
OSTEONECTIN Osteonectin, initially isolated from demineralized bone matrix, was named according to its ability to bind to Ca 2+, hydroxyapatite, and collagen and to nucleate hydroxyapatite deposition [371,372]. The protein has also been called secreted protein, acidic and rich in cysteine (SPARC), basement membrane 40, or "culture shock" protein [373,374]. Osteonectin is an antiadhesive protein because it inhibits cell spreading, induces rounding of cells, and disassembles focal adhesions [375]. Other activities of osteonectin include calcium-dependent binding to collagens and thrombospondin, binding to plateletderived growth factor-AB and-BB, and regulation of cell proliferation and MMP expression [375]. The gene S P A R C or ON is located on chromosome 5q31.3-q32 [376]. The gene is expressed at high levels in tissues undergoing morphogenesis, remodeling, and wound repair. It is also made by cells of osteoblastic lineage and the hypertrophic chondrocytes of the growth plate [377-381]. The S P A R C gene is also expressed in several postnatal nonskeletal tissues, including salivary and renal tubular epithelium [382]. Osteonectin is the most abundant noncollagenous protein in mineralized bone matrix [372]. The S P A R C
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William G. Cole
gene encodes a full-length protein of 303 amino acids, including the signal peptide of 17 amino acid residues. The core molecular weightis 33 kDa [383]. The protein extracted from bone has an apparent molecular weight of 43 kDa due to posttranslational modifications such as glycosylation [372]. The mature human protein consists of 286 amino acid residues divided into three domains [384-387]. An amino-terminal acidic segment (residues 1-52) binds five to eight calcium ions with low affinity and mediates interactions with hydroxyapatite. A follistatin-like domain (residues 53-137) contains five disulfides and an N-linked oligosaccharide at Asn 99. Finally, an oL-helical domain contains two EF hand, high-affinity, extracellular calcium binding sites (EC domain, residues 138-286). Crystal structure analysis showed that the follistatin and extracellular calcium domains interact through a small interface that involves the EF hand pair of the extracellular domain [385,388,389]. The elongated follistatin domain is structurally related to serine protease inhibitors of the Kazal family. Residues implicated in cell binding, inhibition of cell spreading, and disassembly of focal adhesions cluster on one side of osteonectin opposite the binding epitope for collagens and the N-linked oligosaccharide. Crystal structure analysis also showed that the collagen-binding epitope in the helix oLA is partially masked by helix oLC [388]. Deletion of helix oLC produced a 10-fold increase in collagen affinity, similar to that seen after proteolytic cleavage of this helix [390]. Five residues were crucial for collagen binding: R149 and N156 in helix otA and L242, M245, and E246 in a loop region connecting the two EF hands of osteonectin [390]. These residues were spatially close and formed a flat ring of 15 A, which matches the diameter of a triple-helical collagen domain. Nearly identical binding characteristics were displayed by types I and IV collagens. Mice deficient in osteonectin are normal at birth and lack any evidence of connective tissue anomalies. By 6 months of age, the mice develop severe eye pathology [391]. Impaired wound healing is also apparent in osteonectin-deficient mice [392]. The anomalies are consistent with osteonectin normally playing a role in the migration of fibroblasts into the wound and in the formation of granulation tissue.
OSTEOCALCIN Osteocalcin is a small protein that accounts for approximately 10% of the noncollagenous protein of bone [393]. It is also called bone ~/-carboxyglutamate or bone Gla protein. It is one of several Gla proteins found in the skeleton. Matrix Gla protein is synthesized in
cartilage [394]. Other Gla proteins, such as protein S, are made elsewhere and are deposited in the bone matrix from the circulation [395]. The Gla proteins have in common the presence of glutamic acid residues that have been ~/-carboxylated by a specific ~/-carboxylase that requires vitamin K as a cofactor [396]. These ~/-carboxyglutamate residues have a high affinity for mineral ions such as Ca 2+ and for hydroxyapatite crystals. The osteocalcin gene, BGLAP, is located on chromosome 1q25-q31 [397,398]. The gene is very small (< 1 kb) and contains only four exons [399]. Exons 1-3 code for the preproosteocalcin, whereas exon 4 codes for the mature protein. The gene is specifically expressed by osteoblasts and is consequently limited to bone, specifically to regions destined for mineralization [400]. Mice lacking osteocalcin develop a phenotype marked by higher bone mass and bones of improved functional quality [401]. Histomorphometric studies performed before and after ovariectomy showed that the absence of osteocalcin leads to an increase in bone formation without impairing bone resorption. These findings, as well as those from detailed studies of bone mineralization in osteocalcin-deficient mice, provide evidence that osteocalcin is a determinant of bone formation and that it is also needed to stimulate bone mineral maturation [401,402]. However, cloning of the mouse osteocalcin gene has shown that the gene structure is complex. It consists of three genes, all transcribed in the same direction, within a 23-kb region of genomic DNA [403]. The genes were named osteocalcin gene 1, osteocalcin gene 2, and osteocalcin-related gene from the 5' end of the osteocalcin cluster. The osteocalcin 1 and osteocalcin 2 genes each contain four exons, whereas the osteocalcinrelated gene contains, in addition to the four exons typical of osteocalcin, exon 1 that is not translated. There is no translational stop codon in any of these genes, and each can be transcribed. The osteocalcin 1 and osteocalcin 2 genes are expressed only in bone, whereas osteocalcin-related gene is expressed in kidney but not in bone. The protein encoded by the osteocalcinrelated gene is the same as nephrocalcin, a calciumbinding protein of the kidney [404]. The osteocalcin promoter has been studied in detail. Responsive elements for 1,25-dihydroxyvitamin D3, glucocorticoids, and tumor necrosis factor-~ have been identified [405-407]. Other transcriptional regulatory sites such as one that binds MSX1 and MSX2, two homeodomain-containing proteins, have also been identified [408]. Two osteoblast-specific elements, OSE1 and OSE2, are present in the mouse osteocalcin gene. OSE2, which is upstream of OSE1, appears to regulate the expression of the osteocalcin gene by mature and immature osteoblasts, whereas OSE 1 appears to regulate expression of the gene by immature osteoblasts.
1. Structure of Growth Plate and Bone Matrix
The full-length protein contains 100 amino acid residues [394,399]. There is a signal peptide of 23 amino acid residues and a propeptide of 28 residues. The mature protein contains 49 amino acid residues. In humans, the protein contains 2 rather than 3 Gla residues seen in other species [409]. The predicted structure of the protein consists of two antiparallel oL-helical domains connected by a 13 turn [410]. A disulfide bond between cysteines 23 and 29 stabilizes the structure. The Gla residues have affinity for free Ca 2+ and Ca2+-containing proteins. Calcium binds to the carboxyl groups of the Gla residues and to the opposing carboxyl groups of aspartic and glutamic acid residues in the two helical domains of osteocalcin [411].
MATRIX Gla PROTEIN Matrix ~/-carboxyglutamic acid protein, or matrix Gla protein, is a highly insoluble protein of bone. It belongs to a family of vitamin K-dependent Gla proteins of the extracellular matrix. It was initially isolated from bovine bone [412]. The gene encoding matrix Gla protein, MGP, is located on chromosome 12p13.1-12.3 [398]. The human gene spans 3.9 kbs of chromosomal DNA and consists of four exons separated by three large intervening sequences that account for more than 80% of the gene. The N-terminal sequences of the known vitamin Kdependent vertebrate proteins reveal a transmembrane signal peptide, followed by a putative ~/-carboxylation recognition site and a Gla-containing domain. Each of these regions corresponds to a separate exon in MGP. The MGP gene also contains a fourth exon of unknown function that codes for 11 residues and lies between the transmembrane signal peptide and the putative recognition site for the ~/-carboxylase. This four-exon organization is essentially identical to that of bone Gla protein and is quite different from the two-exon organization encoding this region in the other known vitamin Kdependent proteins [398]. Analysis of the MGP gene promoter revealed, in addition to the typical TATA and CAT boxes, the presence of a number of putative regulatory sequences homologous to previously identified hormone and transcription factor responsive elements. In particular, two regions of the promoter were delineated containing possible binding sites for retinoic acid and vitamin D receptors [398]. The MGP gene is expressed in many tissues, particularly in lung and cartilage [413,414]. Despite the widespread expression of MGP, gene expression is limited to smooth muscle cells of the arteries and the chondrocytes
25
[414]. In the growth plate, MGP is most highly expressed in proliferative chondrocytes and in the lower hypertrophic chondrocytes at the site where cartilage will calcify [414-416]. MGP expression was not detected in osteoblasts and to only a low level in fibroblasts of the periosteum and perichondrium [417]. The full-length matrix Gla protein contains 103 amino acid residues [394]. It contains a signal peptide of 19 amino acid residues. The mature protein contains residues 20-96. The C-propeptide from residues 97-103 is cleaved. It has been proposed that the amino acid substitution of Lys 79 for Gln 79 in the human protein allows removal of additional basic residues from the C terminus by a carboxypeptidase B-like activity [418]. ~/-Carboxylation of Glu occurs at residues 21,56,60,67, and 71 of the full-length protein [394]. Serine residues at positions 22,25, and 28 of the full-length protein, located in the amino terminus of the mature protein, are partially phosphorylated. Phosphorylation occurs in the recognition motif Ser-X-Glu/Ser(p) [419]. The importance of matrix Gla protein as an inhibitor of mineralization is demonstrated in mice that lack matrix Gla protein. The phenotye consists of calcification of arteries and the inappropriate calcification of cartilages, including growth plates, resulting in short stature, osteopenia, and fractures [417].
BONE SIALOPROTEIN Bone sialoprotein is the second major sialoprotein of bone [2]. It is also referred to as bone sialoprotein-2 or integrin-binding sialoprotein. It constitutes approximately 12% of the noncollagenous protein of human bone [2]. It binds to calcium and hydroxyapatite, cells, and collagens. The gene for bone sialoprotein or integrin-binding sialoprotein (IBSP) is located on chromosome 4q21-q25, which is also the location of the distinct SPP1 gene for osteopontin. Gene expression is more limited than that for osteopontin [243]. The IBSP gene is expressed by hypertrophic chondrocytes in the growth plate, in a subset of osteoblasts at the onset of matrix mineralization, and in osteoclasts [420]. Outside of the skeleton, bone sialoprotein is expressed in odontoblasts and in trophoblast of the placenta. The IBSP m R N A encodes a full-length protein of 317 amino acid residues and a signal peptide of 16 amino acid residues. The mature protein has a deduced core molecular weight of 33,600 [243,421]. Bone sialoprotein purified from bovine bone has a molecular weight of 59 kDa due to its high content of carbohydrate. It is rich in sialic acid and O-linked glycosidically linked
26
William G. Cole
oligosaccharides. Bone sialoprotein also contains stretches of polyglutamic acid as opposed to polyaspartic acid in osteopontin. The stretches of up to 10 glutamic acid residues provide high-affinity binding to Ca 2+. This characteristic is likely important in the role of bone sialoprotein in matrix mineralization. In addition, approximately half of the serine residues in the protein carry phosphate groups. An RGD sequence is located at the C terminus, in contrast to the central location in osteopontin. It enables bone sialoprotein to bind to cells via an integrin receptor of the vitronectin type (OLv[33). The RGD sequence is surrounded by tyrosine sulfation consensus sequences, although it is unclear whether sulfation affects the kinetics of binding. Bone cells attach to intact bone sialoprotein in an RGD-dependent manner, but frag'ments of bone sialoprotein can bind to cells in an RGD-independent manner.
BONE ACIDIC GLYCOPROTEIN-75 Bone acidic glycoprotein-75 (BAG-75) is another sialoprotein of mineralizing tissue. The function of the protein is unclear, but based on its affinity for hydroxyapatite, it is likely involved in mineral nucleation or growth or both. Its amino-terminal amino acid sequence is similar to that of osteopontin and dentin matrix protein-1. Although the complete sequence of BAG-75 and its gene location are unknown, it appears from antibody studies that BAG-75 and dentin matrix protein-1 and osteopontin are distinct proteins [422].
DENTIN MATRIX ACIDIC PHOSPHOPROTEIN- 1 Dentin matrix protein-1 was first isolated by cDNA cloning using a rat odontoblast mRNA library [423]. It was initially postulated to be specific to dentin but later was detected in calvaria and long bones [424]. The gene encoding dentin matrix acidic phosphoprotein-1, DMP1, is located within 150kbs of the bone sialoprotein locus and within 490 kbs of the SPP1 locus for osteopontin on chromosome 4q21. The gene contains six exons [422]. The gene encodes a protein chain of 513 amino acids, including a signal peptide [423]. The amino acid sequence is highly acidic and serine rich. Approximately 55 of the 107 serine residues of rat dentin matrix acidic phosphoprotein-1 are potential sites for phosphorylation [423]. The protein is very difficult to extract from bone. The full-length protein, with its posttranslational modifica-
tions, likely has a molecular weight of 150 kDa [422]. There is also one RGD site for cell binding.
OSTEOPONTIN Osteopontin, also known as secreted phosphoprotein 1, is a secreted glycoprotein with the characteristics of a matricellular protein [425]. It was named osteopontin because it was proposed to act as a "bone bridge" from bone cells to hydroxyapatite [426]. It is highly expressed in bone but also by many cell types, including hypertrophic chondrocytes, odontoblasts, macrophages, as well as endothelial, smooth muscle, and epithelial cells [273,427]. Osteopontin is involved in a diverse range of biological processes, including biomineralization, cell attachment and cell signaling, cell migration, inflammation, and leukocyte recruitment. Osteopontin is a potent inhibitor of apatite formation and growth [428]. The inhibition was dose dependent and was abolished when phosphate groups were removed from the osteopontin [428,429]. Osteopontin blocked crystal elongation [428]. The osteopontin gene, SPP1 (secreted phosphoprotein 1), maps to chromosome 4q21-q25 [421,430]. It contains seven exons spanning approximately 9kbs [431,432]. Differential RNA splicing involving 42 nudeotides of exon 5 generates two alternatively spliced products in humans [430]. Specific sequences in the Y region of the SPP1 gene have been found to be regulated by hormones and growth factors associated with bone formation and bone remodeling. Some of the regulatory factors include TGF-[31, TGF-[32, retinoic acid, PTH, endothelin, proinflammatory cytokines, some BMPs, and 1,25-dihydroxyvitamin D3 [433-438]. Bone cells are a major site of synthesis and secretion of phosphorylated osteopontin [439]. In bone, osteopontin was localized to osteoblasts, lining cells, osteocytes, and cells with fibroblastic morphology associated with the periosteum [440,441]. This protein is also synthesized by hypertrophic chondrocytes [439]. The full-length human osteopontin protein contains 300 amino acid residues, including a signal peptide of 16 amino acid residues [442]. The protein is acidic, phosphorylated, and rich in sialic acid [443]. Three regions of the protein are highly conserved, including the aminoterminal one-fourth of the chain, a segment around the RGD integrin-binding sequence, and the extreme C terminus [442]. Four of the 9 or 10 residues in the poly-Asp region (residues 70-80) are strictly conserved. This sequence may be involved in the attachment of the protein to calcium phosphate apatite and in the regulation of growth of the bone crystals [429].
2,7
1. Structure of Growth Plate and Bone Matrix
Osteopontin has an R G D cell binding site, a calcium binding site, and two heparin-binding domains [444]. The RGD domain (residues 128-130) interacts with cell surface integrins OLv[33, e~v[31, and OLv[35to regulate cell attachment and spreading, intracellular signaling, and cell migration [445,446]. A cryptic SVVYGLRcontaining domain interacts with OL9~1 integrins after thrombin cleavage at residues 137 and 138 [447]. Osteopontin does not bind the standard form of CD44 (hyaluronic acid receptor) but may bind various isoforms of CD44 [448]. Osteopontin contains two conserved amino-terminal domains with heparin-binding homology that are likely to regulate its binding to the extracellular matrix. Osteopontin also binds directly to fibronectin, collagen, and osteocalcin [425]. Serine residues are also highly conserved. Many of them are phosphorylated (Pse) in Ser-X-Glu/Pse/ Asp or Ser-X-X/Glu/Pse motifs [443]. The highly conserved threonines found at positions 107,116, and 121 contain O-linked oligosaccharides [449]. A potential N-glycosylation site is present at residues 63-65. Unidentified sites of sulfation also exist in osteopontin. Extracted osteopontin peptides from bone show that the protein exists as a heterogeneous mixture of molecules that differ in their extent of posttranslational modification [450]. In particular, bone contains osteopontin molecules that differ widely in the extent of serine and threonine phosphorylation. Similar findings have been observed in the osteopontins produced by cultured osteoblasts. In one study, a 55-kDa form, produced by immature osteoblasts, was less phosphorylated and sulfated than a 44-kDa form produced by more mature osteoblasts [451]. The 55-kDa form may have a localized function because it was secreted into cement lines, whereas the 44-kDa form may have a more widespread role in the regulation of mineralization throughout the bone. Glutamines at positions 34 and 36 are substrates for transglutaminase [452]. Transglutaminase-promoted cross-linking may account for the higher-molecularweight aggregates of osteopontin in bone extracts [453]. Mice that lack osteopontin show no evidence of skeletal developmental anomalies in the neonatal period [454]. However, spleen cells from the deficient mice were better able to form osteoclasts than wild-type spleen cells. The phenotype became more pronounced with age. At 4-6 months of age, the osteopontin-deficient mice had two times the trabecular bone volume and three times the number of osteoclasts than wild-type mice [455]. The osteopontin-deficient mice were also resistant to ovariectomy-induced bone resorption, although the osteoclast numbers were not significantly different than those in sham-operated wild-type mice. This finding supports
the proposal that osteopontin is an important regulator of osteoclast activity [455].
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446. Liaw, L., Lindner, V., Schwartz, S. M., Chambers, A. F., and Giachelli, C. M. (1995). Osteopontin and beta 3 integrin are coordinately expressed in regenerating endothelium in vivo and stimulate Arg-Gly-Asp-dependent endothelial migration in vitro. Circ. Res. 77, 665-672. 447. Smith, L. L., Cheung, H. K., Ling, L. E., Chen, J., Sheppard, D., Pytela, R., and Giachelli, C. M. (1996). Osteopontin N-terminal domain contains a cryptic adhesive sequence recognized by alpha9betal integrin. J. Biol. Chem. 271, 28485-28491. 448. Weber, G. F., Ashkar, S., Glimcher, M. J., and Cantor, H. (1996). Receptor-ligand interaction between CD44 and osteopontin (Eta-l). Science 271, 509-512. 449. Butler, W. T. (1989). The nature and significance of osteopontin. Connect. Tissue Res. 23, 123-136. 450. Neame, P. J., and Butler, W. T. (1996). Posttranslational modification in rat bone osteopontin. Connect. Tissue Res. 35, 145-150. 451. Sodek, J., Chen, J., Nagata, T., Kasugai, S., Todescan, R., Jr., Li, I. W., and Kim, R. H. (1995). Regulation of osteopontin expression in osteoblasts. Ann. N. Y. Acad. Sci. 760, 223-241. 452. Prince, C. W., Dickie, D., and Krumdieck, C. L. (1991). Osteopontin, a substrate for transglutaminase and factor XIII activity. Biochem. Biophys. Res. Commun. 177, 1205-1210. 453. Sorensen, E. S., Rasmussen, L. K., Moiler, L., Jensen, P. H., Hojrup, P., and Petersen, T. E. (1994). Localization of transglutaminase-reactive glutamine residues in bovine osteopontin. Biochem. J. 304 (Pt. 1), 13-16. 454. Rittling, S. R., Matsumoto, H. N., McKee, M. D., Nanci, A., An, X. R., Novick, K. E., Kowalski, A. J., Noda, M., and Denhardt, D. T. (1998). Mice lacking osteopontin show normal development and bone structure but display altered osteoclast formation in vitro. J. Bone Miner. Res. 13, 1101-1111. 455. Yoshitake, H., Rittling, S. R., Denhardt, D. T., and Noda, M. (1999). Osteopontin-deficient mice are resistant to ovariectomyinduced bone resorption. Proc. Natl. Acad. Sci. USA 96, 8156-8160.
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2 I Bone Cell Biology Osteoblasts, Osteocytes, and Osteoclasts JANE E. AUBIN* and JOHANN. M. HEERSCHE t *Department of Medical Genetics and Microbiology, Faculty of Medicine, Toronto, Ontario, Canada tDental Research Institute, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
latory mechanisms controlling osteoblast development, a process comprising multiple proliferation and differentiation stages, and their activities and lifetimes. In addition, the mesenchymal stem and progenitor cell pools from which osteoblasts derivemwhich are different from those that produce osteoclasts early in mammalian development [5]mare currently the subject of exciting new research with considerable potential clinical application. Osteoclasts are mononuclear or multinuclear cells located on or adjacent to bone surfaces that stain positive for tartrate-resistant acid phosphatase (TRAP), have receptors for the calcium-regulating hormone calcitonin (CT), and are able to resorb bone [6]. They also contain high amounts of proton pumps (V-H+-ATPase) and of the proteolytic enzymes matrix metalloproteinase-9 (MMP-9) and cathepsin K [7]. Osteoclasts are terminally differentiated cells of hemopoietic origin and do not undergo mitosis. Their size and number increase due to differentiation and fusion of mononuclear precursor cells either with each other or with existing osteoclasts. Several stages have been identified in the activity cycle of multinucleated osteoclasts: migration to the bone surface, attachment to bone, followed by formation of a sealing zone delineating and surrounding an area in which extensive membrane ruffling is observed. Once established, this sealed-off area between ruffled border and bone surface is acidified by the plasmalemmal vacuolar-type H+-ATPase (V-ATPase) located on the osteoclast ruffled border membrane [8]. The mineral component of bone is dissolved by protons that are generated by the enzyme carbonic anhydrase II and
INTRODUCTION Bone tissue consists of a mineralized organic matrix formed and maintained by cells that are continuously engaged in modeling and remodeling to adapt the tissue to the demands placed on it. The demands are both mechanical (e.g., adaptation to stress and strain) and physiological (e.g., related to bone being the predominant reservoir of calcium available for instant mobilization). The cells that produce the bone matrix are osteoblasts, which are cuboidal, polar, basophilic cells that line bone surfaces and synthesize and subsequently mineralize the organic bone matrix. They express high levels of alkaline phosphatase (ALP) and synthesize and secrete collagen and a diverse set of noncollagenous proteins that comprise the organic matrix of bone. When their matrixproducing phase terminates, some osteoblasts disappear through programmed cell death (apoptosis), whereas others differentiate into fiat cells lining the bone surface (lining cells) or become cells enclosed in the bone matrix in small lacunae (osteocytes) [1]. Osteocyte access to nutrients is provided and connections to other cells are made via thin cell processes that are located in a canalicular network permeating all bone tissues. In adult humans, approximately 80% of cancellous bone surfaces and 95% of intracortical bone surfaces are covered by lining cells. The remainder are occupied by osteoblasts or by bone resorbing cells called osteoclasts. The regulatory mechanisms that govern which osteoblasts assume which fate are only beginning to be elucidated [2-4]. However, information is rapidly accumulating on genes and regu-
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Jane E. Aubin and Johan N. M. Heersche
extruded into the extracellular space by V-ATPases. Next, the organic matrix of bone is degraded by several enzymes secreted by osteoclasts, mainly MMPs and cysteine proteinases [9,10]. As mentioned previously, the skeleton adapts to the demands placed on it by forming and/or resorbing bone tissue at appropriate sites. Maintaining the integrity of the adult skeleton requires an equilibrium between the amount of bone formed and the amount resorbed, which appears to be accomplished by close cooperation between osteoclasts resorbing bone and osteoblasts forming it. This coordinated activity is most obvious in the microscopic sites in which bone formation occurs after prior bone resorption (the activation-resorption formation sequence) [1,11]. The first stage, activation, represents the migration of osteoclast precursors to an area of the bone surface to be resorbed, attachment of these precursor cells, and subsequent fusion of these cells into multinuclear osteoclasts. During the resorptive phase, bone is resorbed by osteoclasts, possibly with the help of numerous mononuclear phagocytes present in the area. In the reversal phase [12,13], cells resembling mononuclear phagocytes and/or other cell types modify the resorbed surface and deposit a structure morphologically identified as a cement line. This phase is followed by a formation phase, during which osteoblasts differentiate at the sites previously resorbed and start to deposit osteoid and bone. Upon completion of this phase, the site enters a resting phase, with no discernable osteoid remaining between the lining cells and the mineralized bone. The sites at which this type of remodeling occurs have been defined as bone remodeling units or basic multicellular units (BMUs). In rat cancellous bone, the total duration of a complete remodeling cycle is approximately 1 month, and in humans it is estimated to be approximately 3 months [1]. The cells participating in the BMUs are osteoclasts and their progenitors, osteoblasts and their progenitors, and, importantly, endothelial cells from the capillary vessels associated with this remodeling. These interact in a variety of ways through autocrine and paracrine mechanisms involving various cytokines, growth factors, and other mediators. Despite the close physiological interactions of the two main cellular systems in bone, there are effectively separate and distinct origins of osteoclasts (hemopoietic cell origin) and stromal/osteoblast lineages in the developing fetus and throughout mammalian development [5]. Little is known about the early phenotypic stages of the osteogenic cells, the basic mechanisms governing the stem cell cycle, or the activation mechanisms relating to their physiological recruitment. It is known, however, that they are present on all bone surfaces. The suggestion that the osteoblast and related stem cells circulate
systemically has been revived recently with negligible evidence for relevance in normal physiological bone anabolic processes.
ONTOGENY OF OSTEOBLASTS AND CONTROL OF OSTEOBLAST DEVELOPMENT There are no unique identifying markers for the multipotential stem cells that give rise to a number of mesenchymal tissues, including bone and cartilage; therefore, their existence and presence are usually defined by a variety of phenotypic and functional or retrospective outcome assays [14]. This has led to an often confusing and still evolving nomenclature for these cells, with terms that include connective tissue stem cells, stromal stem cells, marrow stromal cells, stromal fibroblastic stem cells, mesodermal progenitor cells (MPCs), multipotent adult progenitor cells (MAPCs), and mesenchymal stem cells (MSCs). The most commonly used term is MSC, but this term is often used with implications of the presence of a pure population when clearly heterogeneous populations of cells are being assayed and studied. Although the term also obscures distinctions between cellular origin and developmental potential, based on its widespread use in the field the term MSC is used in this chapter. Within this system, there may be a hierarchy of stem and progenitor cells [15], as there is in the hemopoietic system, in which the cells yielding bone are the osteogenic cells. Their development into osteoblasts occurs through a sequence of phenotypic transitions that have been described using morphological and molecular criteria, as discussed later.
S t e m Cells, O s t e o p r o g e n i t o r s , and Plasticity Friedenstein first showed that postnatal bone marrow stroma contains cells that have both significant proliferative capacity and the capacity to form bone when transplanted in vivo in diffusion chambers. Subsequently, he and others demonstrated that in addition to bone, cartilage, adipocytes, and fibrous tissue also form in vivo and all the tissues can arise from transplanted single colonies or colony-forming units-fibroblasts (CFU-Fs) [16-19]. In vivo analyses of stromal cells have been augmented by functional assays in vitro that show formation of a range of differentiated cell phenotypes that include not only osteoblasts, chondrocytes, and adipocytes but also muscle cells and have led many to identify stromal populations as MSCs. However, experiments needed to address whether marrow stroma contains a definitive stem cellmby the definition of self-renewal capacity and
2. Bone Cell Biology
ability to repopulate all the appropriate differentiated lineages or even by less stringent definitions [20]--are only beginning to be done. For example, although expanded populations of human stromal cells are routinely reported to express a capacity to undergo differentiation along multiple mesenchymal lineages, when individual colonies were assessed in one study, only two of six appeared to express multilineage capacity and none were explicitly tested for self-renewal capacity [21]. This supports Friedenstein's original work in which he estimated that approximately 15% of the total CFU-Fs have stem cell-like properties [16,22]. Recent studies also show that CFU-Fs are heterogeneous in size, morphology, and potential for differentiation in vivo [23]. Limiting dilution and very low-density plating experiments have also revealed that only a proportion of CFU-Fs are CFU-alkaline phosphatase (CFU-ALP) and that only a proportion of these are CFU-osteogenic (CFU-O) (clonogenic bone colonies or bone nodules), although some variations are reported between different species. CFU-adipocytic (CFU-A) also comprise only a subset of CFU-Fs [24,25]. These data are consistent with the view that CFU-Fs belong to a lineage hierarchy in which only some of the cells are multipotential stem or primitive progenitors, whereas others are more restricted [15] (Fig. 1). A variety of studies on clonally derived immortalized (e.g., spontaneously or via large T antigen expression) cell lines derived from stroma, bone-derived cells, or other mesenchymal/mesodermal tissues, such as the mouse embryonic fibroblast line C3H10T1/2, the fetal rat calvaria-derived cell lines RCJ3.1 and ROB-C26, and the mesodermally derived C1 line, have also provided evidence for the existence of multipotential mesenchymal progenitor or stem cells capable of giving rise to multiple differentiated cell phenotypes, including osteoblasts, chondroblasts, myoblasts, and adipocytes [15]. However, two different kinds of events have been proposed to underlie MSC commitment. The first is a nonrandom, single-step process in which multipotential progenitors become restricted to a single mesenchymal lineage by particular environmental or culture conditions (e.g., soluble inducers, different substrates, and cell density), such as in C1 [26] (Fig. 1A). A second model comprising a stochastic process with an expanding hierarchy of increasingly restricted progeny has also been proposed (e.g., RCJ3.1) [15] (Fig. 1B). In keeping with the latter concept is the previously mentioned fact that CFU-Fs are heterogeneous with respect to phenotype and function. For examPle, there is evidence that single committed progenitors (i.e., progenitor cells restricted to particular lineages) can be identified by functional assays of their differentiation capacity in vitro (i.e., CFU-O or CFU-A assays). However, a number of studies on rodent
45
FIGURE 1 Schematic of stem cell commitment via either a single-step process (A) or a multistep hierarchical process with increasing lineage restriction (B) to various end-stage mesenchymal cell types. Also depicted is apparent plasticity between osteoblasts and adipocytes. Known transcription factors playing regulatory roles in the mesenchymal lineages are indicated.
and human trabecular bone cells and clonally derived lines of bone marrow stromal cells suggest that bipotential adipocyte-osteoblast precursor cells also exist [27-29]. It has also been suggested that the inverse relationship sometimes seen between expression of the osteoblast and adipocytic phenotypes in marrow stroma (e.g., in osteoporosis or in some culture manipulations) may reflect the ability of single or combinations of agents to alter the commitment or at least the differentiation pathway that these bipotential cells transit [28,30]. In some cases, individual colonies are seen in which both
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Jane E. Aubin and Johan N. M. Heersche
osteoblast and adipocyte markers are present simultaneously [31]. However, whether a clearly distinguishable bipotential adipo-osteoprogenitor can be identified or other developmental paradigms such as plasticity or transdifferentiation underlie expression in these two lineages needs to be further analyzed. For example, dedifferentiation has been posited to account for observations in some cultures of marrow stroma in which highly differentiated adipocytes are thought to revert to a less differentiated, more proliferative fibroblastic precursor phenotype and then to osteogenic phenotype (Park, 1999 #771]). On the other hand, osteoblasts differentiated to the point of expressing osteocalcin (OCN) are able to undergo rapid differentiation events that lead to essentially 100% of the formerly osteoblastic cells expressing adipogenesis when they are transfected with the nuclear receptor family member peroxisome proliferator activated receptor '~2 (PPAR~/2)[32]. Thus, although OCN is a very late marker of osteoblast maturation, data are consistent with osteoblastic cells being able to transdifferentiate into an adipogenic phenotype, an outcome of significant clinical interest in osteoporosis and the aging or immobilized skeleton [27,28]). A variety of observations suggest that a bipotential osteochondroprogenitor may also exist. However, as for the adipocyte-osteoblast phenotypes, the ability to transdifferentiate or change expression profiles may also characterize the osteoblast-chondroblast lineages [15]. Such observations and their cellular and molecular basis have led to considerable interest in the concept of "plasticity" of stromal and other cell types [33-38]. However, the possible presence of low frequencies of undifferentiated/uncommitted stem cells and multi- and bipotential progenitors in cultures that may also contain higher frequencies of monopotential progenitors with developmental plasticity often complicates the ability to unambiguously determine the nature of the cells being affected. Difficulties in establishing unambiguous evidence for multipotentiality, together with certain discrepancies between results in calvaria versus stromal and other populations, highlight the need for more markers and experiments to distinguish the molecular mechanisms underlying the ability of cells to express multipotentiality, the number and nature of commitment steps to restricted phenotype(s), and both physiological and pathological mechanisms that may govern plasticity. Stem cells in the adult organism (adult stem cells) are receiving much attention for most lineages, including those of the skeleton. Established paradigms suggest that tissue-specific stem cells have the capacity to differentiate only into cells of the tissue of origin; have less selfrenewal ability than, for example, embryonic stem (ES) cells; and, although they differentiate into multiple lineages, do not have pluripotency. Recent work has
challenged these conclusions. For example, in addition to the differentiation capacity summarized previously, transplanted MSCs have been found in the brain, in which they have been suggested to differentiate into astrocytes [39] and to form functional neurons [40,41]. After transplantation of bone marrow or enriched hematopoietic stem cells (HSCs), skeletal myoblasts, cardiac myoblasts, endothelium, hepatic and biliary duct epithelium, lung, gut and skin epithelia, and neuroectodermal cells of donor origin have been detected, suggesting that tissue-specific stem cells can differentiate into lineages other than the tissue of origin [42-44]. Such studies have helped to rekindle interest in concepts of plasticity or transdifferentiation of stem and progenitor populations. However, as noted previously, most studies have not addressed, let alone demonstrated, whether a single tissue-specific stem cell differentiates into functional cells of multiple tissues [44]. The complexities of both the issue and the requirement for sophisticated experimental controls were highlighted by recent reports that at least some of what has been heralded as intrinsic plasticity may actually reflect fusion of donor and recipient cells [45,-47]. Notable are recent studies from Verfaillie and colleague, who identified among populations isolated from the marrow that contain stem cells with the capacity to generate osteoblasts cells they termed multipotent adult progenitor cells (MAPCs), which copurify with MSCs [48,49]. These cells have an extremely high proliferative capacity in vitro and differentiate at the single cell level not only into mesenchymal cells [48] but also into cells with visceral mesoderm, neuroectoderm, and endoderm characteristics in vitro. Strikingly, when injected into early blastocysts, single MAPCs contribute to most, if not all, somatic cell types [49]. Although many questions remain, including whether MAPCs are a rare population of ES cells that have "escaped" into adulthood, cells functionally characterized as MAPCs have been isolated from mouse, rat, and human bone marrow, which should allow comparisons between powerful rodent experimental models, including the classically pluripotent ES cells, and human-derived MAPCs. More transplantation assays, with assay of both longterm culture-initiating cells and cells capable of long-term repopulating ability determined by their ability to serially repopulate mice at limiting dilution, may advance the field [50]. Although such assays may be difficult to achieve for MSCs, especially in vivo, the results for MAPCs lend confidence to the view that strategies are available. Clear quantitation and understanding of the clonality of mesenchymal cell progeny, the ratios of stem to other more restricted progenitors in various stromal populations, the identifiable commitment and restriction points in the stromal cell hierarchy, the self-renewal capacity, and the repopulation capacity
2. Bone Cell Biology
of individual precursor cells should be goals. These issues are also increasingly important as work on stromal and related populations increases based on their proposed utility for tissue regeneration and as vehicles for gene therapy. It is also clear that parallel efforts must continue on alternate sources of cells with MSC and MAPC characteristics. Cells with features similar to adult bone marrow MSC have also been isolated from adult peripheral blood [51,52], fetal cord blood [53], and fetal liver, blood, and bone marrow [54]. Muscle satellite cells have also been found to be multipotential, undergoing myogenic, osteogenic, and adipogenic differentiation in vitro [55]. Cells with MSC-like properties, including the capacity for osteochondrogenic differentiation in vitro, have also been isolated from extramedullary adipose tissue [56,57], including the inguinal fat pad [58]. The relationship of these cells to each other developmentally, phenotypically, and functionally must be investigated further, but the fact that seemingly diverse populations of cells exist with apparently similar regenerative and developmental potential is important for development of cell and gene therapy approaches in the skeleton. Control of O s t e o b l a s t D e v e l o p m e n t In the past several years, significant advances have been made in identifying regulatory mechanisms underlying lineage restriction, commitment, and/or differentiation within some of the mesenchymal lineages (Fig. 1). The presence of master genes, exemplified by the MyoD, myogenin, and Myf-5 helix-loop-helix transcription factors in muscle lineages, is one paradigm in which one transcription factor is induced and starts a cascade that leads to sequential expression of other transcription factors and phenotype-specific genes [59]. A factor of a different transcription factor family, PPAR~/2, together with other transcription factors including the CCAAT/ enhancer binding (C/EBP) protein family, plays a key role in adipocyte differentiation [60]. Sox9, a member of yet another transcription factor family, is essential for chondrocyte differentiation, expression of various chondrocyte genes, and cartilage formation [61,62]. Understanding of genetic control mechanisms in skeletal development is advancing rapidly [63]. Runx2, a member of the runt homology domain transcription factor family, plays a crucial role in osteoblast development. Runx2 was identified in part based on its ability to regulate OCN [64,65]; ectopic expression of Runx2 in nonosteoblastic cells leads to expression of osteoblastspecific genes including OCN, and, strikingly, deletion of Runx2 in mice leads to animals in which the skeleton comprises only chondrocytes and cartilage without any evidence of bone [66] and in which bone is formed post-
47
natally [67]. Haploinsufficiency in mice [66] and in humans leads to the cleidocranial dysplasia phenotype (Mundlos, 1997 #539; Otto, 1997 #538) [68,69]. Runx2 is the earliest osteoblast differentiation marker currently known; its expression during development and after birth is high in osteoblasts, and it is upregulated in cultures treated with bone morphogenetic proteins (BMPs) and other factors that stimulate bone formation [70]. However, in contrast to MyoD and PPAR~/2, Runx2 is not a master gene; it is necessary but not sufficient to support differentiation to the mature osteoblast phenotype [71,72]. Recent data also suggest that a fine balance of Runx2 expression levels may be required for skeletal health since overexpression ofRunx2 in osteoblasts appears to block osteoblast maturation, leading to an osteoporotic phenotype [73]. Interestingly, recent studies have also suggested that at least some hypertrophic chondrocytes express Runx2 and the development/maturation of at least some chondrocytes is aberrant in Runx2-deficient mice, although the skeletal site-specific nature of the defects suggests that much more information is needed [69,74-77];. With respect to the issue of plasticity, it is interesting that PPAR~/2 may transdifferentiate osteoblastic cells to adipocytes via its ability to downregulate Runx2 [78]. The recently identified novel zinc finger-containing transcription factor Osterix (Osx) is also required for osteoblast development and bone formation and lies downstream of Runx2 in osteoblast development [79]. Because Osx null preosteoblasts express typical chondrocyte marker genes, it has been proposed that Runx2-expressing osteoblast precursors are still bipotential for osteoblast-chondroblast development. Ablation of Indian hedgehog (Ihh), a member of the Hedgehog family of secreted growth factors, leads to mice with a disorganized growth plate, as expected based on data indicating that Ihh regulates chondrocyte differentiation. However, Ihh null mice also have no osteoblasts in bone formed by endochondral ossification [80]. Since the aberrant chondrocyte differentiation phenotype can be mimicked by HIP (hedgehog interacting protein) overexpression in transgenic mice that have osteoblasts [81], the data suggest that the failure of osteoblast development in endochondral sites is due specifically to lack of Ihh rather than a secondary effect due to aberrant chondrocytes. Interestingly, Runx2 expression, which is normally high in osteoblasts in endochondral bones, is absent in Ihh -/- mice. Also, consistent with the fact that Ihh is not normally expressed in intramembranous bones, osteoblast differentiation occurs normally in these bones in Ihh -/- animals. These data support the concept that Ihh may be a regulator of Runx2 and osteoblast development but in a skeletal site-specific manner.
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The recent elucidation of the osteoprotegerinR A N K - R A N K L pathway underlying the stromal cellhemopoietic cell interactions regulating osteoclast formation and activity [82] also makes it tempting to speculate that a reciprocal or related pathway may regulate osteoblast formation and activity. The fact that hemopoietic cells influence osteoblast development in the bone marrow stromal cell model in vitro [24,83] lends support for this hypothesis. Also, the fact that other apparently osteoblast-specific cis-acting elements have been found in several genes suggests that there are other important osteoblast-associated or-specific transcription factors still to be elucidated. Later, a variety of other transcription factors and receptor signaling pathways that clearly also influence the rate and amount of bone formed are discussed. Antibodies a n d O t h e r Tools for MSC/MAPC Biology Identification and enrichment of stem and progenitor cells in hematopoietic cell populations have been aided enormously by the availability of a large and expanding number of antibodies [84]. Some of the same reagents, and others prepared specifically against osteoblastic and stromal populations, are beginning to be used in MSC and osteoblast biology [14]. In this section, the focus is on antibodies that show promise for identification of stem and precursor populations; later, those more useful in studying cells and gene products of more mature osteoblast lineage cells are discussed. It is also important to recognize that none of the available antibodies is absolutely lineage and cell stage specific. In addition, no single marker or antibody is available by which unambiguously or solely to isolate definitive stem cells in any lineage, including HSCs, and it is combinations of markers that have yielded the greatest advances. Several antibodies reacting with surface antigens on human MSCs in vitro have been generated, including the antibodies STR0-1 [85], SH-2, SB-10 [86,87], and HOP-26 [88] (Fig. 2). The cell surface antigen recognized by STRO-1 is unknown, but its expression is restricted to a subpopulation of cells in fresh adult human bone marrow, including CFU-F, and the STRO-1 + fraction of bone marrow contains osteogenic precursors [89]. Among hybridoma cell lines prepared by Bruder and colleagues [86] by injecting human stromal cells induced with osteogenic medium into mice, SB-10 reacts with marrow stromal cells and osteoprogenitors but not with more differentiated cells (i.e., those already expressing ALP). It is now known that SB- 10 recognizes activated leukocyte cell adhesion molecule (ALCAM) [87], and although culture-expanded human MSCs homogeneously express SB10, ALCAM is not restricted to MSCs or osteoprogenitor
cells. ALCAM is an immunoglobulin (Ig) superfamily ligand for CD6 antigen [90] that is present in lymphoid tissue and may be involved in homing ofhemopoietic cells [91,92]. Nevertheless, ALCAM expression may be useful in combination with other reagents for cell subfractionation, and it is worth considering its role in osteoblast development in more detail since treating human MSCs in vitro with SB-10 accelerates osteogenic differentiation, implicating ALCAM in this process [87]. The SH-2 antibody recognizes endoglin (CD 105), which is a transforming growth factor-13 (TGF-13) receptor accessory molecule (sometimes called TGF-13 receptor III) that is present in many cell types, including connective tissue stromal cells, endothelial cells, syncytiotrophoblasts, and macrophages [93]. Its functions in TGF-13 receptor-ligand interactions and signaling are poorly understood, but it has been proposed to control chondrogenic differentiation of MSCs and interactions between these cells and hematopoietic cells in the bone marrow as well as in dermal embryogenesis and angiogenesis [94]. The HOP-26 antibody, raised against human bone marrow fibroblasts at an early stage of culture, reacts with cells close to newly forming bone in the periosteum and in trabeculae of developing human fetal limb and in adult trabecular bone, with a small proportion of cells located within the bone marrow spaces [88]. Immunopanning with HOP-26 isolates the majority of CFU-F and it is thus a useful antibody for selecting these cells to enrich osteoprogenitor populations from marrow [95]. These examples emphasize the caution needed when using monoclonal antibodies on viable cells versus fixed tissues in which specific epitopes may be masked or unmasked depending on treatment modalities and section site. As mentioned previously, combinations of antibodies, often coupled with other phenotypic traits, are frequently used for enrichment and isolation of progenitors. The STRO-1 bright/VCAM-1 +/COLL-I + population of human bone marrow has extensive proliferative capacity and differentiation capacity for osteogenesis and adipogenesis [96]. The bone/liver/kidney isoform of ALP has been used in a number of studies both in combination with other antibodies and alone [97]. Dual-color fluorescenceactivated cell sorting of human trabecular bone-derived cells with antibodies against STRO-1 and ALP indicates that early osteoblast precursors reside in the STROI+ALP - population, differentiated osteoblasts in the STRO-1-ALP + and STRO-1-ALP- populations, and cells of an intermediate developmental stage in the STRO-I+ALP + population [98]. Similar results have been obtained with human bone marrow stromal cells [99]. Osteogenic cells were sorted from mouse bone marrow based on light scatter characteristics, Sca-1 expression, and their binding to wheat germ agglutinin (WGA) [100]. Cells from the S c a - I + W G A bright gate, but
2.
BoneCell Biology
49 Lidell Apoptosis
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Collagen type I Osteocalcin Bone sialoprotein Osteopontin Factors
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and receptors
PTH/PTHrP-R PTHrP PDGF-Rc~
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++ ++ +++
+++ ++ +++
++ +++
FIGURE 2 P o s t u l a t e d transitions in the osteoblast lineage with expression profiles o f several well-established markers. The list is n o t exhaustive b u t does include some i m p o r t a n t categories o f molecules in the lineage and their utility to help define transitions in osteoblast differentiation and heterogeneity a m o n g osteoblast populations. Little is k n o w n a b o u t the abruptness o f t u r n - o n or t u r n - o f f o f these markers, and in m a n y cases expression levels m a y vary as changes to a continuum. - , no detectable expression; - / + , + , + + , and + + + , expression ranging f r o m detectable to very high; ~ + + + , heterogeneous expression in individual cells.
not from other gates,synthesized bone proteins and formed a mineralized matrix but lost this capacity when subcultured. Further immunophenotypic characterization showed that FsChighsschighLin-Sca 1+WGA bright cells expressed stromal (KM 16) markers and endothelial (Sab-1 and Sab-2) markers but not hemopoietic cell markers such as c-kit and Thyl.2. Sorted FSChighsschighLin-Sca-1 +WGA bright cells formed bone nodules in vitro. In contradiction to data showing the absence of osteopontin (OPN) expression in the STROI+ALP - population defined previously as comprising stem cells (Gronthos, 1999 # 1107), OPN expression combined with cell size and granularity was used to sort rat calvaria and rat bone marrow stromal cells to attempt to enrich for cells responsive to BMP-7; these were said to
have stem-like properties [101,102]. In any case, studies suggest that combinations of antibodies, with and without other cellular traits, permit the identification of cells of the osteoblast lineage at different stages of differentiation and support previous studies suggesting a hierarchy of marker expression during osteoblastic development in vitro [103] (Fig. 2). The studies discussed previously have clearly fractionated populations into subpopulations with different characteristics and differentiation potentials. Extensive immunophenotyping combined with quantification of the most primitive osteoprogenitors or MSCs and their progeny at single cell or colony levels so that clear lineage relationships and hierarchies can be established analogous to those that have been achieved in hemopoietic
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populations is just under way. Pittenger and colleagues reported that culture-expanded human MSCs are uniformly positive for SH2, SH3, CD29, CD44, CD71, CD90, CD106, CD 120a, and CD 124 but negative for CD14, CD34, and the leukocyte common antigen CD45 during culture expansion [21]. The phenotypes of mouse MAPCs (mMAPCs) in fresh bone marrow are unknown, but after extensive culture (in which their pluripotent stem cell properties are functionally tested, as described previously), they are CD34-CD44-CD45-c-Kit- and MHC class I- and II-; mMAPCs express low levels of Flk-1, Sca-1, and Thy-1 and higher levels of CD13 and stagespecific antigen-I [49]. Among other novel new strategies that hold promise are those in which promoters of osteoblast-associated genes expressed at different differentiation stages have been used to drive expression of green fluorescent protein in mice [104] and to fractionate subpopulations of osteoblastic cells from mice [105]. Such approaches are in their infancy but have potential to contribute new understanding of osteoblast lineage and of bone cell activity in general in health and disease [106].
OSTEOPROGENITOR CELLS AND REGULATION OF OSTEOBLAST DIFFERENTIATION AND ACTIVITY O s t e o p r o g e n i t o r Cells The morphological and histological criteria by which osteoblastic cells, including osteoprogenitors, preosteoblasts, osteoblasts, and lining cells or osteocytes, are identified has been extensively reviewed and will not be reiterated in detail here [103]. Morphological definitions are routinely supplemented by analysis of expression of osteoblast and bone-associated macromolecules, including the bone matrix molecules COLL-I, OCN, OPN, and bone sialoprotein (BSP),and transcription factors that regulate them and commitment/differentiation events (e.g., Runx2, Osx, AP-1 family members, Msx-2, and Dlx-5) (Fig. 2). Committed osteoprogenitors (i.e., progenitor cells restricted to osteoblast development and bone formation under default differentiation conditions) can be identified in bone marrow stromal cell populations and populations derived from calvaria and other bones by functional assays of their proliferation and differentiation capacity in vitro or, as often designated, the CFU-O assay. CFU-Os appear to comprise a subset of CFU-F and CFU-ALP [24,25,107,108]. With appropriate culture conditions, cells morphologically identical to cells described in vivo and subject to many of the same regulatory activities can be identified, and the deposited matrix contains the major bone matrix proteins.
Much has been learned from the in vitro bone nodule assay, in which the nature of both the osteoprogenitors and their more differentiated progeny has been investigated. Bone nodules represent the end product of the proliferation and differentiation of CFU-O or osteoprogenitor cells present in the beginning cell population. Estimates by limiting dilution indicate that these osteoprogenitor cells are relatively rare in cell populations digested from fetal rat calvaria (i.e., <1%) [109] and rat [24] and mouse [110] bone marrow stroma (i.e., 0.5-1 • 10-5 of the nucleated cells of unfractionated marrow or <1% of the stromal layer) under standard isolation and culture conditions. The number of nodules or colonies forming bone can be counted for an assessment of osteoprogenitor numbers recoverable from fetal calvaria or other bones (e.g., vertebrae [111,112] and the primary spongiosa of the femur metaphysis [113]) and bone marrow stroma [17] under particular assay conditions. However, evidence from rat calvaria cell bone nodule assays indicates the existence of at least two populations of osteoprogenitors. One population appears capable of constitutive or default differentiation in vitro: that is, in standard differentiation conditions (ascorbic acid, [3-glycerophosphate, and fetal calf serum), differentiation leading to the mature osteoblast phenotype appears to be a default pathway, whereas the other population, apparently more primitive based on cell sorting and immunopanning with ALP antibodies [114], undergoes osteoblastic differentiation only following the addition of specific inductive stimuli (Fig. 3). Thus, the addition of glucocorticoids (often dexamethasone, but natural corticosteroids have also been used [115]), other steroids (e.g., progesterone [116]), or other kinds of factors (e.g., BMPs [117] or bFGF [118]) increases the number of bone nodules or bone colonies in calvariaderived and bone marrow stromal cell cultures, suggesting the presence of "inducible" osteoprogenitor cell populations as well. Whether other precursor stages in addition to the multipotential or committed progenitors discussed previously can also be identified by combinations of assays in vitro remains to be explicitly tested. Whether all progenitors that differentiate to osteoblasts and make bone belong to the same unidirectional lineage pathway (i.e., immature progenitors induced by a variety of agents to undergo differentiation to mature osteoblasts), whether under all developmental situations osteoprogenitor cells must transit all recognizable differentiation stages (or may skip steps under appropriate conditions), and whether recruitment from other parallel lineages and pathways can result in functional osteoblasts remain to be established. However, recently we developed a statistically rigorous map, based on gene expression profiles, of the cell fate decisions that occur during osteoprogenitor differentiation in RC cultures
2. Bone Cell Biology
51
Decreasing Proliferative Capacityand Increasing Differentiation v
Transit amplifyingpopulations
Limited self-renewal Extensive proliferation Ik Asymmetricand/or symmetric divisions?
Unlimited self-renewal Asymmetric divisions Runx2/Cbfal,Osx Multipotential stem cell
Limited proliferation ~
O
Q
O-,-
Immature Mature "//" 0 osteoprogenitor I ~_ osteoprogeni I tor ~
~
O
0
~
~
Post-mitotic ~O O
@
O
Lining Cell
0
Apoptosis
/
O
Requiresstimulus, Defaultdifferentiation Osteocyte e.g.,dexamethasone,in vitrounderstandard Preosteoblast Mature fordifferentiation differentiationconditions osteoblast in vitro FIGURE 3 Postulatedtransitions in the osteoblastlineagewith changesin self-renewalcapacity,proliferation, and differentiationas detectablefrom in vitro colonyformation assaysand in vivo analysis. and showed that different developmental routes can be taken to achieve the same end point osteoblast phenotype [119]. These routes appear to involve both developmental dead ends (leading to the expression of genes not correlated with osteoblast-associated genes or the mature osteoblast phenotype) and developmental flexibility (the existence of multiple gene expression routes to the same developmental end point). Whether such developmental flexibility is an entirely stochastic process is not known, but it seems plausible that various regulatory molecules, including hormones, growth factors, and cytokines, may alter fate choices at specific bifurcation points on these pathways. In addition, as previously discussed, plasticity between mature cell phenotypes normally considered indicative of terminal differentiation can contribute to osteoblast pools at least in vitro. It is also worth considering whether the osteoprogenitors in calvaria, other bones and bone marrow stroma, blood, and other sources (e.g., pericytes) are the same [36,120-122]. As discussed in more detail later, they do appear to reach similar end points with respect to the ability to make and mineralize a bone matrix, but they may not be identical. Recent data have also indicated that in rat stromal populations, as in rat calvaria-derived populations, there are two pools of osteoprogenitors: those that differentiate in the absence of added glucocorticoids (assumed to be more mature) and those that do so only in its presence of glucocorticoids (assumed to be more primitive). However, the number of the former type is relatively low and thus detectable only at relatively high plating cell densities, and the latter type comprises the majority in stromal
populations [24]. Whether the two types or stages of progenitors are identical in other features to the progenitors in calvaria remains to be assessed rigorously, as must their relationship to multilineage CFU-F. It is also worth noting that in rat stroma, unlike in rat calvaria, limiting dilution analysis indicates that more than one cell type is limiting for nodule formation in vitro, suggesting a cell nonautonomous aspect to differentiation of the stromal CFU-O; osteogenic differentiation is enhanced, for example, when the nonadherent fraction of bone marrow or its conditioned medium is added to the adherent stromal layer [24]. A role for accessory cells in osteogenesis of human bone marrow-derived osteoprogenitors has also been reported [83]. The relationship of inducible osteoprogenitors that apparently reside in the nonadherent fraction of bone marrow and that are assayable under particular culture conditions (e.g., in the presence of PGE2 in rat [123] or as colonies in soft agar or methylcellulose in humans [124]) also remains to be determined. Direct and unambiguous comparisons have not been done but should be advanced as more markers for the most primitive progenitors, including stem cells, become available. Morphologically recognizable osteoblasts associated with bone nodules appear in long-term bone cell cultures at predictable and reproducible periods after plating. Recent time-lapse cinematography of individual progenitors forming colonies in low-density rat calvaria cultures indicated that primitive (glucocorticoid-requiring) osteoprogenitors divide approximately eight times prior to achieving cuboidal morphology and matrix deposition, suggesting they are a transit amplifying population
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Jane E. Aubinand lohan N. M. Heersche
[103,125] (Fig. 3). Interestingly, however, measurement of large numbers of individual bone colonies in lowdensity cultures shows that the size distribution of fully formed bone colonies covers a large range but is unimodal, suggesting that coupling between proliferation and differentiation of osteoprogenitor cells may be governed by a stochastic element but distributed around an optimum corresponding to the peak colony size/division potential [125]. The osteoprogenitors measurable in functional bone nodule assays also appear to have a limited capacity for self-renewal in both calvaria [126] and stromal [127] populations, consistent with the fact that they are committed progenitors with a finite life span [15]. However, in comparison to certain other lineages, most notably hemopoietic cells, relatively little has been done to assess regulation of self-renewal in different osteogenic populations, beyond the effects of glucocorticoids [126,127]. According to signaling threshold models, differentiation of hemopoietic stem cells is suppressed when certain receptor-ligand (soluble or matrixbound) interactions are maintained above a particular threshold and it is increased or more probable when levels are reduced [50,128]; this regulation is sometimes a proliferation-differentiation switch but in some cases is independent of proliferation [129]. Very little has been done to assess comparable issues in the osteoblast lineage, but the differential expression of a variety of receptors for cytokines, hormones, and growth factors during osteoblast development and in different cohorts of osteoblasts indicates that similar mechanisms may play a role in bone formation. Differentiation of O s t e o p r o g e n i t o r Cells to O s t e o b l a s t s and O s t e o c y t e s A fundamental question of osteoblast development remains how progenitors progress from a stem or primitive state to a fully functional matrix-synthesizing osteoblast. Based on bone nodule formation in vitro, the process has been subdivided into three stages: proliferation, extracellular matrix development and maturation, and mineralization, with characteristic changes in gene expression at each stage. Some apoptosis can also be seen in mature nodules. In many studies, it has been found that genes associated with proliferative stages (e.g., histones and protooncogenes such as c-fos and c-myc) characterize the first phase, whereas certain cyclins (e.g., cyclins B and E) are upregulated postproliferatively [130]. Expression of the most frequently assayed osteoblast-associated genes COLL-I, ALP, OPN, OCN, BSP, and parathyroid hormone/parathyroid hormone-related protein (PTHrP) receptor (PTH1R) is asynchronously upregulated, acquired, and/or lost as the progenitor cells differentiate and the matrix matures and mineralizes
[15,130]. In general, ALP increases and then decreases when mineralization is well progressed; OPN peaks twice during proliferation and then again later, prior to certain other matrix proteins, including BSP and OCN; BSP is transiently expressed very early and then upregulated again in differentiated osteoblasts forming bone; and OCN appears approximately concomitantly with mineralization [15]. Notably, however, use of global amplification poly(A) polymerase chain reaction (PCR) combined with replica plating and immunolabeling showed that all these osteoblast-associated markers are upregulated prior to cessation of proliferation in osteoblast precursors except OCN, which is upregulated only in postproliferative osteoblasts. In other words, differentiation is well progressed before osteoblast precursors leave the proliferative cycle. Based on the simultaneous expression patterns of up to 12 markers, osteoblast differentiation can be categorized into a minimum of seven transitional stages [103,131-133] (Fig. 2), not just the three stages mentioned earlier. An interesting issue is whether osteoprogenitor cells in all normal circumstances must transit all stages or can skip some steps under control of particular stimuli or regulatory agents. Although many cell systems have been reported to follow the general proliferation-differentiation outline discussed previously, some discrepancies are not always noted in detail. At least some of the variations may reflect inherent differences in the populations being analyzed (e. g., species differences or different mixtures of more or less primitive progenitors and more mature cells). However, there is growing evidence from both in vitro and in vivo observations that different gene expression profiles for both proliferation and differentiation and regulatory markers may underlie developmental and maturational events in different osteoblasts [14,134]. In other words, high levels of genes typical of some normal osteoblastic cells may not be required in others, leading to heterogeneity among osteoblast developmental pathways and/or the resulting osteoblasts. For example, as already discussed, Ihh is expressed in and required for development of osteoblasts associated with endochondral bones but not other osteoblast populations [80]. It has been evident for some time that not all osteoblasts associated with bone nodules in vitro are identical [135-137]. Single-cell analysis of the most mature cells in mineralizing bone colonies in vitro showed that the heterogeneity of expression of markers by cells classed as mature osteoblasts is extensive and appears not to be related to cell cycle differences [138] (Fig. 2). That extensive diversity in expressed gene repertoires is not a consequence or an artifact of the in vitro environment was confirmed by analysis of osteoblastic cells in vivo. When individual osteoblasts in 21-day-old fetal rat calvaria were analyzed, only two of nine markers
53
2. Bone Cell Biology
sampled (ALP and PTH1R) appeared to be global or ubiquitous markers expressed by all osteoblasts in vivo. Strikingly, all other markers analyzed (including OPN, BSP, OCN, PTHrP, c-fos, Msx-2, and E11) were differentially expressed at both m R N A and protein levels in only subsets of osteoblasts, depending on the maturational state of the bone, the age of the osteoblast, and the environment (endocranium and ectocranium) and the microenvironment (adjacent cells in particular zones) in which the osteoblasts reside [139]. The biological or physiological consequences of the observed differences are not known, but a few differences in expression profiles between younger versus older osteoblasts were reported earlier at other skeletal sites. In the primary versus secondary spongiosa of rat tibiae, collagen type I expression was equivalent in newly differentiated versus more mature osteoblasts, but osteocalcin m R N A was detectable only in the latter cells [140]. Bianco et al. [141] reported differences in BSP expression at the beginning of bone deposition in nascent osteoblasts and its absence in most mature osteoblasts in rat femurs. However, the fact that even osteoblasts of apparently similar age are heterogeneous suggests that different phenotypes may be required for different bone functions and supports the notion that not all mature osteoblasts develop via the same regulatory mechanisms nor are they molecularly or functionally identical. They predict that the makeup of different parts of bones may be significantly different, as previously suggested by the observation that the presence of and amounts of extractable noncollagenous bone proteins are different in trabecular versus cortical bone and in different parts of the human skeleton [134,139,142,143]. They also suggest that global or ubiquitously expressed molecules (e.g., COLL-I and ALP) serve common and nonredundant functions in all osteoblasts, and that only small variations in expression of these molecules may be tolerable; for example, all bones display mineralization defects in ALP knockout mice [144,145]. On the other hand, differentially expressed lineage markers (e.g., BSP, OCN, and OPN) vary much more, both between osteoblasts in different zones and between adjacent cells in the same zone, and may have specific functions associated with only some positionally or maturationally defined osteoblasts. In this regard, it is striking that all the noncollagenous bone matrix molecules are extremely heterogeneously expressed, and that ablation of many of those studied to date (e.g., OCN [146], OPN [147], and BSP [148]) does not result in complete failure of osteoblast differentiation and maturation, although the amount, quality, and remodeling of the bone formed may differ from normal. The observed differences in m R N A and protein expression repertoires in different osteoblasts may also
contribute to the heterogeneity in trabecular microarchitecture seen at different skeletal sites [149], to sitespecific differences in disease manifestation such as seen in osteoporosis [150,151], and to regional variations in the ability of osteoblasts to respond to therapeutic agents. In this regard, recent observations on transgenic mice overexpressing constitutively active PTH1R are interesting [152]. The opposite effects observed in trabecular and endosteal osteoblast populations versus periosteal osteoblast populations are reminiscent of the differential effects of PTH in trabecular versus cortical bone in primary hyperparathyroidism [152,153]. However, consistent with our observations on global expression of PTH1R in all osteoblast populations [139], Calvi et al. [152] found similar levels of transgene m R N A expression in both compartments, suggesting that in this case differential receptor expression cannot account for the different responses elicited by the ligand/ PTH. These data predict exquisite control of a signaling threshold as discussed earlier, other intrinsic differences downstream of the PTH1R in these different osteoblast populations, or that the different bone microenvironments in these sites modulate the osteoblast response. The nature of these and other signals leading to diversity of osteoblast gene expression profiles is not known [134,138]. However, the fact that heterogeneity is apparently controlled both transcriptionally and posttranscriptionally implies that the regulation is complex. Observations also suggest that it will be important to analyze the expression of regulatory molecules, including more transcription factors, not only globally but also at the individual osteoblast level. Another unanswered question is whether the striking diversity of marker expression in different osteoblasts is nonreversible or reversible in a stochastic manner or governed by changes in a microenvironmental signal or receipt of hormonal or growth factor cues or both. Since the heterogeneity observed extends to the expression of regulatory molecules such as cytokines and their receptors, it suggests that autocrine and paracrine effects may be elicited on or by only a subset of cells at any one time and the responses to such stimuli could be varied [134]. The possibility that the heterogeneity can be subdivided further seems likely as new markers for the lineage are identified. O t h e r M a r k e r s of O s t e o b l a s t s and Osteocytes As summarized previously [103], many other molecules are known to be made by osteoblast lineage cells, often with differentiation stage-specific changes in expression levels, but sometimes without known function in bone. Several molecules have recently been identified
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to be particularly highly expressed in osteoblasts and osteocytes, suggesting that they may play roles in mechanosensing or other activities in bone. For example, a novel osteocyte factor, termed OF45, was recently identified by subtractive hybridization based on its high expression in bone marrow stromal cells [154]. Northern blot analysis detected the mRNA in bone but not other tissues, and immunohistochemistry revealed that the protein was expressed highly in osteocytes within trabecular and cortical bone. The cDNA was predicted to encode a serine/glycine-rich secreted peptide of 45 kDa containing numerous potential phosphorylation sites and one RGD sequence motif, which may suggest a role for this new protein in integrin binding and osteoblast (or osteoclast) recruitment, attachment, and differentiation. The mRNA for Phex, a phosphate-regulating gene with homology to endopeptidases on the X chromosome that is mutated in X-linked hypophosphatemia, is differentially expressed in osteoblasts as they differentiate [155-157], and the protein is detectable only in osteoblasts and osteocytes [158] (Fig. 2). Two cell surface multifunctional molecules that are regulated by osteotropic hormones and expressed throughout osteoblast differentiation, but most highly in osteoblasts and osteocytes, are galectin-3 [159] and CD44 [160]. Galectin- 1 has also been reported in osteoblasts, with differences in nuclear matrix-binding capacity in proliferative precursors versus mature osteoblasts [161]. Consistent with one of its earlier names (Mac-2) and role as a macrophage marker, ablation of galectin-3, which is thought to have pleiotropic effects in cells in which it is expressed, with diverse roles in adhesion, apoptosis, and other cellular functions, alters monocyte-macrophage function and survival [162]. In addition, growth plate and chondrocyte defects with altered coupling between chondrocyte death and vascular invasion have been reported in galectin-3 null mice [163]; notably, most hypertrophic chondrocyte-osteoblast markers studied (PTH1R, OPN, and Runx2) appeared to be relatively normally distributed, although Ihh expression was altered. Additional work is required to determine whether osteoblasts and bone metabolism are changed in these animals. In contrast, simultaneous ablation of all known CD44 isoforms, some of which are known to bind OPN, altered tissue distribution of myeloid progenitors, with evidence of defective progenitor egress from bone marrow and highly tumorigenic fibroblasts, but there were no detectable effects on bone [Schmits, 1997 # 1186]. With completion of the human genome sequence project and those of other species and significant progress on the mouse and rat, and also with novel new methods for cell-specific gene identification and functional genomics, we can expect continued increases in the number of known osteoblast gene products [164]. For example, in
a small-scale cDNA fingerprinting screen from globally PCR-amplified rat calvaria-derived osteoblast colonies, Candeliere et al. [133] identified several new markers with differential expression during osteoblast differentiation, including glycyl tRNA synthetase and cystatin C, among other novel, previously unknown molecules. Highthroughput serial analysis of gene expression and microarray hybridization of MC3T3-E1 cells at different times after induction of osteoblast differentiation yielded a large number of known and novel osteoblast markers whose expression is differentially regulated during osteoblast maturation [165,166]. Rab24, calponin, calcyclin, cystatin-3, and lumican, among other mRNAs, were upregulated, whereas levels of MSY-1, SH3P2, fibronectin, cx-collagen, procollagen, and LAMPI mRNAs decreased with differentiation in this model. Among unexpected mRNAs identified was the TGF-[3 superfamily member Lefty-1, which in preliminary blocking studies appears to play a role in osteoblast differentiation [165]. In another gene array analysis of differentiating MC3T3-E1 cells [167], the antiproliferative factor Tob was identified, which is known to alter bone formation [168]. Among other new gene products identified were several transcription factors (e.g., SEF2) whose functions have not been studied in osteoblasts.
REGULATION OF OSTEOBLAST DIFFERENTIATION AND ACTIVITY Transcription factors, hormones, cytokines, and growth factors and their receptors can serve both as markers and as stage-specific regulators of osteoblast development and differentiation. It is beyond the scope of this chapter to review every factor that known to influence osteoblast differentiation and bone formation at some level because many will be discussed elsewhere in this book. However, we have chosen examples that emphasize other issues we discuss, including heterogeneity of osteoblast response, proliferation-differentiation coupling, and differentiation stage-specific regulatory mechanisms. Regulation by Transcription Factors It is evident from the preceding discussion that Cbfal/ Runx2 is necessary for osteoblast development. However, several other issues related to Runx2 are of interest, including the role of the multiple isoforms [70]. At least three appear to be able to regulate OCN expression [169] and osteoblast differentiation in/n vitro models [170], but with different efficacies/activities. Identification of the regulatory molecules lying upstream of Runx2 would
2. Bone Cell Biology also be informative. Runx2 regulates itself directly via binding on its own promoter [67,171]. As discussed earlier, regulation of Runx2 by other transcription factors is yielding interesting information on skeletal site-specific regulatory mechanisms for Runx2 specifically and bone development generally. Downregulation of Runx2 expression was observed in both Msx2-deficient [172] and Bapxl-deficient [173] mice. A particularly interesting aspect of the phenotypes was that Msx-2 deficiency caused delayed growth and ossification of the skull and long bones, whereas the axial skeleton was affected in Bapx-l-deficient mice. In addition, Hoxa-2deficient mice exhibit ectopic bone formation associated with ectopic expression of Runx2 in the second branchial arch [174], suggesting that Hoxa-2 may normally inhibit expression of Runx2 and bone formation in this area. Recently, mice carrying a mutation in the F G F receptor 1 (FGFR1) were generated and found to have craniosynostosis (premature fusion of cranial sutures) with increased Runx2 expression and accelerated osteoblast differentiation in the sutures [175]. With respect to other mechanisms of Runx2 regulation, relatively little is known, but inhibitory cofactors (TLE2/Groucho and HES1), phosphorylation via the MAPK pathway, and cAMP-induced Runx2 proteolytic degradation through a ubiquitin/proteosome-dependent mechanism have all been described in vitro [69]. The number of transcription factors that appear to regulate osteoblast recruitment, osteoblast number, and the rate and duration of osteoblast activity is increasing. Ducy et al. [67] used a dominant-negative strategy in transgenic mice to show that Runx2 plays a role beyond osteoblast development in that it appears to regulate the amount of matrix deposited by osteoblasts in postnatal animals. Tob, a member of an emerging family of antiproliferative proteins, has recently been shown to be a negative regulator of osteoblast production through regulation of BMP/Smad signaling [168]. Bone histomorphometry showed that numbers of osteoclasts in tob -/mice are equivalent to those in wild-type littermates, but the osteoblast surface and bone formation rate are increased significantly. The data are consistent with the view that Tob may normally function to inhibit proliferation of osteoblast precursors and their differentiation into mature ALP-positive osteoblasts, although further studies are needed to address the possibility that Tob inhibits the function of mature osteoblasts. Although it has been studied most frequently in the context of craniofacial development [176], Msx-2 appears to play a role in osteoblast differentiation in other bones, as evidenced by the broadly distributed (but not universal) bone abnormalities described in Msx-2 -/- mice [172]. Msx-2 functional haploinsufficiency also causes defects in skull ossification in humans
55
[177]. However, not only Msx-2 loss-of-function studies but also gain-of-function studies have been informative. Mutations in Msx-2 that increase its D N A binding activity [178,179] and overexpression of Msx-2 under the control of a segment of the mouse Msx-2 promoter that drives expression in a subpopulation of cells in the skull and the sutures [180,181] result in enhanced calvarial bone growth and craniosynostosis. The latter data, together with the findings of Dodig et al. [182] on the effect of forced over- or underexpression of Msx-2 on osteogenic cell differentiation in vitro, are consistent with the hypothesis that enhanced expression of Msx-2 keeps osteoblast precursors transiently in a proliferative state, delaying osteoblast differentiation and resulting in an increase in the osteoblast pool and ultimately in an increase in bone growth. More generally, these studies show that perturbations in the timing of proliferation and differentiation of osteoprogenitors have profound consequences on the number and activity of osteoblasts and bone morphogenesis. It is also clear that further analysis of other homeodomain transcription factor expressed in osteoblasts is required. For example, Dlx-5 is differentially expressed as osteoblasts differentiate in vitro [183,184] and in different cohorts of osteoblasts in vivo [185]. The phenotype of Dlx-5 -/- mice is complex, with many tissue abnormalities, but with respect to bone it is characterized by craniofacial abnormalities affecting derivatives of the first four branchial arches, delayed ossification of the roof of the skull, and abnormal bone formation in endochondral bones, the latter perhaps reflecting the differential expression of Dlx-5 at different skeletal sites [185]. Osteosclerosis results when either of two different members of the AP-1 subfamily of leucine zippercontaining transcription factors is overexpressed in transgenic mice: Fra-1 [186], a Fos-related protein encoded by the c-Fos target gene Fosll (referred to as fra-1), and AFosB, a naturally occurring splice variant of FosB [187]. In both cases, the transgenic mice appear normal at birth, but with time much increased bone formation is evident throughout the skeleton (endochondral and intramembranous bones). The osteosclerotic phenotype derives from a cell autonomous modulation of osteoblast lineage cells that is characterized by accelerated and more osteoblast differentiation and bone nodule formation in vitro. Interestingly, it is the further truncated A2AFosB isoform, rather than hFosB, that appears to be responsible for the increased bone formation seen in hFosB transgenic mice. This suggests that further study of differentiation stage-specific aspects of the mechanism may shed additional light on the underlying lineage perturbation since differentiation stage-specific alternative splicing of fosB m R N A and selective initiation site use of AFosB appear to be involved in regulation of osteoblast
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development and the increased bone formation seen in AFosB transgenic mice [187]. Notably, since neither Fra-1 nor AFosB (or A2AFosB) possess the typical C-terminal transactivation domain, it seems likely that the transcriptional potential of either resides in or depends on specific heterodimerizing partners or coactivators, either activators or repressors, that remain to be elucidated [188,189]. A decrease in adipocyte development also occurs in AFosB transgenic mice, but the bone phenotype appears not to be due to changes in leptin levels. This is interesting given that mice with leptin or leptin receptor deficiency have increased bone formation, suggesting that leptin may normally be an inhibitor of bone formation acting through the central nervous system [190]. Mice homozygous for mutations in the gene (AIM) encoding the nonreceptor tyrosine kinase c-Abl also have a bone phenotype, including osteoporotic (both thinner cortical and reduced trabecular) bones and reduced mineral apposition rates [191], apparently reflecting no change in osteoclast number or activity but an osteoblast defect manifested by delayed maturation in vitro. Whether a cell-autonomous defect in osteoblasts is responsible for the osteopenia seen in the spontaneous mouse mutant staggerer (sg/sg) [192], which carries a deletion within the retinioic acid receptor-related orphan receptor alpha (RORot) gene, remains to be determined, but since RORet appears to transcriptionally regulate BSP and OCN, this is clearly a possibility, given the increased bone formation seen in OCN null mice [146]. Interestingly, another steroid orphan receptor, estrogen receptor-related receptor alpha (ERRet), appears to play a positive regulatory role in proliferation and differentiation of osteoprogenitor cells, at least in vitro [193]. Recently, it was found that mice with a targeted disruption of the low-density lipoprotein receptor-related protein (Lrp5; a Wnt coreceptor) develop a low bone mass phenotype postnatally, apparently secondary to decreased osteoblast proliferation and functioning in a Cbfal-independent manner [194]. Lrp5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts [194]. Notably, the phenotypic characteristics of Lrp -/- mice, including persistent embryonic eye vascularization, recapitulate human osteoporosis-pseudoglioma syndrome caused by LRP5 inactivation [195] and are consistent with other recent evidence that certain mutations in Lrp5 are associated with a high bone mass trait in humans [196,197]. These studies indicate the need for further studies of the Wnt signaling pathway in osteoblast lineage cells [198]. It is beyond the scope of this chapter to review all the genes in which mutations or ablation appear to perturb bone formation or cause differences in formation of the craniofacial, appendicular, and axial skeletons [199], but
the available data indicate regulatory paradigms of significant complexity that are likely to become more complex as the roles of other related factors are elucidated. Regulation by H o r m o n e s , Growth Factors, a n d Cytokines In both normal development and bone marrow injury associated with local bleeding, clotting, and neovascularization, there is induction of an environment rich in growth factors [e.g., platelet-derived growth factor (PDGF), FGF, TGF-13, and vascular endothelial growth factor (VEGF)] followed by a process of very active bone formation [200,201]. To elucidate the responding target cells (stem cells, mesenchymal precursors, and committed progenitors) and the precise nature of the responses to these and other systemic or local growth factors, cytokines, and hormones, much attention has focused on in vitro studies. However, definitive conclusions have often been elusive, with differing requirements proposed depending on species studied [202,203] and conflicting results depending on the model cell system under study, the outcome assays used (e.g., total CFU-F or specific subpopulations of CFU-ALP or CFU-O), and the presence or absence of other factors. It is beyond the scope of this chapter to exhaustively summarize all such data. Instead, some examples that have helped establish some important conceptual principles are given. There is growing evidence that at least some of the actions of growth and differentiation factors are dependent on the relative stage of differentiation (either more or less mature) of the target cells, with stimulatory/ mitogenic or inhibitory responses when test factors are added to proliferative/progenitor stages and stimulation or inhibition of differentiation stage-specific precursors and mature osteoblasts when the same factors are added later. This is true, for example, for the inflammatory cytokine interluekin-1 (IL-1), which is stimulatory to CFU-O formation when calvaria-derived cultures are exposed transiently during proliferative culture stages but inhibitory when cells are exposed transiently through differentiation stages or chronically through proliferation and differentiation stages [204]. Many other factors of current interest also have biphasic or multiphasic effects on CFU-O in vitro, including EGF [205], TGF-[3 [206-208], and PDGF [209]. Another example of clinical significance is the reported catabolic versus anabolic effects of PTH. PTH1R is expressed throughout osteoblast differentiation, although levels of expression and activity appear to increase as osteoblasts mature [210]. Chronic exposure to PTH inhibits osteoblast differentiation in an apparently reversible manner at a relatively late preosteoblast stage in rat calvaria cells [211]. However, when the cells
2. Bone Cell Biolosb,
were treated with 1-hr versus 6-hr pulses in 48-hr cycles during a 2- or 3-week culture period, either inhibition (1-hr pulse; apparently related to cAMP/PKA pathways) or stimulation (6-hr pulse; apparently related to cAMP/ PKA, Ca2+/PKC, and IGF-1) in osteoblast differentiation and bone nodule formation was seen [212]. In mice deficient in PTH1R, not only was a well-studied defect in chondrocyte differentiation seen (as seen also in PTHrP knockout mice) [213] but also increased osteoblast number and increased bone mass were seen (a phenotype not seen in PTHrP-deficient mice) [214], supporting the view that PTH plays an important role in regulation of osteoblast number and bone volume. Consistent with this latter view are studies showing that PTH may increase osteoblast lifetime by decreasing osteoblast apoptosis [215]. As already mentioned, when Calvi et al. [152] expressed constitutively active PTH1R in bone [216], osteoblastic function was increased in the trabecular and endosteal compartments but decreased in the periosteum of both long bones and calvaria; interestingly, an apparent increase in both osteoblast precursors and mature osteoblasts was seen in trabecular bone. Because of their ability to induce de novo bone formation at ectopic sites, BMPs, which are members of the TGF-13 superfamily, have been extensively studied in vivo and in vitro as regulators of osteoblast development [70]. Although many reports document a stimulatory effect of BMPs on osteoblast differentiation, a few show inhibitory effects. Interestingly, although mouse knockout experiments have clearly indicated a role for BMPs in skeletal patterning and joint formation, ablation experiments have not provided evidence for a role of BMPs in osteoblast differentiation in vivo [68,217]. This may be the result of functional redundancy among members of this family (there are currently more than 30), and further experiments are required to elucidate unequivocally the role of BMP family members in osteoblast differentiation. However, TGF-13 has been shown to have biphasic effects on osteoblast development in vitro, inhibiting early stage progenitors while stimulating matrix production by more mature cells in the lineage, including osteoblasts [103,218]. These diverse effects may help account for the complex effects seen when TGF-I3 is overexpressed via the OCN promoter in transgenic mice; these mice have low bone mass, with increased resorption, but increased osteocyte numbers and hypomineralized matrix [219]. Studies from Derynck's group showed that when a dominant-negative TGF-[3 type II receptor was expressed in osteoblasts, osteocyte number, bone mass, and bone remodeling were all influenced in a manner suggesting that TGF-I3 increases the steadystate rate of osteoblastic differentiation from osteoprogenitor cell to terminally differentiated osteocyte [220]
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while also regulating bone remodeling, structure, and biomechanical properties [221]. Given their inclusion in the majority of CFU-F and CFU-O in vitro assays, it is worth considering glucocorticoids (most often dexamethasone in in vitro assays) in more detail. Glucocorticoid effects in vivo and in vitro are complex and often oppositemthat is, stimulation of osteoblast differentiation in vitro [103] but glucocorticoidinduced osteoporosis in vivo [222]. An emerging picture of glucocorticoid-induced stimulation of osteoprogenitor cell recruitment, self-renewal, and differentiation [103] opposed by glucocorticoid-induced inhibition of several molecules synthesized by the mature osteoblast [223] and a glucocorticoid-induced increase in osteoblast apoptosis [222] may account for some of the discrepancy. An area of considerable interest is the stimulatory activity of glucocorticoids on osteoprogenitors [15]. One mechanism by which dexamethasone or other glucocorticoids may act is through autocrine or paracrine regulatory feedback loops in which production of other factors is modulated, including growth factors and cytokines that regulate the differentiation pathway. For example, in rat calvaria cultures, glucocorticoids downregulate endogenous production of LIF, which is known to be inhibitory to bone nodule formation when cells are treated at a late progenitor/preosteoblast stage [224], and upregulate BMP-6, which is stimulatory [225] possibly through LMP-1, a LIM-domain protein [226]. These are but two of a growing list of examples of dexamethasone regulation of endogenously produced factors with apparently autocrine or paracrine activities on osteoblast lineage cells. Many factors of interest have effects on gene expression in mature osteoblasts that may or may not correlate with effects on the differentiation process and may be opposite for different osteoblast genes, with glucocorticoids being a case in point. The molecular mechanisms mediating these complex effects are generally poorly understood. However, the ability to form particular transcription factor complexes, localization and levels of endogenous expression of cytokine/hormone/growth factor receptors, and expression of cognate or other regulatory ligands within specific subgroups of osteogenic cells as they progress from a less to a more differentiated state may all be involved. As noted previously, there is increasing evidence that the probability for selfrenewal versus differentiation of hemopoietic stem cells is regulated, at least in part, by maintenance of required/ critical signaling ligands (soluble or matrix or cellbound) above a threshold level. Although there are few explicit data or experiments examining these issues in MSCs or osteoprogenitor populations, it seems likely that similar threshold controls may apply. It must also be kept in mind that many growth factors, cytokines,
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and other soluble regulatory factors may have much more widespread effects and activities on cells not normally considered their targets. For example, although most often considered as osteoclast regulatory factors, granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-3, as well as M-CSF, appear to stimulate the proliferation and/ or differentiation of bone marrow fibroblastic precursors [227].
unique activities in osteocytes, including alteration of connexin 43 expression and stimulation of apoptosis via the CPTHR. This list of gene products expressed in osteocytes is not exhaustive, but it exemplifies the diversity of regulatory activities and functions that one can begin to ascribe to osteocytes and some possible pathways by which they respond to both mechanical and hormonal regulation of bone.
OSTEOCYTES
O s t e o c y t e s as M e c h a n o s e n s o r s a n d Signal Transducers
Markers and Activity Nijweide and colleagues [228] pioneered approaches to the isolation of osteocytes from chick calvariae using a monoclonal antibody to a cell surface molecule that only recently was identified as recognizing Phex [229]. Over several studies [228,230,231], they established a number of osteocyte characteristics, including cell postmitotic status, a dendritic shape maintained in vitro, relatively low expression of ALP and several bone matrix proteins (e.g., collagen type I and fibronectin), and induction of nitrous oxide (NO), Cox-2, and prostaglandin production in response to mechanical strain [232,233]. Recently, Bonewald and colleagues established the mouse MLO-Y4 osteocytic cell line [234,235], which has similar properties, but as an SV40 large T antigenimmortalized line; fit offers some advantages for studies requiring large numbers of cells. These studies, in addition to many other in vitro and in vivo studies in which both gene products and responses to mechanical stimulation have been assessed, have helped to dispel the notion that osteocytes are relatively inactive cells (Fig. 2). It is now clear that osteocytes express a wide variety of molecules also.expressed at high levels in osteoblasts, although the expressed gene repertoires are not identical given that many molecules (e.g., ALP, collagen, and fibronectin) expressed highly in osteoblasts appear to be downregulated in osteocytes. Among those molecules expressed at high levels in both osteoblasts and at least some osteocytes are transcription factors such as ERs [236,237] and E R R s [193], adhesion and signaling molecules such as galectin-3 [159] and CD44 [160,238-241], growth factors including IGF-1 [242,243], the gap junction hemichannel connexin 43 [234,244], clinically relevant enzymes such as Phex [158,229], and some but not all of the bone matrix molecules including OPN [234]. Notably, given-evidence that several hormones, including PTH, modify osteoblastic cell response to mechanical stimulation, Divieti et al. [245] reported that osteocytes also express much higher levels of the receptor for C-terminal PTH fragments (CPTHR) than do osteoblasts and that both PTH(1-84) and PTH(39-84) elicit
It is well established that bone tissue is sensitive to the mechanical demands made on it and that abnormally low mechanical stress (bed rest, immobilization, or lack of gravity such as occurs in space flight) results in decreased bone mass and disuse osteoporosis [Houde, 1995 #1392; Zerwekh, 1998 #1396] [246,247]. As summarized earlier, osteocytes derive from osteoblasts that have become entrapped in the deposited bone matrix. A network of canaliculi comprising unmineralized matrix houses long osteocyte processes that allow intercellular communication via connexin 43-containing gap junctions between osteocytes; the network also connects with osteoblasts and the lining cells that cover the bone surface, which also derive from osteoblasts. Thus, osteoblasts, osteocytes, and lining cells form a network in which cells may act together as signal transducers of changes in strain or mechanical load [248]. Among cells making up the syncytium, osteocytes have long been postulated to be the major force transducers [247,249,250]. Osteocytes make up a large proportion of the cells in adult bone and are extremely sensitive to mechanical stress, which is likely imparted by the flow of interstitial fluid along the surface of the osteocytes and lining cells [249-251]. Huiskes et al. [248] reported an interesting computational model with simulations of bone remodeling as a regulatory process, governed by mechanical usage and orchestrated by osteocyte mechanosensitivity. In the model, they made several assumptions based on known experimental results, including that osteocytes react very quickly to load [252] with production of biochemical messengers such as prostaglandins [253] that are dissipated through canaliculi to the bone surface, where they trigger osteoblast precursor recruitment. The model included a resorption component based on the probability of osteoclast activation per surface site at any time. This computational model is interesting because it predicts the emergence and maintenance of bone trabecular architecture as an optimal mechanical structure as well as its adaptation via cell coupling or feedback from mechanical load [248]. Disruption of the actin-containing cytoskeleton abolishes osteoblast-osteocyte response to stress, including
2. Bone Cell Biology
production of prostaglandins [254], suggesting that the cytoskeleton is involved in mechanotransduction in bone as in other cell types [255-257]. In addition, a variety of hormones, including PTH [258,259] and estrogen [260], modulate bone cell responses to mechanical loading, although the mechanisms by which they do so are not clear but may be multifactorial. Enzymes participating in oxidative responses, including glucose 6-phosphate dehydrogenase (G6PD), were among the first molecules found to be upregulated rapidly in mechanically stimulated osteocytes and osteoblasts [252]. Other upregulated molecules include phospholipase A2, Cox-2 and prostaglandins, NO, protein kinase C, IGF-1, multiple glutamate receptors, and AP-1 family members [249,261]. However, an unambiguous role for any of these in the mechanotransduction response is not known, but recent approaches in which gain-of-function and loss-of-function mutations in some of these and other genes are being studied for their impact on bone cell responses to mechanical loading should help. For example, using a novel device that enables the noninvasive application of controlled mechanical loads to the murine tibia of IGF-1overexpressing transgenic mice, Gross and colleagues [262] concluded that IGF-1 is a synergistic factor required for the periosteal bone formation response to mechanical loading. Data from mice lacking iNOS suggest that NO generated by iNOS plays a critical role in adjusting bone turnover and increasing osteogenic activity in response to an acute increase in mechanical loading after tail suspension [263]. Similarly, the increase in osteoclastic bone resorption and suppression of osteoblastic bone formation in response to reduced mechanical stress was not seen in mice lacking OPN [264]. p53-p21 signaling has also been suggested to play a role in bone loss occurring with mechanical unloading [265]. However, the complexities and caveats in interpretations of data from knockout animals were also well demonstrated in the commentaries elicited in response to the conclusion that glutamate plays no role in bone growth based on studies in mice with GLAST glutamate transporters knocked out [266-268]. The availability of the recently established mouse MLO-Y4 osteocytic cell [234,235] should also help advance the field by aiding with the identification of novel osteocyte markers, molecular studies of osteocyte junctional complexes, and the response to mechanical forces and other stimuli. For example, it has been useful in increasing the understanding of the role of prostaglandins in cellular response to mechanical strain and gap junction-mediated intercellular communication [269-271]. Following up studies by Kumegawa and colleagues [273],who showed that osteocytes produce a small protein inhibitor of osteoclastic bone resorption, Vaananen and colleagues [272] found that MLO-Y4
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osteocytic cells secrete significant amounts of TGF-[3, which inhibits bone resorption and is modulated by estrogen. This suggests that osteocytes have an active, inhibitory role in the regulation of bone resorption and may participate in the antiresorptive effects of estrogen.
MORPHOLOGICAL FEATURES OF OSTEOCLASTS Osteoclasts are easily recognized in histological sections of bone tissue as large multinucleated cells with up to 25 nuclei and are found in close association with bone surfaces. They are often located in slight indentations on the bone surface called Howship's lacunae, which result from osteoclasts having dissolved a small area of bone underneath the cell. Osteoclasts contain large amounts of the enzyme tartrate-resistant acid phosphatase (TRAP), and when stained histochemically for this enzyme they become quite conspicuous. However, not all osteoclasts contain large numbers of nuclei: Mononuclear TRAP-positive cells can frequently be seen, and osteoclasts containing two to four nuclei are not uncommon. Electron microscopic examination of cross sections through actively resorbing osteoclasts reveals several more morphological features. The area in which the cells closely adhere to bone, the clear zone, is characterized by an abundance of microfilaments and the absence of cellular organelles. The cytoplasm of osteoclasts contains an abundance of vesicular structures: Endocytotic vesicles are located adjacent to the ruffled border, and primary and secondary lysosomes and a variety of vesicles involved in transcytotic transport (from the ruffled border area to the basal surface) of endocytosed matrix degradation products and an extensive Golgi system are located adjacent to the nuclei [274]. When actively resorbing osteoclasts in situ are fixed, stained immunohistochemically with antibodies against F-actin, and then viewed under fluorescence microscopy, the clear zone area appears as a ring-like structure surrounding the ruffled border area [275]. This ring of actin-containing filaments delineates the area where the osteoclast closely adheres to the bone surface, likely via otv[33 integrin receptors, and forms a sealed-offextracellular compartment between the ruffled border and the bone surface in which bone resorption occurs [276]. When resorbing osteoclasts are viewed in situ using scanning electron microscopy, they appear as large, bulging cells with a surface covered with numerous filopodia. Where the osteoclast has moved away from an area it resorbed previously, exposed collagen fibers can frequently be seen, representing remains of the organic matrix after
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the osteoclast has removed the mineral component of the bone matrix.
MECHANISMS OF OSTEOCLASTIC BONE RESORPTION Establishment of t h e Extracellular Resorption Z o n e The generally accepted view is that osteoclasts create an extracellular sealed-off compartment in which protons and proteolytic enzymes are secreted. Essential for this is the creation of a tight sealing zone, preventing leakage of ions and enzymes. Evidence indicates that the av[33 integrin is instrumental in establishing this sealing zone [277]. It is unclear, however, whether ctv[33 integrinmediated adhesion is essential for maintaining this zone: Immunohistochemical evidence suggests that this integrin is expressed on both the basolateral aspect and the ruffled border part of the osteoclast plasma membrane but not on the plasma membrane in the area of the sealing zone. However, blocking ctv[33 with RGDcontaining peptides or antibodies to the oLv[33 integrin inhibits osteoclastic bone resorption in vivo and in vitro, strongly suggesting that oLvl33-mediated processes are essential for osteoclastic resorption.
Demineralization Under normal conditions, osteoclasts are solely responsible for removing approximately 500 mg of calcium per day from the adult human skeleton. This is accomplished through an exchange mechanism, whereby calcium ions in the hydroxyapatite crystals are replaced by hydrogen ions. The H § ions required to dissolve the hydroxyapatite crystals comprising the mineral component of the bone matrix during the resorption process are generated by the activity of carbonic anhydrase type II (CAII), an enzyme that uses CO2 and H20 to generate H2CO3, which subsequently dissociates into H + ions and HCO3- ions. The protons thus generated are extruded into the extracellular space by a vacuolar-type H +ATPase (V-ATPase) located on the osteoclast ruffled border membrane and dissolve the bone mineral by H+/Ca 2+ exchange [278]. It is interesting that the osteoclast appears to be essentially self-sufficient in generating the components required to complete this part of the resorptive process: Protons generated by CAII activity, using CO2 produced during the oxidative phosphorylation of glucose in the mitochondria, are extruded by the activity of a V-ATPase requiring ATP, which is similarly generated by mitochondrial oxidative phosphorylation of glucose. The required glucose enters the osteoclast
through a GLUT-2 transporter [279], and all components of the system (mitochondria, the proton pump, the GLUT-2 transporter, and CAII) are abundant in osteoclasts. Proton extrusion into the extracellular resorption zone is accompanied by C1 extrusion via a chloride channel [280]. The driving force behind C1 extrusion is the potential difference that arises from electrogenic proton transport across the ruffled border membrane. In keeping with the self-sufficiency of the osteoclast, the required C1 ions enter the cell through a C1-/HCO3- exchanger located on the basolateral surface of the cell, which is driven by HCO3 accumulated by CAII activity combined with proton extrusion [281]. D e g r a d a t i o n of t h e Organic Matrix After demineralization, the remaining organic matrix of bone (90% type I collagen and 10% other proteins and proteoglycans) is partly degraded outside the cell in the extracellular resorption zone and partly inside the cell in various vesicular structures. Two classes of enzymes, matrix metalloproteinases and cysteine proteinases, play a major role in the degradation of the organic component of the bone matrix. Inhibition of the activity of these two classes of enzymes results in the accumulation of collagen fibers in the extracellular resorption zone [282]. Several recent studies have shown that cathepsin K is the predominant protease involved in bone matrix degradation [10,283-286]. Cathepsin K is secreted into the resorption pits by osteoclasts in its mature form, where it cleaves type I and type II collagens at their helical domains. Upon cleavage of the triple helix, collagen quickly unwinds and becomes susceptible to any proteinase with gelatinolytic activity. The ability to cleave the native triple helix of collagen at multiple sites makes cathepsin K unique among mammalian proteases [287]. The role ofmetalloproteinases in osteoclast function is less clear, although tissue culture experiments have demonstrated that MMP inhibitors have a strong inhibitory effect on collagen type I resorption by osteoclasts [288]. In addition, inhibition of MMPs in osteoclasts plated on bone slices has been shown to result in accumulation of demineralized bone matrix underneath the osteoclasts, further implicating MMPs in organic matrix degradation [282,289]. Also in support of a role for MMPs in the resorptive process is the observation that bone resorption is attenuated in mice carrying a mutation in type I collagen at the site that is targeted by neutral collagenases [290]. In fact, both cysteine proteinases and metalloproteinases may play a role in the resorptive process" Cysteine proteinases may degrade the collagen matrix prior to
2. Bone Cell Biology
subsequent degradation by MMPs [291]. Interestingly, osteoclasts in different parts of the skeleton may differ in terms of the relative importance of MMPs and cysteine proteinases in the resorption process: MMPs could be largely responsible for organic matrix degradation by osteoclasts in intramembranous bone (e.g., the bone of the skull and the scapula), whereas cysteine proteinases appear to be involved in osteoclastic resorption of both long bones and skull bones [289]. Processing of Degradation Products The way in which degradation products are removed from the sealed-off space between ruffled border and bone surface was recently determined by marking the bone surfaces being resorbed with tags that could be traced using either fluorescence or electron microscopy. It has thus become clear that osteoclasts endocytose degraded bone matrix components and solubilized mineral components at the ruffled border, transport it across the cell enclosed in endocytotic vesicles, and subsequently release the vesicle content at the opposite surface by merging the vesicles with the plasma membrane [274,292,293]. Thus, matrix degradation products can be disposed of continuously, without the osteoclasts losing its seal. An alternative mechanism for releasing degradation products from the extraceUular resorption zone is that osteoclasts move away from the resorption area temporarily, thereby allowing accumulated degradation products to diffuse away. Using time-lapse videomicroscopy, resorbing osteoclasts have indeed been observed to move back and forth within a clearly delineated resorption space [294]. A combination of the two mechanisms certainly seems possible.
ORIGIN OF OSTEOCLASTS As mentioned previously, osteoclasts are multinucleated cells of hematopoietic origin. This was first demonstrated by G6thlin and Ericsson using parabiotic rats [295]. They found that osteoclasts were derived from a bloodborne hemopoietic precursor, whereas the osteoblasts were derived from a local progenitor cell of connective tissue origin. Subsequent experiments by Walker [296] proved that osteoclasts are cells from the hemopoietic lineage: He transplanted spleen cells or bone marrow cells from normal mice into irradiated osteopetrotic littermates, which resulted in the formation of normal osteoclasts. This was confirmed by Scheven et al. [297], who cocultured purified bone marrow fractions with living osteoclast-free embryonic mouse longbone rudiments and found that the fraction containing
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hemopoietic stem cells gave rise to osteoclasts. These authors also demonstrated that, at least in embryonic mouse long bones, proliferating osteoclast progenitors are seeded via the bloodstream in the mesenchyme surrounding the bone rudiments, where they then proliferate, differentiate, and fuse into multinucleated boneresorbing osteoclasts.
REGULATION OF OSTEOCLAST ACTIVITY AND DIFFERENTIATION Stimulation or inhibition of osteoclastic bone resorption may involve changes in activity of existing osteoclasts, changes in the total number of osteoclasts, or changes in size of the osteoclasts. It is often difficult to separate regulation of differentiation and regulation of activity because many factors affecting one of these processes also simultaneously affect the other. From studies on the effects of the bone-resorbing hormones PTH and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] in vivo and in in vitro bone organ culture systems, it is known that these hormones increase osteoclastic activity. When isolated osteoclast cultures became available, and results obtained with these systems were compared with those obtained using cultures containing both dispersed osteoclasts and stromal cells, it quickly became clear that these hormones did not activate osteoclasts directly, but that the signals were transmitted by other cell types in bone tissue. The first breakthrough toward elucidating this indirect pathway came from studies analyzing the cause of bone defects in the osteopetrotic op/op mouse [298]. These mice almost completely lack osteoclasts and tissue macrophages as a result of a single point mutation in the gene encoding M-CSF. Marrow cells from op/op mice cocultured with osteoblast-like cells or stromal cells from normal littermates (or vice versa) revealed that the defect in osteoclast differentiation in op/op mice resulted from the failure of osteoblastic/stromal cells to produce M-CSF required for osteoclast differentiation. Indeed, PTH and 1,25(OH)2D3 have been shown to upregulate M-CSF production by stromal cells [299] and SAOS-2 cells transfected with the PTH/PTHrP receptor respond to PTH with an increase in M-CSF production [300]. Osteoclast formation and activity in in vivo and in vitro systems containing osteoclasts, osteoclast precursors, and cells from the bone stromal compartment are affected not only by the osteotropic hormones PTH and 1,25(OH)2D3 but also by prostaglandins of the E series, estrogens, androgens, cytokines, and growth factors [e.g., IL-6, IL-11, interferon-~/(IFN-~/), tumor necrosis factor-tx (TNF-et), TGF-[3, BMP-2, oncostatin M, and LIF]. By using a coculture system consisting of spleen
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cells and either osteoblastic cells from calvariae or established marrow-derived stromal cell lines, it was discovered that effects on osteoclast differentiation of some of these factors were mediated through the osteoblastic or stromal cell component of the cultures, that others were acting directly on the osteoclast on their precursors, and that still others were likely acting on both. The mediator generated by the stromal cells was discovered relatively recently and initially named osteoclast differentiation factor (ODF) [301]. This factor proved to be identical to the previously identified protein osteoprotegerin ligand (OPGL) [302], which in turn was identical to the previously discovered ligand for the receptor activator of NF-KB (RANKL). Since the receptor on the osteoclast recognizing ODF (= OPGL = RANKL) is the R A N K receptor [303], the ligand is now commonly called RANKL. RANKL is an absolute requirement for osteoclast formation, and this was proven conclusively by the generation of rankl knockout mice that had severe osteopetrosis due to a complete lack of osteoclasts. That R A N K L was indeed produced by cells other than the osteoclasts or their precursors was confirmed by analyzing cocultures of spleen cells from opgl -/- mice with osteoblastic cells from normal mice. Functional osteoclasts were generated in these cocultures, whereas cocultures of spleen cells from normal mice with osteoblastic cells from opgl -/- mice did not form osteoclasts. Thus, lack of R A N K L production in osteoblastic cells of opgl - / - mice was the cause of the osteoclast deficiency. It was discovered virtually simultaneously that osteoclast formation could be inhibited by a soluble receptor for RANKL, OPG, which is secreted by a large variety of cells and organs, including fibroblasts, osteoblasts, lung, heart, kidney, and intestine [304]. OPG-deficient mice are severely osteoporotic [305], whereas mice overexpressing OPG have an osteopetrotic phenotype. OPG acts by binding RANKL, thereby preventing interaction of R A N K L with its receptor and thus inhibiting osteoclast differentiation. The consensus is that osteoclast formation in the bone microenvironment is determined by the amount of unbound R A N K L available to interact with the R A N K receptor on osteoclasts or osteoclast precursors. RANKL-induced osteoclast differentiation in stromal cell-free systems requires the presence of M-CSF. The mechanisms responsible for this were determined by Cappellen et al. [306], who performed a microarray analysis of osteoclast differentiation induced by M-CSF and R A N K L in a purified mouse bone marrow cell culture. They discovered that early stages of differentiation of osteoclast precursors were associated with M-CSF-induced upregulation of m R N A for R A N K and R A N K pathway components (i.e., TNF receptorassociated factor 2A, PI3 kinase, etc.) Interestingly,
M-CSF also upregulated m R N A levels for several cytokine receptors (e.g., IL-11RoL2, gpl30, IFN-~/R) and cytokines (e.g., IL-loL, IL-18, and IFN-[3). This strongly suggests that osteoclasts and their precursors can respond directly to IL-11, IL-6, and IFN-~/and that they also produce cytokines that affect not only their own metabolism (autocrine) but also cells in their immediate environment (paracrine), such as osteoblastic stromal cells and endothelial cells associated with capillary ingrowth. In a separate series of experiments, the same authors demonstrated that a combination of IL-loL, IL6, and IL-11 directly stimulated osteoclastic activity in their culture system. When R A N K L was added together with M-CSF, approximately 70 novel target genes were found to be upregulated in addition to the already known target genes Jun and F O S - C 1 [307]. Upregulated target genes were mostly transcription factors but also genes encoding signaling molecules, chemokines, cytokines growth factors, and their receptors. Interestingly, among these were PDGF-oL and IGF-1 (implicated in the regulation of osteoblast differentiation) and the EP-2 prostaglandin E2 receptor. These studies emphasize that although much more is now known than only a few years ago about the regulation of osteoclast differentiation and function by interactions between osteoblast and osteoclast lineage cells, many aspects of regulation of their activity, differentiation, and interaction remain to be discovered. Recent evidence supports the idea that endothelial cells also participate in the regulation of osteoclast differentiation, as indicated by the observations that both OPG and R A N K L expression in cultured vascular endothelial cells are upregulated by IL-loL and TNF-oL [308]. Also, endothelin-1 (ET-1) produced by endothelial cells has been shown to stimulate proliferation and differentiation of osteoblastic cells [309], whereas VEGF has been shown to stimulate osteoclast formation in osteopetrotic M-CSF-deficient mice [310]. This further illustrates the complexity of the regulatory processes in the previously mentioned BMU: IL-loL, for example, directly affects osteoblastic cells, osteoclast lineage cells, and endothelial cells, and it seems certain that numerous other agonists will be identified in the near future that similarly affect all three cell types (e.g., PGE-2, IL-6, TNF-oL, TGF-[3, and several BMPs) [311,312]. Among factors that downregulate osteoclast activity and/or differentiation, estrogen figures prominently. Estrogen deficiency causes osteoporosis [313]. Closer examination of estrogen deficiency in humans and rats reveals that the major effect of estrogen deficiency in terms of osteoclastic activity is an increase in the rate of bone remodeling. It is also of interest that bone loss in estrogen-deficient rats is not uniform: The amount of cancellous bone in the epiphysis does not decrease significantly,
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whereas the amount of cancellous bone in the metaphysis of the long bones decreases dramatically [Westerlind, 1997 #1469]. Estrogen treatment of estrogen-deficient postmenopausal women does not change the average depth of the osteoclastic resorption lacunae [313], which suggests that the resorptive activity of individual osteoclasts is not affected by estrogen. Rassi et al. [314] showed that estrogen treatment decreased 1,25(OH)zD3induced osteoclast formation in porcine bone marrow cultures by 50%, possibly through increased apoptosis of osteoclast precursor cells. Alternatively, estrogen may cause the decrease in 1,25(OH)2D3 responsiveness indirectly through downregulation of RANKL-mediated JNK1 activation, as found in cultures of both murine marrow cells and RAW cells [315].
OSTEOCLAST SIZE: MULTINUCLEATION AND FUNCTION The observation that osteoclast size is generally increased in diseases characterized by increased bone resorption (e.g., end-stage renal disease, Paget's disease, periodontal disease, and rheumatoid arthritis) suggests that multinucleation confers increased capacity for resorptive activity. We were able to show in an in vitro culture system that large osteoclasts are indeed more effective resorbers than small osteoclasts because the proportion of large osteoclasts that are resorbing is much greater than the proportion of small osteoclasts that are resorbing [316]. Thus, many small osteoclasts are not as effective as a smaller number of large osteoclasts with the same total number of nuclei. In several other multinucleated cells, the gain or loss of certain characteristics has been associated with cells becoming multinucleated. For example, Vignery and colleagues [317] reported that in multinucleated giant cells (MNGs) gene expression changed with multinuclearity: Mononuclear rat macrophages expressed the oL1 isoform of the Na+/K+-ATPase only, whereas the or3 isoform appeared when these cells fused to produce multinuclear cells. Enelow et al. [318] found that although microbicidal activity of human MNGs was enhanced in parallel with increased size and number of nuclei, the oxidative activity per unit cytoplasmic protein was significantly increased in the larger cells. With regard to osteoclast-like cells, it was reported that chick macrophages expressed high levels of m R N A for retinoic acid receptors alpha and gamma as well as the vitamin D receptor, whereas the osteoclast-like MNGs that formed in long-term cultures of these same cells had lower expression of these transcripts [319]. Formation of large
multinucleated cells has been studied in most detail in the formation of muscle, in which fusion of myoblasts leads to formation of multinucleated myotubes and subsequently striated muscle fibers. Recent evidence clearly indicates that fusion is a multistep process involving differentiation of muscle progenitors into founder cells and of surrounding mesenchymal progenitors into fusion-competent myoblasts [320]. One could speculate that similar processes occur with fusion of osteoclast precursors, and we are only starting to be able to analyze very limited aspects of the complicated mechanisms involved in this process. The mechanisms underlying several other processes of crucial importance to osteoclast function (e.g., exocytosis, proton transport, and cell fusion) are extremely complex and unlikely to be dissected first in osteoclast systems. For example, the processes of exocytosis and acidification of cellular compartments have been and are currently studied extensively in yeast [321]. Evaluation of the mechanisms discovered in other systems by searching for parallel mechanisms in the osteoclast will likely be the major avenue whereby such processes will be elucidated.
STEM CELL, OSTEOBLAST, AND OSTEOCLAST CHANGES IN DISEASE Many studies are beginning to be performed in which cells from patients with skeletal disorders or animal models of skeletal diseases are manipulated in vitro and in vivo to extend genetic understanding to that of the underlying cellular and molecular basis of bone diseases. For example, as described earlier, quantification of colony formation (for stromal cells: CFU-F, CFU-O, etc.; for osteoclasts: CFU-GM, etc.)can be used as an assessment of the stem/progenitor cell pools of an individual or a tissue site. In addition, cell transplantation studies are highly informative, and both kinds of assays help to clarify issues about the cell autonomous or nonautonomous nature of particular diseases. Regarding the latter, some studies showed normal osteoclast formation and cure of osteopetrosis when spleen or bone marrow cells were transplanted from normal mice into irradiated osteopetrotic littermates [296], whereas others did not [322,323]. Of course, these and other studies have led not only to elucidation of the hematopoietic origin of osteoclasts but also to elucidation of the role for cell nonautonomous stromal cell-hematopoietic cell interactions in osteoclastogenesis. Manifestations of skeletal growth and density abnormalities, especially in mice and rats, are routinely
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dissected in vitro for clarification of the intrinsic roles of osteoclast lineage or osteoblast lineage cells or both. For example, mutations in mice in several different kinds of molecules (e.g., transcription factors, Wnt and other signaling pathways, and many others) alter bone formation in vivo with cell autonomous effects in osteoblast lineage cells determined by in vitro manipulations. The same kinds of analyses are being done for osteoclast lineage cells (e.g., transcription factors, ligand-receptor signaling pathways, and enzymes). However, far fewer studies have been done on cells from patients with skeletal disease. One exception relates to continuing analysis of the underlying osteoclast defects in malignant juvenile osteopetrosis [324]. Another exception is in quantification of stem and osteoprogenitor pools with aging and in age-related bone disorders. Discrepant data exist regarding whether CFU-F, CFU-O, and CFU-ALP colony size and number decrease or remain the same in humans, mice, and rats with aging and disease status (i.e., osteoporosis and osteoarthritis) [14]. However, the majority of data indicate that at least the CFU-ALP fraction of the CFU-F population declines (often dramatically) in number and/ or size with aging, suggesting that osteogenic capacity declines with age [325-332]. In addition, the responsiveness of CFU-F to systemic or locally released osteotropic growth factors has also been reported to decrease with age, as suggested earlier [333]. For example, the stimulatory effect of TGF-I3 on colony number and cells per colony in human osteoprogenitor cells derived from 98 iliac crest biopsies significantly declined with donor age [334]. Gazit et al. [332] suggest that changes in the osteoprogenitor cell/CFU-F compartment occur with aging in BALB/c mice because of a reduction in the amount and/ or activity of TGF-131. Ligand concentration-dependent ERa induction and loss of receptor regulation and diminution of ligand-receptor signal transduction with increasing donor age have also been reported [335]. In other recent studies, PGE2 was found to exert stimulatory and inhibitory effects on osteoblast differentiation and bone nodule formation through the EP-1/IP-3 pathway and EP-2/EP-4-cAMP pathway, respectively, in cells from young rats. However, the EP-1/IP-3 pathway was reported to be inactive in cells isolated from aged rats [336]. Thus, the known loss of bone with aging or menopause may be due to reduced responsiveness of osteoprogenitor cells to biological factors resulting in alteration of their subsequent differentiation potentials or to local changes in these factors. Bianco et al. [337,338] reported that stromal cells isolated from patients with fibrous dysplasia (FD) and McCune-Albright syndrome FD fail to recapitulate a normal ossicle in an in vivo transplantation assay; instead, they generate a miniature replica of fibrous dys-
plasia with ossicles that fail to establish a hematopoiesissupporting stroma, including adipocytes, and form less structurally sound and/or less abundant bone. Further analysis of these cells should prove useful for the design of effective therapeutic strategies.
TISSUE ENGINEERING AND STEM CELL THERAPY FOR SKELETAL DISEASES During the past few years, there has been much interest in the potential use of stem cells for cell and gene therapy in many disorders of metabolic, environmental, and genetic origins, including diseases of the skeleton. Studies with embryonic stem (ES) cells [339] and postnatal, tissue-derived stem cells, including HSCs and MSCs, are attracting considerable attention [340-343]. As is already evident, bone marrow and HSC transplantation is one paradigm by which osteoclast biology has been advanced. However, for many years, lack of evidence for significant donor stromal cell engraftment in host animals and patients receiving bone marrow transplants led to the conclusion that transplantation of osteogenic cells, particularly by a systemic route of delivery, was an unachievable goal. As already summarized and notwithstanding significant caveats, recent data challenge that conclusion. As outlined previously, MSCs and MAPCs are notable not only because of their potential for significant expansion but also for their apparently robust and multilineage differentiation potential, at least in vitro. They have therefore been proposed as suitable for both cell and gene therapy for skeletal diseases such as osteoporosis and osteoarthritis in which replacement of skeletal mass and refurbishing bone and joint structures are goals [344]. Another goal is replacement of missing or damaged tissue in a number of diseases affecting younger individuals. For example, some forms of osteogenesis imperfecta (OI) may be amenable to cell and gene therapy approaches, and both mouse OI and human studies are under way. Particularly notable are results from Horwitz and colleagues, who found evidence for a beneficial bone effect in OI children transplanted with HLAcompatible sibling bone marrow [345] or allogeneic stromal fibroblasts transplanted after bone marrow transplantation [346]. On the other hand, no conclusive effects have been seen in children transplanted with allogeneic stromal fibroblasts alone [Horwitz, 1999 # 1141]. In these latter studies, few or no donor cells were detectable 6 months posttransplantation, consistent with the low donor cell engraftment seen in most studies in humans and mice in which a low degree (1-5%) of
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engraftment of bone or bone marrow stroma is seen, as assessed by a transgenic or unique endogenous genetic marker [347]. As discussed previously, however, in most cases the marker genes used do not discriminate whether the donor cell arises from a mesenchymal or, for example, from a macrophage lineage cell or donor-recipient cell fusion. In this regard, only one study in which whole bone marrow was delivered systemically reported the presence of donor osteoblasts and other osteoblast lineage cells, although the degree of engraftment and contribution of donor cells to bone formation was not assessed [348]. Nevertheless, the studies offer considerable promise and highlight the need for many more detailed studies in which engraftment rates, engraftment longevity, and the underlying mechanisms by which clinically relevant improvements in outcome are achieved are all assessed.
Acknowledgments We thank many members of our labs and other colleagues for valuable input and discussions over many years. This work was supported by CIHR Grants MT-12390 and MOP-49419 to JEA and MT-15654 and MT-14655 to JNMH and a grant from the Stem Cell Network of Centres of Excellence (JEA).
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3 Prenatal Bone Development Ontogeny and Regulation BENOIT ST.-JACQUES*and JILL A. HELMS t *Department of Human Genetics, McGill University, and Genetics Unit, Shriners Hospital for Children, Montreal Quebec, Canada tDepartment of Orthopedic Surgery, University of California, San Francisco, San Francisco, California
INTRODUCTION
local level. Thus, the division of skeletal development in pre- and postnatal periods, while understandable from a clinical standpoint, is somewhat artificial from a developmental standpoint. Due to space limitation, this chapter focuses primarily on local control of the early steps of skeletogenesis. A more complete treatment of hormonal regulation of bone growth is provided in Chapter 4. Skeletogenesis in the human embryo has been well described at the anatomical and histological level. However, for obvious reasons, our current understanding of the regulation of bone formation at the molecular level derives almost completely from experimental approaches using other vertebrate embryos. In particular, advances in genetic manipulations in the mouse have allowed the identification of many genes regulating skeletogenesis [2]. Improved techniques to modulate and detect gene expression, combined with the ease of surgical manipulations, have also made the chick embryo a very powerful tool to study questions related to early patterning and organogenesis. Finally, the recent identification of the genetic basis for several hereditary skeletal disorders in human has yielded many significant insights [3,4]. This chapter emphasizes results obtained from such in vivo systems as well as from organotypic culture systems at the expense of the vast and sometimes confusing literature reporting observations on immortalized cell lines. Formation of the skeleton can best be understood as a series of steps [5] contributing to one of two processes, namely skeletal patterning or bone formation [6]. Migration of undifferentiated mesenchymal cells to their ultimate location in the embryo, their interaction with a local
"The toolbox used to pattern an organism is at hand. Add a pinch of BMP, sprinkle some Hedgehog, a touch of Wnt, and a handful of F G F and you can pattern an embryo, a limb, or an organ. The secret lies not in the ingredients themselves, but in the order and amount in which they are provided, the way they are distributed in space and time, their cellular context, and the intricate cross-regulatory interactions among different signaling pathways." [1]
The passage quoted previously is obviously an exaggeration in that it offers a very generous assessment of our current understanding of the molecular mechanisms controlling organogenesis. Nevertheless, it rightly highlights the remarkable progress made during the past 10 years in identifying many of the key molecules involved in these control mechanisms. It also illustrates the fact that in formation of the diverse skeletal elements, like any other organ, the early steps depend largely on local cell-cell and cell-matrix interactions mediated by factors acting in a paracrine or autocrine manner. Effectors of these interactions include specific receptors, molecules of the intracellular signal transduction pathways, transcription factors, and specialized extracellular matrix components. Most of these signaling pathways are also involved in formation of other organs, and most of them play multiple roles at different stages of skeletogenesis. At later developmental stages, however, many of the mechanisms and pathways that will control postnatal bone growth are also acting. These include regulatory mechanisms at the systemic (endocrine) as well as the
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epithelium, and their condensation into cartilage and bone precursors lead to the specification of the position, number, and shape of all skeletal elements and define skeleton patterning. Mutations in genes essential for these early steps most often result in dysostoses, disorders that affect specific skeletal elements while the rest of the skeleton is normal [7]. Differentiation of the skeletal-specific cell types (chondrogenesis and osteogenesis) and the precise coordination of cellular proliferation, extracellular matrix production and remodeling, programmed cell death, and angiogenesis define bone formation. Mutations in genes controlling these processes result in dysplasias, conditions affecting cartilage and bone tissues generally [7]. Following this logical dichotomy, this chapter first presents a general overview of skeletogenesis at the cellular level and describes the embryonic origins of the skeleton as well as some of the molecular mechanisms controlling early patterning. Then, we discuss in more detail our current view of the molecular mechanisms controlling processes common to the formation of all bones, including condensation, differentiation, and growth. Reviews covering many aspects of the broad subject treated in this chapter have appeared recently and the reader is urged to refer to these for complementary information [2-15]. A recent compilation of more than 200 genes expressed in skeletal tissues also provides useful information on cellular function and associated diseases [16].
SKELETOGENESIS In this chapter, the term skeletogenesis is used when referring to the complex multistep process by which the different elements of the skeleton are formed. Thus, skeletogenesis encompasses patterning, differentiation, and growth. Bone formation is a general term used to describe the steps common to the formation of all bones and thus excludes specific patterning mechanisms. We reserve the term ossification to designate specifically the process whereby osteoblasts differentiate and the bone matrix they secrete becomes a significant component of a given skeletal element. Similarly, chondrification refers to the appearance of chondroblasts and the production of a cartilage matrix. Finally, calcification refers specifically to the process by which the original bone matrix (osteoid) or the cartilage matrix produced by hypertrophic chondrocytes become calcified~that is, enriched in calcium phosphate deposits in the form of hydroxyhapatite crystals.
M o d e s of B o n e Formation Most members of the animal kingdom have either an exoskeleton or an endoskeleton depending on whether their skeletal tissues form on the outside or inside of the body. Primitive vertebrates had both a true exoskeleton and an endoskeleton. The exoskeleton is believed to have originated early during vertebrate evolution as a series of dermal plates that covered the body and served as protective armor. Internal support for the gills, sense organs, neural tube, appendages, and organs of the trunk was provided by a cartilaginous endoskeleton, which later in evolution also came to utilize bone [17]. In modern vertebrates, most skeletal elements derive from the primitive endoskeleton. Vestiges of the exoskeleton, however, are present in the skull and pectoral girdle. Although such exoskeletal derivatives have become thoroughly integrated with elements of the endoskeleton, they still form in a manner unlike that of the endoskeleton. These bones are termed dermal based on their historical association with the skin and ectoderm. They form by direct differentiation of mesenchymal cells into osteoblasts in a process called membranous (or intramembranous) ossification in reference to their association with membranes covering the developing brain in formation of the skull vault. On the other hand, bones deriving from the primitive endoskeleton develop first as cartilage elements that are subsequently replaced by bone. Therefore, they are known as cartilage replacement bones [18], and the process by which they form is called endochondral ossification. It should be recognized, however, that in the complex process of endochondral ossification of a typical long bone, formation of the trabecular bone of the metaphysis and of the cortical bone of the diaphysis takes place by two very different mechanisms. In the first case, cartilage is replaced by bone in a true "endochondral" process. In the case of the cortical bone, perichondrial cells differentiate into osteoblasts without replacement of a cartilage intermediate. Consequently, this process is often described in the scientific literature as a type of intramembranous ossification. However, it is clear that at the molecular level, as well as from a developmental standpoint, the regulation of this type of"subperiosteal" ossification and true intramembranous ossification are significantly different. Thus, there are three different modes of bone formation: intramembranous, endochondral, and subperiosteal. Intramembranous Ossification
Intramembranous ossification begins toward the end of the second month of gestation in humans. The process has been most extensively studied in the developing
3. Prenatal Bone Development cranium, where it is often preceded by a cellular proliferation at specific sites in the mesenchyme [19]. It becomes histologically evident when a cluster of pale-staining stellate mesenchymal cells aggregate and take on a rounded basophilic appearance characteristic of osteoprogenitor cells. These gradually differentiate into mature secretory osteoblasts that actively produce the extracellular matrix rich in collagen type I that is characteristic of bone. Sites where this process occurs are called ossification centers, and most dermal bones originate from a small number of such centers. It is important to note that although the initial condensation occurs in an avascular milieu, the differentiation of osteoblasts and the onset of mineralization are intimately related to blood vessel invasion, first in the surrounding mesenchyme and ultimately in the bone rudiment [20]. The first bit of bone (spicule) is irregularly shaped and completely surrounded by the osteoblasts that secreted it. Some of the osteoblasts soon become enclosed in the matrix being formed around them and thereafter become known as osteocytes. Each osteocyte is enclosed in its own lacuna but extends projections through small channels called canaliculi to maintain contact with neighboring osteocytes and intercellular fluids outside of the ossification center. Less differentiated osteoprogenitor cells present at the periphery proliferate to supply new osteoblasts, which continue to add bone to form a well-defined longer structure called a trabecula (beam). Similar changes occur in the immediate vicinity so that new trabeculae are formed and the center of ossification expands. Some trabeculae radiate from the center. These primary ones soon become connected by secondary trabeculae to form a scaffolding characteristic of cancellous (or spongy) bone. All trabeculae increase in thickness as new bone is added on their surfaces and the primary ones increase in length by accretion on their free ends. The pattern of growth and orientation of bony trabeculae depends on the type of bone. In the case of "fiat" bones, such as the parietal or frontal, the primary trabeculae soon form a radiating network mainly parallel to the surface of the skull, although some are directed at right angles and thus add to the thickness of the center [19]. The first bone formed in the embryo is of an immature type, displaying relatively high cellularity and an almost random orientation of collagen fibers [21]. At approximately the time of birth, this woven bone is gradually replaced by the more mature lamellar bone characterized by successive layers of uniformly oriented collagen fibers. Almost all the immature bone that forms during embryonic life is eventually replaced, although some persists mixed with mature bone in the tooth sockets, near cranial sutures, in the osseous labyrinth, and near tendon and ligament attachments [22]. At an early stage, the intramembranous bone has the
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structure of a scaffold of trabeculae with relatively little bone and with large spaces invaded by blood vessels. As remodeling occurs, the continued deposition of fresh bone lamellae on trabeculae changes the nature of the bone from cancellous (spongy) to compact (dense), with only narrow canals occupied by blood vessels [22]. Endochondral Ossification
This process also initiates with the aggregation of undifferentiated mesenchymal cells to form condensations~ Unlike in intramembranous ossification, however, cellular proliferation does not play an important part in this process. The condensation step is the result of an increase in cell packing mediated by changes in the extracellular matrix and cell-cell adhesion molecules [23]. By their positions, shapes, and sizes, these prechondrogenic condensations prefigure the different skeletal elements [24] (Fig. 1A). As in intramembranous ossification, the initial condensation forms in an avascular environment, but here it remains avascular. In the core of these condensations, cells differentiate into chondroblasts (Fig. 1B) that secrete a cartilage matrix characterized by the presence of types II, IX, and XI collagen and specific proteoglycans such as aggrecan. At the periphery of the condensation, cells surrounding the cartilage core flatten and form a thin membrane of stacked cells called the perichondrium, which insulates the cartilage from the surrounding mesenchyme [25] (Fig. 1C). Perichondrial cells retain chondrogenic potential and probably contribute to the radial expansion of the cartilage by appositional growth. As new matrix is produced, the central cells become enclosed in it and become known as chondrocytes. At this early stage, the chondrocytes and perichondrial cells proliferate rapidly, and this proliferation together with the deposition of new matrix drives the growth of the elements. At a certain stage, which is specific for each element, chondrocytes in the center undergo progressive maturation. They acquire a flattened appearance and become organized in columns along the longitudinal axis of the developing skeletal element (Fig. 1D). Columns are separated by relatively thick lateral partitions of cartilage, the longitudinal septa, whereas chondrocytes within a column are separated by thin transverse septa. Further maturation of these cells leads to the development of a hypertrophic chondrocyte phenotype characterized by cell enlargement, cessation of proliferation, and secretion of a distinct extracellular matrix rich in type X collagen that becomes progressively calcified [26] (Fig. 1D). These changes are accompanied by vascularization of the perichondrium tissue and soon after by differentiation of theinner perichondrium cells into osteoblasts, which secrete a layer of primary bone, the
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FIGURE 1 Endochondral bone formation. Schematic representations of the different steps in the process of endochondral bone formation. (A) Mesenchymalcondensation. (B) Chondroblastdifferentiation and formation of a perichondrium (pe). (C) Maturation ofchondrocytesand appearance of enlarged (hypertrophic)cellsin the center of the element. Most chondrocytesand perichondrial cells proliferate rapidly at this stage and the element enlarges by interstitial and appositional growth. (D) Vascularization of the perichondriumand organization of an embryonic growthplate with zonesof proliferation (p), maturation (m), and hypertrophy(h). (E) Depositionof a bone collar by periosteal osteoblasts (not shown), vascular invasion of the calcified cartilage of the hypertrophic zone, and apoptosis of terminally differentiated hypertrophic chondrocytes. (F) Resorbtion of the cartilage matrix by "septoclasts" and appearance of the trabecular osteoblasts establishing the primary ossification center and the marrow space (ma).
bone collar, surrounding the hypertrophic region of the cartilage [25] (Fig. 1E). At this stage, the thin membrane covering the newly formed bone becomes known as the periosteum and continues to supply osteoblasts that produce the bone matrix of the diaphysis. Changes in the composition and properties of the cartilage matrix in the hypertrophic zone, including calcification of the longitudinal septa and the release of angiogenic factors, trigger its invasion by capillaries [27] (Fig. 1E). This results in death of the terminally differentiated hypertrophic chondrocytes and degradation of the uncalcified transverse
cartilage septa by invading septoclasts (sometimes called chondroclasts), a cell type of ill-defined origin associated with the invading capillary sprouts [28]. Remnants of the calcified longitudinal septa act as templates on which invading osteoblasts secrete the bone matrix of the first trabeculae, thus establishing the primary ossification center (Fig. 1F). Osteoclasts, which are bone-resorbing cells of hematopoietic origin, subsequently remove the newly formed bony trabeculae to generate the marrow space. The synchronous maturation and columnar organization of the chondrocytes result in a dynamic histo-
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logical structure known as the embryonic growth plate in which zones of proliferation, maturation, hypertrophy, and bone formation can be identified, proceeding from the articular ends (epiphysis) to the midshaft (diaphysis) of the element. Continued proliferation of the less mature chondrocytes at the extremities, their differentiation into hypertrophic chondrocytes, and their replacement by trabecular bone near the center result in distal displacement of the growth plate and longitudinal growth of the bone. In a typical long bone, this growth is accompanied by resorbtion of the older trabeculae to generate the marrow space of the diaphysis, thus leaving significant trabecular bone only at the extremities (metaphysis). Continued deposition of cortical bone by the periosteum (subperiosteal bone) leads to radial growth. Eventually, a secondary ossification center forms in the epiphysis distal to the growth plate, from which ossification proceeds in all directions. However, this does not modify the general organization of the growth plate, which remains active until cessation of growth after puberty. In the long bones of many species, including human, the growth plate eventually disappears and the only cartilage that remains is the articular cartilage, which forms a protective, low-friction surface at the extremities of the bones to allow smooth joint movement. As in intramembranous ossification, the first bone produced in the trabeculae and the diaphysis of the long bones is of a woven type. Removal of this immature bone by osteoclasts leads progressively to its replacement by mature lamellar bone [22]. Coordinating the different steps of the endochondral ossification process is critical in determining the size, shape, microarchitecture, and ultimately the mechanical properties of the bones. This coordination is achieved to a large extent by the action of a number of signaling pathways mediating cell-cell communication. Timing a n d S e q u e n c e of B o n e Formation in H u m a n s All skeletal elements do not start forming simultaneously in the embryo. Generally, chondrification proceeds in a rostral-caudal (head-to-tail) fashion in the axial skeleton and in a proximal-distal (shoulder-tofingertips) fashion in the limbs skeleton [19]. This is a direct consequence of the developmental origin of the different elements from the progressively forming somites and the outwardly elongating limb buds, respectively. The chondrification sequence is rapid, and early in the seventh gestational week the cartilage primordia Of all the elements of the axial and appendicular skeleton are present [29]. In the cephalic regions, cartilage formation proceeds in a caudal-to-rostral direction. It starts at the beginning of Week 7 and continues well into Week 8
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[30]. Thus, the appearance of the cartilage primordia of the skull base occurs after that of most axial and appendicular elements, but it precedes ossification of the membranous bones of the skull vault. There is relatively little information on the timing of the formation of the mesenchymal condensations that will initiate formation of the different endochondral bones in the human embryo. On the other hand, the sequence of ossification of the different skeletal elements has been extensively studied [30-32]. The sequence of ossification in the mouse is also well-known [33] and generally resembles that in human. The following discussion highlights some general observations. Almost all primary ossification centers appear between Weeks 7 and 12 of embryonic life. In contrast, the secondary ossification centers such as in the epiphyses appear over a long period, from late fetal life until puberty. The clavicle is the first bone in the body to ossify, with the mandible and maxilla following almost immediately. The premaxilla forms in continuity with the maxilla proper, either as a mesenchymal condensation or as a center of ossification. Generally, the facial and calvarial centers appear before the basicranial centers, followed by the hyoid centers [31]. More than 100 centers of ossification appear during human skull formation [34]. Extensive fusion will take place between many of them, such that the number is reduced to 45 by the time of birth [35]. Postnatal fusion leads to further reductions. For instance, the human occipital bone ossifies from at least seven centers, two of which are intramembranous and responsible for the "interparietal" (squamous) part of the bone. Thus, in its definitive form, the occipital bone is a composite structure, and the different parts of the occipital do not completely fuse until several years after birth. In the axial skeleton, the costal centers appear first, followed by the primary vertebral centers and the sternal centers. The second to 11th rib centers appear at approximately the same time. The centers in the first and 12th ribs appear later in that order. The vertebrae ossify from three primary centers, one for the body and one for each neural arch. In the spine as in the thoracic cage, the sequence is not strictly cephalocaudal because ossification of the vertebral body starts first in the lower thoracic and upper lumbar regions and propagates in both directions from this area. Some delay is observed before the first cervical and the last two sacral centers appear [31]. Ossification centers in the neural arches appear in a cephalocaudal sequence except in the atlas and axis, in which ossification is slightly delayed [31]. The sternum arises as a pair of cartilaginous bands that fuse along the midline as the ventral body wall develops. This fused cartilage precursor subsequently subdivides into craniocaudal elements, most of which will fuse again and will ossify after the fifth month to form the body of the sternum [36].
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The primary ossification centers of the pectoral girdle appear before those of the pelvis in the following order: clavicle, scapula, ilium, ischium, and pubis. The centers in the humerus, femur, and then radius, ulna, and tibia appear at approximately the same time. The fibula differentiates slightly later. The bones of the hand invariably appear before their counterparts in the foot. These differ in that centers in the distal phalanges of the hand appear before those of the metacarpals, whereas the distal phalanges centers of the foot appear after the metatarsal centers [31]. The proximal and middle phalanges ossify last. Of the tarsal and carpal bones, only the talus and calcaneous generally begin to ossify before birth, with the cuboid sometimes and the lateral cuneiform rarely ossifying before birth [19].
SKELETAL ORGANIZATION AND EMBRYONIC ORIGIN OF BONES The human skeleton can be divided into three components based on the anatomical location of the elements: the craniofacial (skull), the axial (vertebrae and ribs plus sternum and sacrum), and the appendicular (limbs, including the shoulder and pelvic girdles) skeletons. These subdivisions also reflect the different embryonic origins of the bones. Whereas the axial and appendicular bones derive from somitic and lateral plate mesoderm, respectively, the craniofacial bones develop largely from the neuroectodermally derived neural crest cells with some contribution from cephalic paraxial mesoderm. This difference in germ layer of origin may be of considerable importance and recent data indicate that the molecular pathways leading to formation of these tissues are distinct. Nonetheless, it is important to note that regardless of developmental origins, all skeletal elements will eventually share similar histological structures and comprise the same few cell types: the chondrocytes in cartilage, the osteoblasts (and osteocytes) or bone-forming cells, and the osteoclasts or bone-resorbing cells, which are of a completely different origin (see Chapter 2).
Skull Anatomically, the skull is divided into the neurocranium, the part surrounding the central nervous system, and the viscerocranium, the skeleton of the face and branchial arches (also called pharyngeal arches in human). Each of these can also be subdivided into a chondrocranuim, composed of the bones that undergo endochondral ossification, and a desmocranium (or dermatocranium), composed of the bones that undergo membranous ossification.
Cartilaginous Neurocranium Formation of the skull base is one of the most complex parts of skeletogenesis and is only briefly described here. Thorough treatment of this important issue can be found in the authoritative review of Dixon [37]. The cartilage of the cranial base appears early in development as a number of condensations that follow a similar pattern in all vertebrates [38]. These centers of cartilage formation can be grouped according to their position relative to the notochord, the rod-like axial mesoderm structure that runs along the rostral-caudal axis of the early embryo. These is a parachordal region centered around the cephalic end of the notochord, a prechordal (or trabecular) region that lies anterior to the notochord, and centers associated with the sense capsules (otic, optic, and nasal) (Fig. 2A). These primordia undergo a complex pattern of growth and fusion to form an elaborate shelf-like base beneath the developing brain and ultimately form most of the bones of the cranial base as well as the deep bony support of the nasal cavity (Figs. 2B and 2C). In humans, the first chondrification centers appear on each side of the cranial end of the notochord on approximately Day 40 and soon fuse to form the rectangularshaped parachordal or basal plate destined to form the base of the occipital bone. At this early stage, the prechordal cartilages are represented by a pair of small hypophyseal cartilages and more rostrally by a pair of elongated trabecular cartilages (trabeculae cranii). The hypophyseal cartilages will fuse to form the basisphenoid cartilage that will give rise to the sella turcica and posterior part of the sphenoid bone. The trabecular cartilages also fuse to form the precursor of the presphenoid and an interorbital septum in which the perpendicular plate of the ethmoid bone will develop. Immediately caudal to the basal plate and rostral to the first cervical vertebra, four occipital somites give rise to three mesodermal condensations that will fuse with the caudal aspect of the basal plate to give rise to the part of the bassioccipital defining the foramen magnum. Chondrification centers later appear laterally to form the primordia of the condylar and supraoccipital parts of the occipital bone. Two other pairs of prechordal cartilages, the ala orbitalis (or orbitoshenoid) and the ala temporalis (or alisphenoid), also form on the ventrolateral aspects of the brain. These will grow and fuse with the main basal stem to develop into the lesser and greater wings of the sphenoid, respectively. At the same time, the cartilaginous capsules of the sense organs develop. The otic capsules are major features of the lateral part of the chondrocranium and contain the developing inner ear apparatus. They will fuse anteriorly with the parachordal region and will
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FIGURE 2 Development and organization of the human skull. The diagrams present lateral views of the human embryonic skull at different developmental stages: (A) 6 weeks, (B) 8 weeks, and (C) 12 weeks. The thin continuous line represents the contour of the head. The dashed line represents the contour and position of the developing brain. The different portions of the skull are represented by different shadings: gray, cartilaginous neurocranium; black, cartilaginous viscerocranium; striped, intramembranous neurocranium; white, intramembranous viscerocranium. 1, Nasal capsule; 2, trabecula crani; 3, optic capsule; 4, ala orbitalis; 5, ala temporalis; 6, hypophyseal cartilage; 7, otic capsule; 8, parachordal cartilage; 9, occipital sclerotomes; 10, first cervical sclerotome; 11, sphenoid; 12, Meckel's cartilage; 13, hyoid; 14, thyroid cartilage; 15, cricoid cartilage; 16, malleus; 17, incus; 18, Reichert's cartilage; 19, occipital; 20, petrous part of temporal; 21, lacrymal; 22, nasal; 23, premaxilla; 24, maxilla; 25, mandible; 26, tympanic ring; 27, styloid process of temporal; 28, stapes; 29, zygomatic; 30, zygomatic arch; 31, squamous part of temporal; 32, greater wing of sphenoid; 33, interparietal part of occipital; 34, parietal; 35, frontal.
ossify as the petromastoid part of the temporal bones. The nasal capsule (ectethmoid) develops as an extension of the trabecular cartilages and becomes the primary skeleton of the upper face.
spheres, these come to define the zones where bone growth slows down and the coronal, lamboid, and saggital sutures develop.
Cartilaginous Viscerocranium Dermal Neurocranium Early in human development, a delicate membrane of mesodermal origin, the meninx primitiva, encapsulates the neural tube [34,39]. This membrane later becomes divided into an inner endomeninx, which differentiates into the deeper meninges, the pia mater, and the arachnoid, and an outer ectomeninx, which gives rise to the dura mater and an outer superficial membrane with osteogenic potential. The bones of the calvaria--the frontals, parietals, and interparietal part of the occipital bone--develop by intramembranous ossification in the ectomeninx that forms the roof of the neurocranium. Ossification centers first appear in areas corresponding to the future eminences and bone formation spreads centrifugally. As development progresses, the dura mater thickens and becomes firmly attached at the dural stretches to the skull base. The dural stretches continue dorsally as frontal, parietal, and occipital dural girdles. In conjunction with a saggital reflection between the cerebral hemi-
The cranial region of an early human embryo resembles that of a fish embryo of a comparable stage. The branchial or pharyngeal arches, which appear during Weeks 4 and 5 of development, contribute to this characteristic appearance of the embryo. These represent evolutionary remnants of the gill arches of more primitive vertebrates. There are usually five identifiable pairs of arches (1-4 and 6), although only the first four are visible on the surface (Fig. 3A). They form during a period of rapid growth of the face and head, with the first arch appearing at approximately 24 days and the others in sequence at approximately 2-day intervals. Each pharyngeal arch consists of a core of mesenchymal tissue covered on the outside by the surface ectoderm and on the inside by epithelium of endodermal origin. Cartilage elements of the viscerocranium form in the mesenchyme of these pharyngeal arches. The first pharyngeal arch, or mandibular arch, develops two elevations called the mandibular and the maxillary prominences (Figs. 3A and 3B). The mandibular prominence gives rise to paired
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FIGURE 3 Cephalicregion of a 28-day human embryo. (A) Lateral view showing the pharyngeal (branchial) arches 1-4. (B) Frontal view showing the division of the first arch into maxillary and mandibular prominences. (C) View in saggital section of the embryo depicted in A showing the contribution of neural crest cells from different axial levels to the mesenchymeof the pharyngeal arches. D, diencephalon; M, mesencephalon; RI-R8, rhombomeres 1-8 of the hindbrain; T, telencephalon; dark gray, central nervous system(CNS).
rod-like structures called Meckel's cartilages that pass ventrally from the region of the otic capsule downward and forward and meet at the midline of the future chin (Fig. 2B). They are one of the most recognizable histological features of the developing head. Meckel's cartilage is a transient structure and is not a cartilaginous precursor of the mandibular bone, which develops by intramembranous ossification. However, it may play a role in morphogenesis of the jaw by guiding the spread of the mandibular bone in anterior and transverse directions from the original center of ossification, and disruption of Meckel's cartilage does affect the shape of the mandible. It may also contribute actively to secondary palate formation by displacing the tongue from between the palatal shelves. At the very proximal or tympanic portion of the cartilage, two small condensations separate and undergo endochondral ossification to give rise to the malleus and incus of the inner ear (Fig. 2B). In human, the rostral (ventral) extremities of the left and right cartilages come in close contact but do not fuse. They remain as cartilage nodules (the chondriola symphysea) in the fibrous tissue of the symphysis of the jaw [40]. The central portion of Meckel's cartilage disappears. In the rostral half, vascular invasion occurs and resorbtion takes place by a process similar to endochondral ossification [41]. In the caudal half, cells acquire a fibroblastic phenotype and give rise to the sphenomandibular ligament [41]. Programmed cell death may also contribute to the disappear-
ance of this structure, although its importance in this process is controversial [41,42]. An elongated cartilage (Reichert's cartilage) also forms in the second pharyngeal arch (Fig. 2B). The dorsal part of this cartilage will ossify to give rise to the stapes of the middle ear and the styloid process of the temporal bone. The ventral end will form part of the hyoid bone (lesser cornu and superior part of the body), whereas the central portion regresses and forms the stylohyoid ligament. The third arch cartilage produces the remainder of the hyoid (greater cornu and inferior part of the body), whereas the fourth and sixth arches form the laryngeal cartilages, including the thyroid and cricoid cartilages. Dermal Viscerocranium
The bones of the face also derive from the mesenchyme of the pharyngeal arches but form via intramembranous ossification (Fig. 2C). The maxillary prominence of the first arch gives rise to the maxilla, which appears as an ossification center below and lateral to the cartilage of the nasal capsule. From this initial center, ossification spreads in the three dimensions to form the alveolar, frontal, and zygomatic processes. From the maxilla, bone gradually develops in the primary palate, forming the premaxillary part, which carries the incisors. Outgrowths of the maxillary prominences also form the palatine shelves, which fuse to form the secondary palate
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within which the palatine bone forms. The zygomatic and the squamous part of the temporal bone also derive from the maxillary prominence. The mandible forms by intramembranous ossification in the mesenchymal core of the ventral part of the mandibular prominence. Contrary to what occurs in other vertebrates, each half of the lower jaw develops as a single element in human. It first appears as an ossification center on the outer aspect of the ventral half of Meckel's cartilage and spreads rapidly anteriorly and posteriorly along its lateral side. Ossification later proceeds medially above and below to form a bony trough that surrounds the cartilage. Thus, Meckel's cartilage contributeslittle to the definitive mandible and is not involved in formation of the condylar cartilages.
Sutures and Synchondroses Sutures (syndesmoses) are craniofacial articulations in which contiguous margins of bones approximate each other and are united by a thin layer of fibrous tissue that permits minor movement [43]. Sutures develop initially by a wedge-shaped proliferation of cells, the osteogenic front (OF), at the periphery of the growing bones [44,45]. OFs may approximate each other in the same plane, leading to the development of end-to-end sutures (the saggital and midpalatal sutures in human), or more commonly may overlap each other, leading to the development of overlapping sutures (all other sutures in humans). Once a suture has formed, five distinct layers can be recognized histologically in most sutures: two cambial and two capsular layers of each adjoining bone, separated by a middle vascular layer [46]. With maturation, the cambial layer becomes reduced to a single layer of osteoblasts, the capsular layer thickens and its fibers become parallel to the sutural surface of the bone, and the middle layer becomes increasingly vascularized [43]. In humans, most sutures become obliterated by fusion of the bones they separate only in young adulthood or later, except for the metopic suture (between the frontal bones) that is usually closed by the third year of life. Synchondroses are remnants of the chondrocranium that persist between ossifying elements and are the principal site of cranial base growth [47]. Structurally, they have the appearance of a bipolar epiphyseal growth plate characterized by a central "germinative" area, on each side of which can be recognized zones of chondrocyte proliferation, maturation, and hypertrophy followed by a zone of primary ossification. The growth of synchondroses is controlled by the same factors regulating epiphyseal growth plate physiology and their function is severely affected in diseases of the endochondral skeleton, leading to a range of craniofacial abnormalities characteristic of many chondrodysplasias.
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Embryonic Origin of Skull Cells Largely due to technical limitations, studies of skull development are much better established in birds than in mammals. Avian data have therefore been used for extrapolation to mammalian skull origins, including the human skull. It is clear that in both birds and mammals, head mesenchyme originates from two principal sources--the cephalic (or cranial) neural crests and the cephalic paraxial mesoderm [48] (Fig. 4A). The neural crest is a transient pluripotent cell population that originates in the folds of the developing neural tube, between the future neural and epidermal ectoderm (Figs. 4B and 4C). As the neural tube develops, neural crest cells delaminate from the prospective dorsal neural tube, undergo an epithelial-to-mesenchymal transition, and migrate in a ventral and lateral direction following a precisely determined pattern both spatially and temporally [49] (Fig. 4D). This migration is rapid and in humans probably takes place between Days 19 and 38 after fertilization, a critical period during which many teratogenic agents can induce craniofacial malformations [47]. The mesenchyme formed by neural crest cells is designated ectomesenchyme to indicate its different embryonic origin (from the ectoderm). Crest cells appear along the entire length of the neural tube and differentiate into a large number of cell types, including neurons and glial cells, melanocytes, smooth muscle, and some endocrine cells. Only cephalic neural crest cells, however, have the additional capability of forming bone, cartilage, and odontoblasts, which are the dentine-secreting cells of the teeth [49]. The contribution of crest cells from different levels of the neural tube has been precisely mapped in cell lineage studies using short-term labeling techniques. Cells originating at the forebrain and midbrain levels will contribute to the frontonasal mass to give rise to all the connective tissue of the face. Cells originating at the level of the rhombomeres (the segmented divisions of the hindbrain) migrate according to their rhombomeric origin to populate the branchial arches and will give rise to all the skeletal elements of the dermal and chondral viscerocranium (Fig. 3C). Crest cells from the posterior midbrain and rhombomeres 1 and 2 populate the first pharyngeal arch (mandibular). Cells from rhombomeres 4 6, and 7 and 8 migrate within arches 2, 3, and 4-6, respectively [49]. Although most of the cephalic mesoderm yields muscles, a detailed analysis of the contribution of cephalic mesoderm in the chick indicates that it also gives rise to the parachordal elements of the skull base. Thus, in the chick, the basipostsphenoid and orbitosphenoid, part of the otic capsule, and the supraoccipital derive from medial cephalic mesoderm [50]. Finally, as indicated previously, the bassioccipital has a different origin
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Benoit St.-Jacques and Jill A. Helms altogether, deriving in part from the somitic m e s o d e r m of the occipital somites (paraxial mesoderm). The origin of the skull vault remains controversial. Despite discrepancies in early experimental results [49], it is likely that the entire cranial vault in the chick is neural crest derived [50,51]. However, extrapolation of the results to mouse and h u m a n skull development m u s t be regarded with caution. In rodent embryos, neural crest migration pathways at early stages were elucidated using dye injection techniques, but their contribution to m a t u r e structures could not be assessed. Recently, however, a transgenic mouse was engineered that has a p e r m a n e n t neural crest cell m a r k e r [52,53]. Analysis of neural crest contribution to different tissues in these animals generally confirmed observations in birds but also indicated that although the frontal and s q u a m o s a l bones are neural crest derived, the parietal and interparietal bones are p r o b a b l y of m e s o d e r m a l origin [54]. This interpretation is incorporated in the representation of the embryonic origin of the n e w b o r n skull elements depicted in Fig. 5. However, further studies in m a m m a l s will be required to confirm this interpretation.
Patterning of the Craniofacial Skeleton To date, a gene or c h r o m o s o m a l location have been linked to approximately 100 craniofacial m a l f o r m a t i o n s in humans, and gene targeting in mice has generated more than 90 loss-of-function m u t a n t s that show craniofacial m a l f o r m a t i o n s [55]. In m o s t of these phenotypes, skeletal elements are affected but in m a n y cases these are secondary defects resulting from axial truncation or neural tube malformation. N o t surprisingly, however, a large proportion of these m u t a t i o n s affect the specification, migration,
FIGURE 4 Origins of skull cells and neural crest cell formation. (A) Dorsal view of the cephalic region of a four-somite chick embryo showing the regions contributing cells to the skull, including the neural folds (nf), in which neural crest cells originate, and the medial head mesoderm (hm, outlined in gray), np, neural plate; nt, neural tube; s, somites. (B) Diagram of tissue interactions leading to specification of the neural ectoderm borders. The neural plate (np) is light gray and the edges of the neural plate are dark gray. Signals emanate from the nonneural ectoderm (n-ne, the prospective epidermis), the node (no), and the paraxial mesoderm (pm). The nature of the ectodermal signal is not known. Signals from the other sources probably include some FGFs and induce M s x l expression in a restricted domain. (C) Neural crest cell differentiation. M s x l expression in the prospective neural crest region (nc) induces BMP4 expression and a positive regulatory loop is established to maintain expression of both factors in the nc region and specify the neural crest cell phenotype, n, notochord; ne, neural ectoderm. (D) Neural crest cell migration. Neural crest cells (ncc) delaminate from the edges of the neural ectoderm and migrate away from the closing neural tube (nt) in a ventrolateral pattern, n, notochord; se, surface ectoderm.
FIGURE 5 Origins of the skull bones. The diagram represents the different bones of a human skull at birth. Embryonic origins of the skeletal elements are depicted by different shadings. Light gray, cephalic neural crest derived; dark gray, possibly medial head mesoderm derived; black, paraxial mesoderm derived (occipital somites).
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or differentiation of the neural crest cells that will give rise to the head skeleton. Rapid progress is being made in the identification of mechanisms controlling these processes [56-59]. Specification of the crest cell population depends on interactions between the neural plate and nonneural ectoderm (prospective epidermis) preceding neural tube closure. Creating contact between these two cell types, either in vivo or in vitro, induces neural crest cell differentiation. There is also evidence that signals from the nonaxial mesoderm are required for neural crest formation. The molecular nature of the signals involved is not completely understood but probably involves the coordinated action of members of the bone morphogenic protein (BMP) and fibroblast growth factor (FGF) families of secreted signaling molecules [56]. According to one model, lateral signals from the nonneural ectoderm (unidentified), paraxial mesoderm, and Hensen's node (possibly an FGF signal) specify the neural crestforming regions at the lateral edges of the neural plate and induce expression of the homeobox-containing transcription factor M s x l (Fig. 4B). This factor activates BMP-4 transcription to establish a positive feedback loop that restricts and maintains high expression of both genes at the border [60] (Fig. 4C). Bmp-4 signaling within the neural fold then induces or maintains the neural crest precursors as shown by the fact that inhibition of BMP signaling in the closing neural folds prevents neural crest formation [61]. Once specified, expansion of the prospective crest population depends on signals of the Wingless/Int-1 (Wnt)-related family of secreted glycoproteins expressed in the dorsal neural tube [62]. Differentiating crest cells express the gene Slug in chick and frog as well as the related gene Snail in mouse [63,64]. These encode transcription factors that are essential for the delamination process probably by regulating the expression of adhesion molecules [58]. The initiation of migration correlates with a decline in production of the adhesion molecules N-cadherin (N-CAM) and cadherin 6B. At the same time, however, cadherins 7 and 11 are upregulated, indicating that a fine regulation of cadherin expression is fundamental for this process [59,65]. The migration routes followed by crest cells are dictated by the composition of the extracellular matrix (ECM). Neural crest cells appear to be prohibited from entering particular territories in the embryo by the presence of inhibitory molecules and to be guided along specific routes by the presence of permissive, migration-promoting factors. Hyaluronic acid, fibronectin, laminins, tenascins, and various collagens and proteoglycans are found in this matrix along the routes taken by migrating crest cells. A large number of these ECM components have been tested in vitro and demonstrate permissive or nonpermissive activities [49]. Evidence of
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a role in vivo has been obtained from gene inactivation studies and injection of blocking antibodies or antisense oligonucleotides. These studies indicate that six ECM components are pivotal determinants of crest cell migration: fibronectin, laminin isoforms 1 and 8, aggrecan, and the PG-M/versican isoforms V0 and V1 [66]. Currently, almost nothing is known about the specific signals that are responsible for target localization and cessation of neural crest migration. Early studies in the chick embryo suggested that neural crest cells were preprogrammed before their migration to give rise to different craniofacial structures and that they passively carried this positional information from the neural tube to the periphery [48,67]. However, a number of studies also contradicted this model and highlighted the plasticity of cranial neural crest populations [68]. Trainor and colleagues [69] resolved this paradox and clearly established that the migrating crest cells that will give rise to the craniofacial structures are not prespecified but respond to local influences to form different structures. For instance, the endoderm of the foregut, which lines the pharyngeal arches, appears to play a major role in instructing the neural crest cells as to the size, shape, and position of the facial skeletal elements [70]. The molecular nature of the signals produced by the endoderm is unknown. On the other hand, known signaling molecules that control patterning and growth in the central nervous system and the limb have also been implicated in patterning of the face [71]. The face forms from the outgrowth of five primordia: the frontonasal process (FNP), and the paired maxillary and mandibular processes, which surround the primitive mouth. These primordia initially consist of buds of undifferentiated mesenchyme, covered by surface ectoderm, that will undergo a complex process of growth, fusion, and differentiation to give rise to the different tissues of the face, including the different bones and cartilages. The vitamin A derivative retinoic acid (RA), the secreted signaling peptide Sonic hedgehog (Shh), BMP-2, -4, and -7, as well as members of the FGF family present in the ectoderm and the mesenchyme of the developing face influence the size, shape, and identity of these facial primordia and ultimately the facial skeleton [72-77]. Genes-encoding homeobox-containing transcription factors of the Hox family are also differentially expressed in the pharyngeal arches and determine pharyngeal arch identity [78]. The signals that control the appearance and patterning of the calvarial bones are not known. However, removal of ectoderm from the presumptive cranial aspect of the chick head or ablation of underlying neuroepithelium result in the absence of calvarial bones, implicating both tissues in an epithelial-mesenchymal inductive interaction [79,80].
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Benoit St.-Jacques and Jill A. Helms Axial S k e l e t o n
The axial skeleton of vertebrates is the most clearly segmented structure in the body and this organization reflects its origin in the embryonic somites. Somites are transient blocks of paraxial mesoderm that form alongside the neural tube and the notochord (Fig. 6A). They bud off in a precisely timed cephalocaudal sequence from the anterior end of the unsegmented presomitic mesoderm (PSM) [81] (Fig. 6B). The segmentation process initially forms undifferentiated epithelial somites (Fig. 7A) that soon become patterned in response to signals from the surrounding tissues [82] (Fig. 7B). Cells facing the surface ectoderm differentiate into the dermomyotome, which retains an epithelial structure and is the source of trunk and limb musculature as well as the dermis of the back. The ventral half of the somite undergoes an epithelial-to-mesenchymal transition to form the sclerotome, which will give rise to the ribs and vertebrae [83,84] (Fig. 7C). Sclerotomal cells undergo extensive proliferation and migration to form condensations that prefigure the axial skeleton elements. Cells from the medial part of the sclerotome condensate around the notochord and ven-
tral neural tube to give rise to the vertebral bodies and intervertebral disks. The lateral regions of the sclerotome migrate dorsally to form the vertebral arches and in the thoracic region also ventrally to form the ribs [85] (Fig. 7D). More caudally, the sacrum derives from the upper sacral vertebrae by projections similar to those that give rise to the ribs in the thoracic region. These extend, flatten, and fuse to form the lateral mass of the sacrum [86]. In addition, in the sclerotomes, a process of resegmentation takes place whereby rostral and caudal halves of each sclerotome segregate and re-fuse with halves of neighboring somites. Thus, each vertebra and rib are derived from the caudal half of one somite fused with the rostral half of the following somite (Fig. 6A) [87]. Because the resegmentation process does not take place in the muscle compartment, the segmental muscle derived from a single somite attaches to two adjacent vertebrae [85].
Somite Formation Segmentation of the paraxial mesoderm is believed to be under the control of a "segmentation clock"
FIGURE 6 Patterningof the paraxial mesoderm and somite formation. (A) Dorsal view of a human embryo in early week 4. The neural groove is still opened at the rostral and caudal ends where the neural folds (nf) are visible, but the neural tube (nt) has closed in the center. Eight somites (s) have formed on either side of the neural tube. A ninth somite is in the process of segmenting from the presomitic mesoderm (psm). (B) Schematic representation of somite formation. Blocks of paraxial mesoderm bud off the rostral end of the presomitic mesoderm (psm) at fixed intervals as epithelial somites. Simultaneously,new mesenchymalcells are recruited into the caudal end of the psm by the process of gastrulation to maintain elongation of the psm in a caudal direction. Rostrocaudal somite polarity is established in the anterior psm before overt segmentation (SO).C, caudal half of each somite; R, rostral half of each somite; SO, newlyforming somite; S1, most recently segmented somite. Dark gray bands represent the differentiating dermomyotomes. (C) Resegmentation. Somite formation requires two types of segmentation. The initial segmentation separates the forming somite from the psm (S1). Once somites have formed, differentiation into sclerotomal and dermomyotomal compartments (DM) occurs (see also Fig. 7). In the sclerotome only, a second segmentation (resegmentation) event occurs in which rostral (R) and caudal (C) halves of each somite segregate and re-fuse with their neighboring halves to form the vertebrae and ribs.
3. Prenatal Bone Development
FIGURE 7 Sclerotome differentiation and formation of vertebrae and rib condensations. The diagrams represent transverse sections through a developing chick embryo at early stages of formation of the axial skeleton. (A) Epithelial somites. The diagram shows the position of the differentiating paraxial mesoderm [epithelial somites (es)] relative to the other tissues, im, intermediate mesoderm; lpm, lateral plate mesoderm; n, notochord; nt, neural tube; se, surface ectoderm. (B) Patterning. The ventromedial part of the somite undergoes an epithelial-to-mesenchymal transition (light gray) and initiates expression of sclerotome-specific genes such as Pax1. Shh signal from the ventral neural tube and notochord is essential to maintain this differentiating population. Noggin also acts by antagonizing BMP activity from the lateral plate mesoderm. Note that at later stages, after the cells have been exposed to Shh, a BMP signal induces chondrogenic differentiation of sclerotomal cells. In the lateral somite, Shh is required in combination with Wnt factors from the dorsal neural tube and surface ectoderm to induce myogenic differentiation. (C) Expansion. Cells of the sclerotome (s) migrate dorsally and medially to surround the notochord and neural tube. The lateral somite forms the
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responsible for the oscillating expression of a gene encoding a basic helix-loop-helix ( b H L H ) transcription factor related to Drosophila hairy called c-hairy-1 in chick [88]. Expression of this gene appears as a wave that sweeps through the PSM once during formation of each somite. This wave does not result from cell displacement or from signal propagation but rather reflects a coordinated pulse of expression in neighboring cells [89]. In the anterior PSM, oscillations cease, resulting in the establishment of alternating bands of cells corresponding to presumptive somites. Thus, the temporal periodicity of expression (the clock) is translated into the spatial periodicity of the somites. It was recently found that the expression of a m e m b e r of the Notch l signaling pathway called lunatic fringe (Lfng, a Golgi enzyme that modifies the extracellular domain of the N o t c h l receptor) is also cyclic in the PSM, thus linking Notch signaling with the segmentation clock [90]. The importance of this signaling pathway in somitogenesis is illustrated by the perturbation of somite formation and defects in vertebrae and ribs seen in mouse embryos with null mutations in several genes encoding members of the Notch pathway. These include Notchl and Notch4, the Notch ligands delta-like 1 (Dlll) and delta-like 3 (Dll3), preselinin 1 and 2 (Psenl and Psen2), recombining binding protein suppressor of hairless (Rbpsuh; also known as Rbp-jK), Lfng, and the b H L H transcription factor mesoderm posterior 2 (Mesp2) [91-95]. In addition, deletion ofpMesogenin, encoding a b H L H transcription factor, in the mouse leads to a loss of Notch/delta pathway components as well as oscillating gene expression in the PSM and results in the complete absence of all trunk paraxial mesoderm derivatives, including vertebrae and ribs [96]. Some axial abnormalities in humans resemble the defects seen in some of these mouse mutants, suggesting that they may also be caused by mutations in genes encoding members of the N o t c h pathway [7]. At least one form of hereditary axial dysostosis has been linked to mutations in the h u m a n Dll-3 homolog, DLL3 [97]. In addition, mutations in another Notch ligand, JAGGED1, cause Alagille syndrome, a condition frequently associated with vertebral malformations [98,99]. The molecular nature of the mechanism responsible for the cyclic expression of hairy and Lnfg is unknown. It is a l s o n o t clear how segmentation is actually achieved,
dermomyotome (dm). ao, aorta. (D) Condensation. Sclerotomal cells form mesenchymal condensations surrounding the notochord and ventral neural tube. The dorsal condensation (dc) will form the neural arches of the vertebra. The ventral condensation (vc) forms the vertebral body. Lateral condensations (lc) will form the ribs (in the thoracic region). At this stage, the lateral somite has differentiated into two compartments, the dermatome (d) and the myotome (m).
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although it probably reflects differences in cell adhesion properties between the rostral and caudal parts of each somite and involves the Eph/ephrin signaling pathway [100-102]. Recently, the level of FGF was also shown to control the transition from immature PSM to a mature state in which cells initiate a segmentation program [103,104], and fibroblast growth factor receptor 1 (Fgfrl)-deficient mice do not form somites [105]. Segmentation of the paraxial mesoderm initially forms undifferentiated epithelial somites [81]. This epithelialization process specifically requires the activity of the bHLH) transcription factor Paraxis [106]. Sclerotome Differentiation
Differentiation of the sclerotome is controlled by the synergistic action of at least two factors, Shh and Noggin, secreted by the notochord and the floor plate of the neural tube [82]. Shh belongs to a small family of genes related to the Drosophila segment-polarity gene hedgehog (hh). Members of this family encode signaling proteins that play a fundamental role as long- and short-range signals in many aspects of embryonic development in both vertebrates and invertebrates [107]. The secreted polypeptide Noggin binds some BMP ligands with high affinity, thereby preventing interaction with their cognate receptors and signal activation [108]. Noggin is expressed in multiple sites including developing bones and is an essential modulator of BMP activity. In the most medial part of the somite, Shh antagonizes the dorsalizing activity of Wnt factors (Wnt 1, 3, and 7) secreted by cells of the dorsal neural tube and surface ectoderm to induce or maintain expression of sclerotomal markers [109-115] (Fig. 7B). Farther from the source of Shh, the combination of lower Shh concentrations and Wnt signals from dorsal tissues specifies myotome differentiation [116]. In addition to establishing the compartments of the somite, Shh signaling is also essential for cell survival in both the myotome and the sclerotome [115]. Noggin probably acts in specification of the sclerotome by antagonizing BMP-4 produced by the lateral plate mesoderm [117] (Fig. 7B). Both Shh and Noggin maintain expression of sclerotomal-specific markers, such as the paired-box transcription factors Paxl and Pax9 [110-115]. Paxl and Pax9 act in concert to maintain proliferation of sclerotomal cells [118]. Sclerotomes do not form in mice homozygous for a null allele of Shh, leading to the complete absence of vertebrae and most of the ribs [113]. Mutations in genes encoding other members of the hedgehog signaling pathway, most notably members of the Gli family of zinc finger transcription factors, also lead to severe skeletal abnormalities of the craniofacial, appendicular, and axial skeletal elements in the mouse [119]. These resemble the
spectrum of skeletal abnormalities observed in Gorlin's syndrome (nevoid basal cell carcinoma syndrome), which is caused by mutations in the human gene PATCHED1 (PTCH1) encoding the Hedgehog receptor [120,121]. Interestingly, the mouse mutant open brain (opb), which displays prominent neural tube and axial skeletal defects [122,123], was recently shown to be caused by mutation of a negative regulator of Shh signaling called Rab23 [124]. Noggin-deficient embryos also display a severe reduction of both the sclerotomal and the myotomal compartments, leading to axial defects that increase in severity in a rostral-to-caudal fashion, with the complete absence of lumbar and tail vertebrae [114,125]. Mutations in Paxl lead to severe defects in the axial skeleton [118,126-131]. Mutations in Pax9 in the mouse or PAX9 in humans result in multiple defects, reflecting expression in many tissues but without significant axial skeleton abnormalities [132,133], probably because Paxl and Pax9 serve partially redundant functions. The phenotype of compound Paxl/Pax9 mouse mutants is much more severe than that of single Paxl or Pax9 mutants, displaying a complete lack of the medial derivatives of the sclerotomes, the vertebral bodies, the intervertebral disks, and the proximal part of the ribs [118]. The Bagpipe homeobox gene 1 (Bapxl; also called Nkx3.2), which encodes a homeobox transcription factor related to Drosophila bagpipe (bap), is expressed in the sclerotome in a pattern similar to that of Paxl under the influence of SHH [134]. Inactivation of Bapxl in the mouse also leads to a specific loss of midline components of each vertebra (centra and intervertebral disks) [135-137]. On the other hand, the paired homeobox gene Uncx4.1 acts upstream of Pax9 and is specifically required for the formation of the pedicles and transverse processes of vertebrae as well as the proximal ribs [138,139]. The fact that formation of different parts of a vertebra depends on different factors probably reflects the evolutionary history of these structures whereby individual elements of the vertebra arose at different times in evolution [140]. The gene Mesenehymalforkhead-1 (Mfhl; also called Foxc2), which encodes a member of the winged-helix/ forkhead family of transcription factors, is also expressed in the sclerotome under the influence of Shh [141] and is required for normal proliferation of sclerotomal cells [142-144]. Inactivating mutations in human FOXC2 lead to hereditary lymphedema-distichiasis, a complex syndrome that shares many of the features of the Mfhl/Foxc2 null mice, including vertebral abnormalities [142,145]. As is the case for the Pax genes mentioned previously, Mfhl/Foxc2 and the related factor Mfl/ Foxcl appear to have partly overlapping functions. The absence of Mfl/Foxcl in the mouse does not lead to obvious axial defects [144] but compound Foxcl/Foxc2 mutants die early in development with more severe
3. Prenatal Bone Development defects than either single homozygote [146]. In addition to being expressed in the sclerotome, Foxcl and Foxc2 are also expressed in the PSM and the simultaneous absence of these two factors leads to downregulation of many genes related to Notch signaling and failure of somitogenesis [146]. The bHLH transcription factor Scleraxis, which is closely related to Paraxis, also appears to play an important role in the formation of the axial skeleton. Scleraxis is expressed throughout the ventral sclerotome and subsequently in the cartilage of developing vertebrae and ribs as well as in craniofacial cartilage and precursors of the long bones [147]. Scleraxis-deficient mouse embryos die before gastrulation, preventing the analysis of its potential role in cartilage formation [148]. However, using a chimeric approach, it was possible to show that Scleraxis-null cells can contribute to cartilage in the limbs and craniofacial region but not to the sclerotome [148]. This suggests a specific role for Scleraxis in specification or survival of sclerotomal cells. Differentiation of the dorsal part of vertebrae is controlled by different molecular mechanisms than those for the rest of the axial structures. Cells in the dorsomedial angle of the sclerotome migrate between the surface ectoderm and the dorsal neural tube to form the dorsal mesenchyme, from which the dorsal part of the neural arch and spinous process of each vertebra will form (Fig. 7). These cells do not maintain expression of Pax1 or Pax9 but instead, under the influence of BMP-4 (secreted by the surface ectoderm and roof plate of the neural tube), express the transcription factors Msxl and Msx2, leading to bone formation in this region [149-151]. Mutations in genes required for normal development of the myotome and that are not normally expressed in the sclerotome, including Pax3 and Myf5, also lead to abnormalities of the ribs and neural arches [152-154]. This indicates that formation of structures derived from the lateral sclerotome also depends on interactions between the sclerotome and the myotome. Platelet-derived growth factor A (PDGFA) expressed in the myotome and its receptor PDGFRA expressed in the sclerotome act downstream of Pax3 and Myf5 and appear to mediate signaling between myotome and lateral sclerotome [155,156]. The absence of Fgf-4 and Fgf-6 expression in the myotomes of Myf-5-deficient mouse embryos also suggests that members of the FGF family may be involved in this interaction [157]. Significant movement ofsclerotomal cells is involved in the formation of axial structures, but little is known about the mechanisms initiating and coordinating this migration. However, downregulation of the adhesion molecule NCAM can be seen in the mesenchymal sclerotome while high expression is maintained in the epithelial dermomyotome, suggesting a role in this process [158,159].
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Patterning of the Axial Skeleton The bones of the axial skeleton have distinct morphological features characteristic of their position along the rostral-caudal axis. The determination of this axial identity is under the control of the homeotic genes of the HOX complex [160-164]. These genes encode a highly conserved family of homeobox-containing transcription factors. In humans, as in most vertebrates, there are 39 HOX genes organized into four clusters of 9-11 genes, named HO2fA-HOXD, that are located on different chromosomes. At the molecular level, HOX genes are believed to act by controlling the transcription of specific sets of target genes, although few such genes have been identified. In general, the order of the genes on the chromosome, within each cluster, corresponds closely to their spatial expression patterns during development [160]. Furthermore, there is extensive overlap between the expression domains of many HO2(genes at any given axial level. The specific subset of genes expressed at each level (the HOX code) determines the identity of the skeletal elements [165]. Consequently, ectopic expression and targeted deletion of Hox genes in the mouse often lead to changes in the number of vertebral elements or to homeotic transformations (changes in the shape of one element that make it resemble a different element) [161,163,166]. In the mouse, mutations in a number of genes that result in misexpression of certain Hox genes also lead to abnormal vertebral patterning. These include mutations in Fgf-rl [167], activin receptor IIB (ActRIIB) [168], the mixed-lineage leukemia (Mll) gene [169], the B lymphoma Mo-Mlv insertion 1 (bmil) protooncogene [170], the caudal-type homeobox 1 (Cdxl) gene [171], growth and differentiation factor 11 (Gdf-11) [172], and the promyelocytic leukemia zinc finger (PlzJ) gene [173]. Administration of exogenous RA during early gestation also leads to changes in the anterior limits of Hox gene expression associated with vertebral transformations [166,174,175]. Consistent with a role for RA in axial patterning, a number of retinoic acid receptor (RAR) null mutant mice exhibit vertebral homeosis, primarily in the cervical region [176-179]. However, the effect of RA on Hox genes controlling axial patterning may not be direct and was recently shown to be mediated at least in part through the activity of Cdxl, a direct RA target [180-182]. How Hox gene activity ultimately controls the shape of different axial elements is not clear, but the different phenotypes could be explained by a deficit in the formation of precartilaginous condensations and/or cellular proliferation [183]. Misexpression of some Hox genes in vivo has been shown to affect the condensation process,
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cellular adhesiveness, chondrocyte proliferation, and chondrocyte maturation [184-186]. Mutations in only three H O X genes have been associated with skeletal defects in humans, and in all cases it is the patterning of the limbs that is affected. Recently, however, a translocation located 60 kb from the 3' end of the HOXD complex was shown to be associated with mesomelic dysplasia (severe shortening of the forearms and lower legs) and vertebral defects [187]. This translocation does not disrupt any coding sequence but most likely exerts its deleterious effect by modifying H O X gene expression [187]. A p p e n d i c u l a r Skeleton The appendicular skeleton consists of the pectoral (shoulder) and pelvic girdles and the limb bones. The basic structure of the appendicular skeleton is remarkably conserved among amniote tetrapods and consists of four segments: a root (zonoskeleton), a proximal segment (stylopodium) consisting of a single bone (humerus or femur), a medial segment (zeugopodium) consisting of two bones (radius/ulna or tibia/fibula), and a distal and more complex part (autopodium) composed of carpus or tarsus and a variable number of digits (hand or foot). With the exception of the clavicle, which forms by a combination of intramembranous and endochondral ossification [188], all other elements of the appendicular skeleton form by endochondral ossification. In humans, limbs first appear as elevations of the ventrolateral body wall (the limb buds) toward the end of the fourth week. The upper limbs develop at the level of the caudal cervical segments and the lower limbs opposite the lumbar and rostral sacral segments [189]. The appearance of the upper limb buds precedes that of the lower limb buds by a few days. Originally, the growing limb buds consist of a central core of undifferentiated mesenchyme encased in an ectodermal jacket. As the bud elongates out of the flank, ectoderm at the distal end of the limb thickens and forms the apical ectodermal ridge (AER). The AER is one of the main regulatory centers of limb growth and patterning. Signals emanating from the AER maintain the subjacent mesenchyme as a population of undifferentiated, rapidly proliferating cells called the progress zone (PZ) (Fig. 8B). This cell population is essential for outgrowth of the limb, and ablation of the AER leads to cessation of growth and distal truncation of the limb [190]. As the limb grows, cells farther from the influence of the AER leave the PZ and begin to differentiate into cartilage and muscle. In this manner, development of the limb proceeds proximal to distal. By the sixth week of development in humans, mesenchymal condensations within the limb differentiate to form the first cartilage precursors of the long bones. The cartilages
first form as continuous rods that through a series of bifurcations and segmentations give rise to the characteristic limb skeleton [191]. At approximately Day 32, the terminal portion of the limb buds becomes flattened to form the handplates and the footplates [189]. Fingers and toes become apparent as programmed cell death separates the AER into five parts leading to continued outgrowth of the cartilaginous digital rays, accompanied by cell death of the interdigital mesenchyme. In humans, this process takes place between Days 48 and 56 of development [189]. Two sources of mesenchyme contribute to the limb. Cells from the lateral plate mesoderm (LPM) give rise to connective tissue and cartilage, whereas cells migrating from the myotomal compartment of the somites give rise to all the muscles. Of these two populations, the connective tissue/cartilage precursors determine the primary limb pattern [192]. Other components of the limb include motor axons emanating from the spinal cord and sensory axons and Schwann cells of neural crest origin. In addition, the earliest vasculature of the limb is derived from endothelial cells arising from segmental branches of the aorta and cardinal veins as well as from angioblasts (endothelial cell precursors) endogenous to the limb mesoderm [36]. Limb Bud Positioning and Induction The first step in limb development is the determination at specific levels of the flank LPM of groups of cells, the limb fields, which acquire the potential to form a limb bud. Once specified, the limb field mesenchyme can be transplanted elsewhere and form a limb bud, irrespective of the origin of the ectoderm that covers it at the transplantation site [192]. How this specification is achieved is not completely understood, but several lines of evidence support the involvement of a H o x code in positioning the vertebrate limb field. Indeed, in many vertebrates, the anterior limit of expression of specific H o x genes corresponds exactly to the level of the prospective forelimbs and differences in the position of forelimbs between chick and mouse correspond to shifts in H o x expression [162,193]. In addition, the absence of limbs in snakes also correlates well with specific changes in H o x gene expression [194]. Finally, some mutations affecting the pattern of expression of some H o x genes lead to displacement of the foreor hindlimbs [172,195]. The prepattern that sets up the limb field is then interpreted by several key tissues that together induce limb budding. In this respect, the intermediate mesoderm (IM), which lies between the somites and the LPM (Fig. 7A), appears to play an important role. In the chick embryo, extirpation of the IM leads to limb reduc-
3. Prenatal Bone Development
tion, whereas a barrier placed between IM and LPM inhibits limb induction [196,197]. These observations suggest that the IM is the source of a diffusible limb inducer that acts on the LPM. A large body of work recently led to the identification of FGF-8 as a mediator of this activity. FGF-8 protein, like many other FGFs, can induce ectopic limb formation when implanted at the interlimb level into the flank of a chick embryo. Furthermore, Fgf-8 is expressed transiently in the IM in a domain first restricted to the level of the prospective forelimb and later at the hindlimb level as well [196]. At later stages, Fgf-8 is also expressed in the limb ectoderm and contributes to the function of the AER. Interestingly, another gene of the same family, Fgf-lO, appears to mediate the limb-inducing activity of Fgf-8. Fgf-lO is initially widely expressed in the LPM, but prior to limb bud induction it becomes restricted to the LPM of the prospective limb territories. Fgf-lO-expressing cells can induce ectopic limb formation [198] and mice deficient for Fgf-lO or its receptor (an isoform of Fgf-r2) are limbless [199-201]. In these mutants, the apical ectoderm of the limb bud never expresses Fgf-8 and the AER fails to form. Moreover, F G F bead implants induce Fgf-lO expression in the LPM prior to ectopic limb initiation and the appearance of Fgf8 expression in the surface ectoderm [202]. All these observations have contributed to the current molecular model of limb induction in which FGF-8 from the IM restricts Fgf-lO expression to the LPM of the prospective limb regions, and FGF-10 subsequently induces Fgf-8 expression in the ectoderm, thereby triggering limb outgrowth [196,197,203-205]. It now appears that signaling through the Wnt pathway mediates these inductive interactions between Fgf-8 and Fgf-lO. Indeed, Wnt-2b is expressed in the LPM of the prospective forelimb area, and its ectopic expression is sufficient to induce ectopic Fgf-lO expression and ectopic limb outgrowth in the flank [204]. Expression of another Wnt gene, Wnt8c, may restrict Fgf-lO expression to the prospective hindlimb area [204]. Finally, induction of Fgf-8 expression in the ectoderm requires Wnt signaling, probably via expression of the Wnt3a gene [204,206]. Determination of Limb Identity Determination of the type of limb to be produced (forelimb vs hindlimb) appears to take place at the earliest stages of limb induction, prior to limb bud formation. For instance, prebud LPM cells taken from the forelimb field develop as a forelimb when transplanted ectopically [207]. Furthermore, the mesoderm is clearly the determinant of limb identity ([190,207,208]. Members of the T-box (Tbx) and paired-type homeobox (Pitx) gene families have been identified that are expressed exclusively in the LPM of the fore- or hindlimb
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prior to bud formation. Tbx5 is expressed in the presumptive forelimb area and Tbx4 and Pitxl in the presumptive hindlimb area in a number of organisms [209-211]. In addition, Pitxl is an upstream regulator of Tbx4 expression because misexpression of Pitxl induces ectopic expression of Tbx4 and inactivation of Pitxl leads to reduced Tbx4 expression [212-214]. Ectopic expression of Tbx4 and Pitxl in chick forelimbs (wings) results in partial transformations to hindlimbs (legs) [212,215,216], whereas ectopic expression of Tbx5 in leg fields results in some respecification to wing identity [215,216]. Although the transformations are not complete, these data indicate that Tbx4 and Pitxl are selectors for hindlimb identity and Tbx5 is a selector of forelimb identity in chick. Furthermore, inactivation of Pitxl in the mouse leads to hindlimb skeletal elements that bear some resemblance to the corresponding bones in the forelimb [213,214]. Again, the transformation is not complete, possibly because Tbx5 is not regulated by Pitxl and its expression remains forelimb specific in mutant mice. How these selector genes interact with the FGF-8/FGF-10 regulatory loop and other possible downstream targets remains to be determined. Two mutations in human TBX genes have been linked to defects of the limb skeleton. Mutations in TBX3 cause ulnar-mammary syndrome, in which patients present with a range of alterations of the ulnar ray in the upper and lower limbs associated with apocrine, cardiac, and genital anomalies [217,218]. Holt-Oram syndrome in which patients display malformations of the radial ray of the upper limbs, associated with varied cardiac anomalies, is due to mutations in TBX5 [219,220]. Patterning of the Limb The limb is probably the part of the vertebrate body for which patterning mechanisms are best understood [197]. Indeed, a systematic study of limb patterning in chicks and mice led to the elucidation of a series of regulatory pathways that govern the establishment of the three axes of the developing limb: the proximaldistal (shoulder-to-digits) axis, the anterior-posterior (thumb-to-little finger) axis, and the dorsal-ventral (knuckles-to-palm) axis (Fig. 8A). Although the mechanisms regulating patterning along these three axes are different, they are not independent from one another and interactions between the regulatory pathways have also been identified. These interactions ensure coordination of growth and patterning along the three axes of the limb. As indicated previously, outgrowth of the limb bud and its elongation in the proximal-distal axis is dependent on the integrity of the AER (Fig. 8B). Surgical removal of the AER stops bud outgrowth and results in a
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FIGLIRE 8 Limbbud patterning. (A) The three axes of limb growth and patterning: lateral view at the limb bud stage. A-P, anteriorposterior axis; Pr-Di, proximal-distal axis; Do-V, dorsal-ventral axis. (B) Growth and patterning centers in the elongating limb bud. A, anterior; AER, apical ectodermal ridge; P, posterior; PZ, progress zone; ZPA, zone of polarizing activity. (C) Regulatory signals in growth and patterning of the limb bud. FGFs produced by the AER induce FGF10 in the subjacent mesenchyme to maintain proliferation and prevent differentiation of cells in the PZ. FGF signals also maintain Shh expression in the ZPA. Shh induces BMP2 expression, which can inhibit AER expression of FGFs. Shh also acts through formins to induce expression of the BMP antagonist Gremlin. BMP2 maintains Gremlin expression, completing the negative feedback loop that controis BMP activity in the autopod. (D) Molecules regulating dorsalventral patterning in the limb bud. The secreted factor Wnt7a is present in the dorsal ectoderm and directs expression of Lmxl in the dorsal mesenchyme. Enl acts by inhibiting expression of Wnt7a, and therefore Lmxl, in the ventral half of the limb bud. Abutting domains of Rfngexpression in the dorsal bud and Enl expression in the ventral bud determine the position of AER formation.
truncated limb [221-223], whereas ectopic implantation of an A E R to an initiated limb bud results in the production of a second proximal-distal axis [222]. An important activity of the A E R is to keep cells of the underlying mesenchyme in a proliferative state. Members of the F G F family of growth factors appear to mediate this activity. Indeed, of all the factors expressed in the AER, only F G F s are capable of substituting for the A E R after its surgical removal [196]. However, owing to functional redundancy, it is difficult to assign a specific role to particular F G F s expressed in this tissue. Loss of function for Fgf-4 [224,225], Fgf-9 [226], or Fgf-17 [227] has no effect on limb formation. Following inactivation of Fgf-8 in the prospective limb ectoderm, patterning
and growth are abnormal but the A E R does form and a limb also develops [228,229]. Thus, it is likely that no individual Fgf expressed in the A E R is solely necessary for its function but that a combination of two or more F G F s mediate its activity. AER-produced F G F s maintain Fgf-lO expression in the limb bud mesenchyme [198], which appears essential for continued proliferation of mesenchymal cells and elongation (Fig. 8C). A E R maintenance is also controlled by BMP signaling [230,231]). In the chick, BMP-soaked beads placed under the A E R induce cell death and A E R regression resulting in truncations of the limb skeleton [232,233]. Conversely, BMP inhibition results in extension and persistence of the A E R and prolonged Fgfexpression [234]. The secreted product of the gene Gremlin was identified as the factor antagonizing BMP activity in the distal limb bud in vivo [235-237]. However, to ensure a minimum level of BMP signaling, also required as part of the distal signaling program, Gremlin expression is in turn controlled by BMP signaling in a regulatory feedback loop [235,236] (Fig. 8C). The formation and proper patterning of the different segments of the limb skeleton require the activity of Hox genes of the HoxA and HoxD clusters [238-240]. Genes of the 5~end of the HoxA cluster are sequentially induced during outgrowth of the bud and come to be expressed in domains restricted along the proximal-distal axis with borders corresponding to the major segments of the limb [241,242]. Genes of the 5~ end of the HoxD cluster are expressed in a set of overlapping domains progressively restricted along the anterior-posterior axis [242,243]. Based largely on these observations, it was first proposed that Hoxa genes determine the proximal-distal identity of the skeletal elements, whereas Hoxd genes determine anterior-posterior identity. In this model, the identity of each element is set by a combinatorial code of Hox gene expression, with each element specified by a unique code [238]. A large number of experiments involving ectopic expression and targeted disruption of Hox in the chick and the mouse have shown that this simple model is not correct. By using a variety of loss-of-function alleles in the mouse, it was possible to show that the localizations of the limb alterations reflect the temporal and structural colineraity of Hox genes of both the HoxA and -D clusters such that inactivation of 3r genes has a more proximal phenotypic boundary than that of the 5t genes. For instance, mutations in Hoxa9 and Hoxd9 affect the stylopodium, whereas mutations in groups 10 and 11 genes affect primarily the zeugopodium and mutations in groups 12 and 13 affect the autopodium [239,240]. A second observation is that, in these different domains, individual gene products quantitatively contribute to an overall protein dose and reduction in the gene dose in each set results in truncation of the corresponding
3. Prenatal Bone Development anatomical region. For instance, a progressive reduction in the dose of groups 11-13 Hox genes leads to a proportional reduction in the length and number of digits [244]. A striking example is also provided by mutations in group l I genes. Mice with individual mutations in the paralogous genes Hoxal! and Hoxdl! have relatively moderate malformations of the radius and ulna [245-247], but in the double mutant both the radius and the ulna are almost entirely eliminated [248]. Interestingly, in humans a distinct alteration of the forearm (radioulnar synostosis) was described as resulting from a heterozygous mutation in H O X A l l [249]. Recently, a balance translocation that appears to deregulate HOXD genes expression was also associated with mesomelic dysplasia (severe shortening of the zeugopods) and vertebral defects [187]. Patterning along the anterior-posterior axis is mediated by a small region of cells in the posterior mesenchyme of the limb bud known as the zone of polarizing activity (ZPA) [250] (Fig. 8B). Extirpation of the ZPA results in posterior skeletal defects [251], whereas implantation of an ectopic ZPA in the anterior mesenchyme causes mirror-image duplication of the digits [250]. This observation has long been interpreted in terms of diffusion of a morphogen from the ZPA, which sets up a gradient across the limb bud, specifying digits pattern in a concentration-dependent manner [194]. It is now known that the signaling peptide Shh mediates the polarizing activity of the ZPA. Shh expression in the limb bud colocalizes with the ZPA in many organisms [252255], and the Shh signaling peptide spreads away from its restricted source within the ZPA in a graded fashion [203,256,257] (Fig. 8C). Finally, implantation of ectopic Shh in the anterior limb bud mesenchyme leads to mirror-image duplication of digits [252,258,259]. RA can also induce digit duplication and compound mutants for different RA receptors of the RAR subfamily exhibit a range of severe limb abnormalities from reductions to duplications [177]. RA appears to act in limb patterning by inducing Shh expression, and complete inhibition of RA signaling prevents the establishment of the ZPA and the appearance of Shh expression [260-262]. It should be noted that Shh is required for growth and patterning of the intermediate and distal limb skeletal elements but not the most proximal limb structures [113,263]. In addition, even in the absence of Shh, some anterior-posterior polarity is maintained [264,265], indicating that Shh is not required for the initiation of limb outgrowth or establishment of an initial polarity within the limb field. Hedgehog signaling regulates the expression and activity of transcription factors belonging to the Ci/Gli zinc finger family [107]. In limb bud mesenchyme, Gli3 acts as a potent repressor of transcription, but this activity is antagonized by the long-range signaling of posteriorly
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produced Shh [266]. The result is an anterior-posterior gradient of Gli3 repressor activity across the limb bud mesenchyme [266]. The importance of this patterning mechanism is illustrated by several syndromes caused by mutations affecting Shh signaling that are associated with carniofacial anomalies and malformation of the autopodium skeleton. These include Smith-LemliOpitz syndrome, attributed to a defect in sterol D7-reductase; Gorlin syndrome (basal cell nevus syndrome), caused by haploinsufficiency for the Shh receptor PATCHED1 (PTCH1); and Greig cephalopolysyndactyly, Pallister-Hall syndrome, and postaxial polydactyly type A, in which different mutations in GLI3 have been identified [267]. Mutations in the gene encoding CBP, a coactivator of the Gli transcription factor, have also been detected in Rubinstein-Taybi syndrome [268]. Mutations in SALL1, another zinc finger transcription factor gene expressed in Hedgehog signaling domains, lead to Townes-Brocks syndrome [269]. Finally, in the mouse, mutations have been identified in genes encoding factors that act to confine Shh expression to the ZPA, including Ptchl [270]; Gli3, mutated in the extra-toe (xt) [271,272] and polydactyly Nagoya (Pdn) mice [273]; the aristalless-like gene Alx4, mutated in the Strong's luxoid mouse [274,275]; and the related gene Cart 1 [276]. The bHLH transcription factor dHAND [277,278] and some HOX proteins [279-281] act as positive regulators of Shh expression. Maintenance of Shh expression in the ZPA also depends on signals from the AER and the surface ectoderm. It is not known how Shh signaling specifies the pattern of skeletal elements in the autopodium, but evidence points to BMPs as downstream signals. Indeed, Shh appears to induce dose-dependent mirror-image duplication of digits even when provided in a nondiffusible form [282]. This observation suggests the existence of secondary long-range signals, and BMPs are plausible candidates. Some Bmps are expressed in the polarizing region following Shh expression, and Bmp-2, -4, and -7 can also be ectopically induced in anterior mesenchyme by RA or Shh [282-285] (Fig. 8C). In addition, BMP signal is required for polarizing activity since in the absence of BMP signaling, ectopic Shh still induces formation of additional digits but they all have the same identity [285]. However, BMPs alone cannot reproduce all the effects of Shh on digit patterning [286]. A model accounting for these different observations was recently proposed [285] that states that Shh initially acts long range to "prime" the region of the limb competent to form digits and determines digit number. Later, Shh acts short range to induce expression of BMPs, whose morphogenetic action specifies digit identity. This model is supported by the recent demonstration that digit identity is actually specified by the interdigital mesenchyme at
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relatively late stages of limb bud development in a process dependent on BMP signaling [287]. BMP signaling determines the fate between digital (chondrogenic) and interdigital (apoptosis) in the distal autopod. Exogenous BMPs induce premature cell death in the interdigital mesenchyme but stimulate chondrogenesis when placed close to developing digits. Interfering with BMP signaling has the opposite effect [288]. As indicated previously, another member of the transforming growth factor-J3 (TGF-[3) superfamily, Tgf-~2, is expressed in the prechondrogenic condensations of the digits and appears to act as an inducer of chondrogenesis. TGF-[31_3 are potent inducers of chondrogenesis in limb mesenchyme micromass cultures and implantation of exogenous TGF-[3 in the interdigital mesenchyme induces formation of extra digits [288]. This is preceded by induction of Bmp-rlB in the condensations, suggesting that TGF-[32 acts by sensitizing these cells to the action of a BMP signal. However, TGF-[32 is not expressed in the more proximal elements of the stylopodium and zeugopodium, and implantation of ectopic TGF-[32 in domains destined to form these elements does not induce formation of ectopic cartilage [288]. Thus, there appears to be specific inductive signals for chondrogenesis along the proximodistal axis. Various 5' Hoxa and Hoxd genes are also involved in digit patterning. Ectopic polarizing activity (Shh or RA) induces ectopic expression of Hoxdll and -13 [282284,286]. In the chick, misexpression of Hoxdll and Hoxal3 resulted in transformations compatible with a change in identity of cartilage primordia [184,289]. In humans, mutations in only a few H O X genes have been shown to cause congenital malformations, and they affect primarily the autopodium. HoxD13 is mutated in synpolydactyly, and HoxA13 is mutated in hand-footgenital syndrome [267,290]. Classical embryology in the chick has shown that once the limb bud has formed, the surface ectoderm is responsible for specification of cell fate along the dorsal-ventral axis [291]. A number of genes are expressed specifically in either the dorsal or the ventral half of the limb bud and are candidates for determinants of dorsal-ventral patterning. Wnt7a, which encodes a secreted signaling factor, is expressed in the dorsal ectoderm and directs expression of the LIM homeodomain transcription factor Lmxl to the dorsal mesenchyme [292] (Fig. 8D). Loss-of-function mutations in either Wnt7a or Lmxl transform dorsal limb structures to a ventral fate [293,294]. Conversely, ectopic expression of either gene in the ventral limb causes dorsalization of ventral structures [292,295]. The transcription factor Engrailedl (Enl), whose expression is restricted to the ventral ectoderm, specifies ventral fates since limbs of Enl -/- mice display a double-dorsal phenotype [296]. Enl appears to
act by inhibiting expression of Wnt7a, and therefore Lmxl, in the ventral half of the limb bud [291] (Fig. 8D). Mutations in LMX1B in humans cause nail-patella syndrome, characterized by hypoplasia or aplasia of dorsally derived structures [291]. Enl also restricts expression of Radical fringe (Rfng) to the dorsal ectoderm and maintains a sharp boundary between Rfng-expressing and -nonexpressing cells. This interface determines the position of the forming AER (Fig. 8D). When Enl is ectopically expressed in the dorsal ectoderm or Rfng is ectopically produced in the ventral ectoderm, new interfaces between Rfng-expressing and -nonexpressing cells are created, giving rise to ectopic AERs and outgrowth [297-299]. Growth and patterning along the three axes of the limb are regulated by interactions between the molecular pathways defining each axis. For instance, Fgfs expression in the AER, which stimulates proximal-distal growth, is also essential to maintain Shh expression in the ZPA and therefore anterior-posterior patterning. In turn, Shh affects outgrowth by inducing BMP expression, which at high levels can induce AER regression. However, Shh also indirectly promotes expression of the BMP antagonist Gremlin in the mesenchyme. This activity of Shh requires the nuclear protein Formin, which is also essential to maintain AER expression of Fgf4 [237] and is mutated in the limb deformity (ld) mouse [300]. Thus, Shh appears to act through Formin to promote Gremlin expression and indirectly maintain Fgf4 expression in the AER (Fig. 8D). Finally, maintenance of Shh expression and development of posterior elements also require the Wnt7a signal in the dorsal ectoderm, providing a link between anterior-posterior and dorsal-ventral axes [293,301,302]. Thus, all three axes are intimately linked by the respective signals Wnt7a, FGF-4, and Shh during limb outgrowth and patterning.
MOLECULAR REGULATION OF BONE FORMATION Initiation Whether a bone forms by intramembranous or endochondral ossification, the initial steps in the process are similar [5]. After mesenchymal cell migration to the site of bone formation, an interaction between epithelial and mesenchymal tissues takes place. This molecular interaction is essential to induce the next phase, the formation of mesenchymal condensations. The final phase is the overt differentiation (osteogenesis or chondrogenesis) of cells in these condensations. The following sections review the nature of the epithelial-mesenchymal interactions and the process of condensation.
3. Prenatal Bone Development
Epithelial-Mesenchymal Interactions The requirement for an early tissue interaction to induce mesenchymal condensation is demonstrated by tissue dissociation-recombination experiments. For instance, embryonic mandibular mesenchyme can be separated from the mandibular epithelium and cultured in vitro. Mesenchyme isolated before migration or at an early stage after migration of mesenchymal cells to the final site of bone formation fails to form bone in culture. However, if early mesenchyme is combined in vitro with mandibular epithelium or if mesenchyme is isolated at a late stage, then osteogenesis takes place, indicating a requirement for epithelial-mesenchymal contact [303]. The signal from the epithelium appears to be permissive in nature. First, mandibular mesenchyme forms bone in culture when recombined with epithelia from other regions of the embryo as well, provided that they are of the correct age. Furthermore, the shape of the bone produced is independent of the source of epithelium but is dictated by the nature of the mesenchyme [303]. Finally, an epithelium such as the mandibular is incapable of inducing bone formation in a mesenchyme that is normally nonosteogenic. Such interactions have been studied in detail mostly in craniofacial structures and have been shown to be essential to induce formation of scleral ossicles and scleral cartilage, mandible and Meckel's cartilage, maxilla, palatine bone, otic vesicle, and calvarial bones, and also clavicle and limb cartilage [35,303-3061. A series of elegant studies by Hall and colleagues [303] have demonstrated that in most cases these epithelialmesenchymal interactions are short range and matrix mediated. In particular, the activity appears to reside in the ECM of the epithelial basal lamina. The molecular nature of the epithelial signal(s) is unclear but probably involves in many cases the BMPs, which form a subfamily of secreted proteins among the TGF-~ superfamily. Members of this group are characterized by a conserved C-terminal domain with several cysteine residues and undergo cellular processing by cleavage of an inactivating N-terminal domain. They act as dimers (probably heterodimers in vivo [307]) to bind a type II transmembrane receptor and induce recruitment and phosphorylation of the cytoplasmic tail of one of two type I receptors (types IA and IB). Phosphorylation of the type I receptor initiates signal transduction through various pathways, including the SMAD family of intracellular signaling molecules. At least 20 different BMPs and the related growth and differentiation factors (GDFs) have been identified in vertebrates [308]. Members of this family of growth factors were first identified as proteins present in demineralized bone matrix, which has the remarkable capacity to induce ectopic
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bone formation in subcutaneous implants [309,310]. Purified recombinant BMPs share this capacity and are able to potentiate chondrocyte and osteoblast differentiation of cells in vitro, including that of mouse embryonic stem cells [311], bone marrow stromal stem cells, limb bud mesenchyme, periosteum, fetal calvaria, and a number of mesenchymal or preosteogenic and prechondrogenic cell lines [312]. Consequently, BMPs have long been considered in vivo "bone inducers." However, BMPs induce ectopic bone by recapitulating the events occurring during endochondral bone formation [313], and it is likely that in development they do not act by inducing ossification (production of bone matrix) per se. Indeed, the developing skeleton is neither the sole nor the main site of B M P expression, and BMPs control organogenesis in many tissues [314]. Instead, the boneforming ability of BMPs probably reflects their role in the induction of mesenchymal condensations [315]. Several BMPs known to have cartilage/bone-inducing potential accumulate in the ECM [316-319], including epithelial basal lamina [316,320], and BMP signaling has been implicated in epithelial-mesenchymal interactions in other organs [314,321]. In the case of the mandible, an epithelial-mesenchymal interaction involving BMP signaling has been documented. BMP-2, -4, and -7 are found in distal mandibular epithelium and the homeobox-containing transcription factor Msxl is present in the mandibular mesenchyme. M s x l function in the mesenchyme is essential, as demonstrated by M s x l knockout mice, which exhibit mandibular defects [322]. Expression of M s x l requires mandibular epithelium [323] and can be induced by ectopic BMPs [73], thus implicating these molecules in the early epithelial-mesenchymal interaction required for mandible formation.
Condensations Skeletal condensations have been studied most intensely in the developing limb in vivo and by means of micromass cultures of limb mesenchyme in vitro. The condensation stage is a very transient phase, rapidly followed by differentiation of the aggregated cells. At the histological level, it is characterized by a significant increase in cell-packing density in specific areas of the mesenchyme. Cells of the precartilaginous condensations can also be visualized by their affinity for the lectin peanut agglutinin (PNA) and their transient upregulation of a number of markers, including versican, tenascin, syndecan, N-CAM, N-cadherin, thrombospondin-4, type I collagen, and heparan and chondroitin sulfate proteoglycans [5]. Cell proliferation plays a role in condensation of the calvarial, scleral, and mandibular primordia but not in the precartilaginous condensations of the limb, where cell movements appear to be more
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important [24]. Regardless of the mechanism, the high cell density generated in the condensations leads to increased cell-cell contact as well as the formation of gap junctions facilitating intercellular communication [23,324,325]. These appear to be essential for differentiation to take place, and the extent of cellular condensation correlates with the level of subsequent chondrogenesis both in vitro and in vivo [10]. Two cell adhesion molecules implicated in the condensation process are N-cadherin and N-CAM. N-cadherin belongs to a group of calcium-dependent transmembrane glycoproteins that mediate cell-cell adhesion by homotypic interactions through their extracellular domains. The protein cytoplasmic domain interacts with the actin cytoskeleton via the catenin molecules. N-CAM is a member of the large immunoglobulin superfamily of membrane glycoproteins and also mediates cell-cell adhesion via homotypic interactions but in a calcium-independent manner. These molecules are expressed at high levels in condensing mesenchyme but disappear in differentiating cartilage and can be detected later only in the perichondrium. Much evidence shows that perturbing the functions of N-cadherin and N-CAM causes reduction or alteration of chondrogenesis both in vitro and in vivo [10]. Conversely, overexpression of N-cadherin and N-CAM in micromass cultures stimulates chondrogenesis [326,327]. Recently, cell-cell adhesion in the digit condensations was also shown to depend on Eph-ephrins interactions. The Eph receptors constitute the largest known subfamily of receptor tyrosine kinases. They are characterized by a unique cysteinerich motif in their extraceUular domain, followed by two fibronectin type III motifs. They interact with a family of at least eight ephrin ligands associated with the cell membranes via a transmembrane domain or a GPI anchor [328]. Two Eph receptors were recently implicated in the sorting and adhesion of limb mesenchymal cells in the mouse and the chick [329,330]. Furthermore, in Hoxal3 mutant mice, expression of one of these, EphA7, is reduced in mesenchymal condensations correlating with altered adhesiveness, poorly resolved condensations, and defective chondrogenesis [330]. Interestingly, misexpression of Hoxdll or Hoxal3 in the developing chick limb also affects condensation size and cell adhesiveness [184,185]. These results suggest that one of the mechanisms by which Hox transcription factors control skeletal patterning is via the regulation of cell adhesion molecules, particularly members of the Eph receptor family, at the condensation stage. Interactions between cells and the ECM also significantly affect condensation. Prior to condensation, mesenchymal cells secrete an ECM rich in hyaluronan (HA) that facilitates cell movement but prevents close cell-cell interactions. As condensation begins, a transient
increase in hyaluronidase activity leads to controlled hydrolysis of HA and reduced intercellular space, thus favoring cell-cell interactions [331,332]. Syndecan-3 is an integral membrane protein transiently expressed in large amounts during formation of the precartilage condensations and downregulated (except in the perichondrium) after chondrocyte differentiation [333]. It is a heparan sulfate proteoglycan that can interact with ECM components and heparin-binding growth factors and signaling molecules [333]. Antibodies against syndecan-3 impair the formation of precartilaginous condensations in micromass cultures of chick limb mesenchyme [334], but the mechanism of this inhibition is not clear. Condensation also coincides with upregulation of several ECM proteins, including type I collagen, fibronectin, and various proteoglycans [10]. Fibronectin is a dimeric proteoglycan present in the ECM of many tissues that plays an important role in cell migration and differentiation. Fibronectin interaction with the cellular integrin receptors can activate signaling through the focal adhesion kinase and the integrin-linked kinase pathways. Prior to condensation, it is distributed throughout the intercellular space of the mesenchyme. It accumulates in the condensations and reaches its maximal level of expression just prior to overt chondrogenesis [335]. Interaction between extracellular fibronectin and heparin-like molecules of the mesenchymal cell surface is crucial for the formation of precartilage condensations [336,337]. Antibodies specific to a certain isoform of fibronectin perturb chondrogenesis both in vitro and in vivo [338]. One mode of action of fibronectin is to stimulate N-CAM expression. Expression of the different cell-cell and cell-matrix adhesion molecules mentioned previously is modulated by signaling factors, and these are therefore also important regulators of condensation. For instance, treatment of pluripotential cells or limb bud mesenchyme with TGF-[3 stimulates expression of fibronectin, N-CAM, N-cadherin, and tenascin [339,340]. Exposure of limb bud mesenchymal cells to some BMPs also correlates with an increase in N-cadherin and N-CAM expression and stimulates chondrogenesis [5,10]. Despite these in vitro observations, it has been difficult to confirm a specific role for these signaling factors in general skeletogenesis in vivo or to assign a specific role(s) to different BMPs. Gene inactivation studies in the mouse have not been very informative in this respect. For instance, null mutations in Bmp-2 or -4, as well as in the BMP receptor type 1A gene (Bmp-rlA), lead to early embryonic lethality [341-343], whereas mice null for Bmp-3 or Bmp-3b (also known as GdflO) display no obvious embryonic phenotype in the skeleton [344,345]. Mutations in Bmp-5-7 as well as in Gdf5 [also called cartilage-derived morphogenic protein 1 (Cdmpl)] and the gene encoding the
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BMP receptor type 1B (Bmp-rlB) do not perturb general skeletogenesis but affect only a subset of specific skeletal elements [346,347]. The analysis of mice carrying null mutations in two of these genes found additional defects not observed in either single mutant, supporting the idea that a significant level of functional redundancy exists in this signaling pathway [347-349]. As in the mouse, mutations affecting BMP signaling in humans also lead to defects in restricted sets of skeletal elements. Mutations in only one BMP-related gene have been linked to human skeletal defects. Based on similarities with the murine braehypodism (bp) phenotype, HunterThompson acromesomelic dysplasia, in which patients present with shortened middle and distal limb segments as well as joint defects, was found to be caused by mutations in GDF5/CDMP1 [267,350]. A mutation creating a dominant negative form of CDMP1 was also found in the more severe form of acromelic dysplasia, Grebe syndrome, whereas a CDMP1 mutation creating a premature stop codon was found in a family displaying a novel type of brachydactyly [267,350]. Mutations in the gene encoding the BMP antagonist Noggin (NOG) also cause two autosomal dominant disorders--proximal symphalangism (SYM1) and multiple synostose syndrome (SYNS1), both characterized by multiple joint fusions [351]. BMP/GDF gain-of-function experiments in chick [232,286,352,353] and mouse embryos [125,354,355] lead to hyperplasia and in some cases fusion of limb cartilage elements. Conversely, broad repression of BMP signaling inhibits cartilage development [355-357]. Some of the phenotypes observed in these in vivo studies suggest that BMP/GDF signaling controls recruitment of cells into the early condensations [125,353,354]. In addition, exposure of limb bud mesenchymal cells to GDF5 increases cell-cell adhesiveness [353], supporting a role in condensation. Insightful analyses of chondrogenesis in micromass cultures of limb bud mesenchyme established that BMP signaling is indeed required for formation of prechondrogenic condensations but also for differentiation of mesenchymal cells into chondrocytes [358-360]. Interactions of the mesenchymal cells with other cell types also affect condensation. For instance, in the developing limb, there is an inverse relationship between condensations and blood vessel distribution. Vessels are initially present throughout the limb mesenchyme but undergo local regression from sites at which precartilaginous condensations will form shortly thereafter [361]. It was recently shown that this vascular regression is essential for the condensation step and subsequent chondrogenesis in the chick developing limb [361]. How vascularization interferes with the condensation process is not clear. In principle, blood vessels could represent a physical barrier to condensation, produce or transport
factors that inhibit this process, or simply create high oxygen levels incompatible with the formation of prechondrogenic condensations [361]. Interestingly, the osteogenic condensations initiating the formation of the intramembranous bones of the calvaria in the chick embryo also form in an avascular milieu [20]. I n t r a m e m b r a n o u s Bone Formation Differentiation
Osteoblasts differentiation is reviewed in Chapter 2. This section only briefly mentions the most important recent developments concerning this issue. A key regulator of osteoblast differentiation and function is Runt domain factor 2/core binding factor oL 1 (Runx2/Cbfal). Runx2/Cbfal is a transcription factor isolated by virtue of its binding to cis-regulatory elements of the gene encoding osteocalcin, the most osteoblast-specific protein known [362]. Runx2/Cbfal is absolutely necessary for osteoblast differentiation, as was unambiguously demonstrated by gene inactivation in the mouse. The skeleton of Runx2/Cbfal-deficient mice is completely devoid of osteoblasts due to a differentiation arrest as early as E 12.5--that is, almost 2 days before the normal appearance of the first mature osteoblasts in wildtype limbs [363,364]. Runx2/Cbfal acts as a positive regulator of osteoblast-specific gene expression. Forced expression of this factor is sufficient to induce osteoblastspecific gene expression in skin fibroblasts and to induce ectopic bone formation in vivo [362,365,366]. In addition, Runx2/Cbfal is required for osteoblast function beyond differentiation [367,368]. Haploinsufficiency of Cbfal causes cleidocranial dysplasia (CCD) in both mice and humans [364]. CCD is an autosomal dominant disorder characterized primarily by a delay in closure of cranial sutures, reduced or absent clavicle, and dental anomalies [364]. Runx2/Cbfal is first expressed in cells of the mesenchymal condensations believed to contain common osteochondroprogenitor cells. With development, its expression progressively increases in preosteoblasts and then in true osteoblasts but is downregulated in all nonhypertrophic chondrocytes [362]. However, Runx2/Cbfal expression is also detectable in hypertrophic chondrocytes of endochondral bones, and Runx2/Cbfal null mice display deficient chondrocyte hypertrophy, supporting a role for this factor as a regulator of chondrocyte maturation [369,370]. E n d o c h o n d r a i Bone Formation Differentiation
The overt differentiation of condensed mesenchymal cells into cartilage-producing cells is characterized by a
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change in cell shape and a shift in production of ECM and adhesion molecules. Differentiating cells enlarge, become rounded, and develop extensive ER and Golgi complex for high-level synthesis and secretion of proteins. Production of N-CAM, N-cadherin, and type I collagen is downregulated, whereas that of types II, IX, and XI collagen as well as the large proteoglycan aggrecan and associated proteins is upregulated. One general observation is that differentiation seems to require a minimal condensation size. For instance, in vitro chondrogenesis depends on the initial cell density of the cultures and also correlates with the extent of condensation [10]. Furthermore, a number of null mutations in the mouse lead to ectopic cartilage formation as the result of accumulation of mesenchyme at sites where condensations normally form but are too small to chondrify [371]. A key regulator of chondroblast differentiation was identified through the discovery that mutations in the gene encoding the HMG domain DNA-binding protein SOX9 cause the rare and severe dwarfism campomelic dysplasia in humans [148,372]. During embryogenesis, Sox9 is expressed in all prechondrogenic condensations and cartilages, where it precedes the expression of the Col2al gene (type II collagen). The function of Sox9 in chondrogenesis was studied by analyzing the fate of Sox9_/_ ES cells in chimeric mouse embryos. It was shown that the mutant cells were excluded from prechondrogenic condensations and failed to express any chondrocyte-specific markers [373]. In addition, teratomas derived from these cells failed to produce any cartilage [373]. Thus, Sox9 is essential in a cell-autonomous manner at the condensation step. However, its best characterized function is in the induction of the chondrocytic phenotype. Sox9 appears to work by direct regulation of transcription of chondrocyte-specific genes. For instance, Sox9 binds to and activates an enhancer element of the Col2al gene, thus inducing production of type II collagen in a chondrocyte-specific manner [374]. Two other members of the Sox family, L-Sox5 and Sox6, interact with the same sequences and together with Sox9 activate transcription of the Col2al and aggrecan genes [375]. L-Sox5 and Sox6 have partly redundant functions and single mutants display limited but different skeletal abnormalities [376]. The Sox5;Sox6 double mutants, however, show severe generalized chondrodysplasia characterized by poor differentiation of chondroblasts that express cartilage matrix components at much lower levels than wild-type cells [376]. Thus, L-Sox5 and Sox6 are potent enhancers of chondroblast differentiation. As indicated previously, BMP signaling stimulates chondrocyte differentiation after the condensation stage [358,360]. In the sclerotome, Shh changes the compe-
tence of target cells to respond to BMP signaling. In the absence of Shh, BMP administration induces lateral plate gene expression in cultured PSM. After exposure to Shh, BMP signaling induces robust chondrogenesis instead [377]. It was recently shown that this Shh effect is mediated by the Bapxl transcription factor, which is essential for formation of the ventromedial elements of vertebrae [134]. Interestingly, Bapxl is a transcriptional repressor and probably acts by preventing expression of factors that interfere with the chondrogenic activity of BMPs [134]. Whether unrelated factors modulate a similar effect in ribs and limb cartilage elements is unknown. BMP signaling may stimulate chondrogenesis by stimulating Sox9 expression [378] or indirectly increasing Sox9 activity via regulation of the cAMP-dependent protein kinase (PKA). Indeed, cells of the prechondrogenic condensations are characterized by an increased level of intracellular cyclic AMP (cAMP) and activated PKA, and modulation of this intracellular signaling pathway has long been known to affect chondrogenesis [10]. Importantly, although this increase is seen at the condensation step, modulation of PKA activity does not affect the process of condensation but the subsequent progression from precartilage condensation to cartilage nodule [379]. Furthermore, PKA activation is induced by BMP signaling, and this activation is essential for the BMP-2-induced stimulation of chondogenesis to occur in vitro [380]. Finally, PKA phosphorylation of Sox9 increases its DNA binding and transcriptional activity [381]. Thus, one can hypothesize a molecular pathway in which BMP signaling stimulates PKA activity, which in turn increases Sox9 activity, thereby stimulating the transcription of chondrocyte-specific genes. However, Sox9 is not the only PKA target and other mechanisms are likely. For instance, PKA also phosphorylates the cAMP-response element (CRE)-binding protein, allowing it to bind the CRE and activate transcription of genes containing this regulatory sequence. During chondrogenesis, PKA also activates protein kinase Ca (PKCo0, and this activation leads to the downregulation of N-cadherin and fibronectin seen in differentiating chondrocytes [379]. PKC may also be involved in Wnt signaling and in this capacity may regulate chondrogenesis. The Writ genes encode a large family (19 members in mouse and humans) of cysteinerich secreted glycoproteins sharing homology with the Drosophila signaling factor wingless. Wnt proteins control morphogenesis and tissue patterning in a wide variety of organs [382]. Based on their biological activities in specific assays, Wnts have been divided into two functional groups, which sometimes display opposite effects. Wnt ligands interact with membrane receptors of the Frizzled (Fz) family and signal via one of two intracellular pathways. In the so-called canonical Wnt/~-catenin pathway, regulation of a number of intermediate effect-
3. Prenatal Bone Development
ors upon binding of the ligand to the receptor leads to cytoplasmic accumulation of [3-catenin. This protein eventually translocates to the nucleus to form an active transcription complex with LEF-1/TCFs factors. [3-Catenin transcriptional activity may also be regulated by adhesion molecules such as cadherins, which sequester catenins. In the Wnt/Ca 2+ pathway, signaling results in intracellular Ca 2§ release and activation of Ca2+-sensitive enzymes including PKC [383]. Ectopic expression of Wntl in developing chick limbs resulted in skeletal abnormalities consistent with a localized failure of cartilage formation [384]. Studies of Wnt activity in chick limb bud micromass cultures confirmed that Wnt l and Wnt7a inhibit chondrogenesis and that this inhibition occurs during the chondroblast differentiation stage [384,385]. However, it was also shown that BMP-induced chondrogenesis repressed WntTa expression but upregulated Wnt3a expression [386]. Thus, it is possible that different Wnt ligands will demonstrate opposite effects on chondrocyte differentiation. Two recent studies indicate that retinoid signaling via the retinoic acid receptor ot (RARo0 also acts to inhibit early chondrocyte differentiation [359,387]. These studies used a transgenic approach in the mouse to express a constitutively active RARot in the limb mesenchyme. Cells expressing the transgene did participate in mesenchymal condensations in vivo and in vitro but failed to differentiate into chondroblasts and maintained instead a prechondrogenic phenotype. In contrast, the addition of a RARoL-selective antagonist to cultures of these cells was sufficient to stimulate chondroblast differentiation and cartilage formation, even when BMP signaling was repressed. Furthermore, inhibition of RAR activity correlated with PKA activation, increased Sox9 expression, and enhanced Sox9 transcriptional activity [388]. In the developing mandibular process, Sox9 and the homeobox transcription factor Msx2 are induced by BMP-4, and the relative amount of these two factors appears to determine where chondrogenesis takes place [389]. These two factors are also expressed in a subpopulation of cranial neural crests cells that will populate the mandibular area. In this population, Msx2 represses chondrogenic differentiation until cell migration is completed within the mandibular primordium [390]. Msx factors generally act as repressors of transcription, but it is not known whether the antagonistic interaction between Sox9 and Msx2 is direct or mediated by other factors. A number of other factors inhibit chondrogenesis, including GDF11 in the limb [391], Hoxa2 in the second branchial arch [392], activin, and PDGF [9],but in most cases the stage at which inhibition takes place has not been precisely established.
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Growth/Profiferation
The insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) are part of a general growth-promoting system acting in utero and in postnatal development. These factors and their receptor (IGF-IR) are closely related in sequence and structure to insulin and the insulin receptor (IR), respectively. IR and IGF-1R are tyrosine kinase receptors, activated as heterotetrameric complexes, which act via the docking protein insulin receptor substrate 1 (IRS-1) to initiate signal transduction by the mitogen-activate protein kinase or the phosphatidyl-inositol 3 kinase pathways [393]. IGF-2 (and to a lesser degree IGF-1) also interacts with a second receptor, the mannose-6-phosphate/IGF-2 receptor (IGF-2R). This is a monomeric receptor devoid of a signaling domain. It essentially acts as a scavenger to prevent accumulation of toxic levels of IGF-2 (and possibly IGF-1) [393]. These receptors are ubiquitously expressed, indicating that insulin/IGF signaling acts on almost all cell types in the body, including the developing skeleton. IGF-1 is also produced in the liver, the main source of plasma IGF-1, and acts as an endocrine factor as well. However, recent studies showed that mouse embryos are not affected by a dramatic reduction of circulating levels of IGF-1, indicating the importance of the autocrine/paracrine role of local IGF signaling in prenatal development and growth [394,395]. In the growth plate of developing bones, IGF expression is stimulated by growth hormone. Gene inactivation studies in the mouse have confirmed the essential role of the IGF signaling pathway in development [396,397]. Igfl - / - mice are only 60% the size of their wild-type littermates at birth and most die soon after birth. The few mutants that survive remain severely growth retarded and exhibit developmental defects, supporting the involvement of IGF-1 signaling in prenatal and postnatal development. Null mutants for igf-2 display similar small size at birth but grow normally after birth, suggesting a more important role in intrauterine growth. Inactivation of the igf-lr gene leads to a more severe growth retardation (45% of normal birth weight) and perinatal lethality due to the loss of both IGF-1 and IGF-2 action. Finally, mice null for igf-2r exhibit a lethal fetal overgrowth syndrome. This lethal phenotype can be partially rescued by crossing the mutation onto a igf-2 null background, indicating that excess IGF-2 is detrimental to fetal development and that IGF-2R functions to prevents this from occurring. Although these effects are not skeletal specific, it is clear that chondrocyte proliferation in the developing endochondral bones is critically dependent on IGF signaling. FGF signaling is also recognized as an essential component of the control of chondrocyte proliferation [398]. The importance of this signaling pathway was first
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revealed with the discovery that an autosomal dominant mutation in the transmembrane domain of the receptor FGF-R3 causes achondroplasia (ACH), the most common inheritable form of dwarfism in humans [399,400]. ACH is characterized by reduced growth of long bones, craniosomatic disproportions, and frontal bossing of the cranium. Interestingly, the mutation resulted in constitutive (ligand-independent) activation of FGF-R3. The related syndromes hypochondroplasia and thanatophoric dysplasia were later shown to also result from different activating mutations in FGF-R3 [398]. Furthermore, the degree of activation of the receptor was found to correlate with the severity of the chondrodysplasia. A number of mouse models harboring activating mutations in Fgf-r3 phenocopy many aspects of these diseases and confirm that reduced chondrocyte proliferation is the main cellular defect [401-407]. Conversely, Fgf-r3 inactivation in the mouse leads to increased proliferation and postnatal skeletal overgrowth [408,409]. Thus, a normal function of FGF-R3 signaling is to limit bone growth by negatively controlling chondrocyte proliferation in the growth plate. This is surprising in view of the fact that FGF signaling promotes proliferation in most cell types. This inhibitory signal could be a unique property of the FGF-R3 receptor or a peculiarity of the chondrocyte response to FGF signaling. Chondrocyte-specific expression of an activated chimeric FGF receptor containing the intracellular domain of FGF-R1 also resulted in an ACH phenotype [410]. This indicates that the response is not receptor but celltype specific. Several observations suggest that transcription factors of the signal transducer and activator of transcription (STAT) family are mediators of FGF action on chondrocytes. Activating mutations in FGF-R3 or treatment of cells with FGF induce nuclear translocation of STAT1, which in turn increases the expression of the cell cycle inhibitor p21 [411-413]. The Fgf-r3-induced ACH-like phenotypes created in mice also correlate with activation of STAT proteins and expression of cell cycle inhibitors [402,404]. Finally, in bone rudiments derived from STAT -/- animals, FGF treatment does not result in growth inhibition and crossing transgenic animals overexpressing FGF-2 into a STAT -/- background rescues the chondrodysplasia characteristic of this transgenic line [412,414]. Identification of the in vivo ligand(s) of FGF-R3 has been difficult. There are 22 known FGF ligands, many of which are expressed during endochondral ossification [398]. Furthermore, ectopic expression of Fgf-2 and Fgf-9 in the mouse led to skeletal dwarfism [415,416] but inactivation of these two genes does not affect the growth plate [226,417]. However, recent data show that growth plate histology of mice lacking Fgf-18 is similar to
that of mice lacking Fgf-r3, providing evidence for a ligand receptor relationship in the growth plate [418,419]. Part of the inhibitory effect of FGF-R3 signaling on growth plate chondrocytes is likely to be indirect and involve the regulation of expression of Indian hedgehog (Ihh). Ihh is a secreted protein closely related to Shh and signals via the same surface receptor complex composed of the multiple transmembrane proteins Patched- 1 (Ptch- 1) and Smoothened (Smo) [107]. Soon after mesenchymal condensation and the initiation of chondrocyte differentiation, Ihh becomes expressed in a broad domain in the center of each cartilage element. At that stage, Ptchl is expressed in a broader domain that encompasses almost all the chondrocytes, the future perichondrium, and some surrounding mesenchyme [420,421]. As chondrocytes in the center of each element mature and hypertrophy, Ihh expression becomes gradually restricted to the prehypertrophic chondrocytes, the more mature chondrocytes about to transition to hypertrophy and CollO expression, as well as some of the upper hypertrophic cells (Fig. 9). High levels of expression of Ptch-1 and Smo become restricted to the perichondrium but proliferating chondrocytes adjacent to the Ihh-expressing cells also continue to express the receptor complex [420-422]. Gene inactivation of Ihh leads to severe prenatal dwarfism due to a 50% decrease in chondrocyte proliferation in utero, suggesting that the prehypertrophic cells signal to maintain the high proliferation rate of Ptch-1/Smo-expressing chondrocytes [421] (Fig. 9). Recent results confirm that this interaction is direct. Chondrocyte-specific inactivation of Smo phenocopies the proliferation defect of the Ihh-deficient mice, whereas constitutive activation of Smo promotes chondrocyte proliferation [423]. Expression of the cell cycle regulator cyclin D1 is markedly downregulated when either Ihh or Smo activity is removed from chondrocytes, again supporting a direct regulation of proliferation by Ihh signaling [424]. In addition, although Ihh also controls chondrocyte maturation by regulating parathyroid hormone-related peptide (PTHrP) expression, its action on proliferation is independent of PTHrP [425]. Missense mutations in human IHH were found in three families segregating the autosomal dominant brachydactyly type A-l, characterized by shortening or the absence of the middle phalanges [426]. In addition, affected individuals in one family were significantly shorter than their nonaffected relatives, suggesting a defect in bone growth. A link between Ihh and FGF-R3 signaling is indicated by the observation that mice harboring an activating mutation in Fgf-r3 have decreased expression of Ihh, Ptch-1, and Bmp-4, whereas in Fgf-r3-deficient mice Ihh, Ptch-1, and Bmp-4 expression is upregulated [401,403,404].
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Chondrocyte Maturation PTHrP is a paracrine factor synthesized in a number of tissues that shares homology with PTH in its Nterminal domain and binds a common receptor, the PTH/PTHrP receptor 1 (PTH-R1) [196]. PTHrP was one of the first factors identified as a regulator of growth plate chondrocyte maturation. Targeting inactivation of the gene encoding either PTHrP or its receptor in the mouse resulted in a lethal phenotype characterized by skeletal dysplasia in which premature maturation of
FIGURE 9 Schematicrepresentation of the Ihh/PTHrP regulatory feedback loop controlling chondrocyte maturation in the growth plate. The diagram depicts the early growth plate of a long bone in a mouse embryo. The cartilage is surrounded by a perichondrium (pe), and zones of chondrocyte proliferation (p), maturation (m), prehypertrophy (p-hy), and hypertrophy (hy) can be seen. The cartilage matrix in the hypertrophic zone is calcified (gray shading). The primary ossification center where bone (b) is deposited by invading osteoblasts is also depicted at the bottom of the figure. Prehypertrophic chondrocytes express Ihh (dark gray shading). The Ihh signal acts directly on maturing and proliferating chondrocytes(1) expressingthe hedgehogreceptor complex Ptchl/Smo (light gray shading) and stimulates proliferation of these chondrocytes. Ihh also signals directly to perichondrial cells adjacent to the Ihh expression domain (2), and via the hedgehog signal transduction pathway it induces expression of TGF[32 in the perichondrium (3). Elevated TGF-[32upregulates Pthrp expression in cells of the articular perichondrium (4). PTHrP acts at a distance on proliferating chondrocytes expressing the Pthrl receptor (5) and slows their maturation into prehypertrophic chondrocytes (6), thus completing the negative feedback loop.
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chondrocytes led to short-limb dwarfism and excessive bone formation at birth [427-429]. Conversely, misexpression of P T H r P in a mouse transgenic system significantly delayed chondrocyte maturation and bone formation such that mice were born with a completely cartilaginous endochondral skeleton [430]. In humans, inactivating mutations in P T H - R 1 cause Blomstrand chondrodysplasia characterized by advanced skeletal maturation with shortness of long bones and increased bone density [431-434], whereas mutations in PTH-R1 resulting in constitutive signaling cause a rare disorder, Jansen metaphyseal chondrodysplasia, characterized by widespread growth plate abnormalities including delayed mineralization [435,436]. Overexpression of a constitutively active receptor in the mouse reproduces several aspects of this disease [437]. A novel activating mutation in PTH-R1 was also identified in enchondromatosis, a condition characterized by the presence of benign cartilage tumors adjoining the growth plate [438]. Finally, PTHrP is a potent inhibitor of chondrocyte maturation in vitro [439,440]. In developing long bones in the mouse, Pthrp is expressed at highest levels by cells of the periarticular perichondrium, at the end of the bones, and at lower levels by the proliferating chondrocytes [28,428, 441443]. The Pthr-1 gene is expressed at low levels by proliferating chondrocytes and at high levels by the maturing chondrocytes adjacent to the Ihh-secreting cells [28,420,421,428,441,443--445]. Interestingly, this is where immunoreactivity for the PTHrP protein is highest [428,441], suggesting that Pthr-1 present on these maturing cells acts as a sink to prevent diffusion of the ligand and establish a sharp transition to below threshold levels of the ligand. These different observations suggested a model in which PTHrP, secreted mostly by cells of the periarticular perichondrium at the end of the developing long bones, diffuses through the zone of proliferating chondrocytes and acts on Pthr-l-expressing cells to prevent or slow their maturation into postmitotic hypertrophic chondrocytes [437,446]) (Fig. 9). A recent analysis of the fate of Pthr-1 - / - cells in chimeric mice supports this interpretation. Pthr-1 null cells undergo hypertrophy prematurely while still in the proliferative zone, surrounded by immature wild-type chondrocytes [422,447]. This confirms that Pthr-l-expressing proliferating chondrocytes are the direct target of PTHrP signaling in the developing growth plate and signaling through Pthr-1 acts in a cell autonomous manner to prevent hypertrophy. PTHrP acts, at least in part, by increasing PKA-dependent phosphorylation and transcriptional activity of Sox9 [448]. Although Sox9 is present in all nonhypertrophic chondrocytes of the growth plate, PKA-phosphorylated Sox9 is detected almost exclusively in the prehypertrophic cells expressing
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high levels of Pthr-1 and is not detectable in Pthr-1 null growth plates [448]. Interestingly, Pthrp expression appears to be controlled by Ihh signaling. The role of Ihh in skeletogenesis was first addressed experimentally in the chick embryo, in which it was shown that ectopic expression of Ihh prevented maturation of chondrocytes and the appearance of hypertrophic cells [445]. Moreover, the misexpression of Ihh was found to prevent the appearance of Ihh-positive chondrocytes, indicating that chondrocyte maturation was blocked early, before the transition to the prehypertrophic phenotype [445]. These phenotypic consequences of Ihh misexpression are the opposite of the phenotype resulting from inactivation of Pthrp in the mouse [28,427,429], suggesting a functional relationship between the two signaling pathways. Vortkamp and collaborators [445] showed that Ihh misexpression in the developing wing bones of the chick in fact led to upregulation of Pthrp expression by perichondrial cells adjacent to the articular surface. Using a murine limb ex vivo culture system [28], it was further shown that Hedgehog protein, like PTHrP, could prevent chondrocyte hypertrophy when added to the culture medium, but only when PTHrP signaling in the explants was intact [445]. Addition of Hedgehog protein to the culture medium of Pthrp -/- or Pthr-1 -/- explants did not prevent chondrocyte maturation. Thus, Ihh signaling appears to regulate chondrocyte maturation via its action on the PTHrP paracrine system by regulating Pthrp transcription. These observations led to a model in which Ihh and PTHrP regulate chondrocyte maturation through the establishment of a negative feedback mechanism [445] (Fig. 9). In this model, production of Ihh by prehypertrophic chondrocytes induces Pthrp expression at the periarticular surface. In turn, PTHrP signals to Pthr/-positive chondrocytes, preventing additional proliferating chondrocytes from initiating maturation and delaying the production of new cells expressing Ihh. As prehypertrophic chondrocytes progress to the hypertrophic state, they stop expressing Ihh, thereby attenuating the negative feedback mechanism. This allows new proliferating chondrocytes to initiate maturation and exit proliferation. This mechanism ensures coordination of the rates of chondrocyte proliferation and maturation throughout development. Inactivation of the Ihh gene in the mouse results in the absence of Pthrp expression leading to ectopic chondrocyte maturation, thus confirming the main features of the model [421]. There is evidence that this regulatory network is also active in the postnatal growth plate [449,450]. Because the Ihh receptor complex Ptch-1/Smo is expressed at high levels in the perichondrium, it was suggested that Ihh signals through this tissue to control Pthrp expression [445]. Recently, TGF-[32 was identified
as a perichondrial relay between these two factors [451-454].
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the parathyroid hormone-related peptide gene. Genes Dev. 8, 277-289. Lee, K., Lanske, B., Karaplis, A. C., Deeds, J. D., Kohno, H., Nissenson, R. A., Kronenberg, H. M., and Segre, G. V. (1996). Parathyroid hormone-related peptide delays terminal differentiation of chondrocytes during endochondral bone development. Endocrinology 137, 5109-5118. Lanske, B., Karaplis, A. C., Lee, K., Luz, A., Vortkamp, A., Pirro, A., Karperien, M., Defize, L. H., Ho, C., Mulligan, R. C., Abou-Samra, A. B., Juppner, H., Segre, G. V., and Kronenberg, H. M. (1996). PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273, 663-666. Weir, E. C., Philbrick, W. M., Amling, M., Neff, L. A., Baron, R., and Broadus, A. E. (1996). Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation. Proc. Natl. Acad. Sci. USA 93, 10240-10245. Karperien, M., van der Harten, H. J., van Schooten, R., FarihSips, H., den Hollander, N. S., Kneppers, S. L., Nijweide, P., Papapoulos, S. E., and Lowik, C. W. (1999). A frame-shift mutation in the type I parathyroid hormone (PTH)/PTH-related peptide receptor causing Blomstrand lethal osteochondrodysplasia. J. Clin. Endocrinol. Metab. 84, 3713-3720. Karaplis, A. C., He, B., Nguyen, M. T., Young, I. D., Semeraro, D., Ozawa, H., and Amizuka, N. (1998). Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia. Endocrinology 139, 5255-5258. Zhang, N., and Gridley, T. (1998). Defects in somite formation in lunatic fringe-deficient mice. Nature 394, 374-377. Jobert, A. S., Zhang, P., Couvineau, A., Bonaventure, J., Roume, J., Le Merrer, M., and Silve, C. (1998). Absence of functional receptors for parathyroid hormone and parathyroid hormone-related peptide in Blomstrand chondrodysplasia. J. Clin. Invest. 102, 34-40. Schipani, E., Kruse, K., and Juppner, H. (1995). A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science 268, 98-100. Schipani, E., Langman, C. B., Parfitt, A. M., Jensen, G. S., Kikuchi, S., Kooh, S. W., Cole, W. G., and Juppner, H. (1996). Constitutively activated receptors for parathyroid hormone and parathyroid hormone-related peptide in Jansen's metaphyseal chondrodysplasia. N. Engl. J. Med. 335, 708-714. Schipani, E., Lanske, B., Hunzelman, J., Luz, A., Kovacs, C. S., Lee, K., Pirro, A., Kronenberg, H. M., and Juppner, H. (1997). Targeted expression of constitutively active receptors for parathyroid hormone and parathyroid hormone-related peptide delays endochondral bone formation and rescues mice that lack parathyroid hormone-related peptide. Proc. Natl. Acad. Sci. USA 94, 13689-13694. Hopyan, S., Gokgoz, N., Poon, R., Gensure, R. C., Yu, C., Cole, W. G., Bell, R. S., Juppner, H., Andrulis, I. L., Wunder, J. S., and Alman, B. A. (2002). A mutant PTH/PTHrP type I receptor in enchondromatosis. Nature Genet. 30, 306-310. Grimsrud, C. D., Romano, P. R., D'Souza, M., Puzas, J. E., Reynolds, P. R., Rosier, R. N., and O'Keefe, R. J. (1999). BMP-6 is an autocrine stimulator of chondrocyte differentiation. J. Bone Miner. Res. 14, 475-482. Zerega, B., Cermelli, S., Bianco, P., Cancedda, R., and Cancedda, F. D. (1999). Parathyroid hormone [PTH(1-34)] and parathyroid hormone-related protein [PTHrP(1-34)] promote reversion of hypertrophic chondrocytes to a prehypertrophic proliferating phenotype and prevent terminal differentiation of osteoblast-like cells. J. Bone Miner. Res. 14, 1281-1289.
3. Prenatal Bone Development 441. Amizuka, N., Warshawsky, H., Henderson, J. E., Goltzman, D., and Karaplis, A. C. (1994). Parathyroid hormone-related peptidedepleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation. J. Cell Biol. 126, 1611-1623. 442. Amizuka, N., Karaplis, A. C., Henderson, J. E., Warshawsky, H., Lipman, M. L., Matsuki, Y., Ejiri, S., Tanaka, M., Izumi, N., Ozawa, H., and Goltzman, D. (1996). Haploinsufficiency of parathyroid hormone-related peptide (PTHrP) results in abnormal postnatal bone development. Dev. Biol. 175, 166-176. 443. Lee, K., Deeds, J. D., Chiba, S., Un-No, M., Bond, A. T., and Segre, G. V. (1994). Parathyroid hormone induces sequential c-fos expression in bone cells in vivo: In situ localization of its receptor and c-fos messenger ribonucleic acids. Endocrinology 134, 441-450. 444. Amizuka, N., Henderson, J. E., Hoshi, K., Warshawsky, H., Ozawa, H., Goltzman, D., and Karaplis, A. C. (1996). Programmed cell death of chondrocytes and aberrant chondrogenesis in mice homozygous for parathyroid hormone-related peptide gene deletion. Endocrinology 137, 5055-5067. 445. Vortkamp, A., Lee, K., Lanske, B., Segre, G. V., Kronenberg, H. M., and Tabin, C. J. (1996). Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273, 613-622. 446. Wallis, G. A. (1996). Bone growth: Coordinating chondrocyte differentiation. Curr. Biol. 6, 1577-1580. 447. Chung, U. I., Schipani, E., McMahon, A. P., and Kronenberg, H. M. (2001). Indian hedgehog couples chondrogenesis to osteogenesis in endochondral bone development. J. Clin. lnvest. 107, 295-304. 448. Huang, W., Chung, U. I., Kronenberg, H. M., and de Crombrugghe, B. (2001). The chondrogenic transcription factor Sox9
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is a target of signaling by the parathyroid hormone-related peptide in the growth plate of endochondral bones. Proc. Natl. Acad. Sci. USA 98, 160-165. van der Eerden, B. C., Karperien, M., Gevers, E. F., Lowik, C. W., and Wit, J. M. (2000). Expression of Indian hedgehog, parathyroid hormone-related protein, and their receptors in the postnatal growth plate of the rat: Evidence for a locally acting growth restraining feedback loop after birth. J. Bone Miner. Res. 15, 1045-1055. Farquharson, C., Jefferies, D., Seawright, E., and Houston, B. (2001). Regulation of chondrocyte terminal differentiation in the postembryonic growth plate: The role of the PTHrP-Indian hedgehog axis. Endocrinology 142, 4131-4140. Alvarez, J., Sohn, P., Zeng, X., Doetschman, T., Robbins, D. J., and Serra, R. (2002). TGFbeta2 mediates the effects of hedgehog on hypertrophic differentiation and PTHrP expression. Development 129, 1913-1924. Alvarez, J., Horton, J., Sohn, P., and Serra, R. (2001). The perichondrium plays an important role in mediating the effects of TGF-betal on endochondral bone formation. Dev. Dyn. 221, 311-321. Serra, R., Karaplis, A., and Sohn, P. (1999). Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation. J. Cell Biol. 145, 783-794. Serra, R., Johnson, M., Filvaroff, E. H., LaBorde, J., Sheehan, D. M., Derynck, R., and Moses, H. L. (1997). Expression of a truncated, kinase-defective TGF-beta type II receptor in mouse skeletal tissue promotes terminal chondrocyte differentiation and osteoarthritis. J. Cell Biol. 139, 541-552.
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Postnatal Bone Growth Growth Plate Biology, Modeling, and Remodeling GERARD KARSENTYand HENRY M. KRONENBERG
Despite the great variety of shapes and sizes, bones grow through the use of a small number of cellular mechanisms. The most common strategy is called endochondral bone formation, which indicates that the cells and matrix of bone are organized on a matrix first synthesized by chondrocytes. The second strategy, intramembranous bone formation, involves the generation of bone more directly by differentiation of mesenchymal cells into osteoblasts without the use of a cartilage-based matrix. In Chapter 3, the mechanism used to establish the initial growth of bone was reviewed. Here, we discuss how these strategies are extended in postnatal life to ensure effective growth of bone postnatally.
ENDOCHONDRAL AND INTRAMEMBRANOUS BONE FORMATION
FIGURE 1
Although most bones use the same cellular sequence in endochondral bone formation, the dramatic, asymmetric lengthening of the appendicular long bones leads to a geometric arrangement of chondrocytes that is particularly easy to follow and analyze (Fig. 1). The initial cartilage mold forms when mesenchymal cells condense and then become recognizable chondrocytes, synthesizing collagens II, IX, and XI and aggrecan, among other matrix molecules, under the direction of transcription factors including SOX-9 [1]. The cartilage mold enlarges both through proliferation along the entire length of the mold and through secretion of a characteristic matrix. In the center of the mold, chondrocytes stop proliferating, enlarge (hypertrophy), and change the constituents of the matrix to include, for example, collagen X. The matrix is subsequently mineralized, specialized osteoclasts called
Pediatric Bone
Serial stages in e n d o c h o n d r a l b o n e f o r m a t i o n .
chondroclasts invade the region of hypertrophic chondrocytes along with blood vessels and preosteoblasts, and the hypertrophic chondrocytes undergo apoptosis [2]. The preosteoblasts become true osteoblasts and lay down a matrix dominated by collagen type I on top of the matrix left behind by the hypertrophic chondrocytes. This is the primary spongiosa, which is subsequently remodeled by further waves of osteoclasts and osteoblasts to form the secondary spongiosa, the forerunner of mature trabecular bone. The marrow space is enlarged by continual osteoclast activity, and hematopoietic precursors migrate from the fetal liver to establish marrowbased hematopoiesis. Near the ends of the bone, the same cellular sequence of chondrocyte hypertrophy, vascular invasion, and bone formation subsequently occurs at so-called secondary ossification centers. The remaining
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Copyright 2003, Elsevier Science (USA). All rights reserved.
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growth cartilage between the expanding bone in the primary and secondary ossification centers is then called the growth plate. Surrounding the cartilage mold, perichondrial cells carry out a distinct genetic program that both regulates the cartilage mold and, in turn, is regulated by it. These perichondrial cells, originally distinguished from chondrocytes by the absence of SOX9 expression, secrete bone morphogenic proteins (BMPs), parathyroid hormonerelated peptide (PTHrP), and other signaling molecules that regulate chondrocyte proliferation and differentiation. Perichondrial cells adjacent to prehypertrophic/ hypertrophic chondrocytes respond to Indian hedgehog and other signals from those cells to become preosteoblasts and then true osteoblasts [3,4]. These osteoblasts lay down a collagen I-containing matrix that forms intramembranous bone adjacent to the cartilage mold. After the chondrocytes disappear, the intramembranous bone remains and is remodeled by endosteal osteoclastic bone resorption and periosteal new bone formation, as preosteoblastic cells proliferate and differentiate. In humans, Haversian systems formed around invading blood vessels further enlarge the bony envelope, called cortical bone. The events just summarized indicate several sources of bone enlargement. Lengthening of the appendicular bones is primarily driven by growth plate activity. In fetal life all nonhypertrophic chondrocytes vigorously proliferate. As a formal growth plate forms, however, the chondrocytes near the top of the growth plate slow down their rate of proliferation dramatically and become "reserve" or "resting" cells that may serve as stem cells for all other chondrocytes. Under controls that are poorly understood, resting cells periodically become proliferating chondrocytes that undergo several rounds of prolifer-
ation. These proliferating cells flatten out, with their short axis parallel to the long axis of the long bone, and form columns of stacked proliferating chondrocytes, with each column representing descendants of a distinct clone of cells. The lengthening of the bone derives primarily from the substantial enlargement that each chondrocyte undergoes when it hypertrophies [5]. The large number of proliferating chondrocytes and the matrix secreted by all of the chondrocytes also contribute to bone lengthening. Because the chondrocytes and their matrix ultimately disappear, however, normal osteoblast function in the primary spongiosa is also essential to allow the expansion of the bone length generated by chondrocyte activity to be translated into permanent lengthening. The important role of the bone adjacent to the growth plate is illustrated, for example, by the short bones found in mice in which the transcription factor Cbfal (Runx2) is inactivated in osteoblasts by a dominant-negative strategy [6]. Lengthening of bone ceases when growth plate chondrocytes disappear during puberty. A small number of bones, primarily the flat bones of the skull, form by intramembranous bone formation without a cartilage mold [7]. These bones form when mesenchymal cells differentiate directly into preosteoblasts and then osteoblasts. The growth of these bones occurs by waves of proliferation and differentiation of preosteoblasts at the perimeter of the growing bones. As the perimeters of adjacent bones approach each other, a specialized structure, the suture, forms. Normal development of the cranium requires that the skull expand in close coordination with the growth of the underlying brain. At the suture, proliferating mesenchymal cells differentiate into osteoblasts; sutures normally close only when brain expansion ceases (Fig. 2) [8].
FIGURE 2 Normalrat coronal suture. Hematoxylinand eosin-stainedsectionswith periosteoal surfaceat top and dura mater at bottom. El9, EmbryonicDay 19, soon beforebirth; P1, P5, and P21, daysafter birth; b, frontal and parietal bones;s, cellularsutureblastema;ps, presumptivesuture.Arrowsindicateleadingedgesof bonefronts [reprinted with permissionfrom Opperman, L. A. (2000).Cranial sutures as intramembranousbone growthsites. Dev. Dyn.219(4), 472-485].
4. Postnatal Bone Growth
Bone growth is regulated by a large variety of paracrine and endocrine signaling systems. In this chapter, we focus on a few such systems, chosen because of their established importance in normal human physiology and disease. Regulators of bone growth are first considered, followed by a discussion of osteoblast and osteoclast biology.
GROWTH H O R M O N E AND INSULIN-LIKE GROWTH FACTOR-1 Growth hormone (GH) is a major regulator of postnatal growth of most organs and through its actions on the growth plate, GH has a dramatic effect on postnatal bone growth. GH is a protein synthesized in the anterior pituitary gland. It acts on a receptor in the cytokine receptor family found in many organs of the body. Many of the actions of GH result from its stimulation of synthesis of insulin-like growth factor-1 (IGF-1). In a classic experiment, Salmon and Daughaday found that administration of GH to hypophysectomized rats stimulated incorporation of sulfate into cartilage proteoglycans [9]. Serum from such animals could also stimulate sulfate incorporation into proteoglycans in bones cultured in vitro, but GH added to serum from hypophysectomized rats could not do so. Thus, Salmon and Daughaday concluded that GH's action on bone was indirect, through the synthesis of a factor that traveled from elsewhere to work on bone. In the strongest version of the somatomedin hypothesis [10], it was proposed that GH increased the production of what is now known as IGF-1 in the liver, and that this IGF-1 then circulated and acted on the growth plate to cause bone growth. As detailed later, this hypothesis has required substantial modification over the years. (Even the initial experiment may have missed a direct effect of GH on sulfation of proteoglycans simply because the assay was not optimal [11].) Nevertheless, the observation that GH and IGF-1 are key regulators of bone growth has stood the test of time and has led to substantial understanding of the actions of these two important factors. The crucial role of GH in controlling bone growth dates from the isolation of GH and the evidence that GH administration corrects the growth defects in patients with genetic or acquired GH deficiency. Patients and mice with mutations in the GH receptor gene (Laron dwarfism) have a similar defect in bone growth [12-18]. Studies of mice missing the GH receptor show that the mice, like people with Laron dwarfism, are of normal size at birth but have defective growth. In mice, the defect is detected Days 10-40 after birth [17]. These mice have growth plates with short proliferative columns, fewer
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proliferative chondrocytes than normal, and smaller hypertrophic chondrocytes than normal. The chondrocytes in the proliferative columns proliferate more slowly than normal, as judged by incorporation of DNA precursors [17]. The formation of secondary ossification centers is delayed. Thus, these mice display defects both in proliferation of chondrocytes and in differentiation (small hypertrophic chondrocytes). IGF-1 is a member of the insulin family of growth factors and activates a distinct receptor that also mediates actions of a closely related ligand, IGF-2. Clear demonstration of the physiologic importance of IGF-1 in bone growth awaited gene ablation studies [19-21]; subsequently, one human with IGF-1 gene mutation and a phenotype analogous to that of the knockout mice was reported [22]. Unlike the fairly normal prenatal phenotype of mice missing the GH receptor, mice missing the IGF-1 gene are only 60% of normal weight at birth and have a high perinatal mortality, which varies depending on the genetic background of the particular mouse strain. These mice, like the GH receptor knockout mice, have small hypertrophic chondrocytes [17,23]. Wang et al. [23] found that proliferative columns are of normal size and that the chondrocytes proliferate at a normal rate, whereas Lupu et al. [17] found that the proliferative columns are short, with a low rate of proliferation. Although the studies differ in several respects, probably the most important difference that might explain the varying results is that the mice have distinct genetic backgrounds. As noted earlier, the observation that GH stimulates the production of IGF-1 in many cell types and the partial overlap in actions of these two factors has led to the hypothesis that many of the growth-promoting actions of GH are mediated by IGF-1. The small size of the IGF-1 knockout mice, despite their high GH levels, is consistent with this idea. Nevertheless, the finding that mice missing both the GH receptor and IGF-1 genes have a greater growth defect than either individual knockout mouse is strong genetic evidence for independent actions of GH and IGF-1 on growth. The double knockout mice have columns of proliferating chondrocytes that are substantially shorter than those of either individual knockout mouse and hypertrophic chondrocytes that are of the same small size found in both individual knockouts. The functional relationships between GH and IGF1 actions on the growth plate remain unsettled. IGF-1 can certainly increase bone growth in the absence of GH action, as best exemplified by the use of IGF-1 to treat Laron dwarfism [13,18]. Similarly, as already noted, mice with normal GH signaling and ablated IGF-1 genes have longer bones than those missing both the GH receptor and IGF-1. In fact, comparisons of the bones of GH
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receptor, IGF-1, and double knockout mice suggest that the actions of GH and IGF-1 are predominantly independent and additive [17]. The presence of mRNA encoding both IGF-1 and the GH receptor in the same proliferating chondrocytes suggests that these chondrocytes are the primary target of each ligand. On the other hand, functional studies suggest that a distinct population of chondrocytes might be unique targets of GH. Although both GH and IGF-1 stimulated clonal growth of chondrocyte colonies in agar, for example, GH stimulated a distinct large population of such colonies [24]. Perhaps analogously, in vivo GH stimulated the expansion of a population of cells at the top of the growth plate that may represent prechondrocytes or chondrocyte stem cells [25]. The possibility that part of the action of GH on chondrocytes might be mediated by IGF-1 is supported by studies showing that GH stimulates production of IGF-1 mRNA in the growth plates of hypophysectomized mice given GH [26]. Furthermore, stimulation of IGF-1 mRNA in the liver after GH administration and increased circulating IGF-1 levels, as well as the decrease or virtual absence of IGF-1 in the blood of GH receptor knockout mice [14,15,17], suggest that part of the action of GH may be via IGF-1. Of course, regulation of IGF-1 by GH gives no clear indication of the functional importance of this regulation on the growth plate. Recent studies suggest that IGF-1 made by the liver may be unimportant for bone growth. Using two distinct promoters to express the Cre recombinase, the IGF-1 gene was ablated specifically from liver in vivo using the Cre-Lox approach [27,28]. In each case, circulating levels of IGF-1 decreased to approximately 20% of baseline, but the animals grew normally. Although these studies demonstrate that a large fraction of circulating IGF-1 derives from the liver, the presence of residual IGF-1 in the circulation and the possibility that liver IGF-1 gene ablation may have been incomplete during the time of greatest IGF-1-dependent growth [17] leave open the possibility that circulating IGF-1 may contribute to bone growth. IGF-2 is a close relative of IGF-1. IGF-2 in mice is synthesized only during fetal life, whereas in humans IGF-2 is synthesized by many tissues throughout life. IGF-2 acts predominantly through the IGF-1 receptor, but when this receptor is missing, IGF-2 can also act through the insulin receptor. Knockout of the IGF-2 gene leads to decreased fetal growth in analogous fashion to knockout of the IGF-1 gene [29]. Because of the action of IGF-2 on the insulin receptor, knockout of IGF-2 and the IGF-1 receptor results in a more severe growth abnormality than knockout of the IGF-1 receptor alone [30]. Since IGF-2 acts predominantly in fetal life and GH acts on growth only postnatally, there are no expected
interactions between IGF-2 and GH in mice. In humans, the possible regulation of IGF-2 by GH has not been extensively analyzed; however, GH administration lowers circulating IGF-2 levels but stimulates production of IGF-2 mRNA in human liver [31]. Mice missing both the GH receptor and IGF-1 are only 17% of normal size [17]. Presumably, if residual IGF-2 action were also blocked, size would be diminished further, were that compatible with life. These dramatic quantitative effects show that GH and IGFs importantly control bone growth in mammals. Further dissection of the important target cells within the growth plate and possible indirect effects of these endocrine/ paracrine systems, which act on multiple organs, awaits additional analysis of mice with genes knocked out of specific cell types.
FIBROBLAST GROWTH FACTORS Fibroblast growth factors (FGFs) and their receptors regulate skeletal patterning and bone growth at virtually every step from the initial outgrowth of the limb to postnatal lengthening of long bones and intramembranous bones of the skull [32]. Here, we focus on the roles of FGFs in bone growth after the initial patterning of the skeleton. Humans and mice each have 22 FGFs encoded in their genomes. A total of 23 different FGFs have been identified, although a human counterpart to mouse FGF-15 and a mouse counterpart to human FGF-19 have not been found [33]. Expression of FGF-7,-8, -17, and -18 occurs in the perichondrium surrounding the growth plate, whereas FGF-2 is expressed in the periosteum of bone. FGF-2 a n d - 9 are both expressed by chondrocytes, whereas FGF-2 is also expressed by osteoblasts. In the growing skull, FGF-2, -4, and-9 are expressed in the mesenchyme of the suture, whereas FGF-18 and -20 are expressed by differentiating osteoblasts. Four genes encode receptors for the FGF family, although the actual number of receptors is larger because of splice variants of these genes [34]. Each encodes a transmembrane protein with tyrosine kinase activity that is triggered by ligand binding at the plasma membrane. The extracellular domain contains three immunoglobulin domains that contribute to ligand-binding specificity and affinity. Splice variants of FGF-R1-3 each substitute alternative sequences in the carboxyterminal portion of the third immunoglobulin domain. Splice variants 1b-3b are expressed in epithelial cells, and splice variants lc-3c are expressed in mesenchymal cells. FGF-R3 is expressed by proliferating chondrocytes,
4. Postnatal Bone Growth
FGF-R1 is expressed by prehypertrophic and hypertrophic chondrocytes, and FGF-R2 is expressed by perichondrial cells. In the sutures of the growing, intramembranous bones of the skull, FGF-R1 is found in the mesenchyme, whereas both FGF-R1 and-2 are in the differentiating osteoblasts of sutures. FGF-R3 is expressed late at the osteoblastic front of sutures. A series of inherited human diseases and mutations in mice have demonstrated the importance of individual components of this complicated network of ligands and receptors in bone development. Genetic analysis of the role of FGF signaling in the growth plate have been complicated by the multiple roles of FGF signaling during early stages of development [34]. Nevertheless, much has been learned. Mice missing the FGF-R3 gene, normally expressed in proliferative chondrocyte columns, have elongated columns and longer bones than normal [35,36]. Humans with constitutively active FGF-R3, due to point mutations such as a glycine-toarginine mutation at residue 380 in the receptor's transmembrane domain, have a form of short-limbed dwarfism called achondroplasia. Both milder and more severe chondrodysplasias are caused by other activating mutations of FGF-R3. Corresponding transgenic animal models have helped clarify the mechanisms whereby activation of FGF-R3 leads to dwarfism. Activation of FGF-R3 in humans and mice or overexpression of FGF-2 in transgenic mice lead to a decrease in chondrocyte proliferation in the growth plate and increased apoptosis [32]. This phenotype is paradoxical since activation of FGF receptor tyrosine kinases typically leads to cellular proliferation. Substantial evidence, however, suggests that FGF-R3 can activate STAT1 in chondrocytes and thereby decrease chondrocyte proliferation. FGF-R3 activation leads to activation of STAT1, and the decrease in chondrocyte proliferation caused by FGF signaling in vivo and in vitro is blocked in chondrocytes from STAT1 -/- mice [37,38]. In addition to providing a molecular explanation for the paradoxical actions of FGF signaling in chondrocytes, these findings argue that the decrease in proliferation is a direct consequence of FGF-R3 activation in chondrocytes. In addition, in transgenic mice, activation of FGF-R3 leads to a decrease in expression of Indian hedgehog (Ihh). Since Ihh stimulates chondrocyte proliferation, this provides a further, indirect mechanism for the decrease in chondrocyte proliferation after activation of FGF-R3. Due to the plethora of FGFs expressed in bone and the many actions of FGFs early in development, it is difficult to identify the specific FGF ligands that normally activate FGF-R3 in the growth plate. In this context, the observation that mice missing FGF-18 have a growth plate phenotype that closely resembles that of the FGF-R3 knockout mouse is somewhat surprising
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[39,40]. This observation suggests that FGF-18, synthesized in the perichondrium surrounding the growth plate, is the primary activator of FGF-R3 in chondrocytes. In the growth plate, the role of FGF-R2, expressed in the perichondrium and in osteoblasts, has been difficult to discern. The FGF-R2 knockout mouse dies in the periimplantation period [41], and chimeras in which FGF-R2 is missing from most nonplacental tissues demonstrate the essential role of FGF-R2 in initial limb outgrowth [42]. These early actions of FGF-R2 have limited analysis of the role of FGF-R2 after formation of the growth plate, however. The recent preliminary description of the conditional knockout of FGF-R2 from bone, using the Cre-Lox approach, makes such an analysis possible [43]. These mice are dwarfed, with a decrease in BrdU incorporation in proliferative chondrocytes and a smaller than normal hypertrophic zone. Presumably, the FGF-R2 in the perichondrium receives a signal that, in turn, triggers regulation of adjacent growth plate chondrocytes to increase their proliferation. The precise relevant ligands for these actions have not been identified, given the multiple FGFs expressed in chondrocytes and perichondrium. FGF-R1 is expressed in prehypertrophic and hypertrophic chondrocytes, although none of the possible functions suggested by its pattern of expression have been demonstrated. Similarly, the roles of the other FGFs expressed in the growth plate remain to be established. FGFs and their receptors have important roles in the growth of intramembranous bones and the bone adjacent to growth plates in endochondral bone formation. In the cranial vault, inappropriately rapid differentiation of osteoblasts at the sutures, when combined with normal or increased cell proliferation, can lead to craniosynostosis, the premature fusion of sutures. This is a serious condition because it does not allow the normal growth of the brain and neural structures. As noted earlier, FGFs and their receptors are expressed in sutures. Activating mutations of FGF-R1-3 in humans can cause craniosynostosis [32,44]. A mouse model of one form of craniosynostosis caused by activating mutation of FGF-R1 has been analyzed [45]. In these mice, increased incorporation of BrdU was found in osteoblasts at the suture, along with acceleration of differentiation of osteoblasts. The activated FGF-R1 caused an increase in Cbfal expression. Since Cbfal is a transcription factor essential for osteoblast differentiation and activates a large number of genes expressed in differentiated osteoblasts [46,47], the increase in cbfal expression is probably a crucial aspect of this phenotype. Humans with an activating mutation in FGF-R2 also have evidence of increased apoptosis of osteoblasts in the prematurely fusing sutures [48]. The ligands most crucial for
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activating FGF receptors in sutures and osteoblasts of the limb are uncertain. However, genetic evidence in mice establishes that both FGF-2 and FGF-18 are important. Mice missing FGF-2 are osteoporotic, with a decrease in osteoblast number, decreased bone formation rate, and, in vitro, a decrease in the number of bone nodules and a decrease in the differentiation of osteoblasts in those nodules [49]. Mice missing FGF-18, in addition to the growth plate abnormalities noted earlier, also have a decrease in osteoblast number in the bones of limbs and a delay in growth of cranial bones [39,40]. Proliferation of preosteoblasts is transiently decreased in cranial sutures, and markers of differentiated osteoblasts, such as osteocalcin and osteopontin, were dramatically decreased. Thus, multiple FGFs and FGF receptors are needed for normal osteoblast production and differentiation.
THYROID HORMONE Children and rodents with hypothyroidism have decreased rates of bone lengthening. Although some of this decrease in growth plate function may be caused by associated decreases in GH levels, GH cannot fully correct abnormalities seen in rats and mice with hypothyroidism. These abnormalities include both shortened proliferative layers of chondrocytes and decreased numbers of hypertrophic chondrocytes [50]. At least some of the effects of thyroid hormone are likely to be direct effects on chondrocytes because both growth plates and isolated chondrocytes respond to T3 in vitro by increasing the conversion of proliferating to hypertrophic chondrocytes [51-53]. Genetically engineered mice missing all transcripts from both the thyroid hormone receptor 0~and b loci have a delay in development of secondary ossification centers, a decrease in the proliferative layer of chondrocytes, and a particularly dramatic decrease in hypertrophic chondrocytes [54]. This phenotype is not as severe as that in mice with congenital absence of thyroid glands, as in mice missing the pax8 gene [55]. These comparisons, as well as comparisons with mice missing selected transcripts from the complicated thyroid hormone receptor loci, suggest that nonligand-binding variants of these receptors function in the absence of thyroid hormone to generate a growth plate phenotype more severe than that in mice missing all transcripts from these loci [54]. Both receptor loci contribute to growth plate function. Some children with thyroid hormone resistance due to dominant negative mutations in the thyroid hormone receptor b gene have short stature, presumably partly through blockade of receptor action in the growth plate [56].
ESTROGEN AND ANDROGEN The effects of sex hormones on growth appear to be very species specific, so lessons from rodents cannot be easily applied to humans. In association with the increase in sex hormones in boys and girls at the time of puberty, linear bone growth accelerates, followed by the disappearance of the growth plate and permanent lack of further growth. Remarkably, several patients with defective estrogen receptor ~ [57] or defective aromatase (the enzyme that converts testosterone to estradiol) [58,59] have been identified. Studies of these patients have clarified certain aspects of the role of sex hormones in human bone growth, although little is known about the regulation of growth plate function at the molecular or even cellular level. Two girls without aromatase presented with signs of androgen excess but lack of a pubertal growth spurt until they were treated with estrogen [60]. Besides cartilage, bone cells and extracellular matrix cells are also required for skeletal growth. More specifically, the osteoblast or bone-forming cell contributes along with the chondrocyte to the longitudinal growth of the skeleton [61]. During skeletal growth, chondrocytes from the growth plate produce multiple spicules of cartilaginous extracellular matrix. These spicules enter the metaphysis, where the osteoblast adheres to them, adding a bony matrix to the cartilage to form mixed spicules. Mixed spicules located at the periphery of a given skeletal element are thought to anchor the metaphysis to the epiphysis. These more peripheral spicules grow in length, and this moves the growth plate centrifugally. This histologic observation suggests a role for the osteoblasts in the control of skeletal growth and has been confirmed by medical observations as well as by the generation and analysis of multiple mouse mutant strains.
OSTEOBLASTS AND BONE GROWTH The best human example of the critical role played by the osteoblasts in the control of longitudinal growth is provided by osteogenesis imperfecta (OI) patients. OI is a genetic disease characterized by synthesis and secretion by the osteoblasts of defective type I collagen molecules [62]. This can be viewed as a near complete arrest of osteoblast function in the most severe forms. Interestingly, all patients with severe forms of OI are shorter than expected given the existence of fractures (P. Byers, personal communication). Similar observations have been made in various mutant mouse strains that are obviously more amenable to a dissection of the cellular
4. Postnatal Bone Growth and molecular events taking place [63]. This critical role that the osteoblasts play in the control of growth illustrates the need to understand their biology during childhood and, by extension, the biology of the cells with which they interact during growth and remodeling, namely the osteoclasts. Indeed, bone, like many other organs in the body, goes through a tremendous amount of destruction and growth during childhood. Specific to bone, however, is that it is the only tissue or organ that contains a cell type (i.e., the osteoclast) whose only function is to destroy the organ hosting it. Thus, the interplay between osteoblasts and osteoclasts is particularly important. We review the current knowledge of the control of osteoblast and osteoclast differentiation and function. G e n e t i c Control of O s t e o b l a s t Differentiation a n d Function Like the chondrocyte, the osteoblast is a cell type of mesenchymal origin; however, chondrocytes may have multiple phenotypes and genotypes. Osteoblasts can be defined by several molecular criteria as those cells that express the transcription factor Cbfal, also called Runx2, and two structural proteins--~(I) collagen and osteocalcin. Transcription factors, as well as extracellular signaling molecules, have been shown to control various aspects of osteoblast biology; their numbers and variety have increased substantially in the past few years. Transcriptional Control of O s t e o b l a s t Differentiation a n d Function
Cbfal is the earliest and most specific marker of osteoblast differentiation. Functional studies have shown that it is a central regulator of osteoblast differentiation and function. The names given to this gene have changed several times since it was isolated. It was originally called PEBP2al, then AML3, Cbfal, and now Runx2 [64-66]. Cbfal is one of the mammalian homologs of the runt Drosophila transcription factor [67]. Three different lines of evidence indicate that Cbfal plays a dominant role during osteoblast differentiation, a function apparently not redundant with the function of any other gene. Following a molecular biology approach initially using the osteocalcin promoter, the only known osteoblastspecific gene, as a tool, Cbfal was identified as the factor binding to an osteoblast-specific cis-acting element present in the promoter of most genes expressed in osteoblasts [68-72]. Its expression during development and after birth is highly osteoblast specific and regulated by the bone morphogenetic proteins and other growth factors [73]. Lastly, and more important, its ectopic expression in nonosteoblastic cells leads to the expression
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of osteoblast-specific genes in vitro and/n vivo [73]. Cbfal has been demonstrated to function as an osteoblast differentiation factor. Mouse and human genetic experiments demonstrated that this is its only function
in vivo. In a genetic approach, deletion of Cbfal in mice led to mutant animals in which the skeleton was made with chondrocytes producing cartilaginous matrix without any osteoblast, indicating that Cbfal is necessary for osteoblast differentiation [74,75]. Cbfal is also sufficient for osteoblast differentiation. This was proven by ectopic expression in primary fibroblast in vitro and in nonhypertrophic chondrocytes in vivo [76]. One study in which Cbfal deletion was performed examined Cbfa+/- mice and determined that they had hypoplastic clavicles and a delay in the structure of the fontannelles, two bone structures formed through intramembranous ossification [75]. These abnormalities are identical to those observed in a classical mouse mutation called cleidocranial dysplasia (CCD) [77]. Cbfal maps at the same location as does CCD and the two mutations are allelic [75]. The third line of evidence demonstrating the role of Cbfal in osteoblast differentiation derives from human genetic studies. Two groups, one searching for a gene causing human CCD and one searching for a disease in which Cbfal is mutated, simultaneously identified deletions, stop codon insertions, and missense mutations in CBFA1 in patients affected with CCD [78,79]. These different lines of investigation can be summarized as follows: Cbfal is expressed specifically in osteoblast progenitors, osteoblast, and, to a lesser extent, prehypertrophic chondrocytes; it is sufficient and necessary for osteoblast differentiation; and its function is dominant during osteoblast differentiation. It is not known what turns on Cbfal expression in vivo. Although BMPs can induce its expression in vitro, the time course needed for this induction is too long to be a direct effect and other signaling molecules must be involved. Moreover, there is no evidence that the BMPs control the late stage of skeletogenesis (i.e., osteoblast differentiation). Cbfal function in osteoblasts is not limited to cell differentiation during embryonic development. At least one in vivo study has shown that, in addition to development, Cbfal regulates the level of bone matrix deposited by previously differentiated osteoblasts [80]. Experiments are under way to demonstrate this second function of Cbfal, but one implication of this finding is that increasing the level of transcription of Cbfal in differentiated osteoblasts could be a treatment for osteoporosis. Several important aspects of Cbfal biology remain to be elucidated. Chief among these is whether or not Cbfal directly or indirectly controls the expression of other osteoblast-specific transcription factors. Other osteoblast-specific cis-acting elements have been described in
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various genes that are expressed exclusively or mostly in osteoblasts, suggesting that other osteoblast-specific transcription factors have yet to be identified. Where these transcription factors lie in the pathway leading to osteoblast differentiation is unknown. Control of O s t e o b l a s t Differentiation by Secreted Molecules Indian hedgehog (Ihh) is one member of the hedgehog family of growth factors that is expressed in developing skeleton [81]. Misexpression experiments in chick first identified Ihh as a regulator of chondrocyte differentiation [82]. Ihh-deficient mice that survive to birth have a disorganized growth plate, as expected; however, they also have no osteoblasts in bone formed through endochondral ossification, but these appear to be present in the skull, the mandibles, and the clavicles~bones that form by intramembranous ossification [83]. It is not known whether this is a direct consequence of the absence oflhh signaling or whether it is an indirect effect. It is also unknown if this is simply a delay in osteoblast generation or an irreversible arrest of differentiation because Ihhdeficient mice die at birth. It seems clear, however, that the failure of osteoblast differentiation in bones forming through endochondral ossification may not be a consequence of the defect in chondrocyte differentiation. Transgenic mice overexpressing hedgehog-interacting protein (HIP) in choncrocytes have also been generated. HIP is a cell surface protein that binds Ihh with the same affinity as patched and antagonizes Ihh signaling [84]. Transgenic mice that overexpress HIP in chondrocytes show a chondrocyte phenotype similar to that of Ihhdeficient mice but have a large number of osteoblasts. The families of growth factors that have received a tremendous amount of attention are the BMPs. These are members of the transforming growth factor-b (TGF-b) superfamily of growth factors that can, when applied locally, induce de novo bone formation by recapitulating all the events that occur during skeleton development. The analysis of their functions in vivo has relied mostly on gene deletion experiments [85]. Although several BMPs affect skeletal patterning and joint formation, it has not been possible using this approach to show that they affect osteoblast differentiation. The best evidence that BMPs control osteoblast differentiation is indirect. Deletion of Tob, a gene encoding an antiproliferative protein, results in higher bone mass due to increased osteoblast numbers. Tob, which can associate with Smadl, -5, and-8, can repress BMP-2-induced, Smadmediated gene expression. BMP-2-induced bone formation is increased in Tob-deficient mice [86]. Interestingly, one of the BMPs, BMP-1, does not belong to the TGF-b superfamily but rather has homology to Tolloid, a gene
thought to be a protease in Drosophila based on genetic data [87]. In mouse, Bmp-1 has been demonstrated to encode a protease capable of releasing the C-terminal propeptide from the type I procollagen molecule, the most abundant protein of the bone extracellular matrix. Deletion of Bmp-1 has revealed the existence of other Bmp-l-like or Tolloid-like proteins that may also be involved in type I collagen processing [88,89]. TGF-b plays a complex role in bone remodeling. In vitro, TGF-b induces extracellular matrix synthesis by osteoblasts and affects osteoblast differentiation. In vivo, Erlebracher and Derynck [90] used a local overexpression strategy to study TGF-b function. Osteocalcin TGF-b2 transgenic mice developed a complex low bone mass phenotype characterized by an overall increase in bone resorption, a large increase of osteocyte numbers, and the presence of a hypomineralized matrix [90]. Additional mouse models overexpressing dominant negative forms of TGF-b receptors in osteoblasts or affecting the level of expression of TGF-b binding proteins will allow further determination of the role of TGF-b in osteoblast function [91]. Recently, based on genetic evidence another family of growth factors has been proposed to contribute to the control of osteoblast differentiation and proliferation the Wnt proteins [92]. Indeed, it was recently shown that the same gene is mutated in osteoporosis-pseudoglioma syndrome, an autosomal recessive form of osteoporosis [93], and in a familial form of high bone mass [94]. This gene encodes LDL receptor-related protein-5 (LRP-5). The former disease is due to the loss-of-function mutations, whereas the latter is caused by a gain-of-function mutation. Both diseases manifest after birth, indicating that the gene mutated and the pathway affected are active after birth. Lrp-5 is a vertebrate homolog of the Drosophila gene arrow, which is a coreceptor for wingless [95]. Lrp-5 acts as a corecepetor for the Wnt proteins in a cell culture assay. This finding, which is currently being investigated at the molecular level, raises the following question: Are the Wnt proteins controlling osteoblast differentiation and proliferation using as a mediator Lef/Tcf transcription factors and/or do they act in a Cbfal-dependent manner? Regardless of the answer, it seems likely, given the postnatal nature of these two phenotypes, that some Wnt proteins control osteoblast function and proliferation.
ENDOCRINE REGULATION OF BONE FORMATION The fact that bone remodeling occurs simultaneously in multiple skeletal locations is generally viewed as
4. Postnatal Bone Growth
evidence that it is controlled locally, through autocrine and/or paracrine mechanisms [96]. However, this observation is also consistent with the possibility that bone remodeling is under endocrine control. This would not be surprising since most other homeostatic functions are known to be under endocrine, if notneuroendocrine, control. Accordingly, several hormones are known to control bone remodeling. For instance, sex steroid hormones such as estradiol are clearly involved in the control of bone remodeling by affecting osteoclast differentiation and thereby bone resorption. As a result, the declining level of sex steroid hormones at menopause is a predisposing factor for osteoporosis [96,97]. Likewise, parathyroid hormone also favors bone resorption [98,99]. The hormones mentioned previously act on bone resorption in physiological conditions. This raises the following question: Is bone formation also under endocrine control? Without presaging on which aspect of bone remodeling they may act, two well-known clinical observations suggest that other hormones besides sex steroids and PTH regulate bone remodeling. The first observation is that obesity protects from bone loss [40], and the second is that menopause favors bone loss. Together, they suggest that bone mass, body weight, and reproduction are regulated by the same hormone(s). Leptin as a Regulator of Bone Formation Leptin is an attractive candidate for such a hormone. Leptin is synthesized by adipocytes and functions as a starvation and adiposity signal through its binding to the long form of its receptor located primarily in the hypothalamus [100,101]. Rodents and humans genetically deficient in leptin signaling are massively obese. The absence of leptin signaling also causes sterility (i.e., hypogonadism), a condition usually causing bone loss. Surprisingly, mice deficient in leptin (ob/ob) or its receptor (db/db) were found to have a two- or threefold higher bone mass than wild-type mice [102]. The ob/ob and db/ db mice are the only known animal models with both hypogonadism and high bone mass; therefore, they are invaluable resources for studying the molecular basis of bone remodeling. Bone histomorphometry analysis performed before and after correction of the hypogonadism of ob/ob mice showed that leptin inhibits bone formation through its action on osteoblasts, whereas it has no overt effect on osteoclast differentiation and function. Importantly, leptin action on bone formation did not involve osteoblast differentiation because ob/ob and db/db mice have a normal number of osteoblasts. This latter observation indicates that any local mode of action of leptin on bone that exists must affect previously differentiated osteoblasts and not their progenitors. The leptin receptor
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does not appear to be expressed on differentiated osteoblasts, and in cell culture studies osteoblasts from db/db mice behave like wild-type osteoblasts. These data indicate that leptin does not directly target osteoblasts. Moreover, intracerebroventricular (ICV) infusion of leptin in ob/ob mice completely rescues their bone phenotype. The fact that this occurs without any circulating leptin in the serum of these animals demonstrates that bone formation, and therefore bone remodeling, is centrally controlled probably via the hypothalamus (Fig. 3). ICV infusion of leptin in wild-type mice causes bone loss, indicating that this regulatory loop has physiological relevance in wild-type animals [102]. By definition, if bone remodeling is a physiologic process with a hypothalamic or central component, then diseases of bone remodeling such as osteoporosis may also have a hypothalamic or central component. As is the case for the control of body weight, the role of leptin in bone formation is not merely a "mouse story." Rats deficient in leptin signaling also have a high bone mass phenotype (M. Amling and G. Karsenty, unpublished observation). Patients with generalized lipodystrophy, a condition marked by a near complete absence of adipocytes and white fat, also exhibit osteosclerosis (increased bone formation) and accelerated bone growth [103]. It is difficult to assess whether leptin-deficient patients do or do not have a high bone mass because these patients are treated with leptin early. It is known from work on ob/ob and db/db mice that the high bone mass phenotype becomes more severe as the animals age. Finally, leptin's role in bone formation may help explain why obese individuals, who are often leptin resistant [104], are protected from bone loss. These results also demonstrate the importance of leptin by showing that, like other major hormones such as thyroid hormones or cortisol, it has multiple target organs and functions (bone mass, body weight, and reproduction) without a necessary hierarchy between them. However, leptin is unlikely to be the sole central regulator of bone formation. Other undiscovered centrally acting hormones may positively or negatively regulate bone formation and possibly other aspects of bone physiology. Transcription Control of O s t e o c l a s t Differentiation a n d Function A recurrent theme in bone biology is that many of the genes identified as regulators of cell differentiation or function in the osteoclast and osteoblast lineage have been known for a long time. However, possibly because bone cells are more difficult to isolate, or possibly because skeletal development initially appeared less attractive than other organogenesis processes, they were not
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originally studied in the context of the skeleton.
Pu-1 function in osteoclast differentiation is one of the best examples. Pu-1 is an ETS domain-containing transcription factor that is expressed specifically in the monocytic and B lymphoid lineages [105]. Deletion of Pu-1 results in a multilineage defect in the generation of progenitors for B and T lymphocytes, monocytes, and granulocytes. The fact that Pu-1 was thought to regulate transcription of c-fms, which plays an important role in osteoclast biology, led Tondravi et al. [106] to explore the possibility that Pu-1 might also control osteoclast development. As hypothesized by this group, Pu-l-deficient mice exhibit a classical osteopetrosis phenotype. There are no osteoclasts and no macrophages in the bone marrow of Pu-l-deficient mice. This is a cell autonomous defect that can be corrected by bone marrow transplantation [106]. Pu-1 is the earliest marker of osteoclast differentiation because it controls both macrophage and osteoclast differentiation. However, no cell type decides in an autonomous manner to differentiate along a particular lineage; thus, one of the challenges in the field is to identify the extracellular signals that induce the expression of PU-1 and of other cell-specific transcription factors involved in osteoclast differentiation. Another transcription factor that plays a critical role during osteoclast differentiation is c-fos [107], which is the cellular homolog of the v-fos oncogene and a major component of the AP-1 transcription factor. The first indication that c-fos might play a role in bone cell differentiation came from the observation that v-fos-containing constructs injected into rodents led to the appearance of osteosarcomas (malignant tumors of the osteoblast) [Ward, 1976 #352]. Likewise, transgenic mice expressing high levels of c-fos in multiple tissues and cell types eventually developed only one type of tumor---vhondroblastic osteosarcoma--and cloned cell lines derived from these transgenic mice have altered osteoblastic gene expression. Given the results of these studies, it came as a surprise that the deletion of c-fos in mice led to an early arrest of osteoclast differentiation without any overt consequences on osteoblast differentiation. As a result of this block in osteoclast differentiation, the main phenotype of c-fosdeficient mice is osteopetrosis [108,109]. The presence of a large number of macrophages in c-fos-deficient mice places c-Fos downstream of Pu-1 in the genetic pathway controlling osteoclast differentiation. The osteopetrotic phenotype of the c-fos-deficient mice was rescued by bone marrow transplantation and by expression of a c-fos transgene [110]. Although other c-fos-related proteins are expressed in osteoclasts, c-fos fulfills a unique function because no other proteins can substitute in vivo for its loss of function. However, c-fos may not be the only member of thefos gene family to contribute to osteoclast differen-
tiation because the fos-related protein Fra-1 can favor osteoclast differentiation in osteoclast macrophage precursor cell lines. A third transcription factor, NF-•B, appears to play a role early during osteoclast differentiation. NF-~:B is formed as a dimer composed of various combinations of proteins: p50, p52, p65, c-Rel, and RelB [111]. These proteins are all related by the Rel homology domain that contains the DNA-binding motif. Through genetic analysis in mice, it was shown that each of these proteins plays an essential and unique role in immune response, but no defects in bone cell differentiation were observed [112,113]. In contrast, mice deficient in both p50 and p52 harbor an osteopetrosis phenotype due to an arrest of osteoclast differentiation. These findings are also important because NF-KB seems to be one of the targets of recently identified growth factors that regulate osteoclast differentiation. The last transcription factor that plays a role in osteoclast differentiation was identified by searching the gene mutated in the mouse mutant microphtalmia (mi). mi mice have the following defects: loss of pigmentation, reduced eye size, and failure of secondary bone resorption [114]. The gene mutated in these mice encodes a bHLH transcription factor called mi [115]. In mi mice, osteoclasts differentiate normally but they fail to resorb bones, thus placing mi downstream of the transcription factors mentioned previously (Fig. 1). The wealth of transcription factors controlling osteoclast differentiation contrasting with the apparent paucity of known osteoclast-specific factors was unexpected. It suggests that many of the genes involved in bone resorption and expressed specifically in osteoclasts have not been identified. Another observation common to most of these osteopetrotic mutants is that although bone resorption was arrested, bone formation was not halted; otherwise, the osteopetrosis phenotype would not exist. In some cases, bone formation was even accelerated--a genetic indication that these two functions may be regulated by different growth factors or hormones
in vivo. Control of O s t e o c l a s t Differentiation a n d Function by S e c r e t e d M o l e c u l e s The requirement for secreted molecules to control osteoclast differentiation was first documented with the genetic elucidation of a classical mouse mutation called op/op [116]. Mice homozygous for this recessive mutation lack osteoclasts and macrophages. The osteopetrotic phenotype of op/op mice is not cured by bone marrow transplantation, indicating that it is non-cell-autonomous defect [116]. The gene mutated in op/op mice encodes the growth factor macrophage colony-stimulating factor
4. Postnatal Bone Growth
(m-CSF), a gene thought to be regulated by PU-1 [117]. Forced expression of Bcl-2, a gene that prevents apoptosis in monocytes, can partially reverse osteopetrosis of the op/op mice, suggesting that the function of m-CSF may be to favor survival of osteoclast progenitors [118]. In the past 3 years, a group of secreted molecules regulating positive or negative osteoclast differentiation have been identified. The genetic and molecular analysis of these molecules has profoundly modified our understanding of bone resorption. In a large genomic screen for novel secreted molecules, a group at Amgenidentified a novel member of the TNF receptor superfamily [119]. This novel molecule contains no hydrophobic transmembrane-spanning sequence, indicating that it is a soluble receptor. Overexpression in transgenic mice of this molecule, called osteoprotegerin (OPG), resulted in severe osteopetrosis due to an arrest of osteoclast differentiation. This molecule is identical to osteoclastogenesis inhibitory factor (OCIF), which was purified and subsequently cloned by another group at Snowbrand Pharmaceutical in Japan using a different approach [120]. The identification and the functional study of OPG/OCIF are very important for another reason. They showed that, in addition to steroid and peptide hormones, there are novel secreted molecules that can act systematically, not in a paracrine/autocrine fashion, to control osteoclast differentiation. The specificity of the function of OPG/OCIF in inhibiting osteoclast differentiation was demonstrated by the phenotype of OPG/OCIF-deficient mice. These mice develop an osteoporosis due to an increase in osteoclasts number [121,122]. The identification of a soluble receptor with such a powerful inhibitory effect on osteoclast differentiation suggested that it may be titering out an osteoclast differentiation activity. This factor was cloned by the same two groups at approximately the same time. The Amgen group used recombinant OPG/OCIF to screen for OPG/ OCIF ligand on the surface of various cell lines. They replaced the criteria of absolute novelty with the criteria of specificity of interaction with OPG. The protein they isolated had previously been cloned and called TRANCE or R A N K ligand (RANKL). RANKL is present on the membrane of the osteoblast progenitor but also as a soluble molecule in bone microenvironment. In vitro, RANKL/osteoclast differentiation factor (ODF) has all the attributes of a real osteoclast differentiation factor: It favors osteoclast differentiation in conjunction with m-CSF, it bypasses the need for stromal cells and 1,25(OH)2 vitamin D3 to induce osteoclast differentiation, and it activates mature osteoclasts to resorb mineralized bone [123]. Consistent with this cell and molecular biology data, RANKL-deficient mice lack osteoclasts and develop a severe osteopetrosis phenotype
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in addition to an immunological phenotype. At the same time, Yasuda et al. [120], pursuing their systematic biochemistry and molecular biology effort, purified to homogeneity the ODF and showed that it was the R A N K ligand [120]. RANKL/ODF is also expressed and synthesized by T cells. Conceivably, the production and secretion of RANKL/ODF systematically or locally by T cells may explain some of the bone abnormalities observed in autoimmune disorders. RANKL binds to RANK because of a cellular receptor present on T cells and bone marrow stromal cells [124,125]. Transgenic mice expressing a soluble form of R A N K develop an osteopetrosis similar to the one observed in OPGL/ODF-deficient mice and polyclonal antibody against R A N K extracellular domain promotes osteoclastogenesis in bone marrow culture, suggesting that R A N K activation mediates the effect of OPGL. What signal transduction pathway is initiated following binding of RANKL to RANK? R A N K intracellular domain contains two binding sites for members of a family of proteins called TNF receptor-associated factors (TRAFs) [126]. TRAFs have been implicated in mediating signals induced by a subset of TNF receptor family members. R A N K contains a binding site for TRAF6 in the middle of its intracellular region [127]. Importantly, TRAF6-deficient mice exhibit an osteopetrotic phenotype, thus providing the beginning of a signal transduction cascade leading to osteoclast differentiation [128]. This observation is also important because TRAFs seem to control the activation of NF-•B, a transcription factor required for osteoclast differentiation. Given all the information available, it is now possible to summarize the role of each component of this signal transduction pathway (Figs. 2 and 3). Other cytokines, such as interleukin-1 (IL-1), IL-2, IL-6, and oncostatin M,, all transduce their signals through gp 130 and can induce osteoclast differentiation in vitro. Their role in vivo has been difficult to establish, possibly because of functional redundancy; nevertheless, unpublished observations from several laboratories indicate that signal transduction through the gp 130 protein plays an important role in osteoclast and osteoblast physiology [129]. Another regulatory gene whose functional skeletal identity was revealed by mouse genetics in c-src, the cellular homolog of the v-src oncogene, which belongs to a large family of tyrosine kinases, c-src is ubiquitously expressed, although its highest level of expression is in platelet and neurons [130]. However, its deletion through gene targeting led to an osteopetrotic phenotype [131]. In c-src-deficient mice, osteoclasts are present but fail to resorb mineralized bone. The mechanisms by which c-src genes control osteoclast function have been and remain the object of multiple investigations. In 1997,
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Schwarzberg et al. used a transgenic approach that showed that rescue of the phenotype was not dependent of the kinase domain. In other lines of investigation, Tanaka et al. identified c-cbl as a downstream gene in a signaling pathway required for bone resorption. In 1998, Duong et al. presented evidence suggesting that phosphorylation of Pyk2, a cytoplasmic kinase activated by Src, is required for osteoclastic bone resorption. The existence of an osteopetrotic phenotype in Pyk2-deficient mice lends further credence to this finding. The list of regulatory genes controlling osteoclast differentiation and function is already surprisingly long, but it may not be complete because there are two mouse mutations causing osteopetrosis for which the mutated gene has still not been identified: oc/oc and gray lethal [132].
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118. Lagasse, E., and Weissman, I. L. (1997). Enforced expression of Bcl-2 in monocytes rescues macrophages and partially reverses osteopetrosis in op/op mice. Cell 89, 1021-1031. 119. Simonet, S., Lacey, D. L., Dunstan, C. R., Kelley, M., Chang, M. S., Luthy, R., et al. (1997). Osteoprotegerin: A novel secreted protein involved in the regulation of bone density. Cell 89, 309-319. 120. Yasuda, H., Shima, N., Nakagawa, N., Mochizuki, S. I., Yano, K., Fujise, N., et al. (1998). Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): A mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 139, 442-444. 121. Bucay, N., Sarosi, I., Dunstan, C. R., Morony, S., Tarpley, J., Capparelli, C., et al. (1998). Osteoprotegrin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 12, 1260-1268. 122. Mizuno, A., Amizuka, N., Irie, K., Murakami, A., Fujise, N., Kanno, T., et al. (1998). Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin. Biochem. Biophys. Res. Commun. 247, 610-615. 123. Burgess, T. L., Qian, Y., Kaufman, S., Ring, B. D., Van, G., Capparelli, C., et al. (1999). The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J. Cell Biol. 145, 527-538. 124. Dougall, W. C., Mglaccum, M., Charrier, K., Rohrbach, K., Brasel, K., De Smedt, T., et al. (1999). RANK is essential for osteoclast and lymph node development. Genes Dev. 13, 2412-2424. 125. Anderson, D. M., Maraskovsky, E., Billingsley, W. L., Dougall, W. C., Tometsko, M. E., Roux, E. R., et al. (1997). A homologue of the TNF receptor and it ligand enhance T-cell growth and dendritic-cell function. Nature 390, 175-179. 126. Wong, B. R., Josien, R., Lee, S. Y., Vologodskaia, M., Steinman, R. M., and Choi, Y. (1998). The TRAF family of signal transducers mediates NF-kB activation by the TRANCE receptor. J. Biol. Chem. 273, 28355-28359. 127. Darnay, B. J., Ni, J., Moore, P. A., and Aggarwal, B. B. (1999). Activation of NF-~:B by RANK requires tumor necrosis factor receptor-associated factor (TRAF) 6 and NF-~cB inducing kinase. Identification of a novel TRAF6 interaction motif. J. Biol. Chem. 274, 7724-7731. 128. Lomaga, M. A., Yeh, W. C., Sarosi, I., et al. (1999). TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev. 13, 1015-1024. 129. Kawasaki, K., Gao, H., Yokose, S., Kaji, Y., Nakamura, T., Suda, T., et al. (1997). Osteoclasts are present in gp 130-deficient mice. Endocrinology 138, 4959-4965. 130. Brugge, J., Cotton, P., Lustig, A., Yonemoto, W., Lipsich, L., Coussens, P., et al. (1987). Characterization of the altered form of the c-src gene product in neuronal cells. Genes Dev. 1, 287-296. 131. Boyce, B., Yoneda, T., Lowe, C., Soriano, P., and Mundy, G. R. (1992). Requirement of pp60c-src expression for osteoclasts to form ruffled borders and resorb bone in mice. J. Clin. Invest. 90, 1622-1627. 132. Suda, T., Udagawa, N., and Takahashi, N. (1996). Cells of bone: Osteoclast generation. In Principles o f Bone Biology (J. P. Bilezikian, L. G. Raisz, and G. A. Rodan, Eds.), pp. 87-102. Academic Press, San Diego.
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5 Parathyroid Hormone and Calcium Homeostasis GORDON J. STREWLER Walter Bradford Cannon Society, Harvard Medical School, Boston, Massachusetts
CELLULAR AND EXTRACELLULAR CALCIUM HOMEOSTASIS
where the concentration of Ca 2+ is approximately 10,000fold higher than that in the cytoplasm (Table 1) [2]. One paradigm couples receptors on the cell surface to release of Ca 2+ from the endoplasmic reticulum. For example, engagement of G protein-coupled receptors by their ligands activates phospholipase C[3; engagement of tyrosine kinase receptors by their ligands similarly activates phospholipase C~/. Activated phospholipase C generates the intracellular second messenger inositol1,4,5-trisphosphate [Ins(1,4,5)P3], which diffuses to the endoplasmic reticulum, activates the Ins(1,4,5)P3 receptor (also an ion channel), and thereby releases large amounts of calcium to generate a calcium transient (Fig. 1). In a second paradigm for calcium signaling, extracellular signals direct the influx of Ca 2+ through ion channels in the plasma membrane. Signals can operate plasma membrane ion channels in three ways. First, depolarization of a cell can open voltage-operated calcium channels (e.g., on excitable muscle or nerve cells). Second, receptors such as the N-methyl-D-aspartate receptor can open calcium channels. Third, second messengers can open calcium channels. Typically, these calcium signals are transient, and mechanisms have evolved to govern the offset of calcium signals by reuptake of calcium into intracellular stores and the repletion of intracellular calcium stores from the extracellular compartment [2].
Intracellular Calcium H o m e o s t a s i s The divalent cation calcium serves an essential role in the physiology of virtually all living things. A full description of the diversity of the intracellular metabolism and functions of calcium is beyond the scope of this chapter, but calcium is a universal intracellular messenger that is utilized by virtually every cell [2]. Many enzymes and ion channels are calcium sensitive, and an impressive array of processes are regulated by changes in intracellular calcium via these proteins. These processes include muscle contraction; secretion of proteins, catecholamines, and other vesicle contents; membrane excitability; the intracellular metabolism of nutrients; cellular proliferation; and cell death (Fig. 1). Most cells maintain the baseline level of cytoplasmic ionized calcium (Ca 2+) at approximately 100 nM (Table 1) and generate calcium signals by raising the concentration of Ca 2+, or [Ca2+], in cytoplasm transiently to 500-1000nM. Cells can generate a calcium transient either by release of calcium from intracellular stores or by influx of calcium from the extracellular space,
TABLE 1
C a l c i u m c o n c e n t r a t i o n s in b o d y fluids.
Extracellular Calcium H o m e o s t a s i s
8.5-10.5 mg/dl
2.1-2.6 mmol/1
Total and Ionized Calcium
Ionized serum calcium
4.4-5.2 mg/dl
1.1-1.3 mmol/1
Protein-bound calcium
4.0-4.6 mg/dl
0.9-1.1 mmol/1
The level of extracellular calcium is significant for a variety of processes. Intracellular functions of calcium such as automaticity of nerve and muscle, contraction of muscle, release of neurotransmitters, and secretion of endocrine and exocrine factors are dependent on the
Total serum calcium
Complexed calcium Intracellular free calcium
Pediatric Bone
0.7 mg/dl 0.00018 mmol/1
0.18 mmol/1 180 nmol/1
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Copyright2003,ElsevierScience(USA). All rights reserved.
-
Contraction
CAM-
MLCK
CAMKS Calcinounin Transcription factors
Proliferation Fert~lization Learnlng and rnernor;;
pathways s~gnaling Ins(l.4,5)P3 3-Kinase Ion channels 1 9 Membrane excitab~l~ty Synaptotagmin Phosphorylase kinase Annexin family
S100 family
---
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Vesicle trafficking Cell proliferatton
Cell proliferation cancer and mefastas~s
ATP synthesis Stero~dsvntheeis
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Apoptasis
FIGURE 1 Intracellular calcium signaling. Extracellular signals can activate receptor tyrosine kinases (RTK), G protein-coupled receptors, or cell depolarization (AV). These signals can operate plasma membrane calcium channels or generate Ins(1,4,5)P3 or other second messengers to release calcium from internal stores in the endoplasmic or sarcoplasmic reticulum (ERISR). Proteins that are targets for calcium signals and the calciumsensitive processes they mediate are shown on the right [redrawn with permission from Berridge, M. J., Lipp, P., and Bootman, M. D. (2000). The versatility and universality of calcium signaling. Nature Rev. Mol. Cell. Biol. 1 , 11-21].
5. Parathyroid Hormone and Calcium Homeostasis stability of the extracellular calcium concentration. In addition, cell and platelet adhesion proteins, such as integrins and cadherins, and extracellular enzymes, such as the proteases of the clotting cascade, are calcium dependent. Finally, the mineralization of bone is dependent on maintenance of normal levels of extracellular calcium and phosphate. Because the maintenance of a normal level of extracellular calcium is essential to so many processes, the calcium level is closely regulated by a set of hormones that include parathyroid hormone, vitamin D, and calcitonin. This chapter concentrates on parathyroid hormone (PTH). Although the physiology of vitamin D and calcitonin is the subject of subsequent chapters, their impact on calcium metabolism is briefly discussed. The fraction of extracellular calcium that is active in signaling is the ionized fraction, Ca 2+. In serum, the ionized calcium concentration averages 1.25 + 0.07 m M (Table 1). Only approximately 50% of the total calcium in serum is present in the ionized form. The remainder is either bound to albumin and other proteins (approximately 40%) or complexed with anions such as phosphate or citrate (approximately 10%). The proteinbound and complexed fractions of serum calcium are metabolically inert and are not regulated by hormones. For convenience, however, the total serum calcium level is routinely measured. It is thus important to appreciate that changes in the level of serum albumin, pH, or anions such as citrate or phosphate can substantially affect the total serum calcium level without affecting the biologically active fraction, [Ca2+]. If the albumin is abnormal, the ionized calcium level can be measured directly or estimated. In most clinical settings, it is adequate to correct the measured total serum calcium for changes in albumin. This is done using the following formula: Corrected serum calcium = measured serum calcium + 0.8(4 - measured serum albumin) Thus, in a patient with a serum calcium of 7.8 mg/dl and a serum albumin of 2.0 g/dl, the corrected serum calcium is 7.8 + (0.8)(4 - 2) - 9.4 mg/dl. The corrected serum calcium is in the midnormal range; it is therefore likely that the reduction in measured serum calcium is wholly attributable to hypoalbuminemia. In modern laboratories the determination of ionized calcium uses a calcium-sensitive electrode that is often incorporated in the apparatus used to measure blood gases and pH [3]. Such direct measurements are done manually. They are expensive and are more difficult to quality control than routine determinations of total serum calcium. The ionized calcium concentration will change with pH, and depending on the method, the sample either must be collected anaerobically so that
13 7
the pH of the sample does not change as CO2 escapes or the ionized calcium must be back-calculated to the physiological pH. Changes in the concentration of phosphate or citrate can affect both the total and the ionized serum calcium. For example, massive transfusion of citrated blood (in which citrate is used as an anticoagulant because of its ability to complex calcium and thus paralyze the clotting cascade) can increase the total serum calcium by complexing it while at the same time inducing tetany because of acute complexation of ionized calcium. Here, the challenge to homeostasis is too massive and acute for a successful response. In this circumstance, determination of the ionized calcium concentration is necessary to guide therapy. The product of the concentrations of Ca 2+ and phosphate in extracellular fluid (e.g., [CaZ+][Pi]) is similar to the solubility product of calcium phosphate so that a substantial acute increase in the serum concentration of either can lead to the precipitation of calcium phosphate salts in soft tissues, and this is a source of clinical problems in severe hypercalcemia or hyperphosphatemia. Tetany is most often seen in rhabdomyolysis, where large amounts of phosphate are acutely released from damaged skeletal muscle in the face of severely impaired renal function. Both total calcium and [Ca 2+] are typically low and the serum phosphorus is extremely high in patients with rhabdomyolysis who experience symptoms. Calcium Fluxes and Balance
The extracellular compartment is bounded by three interfaces that are important for systemic calcium homeostasis: the gut, the kidney, and the bone. This chapter first considers the fluxes of calcium across these boundaries in a young adult in a state of zero net calcium balance (Fig. 2), and then calcium balances during skeletal growth are discussed.
FIGURE 2 Externalcalcium balance and calcium fluxes through the extracellular fluid (ECF). Reprinted with permission from Felig, P., Baxter, J. D., Broadus, A. E., Frohman, L. A. (eds.). "Endocrinology and Metabolism, 2nd edition," McGraw-Hill, 1987.
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Gordon i. Strewler
The average diet contains 400-1600mg of calcium (10-40 mmol). From this dietary load, the net absorption in a young healthy adult averages 100-250 mg. Calcium is absorbed throughout the small bowel, with a relative efficiency in animal studies of duodenum > jejunum > ileum. In humans, the average transport rates are similar in jejunum and ileum, but the bulk of calcium is absorbed in the ileum because of the lengthy transit through this long segment of the small intestine. Intestinal transport of calcium is saturable, with an active component that is under the control of vitamin D (see Chapter 7) and a passive component (Fig. 3). These two components can be differentiated in studies of perfused human intestinal segments [4]. The active, saturable component of calcium absorption occurs through the apical membrane of intestinal enterocytes, and calcium is then pumped out of the cell at the basolateral border (see Chapter 7). The passive component of calcium transport is a manifestation of the leakiness of the intestinal epithelium and primarily represents paracellular movement of calcium. The unidirectional flux of calcium from intestinal lumen to the blood is a linear function of the luminal calcium concentration (Fig. 3). The unidirectional backflux of calcium from blood into feces, or endogenous fecal calcium, is largely determined by the level of extracellular calcium and tends to be relatively insensitive to changes in dietary calcium intake. Its magnitude can be determined in calcium kinetic studies in which one calcium isotope is administered orally as a tracer for calcium absorption at the same time a different isotope is administered intravenously as a tracer for the backflux of calcium from blood to intestinal lumen so that unidirectional fluxes can be determined.
,,C
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Calcium kinetic studies were once done with radioactive isotopes of calcium but now are performed with stable isotopes, the concentration of which can be sensitivity to calcium intake, the obligatory loss of endogenous fecal calcium, together with the obligatory loss determined by mass spectrometry. With its relative in of calcium in the urine and sweat, represents the negative side of the external balance equation: If the absorption of dietary calcium is not sufficient to overcome these obligatory losses, net negative calcium balance will ensue. The saturability of calcium absorption (Fig. 3) is important when calcium requirements during growth and adolescence are considered because it effectively defines a threshold of calcium intake below which an increased intake will lead to increased retention in the skeleton and above which further increases in calcium intake will not (Fig. 4). Heaney [5] and others have persuasively argued that the intake corresponding to this point of inflection represents the true minimum daily requirement of calcium, and this reasoning has been used in recent reassessments of recommended calcium intake, such as that of the Food and Nutrition Board of the National Academy of Sciences in 1997 [6]. In addition to its support function, bone is a huge reservoir for calcium and phosphate. More than 99% of body calcium is stored in bone. Bone calcium is in dynamic equilibrium with the extracellular fluid (Fig. 2), with substantial bidirectional fluxes between the extracellular compartment and an exchangeable bone calcium pool that is readily demonstrable by calcium kinetic studies. The human skeleton at birth contains approximately 25 g of calcium, and the mass of calcium in bone will increase to 1000-1200g by adulthood. All of the accretion of calcium to support the growth of bones must come from the diet, so the external balance of calcium must be substantially positive during childhood and adolescence. The net bone balance of calcium will be essentially identical to the external balance since for
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FIGURE 3 Intestinal calcium transport. Reprinted with permission from Felig, P., Baxter, J. D., Broadus, A. E., Frohman, L. A. (eds.). "Endocrinology and Metabolism, 3rd edition," McGraw-Hill, 1995.
FIGURE 4 Thresholdbehavior of calcium intake. Theoretical relationship of bone accumulation to intake. Below a certain value- the threshold (arrow)- bone accumulation is a linear function of intake. Above the threshold (the horizontal line), bone accumulation is limited by other factors and is no longer related to changes in calcium intake. (Copyright Robert P. Heaney, 1992. Reproduced with permission.)
139
5. Parathyroid Hormone and Calcium Homeostasis
practical purposes bone is the only storage site for calcium. These issues are discussed in detail in Chapter 9. The renal handling of calcium involves filtration at the glomerulus and reabsorption in proximal and distal nephron segments [7]. Ionized and complexed calcium is freely filterable; the filtered load of calcium approximates 8 g/day, and 98 or 99% is reabsorbed in the tubule. It was shown by micropuncture that approximately 70% of the filtered Ca 2+ is reabsorbed in the proximal tubule segments, 20% in the thick ascending limb of Henle's loop, 5-10% in the distal tubule, and less than 5% in the collecting duct system. However, it is mainly the distal handling of calcium that is under hormonal control. The renal excretion of calcium is increased markedly during natriuresis, by volume expansion, and in metabolic acidosis. Hypercalcemia generally increases the filtered load of calcium, even though G F R is reduced, reduces tubule reabsorption (by direct effects of hypercalcemia and by suppression of PTH), and usually produces a calciuresis as the net effect. Conversely, the urinary calcium excretion is low in hypocalcemia because of avid reabsorption caused by a marked reduction in the filtered load. However, at normal levels of serum calcium, substantial losses of calcium in the urine of 50-150 mg per day are obligatory, even when the intake of calcium is reduced to zero. Calcium Nutrition in Childhood and Adolescence
Calcium absorption and retention vary markedly during childhood and adolescence, with two major peaks at times of maximal skeletal growth. The first occurs in infancy. Calcium absorption averages 61 + 22% at age 6 months [1] and declines thereafter. A second peak in calcium absorption and retention occurs during puberty [8,9], beginning in the early stages [10] and peaking at 11.4 years in girls and 13.3 years in boys, approximately 1 year after attainment of peak height velocity and coincident with menarche in girls [11,12], and rapidly declining thereafter [9,11,12]. The hormonal factors that provide for absorption and retention of calcium coincident with the maximal demands of skeletal growth are incompletely understood. Calcium balance and calcium kinetic studies have been performed to determine the age dependence of the relationship between calcium intake and net calcium balance [13], as shown schematically in Fig. 4. Many such studies are summarized in Fig. 5, which shows regression lines for calcium intake versus calcium balance for infants, children, adolescents, and young adults. Note that the slopes of the regression lines are reasonably similar across the age groups: The increment in calcium balance for a given increase in calcium intake is independent of age. The minimal intake required to maintain positive
600
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FIGURE 5 Regression lines for the subthreshold regions of the intake-balance relationships in infants, children, adolescents, and young adults from the data of Matkovic and Heaney (1992). Am. J. Clin. Nutr. 55, 992-996. (Copyright Robert P. Heaney, 1992. Reproduced with permission.)
calcium balance increases markedly with age, however. Infants have the highest retention of dietary calcium and also have the lowest obligatory calcium losses. For older age groups, higher levels of calcium intake are required to sustain a positive balance, an effect that is largely accounted for by an increase in urinary calcium. Nutritional Factors That Influence Calcium Balance
Nutritional factors that influence calcium balance include sodium, protein, and vegetable nutrients. The sodium intake has a major influence on the urinary excretion of calcium: Every 100 mmol of sodium (2300 mg) excreted by the kidney increases urinary calcium by approximately 1 mmol (20-60 mg) [5]. Every gram of protein metabolized increases urinary calcium by approximately 1 mg [5]. Part of the effect of protein on calcium excretion occurs due to the use of bone buffers to neutralize acid generated from the metabolism of protein. A diet high in protein and acid ash will induce bone resorption, and the result is loss of calcium in the urine and negative calcium balance, which can be prevented by provision of dietary bicarbonate [14,15]. Overall, approximately half of the variance in calcium balance in normal women is explained by urinary losses [5], indicating that urinary excretion and the influence of sodium and protein intake are critical in determining an individual's calcium balance. The effect of vegetable nutrients on calcium absorption is less dramatic than the effects of dietary sodium and protein on urinary calcium excretion. The effect of dietary fiber is relatively small, but oxalate and phytate substantially reduce the availability of calcium; thus, as Heaney [5] summarized, the calcium in beans is approximately half as available as the calcium in milk, and the calcium in spinach and rhubarb is almost totally unavailable. The binding of calcium to phytate or oxalate in
140
Gordon J. Strewler
vegetables occurs before the food is ingested, and there is little effect of a high oxalate or phytate intake on availability of calcium from dairy sources.
PARATHYROIDS AND SECRETION AND METABOLISM OF PARATHYROID H O R M O N E A n a t o m y a n d D e v e l o p m e n t of the Parathyroid Glands The four parathyroid glands weigh an average of approximately 30mg each. The superior glands are most often found at the posterior aspect of the thyroid capsule; the inferior glands are usually near the inferior thyroid margin. The position of the inferior glands is more variable, and they are sometimes found with the thymus in the anterior mediastinum. Approximately 5% of the population has only three glands; five glands are found in 3-13% of the population, and six or more glands are occasionally present [16]. These are important features of the surgical anatomy of the parathyroids; many are the vagaries of surgery on these tiny, wandering glands. The parathyroid glands first appear during Weeks 5 or 6 of gestation, with the upper glands developing from the fourth branchial pouch. The lower glands originate together with the thymus from a more cephalad location in the third branchial pouch but migrate downward, sometimes accompanying the thymus into the mediastinum. Several transcription factors have been implicated in the development of the parathyroids and adjacent glands, including Hoxa3 [17], ablation of which causes defective development of thyroid, parathyroid, and thymus; GATA3, deficiency of which leads to hypoparathyroidism, sensorineural hearing loss, and renal anomalies [18]; and Tbxl, which is required for normal development of parathyroids, thymus, cardiac outflow tract, and the face [19]. Isolated hypoparathyroidism occurs with the loss of Glial cells missing-2 (GCMB), which is expressed exclusively in the parathyroids [20,21]. Unlike humans with loss of the GCMB gene, mice with a deletion of the Glial cells missing-2 gene have detectable PTH levels, and it has been suggested that the thymus may provide an additional source of parathyroid cells in the mouse [20]. Physiology of Parathyroid H o r m o n e Secretion The primary purpose of PTH is to regulate the serum calcium concentration within the very narrow physiological range of approximately 8.9-10.1 mg/dl; in normal humans, the individual variation of[Ca 2+] from the mean
FIGURE 6 Relationshipbetween serum calcium and simultaneous PTH. The concentration of calcium was modified by infusion of calcium (circles) or citrate (triangles). Original data from Conlin, et al. (1989). J. Clin. Endocrinol. Metab. 69, 593-599. Reprinted with permission from Strewler,G. J. and Greenspan, F. S. (eds.) "Basicand Clinical Endocrinology, 5th edition," Appleton and Large, 1997. is less than 2%. To achieve this aim, the secretion of PTH is directly controlled by the level of calcium in serum in a classical negative feedback loop. The relationship of serum calcium to PTH is steeply sigmoidal, with the steep portion of the curve corresponding to the normal range of serum calcium (Fig. 6). This relationship is required to achieve very tight control of the serum calcium: Small increments in the calcium concentration produce a major change in the level of PTH. The molecular basis of this feat lies primarily in the design of the receptor on parathyroid cells that senses the ambient level of calcium, and is described in the next section. Another feature of the system, as shown in Fig. 6, is that when maximally suppressed, the gland still "leaks" some parathyroid hormone. This leak may be important in the genesis of "tertiary" hyperparathyroidismmfor example, in persistent hypercalcemia following renal transplant in patients who developed secondary hyperparathyroidism on dialysis. Parathyroid Calcium S e n s o r
Structure and Physiologic Functions The calcium sensor on parathyroid cells is a G proteincoupled receptor (GPCR) with a large extracellular domain comprising approximately 600 amino acids, the seven membrane-spanning domains characteristic of this receptor class, and a carboxyl-terminal tail [22] (Fig. 7). It is most closely related to the metabotropic glutamate receptor and other members of family C of GPCRs, which also includes the GABAB receptors and a group of putative pheromone receptors [23]. The calciumsensing receptor is also expressed on C cells of the thyroid, where it regulates the positive feedback effects of calcium upon the secretion of calcitonin. In the distal renal
141
5. Parathyroid Hormone and Calcium Homeostasis
SP ..
o Q
*
,t NH2 *~,LL
X
X Inactivating ,
X
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FIGURE 7 Schematic representation of the principal predicted topological features of the human Ca2+-sensing receptor: SP, signal peptide; HS, hydrophobic segment; PKC, protein kinase C. Also delineated are missense and nonsense mutations causing either familial hypocalciuric hypercalcemia or autosomal dominant hypocalcemia. These are indicated using the three-letter amino acid code, with the normal amino acid indicated before and the mutated amino acid shown after the numbers of the relevant codons [reprinted with permission from Brown, E. M., Bai, M., and Pollak, M. (1997). Familial benign hypocalciuric hypercalcemia and other syndromes of altered responsiveness to extracellular calcium. In Metabolic Bone Diseases (S. M. Krane and L. V. Avioli, Eds.), 3rd ed., pp. 479-499.Academic Press, San Diego].
nephron, the calcium-sensing receptor plays an important role in the control of renal calcium excretion within the cortical thick ascending loop of Henle, regulates the renal handling of sodium, potassium, and chloride, and probably causes the impairment of urinary concentrating ability in hypercalcemia [24]. The calcium sensor is also present in bone, cartilage, and other tissues, in which it likely has a number of additional physiological roles [22].
Calcium Binding and Receptor Activation Calcium binds to the extracellular domain of the calcium-sensing receptor [25]. Ser 147 and Ser 17~ a r e involved in calcium binding [26], and the corresponding amino acids in the metabotropic glutamate receptor and the GABAB receptor are also involved in binding of their cognate ligands. For several reasons, however, it is likely that binding of calcium is more complex. First, the steep
slope of the curve describing the relationship of extracellular [Ca 2+] strongly suggests positive cooperativity, raising the question whether multiple calcium ions are bound to a single receptor [25], such that binding of one molecule of C a 2+ increases the affinity of the receptor for binding of subsequent molecules. Second, mutations at many sites in the extracellular domain of the receptor (Fig. 7) shift its sensitivity to calcium and can produce either chronic hypercalcemia or chronic hypocalcemia, indicating that the extracellular domain is broadly involved in calcium binding. Third, the receptor dimerizes through cysteine residues in the extracellular domain [27,28] and there are functional interactions between individual members of a dimer [29]. Mutations in the calcium-sensing receptor that decrease its sensitivity to calcium produce usually lifelong mild hypercalcemia. The syndrome is called familial benign hypocalciuric hypercalcemia [30] because these mutations also reduce the sensitivity to calcium in the
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kidney, resulting in increased calcium reabsorption (see Chapter 20). Loss of both receptor copies eliminates the ability to sense calcium; the resultant syndrome of severe neonatal hypercalcemia requires total parathyroidectomy [30]. Conversely, mutations that increase sensitivity to calcium produce mild lifelong hypocalcemia and relative hypercalciuria [31]. Calcimimetic compounds have been identified that change the sensitivity of the calcium-sensing receptor and the consequent sensitivity of parathyroid hormone secretion. These drugs are used for the treatment of hyperparathyroid disorders [32,33]. The phenylalkalamine NPS R-568 and AMG073 left-shift the response of the parathyroid cell to extracellular [Ca2+], markedly decreasing the rate of PTH secretion at ambient calcium levels. NPS R-568 appears to act by modifying allosterically activation of the receptor by calcium [34]. Calcimimetic agents reduce secondary hyperparathyroidism in rat models [35-37] and preliminary results indicate that AMG073 is effective in the treatment of chronic mild primary hyperparathyroidism [38].
lntracellular Signaling by the Calcium-Sensing Receptor The calcium-sensing receptor is coupled to phospholipase C[3 by pertussis toxin-insensitive G proteins, probably Gq or Gll [25]. In response to an acute increase in extracellular [Ca2+], phospholipase C[3 is activated and the parathyroid cell cleaves the phospholipid substrate phosphoinositol-4,5-bisphosphate in the plasma membrane to produce the intracellular second messenger IP3, which diffuses to the endoplasmic reticulum and induces the transient release of calcium from its stores [25,39,40]. The function of G proteins in receptor coupling to phospholipase C is discussed later [2,41,42]. Activation of phospholipase C[3 also stimulates protein kinase C (PKC) [42a]. Exposure of cells that express the parathyroid calcium sensor to high extracellular Ca 2+ also stimulates the activity of phospholipases A2 and D, but this is probably an indirect effect of the activation of PKC [42b], involving the MAP kinase pathway [42c]. Blockade of phospholipase A2 is reported to inhibit the secretion of PTH [42d]. It is notable that the paradigm of such laboratory experiments, in which a calcium transient is induced by acutely increasing extracellular calcium, does not apply to the physiological situation, in which the secretion of parathyroid hormone is tonically maintained at a level that allows the maintenance of serum [Ca 2+] near its set point. In a manner as yet poorly understood, an intracellular signal from the calciumsensing receptor sets the tonic secretion of parathyroid hormone at a level appropriate to the set point of the gland for [Ca2+]. The signaling events for tonic PTH secretion may not be the same as for acute calcuim transients.
Mg 2+ as a PTH Secretogogue The divalent cation Mg 2+ is also a ligand for the parathyroid calcium sensor and can inhibit the secretion of PTH, although it is approximately threefold less potent than Ca 2+. The parathyroid calcium-sensing receptor probably regulates magnesium levels in vivo because individuals with familial benign hypocalciuric hypercalcemia have mild hypermagnesemia [42e] and those with activating mutations of the receptor tend to present with mild hypomagnesemia. Paradoxically, severe hypomagnesemia "paralyzes" the parathyroid glands, producing a form of reversible hypoparathyroidism. This syndrome apparently results from an effect of hypomagnesemia on the activation of G proteins [42f]. Chemistry, Biosynthesis, and Processing of Parathyroid H o r m o n e
Parathyroid Hormone Peptide Family PTH
The PTH peptide family has three members: PTH, the parathyroid hormone-related peptide (PTHrP), and the tuberoinfundibular peptide of 39 amino acids (TIP39). PTH is an 84-amino acid peptide with a molecular weight of 9300 whose sequence is highly conserved among terrestrial mammals that possess parathyroid glands (Fig. 8). As discussed in detail later, the PTH sequence encompasses an amino-terminal receptor-binding domain (amino acids 1-34) and a carboxyl-terminal domain that may have its own cognate receptor. PTHrP
PTHrP is a peptide of 139-172 amino acids in various isoforms, with limited homology to PTH overall but strong homology in the amino-terminal receptor-binding domain, where 8 of 13 residues are identical. As a consequence of conservation of their receptor-binding domains, the two peptides have essentially identical affinity for a common receptor, the PTH1 receptor (PTH1R). PTHrP is a locally active cytokine rather than a hormone [43,44]. It is present in many tissues, including mesenchymal tissues such as cartilage [45], bone [46], and skeletal, cardiac, and smooth muscle [47]; epithelia such as skin, mammary, the enamel epithelium of the teeth [48], and the intestinal mucosa; endocrine glands such as the parathyroid and pancreatic islets [48]; the placenta [48]; and regions of the central nervous system [47]. During development, PTHrP plays a critical role in endochondral bone formation [43,45]. Under the control of Indian hedgehog, it regulates entry of chondrocytes into the terminally differentiated state and thus the rate at which endochondral bone grows and is mineralized
5. Parathyroid Hormone and Calcium Homeostasis PRE $ PRO $ -31 -6 human MIPAKDNAKVMIVMLAICFLTKSDG KSVKKR bovine MMSAKDMVKVMIVMLAICFLARSDG KSVKKR porcine M M S A K D T V K V N V V N L A I C F L A R S D G K P I K K R rat MMSASTNAKVMILMLAVCLLTQADG KPVKKR canine MMSAKD~IVMFAICFLAKSDG KPVKKR chicken M T S T K N L A K A I V I L Y A I C F F T N S D G R P M N K R
PTH +i +I0 SVSEIQLMHN AVSEIQFMHN SVSEIQLMHN AVSEIQLMHN SVSEIQFMHN SVSEMQLMHN
human bovine porcine rat canine chicken
+20 +30 +40 +50 LGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRK LGKHL S S N E R V E W L R K K L Q D V H N F V A L G A S I A Y R D G S S Q R P R K LGKHL S S L N N V E W L R K K L Q D V H N F V A L G A S I V H R D G G S Q R P R K LGKHLASVERMQWLRKKLQDVHNFVSLGVQMAAREG SYQNPTK LGKHL S S N E N V E W L R K K L Q D V H N F V A L G A P I A H R D G S S Q R P L K L G E H R H T V E R Q D W L Q M K L Q D V H . . S A L E ...... D A R T Q R P R N
human bovine porcine rat canine chicken
+60 +70 +80 KEDNVLVE... S H E K S L G E A .......... D K A D V N V L T K A K S Q KEDNVLVE... S H Q K S L G E A .......... D K A D V D V L I K A K P Q KEDNVLVE... S H Q K S L G E A .......... D K A A V D V L I K A K P Q KEENVLVD... G N S K S L G E G .......... D K A D V D V L V K SQ KEDNVLVE... S Y Q K S L G E A .......... D K A D V D V L T K A K S Q KEDIVLGE I R N R R L L P E H L R A A V Q K K S ID L D K A Y M N V L F K T K P .
FIGURE 8 Sequences of PTH from various species. (Reprinted with permission from "Parathyroids: Basic and Clinical Concepts" (J. P. Bilezikian, R. Marcus, and M. A. Levine, Eds.), 2nd edition, pp. 245260. Academic Press: San Diego (2001).
(see Chapters 3 and 4). It is also clear that PTHrP is necessary for normal mammary development [44,48] and tooth eruption [43], and new physiological actions continue to be identified.
TIP39 Recently identified, TIP39 is a 39-amino acid molecule with limited sequence homology with PTH: 9 of 39 residues are identical in the two peptides [49]. Its functions are not fully understood, although it may act as a releasing factor for ACTH-releasing factor, vasopression, and luteinizing hormone [50]. TIP39 was identified as an endogenous ligand for a hypothalamic receptor, PTH2R [51,52], which is closely related to PTH1R and, at least the case of the human receptor, recognizes PTH, although it has an approximately 10-fold higher affinity for TIP39, its presumed natural ligand [49].
Evolution of the P T H Peptide Family It is not clear when PTH appeared in evolution. The parathyroid glands are present only in terrestrial animals, but three receptors that recognize PTH have been identified in fish. Two are homologous to the mammalian PTH1 and PTH2 receptors, and the third is unique although closely related in sequence to PTH1R [53,54]. All three receptors respond to mammalian PTH, but only PTH1R and PTH2R respond to PTHrP and a fugu PTHrP analog. However, the zebrafish PTH2R binds tuberoinfundibular peptide preferentially. Since all three receptors efficiently recognize one or both of the other members of the family, their presence does not
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make a strong argument for the presence of PTH in the teleost genome.
Biosynthesis and Processing of PTH The human PTH gene is on the short arm of chromosome 11 and contains three exons. The first encodes a short 5' untranslated domain, the second a prepro region of 29 amino acids, and the third the remainder of the coding sequence and the 3' untranslated domain [55,56]. As it emerges from the endoplasmic reticulum, the nascent preproPTH polypeptide is cleaved at position - 7 / - 6 to produce the 90-amino acid proPTH (Fig. 8). In contrast to the evanescence of preproPTH, proPTH has a half-life of approximately 15 min in pulse-chase studies and is converted to PTH coincidentally with arrival in the trans-Golgi. It appears that the cleavage enzyme is the ubiquitous prohormone convertase furin rather than one of the specialized convertases of neuroendocrine secretory granules such as PC2 and PC1 [57], although PC7 has recently been shown to be expressed in parathyroid cells and can cleave PTH [58]. Perhaps the use of furin is a consequence of the evolution of PTH from PTHrP, which was designed for secretion by a diversity of cells, most of which lack the specialized machinery of neuroendocrine cells [56]. The sequence of preproPTH has the general features of signal sequences that direct proteins into the lumen of the endoplasmic reticulum [59]. Deletion of 10 or more residues, including part of the hydrophobic core, prevents entry into the secretory pathway and results in rapid degradation of the nascent peptide [60]. A spontaneous mutation that disrupts the hydrophobic core and blocks translocation of the nascent peptide causes familial hypoparathyroidism [61]. The function of the pro sequence is incompletely understood. Deletion of the pro sequence results in inefficient cleavage of preproPTH, with some molecules cleaved in the pro domain [62,63]. There must be powerful evolutionary pressures to avoid infidelity of processing of the amino terminus, since an intact amino terminus is required for biological activity. It is thus possible, as Kronenberg et al. [56] have argued, that the pro domain evolved as a linker region to permit evolution of the amino terminus of PTH, the primary receptor-activating domain, to proceed at some physical distance from the end of the signal sequence, which may have had incompatible evolutionary constraints on its amino acid sequence. These evolutionary adaptations probably occurred first in PTHrP, which has similar sequence features [64]; the existence of proPTHrP has not been directly shown, however. After biogenesis in the Golgi apparatus, PTH is packaged in dense secretory granules like those of other endocrine cells and stored to await secretion. It has been estimated that glandular stores of PTH are sufficient to
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maintain maximal rates of secretion for approximately 1.5 hr. PTH is stored and secreted with the peptide chromogranin A (formerly called parathyroid secretory protein-l), a molecule that is also cosecreted with other hormones from dense neurosecretory granules. It is now becoming clear why chromogranins are obligatory fellow travelers in dense secretory granules: chromogranin A is an on/off switch that is both necessary and sufficient for biogenesis of dense secretory granules and hormone sequestration within them [65]. The Control of PTH Synthesis and Secretion by Calcium and Other Secretagogues
PTH is secreted in response to the challenge of hypocalcemia. If the challenge were acute, lasting less than 1 hr, stores of preformed PTH that reside in secretory granules would suffice. However, in response to chronic hypocalcemia, the parathyroid glands must mount a threefold response, secreting their stores of PTH; increasing transcription of the PTH gene and the biogenesis of PTH; and, over days and weeks, undergoing hyperplasia to increase the mass of parathyroid tissue available for secretion of its hormone. How are the pathways by which a decrease in the extracellular [Ca 2+] evokes an acute secretory response related to pathways by which hormone synthesis and cell growth are controlled? How is the control of cell growth in states of chronic hypocalcemic challenge (secondary hyperparathyroidism) related to the loss of full control of cell growth that characterizes primary hyperparathyroidism?
Regulation by Calcium Hypocalcemia markedly upregulates the level of PTH mRNA, but hypercalcemia reduces its level only minimally [66,67]. The parathyroid cell is thus much better equipped to respond to its primary challenge, hypocalcemia, than to adapt over hours or days to hypercalcemia, although chronic hypercalcemia will eventually reduce parathyroid gland size. Increasing medium calcium suppresses the transcription of PTH mRNA in cultured cells [68] and a putative calcium response element was identified in the upstream region of the PTH promoter [69]. However, the biological significance of these results is unclear in light of physiological studies showing minimal effects of hypercalcemia on PTH mRNA levels [66,67]. Hypocalcemia increases the level of PTH mRNA primarily by a posttranscriptional effect on the stability of PTH mRNA [70]. The cis-acting locus at which the stability of the mRNA is controlled by calcium and phosphorus is a 26-nucleotide stretch of the Y untranslated region (UTR). In hypocalcemic rats, trans-acting proteins (one of which is AUF1) [71] bind to this locus in vitro and stabilize RNA transcripts [70]. Insertion of the stability
motif from PTH mRNA into growth hormone transcripts is sufficient to confer sensitivity of the chimeric growth hormone transcripts to parathyroid cell proteins that were obtained from animals fed either a low-calcium diet or a low-phosphate diet [72], but it is not known how parathyroid proteins are modified by hypocalcemia.
Regulation by Phosphate Changes in serum phosphate levels were long thought not to regulate the secretion of PTH directly, acting instead by inducing changes in [Ca 2+] or 1,25(OH)2D. However, it is now clear that both hypophosphatemia and hyperphosphatemia influence the parathyroids directly. Hypophosphatemia decreases PTH mRNA levels by a posttranscriptional mechanism [73-75]. This is a direct effect of hypophosphatemia because it occurs under conditions in which neither the serum [Ca 2+] nor the serum 1,25(OH)2D levels are abnormal. The stability of PTH transcripts is decreased by parathyroid proteins from rats on a low-phosphate diet; thus, hypophosphatemia, like hypocalcemia, regulates PTH mRNA levels by regulating the stability of PTH mRNA. Both the cisacting locus in the PTH mRNA and the trans-acting RNA-binding proteins that the cell employs to respond to hypophosphatemia are similar to those it uses to respond to hypocalcemia [70-72]. Hyperphosphatemia increases the level of PTH mRNA from isolated parathyroid glands by a mechanism independent of calcium and 1,25(OH)2D [76,77]. The means by which the parathyroid gland senses the extracellular phosphate level is not understood, but the parathyroid possesses a sodium-coupled phosphate transporter whose levels are regulated by changes in serum phosphate and 1,25(OH)2D [78], and this transporter has been suggested to be a candidate phosphate sensor.
Regulation by 1,25(OH) 2D Parathyroid hormone and vitamin D interact in complex ways to maintain a normal serum calcium concentration. Exposure to 1,25(OH)2D downregulates PTH mRNA levels and PTH secretion in isolated parathyroid glands [79,80] and in the intact rat [81]. Transcriptional inhibition results from binding of the vitamin D receptor (VDR) to a conserved negative regulatory element in the PTH gene promoter. The sequence of this negative regulatory element resembles that of positive vitamin D response elements in other genes [82,83]; binding of the receptor must lead to differences in assembly of the transcriptional complex to induce negative rather than positive regulation of the gene. Hypocalcemia stimulates both the secretion of PTH and the production of 1,25(OH)2D, so the concentrations of the two hormones vary in tandem in most cir-
5. Parathyroid Hormone and Calcium Homeostasis
cumstances and they synergize to protect the serum calcium concentration. Hence, a negative feedback loop in which 1,25(OH)2D inhibits the secretion of PTH does not appear to be physiologically adaptive. When 1,25 (OH)2D is administered to rats made acutely hypocalcemic by the administration of phosphate, the stimulation of PTH mRNA levels is reversed. However, in animals made hypocalcemic by feeding a low-calcium diet, PTH levels increase appropriately in the face of a large increase in serum 1,25(OH)2D [84]. The relative resistance to 1,25(OH)2D during chronic hypocalcemia may be induced by nuclear accumulation of calreticulin, a calcium-binding protein that interferes with the negative regulation of transcription by the VDR [85]. This suggests that a mechanism exists to maintain a physiologically adaptive hierarchy between the contrary parathyroid effects of calcium and 1,25(OH)2D. Whatever the adaptive mechanism may be, it is efficient, since VDR knockout mice in which normocalcemia and normophosphatemia are maintained by manipulation of the diet have normal PTH levels [86]. Regulation in Uremia
The clinical implications of regulation of PTH secretion by phosphate and vitamin D are most important in uremia (see Chapter 28), in which the dual effects ofhyperphosphatemia and reduced 1,25(OH)2D levels acutely increase PTH secretion and chronically promote parathyroid gland growth, thus producing secondary hyperparathyroidism. In early renal failure hyperparathyroidism can be prevented by lowering phosphate through phosphate restriction, thus increasing the level of 1,25(OH)2D [87,88]. In chronic renal failure, dietary phosphate restriction directly ameliorates or prevents secondary hyperparathyroidism, independent of 1,25(OH)2D [76]. As important as the acute effects of dietary phosphate and vitamin D growth in the genesis of secondary hyperparathyroidism are their parallel effects on parathyroid cell. Control of Growth of the Parathyroid Glands
To maintain the appropriate number of parathyroid cells to respond to physiological stimuli, the parathyroid economy has linked the control of parathyroid gland size to the calcium sensor and also to the VDR and a phosphate sensor. These pathways allow parathyroid gland size to change in parallel with changes in synthesis and secretion of PTH so that the acute response is amplified in chronic states. In hyperparathyroid states, the normal feedback regulation of PTH secretion and the normal control of parathyroid cell growth are lost in parallel (see Chapters 20 and 28). Parfitt [89] reviewed the control of growth of the parathyroid glands in normal physiology and hyperparathyroid states.
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The parathyroid glands have a mean weight of only 3 mg at birth but grow rapidly during the first years of life and weigh an average of 18 mg at age 2 years. They achieve their adult weight of approximately 40 mg by age 30 [89]. Thereafter, their rate of proliferation is approximately 5% per year, sufficient to compensate for cell loss and maintain a normal cell number. The glands respond to physiological demands mainly by hyperplasia, not hypertrophy, and shrink sluggishly in response to hypercalcemia. Regulation of Cell Growth by Calcium
The number of parathyroid cells is directly controlled by the calcium-sensing receptor. Loss of the receptor, as in neonatal severe hyperparathyroidism, leads to massive parathyroid hyperplasia in the face of severe hypercalcemia. A shift in the set point of the receptor, as in familial benign hypocalciuric hypercalcemia, probably increases the size of the parathyroids slightly [90]. Treatment with calcimimetic compounds that act directly at the receptor reduces parathyroid gland size and cell number in experimental models of secondary hyperparathyroidism [35-37]. Finally, as Parfitt et al. [91] noted, parathyroid adenomas, although they are clonal neoplasms caused by dysregulation of genes for parathyroid cell growth, do not grow progressively but rather enlarge very little after achieving a certain size. This behavior can be explained by a "set point" hypothesis: Parathyroid adenomas grow until the serum calcium reaches the set point for secretion of PTH and then essentially stop growing. Thus, the negative growth influence of ambient calcium concentrations above the set point dominates the control of parathyroid cell growth, even when the cells have a fundamental abnormality of growth control [89]. If correct, the set point hypothesis has an important consequence for chronic medical therapy of hyperparathyroidism, namely that drugs that reduce the serum calcium level by inhibiting bone resorption, once they lower serum calcium below the set point, will simply cause additional enlargement of the parathyroid adenoma [33]. Calcimimetic drugs potentially avoid this problem by resetting the set point. However, given that the calcium-sensing receptor is expressed in the growth plate, such drugs may affect chondrocyte growth and differentiation and may thus have adverse effects on growth in children. The relationship between parathyroid growth and the set point for hormone secretion is bidirectional: Primary abnormalities in parathyroid growth also produce a shift in the set point for PTH secretion. The set point is rightshifted in both primary and secondary hyperparathyroidism [92]. The shift in set point can be reproduced by transgenic introduction of a growth control gene, cyclin D, into parathyroid cells [93]. Thus, changes in [Ca 2+] regulate the growth of parathyroid cells, and changes in the growth of parathyroid cells reciprocally regulate the [Ca2+].
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Growth Regulation by Phosphate and Vitamin D
Hypocalcemia, hyperphosphatemia, and decreased 1,25(OH)2D levels all contribute to the genesis of parathyroid hyperplasia in uremia. 1,25(OH)zD inhibits the proliferation of parathyroid cells in vitro. Vitamin D and metabolites are used clinically to manage secondary hyperparathyroidism in patients with chronic renal failure [94] (see Chapter 28). However, the importance of 1,25(OH)zD as a regulator of parathyroid gland size in circumstances other than renal failure is less clear because the absence of the VDR does not induce the proliferation of parathyroid cells in the knockout mouse model when normal levels of serum calcium and phosphorus are maintained by dietary means [86]. Hyperphosphatemia is another stimulus to parathyroid growth, independent of changes in [Ca 2+] or 1,25(OH)zD [76]; hence, control of the serum phosphorus level by restricting dietary phosphate and administration of oral phosphate binders [calcium, aluminum-containing antacids, or sevelamer hydrochloride (Renagel)] is crucial to prevention of secondary hyperparathyroidism in patients with chronic renal failure on dialysis. Chronic oral phosphate therapy of the hereditary phosphate-wasting disorders can also lead to the development of secondary hyperparathyroidism (see Chapter 25). The means by which parathyroid cells sense changes in ambient phosphate concentrations has not been established, although the cells express a sodium-coupled phosphate transporter whose level is regulated by the extracellular phosphate concentration [78]. It has recently been suggested that an increase in the parathyroid level of the growth factor transforming growth factor-~ may mediate the growth effects of dietary phosphate and 1,25(OH)zD, which are exerted on the cell growth regulatory protein p21 wafl/cipl, a regulator of cyclin-dependent kinases [95,96]. Peripheral M e t a b o l i s m of PTH PTH is rapidly metabolized after secretion, with a disappearance half-time of approximately 2 min [56,97]. Metabolism occurs mainly in the liver (60-70%), where it is taken up and rapidly degraded by Kupffer cells [98], and in the kidney (20-30%) [56,99,100]. The removal of PTH by the kidneys is almost exclusively by glomerular filtration, after which uptake and degradation by cells of the renal tubule involve the protein megalin [101]. The rapid degradation of PTH ensures that its level in blood is set by the secretory rate. Advantage is taken of its rapid clearance when intraoperative determinations of the PTH level are used during parathyroidectomy to determine when a parathyroid adenoma has been removed [102].
Circulating PTH is a heterogeneous pool because in addition to the full-length, biological molecule, a variety of fragments are present in the circulation [56] (Fig. 9). Most of these are carboxyl-terminal fragments generated by cleavage of PTH at several sites in the region 33-43 [99,100]. They arise both by peripheral metabolism of PTH in Kupffer cells of the liver [98-100] and by direct secretion from the parathyroid gland [103,104]. Although only 10-20% of PTH is converted to carboxyl-terminal fragments, they predominate over full-length PTH in serum because they have a much longer half-life than that of PTH. Carboxyl-terminal fragments are cleared in the kidney and they therefore accumulate in renal failure [56,97]. Because they are present in such abundance, carboxyl-terminal fragments are the easiest form of PTH to measure in serum, and early PTH assays were mostly directed to determinants in the carboxyl-terminal domain. As discussed later, several lines of evidence suggest that there may be a distinct receptor for the carboxyl-terminal region of PTH, which specifies a distinct set of biological actions. Amino-terminal fragments that contain the determinants required for biological activity are presumably produced in the Kupffer cell by cleavage in the region 33-43, but they do not appear to circulate in significant amounts [100]. It has recently been found that PTH appears to be cleaved also at one of the first residues to produce a fragment that may be similar to PTH(7-84) [105-107]. Synthetic PTH(7-84) is not active at the PTH1R, although it has weak activity as a competitive antagonist of PTH(1-84)-stimulated cAMP accumulation, but it has hypocalcemic properties in vivo. These actions appear to be mediated through a putative receptor for carboxyl-terminal PTH [108-111]. Non-PTH(1-84) account for approximately one-third of the circulating species immunoreactive PTH in two-site immunoassays for the whole molecule, both in individuals with normal renal function and in renal failure [106,107,112,113]. A new generation of whole molecule PTH assays have been introduced that are specific for PTH(1-84).
i Blood
arathyroids I
1
Intact
'
t,
Carboxyl-terminal
! ',ver I I FIGURE 9 Schematic representation of the origin and clearance of PTH fragments. (Reprinted with permission from Felig, P., Baxter, J. D., Broadus, A. E., Frohman, L. A. (Eds.) "Endocrinology and Metabolism, 3rd edition," McGraw-Hill, 1995.)
5. Parathyroid Hormone and Calcium Homeostasis
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ASSAY OF PTH For many years, the study of the physiology of the parathyroid gland and its disorders was hampered by technical difficulties in assaying the hormone [114]. PTH circulates at a low concentration (1-6 pmol/liter) compared to many other hormones. Also, PTH in blood is immunologically heterogeneous, an observation first made during pioneering efforts by Berson and Yalow to develop a radioimmunoassay of the hormone [115]. The best of the early PTH assays proved to be those that used antibodies directed at determinants in the midregion of the sequence [116]. These determinants are part of carboxyl-terminal fragments that have a long half-life compared to intact PTH and are thus present at a much higher concentration. Although they could detect PTH in the form of these fragments, midregion assays suffered problems with sensitivity and specificity. It is now recognized that PTH is appropriately suppressed in non parathyroid hypercalcemia, but early assays detected immunological activity in serum, presumably from interfering substances, and this led to considerable confusion about the role of PTH or related molecules in the pathogenesis of hypercalcemia in malignancy [117]. Intact PTH values in midregion assays were also increased in renal failure because of impaired renal clearance of the fragments. Although assays for amino-terminal, midregion, and carboxyl-terminal fragments are still in clinical use, they have no advantages over assays for the whole molecule, which are both more sensitive and more specific. The current generation of PTH assays consist of twosite assays for intact PTH [118-122]. By using two antibodies in combination (Fig. 10), it is possible to improve both the sensitivity and the specificity of the assay. In the assay serum is added to tubes containing a capture antibody that is bound to a bead or some other type of solid phase support. PTH is bound to a solid phase by the capture antibody and is then detected with a labeled second antibody. The sensitivity of two-site assays is high (<1 pmol/liter) because the individual affinities of both antibodies contribute to overall assay sensitivity. The specificity of the assays has also been improved in two ways. Nonspecific interference is reduced to negligible levels because potential interfering substances in serum are removed before the bound PTH is detected. Interference by heterogeneous PTH fragments should be eliminated by choosing the antigenic determinants for the two antibodies from opposite ends of the molecule and thus making the assay specific for the intact PTH(1-84) molecule, although the generation of intact PTH assays currently in clinical use also recognizes an almost full-length form of PTH.
FIGURE | 0 Principles of two-site assay of PTH. (Reprinted with permission from Strewler, G. J. and Greenspan, F. S. (eds.) "Basic and Clinical Endocrinology, 3rd edition," Appleton and Lange, 1997.)
A variety of two-site assay formats are available [118-122]. They differ from one another primarily in the method that is used to detect the second antibody bound to PTH: Immunoradiometric assays use radioactively labeled second antibody, thus requiring facilities that can deal with radiation hazards and the limited shelf-life of the reagent. These have largely been supplanted by enzyme-linked immunosorbent assays and immunochemiluminometric or immunofluorometric assays. Most assay formats can be automated, allowing for easy access by hospital labs, and they can be set up to allow determination of PTH in 1 hr or less so that they can be used intraoperatively to help the parathyroid surgeon determine when a parathyroid adenoma has been removed [102,123]. It was recently found that the first generation of intact PTH assays actually recognize an extended PTH fragment, hitherto unrecognized, that is truncated at the amino terminus and therefore lacks biological activity [105-107]. It is estimated that this fragment comprises approximately one-third of immunoreactive intact PTH in normal subjects (67-79%) and 33-68% in patients with uremia [106,107,112,113]. A new generation of assays have recently been introduced with antibodies that require that the entire amino terminus be present in order to recognize PTH, and they are specific for biologically active PTH(1-84); these are provisionally termed whole PTH assays to distinguish them from first-generation intact PTH assays [112]. One of them has a normal
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range of 7-36 pg/ml [112] (Fig. 11), compared to 10-65 pg/ml in intact assays. The implications of these developments for the clinical use of PTH assays remain to be determined. PTH assays are used clinically in the differential diagnosis of hypercalcemic and hypocalcemic states and in the management of patients with chronic renal failure. Intact PTH assays and whole PTH assays identify elevated PTH levels in 80-90% of patients with primary hyperparathyroidism (Fig. 11); most of the remaining patients have PTH levels in the upper normal range. A high normal PTH in ahypercalcemic patient is consistent with the diagnosis of primary hyperparathyroidism because PTH values of patients with nonparathyroid hypercalcemia are suppressed to the lower normal range or lower [124]. Conversely, a suppressed PTH in a patient with a malignant tumor and hypercalcemia essentially excludes a coincidental parathyroid tumor. Whether the whole PTH assay will improve assay performance remains to be seen. Most hypoparathyroid
FIGURE 1 1 Scatterplotof whole PTH(1-84) values in healthy controls and various patient groups. Shaded area indicates the plasma normal range (7-36pg/ml) of wholePTH(1-84). The y axis is expressed by log2 scale. The whole PTH levels of 10 patients with primary hyperparathyroidism (1~ HPT) were in the upper normal range and the overall diagnostic sensitivity was 93.9% (155/165) [from Gao et al. (2001). J. Bone Miner. Res.16, 605-614. Copyright 2001 American Society for Bone and Mineral Research).
patients have subnormal PTH values, a notable exception being those with inherited activating mutations of the parathyroid calcium-sensing receptor, who typically have mild hypocalcemia and PTH values in the lower normal range [31,125]. To diagnose and manage the two dominant forms of renal osteodystrophy, secondary hyperparathyroidism and adynamic bone disease, it is essential to monitor the PTH level [126,127]. Intact PTH assays are a considerable improvement over older radioimmunoassays for PTH fragments, but they are also interfered with by renal failure, as discussed previously [105,107,112]. Currently, research is focused on whether better clinical performance can be achieved by whole PTH assays, which appear to be minimally affected by renal insufficiency.
PARATHYROID H O R M O N E ACTION PTH a n d Mineral Ion H o m e o s t a s i s To maintain the concentration of calcium within narrow limits, PTH regulates the transfer of calcium across the three interfaces of the extracellular space--the bone, the kidney, and the intestine. The kidney and bone are direct targets of PTH, but the regulation of intestinal calcium absorption by PTH is indirect. PTH regulates the renal synthesis of 1,25(OH)2D, the active metabolite of vitamin D, which in turn controls the active entry of calcium through intestinal enterocytes. In response to hypocalcemia, PTH induces the release of calcium from skeletal stores by stimulating osteoclasts to resorb bone. The hormone thus taps the body's only substantial store of calcium to replete the extracellular compartment, for example, when the intake is low and cannot keep up with urinary losses. In bone, however, PTH targets not only the osteoclast (the bone-resorbing cell) but also the osteoblast (the bone-forming cell). Large net losses of bone mineral are averted by coupling resorption of existing bone to synthesis of new bone. The skeletal reserves of calcium are huge and repletion is efficient, so that only in rare instances (e.g., in chronic renal failure) is the integrity of the skeleton compromised by secondary hyperparathyroidism. PTH signals the kidney to conserve calcium, synthesize 1,25(OH)2D, and waste phosphate in the urine. It makes physiological sense that in order to protect the serum calcium concentration, PTH should conserve calcium by inhibiting its renal excretion. In response to hypocalcemia, PTH also activates the conversion of 25OH-vitamin D, the principal circulating form of the vitamin, to 1,25(OH)2D, its active form. As discussed in Chapter 7, the renal synthesis of 1,25(OH)2D is also regulated by the mineral ions calcium and phosphate;
5. Parathyroid Hormone and Calcium Homeostasis
the kidney thus acts to synthesize information about systemic mineral ion levels and their urinary excretion rates and set the level of 1,25(OH)zD accordingly. Through 1,25(OH)zD, the kidney dictates that the rate of calcium absorption in the intestine responds to the integrated needs of mineral ion homeostasis. PTH is also involved in the regulation of renal phosphate reabsorption. PTH increases urinary phosphate excretion at the same time as it reduces urinary calcium excretion. Why is this? The dissolution of bone mineral (hydroxyapatite) during resorption simultaneously releases calcium and phosphate into the extracellular space. Accumulation of phosphate would impair the maintenance of calcium homeostasis because of the complex interrelationships of mineral ions and the hormones that regulate their levels. Hyperphosphatemia would lower [Ca 2+] by precipitating calcium phosphate (because the normal circulating concentrations of the two ions approach their solubility product) and would inhibit the renal synthesis of 1,25(OH)zD; by these mechanisms, as well as by a direct effect, hyperphosphatemia would stimulate the additional synthesis and secretion of PTH. These entanglements are averted because PTH allows the phosphate that is liberated during bone resorption to be excreted. To understand the importance of this, consider what happens in renal failure. As phosphate accumulates because it cannot be excreted, PTH levels increase sharply. This secondary hyperparathyroidism induces further resorption of bone, creating a positive feedback loop in which bone resorption, via the serum phosphorus and PTH, begets more bone resorption. From this example, it is clear that the phosphaturic effect of PTH is an important part of integrated control of the economy of calcium and phosphate.
PTH R e c e p t o r s a n d Intracellular Signaling
PTH Receptor In its target tissues, bone and kidney, the cellular agent for the actions of PTH is a G protein-coupled receptor (GPCR) whose structure is shown schematically in Fig. 12 [128-130]. The receptor has essentially identical affinities for PTH and PTHrP and is referred to as the PTH1R or the PTH/PTHrP receptor. It has a large extracellular domain, seven membrane-spanning domains, and a cytoplasmic tail. Although the general architecture of the receptor with its seven transmembrane domains is canonical for the GPRC fainily, PTH1R is distinct in sequence from most of this huge receptor family, but it shares structural and functional motifs with a group of other GPRCs that recognize peptide ligands of 30-50 amino acids (class II GPCRs). These include receptors for calcitonin, glucagon, secre-
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tin, vasoactive intestinal peptide, and corticotropinreleasing hormone [131]. A second GPCR, the PTH2R, is the receptor for TIP39, probably the third member of the PTH peptide family [49]; however, the human homolog of this receptor is also activated by PTH but not by PTHrP [51,52]. The PTH2R is present in the central nervous system. A third receptor has been identified in the zebrafish that, like the PTH1R, recognizes PTH and PTHrP, but not TIP39 [54]. Although distinctive structurally, class II GPCRs appear to have a similar kind of interaction with G proteins as the rest of this large family, which has thousands of members in humans. Thus, the problem of coupling PTH to an intracellular signal was solved with an ancient set of molecules whose versatility has resulted in their widespread distribution throughout evolution [132,133]. GPCRs recognize light, odors, calcium, catecholamines, and proteins, and they have existed at least since the appearance of the roundworm Caenorhabditis elegans, which remarkably devotes approximately 10% of its genome to GPRCs.
Hormone-Receptor Interaction Although PTH is an 84-amino acid peptide, all the determinants for receptor binding and activation are present in PTH(1-34). An intact amino terminus is required for activation of adenylyl cyclase, and amino-truncated peptides are weak partial agonists [e.g., PTH(3-34)] or pure antagonists [e.g., PTH(7-34)] [128,130, 134,135]. Minimization of the peptide reveals that PTH(1-11) with some substitutions to increase its activity is a reasonably potent ligand for PTH1R [136,137]; this activation domain is conserved between PTH and its more ancient relative PTHrP, which shares 8 of the first 13 amino acids of PTH. This adenylyl cyclase activation domain in PTH has an inducible s-helical conformation; it is likely that the hydrophobic environment of the membrane induces the active conformation of this domain [136]. The determinants required for activation of a second signaling pathway, phospholipase C, are less certain: Some studies suggest that the tetrapeptide PTH (29-32) is sufficient [138], whereas others suggest that an intact amino terminus is required for efficient signaling [139]. The carboxyl-terminal region of PTH(1-34) is its principal binding domain [140,141]. Several nuclear magnetic resonance structures and a crystal structure [142] indicate that PTH(1-34) and PTHrP(1-34) both possess a similar, stable ~-helical region from approximately residues 17 to 30 [143-145]. In fact, the binding domains of PTH and PTHrP are interchangeable, even though they have no primary sequence homology [140,141]. This suggests that the main structural requirement for binding is simply an amphipathic helix; indeed, insertion of a
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FIGURE 1 2 The human parathyroid hormone type 1 (PTH-1) receptor (593 amino acids), showing its predicted domain organization (the locations of the seven transmembrane s-helical domains in the sequence were predicted by the Protein-Predict computer program). The Cys disulfide linkages (connecting lines) were determined from studies on the N-terminal PTH-1 receptor fragment produced in Escherichia coli. N-linked glycosyl groups are indicated by the forked symbols. Other key residues referred to in the text are indicated by single amino acid code within open circles, some of which are labeled with sequence position number for reference. The human skeletal disease of Blomstrand's chondrodysplasia (receptor inactivity) results from a mutation at Pro132--.Leu and that of Jansen's chondrodysplasia (receptor constitutive activity) results from mutations at Arg233--.His, Thr41~ and Ile458---,Arg, all shown in dark gray. Serine (S) residues shown in dark gray in the cytoplasmic tail are phosphorylation sites. C, Cys; F, Phe; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr [reproduced with permission from Gardella, T. J., and Jfippner, H. (2001). Trends Endocrinol. Metab.12, 210. Copyright Elsevier Science, Ltd.].
"generic" helix into PTH from positions 22 to 31 will also create a potent ligand [146]. The details of PTH binding and activation of its receptor are not fully understood, but considerable insight has been gained by a combination of molecular cross-linking of PTH and the receptor [128,130,147-150], mutations in both ligand and receptor, and modeling based on the structure of PTH. The binding domain of PTH associates with the large extracellular domain of the PTH1R, allowing the amino terminus to be inserted into a shallow binding pocket formed by the transmembrane domains (Fig. 13). This model is consistent with other kinds of data [128]: The amino terminus of PTH can activate a receptor from which nearly all the large extracellular domain has been removed [151], albeit with low affinity, and the
receptor is constitutively active if the amino-terminal activation domain of PTH is tethered to it [152]. Thus, the extracellular domain is required for high-affinity binding but is dispensable for receptor activation. In order to activate the PTH receptor, binding of hormone at the outer face of the plasma membrane must transmit a signal to the cell interior, such as a conformational change in the transmembrane domains that rearranges the intracellular loops of the receptor so that they can turn the G protein switch [128,129]. Direct evidence of an agonist-induced separation of transmembrane domains 3 and 6 of the PTH1R has been reported [153]. This presumably changes the conformation of the intracellular loops that connect the transmembrane domains (Fig. 13). Several residues in
5. Parathyroid Hormone and Calcium Homeostasis
FIGURE 13 Model of hPTH(1-34) binding to the PTH/PTHrP receptor. The receptor is shown in light gray and PTH (1-34) in dark gray. The locations of contact points between the ligand and receptor that were identified by crosslinking are indicated (Serl-M425, Lys13R 186, Trp23-T33/Q 37, Lys27-L261). Reprinted with permission from "The Parathyroids: Basic and Clinical Concepts, 2nd edition," (J. P. Bilezikian, R. Marcus, and M. A. Levine,Eds.) pp. 245-260. Academic Press: San Diego (2001). intracellular loops 2 (Lys 319) and 3 (Va1384,Leu 385,Thr 387, and Lys 388) are necessary for the receptor to efficiently couple to G proteins and are thus candidates as direct sites of interaction between the receptor and Gs to activate adenylyl cyclase or between the receptor and Gq to couple the receptor to phospholipase C [154,155] (Fig. 12). Mutations in Blomstrand chondrodysplasia inactivate PTH1R [156,157], producing an embryonic lethal disorder, and mutations in the second, sixth, and seventh transmembrane domains in Jansen's chondrodysplasia (Fig. 12) render the receptor constitutively active [158], producing a chondrodysplasia and hormone-independent hypercalcemia. In both cases, the chondrodysplasia results mainly from disruption of the developmental regulation of chondrogenesis by the other ligand of the PTH 1R, PTHrP. Chondrogenesis aside, the ability of the constitutively active PTH1R to mimic primary hyperparathyroidism provides strong evidence that the PTH1R is responsible for most of the actions of PTH in bone and kidney (see Chapter 3). Regarding PTH1R, these disorders identify the regions of the receptor that are potentially important for activation, but the precise role of these residues in activation of the receptor is unknown.
1 51
[159,160]. PTH 1R mainly binds to the heterotrimeric G proteins Gs and Gq [161,162]. Each of these G proteins consists of ~, [3, and 7 subunits; in the resting state, the subunit of the G protein is bound to GDP. When PTH binds, the receptor changes the conformation of the G protein such that GDP dissociates from G~ and is replaced by GTP (Fig. 14). GTP binding switches G~ to the "on" position and dissociates it from the [37 complex so that active G~ is freed to turn on effector proteins. The effectors will generate two intracellular second messengers: Gs~ activates the catalytic subunit of adenylyl cyclase to generate cyclic AMP (cAMP) from ATP, and Gq~ activates phospholipase C[3 to cleave inositol phosphates and produce the signaling intermediate 1,4,5inositol trisphosphate (IP3). The G protein cycle is completed when GTP is hydrolyzed to GDP by the intrinsic GTPase activity of G~; this switches off the G protein, terminating its signal, and permits reassociation of G~ with [37 to reconstitute the heterotrimeric resting state (Fig. 14). cAMP generated in response to PTH diffuses through the cell and turns on a specific serine/threonine protein kinase, protein kinase A (PKA), which is localized to signaling complexes within the cell by specific anchor proteins [41,42,163]. The targets for PKA include transporters [164], cytoplasmic signal transduction molecules that produce cross talk between the cAMP signaling pathway and other pathways [165], and the phosphorylation-sensitive transcription factor CRE-binding protein (CREB), whose activity is regulated by PKA [166]. The cAMP signal is terminated by enzymes called cyclic nucleotide phosphodiesterases, which hydrolyze cAMP to AMP [167,168]. cAMP is the primary intracellular second messenger for many of the actions of PTH in kidney and bone. The best evidence for this derives from the disorder pseudohypoparathyroidism (types 1A and 1B), in which mutations that reduce the amount of Gs~ produce target
Inactive Y ~y G proteinGDP-aI
~GTPa+13y Active ~ S ~ GprOtein Activation of AC, PLC, etc.
~
GDP-(x Pi
Intracellular Signaling by the PTH Receptor G proteins are simple switches that couple the PTH receptor to two intracellular signaling pathways
FIGURE 14 The activation cycle of a G protein. Reprinted with permission from "The Parathyroids: Basic and Clinical Concepts, 2nd edition," (J. P. Bilezikian, R. Marcus, and M. A. Levine,Eds.) pp. 245260. AcademicPress: San Diego, (2001).
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organ unresponsiveness to PTH (see Chapter 20). The individual roles of PKA and PKC in the actions of PTH in its target tissues are further discussed later. Phospholipase C[3 cleaves the phospholipid substrate phosphoinositol-4,5-bisphosphate in the plasma membrane to produce the intracellular second messenger IP3, which diffuses to the endoplasmic reticulum and induces the transient release of calcium from its stores [2,41,42]. The cleavage of phosphoinositol-4,5-bisphosphate also produces a diacylglycerol that, together with calcium, activates PKC. In general, the actions of PTH are more often dependent on cAMP than Ca 2+ as an intracellular second messenger. PKC may modulate part of the effect of PTH on sodium-dependent phosphate transport [169] and stimulation of vitamin D-1 ~hydroxylase in the kidney [170], but both are markedly reduced by Gs~ mutations in pseudohypoparathyroidism (see Chapter 20), and this establishes the primacy of cAMP regulation of these actions. Treatment of bone cells with PTH translocates the PKC isozymes PKC~ and PKC[31 to the plasma membrane. Activated PKC has a role with regard to the effects of PTH on interleukin-6 (IL-6) in bone cells [171,172].
Regulation of the PTH Receptor
Regulation of Receptor Protein Exposure to the PTH ligand modifies signaling by the PTH receptor in two ways [129]. The receptor can be uncoupled from its G protein partner; in this process, called desensitization, the response to PTH is considerably dampened by a prior treatment with PTH. Occupancy of the receptor by PTH will eventually lead to the disappearance of receptors from the cell surface, a phenomenon called receptor downregulation. The two processes have independent but related mechanisms. The mechanisms for acute desensitization involve a regulatory system of G protein receptor kinases (GRKs) and arrestin proteins that is also used for desensitization of other GPRCs and is best understood in the case of the [3-adrenergic receptor [173,174]. The PTH 1R is phosphorylated on its carboxyl-terminal tail by GRK2 in response to agonist binding [175,176] and this inhibits signaling [177]. Stable expression of a dominant-negative form of GRK2 suppresses PTH-induced desensitization of PTH 1R [178]. By analogy with other GPRCs, phosphorylation of the cytoplasmic tail of the PTH1R presumably promotes the binding of arrestin proteins to the receptor, which sterically hinder the binding of its G protein partners to the receptor and thereby uncouple it from them. Receptor downregulation begins with clustering of occupied PTH receptors in clathrin-coated pits in the plasma membrane [129,179]. The pits are sites of endo-
cytosis, where bits of plasma membrane with clustered PTH receptors are pinched off and internalized as vesicles. Once in endocytic vesicles, the PTH receptor can be recycled to the cell surface or can progress down the endocytic pathway for eventual destruction in lysosomes. Endocytosis of the PTH receptor requires the sequence Tyr-Gly-Pro-Met in the cytoplasmic tail [179]; this resembles a consensus sequence for internalization of other plasma membrane proteins. The role of receptor phosphorylation and arrestin binding in the internalization process is not fully understood. In the paradigm of the [3-adrenergic receptor, phosphorylation of the receptor induced by agonist binding leads to binding of arrestin, which targets the receptor to clathrincoated pits [173,174]. A PTH1R in which all of the GRK phosphorylation sites have been mutated is internalized normally, however [180]. Expression of a dominant-negative version of GRK impairs internalization [178], and activation of the PTH receptor, whether agonist induced or constitutive, results in translocation of arrestin from cytoplasm to the cell membrane and eventual colocalization with PTH1R in intracellular vesicles [181,182]. Thus, assumption of the active conformation of the PTH1R probably induces arrestin binding and thereby targets the receptor for internalization, but it is not clear whether agonist-induced phosphorylation of the PTH receptor is required for these events. How important is receptor regulation to the biology of parathyroid hormone action? Desensitization and downregulation of renal PTH receptors occur during infusion of PTH in vivo [183] and in models of vitamin D deficiency [184,185] and uremia [186-188]. The downregulation of PTH receptors in chronic vitamin D deficiency can be prevented by parathyroidectomy [189]; however, parathyroidectomy does not prevent receptor loss in uremia, so mechanisms other than agonist-induced downregulation must be operative [190]. When given intermittently, PTH(1-34) has a powerful anabolic effect on bone [191] and will be a valuable therapeutic in osteoporosis [192], but when administered continuously it has a catabolic effect and produces osteoporosis [191]. It is intriguing that downregulation of PTH receptors may account for the profound differences between the net effects of continuous and intermittent administration of PTH, and this issue is of intense interest.
Gene Regulation The PTH1R gene is complex, with 13 introns and at least two promoters, a downstream promoter that is used in all tissues that express the gene (which mediates the effects of PTHrP in many tissues), and an upstream promoter used in the kidney [193,194]. Whether these differences simply reflect opportunities for tissue-specific gene regulation or lead to expression of structurally dif-
5. Parathyroid Hormone and Calcium Homeostasis ferent forms of the P T H 1 R remains to be established [194,195]. Exposure to PTH reduces receptor m R N A levels in osteoblasts [196,197] but not in kidneys of rats with secondary hyperparathyroidism [190,198,199]. However, hypoparathyroidism, induced by either parathyroidectomy or dietary phosphate depletion, strongly upregulates P T H 1 R m R N A levels in rat renal cortex [200]. Whether regulation of PTH receptor gene expression is truly tissue specific, and whether it is significant to systemic calcium homeostasis, awaits more studies of receptor regulation in vivo.
Another PTH Receptor The carboxyl-terminal region of PTH [PTH(35-84)] is as highly conserved during evolution as PTH(1-34), even though it is completely dispensable for activation of PTH1R. Cleavage of PTH(1-84) produces long-lived carboxyl-terminal fragments, including those that are similar to PTH(7-84) [105-107] and a family of fragments whose amino terminus is between residues 34 and 43 [99,201]. Several lines of evidence suggest that there may be a distinct receptor for the carboxyl terminus of PTH that recognizes these fragments but does not recognize the amino-terminal activation domain for the P T H 1 R [e.g., PTH(1-34)]. Saturable, specific binding sites for carboxyl-terminal fragments can be demonstrated on osteoblasts and at high concentrations on an immortalized osteocyte cell line [108-111]. Infusion of such fragments lowers the serum calcium concentration in the parathyroidectomized rat and antagonizes the calcemic effect of PTH(1-84) [110]. Exposure to PTH (7-84) in vitro inhibits calcium release from bone explants in response to either PTH or 1,25(OH)zD and also inhibits the formation of osteoclast induced by vitamin D in murine marrow cultures [111]. Previous studies have reported other effects of carboxyl-terminal PTH fragments on osteoblast function [202-204]. The putative receptor remains to be identified, and the biological significance of cleavage of PTH into carboxyl-terminal fragments that antagonize the biologic effect of its amino-terminal fragments remains to be explained.
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therapeutic dose of hPTH(1-34) in osteoporotic women [205]. Sustained exposure to PTH also activates the process of osteoclastogenesis by which new osteoclasts appear. Chronic exposure to excess PTH produces a characteristic pattern of bone loss: osteoporosis with trabecular thinning and loss, intracortical tunneling, and subperiosteal bone resorption, the combination of which is characteristic of hyperparathyroid states (see Chapter 20). PTH induces bone resorption through activation of the R A N K L / R A N K system [46,206,207]. The receptor activator of NF-•B (RANK) is a member of the tumor necrosis factor (TNF) receptor family, and its ligand, R A N K ligand (RANKL), is a T N F family member. Evidence from genetic experiments indicates that virtually all bone resorption, whether induced by PTH, vitamin D, or a cytokine, utilizes the R A N K L / R A N K system as a final common pathway (see Chapter 2). R A N K L is expressed on the surface of stromal cells or osteoblasts, and it can be secreted as well. Activation of its receptor, R A N K , on monocyte/macrophage lineage cells is necessary, and exposure to M-CSF and R A N K L is sufficient to induce osteoclastogenesis. PTH induces osteoclastic bone resorption through an indirect effect on stromal cells and/or mature osteoblasts to increase the expression of R A N K L [208,209] and simultaneously to decrease the expression of OPG [210,211] (Fig. 15). Stimulation of osteoclastogenesis by PTH is blocked by antibodies to R A N K L [212] or by
P a r a t h y r o i d H o r m o n e Action in B o n e
Stimulation of Bone Resorption To protect the serum calcium concentration, PTH induces the resorption of bone so that calcium and phosphate are released into the blood. The acute increase in the serum calcium concentration occurs within hours and presumably involves the activation of preexisting osteoclasts. For example, serum calcium increases approximately 2 hr after administration of a
FIGURE 15 PTH induces osteoclast formation and activityby effects on RANKL and OPG. Osteoclast precursors proliferate and eventually fuse to form multinucleate osteoclasts in the presence of M-CSF and RANKL, which is presented to them on the surface of stromal cells. PTH simultaneously increases stromal cell expression of RANKL and decreases secretion of the decoy receptor OPG, which inhibits osteoclastogenesis. RANKL not only induces the formation of osteoclasts but also induces multinucleate osteoclasts on the bone surface to become active as bone-resorbing cells.
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OPG [208,213], and infusion of OPG into animals blocks the hypercalcemic response to PTH or PTHrP [214-216]. The role of stromal cells or osteoblasts as the primary recipient of the signal from PTH is consistent with the distribution of PTH receptors in bone: Marrow stromal cells and osteoblasts have receptors for PTH [217-220], but osteoclasts do not [218,221,222]. Whether immature marrow stromal cells or more mature cells of the osteoblast lineage are primarily responsible for nurturing osteoclast precursors is not clear, but RANKL levels decline and OPG levels increase during osteoblast maturation [223]. Bone resorption by mature osteoclasts in response to PTH has long been recognized as requiring coculture with osteoblasts or marrow stromal cells [224]. Highly purified cultures of isolated osteoclasts are unresponsive to PTH, but exposure to soluble RANKL is sufficient to induce bone resorption [225]. Thus, both acute effects of PTH on existing osteoclasts and chronic effects on the production of new osteoclasts are probably mediated by RANKL. It is conceivable that a parallel pathway exists in which other cytokines, such as IL-6 or IL-11, could mediate part of the effect of PTH on bone resorption [226-228]; if so, however, this is likely of secondary importance [229,230]. Stimulation of Bone Formation
Regarding calcium homeostasis, the stimulation of bone formation by PTH serves the function of linking new bone formation to bone resorption, thus restoring bone sacrificed to the demands of hypocalcemia. In the classical model of bone remodeling, bone-resorbing substances activate new teams of osteoclasts to resorb bone. After these osteoclasts have excavated a pit there is a reversal phase and then the formation of new bone in the excavated space (see Chapter 2). The signals that recruit osteoblasts to the remodeling site and control their activity have not been identified but are believed to be chemotactic signals generated by the release of factors from the resorbed bone matrix. Bone formation is coupled in space and time to prior bone resorption. The stimulation of bone formation in primary hyperparathyroidism cannot be completely explained by activation of bone remodeling units [231]. At the level of the bone remodeling unit, not only is activation frequency increased (as could be explained by a primary effect of PTH to activate bone resorption) but also the mineral apposition rate is increased, the duration of the active formation period is prolonged, and the mean wall thickness is increased [232,233]. PTH both increases the daily productivity of osteoblasts and extends their active work life by making each osteoblast more productive, by adding more osteoblasts, or both. As a result, trab-
ecular bone mass is preserved and may even be increased in primary hyperparathyroidism despite marked bone resorption [234,235]. The anabolic effect of PTH is markedly accentuated when the hormone is administered once daily--an observation [236] that has led to the introduction of PTH for osteoporosis therapy. Daily subcutaneous administration 6f PTH(1-34) causes rapid, dramatic expansion of cancellous and cortical bone mass in animals [191,237240] and humans [192,241-243], increases bone strength [244-246], and markedly reduces the incidence of new fractures in postmenopausal women [192]. It is not known why intermittent exposure to PTH has an anabolic effect whereas continuous high-dose PTH often produces net catabolism of bone [247]. It is possible that continuous exposure to PTH induces receptor downregulation and thereby diminishes the stimulation of an anabolic pathway. Intermittent treatment with PTH increases the activation frequency of bone multicellular units and greatly increases osteoblast surface [231,248-250]. The increase in osteoblast number or mineralizing surface is quantitatively greater than the increase in daily mineralization rate [248-250]; that is, there are more osteoblasts at work and/or they work longer, but they also work harder. By increasing bone formation, treatment with PTH and estrogen induces an increase in the connectivity of trabecular bone that does not occur during treatment with antiresorptive agents alone [249,251] and in high doses can virtually replace the marrow space with trabecular bone. Thus, treatment with once-daily PTH magnifies all the effects of primary hyperparathyroidism on osteoblast function. There is also preliminary evidence that PTH treatment, in addition to activating bone remodeling units, can induce bone formation on previously quiescent bone surfaces [252]. The cellular effects of PTH on osteoblasts are many [46] (Table 2), and it is not easy to synthesize a coherent view of the biochemical and cellular basis for the overall effects of PTH on osteoblast recruitment or osteoblast function. Consider osteoblast synthetic activity. PTH induces the expression of the immediate early gene families c-fos (cfos,fra-1, and fra-2) and c-jun (c-jun andjunD) [253-256]. This is mediated by phosphorylation of the transcription factor CREB by PKA [257-259] to induce binding to a CRE in the c-fos promoter [257,258]. PTH also causes cAMP-dependent phosphorylation of the transcription factor cbfal [260]. How these transcriptional effects are related to increases in osteoblast synthetic function is not clear, since the direct effect of acute PTH administration is to inhibit the transcription of the gene for type I collagen, the principal protein of bone matrix [46,261,262]. PTH also increases the number of osteoblasts on the bone surface--at least when given in anabolic regimens.
5. Parathyroid Hormone and Calcium Homeostasis TABLE 2
A.
Effects of PTH on b o n e cells.
Effects on osteoblast precursor cells. Activate immediate early genes
B.
Effects on osteoblasts.
Transcriptionfactors Induce expression of c-fos family (c-los, fra-1, fra-2) Induce expression of c-jun family (c-jun, junD)
Induce phosphorylation of cbfa-1
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Increase secretion of TIMP Increase secretion of tissue-type plasminogen activator, urokinase Decrease secretion of PAI- 1 Effects on osteocytes Required for c-fos response to mechanical stimulation Enhances calcium influx through mechanosensitive channels.
From reference 46.
Activate destruction of cbfa-1 in proteosomes Cytokines
Insulin-like growth factors Induce secretion of IGF-1 (rat) Induce secretion of IGF-1 and IGF-2 (mouse) Induce secretion of IFGBP-4, IGFBP-5 and IGFBP-RP-1
Transforming growth factor-J3 Increase secretion of TGF-[31 and TGF-[32 Interleukin-6 cytokine family Increase secretion of interleukin-6 Increase secretion of interleukin-11 Increase secretion of LIF Other cytokines and prostaglandins Induce expression of RANKL
Inhibit secretion of osteoprotegerin Increase secretion of GM-CSF Induce synthesis of prostaglandin G/H synthase-2 Induce secretion of prostaglandin E2 Ion channels
Depolarize osteoblasts, followed by sustained hyperpolarization Inactivate quinine-sensitive potassium channels Calcium entry through L-type voltage-gated calcium channels Open calcium-sensitive potassium channels Cell shape
Induce retraction of osteoblasts
Gap junctions Increase expression of connnexin-43 Open gap junctions Bone matrix proteins and alkaline phosphatase
Decrease transcription of col(I)A1 gene, inhibit collagen synthesis Increase synthesis and secretion of osteocalcin Increase secretion of bone sialoprotein Inhibit osteopontin gene expression Stimulate or inhibit expression of alkaline phosphatase Proteases and inhibitors z
Increase secretion of stromlysin Increase secretion of gelatinase B Increase expression and secretion of collagenase-3
PTH could increase osteoblast precursors growth in bone marrow, the proliferation of osteoblast precursors, or the recruitment of osteoblast precursors to the forming surface. Intermittent PTH may increase the entry of stromal cells into the osteoblast lineage [263], although the target cell for PTH remains to be found. Transcripts for the PTH/PTHrP receptor are absent or nonabundant in STRO-l-positive, alkaline phosphatase-negative marrow stromal cells [264,265], which are thought to represent early osteoblast precursors. Intermittent PTH does not increase the proliferation of osteoblast precursors: Neither the level of m R N A for the proliferation marker histone H4 [266] nor the number of [3H]thymidine-labeled nuclei on the bone surface are increased [267]. Intermittent exposure to PTH, however, could increase homing to the bone surface of late, postmitotic osteoblast precursors in the bone marrow, which have PTH receptors [217,222]. PTH increases the life span of the active osteoblast [231]. In the mouse, intermittent treatment with a high dose of PTH reduces the rate of osteoblast apoptosis [268], although PTH treatment actually increased the number of apoptotic osteoblasts in metaphyseal bone of young rats [269]. Although inhibition of apoptosis contributes to the anabolic effect of PTH, it is not clear whether the reduction in cell death is quantitatively sufficient to account for all of it. If the life span of an active osteoblast is several months [231], then the turnover rate of osteoblasts resulting in a reduction in the rate of apoptosis would be too slow to account for the rapid expansion of the osteoblast pool, which occurs within 1 week of the onset of PTH administration in rodent models. For example, if the osteoblast life span is 100 days, the turnover rate is 1% per day, and the maximal increase in osteoblast pool size expected from complete abolition of apoptosis is also 1% per day. If a reduction in apoptosis rate were the primary effect of PTH, increases not only in mean wall thickness but also in the duration of the active formation period would be expected. It is reasonably clear that mean wall thickness is increased by anabolic PTH regimens or in primary hyperparathyroidism, but whether the duration of the active formation period is also increased has not been resolved [233,248].
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In addition to increasing the rate of osteoblast precursor formation, the exit from a preosteoblast pool onto the bone surface, orthe active life span of working osteoblasts, PTH treatment may also recruit osteoblasts from the lining cells that cover quiescent bone surfaces [267]. Recruitment from this population would induce bone formation on previously quiescent surfaces [252]. Relatively few lining cells are needed to cover large areas of quiescent surface because the cells are flat; however, it seems unlikely that there are enough cells to account for the increase in osteoblast number following PTH treatment. The fact that so many possibilities exist for the anabolic effect of PTH is symptomatic of our profound ignorance regarding the basic cellular kinetics of the osteoblast population. In order to determine more definitively the mechanisms by which PTH has its anabolic effect, we must learn much more about the origin, life span, and fate of osteoblasts. Other cytokines could mediate the effects of PTH on bone formation [46,191,231]. Continuous exposure to PTH inhibits collagen synthesis by isolated rat calvaria, but exposure to PTH for the first 24 hr of a 72-hr experiment markedly increased collagen synthesis [270]. Stimulation of collagen synthesis by PTH is blocked by antibodies to insulin-like growth factor-1 (IGF-1), but proliferation is not [271]. Treatment of intact rats with PTH increases m R N A for IGF-1 [272] and the bone matrix content of IGF-1 [272a]. PTH also regulates expression of IGF binding proteins in bone [273]. Skeletal unloading causes resistance in vitro to IGF-1 and to the anabolic effect of PTH, suggesting that resistance to IGF1 may account for resistance to PTH [274]. Conditional mutagenesis of the IGF-1 receptor gene in bone, however, produces a high turnover state with markedly increased numbers of osteoblasts--not the depression of bone turnover that might be expected with blockade of PTH action [275]. Intermittent PTH treatment of rats increases the bone matrix content of TGF-[31 [272], raising the possibility that the anabolic effects of PTH observed with intermittent administration may also be mediated, at least in part, by increased secretion of this potent osteoblast growth and differentiation factor. The effect of PTH on TGF-[3~ may be PKC mediated, whereas its effect on TGF-[32 is PKA mediated [276]. The role of PTH in bone will not be understood until the role of PTHrP is determined. PTHrP is expressed in mature osteoblasts [217,277], periosteal cells [278], lining cells [217], and preosteoblasts [279-281]. Intimate interrelationships between paracrine effects of PTHrP and endocrine effects of PTH likely occur through their shared receptor. The phenotype of hypoparathyroidism, a low turnover bone state with preservation of the trabecular bone mass, is quite different from the phenotype
of the PTH 1R knockout, which has opposite changes in both cortical and trabecular bone compartments (with the caveat that the observations were made in late-stage mouse embryos with a severe chondrodysplasia) [282]. That knockout of the ligand has an effect opposite that of knockout of the receptor raises the possibility that a different ligand, PTHrP, dominates in the physiological regulation of trabecular osteoblasts. These issues can only be clarified by tissue-specific ablation or substitution of the genes for PTHrP and the PTH1R in bone using methods such as Cre-Lox technology.
PTH Action in t h e Kidney
Reabsorption of Calcium and Magnesium To protect the serum calcium concentration and prevent urinary calcium wasting in times of scarcity, PTH increases renal calcium reabsorption [283-285]. The major effect of PTH is to increase calcium reabsorption in the distal nephron, primarily the cortical thick ascending loop of Henle (cTAL) and the connecting tubule (CNT) [85,286-289]. Approximately 60% of filtered Ca 2+, but only 20% of filtered Mg 2+, is reabsorbed by the proximal tubule. Calcium reabsorption in the proximal convoluted tubule is passive and paracellular, driven mainly by the reabsorption of sodium, and it is mildly inhibited by PTH [286,290] because PTH inhibits two of its driving forces, sodium proton exchange and sodium phosphate cotransport (Fig. 16). Both Ca 2+ and Mg 2+ are passively reabsorbed in the cTAL [283,284,291]. This is a predominant site of Mg 2+ absorption because 60% of Mg 2+ but only 20% of Ca 2+ is reabsorbed in this segment. Passive paracellular reabsorption of both cations is driven by a lumen-positive transepithelial voltage gradient. This gradient is generated by Na+-K+-Ce2 transport by the NKCC2, C1C-Kb, and R O M K transporters [291] (Fig. 16). Paracellin-1, a member of the claudin family of tight-junction proteins that is expressed only in Henle's loop and the DCT, was recently identified as the cause of an autosomal recessive renal magnesium and calcium-wasting disorder [292] and is necessary for passive paracellular conductance of Ca 2+ and Mg 2+ [293]. PTH increases the conductance of the paracellular pathway and also the driving force for transport [291,294]. It is not known whether paracellin-1 is a direct target for PTH. The calcium-sensing receptor is also expressed in Henle's loop, and activation of this receptor by luminal Ca 2+ or Mg 2+ inhibits reabsorption of these ions by reducing the transepithelial voltage gradient [25]. The major sites of PTH action are the DCT and CNT, where the final 5-10% of Ca 2+ and Mg 2+ is absorbed. Here, PTH increases Ca 2+ uptake across apical
5. Parathyroid Hormone and Calcium Homeostasis
| 57
FIGURE 16 Calcium and magnesium reabsorption in PCT and cTAL. (Left) In the PCT, Ca 2+ and Mg 2+ are passively reabsorbed via paracellular routes at rates driven by the lumen-positive transepithelial voltage and limited by the conductance of the intercellular junctions for these cations. The transpeithelial voltage is generated by paracellular diffusion of C1- ions that are progressively concentrated along the lumen by active transcellular Na + reabsorption. Major mechanisms of Na + reabsorption include Na+/H + exchange and Na+-dependent cotransport of anions (phosphate, amino acids, sulfate, etc.). (Right) In cTAL, Ca 2+ and Mg 2+ reabsorption again occurs via voltage-dependent paracellular transport. Apical NKCC2 cotransporters and ROMK K + channels maintain the lumen-positive transepithelial voltage necessary for cation transport, which is inhibited by Ca2+/MgZ+-dependent activation of the CaSR and by the loop diuretic furosemide. The channel protein paracellin-1 appears to be critical for paracellular cation transport in the cTAL and could be a target for CaSRs and PTHRs, which respectively reduce and augment cation transport in this nephron segment. Reprinted with permission from "The Parathyroids: Basic and Clinical Concepts, 2nd edition," (J. P. Bilezikian, R. Marcus, and M. A. Levine, Eds.) pp. 245-260. Academic Press: San Diego (2001).
membranes of distal tubular cells [283-285,295-297], the likely rate-limiting step in transepithelial calcium transport (Fig. 17). The molecular nature of the apical calcium channel is not entirely established: Closely related (possibly identical) channels [295], CaT2 [298] and ECaC1 [299], are candidate molecules, but neither has been shown to be regulated by PTH. Interestingly, both ECaC1 and another related transporter, CAT1, are expressed in the apical membrane of intestinal enterocytes and both are vitamin D sensitive [300,301]. Thus, in the two epithelia in which external calcium balance is determined, the intestinal enterocyte and the renal tubule epithelium, vitamin D and PTH may operate the same calcium-transporting machinery. Calcium may be ferried across the cell by a vitamin Ddependent calcium-binding protein, calbindin-D28K [302-304]. This would provide a means to move calcium while maintaining a low intracellular [Ca2+]. The import-
ance of the physiological role of calbindin is not fully resolved. A preliminary report indicates that renal calcium wasting occurs in calbindin-D28K knockout mice [304], but urinary calcium wasting does not occur in vitamin D-deficient states, suggesting that efficient calcium resorption in the distal nephron does not absolutely require a vitamin D-dependent transport step. There are two potential mechanisms for extrusion of calcium from renal epithelial cells. Increased Na+/Ca 2+ exchange has been demonstrated following PTH administration in vivo and in vitro [305-307], and PTH's effect is abolished either by elimination of the sodium gradient that drives exchange or by removing extracellular Ca 2+ [308,309]. PTH may also increase Ca 2+ extrusion by activating the basolateral CaZ+-ATPase [310], although this is not observed in all systems [311]. It is not known where or how PTH stimulates calcium transport in the distal nephron. A number of studies
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also be inserted into the apical membrane by means of vesicle trafficking [315]. Finally, PTH reportedly hyperpolarizes distal tubule cells, at least in part by increasing a basolateral chloride conductance [316], and this could secondarily affect either calcium entry or calcium efflux through the sodium-calcium exchanger.
Reabsorption of Phosphate PTH induces phosphaturia, presumably as a mechanism to dump phosphate that is absorbed along with calcium under the influence of vitamin D or that is released from bone along with calcium when bone is resorbed. The absence of the phosphaturic effect of PTH was the principal evidence used by Albright et al. [317] to conclude that target organ unresponsiveness to PTH is the basis of pseudohypoparathyroidism. PTH inhibits phosphate reabsorption by reducing the number of transporters in the apical membrane (Fig. 18)
FIGURE 1 7 PTH regulation of distal tubular calcium reabsorption. In DCT, Ca 2+ reabsorption involves apical Ca 2+ entry via voltagesensitive Ca 2§ channels and subsequent basolateral extrusion by Ca2+-ATPases and Na+/Ca 2+ exchangers driven by Na+-K+-ATPase. Multiple Ca 2+ channels are expressed here. Calbindin D28K binds and shuttles Ca 2§ from the apical membrane to the basolateral sites of active Ca 2+ extrusion, thereby buffering the cytoplasm from high concentrations of transported Ca 2+. Calbindin D28K is induced by 1,25 (OH)2D3 and may directly activate apical Ca 2+ channels, which otherwise are inhibited by intracellular Ca 2+ ions. PTHR activation leads to insertion of additional apical Ca 2+ channels, hyperpolarization of the cell (via enhancing basolateral C1- exit?) and, thus, activation of Ca 2+ channels, increased calbindin D28K expression, and stimulation of the basolateral Ca2-ATPase. The routes and mechanisms of Mg 2+ reabsorption in DCT are unknown. Reprinted with permission from "The Parathyroids: Basic and Clinical Concepts, 2nd edition," (J. P. Bilezikian, R. Marcus, and M. A. Levine, Eds.) pp. 245-260. Academic Press: San Diego (2001).
indicate that cAMP mimics the effect of PTH in isolated renal tubule segments perfused in vitro [296,308]. However, it is also reported that the effect of PTH is blocked by inhibitors of PKC but not PKA [312]. Phosphorylation of MEK by a PKC-dependent pathway has been implicated [313]. Although the rate-limiting calcium entry step is subject to regulation by PTH [285,296,297] and ECaC1 has a putative PKC phosphorylation site, it remains to be determined whether the calcium transporter is directly phosphorylated. An assembly protein such as NHERF [164,314] could be involved in assembly of a multiprotein signaling complex since the calcium channel has a PDZ interaction domain similar to that on the sodium-proton exchanger, for which regulation of a multiprotein complex is important [164]. Additional transporters could
FIGURE 18 Phosphate (Pi) reabsorption in the proximal tubule. Phosphate must be actively transported across the apical membrane of the PCT cell because of the strongly interior-negative potential and the fact that cytosolic Pi concentration (1 mM) is approximately 100fold higher than equilibrium. This transport is accomplished by an electrogenic type IIa NaPi cotransporter that is energized by the steep transmembrane Na § gradient established by the basolateral Na+-K § -ATPase. Activity of this cotransporter is reduced by PTHR activation. Mechanisms of basolateral Pi exit are not well understood, but an anion exchanger could allow Pi to leave the cell passively. Reprinted with permission from "The Parathyroids: Basic and Clinical Concepts, 2nd edition," (J. P. Bilezikian, R. Marcus, and M. A. Levine, Eds.) pp. 245L 260. Academic Press: San Diego (2001).
5. Parathyroid Hormone and Calcium Homeostasis
[169,318]. Three sodium phosphate cotransporters have been cloned. Two, the type I and type IIa NaPi cotransporters, are expressed on apical membranes of the proximal tubule, but the type IIa transporter is the main target of regulation by PTH. The level of the type IIa transporter on the apical membrane is reduced by treatment with PTH [319,320], and disruption of the type IIa cotransporter gene in the mouse largely abolishes hormone-sensitive phosphate reabsorption [321]. The type IIa cotransporter is predicted to have eight transmem brane domains and is responsible for approximately 70% of sodium-coupled phosphate reabsorption in the proximal tubule [322]. Its level on the apical membrane is also regulated by the intake of dietary phosphate, so transporter density and phosphate reclamation are maximized by a low dietary phosphate content [169,318]. The type I NaPi cotransporter is a less specific anion transport protein and the type III cotransporter is expressed in many cell types and may serve cellular housekeeping functions [169]. PTH controls the level of type IIa NaPi cotransporters on the apical membrane by regulating membrane trafficking. PTH increases the retrieval of cotransporters from the apical membrane by targeting them to clathrin-coated pits in the intermicrovillar clefts. Once there, the transporter is incorporated into vesicles together with soluble proteins from tubule fluid and internalized (Fig. 19). Vesicles containing internalized NaPi cotransporters are degraded in lysosomes, and there is no evidence that cotransporters can be recycled onto the cell surface [323,324].
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The signaling pathway most important for inhibition of phosphate reabsorption by PTH is the cAMP pathway. There is compelling clinical evidence for this conclusion in pseudohypoparathyroidism, in which a deficiency of Gs0~ is responsible for renal unresponsiveness to the phosphaturic effect of PTH (see Chapter 20). cAMP mimics the effect of PTH [325]. In cultured renal cells, inhibition of phosphate transport is correlated with activation of PKA [326], and blockade of the cAMP pathway prevents inhibition of phosphate uptake by PTH but blockade of the PKC pathway does not [327,328]. However, it is also clear that PTH can inhibit sodium-coupled phosphate transport by using the PKC signaling pathway. Analogs of PTH with minimal adenylyl cyclase-stimulating activity are effective [329,330], and some workers report that blockade of the PKC pathway can inhibit PTH effects [331]. It thus appears that cAMP is necessary for inhibition of phosphate reabsorption but may not be sufficient, and the PKC pathway can also be used. From studies of perfused tubules it appears that an apical receptor for PTH, which faces the tubule lumen, can inhibit phosphate transport via PKC, whereas the receptor facing the bloodstream on the basolateral membrane of the tubule cell uses PKA [332]. Whether PTH filtered at the glomerulus is biologically active in the tubule remains to be determined. The molecular site targeted by PTH has not been identified. It may well not be the transporter; the type IIa cotransporter has PKC phosphorylation sites, but mutation of these residues does not abolish inhibition of transport by PTH [333,334]. A domain containing two
FIGURE 19 Regulation ofNaPi cotransport by PTH. Activation of the PTH1R on the basolateral membrane of PCT cells stimulates PKA and PKC. Activation of PKA or PKC induces a rapid decrease in activity of NaPi-2 transporters expressed on the apical surface. This may involve phosphorylation of one or more intermediary proteins (X) because consensus PKC phosphorylation sites within the NaPi-2 protein can be eliminated without affecting the regulatory effect of PKC. At least part of the PTH effect involves retrieval of surface NaPi-2 cotransporters by a microtubule-dependent process of endocytosis, lysosomal fusion, and degradation. Reprinted with permission from "The Parathyroids: Basic and Clinical Concepts, 2nd edition," (J. P. Bilezikian, R. Marcus, and M. A. Levine, Eds.) pp. 245-260. Academic Press: San Diego (2001).
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intracellular loops of the transporter is required for the effect of PTH [334]. The type IIa cotransporter interacts with a Na+/H + exchanger regulatory factor family member (NHERF-1) [314,335] and other proteins containing a PDZ domain [336], and this interaction could be important for sorting of receptors to the apical membrane, for retrieval of receptors by PTH, or both. Reabsorption of Bicarbonate
Hyperparathyroid states may be accompanied by a mild renal tubular acidosis. This acidosis is often subtle, but mild hyperchloremia is characteristic of primary hyperparathyroidism and was formerly used as a clinical sign in the differential diagnosis of hypercalcemia. The locus of this effect is the proximal renal tubule, where PTH markedly inhibits sodium bicarbonate reabsorption. Most of the rejected sodium and bicarbonate is reabsorbed in the distal nephron, so the effect on the final urine is small, but increased distal bicarbonate delivery increases distal reabsorption of calcium. PTH inhibits the activity of the sodium-proton exchanger NHE3 [283,337,338]. The receptor is phosphorylated by PKA [339,340], and this leads to an alteration in an allosteric modifier site on the cytoplasmic surface [341] and an acute reduction in sodium-proton exchange, followed 20-30 min later by a loss of receptors from the apical membrane [339]. It appears that the association of PKA with NHE3 requires the formation of a multiprotein signaling complex organized by the adapter protein NHERF-1, which brings together NHE3 and PKA bound to the scaffolding protein ezrin [314,335]. Because NHERF and related PDZ-domain scaffolding proteins are also involved in transporter and receptor trafficking [314,342], it is conceivable that a similar multiprotein complex is also an intermediate in the internalization of NHE3. Although cAMP is clearly the major second messenger for PTH with regard to bicarbonate reabsorption, dual signaling through PKC does occur [138,343]. PTH also inhibits the activity of Na+-K+-ATPase activity in the basolateral border of proximal tubule cells [344]. This inhibition, unlike many of the other transport effects of PTH, appears to be calcium and PKC mediated and to involve the generation of eicosanoids by activation of phospholipase A2 [345,346]. Vitamin D Metabolism
The effects of PTH on renal vitamin D metabolism are closely coordinated with the effects of mineral ions and vitamin D to produce exquisite regulation of the levels of 1,25(OH)2D (see Chapter 7). Two important enzymes of vitamin D metabolism are found in the kidney, both in proximal tubule. The renal 25-hydroxyvitamin D-I~-
hydroxylase (1 ~-hydroxylase) is the final step in the synthesis of the active vitamin D metabolite, and its activity is exquisitely regulated. PTH increases l~-hydroxylase activity by a direct effect on transcription of the gene [22,347] that is primarily mediated by cAMP, although some reports indicate that PKC can also be a second messenger for PTH [170]. Renal 25-hydroxyvitamin D-24-hydroxylase (24hydroxylase) is an enzyme that has the opposite effect of that of l~-hydroxylase. Both 25-hydroxyvitamin D and 1,25(OH)ED are substrates for 24-hydroxylase, and 24hydroxylation is an important degradative pathway for 1,25(OH)ED: Ablation of 24-hydroxylase leads to massive accumulation of 1,25(OH)ED with hypercalcemia and osteomalacia [348]. PTH inhibits the activity of 24-hydroxylase, thus inhibiting the metabolism of 1,25(OH)ED and its production by l~-hydroxylase, cAMP is the main second messenger for this inhibition [349,350], which results from reduced stability ofmRNA for the enzyme [351].
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Taylor, P., Brown, E. M., and Thakker, R. V. (1996). A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the calcium-sensing receptor. N. Engl. J. Med. 335, 1115-1122. 32. Nemeth, E. F., and Fox, J. (1999). Calcimimetic compounds: A direct approach to controlling plasma levels of parathyroid hormone in hyperparathyroidism. Trends Endocrinol. Metab. 10, 66-71. 33. Strewler, G. J. (2000). Medical approaches to primary hyperparathyroidism. Endocrinol. Metab. Clin. North Am. 29, 523-539. 34. Nemeth, E. F., Steffey, M. E., Hammerland, L. G., Hung, B. C., Van Wagenen, B. C., DelMar, E. G., and Balandrin, M. F. (1998). Calcimimetics with potent and selective activity on the parathyroid calcium receptor. Proc. Natl. Acad. Sci. USA 95, 4040-4045. 35. Olgaard, K., and Lewin, E. (2001). Prevention of uremic bone disease using calcimimetic compounds. Annu. Rev. Med. 52, 203-220. 36. Wada, M., Furuya, Y., Sakiyama, J., Kobayashi, N., Miyata, S., Ishii, H., and Nagano, N. (1997). The calcimimetic compound NPS R-568 suppresses parathyroid cell proliferation in rats with renal insufficiency. Control of parathyroid cell growth via a calcium receptor. J. Clin. Invest. 100, 2977-2983. 37. Wada, M., Nagano, N., Furuya, Y., Chin, J., Nemeth, E. F., and Fox, J. (2000). Calcimimetic NPS R-568 prevents parathyroid hyperplasia in rats with severe secondary hyperparathyroidism. Kidney Int. 57, 50-58. 38. Peacock, M., Shoback, D. M., Greth, W. E., Binder, T. A., Graves, T., Brenner, R. M., Turner, S. A., and Marcus, R. (2002). The calcimimetic AMG 073 reduces serum calcium (Ca) in patients with primary hyperparathyroidism (PHPT). J. Bone Miner. Res. 16 (Suppl 1). 39. Shoback, D. M., Thatcher, J., Leombruno, R., and Brown, E. M. (1984). The relationship between PTH secretion and cytosolic calcium concentration in dispersed bovine parathyroid cells. Proc. Natl. Acad. Sci. USA 81, 3113-3117. 40. Shoback, D. M., Membrefio, L. A., and McGhee, J. G. (1988). High calcium and other divalent cations increase inositol triphosphate in bovine parathyroid cells. Endocrinology 123, 382-389. 41. Gardner, D. G. (1997). Mechanisms of hormone action. In Basic and Clinical Endocrinology (F. S. Greenspan and G. J. Strewler, Eds.), 5th ed., pp. 58-75. Appleton & Lange, Stamford, CT. 42. Kahn, C. R., Smith, R. J., and Chin, W. W. (2002). Mechanism of action of hormones that act at the cell surface. In Williams Textbook of Endocrinology (J. D. Wilson, D. W. Foster, H. M. Kronenberg, and P. R. Larsen, Eds.), 9th ed., pp. 95-143. Saunders, Philadelphia. 42a.Membreno, L., Chen, T. H., Woodley, S., Gagucas, R., and Shoback, D. (1989). The effects of protein kinase-C agonists on parathyroid hormone release and intracellular free Ca 2+ in bovine parathyroid cells. Endocrinology 124, 789-797. 42b.Kifor, O., Diaz, R., Butters, R., and Brown, E. M. (1997). The CaZ+-sensing receptor (CAR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells. J. Bone Miner. Res. 12, 715-725. 42c.Kifor, O., MacLeod, R. J., Diaz, R., Bai, M., Yamaguchi, T. Yao, T., Kifor, I., and Brown, E. M. (2001). Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK193 cells. Am. J. Physiol. RenalPhysiol. 280, F291-F302. 42d.Bourdeau, A., Souberbielle, J. C., Bonnet, P., Herviaux, P., Sachs, C., and Lieberherr, M. (1992). Phospholipase-A2 action and arachidonic acid metabolism in calcium-mediated parathyroid hormone secretion. Endocrinology 130, 1339-1344. 42e.Marx, S. J., Attic, M. F., Levine, M. A., Spiegel, A. M., Downs, R. W., Jr., and Lasker, R. D. (1981). The hypocalciuric or benign variant of familial hypercalcemia: Clinical and biochemical features in fifteen kindreds. Medicine (Baltimore) 60, 397.
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5. Parathyroid Hormone and Calcium Homeostasis 312. Hoenderop, J. G., De Pont, J. J., Bindels, R. J., and Willems, P. H. (1999). Hormone-stimulated Ca 2+ reabsorption in rabbit kidney cortical collecting system is cAMP-independent and involves a phorbol ester-insensitive PKC isotype. Kidney Int. 55, 225-233. 313. Sneddon, W. B., Liu, F., Gesek, F. A., and Friedman, P. A. (2000). Obligate mitogen-activated protein kinase activation in parathyroid hormone stimulation of calcium transport but not calcium signaling. Endocrinology 141, 4185-4193. 314. Weinman, E. J. (2001). New functions for the NHERF family of proteins. J. Clin. Invest. 108, 185-186. 315. Bacskai, B. J., and Friedman, P. A. (1990). Activation of latent Ca 2+ channels in renal epithelial cells by parathyroid hormone. Nature 347, 388-391. 316. Garcia-Ocana, A., Galbraith, S. C., Van Why, S. K., Yang, K., Golovyan, L., Dann, P., Zager, R. A., Stewart, A. F., Siegel, N. J., and Orloff, J. J. (1999). Expression and role of parathyroid hormone-related protein in human renal proximal tubule cells during recovery from ATP depletion. J. Am. Soc. Nephrol. 10, 238-244. 317. Albright, F., Burnett, C. H., Smith, P. H., and Parson, W. (1942). Pseudo-hypoparathyroidism--An example of"Seabright-Bantam syndrome." Endocrinology 30, 922-932. 318. Hernando, N., Karim-Jimenez, Z., Biber, J., and Murer, H. (2001). Molecular determinants for apical expression and regulatory membrane retrieval of the type IIa Na/Pi cotransporter. Kidney Int. 60, 431-435. 319. Kempson, S. A., Lotscher, M., Kaissling, B., Biber, J., Murer, H., and Levi, M. (1995). Parathyroid hormone action on phosphate transporter mRNA and protein in rat renal proximal tubules. Am. J. Physiol. 268, 784-791. 320. Lotscher, M., Kaissling, B., Biber, J., Murer, H., and Levi, M. (1997). Role of microtubules in the rapid regulation of renal phosphate transport in response to acute alterations in dietary phosphate content. J. Clin. Invest. 99, 1302-1312. 321. Zhao, N., and Tenenhouse, H. S. (2000). Npt2 gene disruption confers resistance to the inhibitory action of parathyroid hormone on renal sodium-phosphate cotransport. Endocrinology 141, 2159-2165. 322. Beck, L., Karaplis, A. C., Amizuka, N., Hewson, A. S., Ozawa, H., and Tenenhouse, H. S. (1998). Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities. Proc. Natl. Acad. Sci. USA 95, 5372-5377. 323. Malmstrom, K., and Murer, H. (1987). Parathyroid hormone regulates phosphate transport in OK cells via an irreversible inactivation of a membrane protein. FEBS Lett. 216, 257-260. 324. Pfister, M. F., Ruf, I., Stange, G., Ziegler, U., Lederer, E., Biber, J., and Murer, H. (1998). Parathyroid hormone leads to the lysosomal degradation of the renal type II Na/Pi cotransporter. Proc. Natl. Acad. Sci. USA 95, 1909-1914. 325. Agus, Z. S., Puschett, J. B., Senesky, D., and Goldberg, M. (1971). Mode of action of parathyroid hormone and cyclic adenosine 3',5'-monophosphate on renal tubular phosphate reabsorption in the dog. J. Clin. Invest. 50, 617. 326. Martin, K. J., McConkey, C. L., Garcia, J. C., Montani, D., and Betts, C. R. (1989). Protein kinase-A and the effects of parathyroid hormone on phosphate uptake in opposum kidney cells. Endocrinology 125, 295-301. 327. Segal, J. H., and Pollock, A. S. (1990). Transfection-mediated expression of a dominant cAMP-resistant phenotype in the opossum kidney (OK) cell line prevents parathyroid hormone-induced inhibition of Na-phosphate cotransport. A protein kinase-Amediated event. J. Clin. Invest. 86, 1442-1450. 328. Martin, K. J., McConkey, C. L., Jacob, A. K., Gonzalez, E. A., Khan, M., and Baldassare, J. J. (1994). Effect of U-73,122, an
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inhibitor of phospholipase C, on actions of parathyroid hormone in opossum kidney cells. A. J. Physiol. 266, 254-258. Cole, J. A., Forte, L. R., Eber, S., Thorne, P. K., and Poelling, R. E. (1988). Regulation of sodium-dependent phosphate transport by parathyroid hormone in opossum kidney cells: Adenosine 3',5'-monophosphate-dependent and-independent mechanisms. Endocrinology 122, 2981-2989. Cole, J. A., Carnes, D. L., Forte, L. R., Eber, S., Poelling, R. E., and Thorne, P. K. (1989). Structure-activity relationships of parathyroid hormone analogs in the opossum kidney cell line. J. Bone Miner. Res. 4, 723-730. Quamme, G., Pfeilschifter, J., and Murer, H. (1989). Parathyroid hormone inhibition of Na+/phosphate cotransport in OK cells: Requirement of protein kinase C-dependent pathway. Biochim. Biophys. Acta 1013, 159-165. Traebert, M., Volkl, H., Biber, J., Murer, H., and Kaissling, B. (2000). Luminal and contraluminal action of 1-34 and 3-34 PTH peptides on renal type IIa Na-P(i) cotransporter. Am. J. Physiol. Renal Physiol. 278, F792-F798. Hayes, G., Busch, A., Lang, F., Biber, J., and Murer, H. (1995). Protein kinase C consensus sites and the regulation of renal NaPi cotransport (NaPi-2) expressed in Xenopus laevis oocytes. Pflugers Arch. 430, 819-824. Karim-Jimenez, Z., Hernando, N., Biber, J., and Murer, H. (2000). A dibasic motif involved in parathyroid hormone-induced down-regulation of the type IIa NaPi cotransporter. Proc. Natl. Acad. Sci. USA 97, 12896-12901. Zizak, M., Lamprecht, G., Steplock, D., Tariq, N., Shenolikar, S., Donowitz, M., Yun, C. H., and Weinman, E. J. (1999). cAMPinduced phosphorylation and inhibition of Na(+)/H(+) exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE regulatory factor. J. Biol. Chem. 274, 24753-24758. Gisler, S. M., Stagljar, I., Traebert, M., Bacic, D., Biber, J., and Murer, H. (2001). Interaction of the type IIa Na/Pi cotransporter with PDZ proteins. J. Biol. Chem. 276, 9206-9213. Pollock, A. S., Warnock, D. G., and Strewler, G. J. (1986). Parathyroid hormone inhibition of Na+-H + antiporter activity in a cultured renal cell line. Am. J. Physiol. 250, 217-225. Helmle-Kolb, C., Montrose, M. H., and Murer, H. (1990). Parathyroid hormone regulation of Na+/H + exchange in opossum kidney cells: Polarity and mechanisms. Pflugers Archiv. Eur. J. Physiol. 416, 615-623. Zhao, H., Wiederkehr, M. R., Fan, L., Collazo, R. L., Crowder, L. A., and Moe, O. W. (1999). Acute inhibition of Na/H exchanger NHE-3 by cAMP. Role of protein kinase a and NHE-3 phosphoserines 552 and 605. J. Biol. Chem. 274, 3978-3987. Collazo, R., Fan, L., Hu, M. C., Zhao, H., Wiederkehr, M. R., and Moe, O. W. (2000). Acute regulation of Na+/H + exchanger NHE3 by parathyroid hormone via NHE3 phosphorylation and dynamin-dependent endocytosis. J. Biol. Chem. 275, 31601-31608. Miller, R. T., and Pollock, A. S. (1987). Modification of the internal pH sensitivity of the Na+/H + antiporter by parathyroid hormone in a cultured renal cell line. J. Biol. Chem. 262, 9115-9120. Weinman, E. J., Steplock, D., and Shenolikar, S. (2001). Acute regulation of NHE3 by protein kinase A requires a multiprotein signal complex. Kidney Int. 60, 450-454. Azarani, A., Goltzman, D., and Orlowski, J. (1995). Parathyroid hormone and parathyroid hormone-related peptide inhibit the apical Na+/H + exchanger NHE-3 isoform in renal cells (OK) via
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Gordon l-Strewler a dual signaling cascade involving protein kinase A and C. J. Biol. Chem. 270, 20004-20010. Zhang, Y., Norian, J. M., Magyar, C. E., Holstein-Rathlou, N. H., Mircheff, A. K., and McDonough, A. A. (1999). In vivo PTH provokes apical NHE3 and NaPi2 redistribution and NaK-ATPase inhibition. Am. J. Physiol. 276, 711-719. Ominato, M., Satoh, T., and Katz, A. I. (1996). Regulation of NaK-ATPase activity in the proximal tubule: Role of the protein kinase C pathway and of eicosanoids. J. Membrane Biol. 152, 235-243. Derrickson, B. H., and Mandel, L. J. (1997). Parathyroid hormone inhibits Na(+)-K(+)-ATPase through Gq/Gll and the calciumindependent phospholipase A2. Am. J. PhysioL 272, 781-788. Murayama, A., Takeyama, K., Kitanaka, S., Kodera, Y., Kawaguchi, Y., Hosoya, T., and Kato, S. (1999). Positive and negative regulations of the renal 25-hydroxyvitamin D3 lalphahydroxylase gene by parathyroid hormone, calcitonin, and lalpha,25(OH)2D3 in intact animals. Endocrinology 140, 2224-2231.
348. St. Arnaud, R., Arabian, A., Travers, R., Barletta, F., RavalPandya, M., Chapin, K., Depovere, J., Mathieu, C., Christakos, S., Demay, M. B., and Glorieux, F. H. (2000). Deficient mineralization of intramembranous bone in vitamin D-24-hydroxylase-ablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D. Endocrinology 141, 2658-2666. 349. Henry, H. L. (1985). Parathyroid hormone modulation of 25hydroxyvitamin D3 metabolism by cultured chick kidney cells is mimicked and enhanced by forskolin. Endocrinology 116, 503-510. 350. Matsumoto, T., Kawanobe, Y., and Ogata, E. (1985). Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 in a cloned monkey kidney cell line (JTC-12). Biochim. Biophys. Acta 845, 358-365. 351. Zierold, C., Mings, J. A., and DeLuca, H. F. (2001). Parathyroid hormone regulates 25-hydroxyvitamin D(3)-24-hydroxylase mRNA by altering its stability. Proc. Natl. Acad. Sci. USA 98, 13572-13576.
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Phosphate Homeostasis Regulatory Mechanisms JOSEPH CAVERZASIO,* HEINI MURER,t and HARRIET S. TENENHOUSE ~ *Division of Bone Diseases, University Hospital of Geneva, Geneva, Switzerland tinstitute of Physiology, University of Zurich, Zurich, Switzerland ~Departments of Pediatrics and Human Genetics, McGill University Children's Hospital Research Institute, Montreal Quebec, Canada
PHYSIOLOGICAL ASPECTS
other 10-20% is in soft tissues (primarily muscle and internal organs), extracellular fluid, and erythrocytes. In soft tissues, Pi comprises 0.1-0.3% of wet weight and is contained intracellularly as phosphorylated sugars, phospholipids, phosphoproteins, nucleic acids, and Pi. In blood, Pi is present in both erythrocytes and plasma. In plasma, phospholipids comprise the main fraction (2.2-3.1mmol/liter), followed by phosphate esters (0.9-1.5mmol/liter) and Pi (0.7-1.4mmol/liter). The concentration of Pi in plasma is not maintained at a steady-state level like that of calcium. It varies among animal species, with the highest levels in fish and the lowest in the adult human. In the fasting human, the plasma Pi concentration varies with age: 0.711.36 mmol/liter in adults, 1.28-2.0 mmol/liter in children, and 1.39-2.67 mmol/liter in newborns. The amount of Pi in plasma and extracellular fluid, approximately 15 mmol in the adult human, represents less than 0.1% of the total phosphate in the body. Plasma Pi concentrations are a reflection of the various fluxes entering and leaving the extracellular pool. Pi enters this pool from the intestine, soft tissues, and bone. It leaves the extracellular fluid through the urine, as a result of the difference between glomerular filtration and net tubular reabsorption, by back flux into the intestinal lumen, and by transfer into soft tissue and bone.
Distribution of P h o s p h a t e in the Body
Intestinal Absorption of P h o s p h a t e
The adult human body contains 15-20 tool of Pi, 8090% of which is present in bone as hydroxyapatite. The
The Pi content of foodstuffs is variable. The concentration, expressed per 100 grams, is highest in fish,
INTRODUCTION During the development and maturation of vertebrates, selective regulatory mechanisms have evolved to maintain the extracellular concentration of inorganic phosphate (Pi) at a relatively high level compared with that prevailing in the adult organism. This homeostatic process fulfills the increased need for Pi that results from increased rates of cellular growth and skeletal mineral deposition. The mechanisms responsible for the regulation of Pi homeostasis during growth are not completely understood and likely involve growth factors that also affect bone growth directly or indirectly. The importance of Pi in bone growth and mineralization is best illustrated by the occurrence of growth and skeletal abnormalities in children with Mendelian hypophosphatemic disorders. This chapter summarizes the physiological aspects of Pi homeostasis, particularly those related to growth, the molecular mechanisms involved in renal Pi transport, and the role of novel Pi regulatory factors encoded by the P H E X and FGF23 genes that are mutated in X-linked and autosomal dominant hypophosphatemic disorders, respectively.
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liver and other organs, eggs, meat, milk, and cheese (30-200mmol). Furthermore, Pi is present in bread (30-60 mmol), vegetables (10-30 mmol), and fruit (3-10mmol). The presence of Pi in so many foodstuffs explains the rarity of Pi deficiency. Usually, a diet with enough calcium and protein is largely sufficient in Pi. The average Pi intake in humans is approximately 30-50 mmol/day. The minimum requirement is approximately 25 mmol/day for males and slightly higher for children and pregnant women. Pi is absorbed in the small intestine, where the Pi absorptive capacity is highest in the duodenum and lowest in the ileum. Pi absorption occurs by a nonsaturable, paracellular pathway as well as by a saturable energy-requiring Na-dependent process that has been localized to the mucosal surface. This explains why increasing dietary Pi intake results in a proportional increment in net Pi absorption, with no indication of saturation in intact animals. This also explains why inadequate Pi absorption results primarily from decreased Pi availability rather than from changes in the intrinsic capacity of intestinal Pi transport. A large body of evidence indicates that the active transfer of Pi across the mucosal membrane involves a vitamin D-regulated, Na-dependent high-affinity transport system [14,42,92,122]. The vitamin D sterols were shown to activate Na-dependent Pi transport by increasing the maximal velocity (Vmax) of the carrier system. The molecular identification of an intestinal Na/Pi cotransporter that is regulated by dietary Pi is described later. Extrarenal Soft Tissue Handling of P h o s p h a t e In contrast to the growing knowledge of Pi transport in intestine, bone, and particularly kidney, the molecular mechanisms mediating Pi fluxes in soft tissues remain poorly understood. Moreover, their precise role in the maintenance of serum Pi concentration and the regulatory mechanisms governing the rate of Pi transfer from the extracellular and intracellular compartments remain unclear. Evidence suggests that hormones involved in modulating carbohydrate metabolism also play a role in the regulation of soft tissue Pi flux. For example, insulin elicits a rapid increase in Pi uptake by rat hepatocytes in primary culture [25] and in skeletal muscle [123], by increasing the Vmaxof a Na-dependent Pi transport system without influencing the Km. These findings suggest that insulin mediates the insertion of preformed Pi carriers into the plasma membrane. The stimulation of Pi uptake by insulin likely occurs in response to the insulin-dependent increase in glucose metabolism, a process that utilizes ATP and increases the demand for Pi from oxidative phosphorylation.
P h o s p h a t e Transport a n d Bone A major function of bone is to act as a mechanical support. This is achieved by the mineralization of an extracellular matrix formed by osteogenic cells. Pi is essential for osteogenic function not only because it is an integral component of hydroxyapatite crystal but also because it can affect the rate of bone matrix production and bone resorption [32,65] and is essential for intracellular energy production. As mentioned previously, the plasma Pi concentration is higher in growing individuals than in adults, most likely to accommodate the increased requirement for Pi during growth and skeletal development. Although the mechanisms responsible for the regulation of Pi homeostasis during growth are not completely understood, growth factors that also affect bone growth, either directly or indirectly, likely play a role. The importance of Pi in bone development is suggested by abnormalities associated with hypophosphatemic disorders [2,9] such as X-linked hypophosphatemia, a metabolic bone disease occurring in both humans and mice (discussed later). Until recently, little was known about Pi transport in bone cells. However, recent studies demonstrate that this process is a relevant component of bone cell physiology and serves to regulate bone formation and remodeling [32,65].
Osteoclast Function and Phosphate Transport Osteoclasts are polarized cells involved in bone resorption and are exposed to high ambient Pi concentrations during the active resorptive process. Osteoclasts possess specific Pi transport systems, including a Na/Pi cotransporter whose activity is dependent on ATP production and Na+,K+-ATPase activity to maintain the inwardly directed Na gradient driving force for Pi cotransport [65]. When osteoclasts are isolated in vitro and allowed to attach to bone particles, Pi transport is stimulated, without an increment of protein synthesis [65]. The stimulatory effect of bone particles on Pi transport is prevented by peptides containing the ArgGly-Asp cell adhesion motif, implicating the involvement of integrins and cell matrix interaction in the regulation of Pi transport. The osteoclast Na/Pi cotransporter probably belongs to the type IIa family [64]. Pi transport is essential to osteoclast function and provides the substrate for ATP synthesis, which is required to support the high rates of proton transport involved in bone resorption.
Phosphate Transport and Osteogenic Cell Function Like all eukaryotic cells, osteogenic cells (i.e., osteoblasts and chondrocytes) are endowed with
6. Phosphate Homeostasis Regulatory Mechanisms
Na-dependent Pi transport systems driven by an inwardly directed Na gradient. There are several differences between Na-dependent Pi transport in osteoblasts and that in renal epithelial cells. Affinity constants for Pi are significantly higher in osteoblasts (300-500 vs 50100 txM for renal epithelial cells) and Na/Pi cotransport in osteoblasts is stimulated by an acidic extracellular pH [35], whereas renal epithelial Pi transport is stimulated by more alkaline pH. PTH, a potent osteotropic factor, stimulates Na/Pi cotransport in osteoblasts [136] but inhibits transport in renal proximal cells. Additional studies are necessary to understand the cellular and molecular mechanisms underlying these differences. Other factors that stimulate Na/Pi cotransport in osteoblasts, such as insulin-like growth factor-1 (IGF-1), platelet-derived growth factor, and fibroblast growth factor (FGF), also enhance the proliferation and/or the differentiation of bone-forming cells [103,119,143,172]. The cellular mechanisms by which these growth factors increase Pi transport in osteogenic cells are not completely understood but may involve a tyrosine phosphorylation process [32]. In addition, fluoride, another potent
IGF-1
PDGF
~
Fluoride
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activator of bone formation, also stimulates Pi transport in osteoblastic cells via the activation of a tyrosine phosphorylation process [34]. Role of Pi Transport in Matrix Vesicle Function
As illustrated in Fig. 1, Pi transport in osteogenic cells not only provides sufficient Pi for metabolic processes but also serves a specialized function in matrix vesicles, which play a role in the initial events of bone matrix calcification. Primary mineralization is regulated by chondrocytes, osteoblasts, and odontoblasts and occurs in epiphyseal cartilage, embryonic bone, postnatal ossification, and the development of predentin. Two distinct but not mutually exclusive theories--collagen nucleation and matrix vesicles production--have been proposed to explain the mechanism involved in the initiation of calcification in skeletal tissues [168]. The first holds that the collagen fibril is the major site of crystal nucleation and that Ca/Pi crystals are deposited along the entire length of collagen fibrils. The alternative theory involves the participation of matrix vesicles, which are produced by
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FIGURE 1 Schematic representation of the regulatory factors and signal transduction mechanisms involved in the stimulation ofNa/Pi cotransport in osteoblast-like cells. Experimental evidence suggests parallel regulation of Pi transport activity in the plasma membrane of bone-forming cells and their derived matrix vesicles. With the notion that Pi transport is important for accumulation of mineral ions, it is hypothesized that Pi transport in matrix vesicles plays a significant role in the control of processes involved in the calcification of the bone matrix.
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osteogenic cells and serve as nucleation sites for mineralization of the extracellular matrix. Matrix vesicles are small organelles (100-200nm in diameter) that have been observed in mineralizing tissues in contact with newly formed mineral crystals. Matrix vesicles arise from bone-forming cells by a process of budding from elongated tubular extensions that project from the plasma membrane of these cells [4]. The mechanism for matrix vesicle-mediated calcification is not fully understood. Several lines of evidence indicate that uptake of Ca and Pi by matrix vesicles, from the extracellular environment, is required for initiation of calcification [51]. Ca enters matrix vesicles by a protease-sensitive carrier that may be related to the annexins [55]. Matrix vesicles also express a Na-dependent Pi transport system that is similar to that in osteoblasts and chondrocytes [104], further suggesting that these structures are derived from the plasma membrane of osteogenic cells. Formation of Pi-Ca-phospholipid complexes in the vesicular space is believed to maintain a favorable Pi gradient, allowing the continuation of Pi transfer into matrix vesicles. In this system, Na only facilitates translocation of Pi across the membrane of matrix vesicles since a mechanism for maintaining the Na gradient is not present in these structures. Accumulation of Pi through this system allows calcification to occur inside matrix vesicles. The calcified vesicles then serve as nucleators of matrix calcification. Studies have shown that Pi transport activity is low in matrix vesicles released during the proliferative phase of osteoblast development but is significantly increased during osteoblast differentiation, peaking at the time of bone matrix formation [104]. The increase in Pi transport activity in matrix vesicles released during the formation of the collagenous matrix may be explained by an enrichment of Pi carriers during the differentiation process. Because Na/Pi cotransport in osteogenic cells is regulated by calciotropic hormones and growth factors, regulation of Pi transport in released matrix vesicles may represent a mechanism by which osteogenic cells modulate the calcification of extracellular matrix. The previously mentioned in vitro observations suggest that Pi transport plays an important role in osteogenic cell- and matrix vesicle-mediated calcification. To address the relevance of these in vitro findings, the molecular identity and expression of Pi transporter(s) in osteogenic cells in vivo were investigated. Data obtained indicate that the type III Na/phosphate cotransporter Pit- 1 (Glvr- 1) is expressed in osteoblastic and chondrocytic cells and is regulated by osteotropic factors [62,119,120]. In situ hybridization analyses of Pit-1 expression in developing embryonic metatarsals revealed that this Pi transporter is expressed only in a subset of hypertrophic chondrocytes during endochondral bone formation in a region in which matrix mineralization proceeds, an observation consist-
ent with a potential role for this Pi transporter in matrix calcification [121]. The recent description of the murine Pit-1 gene and its promoter region [120] will allow more detailed analyses of the functional role of this transporter in primary bone calcification. Renal Pi Transport Na/Pi cotransport across renal epithelial cells is an important determinant of plasma Pi concentration [15]. In an adult human, the plasma Pi concentration is ~l.2mmol/liter, the glomerular filtration rate ~120ml/ min, and the amount of Pi filtered daily ~210mmol. If 20% of the filtered load is excreted in the urine, the mass of Pi reabsorbed is-168 mmol/day. This net renal reabsorptive flux for Pi is ~10 times greater than the net intestinal absortive flux in adults with a dietary Pi intake of 25 mmol/day and an intestinal fractional absorption of 70%. Furthermore, the renal reabsorptive flux for Pi varies with the dietary Pi intake and the utilization of Pi by the organism [18,19]. Thus, the tubular reabsorption of Pi (TRPi) plays a central role in the regulation of plasma Pi level and Pi homeostasis. By altering the level of Pi in the extracellular fluid, the net TRPi can also influence the intracellular pool of Pi in the various tissues and the amount of Pi in bone mineral.
Overall Tubular Pi Transport In the mammalian kidney, Pi transport is a saturable process with no appreciable simple diffusion component and is characterized by a maximum rate of Pi transfer across the tubular epithelium from lumen to blood. The maximum rate is not an absolute constant but varies with the physiological or pathological condition [13,15,17,18]. The tubular maximum for Pi reabsorption per unit of glomerular filtration rate (TmPi/GFR) represents the most reliable quantitative estimate of the overall tubular Pi transport capacity. Most, if not all, significant regulatory factors or conditions that influence Pi homeostasis by altering the renal handling of Pi have been shown to exert their effect on TmPi/GFR. A very strong correlation exists between TmPi/GFR and fasting plasma Pi concentration [15,17], indicating that the capacity of the renal tubule to transport Pi is a major determinant ofextrarenal Pi homeostasis. Therefore, an understanding of the mechanisms that underlie changes in TmPi/GFR is key to our understanding of Pi homeostasis in health and disease.
Tubular Localization of Pi Reabsorption Under normal conditions, ~80-90% of the Pi filtered load is reabsorbed in the proximal tubule [13,139]. There is evidence for inter- and intranephron heterogeneity in
6. Phosphate Homeostasis Regulatory Mechanisms
proximal tubular Pi transport capacity, with higher transport rates in early ($1/$2) vs late ($3) and in deep (juxtamedullary) vs superficial (cortical) proximal tubular segments [13,139]. A small fraction of Pi reabsorption may also occur in the distal and terminal segments of the nephron [13,17]. The molecular mechanisms of proximal tubule Pi transport are described later.
Regulation of Renal Pi Transport Parathyroid Hormone
Chronic changes in parathyroid hormone (PTH) status, observed clinically or elicited experimentally, are associated with alterations in tubular Pi transport capacity. For example, in chronic hypo- or hyperparathyroid states, TmPi/GFR is increased or decreased, respectively, relative to the normal state. The mass of cAMP produced by the kidney, as estimated by the amount of cAMP excreted in the urine per milliliter of GF, is usually found to be decreased or increased in chronic hypo- and hyperparathyroidism, respectively. These findings suggest that the alterations in renal Pi handling observed in chronic states of hypo- or hyperparathyroidism are cAMP dependent. The molecular mechanism by which cAMP influences tubular Pi transport in the proximal tubule is described later. Renal Adaptation to Changes in Dietary Pi
Dietary Pi intake influences its tubular reabsorption and urinary excretion. Pi reabsorption is increased in animals fed a low Pi diet and reduced in animals on a high Pi diet. The tubular adaptation to dietary Pi is independent of PTH and calcitonin and is evident in thyroparathyroidectomized (TPTX) animals [141,164]. It is also independent of growth hormone production, as shown by studies ofhypophysectomized animals [30]. However, the response to dietary Pi restriction is influenced by growth [28]. Of interest is the finding that Pi restriction causes resistance to the phosphaturic effect of PTH [58]. In vivo micropuncture studies have shown that Pi reabsorption by proximal tubule is markedly enhanced in Pi-deprived animals. They also suggest that the adaptive response to changes in dietary Pi may involve distal nephron sites [106]. The adaptive changes in the tubular capacity to reabsorb Pi are paralleled by changes in the Na-dependent component of Pi transport across the brush-border membrane (BBM) of proximal tubular cells. The response of the BBM to changes in dietary Pi intake is specific for Pi transport. There is no change in the Na-dependent or -independent uptakes of L-proline or D-glucose measured in the same BBM vesicle preparations [110]. The effects of dietary Pi on Pi transport are
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reflected by changes in gma x of the BBM Na/Pi cotransporter, without a change in the affinity (Km) of the transporter for Pi, indicating an increase in the number of transporters in the BBM [110]. The adaptive response to dietary Pi restriction is more pronounced and occurs more rapidly in young animals compared to adults, as demonstrated by clearance studies [28]. Two distinct responses have been observed in the renal adaptation to dietary Pi restriction. An early phase occurs rapidly and is detectable in BBM vesicles within 2-4 hr [27,95]. This is preceded by a decline in serum and urine Pi concentration. It is independent of PTH and does not require de novo protein synthesis. This early phase of adaptation likely involves changes in the rate of endocytosis and exocytosis of Na/Pi cotransporters between the BBM and endosomes. A similarly rapid but opposite adaptation in BBM Pi transport is also observed after an intravenous Pi load [37]. The chronic phase of adaptation to dietary Pi restriction takes several days to complete [164], requires de novo synthesis of new transporters [110], and participates in the long-term maintenance of Pi homeostasis. Dissociation between Na/Pi Cotransport and TmPi/GFR
Several studies have demonstrated that BBM Na/Pi cotransport is not always related to TmPi/GFR. For example, in adult rats fed control and low Pi diets, the diet-induced changes in BBM Na/Pi cotransport do not correspond to those in TmPi/GFR, determined by clearance studies in intact animals [29]. Moreover, in the Hyp mouse, an animal model for X-linked hypophosphatemia, a similar dissociation between BBM Na/Pi cotransport and TmPi/ml GFR was reported [105,151]. Taken together, these findings suggest that the BBM of the proximal tubule cell may not be the only site involved in the adaptive response and are consistent with the demonstration that the distal nephron contributes to the adaptive process [106]. Growth and Renal Pi Transport
The plasma Pi concentration is higher in infants than in children or adults [24]. Although a low GFR contributes to the retention of Pi in infancy [22], it is well documented that tubular Pi reabsorption is higher in infants and plays a major role in the maintenance of high serum Pi concentration during growth. In children, the high serum Pi levels were thought to be related to increased GH levels [60]. However, the difference in plasma GH levels between children and adults was not striking, and in pitiutary dwarfs serum Pi levels are still higher than in adult control patients [40]. Therefore, the difference in serum Pi levels between children and adults cannot be due entirely to differences in plasma GH concentrations.
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Animal studies also demonstrated that both TmPi/ GFR and plasma Pi concentrations are lower in adult when compared to young growing animals [28]. The dependence of renal Pi transport on growth was also shown in thyroparathyroidectomized rats, indicating that the change in renal Pi transport with growth or age is independent of PTH [28]. In addition to the PTH/ adenylate cyclase system, neither calcitonin nor thyroxine appear to be involved in the age-dependent change in tubular Pi transport. Furthermore, the difference in renal Pi handling between young, growing and adult rats could not be attributed to changes in GFR, plasma calcium level, renal handling of Na, alterations in blood carbon dioxide tension, or the acid-base status of the animals [28]. Moreover, the lower TmPi/GFR in adult compared with young rats could not be corrected by chronic administration of GH. The age-related differences in renal Pi handling appear to correlate with the plasma concentration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D], the active hormonal form of vitamin D that stimulates the intestinal absorption of Pi and Ca [28]. However, the finding that chronic administration of 1,25(OH)2D did not increase renal tubular Pi transport capacity in experimental animals [20] strongly suggests that this vitamin D metabolite is not involved in the change of renal Pi transport with growth or age. Rather, it suggests the existence of a common mechanism for the regulation of renal Pi transport and 1,25(OH)2D production with growth or age. The contribution of the growth hormone somatomedin axis to growth- and age-dependent changes in renal Pi transport and serum Pi concentration was also investigated. Chronic administration of IGF-1 increased TmPi/ ml GFR and plasma Pi in both hypophysectomized (HPX) rats and TPTX-HPX rats compared with vehicle-treated animals [31]. In HPX animals, the IGF1-dependent increase in renal Pi handling and plasma Pi was not associated with a consistent change in plasma Ca concentration or in urinary Ca excretion. The change in tubular Pi transport in response to IGF-1 was evident at the level of the BBM, consistent with proximal tubular involvement. IGF-1 also elicited a corresponding increase in plasma concentration of 1,25(OH)2D in HPX rats [31], suggesting that IGF-1 mimics the changes in tubular Pi transport with growth. In addition to its renal effects on the tubular Pi reabsorption and the production of 1,25(OH)2D, IGF-1 is also involved in the regulation of bone growth. In vivo and in vitro studies demonstrate that GH and IGF-1 have important regulatory roles in the rates of differentiation and proliferation of chondrocytes in the epiphyseal growth plate. It has been suggested that local production of IGF-1 is an important mediator of GH action in bone. IGF-1 stimulates both proliferation and differentiation
al.
of cultured osteoblasts and increases type 1 collagen synthesis, alkaline phosphatase activity, and osteocalcin production. Thus, IGF-1 appears to be an important factor in orchestrating essential events in Pi homeostasis for bone growth and development [33].
CELLULAR AND MOLECULAR ASPECTS Cellular M e c h a n i s m s
Secondary Active Transport Pi is taken up from the tubular fluid across the BBM by a Na-dependent Pi transport system(s); transcellular Pi transport is completed at the basolateral membrane by transport pathways that are not well characterized (Fig. 2) [113,114]. Both anion exchange mechanisms and Pi leak pathways have been proposed [113,114]. Na-dependent influx of Pi across the basolateral membrane has also been described and may play a role when apical Pi influx does not meet the cellular requirements.
+
basolateral
luminal
ATP
3Na+ ~ Pi
ADP+Pi
xNa+ org. anions ", I ~
v " Pi
ly
+Pi
I
2K+
I
3Na+
FIGURE 2 Secondary active Na/Pi cotransport. A type IIa Na/Pi cotransporter mediates apical Pi influx. The role of the apical type I Na/Pi cotransporter is unclear. The basolateral membrane contains the primary active Na/K pump and several Pi transport pathways that include a type III Na/Pi cotransport pathway mediating housekeeping Pi influx and poorly defined Pi efflux mechanisms. For further information see text and Refs. [113] and [114].
6. Phosphate Homeostasis RegulatoryMechanisms Studies on the physiological and pathophysiological regulation of overall renal Pi handling suggest that apical (brush-border) Na/Pi cotransport is rate limiting [113,114]. A hallmark of tubular Pi reabsorption, higher transport rates at alkaline pH values, is also a property of BBM Na/Pi cotransport [3,113,114]. Renal NA/P! Cotransporter(s)
Apical (Type I vs Type IIA) Molecular biological approaches led to the structural identification of renal BBM Na/Pi cotransporters [107,108,112-114]. Injection of mRNA, isolated from kidney cortex, led to an increase in Na-dependent Pi uptake in Xenopus laevis oocytes. This observation was the basis for the expression cloning of Na/Pi cotransporter cDNAs that stimulate high-capacity, Na-dependent Pi uptake when expressed in oocytes or in a variety of cell lines in culture [107,108,112-114].
Type I Na/Pi Cotransporter Immunohistochemical analysis of renal sections and RNA localization studies indicated that the type I "transporter" localizes to the proximal tubular BBM. However, the inter- and intranephron heterogeneity described previously was not observed for the type I Na/Pi cotransporter. Furthermore, after expression in X. laevis oocytes, type I-mediated Pi uptake did not exhibit the characteristic pH dependence of proximal tubular Pi transport [113,114]. Detailed expression studies of X. laevis oocytes indicated that the type I transporter protein shows very distinct anion channel properties and that the kinetic properties of induced Na-dependent Pi transport function was similar to that of the intrinsic oocyte Pi transport activity [23,113,114,170]. Furthermore, physiological conditions known to regulate proximal tubular Pi handling altered neither type I protein nor mRNA expression. Thus, the type I Na/Pi cotransporter seems not to be a major determinant of proximal tubular Pi handling [113,114].
Type lla Na/Pi Cotransporter The type IIa Na/Pi cotransporter is highly expressed in the proximal tubular BBM and in subapical vesicular structures. The corresponding mRNA is found only in proximal tubules. The inter- and intranephron heterogeneity of expression corresponds with that of proximal tubular reabsorptive capacities. When expressed in a variety of heterologous systems, its transport properties correspond to those observed in isolated BBM [113,114]. The type IIa Na/Pi cotransporter is a major determinant of proximal tubular Pi transport and thus a key
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player in renal Pi reabsorption [93,107-109,111-114]. Interestingly, the rate of Pi transport is predominantly regulated by altering the abundance of type IIa protein in BBM (e.g., in PTH-induced phosphaturia and in dietary Pi-dependent alterations). In addition, genetically determined Pi wasting disorders are associated with altered expression levels of this transporter. Finally, gene deletion and antisense-induced suppression of corresponding type IIa Na/Pi cotransporter mRNA documented that N80% of BBM Na/Pi cotransport activity can be attributed to the type IIa Na/Pi cotransporter protein [10,118]. A related Na/Pi cotransporter (type IIb) is expressed in small intestinal BBM as well as in other epithelial tissues but not in kidney. It also plays a role in the physiological regulation of intestinal Pi reabsorption [69,71]. The gene encoding the human type IIa cotransporter (NPT2) maps to chromosome region 5q35 and the corresponding mouse gene (Npt2) to chromosome region 13B [66,87,88]. The gene structure is conserved in both species--~16 kb in length consisting of 13 exons and 12 introns [66]. The promoter structure is also conserved in the species analyzed to date (opossum, mouse, human, and rat) and contains sequences suggested to be important for transcriptional regulation by bicarbonate/carbon dioxide tension, vitamin D, and low Pi medium and for restricted expression in proximal tubular cells [67,76,83, 113,144,145]. However, because type IIa-mediated changes in renal Pi transport in vivo are not always associated withcorresponding changes in mRNA levels, the physiological significance of transcriptional regulation of type IIa gene expression remains to be determined [107,108,112-114]. The mechanism for type IIa-mediated Na/Pi cotransport has been studied in great detail [52,113]. The fully loaded carrier translocates three Na ions and one Pi ion, with a preference for the divalent ion species, thus explaining in part the higher transport rates at alkaline pH [3,52,53]. Due to the 3:1 (Na:Pi) stoichiometry, a net inward movement of a positive charge occurs during cotransport, making this transporter sensitive to alterations in the transmembrane electrochemical gradient for Na (i.e., Na concentration gradient and membrane potential). Na binding is sequential: The first Na ion interacts with the negatively charged empty carrier and permits interaction with Pi, followed by interaction with two additional Na ions. Phosphonoformic acid blocks the carrier after the first Na interaction, thereby explaining its inhibitory characteristics [52,113]. In addition to the previously mentioned effects on the mono/divalent Pi ratio, pH dependence is explained by the sensitivity of the translocation step to pH (empty carrier) and by competition with Na interaction [3,53]. The current knowledge of the molecular structure and topology of the type IIa Na/Pi cotransporter is limited.
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The transporter has an even number of transmembrane segments with cytoplasmic-oriented NH2 and COOH termini, which are separated by a large glycosylated extracellular loop into two domains, each essential for transport activity and containing functionally important subdomains [113]. The third predicted intracellular loop (ICL-3) contains amino acid residues that contribute to regulated type IIa internalization/endocytosis, and the COOH-terminal tail contains sequences that are involved in apical delivery/expression [79,80]. Although type IIa transporters might be clustered by interacting proteins [57], each transporter unit is fully capable of mediating Na/Pi cotransport [86]. Basolateral (Type III vs Others)
Type III Na/Pi Cotransporter Two viral receptors have been shown to mediate Na/Pi cotransport after expression in X. laevis oocytes [81,113]. The transporters have been named Pit-1 and Pit-2 (Glvr-1 and Ram-l) and are now designated as type III Na/Pi cotransporters, mRNA localization and immunofluorescence studies demonstrated the ubiquitous expression of type III Na/Pi cotransporters in kidney cell plasma membranes, including the basolateral membrane of proximal tubular cells [154 and unpublished observation]. Type III Na/Pi cotransporters mediate the transport of three Na to one Pi, a process that depends on the driving force (i.e., electrochemical Na gradient) and the influx of Pi from the peritubular interstitium [81,113]. Their role seems to be that of housekeeping Na/Pi cotransport-that is, the maintenance of sufficient cellular Pi if apical delivery is insufficient for the cellular metabolic requirements.
Other Pi Transporters There is an almost complete lack of information on the basolateral Pi exit pathway that completes the transcellular reabsorptive flux of Pi. Studies on intact tubules have provided evidence for a rather nonspecific "leak" pathway, and studies with isolated basolateral membrane vesicles have provided evidence for Na-dependent and Na-independent mechanisms [110,113,114]. The former could be the type III Na/Pi cotransporter and the latter an unidentified stilbene-inhibitable anionexchange mechanism. Regulation: Type lla NA/P~ C o t r a n s p o r t e r as t h e Key Player "Fast" vs "Slow" Mechanisms Changes in the abundance of type IIa Na/Pi cotransporter protein in the renal BBM can account for the
al.
majority of physiological and pathophysiological situations in which renal Pi handling is subject to regulation. For example, an increase in proximal tubular Pi reabsorptive flux is associated with an increase in type IIa protein expression and a decrease in Pi reabsorptive flux is associated with a decrease in protein expression [107,108,112-114]. Both slow (hours, days, or weeks, e.g., in response to chronic changes in dietary Pi intake, different Pi demand/growth, and ontogeny/aging) and fast mechanisms (minutes or hours, e.g., in response to PTH and other peptide hormones) can lead to altered type IIa cotransporter expression and to corresponding changes in Pi reabsorption. The majority of changes in Na/Pi cotransport and type IIa cotransporter expression occur in the absence of corresponding changes in type IIa mRNA levels, perhaps with the exception of prolonged treatment with 1.25-(OH)ED [145], T3 [50], prolonged dietary Pi deprivation or Pi overload [125], and prolonged changes in PTH levels [82]. Obviously, changes in ontogeny/aging are associated with changes in mRNA levels [140,162]. Fast mechanisms leading to decreased or increased type IIa transporter expression and associated transport activity are the result of transporter retrieval (decrease) or transporter insertion (increase) from or into the BBM (Fig. 3) [107,108,111,112]. Membrane Traffic in Fast Regulation
Agonist-induced, rapid retrieval of the type IIa Na/Pi cotransporter from the BBM has been documented in response to increases in PTH levels [82], nitric oxide (NO), and atrial natriuretic peptide (ANP) [6] and also to increases in dietary Pi supply (even in the absence of PTH) [94,125]. Internalization of the transporter occurs primarily at intermicrovillar clefts [113,163]. The microtubular network does not participate in the initial internalization step but rather in the delivery of the internalized protein to lysosomes [96,97,113]. In contrast, rapid insertion (exocytosis) of the type IIa transporter into the apical membrane (e.g., in response to Pi deprivation) depends on an intact microtubular network [96]. Membrane retrieval and reinsertion mechanisms require molecular specificity. For retrieval, the cellular machinery has to recognize the transporters to be internalized, and for reinsertion molecular information is required for apical expression/delivery. The predicted third intracellular loop, ICL-3, contains two basic amino acids required for internalization [79]. The mechanism whereby the internalization signal is recognized has not been delineated. Similarly, the COOH-terminal tail contains signals for apical expression, which include the terminal three amino acids and an internal pair of basic amino acids [80]. Studies have demonstrated that recognition for regulatory internalization and apical insertion
6. Phosphate Homeostasis RegulatoryMechanisms
(
:c
);
c
)
T T
mRNA
(~
FIGURE 3 Membranetraffic in the control of expression of apical type IIa Na/Pi cotransporter. Regulatoryretrieval occurs at intramicrovillar clefts and can be followed by lysosomaldegradation. Reinsertion of the cotransporter can be preceded by de novo synthesis. For further information, see text and Refs. [113]and [114]. Ly, lysosomes; LE, late endosomes; SAC, subapical compartment; O, type IIa transporters.
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Both protein kinase A and protein kinase C activities are involved in PTH-mediated regulation of renal Na/Pi cotransport. PTH signaling occurs via basolateral PTH receptors and preferentially involves the protein kinase A pathway [113,114]. Interestingly, PTH receptors are also present in the BBM, where their effects are preferentially mediated by protein kinase C [113,114]. PTH also activates several extracellular signal-regulated kinases [91]. ANP and NO inhibit Na/Pi cotransport by cGMPmediated internalization. Similar to PTH, ANP can exert its effect in mice from both cell surfaces of the proximal tubules [6]. Changes in the cytosolic Ca concentration do not seem to be a sufficient trigger for fast alterations in membrane expression of the type IIa Na/Pi cotransporter [113,114]. Participation of tyrosine kinase in the regulatory increase in Na/Pi cotransport activity and corresponding cotransporter expression has been suggested. However, the participation of this regulatory mechanism in fast regulation of tubular Pi handling remains to be documented [113,114]. A variety of cellular signaling mechanisms have been proposed to mediate the action of a number of hormones on renal Na/Pi cotransport [113,114]. However, in most studies, tissue culture models were used and molecular information on the transport pathway affected was not always available. Thus, in most cases, it is not possible to conclusively interpret the data.
PATHOPHYSIOLOGICAL ASPECTS requires a specific cellular context and specific interacting protein(s) [57,701.
Signaling Mechanisms Evidence suggests that transporter internalization is associated with Pi transport inhibition. However, it is not clear whether the transporter is first inhibited and then internalized, as was shown for the BBM Na/H exchanger NHE-3 [112]. Moreover, the signaling trigger for internalization, in addition to that described for ICL-3, is not known. Given that different protein kinases participate in regulation, direct protein phosphorylation is an obvious mechanism. However, there is no evidence to suggest that direct phosphorylation plays a role in the regulation of the type IIa Na/Pi cotransporter [75]. These data do not preclude a phosphorylation event at an intermediary step (e.g., at the level of the previously mentioned interacting proteins). Nevertheless, the participation of intracellular protein kinase-mediated regulatory cascades has been well documented for the regulatory internalization of the type IIa Na/Pi cotransporter by PTH.
Hypophosphatemic States Since Pi is sufficiently abundant in the diet, Pi deficiency is unlikely to develop except in unusual circumstances (Table 1) [73,74]. For the purpose of this chapter, the discussion is limited to four well-characterized hypophosphatemic disorders associated with renal Pi wasting. Three, X-linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets (ADHR), and hereditary hypophosphatemic rickets with hypercalciuria (HHRH), are inherited, and the fourth, oncogenic hypophosphatemic osteomalacia (OHO), is acquired. The discussion focuses primarily on the renal Pi transport phenotype in each of these disorders and their underlying pathophysiology.
X-Linked Hypophosphatemia XLH is characterized by growth retardation, rachitic and osteomalacic bone disease, hypophosphatemia, and renal defects in the reabsorption of filtered Pi and the metabolism of vitamin D (Table 2) [146,147]. The features
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Joseph Caverzasio et al. TABLE 1 Causes of Hypophosphatemia 1. Decreased renal reabsorption
Hyperparathyroidism- primary, secondary Renal tubular defects- Fanconi, fructose intolerance Diabetic ketoacidosis Metabolic or respiratory acidosis Renal transplantation ECF fluid expansion Mendelian hypophosphatemias Oncogenic hypophosphatemicosteomalacia 2. Decreased intestinal absorption
Malabsorption- steatorrhea, chronic diarrhea Malnutrition- starvation Administration of phosphate binders, antacid abuse Vitamin D deficiency- nutritional, familial 3. Miscellaneous causes
Nutritional repletion Respiratory alkalosis Recovery from metabolic acidosis (diabetic ketosis) Sepsis, especiallygram-negativebacteremia "Hungry-bone" syndromeafter parathyroidectomy Insulin therapy
that distinguish XLH from other Mendelian hypophosphatemias are its X-linked dominant mode of inheritance, its higher prevalence (1 in 20,000), and the availability of two murine homologs, Hyp [48] and Gy [98]. The latter have served as valuable models to examine the mechanism for renal Pi wasting in XLH [134,147,156]. Lessons from Hyp and Gy Mice
Both Hyp and Gy mice exhibit significant hypophosphatemia, secondary to increased urinary Pi excretion, relative to filtered Pi (Table 2) [48,98]. The defect in Pi
reabsorption was localized to the proximal tubule by micropuncture studies [41,56], persists after parathyroidectomy [41,85], is specific for Pi [84,98,150,155,157], and involves a decrease in the maximal velocity (Vmax) of a high-affinity, low-capacity Na/Pi cotransport system that resides in the renal BBM [149,153]. These findings highlight the importance of the high-affinity Pi transport system in the overall maintenance of Pi homeostasis and suggest that a perturbation of this system is sufficient cause for hypophosphatemia in Hyp and Gy mice. Several approaches have been undertaken to elucidate the mechanism for the decrease in high-affinity Na/Pi cotransport Vmax in the mutant strains. It was demonstrated that the affinity of the cotransporter for Na, the number of Na ions interacting with the cotransporter, and the response of the cotransporter to membrane potential and pH are all normal in Hyp mice [68]. Subsequent studies showed that the decrease in Vmax in Hyp and Gy mice is associated with a corresponding decrease in the renal abundance of type IIa Na/Pi cotransporter (Npt2) m R N A and protein [12,148,158]. Thus, the Pi transport defect in Hyp and Gy mice can be ascribed to a decrease in the number of Npt2 cotransporters in the renal BBM. There is considerable evidence to suggest that the defect in renal Pi transport in Hyp mice is mediated by a circulating factor. When normal mice are parabiosed to Hyp mice, the normal animals develop significant hypophosphatemia, decreased renal Pi reabsorption [100], and a reduction in renal BBM Na/Pi cotransport that is independent of PTH [101]. In addition, using a renal crosstransplantation model, it was demonstrated that the Hyp phenotype is neither corrected nor transferred [116]. Direct evidence for humorally mediated inhibition of renal Pi transport in Hyp mice was provided by the demonstration that serum from Hyp mice inhibits Na/ Pi cotransport in primary mouse renal cell cultures to a significantly greater extent than an equivalent amount of serum from normal littermates [90]. Inhibition of Na/Pi cotransport was also elicited by incubation of target
TABLE 2 Summary of Hypophosphatemic Disorders Disorder
XLH Hyp
G__.yy ADHR OHO HI-IRH Npt2-/-
Renal phosphate wasting
Hypophosphatemia
Elevated serum 1,25-(OH)2D
Hypercalciuria
Primary defect
yes yes yes yes yes yes yes
yes yes yes yes yes yes yes
no no no no no yes yes
no no no no no yes yes
PHEX mutations 3'Phex deletion 5'Phex deletion FGF23 mutations
tumour-mediated unknown Npt2 deletio
6. Phosphate Homeostasis Regulatory Mechanisms
renal cell cultures with conditioned medium derived from cultured Hyp osteoblasts, whereas no inhibition was apparent with conditioned medium from normal osteoblast cultures [90]. These data suggest that the Hyp osteoblast is responsible for the release and/or modification of a humoral factor that inhibits Na/Pi cotransport in renal epithelial cells.
The PHEX Gene: Positional Cloning and Mutation Analysis The gene responsible for XLH was identified by an international consortium using a positional cloning approach [39]. The gene, designated PHEX (formerly PEX) to depict a phosphate-regulating gene with homology to endopeptidases on the X chromosome, encodes a 749amino acid protein [54] that exhibits significant homology to the M13 family of zinc metallopeptidases, which includes neutral endopeptidase 24.11 (NEP) [126] and endothelin-converting enzymes 1 [169] and 2 (ECE-1 and ECE-2) [49]. These are type II membrane glycoproteins characterized by a short NHz-terminal cytoplasmic domain, a single transmembrane hydrophobic region, and a large extracellular domain [165]. The latter includes 10 highly conserved cysteine residues and a zinc-binding motif, which in the case of NEP and ECE- 1 is essential for conformational integrity and catalytic activity, respectively [165]. To date, 157 mutations in the PHEX gene have been identified in XLH patients, and to centralize information on P H E X mutations, an online locus-specific PHEX mutation database has been established (http://data. meh.mcgill, ea/phexdb) [132]. The mutations that are scattered throughout the gene include deletions (from 1 to 55 kb), splice junction and frameshift mutations, as well as duplications, insertions, and missense and nonsense mutations, and they are consistent with loss of function [132]. Recent studies demonstrated that in contrast to the wild-type recombinant PHEX protein, which is targeted to the plasma membrane, disease-causing missense mutations in the PHEX gene result in proteins that are not fully glycosylated and remain trapped in the endoplasmic reticulum, where they are degraded [131]. Of interest is the finding that some mutant proteins can be rescued from the endoplasmic reticulum to the cell surface [131]. Thus, for patients carrying trafficking mutations, these results provide a mechanism for loss of PHEX function and a basis for the development of novel therapeutic approaches. Mutations in the Phex gene have also been identified in the Hyp and Gy models of the human disease. Hyp mice harbor a large 3' deletion [11,142], whereas Gy mice have a deletion in the 5' region that includes the upstream gene, spermine synthase [142]. Gy is thus a contiguous gene deletion syndrome, which may explain why Gy mice
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exhibit phenotypic features that are not apparent in Hyp mice [98,137].
PHEX Tissue Distribution and Endopeptidase Activity The function of PHEX and the mechanisms whereby it regulates skeletal mineralization and renal Pi transport and vitamin D metabolism are not immediately apparent. To address this issue, the pattern of PHEX tissue expression was examined by a variety of experimental approaches. These efforts demonstrated that PHEX is expressed predominantly in osteoblasts [43,63,129,130], osteocytes [102,130], and odontoblasts [129,130], but not in kidney [11,43,61,129,130]. The physiological significance of PHEX m R N A expression, detected by reverse transcriptase polymerase chain reaction, in other tissues, including lung, brain, ovary, and testis [99], remains unclear since these tissues are not involved in the pathophysiology of XLH. Moreover, PHEX protein expression has only been detected in bones and teeth [44,102,130], consistent with the skeletal and dental abnormalities evident in XLH and Hyp and the notion that the renal Pi leak in Hyp mice is dependent on a circulating factor [100,116]. Although it is not immediately apparent how loss of PHEX function leads to the downregulation of renal Npt2 gene expression, it has been suggested that PHEX is involved in the inactivation of a phosphaturic hormone or the activation of a Piconserving hormone [11,39,46] and that loss of PHEX/ Phex function is associated with either excess phosphaturic hormone or a deficiency in Pi-conserving hormone. In either case, renal Pi wasting would ensue. Endogenous PHEX substrates have not been identified. The demonstration and characterization of PHEXmediated endopeptidase activity have been complicated by the plasma membrane localization of PHEX protein. To prevent interference by contaminating endopeptidases in membrane preparations, a soluble and secreted form of PHEX (secPHEX) was genetically engineered [16]. The recombinant protein, recovered from the culture medium, was purified to homogeneity and tested for endopeptidase activity with 14 peptide substrates, including bone-related peptides [16]. Only PTHrP107_139 was degraded by secPHEX. Introduction of a mutation in the catalytic zinc-binding domain of secPHEX completely abrogated the degradation of PTHrP107_139, indicating that a contaminant protease copurifying with secPHEX was not responsible for PTHrP107_139 degradation [16]. Of interest was the finding that endopeptidase activity was inhibited by Pi, pyrophosphate, and osteocalcin, all of which are abundant in bone and may play a role in regulating PHEX activity in vivo [16]. The role of PTHrP107_139 in the pathophysiology of XLH remains to be determined. Although it is also produced by osteoblasts, the function of PTHrP107_139
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is obscure [16]. Moreover, it is not clear whether circulating PTHrP107_139 can inhibit renal Pi reabsorption. Because the PHEX cleavage sites identified in human PTHrP107_139 are not conserved in rat, mouse, and chicken, the data suggest that any biological role for PHEX-mediated degradation of PTHrP107_139 may be confined to humans [16].
Strategies for Treatment XLH patients are treated with oral Pi supplements in four or five doses per day and 1,25(OH)2D [147]. This form of therapy is far from ideal: Long-term compliance is difficult, complete healing of rickets rarely occurs, and careful monitoring is essential to ensure a correct balance of Pi and 1,25(OH)2D. Too much Pi leads to secondary hyperparathyroidism, and an excess of 1,25(OH)2D leads to hypercalcemia, hypercalciuria, and nephrocalcinosis. Although the progression of nephrocalcinosis can be prevented by thiazide diuretics [135] and final height can be improved by the administration of GH to poorly growing XLH children [7], novel therapeutic options based on a knowledge of the mechanism of action of the PHEX gene product are clearly necessary. Enzyme replacement therapy using recombinant secPHEX to clear circulating phosphaturic peptides that accumulate in XLH may be a viable option. In this regard, repeated infusions of recombinant enzyme ([3-glucosidase) have proved effective in the treatment of Gaucher's disease, a Mendelian lysozomal storage disorder characterized by abnormal globoside and ganglioside catabolism [59]. To test the efficacy of enzyme replacement therapy and other approaches, the Hyp mouse will serve as an invaluable model.
Autosomal Dominant Hypophosphatemic Rickets The features of ADHR are similar to those of XLH and include short stature, bone deformities, renal Pi wasting, hypophosphatemia, and inappropriate serum levels of 1,25(OH)zD for the degree of hypophosphatemia (Table 2). ADHR is far less common than XLH. It exhibits male-to-male transmission, consistent with autosomal dominant inheritance, and it is characterized by incomplete penetrance and variable age of onset.
Positional Cloning of the ADHR Gene The ADHR locus was mapped to human chromosome region 12p 13 by linkage studies in a large ADHR kindred [47] and the gene subsequently identified by positional cloning [38]. The gene encodes a new member of the FGF family, FGF23, a 251-amino acid peptide that is secreted and processed to amino- and carboxy-terminal peptides at a consensus pro-protein convertase (furin) site, RHTR (Arg-His-Thr-Arg) [138]. Missense mutations in FGF23, identified in four unrelated ADHR families, in-
volve the two R residues in this proteolytic cleavage site [38] and abrogate peptide processing [21,138].
FGF23, a New Player in the Regulation of Phosphate Homeostasis To determine whether FGF23 can influence renal Pi handling and skeletal mineralization, both of which are defective in ADHR, the recombinant peptide was administered to mice and FGF23-transfected Chinese hamster ovary cells were transplanted into nude mice [138]. These studies, particularly those in nude mice, demonstrated that exogenous FGF23 could elicit the biochemical and clinical features of ADHR, including increased urinary Pi excretion, hypophosphatemia, and bone deformities [138]. In contrast, recombinant FGF23 had no effect on Na/Pi cotransport in opossum kidney (OK) cells [138]. However, other workers demonstrated that conditioned medium from FGF23-transfected cells significantly inhibited Na/Pi cotransport in Opossum Kidney (OK) cells [21]. Clearly, additional work is necessary to determine whether the inhibitory effect of FGF23 on renal Pi transport is direct and involves a downregulation ofNpt2 gene expression. Although FGF23 expression is not detectable in normal tissues, it is abundantly expressed in tumors removed from patients with OHO, an acquired renal Pi wasting disorder with features of XLH and ADHR [21,138,166]. In addition, extracts from OHO tumors also inhibit Na/Pi cotransport in OK cells [26,78,115]. Taken together, the current data are consistent with the notion that FGF23, at least in high doses, can inhibit renal Pi reabsorption and that FGF23 contributes to renal Pi wasting in ADHR and OHO. Additional work is necessary to elucidate the physiological role of FGF23, to define the relative potency of the wild-type and mutant, unprocessed FGF23 peptides, and to determine whether FGF23 is a PHEX substrate. Indeed, a recent study reported that wild-type, but not mutant, FGF23 is degraded by PHEX [21]. Thus, it is tempting to speculate that ADHR and XLH involve mutations in autosomal and X-linked genes, respectively, that function in the same metabolic pathway (Fig. 4).
Strategies for Treatment Therapy for ADHR is similar to that for XLH and consists of a combination of oral Pi and vitamin D. However, once the mechanism of FGF23 processing, action, and degradation is well understood, novel therapeutic approaches will likely be developed.
Oncogenic Hypophosphatemic Osteomalacia OHO, also known as tumor-induced osteomalacia, is an acquired and rare form of renal Pi wasting, with
6. Phosphate Homeostasis Regulatory Mechanisms
1
FGF23
Gain of function
- ---I~
185
Renal phosphate wasting
Pro-protein convertase N-term
PHEX
,~l
C-term I - _ -I~ Function unknown
i
Degradationproducts FIGURE 4 A unifying hypothesis for renal phosphate wasting in X-linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets (ADHR), and oncogenic hypophosphatemic osteomalacia (OHO).
clinical and biochemical features of XLH and A D H R (Table 2). In contrast to XLH and ADHR, OHO patients also exhibit weakness, fatigue, and fractures. Several types of tumors have been associated with this syndrome, but the majority appear to arise from mesenchymal elements [89]. Evidence suggests that the phenotypic features of OHO result from the secretion of a humoral factor designated phosphatonin [45] since removal of the offending tumor results in complete correction of the clinical and biochemical phenotype.
Nature of Phosphatonin and Mechanism of Action Several groups have demonstrated that OHO tumor extracts or conditioned medium derived from cultured OHO tumor cells specifically inhibit Na/Pi cotransport in renal proximal tubular cell cultures [1,26,78,89,115, 128,167]. However, there is little agreement about the nature, stability, and signaling mechanism of the putative tumor factors. Molecular weights ranging from >3 to 55-58 kDa have been reported [26,78,115,128], both heat lability [26,167] and heat stability [78,115] have been documented, and susceptibility [115] as well as resistance [78] to papain digestion have been demonstrated. Moreover, the tumor factors were shown to either stimulate [78] or have no effect [26] on the production of cAMP, a second messenger for PTH-mediated inhibition of Na/Pi cotransport, in target renal cells. These studies suggest that either there are several phosphaturic factors produced by OHO tumors or the measurement of Na/Pi cotransport in target renal cell cultures does not provide
a reliable assay of phosphaturic factor activity. In this regard, the renal cell cultures may be devoid of the receptors and/or signaling pathways necessary to elicit the Pi transport response. Two phosphatonin candidates, FGF23 [21,127,166] and mepe (matrix extracellular phosphoglycoprotein) [127], have been identified as differentially or abundantly expressed genes in OHO tumors. As discussed previously, mutations in the FGF23 gene [38] that prevent processing of the peptide [21,138] are responsible for ADHR. Moreover, FGF23 can elicit the phenotypic features of A D H R and OHO when administered to mice [138]. Currently, there is no information concerning the effect of mepe on renal Na/Pi cotransport. Recent studies demonstrate that mepe is expressed in fully differentiated mouse osteoblasts and that mepe mRNA expression is markedly increased during osteoblast-mediated matrix mineralization [5]. Thus, although there is evidence to suggest that FGF23 contributes to the pathogenesis of OHO, the role of mepe in this disorder remains to be determined. Further work is necessary to establish whether the OHO tumor factor may be related to the circulating phosphaturic factor that is responsible for the renal Pi leak in the Hyp mouse model of XLH.
Strategies for Treatment The obvious therapy for patients with OHO is surgical removal of the tumor, which is the source of the offending phosphaturic factor(s). However, the tumors are small, present in obscure areas, difficult to locate
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without sophisticated imaging procedures, and often inaccessible. If the tumor cannot be found or excised, treatment with combined Pi and 1,25-(OH)2D3 can be initiated and should be continued until the tumor is removed.
A Unifying Hypothes&for Renal Phosphate Wasting in XLH, ADHR, and OHO The simplified model depicted in Fig. 4 invokes a common pathway to explain the underlying basis for renal Pi wasting in XLH, ADHR, and OHO. The hypothesis requires that FGF23 is phosphaturic, either directly or indirectly; that FGF23 and/or its N-terminal and C-terminal fragments are degraded by PHEX; and that mutant FGF23 is not degraded by PHEX but retains its phosphaturic activity. Since the PHEX substrate pocket can accommodate acidic amino acidic residues [16], the PHEX cleavage site is most likely distinct from the furin cleavage in FGF23. In the case of XLH, we speculate that loss of PHEX function results in the accumulation of FGF23 in the circulation and that this in turn leads to the inhibition of renal Pi reabsorption. In the case of ADHR, we propose that mutations in the furin cleavage site, which prevent the processing of FGF23 into N-terminal and C-terminal fragments and their subsequent clearance by PHEX, lead to the accumulation of a "stable" circulating form of the peptide that also inhibits renal Pi reabsorption. In the case of OHO, we suggest that ectopic overproduction of FGF23 overwhelms its processing and degradation by pro-protein convertase and PHEX, respectively, leading to the accumulation of FGF23 in the circulation and inhibition of renal Pi reabsorption. Clearly, additional data, such as the plasma concentration of FGF23 in patients with XLH, ADHR, and OHO relative to that in normal individuals, are required to establish the validity of this hypothesis.
Hereditary Hypophosphatemic Rickets with Hypercalciuria HHRH was first identified in a large Bedouin kindred [160,161] and only a few sporadic cases have been subsequently reported [36,117,124,159]. HHRH shares many of the clinical and biochemical features of XLH and ADHR, including growth retardation, bone deformities, renal Pi wasting, and hypophosphatemia, but it can be distinguished from the latter by appropriately increased serum levels of 1,25-(OH)2D and associated hypercalciuria (Table 2). Pi supplementation alone will correct all the clinical and biochemical abnormalities, with the exception of the renal Pi leak. On the basis of these findings, it was suggested that HHRH is a primary disorder of renal Pi reabsorption [161].
Similarities to Npt2 Knockout Mice As discussed earlier, there is considerable evidence that the type IIa Na/Pi cotransporter Npt2 is an important determinant of Pi homeostasis and a target for its regulation. To determine whether loss of Npt2 function elicits a phenotype similar to HHRH and to define the role of Npt2 in the overall maintenance of Pi homeostasis, mice deficient in the Npt2 gene were generated by targeted mutagenesis [10]. Mice homozygous for the disrupted gene (Npt2 -/-) exhibit increased urinary Pi excretion, an 80% decrease in renal BBM Na/Pi cotransport, hypophosphatemia, and an adaptive increase in the serum concentration [10] and renal production [152] of 1,25-(OH)zD, with associated hypercalcemia and hypercalciuria (Table 2) [10]. Moreover, renal BBM Na/Pi cotransport in Npt2 -/- mice cannot be compensated for by other renal Na/Pi cotransporters and is not responsive to dietary Pi deprivation [72] or to PTH administration [171]. The finding that Npt2 -/- mice have a biochemical phenotype that resembled that in patients with HHRH and the notion that HHRH is a primary renal Pi wasting disorder suggest that mutations in the human ortholog, NPT2, may be responsible for HHRH.
Exclusion of NPT2 as the Gene Responsible for H H R H The NPT2 coding region, and a 120-bp fragment of the NPT2 promoter, in two affected individuals from the Bedouin kindred and in unrelated HHRH patients from four small families were sequenced. No putative diseasecausing mutations were found [77]. Two single nucleotide polymorphisms (SNPs), a silent substitution in exon 7 and a nucleotide substitution in intron 4, were identified and neither segregated with HHRH in the Bedouin kindred [77]. Furthermore, linkage analysis demonstrated that these SNPs, as well as five microsatellite markers flanking NPT2 in the chromosome 5q35 region, were not linked to HHRH in the Bedouin kindred [77]. Taken together, these data excluded NPT2 as a candidate gene for HHRH. H y p e r p h o s p h a t e m i c States
Renal Insufficiency Since the kidney is capable of excreting excess Pi over a wide range of dietary intake, hyperphosphatemia rarely occurs under normal conditions. Hyperphosphatemia is most commonly associated with compromised renal function (Table 3) [2]. As renal insufficiency progresses, the number of functional nephrons is reduced and urinary excretion of Pi is significantly decreased. Hyperphosphatemia can also result from increased intake of Pi or the translocation of Pi from cells to the extracellular fluid, arising from increased tissue breakdown, particularly
6. Phosphate Homeostasis Regulatory Mechanisms TABLE 3
Causes of Hyperphosphatemia
Decreased renal phosphate excretion
Renal insufficiency- chronic, acute Hypoparathyroidism Pseudohypoparathyroidism Tumoral calcinosis Increased phosphate intake
Oral, rectal and intravenous Transcellular shift to extracellular fluid
Catabolic states Rhabdomyolysis Acidosis, metabolic or respiratory Tumour lysis syndrome
in patients with renal insufficiency (Table 3). The s h o r t - t e r m consequences o f h y p e r p h o s p h a t e m i a are h y p o c a l c e m i a a n d tetany, whereas in the long term soft tissue calcification, secondary h y p e r p a r a t h y r o i d i s m , a n d renal o s t e o d y s t r o p h y are manifest. The m o s t effective t r e a t m e n t for h y p e r p h o s p h a t e m i a associated with renal insufficiency is to reduce Pi intake a n d administer Pi binders. P s e u d o h y p o p a r a t h y r o i d i s m a n d T u m o r a l Calcinosis
P s e u d o h y p o p a r a t h y r o i d i s m (PHP) a n d t u m o r a l calcinosis are a u t o s o m a l d o m i n a n t disorders characterized by h y p e r p h o s p h a t e m i a s e c o n d a r y to increased renal t u b u l a r r e a b s o r p t i o n o f Pi. P H P comprises a g r o u p o f disorders characterized by target tissue unresponsiveness to P T H [8]. M u t a t i o n s in the G N A S 1 gene, which encodes four distinct i m p r i n t e d transcripts including the alpha subunit o f the stimulatory G protein, have been identified in patients with P H P type l a a n d p s e u d o - P H P , n o w referred to as Albright's hereditary o s t e o d y s t r o p h y . The d e m o n s t r a t i o n of tissue-specific p a t e r n a l or m a t e r n a l imprinting o f G N A S 1 transcripts m a y explain why individuals with p s e u d o - P H P exhibit neither P T H resistance n o r the skeletal manifestations o f P H P even t h o u g h they carry one m u t a n t G N A S 1 allele. A l t h o u g h other types o f P H P have been described (types Ib, Ic, a n d II), their underlying genetic basis has not been elucidated. T u m o r a l calcinosis, which is frequently seen in y o u n g blacks, is associated with p a t h o l o g i c soft tissue calcification, likely arising f r o m the elevated Ca/Pi p r o d u c t in the b l o o d [133]. Circulating levels o f P T H a n d 1,25-(OH)2D are n o r m a l , despite h y p e r p h o s p h a t e m i a . The p a t h o g e n esis o f this disorder is obscure, a l t h o u g h it has been p o s t u l a t e d that the basic defect resides within the renal proximal t u b u l a r cell.
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6. Phosphate Homeostasis Regulatory Mechanisms 127. Rowe, P. S., de Zoysa, P. A., Dong, R., Wang, H. R., White, K. E., Econs, M. J., and Oudet, C. L. (2000). MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia. Genomics 67, 54-68. 128. Rowe, P. S., Ong, A. C., Cockerill, F. J., Goulding, J. N., and Hewison, M. (1996). Candidate 56 and 58 kDa protein(s) responsible for mediating the renal defects in oncogenic hypophosphatemic osteomalacia. Bone 18, 159-169. 129. Ruchon, A. F., Marcinkiewicz, M., Siegfried, G., Tenenhouse, H. S., DesGroseillers, L., Crine, P., and Boileau, G. (1998). Pex mRNA is localized in developing mouse osteoblasts and odontoblasts. J. Histochem. Cytochem. 46, 459-468. 130. Ruchon, A. F., Tenenhouse, H. S., Marcinkiewicz, M., Siegfried, G., Aubin, J. E., DesGroseillers, L., Crine, P., and Boileau, G. (2000). Developmental expression and tissue distribution of Phex protein: Effect of the Hyp mutation and relationship to bone markers. J. Bone Miner. Res. 15, 1440-1450. 131. Sabbagh, Y., Boileau, G., DesGroseillers, L., and Tenenhouse, H. S. (2001). Disease-causing missense mutations in the PHEX gene interfere with membrane targeting of the recombinant protein. Hum. Mol. Genet. 10, 1539-1546. 132. Sabbagh, Y., Jones, A. O., and Tenenhouse, H. S. (2000). PHEXdb, a locus-specific database for mutations causing X-linked hypophosphatemia. Hum. Mutat. 16, 1-6. 133. Savaci, N., Avunduk, M. C., Tosun, Z., and Hosnuter, M. (2000). Hyperphosphatemic tumoral calcinosis. Plastic Reconstr. Surg. 105, 162-165. 134. Scriver, C. R., and Tenenhouse, H. S. (1990). Conserved loci on the X chromosome confer phosphate homeostasis in mice and humans. Genet. Res. 56, 141-152. 135. Seikaly, M. G., and Baum, M. (2001). Thiazide diuretics arrest the progression of nephrocalcinosis in children with x-linked hypophosphatemia. Pediatrics 108, E6. 136. Selz, T., Caverzasio, J., and Bonjour, J. P. (1989). Regulation of Na-dependent Pi transport by parathyroid hormone in osteoblastlike cells. Am. J. Physiol. 256, E93-E100. 137. Shetty, N. S., and Meyer, R. A., Jr. (1991). Craniofacial abnormalities in mice with X-linked hypophosphatemic genes (Hyp or Gy). Teratology 44, 463-472. 138. Shimada, T., Mizutani, S., Muto, T., Yoneya, T., Hino, R., Takeda, S., Takeuchi, Y., Fujita, T., Fukumoto, S., and Yamashita, T. (2001). Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proc. Natl. Acad. Sci. USA 98, 6500-6505. 139. Silve, C., and Friedlander, G. (2000). Renal regulation of phosphate excretion. In The Kidney: Physiology and Pathophysiology (D. W. Seldin and G. Giebisch, Eds.), pp. 1885-1904. Lippincott Williams & Wilkins, Philadelphia. 140. Sorribas, V., Lotscher, M., Lofting, J., Biber, J., Kaissling, B., Murer, H., and Levi, M. (1996). Cellular mechanisms of the agerelated decrease in renal phosphate reabsorption. Kidney Int. 50, 855-863. 141. Steele, T. H., and DeLuca, H. F. (1976). Influence of dietary phosphorus on renal phosphate reabsorption in the parathyroidectomized rat. J. Clin. Invest. 57, 867-874. 142. Strom, T. M., Francis, F., Lorenz, B., Boddrich, A., Econs, M. J., Lehrach, H., and Meitinger, T. (1997). Pex gene deletions in Gy and Hyp mice provide mouse models for X-linked hypophosphatemia. Hum. Mol. Genet. 6, 165-171. 143. Suzuki, A., Palmer, G., Bonjour, J. P., and Caverzasio, J. (2000). Stimulation of sodium-dependent phosphate transport and signaling mechanisms induced by basic fibroblast growth factor in MC3T3-E1 osteoblast-like cells. J. Bone Miner. Res. 15, 95-102.
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144. Taketani, Y., Miyamoto, K., Tanaka, K., Katai, K., Chikamori, M., Tatsumi, S., Segawa, H., Yamamoto, H., Morita, K., and Takeda, E. (1997). Gene structure and functional analysis of the human Na+/phosphate co-transporter. Biochem. J. 324, 927-934. 145. Taketani, Y., Segawa, H., Chikamori, M., Morita, K., Tanaka, K., Kido, S., Yamamoto, H., Iemori, Y., Tatsumi, S., Tsugawa, N., Okano, T., Kobayashi, T., Miyamoto, K., and Takeda, E. (1998). Regulation of type II renal Na + -dependent inorganic phosphate transporters by 1,25-dihydroxy vitamin D3. Identification of a vitamin D-responsive element in the human NAPi-3 gene. J. Biol. Chem. 273, 14575-14581. 146. Tenehouse, H. S., and Econs, M. J. (2001). Mendelian hypophosphatemia. In The Metabolic and Molecular Basis of Inherited Disease (C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, Eds.), pp. 5039-5067. McGraw-Hill, New York. 147. Tenenhouse, H. S. (1999). X-linked hypophosphataemia: A homologous disorder in humans and mice. Nephrol. Dial. Transplant. 14, 333-341. 148. Tenenhouse, H. S., and Beck, L. (1996). Renal Na(+)-phosphate cotransporter gene expression in X-linked Hyp and Gy mice. Kidney Int. 49, 1027-1032. 149. Tenenhouse, H. S., Klugerman, A. H., and Neal, J. L. (1989). Effect of phosphonoformic acid, dietary phosphate and the Hyp mutation on kinetically distinct phosphate transport processes in mouse kidney. Biochim. Biophys. Acta 984, 207-213. 150. Tenenhouse, H. S., Lee, J., and Harvey, N. (1991). Renal brushborder membrane Na(+)-sulfate cotransport: Stimulation by thyroid hormone. Am. J. Physiol. 261, F420-F426. 151. Tenenhouse, H. S., and Martel, J. (1993). Renal adaptation to phosphate deprivation: Lessons from the X-linked Hyp mouse. Pediatr. Nephrol. 7, 312-318. 152. Tenenhouse, H. S., Martel, J., Gauthier, C., Zhang, M. Y., and Portale, A. A. (2001). Renal expression of the sodium/phosphate cotransporter gene, Npt2, is not required for regulation of renal 1 alpha-hydroxylase by phosphate. Endocrinology 142,1124-1129. 153. Tenenhouse, H. S., Meyer, R. A., Jr., Mandla, S., Meyer, M. H., and Gray, R. W. (1992). Renal phosphate transport and vitamin D metabolism in X-linked hypophosphatemic Gy mice: Responses to phosphate deprivation. Endocrinology 131, 51-56. 154. Tenenhouse, H. S., Roy, S., Martel, J., and Gauthier, C. (1998). Differential expression, abundance, and regulation of Na+-phosphate cotransporter genes in murine kidney. Am. J. Physiol. 275, F527-F534. 155. Tenenhouse, H. S., and Scriver, C. R. (1978). The defect in transcellular transport of phosphate in the nephron is located in brushborder membranes in X-linked hypophosphatemia (Hyp mouse model). Can. J. Biochem. 56, 640-646. 156. Tenenhouse, H. S., and Scriver, C. R. (1992). X-linked hypophosphatemia. A phenotype in search of a cause. Int. J. Biochem. 24, 685-691. 157. Tenenhouse, H. S., Scriver, C. R., McInnes, R. R., and Glorieux, F. H. (1978). Renal handling of phosphate in vivo and in vitro by the X-linked hypophosphatemic male mouse: Evidence for a defect in the brush border membrane. Kidney Int. 14, 236-244. 158. Tenenhouse, H. S., Werner, A., Biber, J., Ma, S., Martel, J., Roy, S., and Murer, H. (1994). Renal Na(+)-phosphate cotransport in murine X-linked hypophosphatemic rickets. Molecular characterization. J. Clin. Invest. 93, 671-676. 159. Tieder, M., Arie, R., Bab, I., Maor, J., and Liberman, U. A. (1992). A new kindred with hereditary hypophosphatemic rickets with hypercalciuria: Implications for correct diagnosis and treatment. Nephron 62, 176-181.
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Strom, T. M., Juppner, H., and Econs, M. J. (2001). The autosomal dominant hypophosphatemic rickets (ADHR) gene is a secreted polypeptide overexpressed by tumors that cause phosphate wasting. J. Clin. Endocrinol. Metab. 86, 497-500. Wilkins, G. E., Granleese, S., Hegele, R. G., Holden, J., Anderson, D. W., and Bondy, G. P. (1995). Oncogenic osteomalacia: Evidence for a humoral phosphaturic factor. J. Clin. Endocrinol. Metab. 80, 1628-1634. Williams, D. C., and Frolik, C. A. (1991). Physiological and pharmacological regulation of biological calcification. Int. Rev. CytoL 126, 195-292. Xu, D., Emoto, N., Giaid, A., Slaughter, C., Kaw, S., deWit, D., and Yanagisawa, M. (1994). ECE-I: A membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell 78, 473-485. Yabuuchi, H., Tamai, I., Morita, K., Kouda, T., Miyamoto, K., Takeda, E., and Tsuji, A. (1998). Hepatic sinusoidal membrane transport of anionic drugs mediated by anion transporter Nptl. J. Pharmacol. Exp. Ther. 286, 1391-1396. Zhao, N., and Tenenhouse, H. S. (2000). Npt2 gene disruption confers resistance to the inhibitory action of parathyroid hormone on renal sodium-phosphate cotransport. Endocrinology 141, 2159-2165. Zhen, X., Bonjour, J. P., and Caverzasio, J. (1997). Plateletderived growth factor stimulates sodium-dependent Pi transport in osteoblastic cells via phospholipase Cgamma and phosphatidylinositol 3'-kinase. J. Bone Miner. Res. 12, 36-44.
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7 Vitamin D Biology RENE ST.-ARNAUD* and MARIE B. DEMAYt *Genetics Unit, Shriners Hospital for Children, Montreal, Quebec, Canada and Departments of Surgery and Human Genetics, McGill University, Montreal Quebec, Canada t Endocrine Unit, Massachusetts General Hospital Harvard Medical School Boston, Massachusetts
METABOLIC ACTIVATION OF VITAMIN D
binds to its specific receptor, the vitamin D receptor (VDR), to regulate the transcription of vitamin D target genes responsible for carrying out the physiological actions of 1,25(OH)2D, mineral homeostasis, skeletal homeostasis, and cellular differentiation. Following exposure to sunlight, both plants and animals are able to synthesize vitamin D. Vitamin D2 (ergocalciferol) is generated in yeast and plants; vitamin D 3 (cholecalciferol) is produced in fish and mammals. The slight differences in the chemical structure of the two compounds (Fig. 1A) do not affect function or metabolism in mammals. The generic term of vitamin D (without subscript) will be used hereafter. This chapter reviews several aspects of vitamin D biology. Vitamin D metabolism is detailed first, followed by a presentation of the physiological roles of the vitamin D hormone. Finally, an in-depth analysis of the mechanism of action of vitamin D is presented. The reader is referred to Chapter 24 for a discussion of the pediatric disorders involving the vitamin D endocrine system.
Following the seminal work that established vitamins (a term derived from the contraction of vital amines) A and B as essential micronutrients, the observation that a fat-soluble component of aerated and heated cod liver oil cured nutritional rickets led to the hypothesis that the healing activity was due to a previously unidentified vitamin that was termed vitamin D [1]. Parallel and subsequent work demonstrating that exposure to sunlight cures rickets in patients and experimental animals [2] challenged the notion that vitamin D is truly a vitamin, but the term initially coined has endured despite the identification of the endocrine system regulating the synthesis and activity of the active form of the D compound. Indeed, vitamin D, produced endogenously in the skin upon exposure to ultraviolet light (sunlight), must be metabolized twice to be activated and function as a key regulator of mineral ion homeostasis. Vitamin D, bound to the vitamin D-binding protein (DBP), is transported to the liver, where the enzyme vitamin D 25-hydroxylase (CYP27) adds a hydroxyl group on carbon 25 to produce 25-hydroxyvitamin D [25(OH)D]. The 25(OH) D metabolite also circulates in the bloodstream bound to DBP. It must be further hydroxylated in the kidney to gain hormonal bioactivity. Hydroxylation at position 1oL by the enzyme 25-hydroxyvitamin D- 1ot-hydroxylase (CYP27B1) converts 25(OH)D to l oL,25-dihydroxyvitamin D [1,25(OH)2D], the active, hormonal form of vitamin D. Upon reaching target tissues, 1,25(OH)2D
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Hepatic 25-Hydroxylation In humans, 80% of the vitamin D requirement can be produced in the skin upon exposure to ultraviolet light (sunlight). The rest must be acquired through dietary sources, such as fish, plants, and grains. Ultraviolet B photons penetrate the epidermis and photolize 7dehydrocholesterol into previtamin D, which rapidly becomes a more thermodynamically stable molecule, vitamin D [3]. Vitamin D then exits the keratinocyte cells
193
Copyright 2003, Elsevier Science (USA). All rights reserved.
194
Ren~ St.-Arnaud and Marie B. Demay
A OH2
HO~
Vitamin D3 (Cholecalciferol)
B
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NADP~
~
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~ FRred ~
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^+ FDX-Fe ~ .. N~
"FDX-Fe 3+
3+ ~ (OH)-D Metabolite P450-FeL ~ " ~ ~ , , ~ p Vitamin D Metabolite
P450-Fe2 + ~
02
P450-Fe2+-CO FIGURE 1 Structure of vitamin D and cytochrome P450 activity. (A) Structure of vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Plants and yeast synthesize vitamin D2; fish and mammals synthesize vitamin D3. The slight differences in the chemical structure of the two compounds do not affect function or metabolism. The open cyclopentanoperhydrophenanthrene nucleus identifies vitamin D metabolites as secosteroids and not steroids. (B) Mechanism of hydroxylation of vitamin D metabolites by cytochrome P450 hydroxylases. NADPH, dihydronicotinamide adenine dinucleotide phosphate; NADP, nicotinamide adenine dinucleotide phosphate; FR, ferredoxin reductase; ox, oxidized; red, reduced; FDX, ferredoxin.
and enters the dermal capillary bed, where it is bound to the DBP. Once associated with DBP in the circulation, vitamin D is transported to the liver, where the cytochrome P450 enzyme vitamin D 25-hydroxylase (CYP27) adds a hydroxyl group on carbon 25 to produce 25-hydroxyvitamin D [25(OH)D]. All vitamin D hydroxylases characterized to date belong to the superfamily of cytochrome P450 enzymes. They are heme-containing, mixed-function oxidases that use molecular oxygen as a terminal electron acceptor. They require the accessory electron transfer proteins, ferredoxin and ferredoxin reductase, to accept reducing equivalents from nicotinamide adenine dinucleotide phosphate (NADPH) and stereospecifically hydroxylate vitamin D metabolites (Fig. 1B). Only one cytochrome P450 molecule has been cloned and shown to hydroxylate vitamin D at position 25--the bifunctional CYP27, which derives its name from its ability to 27-hydroxylate the side chains of cholesterolderived intermediates involved in bile acid biosynthesis [4]. However, some experimental evidence argue for the existence of a second, perhaps more physiologically relevant, vitamin D 25-hydroxylase enzyme [5]. First, mice with a disrupted cyp27 gene have reduced bile acid synthesis but normal vitamin D metabolite levels [6].
Second, patients with the inherited disease cerebrotendinous xanthomatosis, caused by mutations in CYP27 [7, 8], exhibit normal vitamin D metabolism and no obvious 25(OH)D or 1,25(OH)zD deficiency [9]. Third, early studies using perfused rat liver revealed kinetics of vitamin D metabolism supporting two 25-hydroxylase activities~a high-affinity, low-capacity enzyme and a low-affinity, high-capacity form (now presumed to be CYP27) [10]. The subcellular localizations of these two enzymatic activities are thought to differ: CYP27 is a mitochondrial enzyme [11,12], whereas the high-affinity, low-capacity enzyme is associated with the endoplasmic reticulum (microsomes) [13]. Next, the promoter of CYP27 is regulated by bile acids but not by vitamin D metabolites [14], a finding that is not consistent with the weak but demonstratable regulation of the hepatic 25hydroxylase observed following vitamin D intake in previously vitamin D-depleted animals [15]. Lastly, cells transfected with an expression vector for CYP27 are not able to 25-hydroxylate vitamin D2 [16]. The 25hydroxy-D2 metabolite is synthesized in vivo, however, suggesting that an enzyme distinct from CYP27 is involved in its synthesis. Early studies established that the liver was the site of 25-hydroxylation of vitamin D [17]. The 25-hydroxylase
7. Vitamin D Biology
enzymatic activity was almost entirely localized to microsomes, whereas mitochondria exhibited little activity toward vitamin D [18]. Although several groups have reported on the purification of the microsomal enzyme from various sources, the gene encoding the protein has not been cloned to date. It has been speculated that the microsomal vitamin D 25-hydroxylase is C YP2Cll since a purified and sequenced 25-hydroxylase from rat liver microsomes has the same N-terminal amino acid sequence as CYP2Cll [19]. However, this cytochrome P450 is male specific [20], and a microsomal 25hydroxylase with an amino-terminal sequence distinct from CYP2Cll has been purified from pig liver microsomes [21]. Thus, CYP27 remains the only cloned enzyme capable of 25-hydroxylation of vitamin D, but its physiological relevance remains debatable. New data from the human genome sequencing project may help to identify heretofore unrecognized cytochrome P450 molecules, and their characterization may reconcile all the data related to hepatic 25-hydroxylation of vitamin D. Hydroxylation of vitamin D at position 25 has been detected in extrahepatic sites [22]. This is in agreement with the wide tissue distribution of CYP27 [23,24]. Recently, CYP27 expression was reported in keratinocytes [25], which, combined with the photosynthesis of vitamin D in skin and the expression of the 25-hydroxyvitamin D-let-hydroxylase and 24-hydroxylase in epidermis, makes skin an autonomous organ in the synthesis, activation, and degradation of vitamin D. Renal I a-Hydroxylation 25(OH)D must be further hydroxylated to gain hormonal bioactivity. This occurs in the convoluted and straight portions of the proximal kidney tubule. Hydroxylation at position let by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D-let-hydroxylase (CYP27B1; hereafter referred to as 1et-hydroxylase) converts 25(OH)D to 1,25(OH)zD, the active, hormonal form of vitamin D. Like CYP27, the l et-hydroxylase belongs to the class of the mixed-function oxidases. The l et-hydroxylase cDNA was cloned from rat and mouse kidney as well as from human keratinocytes and kidney [26-29]. Cloning was hampered by the low levels of mRNA expression. Several ingenious strategies were developed to clone cDNAs for the l c~-hydroxylase from several species. Reduced stringency hybridization cloning using a probe derived from the heme-binding domain of the rat 25-hydroxyvitamin D-24-hydroxylase (eyp24) cDNA [30] first allowed the identification and characterization of a full-length l et-hydroxylase cDNA from the kidney of vitamin D-deficient rats [27]. Similar strategies, using the polymerase chain reaction (PCR) with degenerate
195
oligonucleotide primers, led to the cloning of the rat kidney cDNA by a different laboratory and also to the cloning of the human cDNA from keratinocytes [26,28]. The PCR primers used were derived from the amino acid sequence of conserved regions of the CYP27 and CYP24 genes. The group of Shideaki Kato took advantage of the VDR knockout mice they had engineered [29,31]. Mice lacking the VDR have extremely high serum concentrations of 1,25(OH)2D because the negative feedback loop through which 1,25(OH)zD downregulates l et-hydroxylase expression is impaired in these animals [31]. The authors reasoned that the elevated 1,25(OH)2D levels reflected excessive let-hydroxylase expression and that the cDNA coding for l et-hydroxylase would be represented at an increased frequency in cDNA libraries prepared from kidneys of VDR-deficient mice. The screening method used a chimeric transcription factor that contained the ligand-binding domain of the VDR. This chimeric factor activates the transcription of a reporter gene only in the presence of 1,25(OH)2D. Cells were transfected with the reporter, the chimeric factor, and clones from the kidney cDNA library and treated with 25(OH)D. Cells expressing l et-hydroxylase activity converted the 25(OH)D precursor to 1,25(OH)2D, activated the chimeric protein, and turned on the reporter gene [29]. This elegant expression cloning strategy led to the identification of the mouse 1et-hydroxylase cDNA. The cDNAs from all species examined to date show high sequence similarity. For example, the sequence of the coding region of the human cDNA is 82% identical to that of mouse 1et-hydroxylase at both the nucleotide and the amino acid levels. The human l et-hydroxylase cDNA is 2469 base pairs (bps) in length and codes for a deduced protein of 508 amino acids containing a ferredoxin-binding domain and a heme-binding domain. The deduced amino acid sequence has substantial homology to members of the mitochondrial P450 family [32], particularly CYP27 (40%) [12]; CYP24 (32%) [30]; P450scC, the cholesterol side chain cleavage enzyme (CYPllA; 33%) [33]; and P450c1113, the steroid l l[3-hydroxylase (CYPllB1; 30%)[341. Every laboratory that isolated the l et-hydroxylase cDNA rapidly obtained the sequence of the let-hydroxylase gene in various species. The gene exists as a single copy in the human genome and contains nine exons spanning 5 kbs. The ferredoxin-binding domain is encoded by sequences contained in exons 6 and 7, whereas the heme-binding domain is contained in exon 8 [35,36]. The human gene has been localized to chromosome 12 using somatic cell hybrids [36] and mapped to 12q13.113.3 by fluorescence in situ hybridization [27,37,38]. The chromosomal location provided further circumstantial evidence that mutations in the gene were responsible for the hereditary disease pseudo-vitamin D deficiency
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rickets (PDDR) (see Chapter 24) since the disease had previously been mapped to this locus by linkage analysis [39,40]. Incontrovertible proof that mutations of the 1oLhydroxylase cause PDDR was provided by the characterization of CYP27B1 mutations in patients with the disease [41]. Animal models of the disease have recently been engineered by gene targeting technology [42,43]. The main site for 1oL-hydroxylation of 25(OH)D is the proximal tubule of the renal cortex [44]. The expression of the enzyme has also been reported in cells from other tissues: osteoblasts [45], keratinocytes [26], and cells of the lymphohematopoeitic system [46]. Identification of these extrarenal sites of expression of 1oL-hydroxylase led investigators to hypothesize that local production of 1,25 (OH)zD could play an important autocrine or paracrine role in the differentiation or function of these cells. In the kidney, expression of the let-hydroxylase gene is subject to complex regulation by parathyroid hormone, calcitonin, calcium, phosphorus, and 1,25(OH)2D. Parathyroid hormone (PTH) has long been known to stimulate l oL-hydroxylase enzymatic activity in kidney [47], and this has been demonstrated to occur at the level of gene transcription [48,49]. The PTH response involves signal transduction through cyclic AMP and protein kinase A [48-51]. Putative cyclic AMP response elements have been identified within the proximal l oLhydroxylase promoter [49]. Additional studies are required to demonstrate the functional relevance of these putative transcriptional response elements. Calcitonin is also a positive regulator of l oLhydroxylase activity [52,53], and this also occurs at the transcriptional level [48]. Interestingly, recent results obtained in a rat model demonstrate that although PTH is mainly responsible for cyp27B1 induction in hypocalcemic animals, calcitonin appears to be the major regulator of the expression of the 1oL-hydroxylase gene in normocalcemic rats [54]. Dietary calcium and phosphate intake are critical in the control of loL-hydroxylase enzymatic activity [55]. Decreases in blood calcium stimulate the synthesis and secretion of PTH, which in turn increases expression of the l oL-hydroxylase gene, as mentioned previously [56]; thus, regulation by calcium is indirect. With respect to the regulation by phosphate, experiments in rats have shown that a low phosphorus diet, leading to hypophosphatemia, results in an increase in l~-hydroxylase mRNA and protein expression [57]. This response was not observed in hypophysectomized hypophosphatemic animals, however, indicating that low serum phosphate concentrations are not sufficient to induce 1et-hydroxylase gene expression. Treatment of hypophysectomized rats fed the low-phosphorus diet with growth hormone (GH) or its mediator, insulin-like growth factor-1 (IGF-1), showed that GH, but not IGF-1,
could partially restore l a-hydroxylase gene expression, suggesting that the effects of dietary phosphorus deprivation are mediated, at least in part, by a GH-dependent mechanism [57]. The regulation of phosphate homeostasis is a complex process, of which renal reabsorption by the sodium-phosphate cotransporter type IIa (NPT2) is a key component [58]. In mice deficient for Npt2, however, low-phosphate diets increase l oL-hydroxylase expression and activity, similar to what is observed in wild-type mice [59]. Thus, changes in serum phosphate concentration per se, and not renal phosphate reabsorption, are sufficient to induce the signaling cascade regulating the expression of the l oL-hydroxylase gene in response to restricted or supplemented dietary phosphate. PTH induces the expression of the l oL-hydroxylase gene, leading to increased 1,25(OH)2D synthesis and intestinal calcium absorption. To prevent sustained production of 1,25(OH)2D that would lead to hypercalcemia, the vitamin D hormone in turn inhibits PTH and 1oL-hydroxylase gene expression [60-64]. A fascinating aspect of the control of the expression of the 1et-hydroxylase gene concerns its repression by 1,25(OH)2D. Analysis of the human 1oL-hydroxylase promoter region that confers responsiveness to 1,25(OH)2D [48] did not reveal the presence of conventional vitamin D response elements (VDREs) or sequences related to the negative VDREs that have been characterized to date (Table 1) [62,65,66]. Promoter-deletion mapping allowed identification of the negative response element that was subsequently used for cloning of the DNA-binding transcriptional regulatory factor using yeast one-hybrid technology [67]. Surprisingly, these experiments identified a novel member of the basic domain/helix-loop-helix (bHLH) family of transcription factors as a regulator of l oL-hydroxylase gene expression [67]. In the absence of 1,25(OH)zD, the bHLH factor stimulates transcription from the loL-hydroxylase promoter. Protein-protein interactions between the bHLH factor and the liganded vitamin D receptor appear responsible for 1,25(OH)zD-mediated repression of l oLhydroxylase transcription. Although these results have only been reported in abstract form [67,68], they identify a novel and heretofore unrecognized mechanism of transcriptional control by the vitamin D hormone and suggest novel targets for drug discovery. 24-Hydroxylation Hypocalcemia results in increased production of 1,25 (OH)2D via PTH. As discussed previously, the expression of the 1ot-hydroxylase gene is controlled in a classic feedback loop to avoid sustained production of 1,25 (OH)2D leading to hypercalcemia. To provide for an even faster "shut-off" mechanism, the 1,25(OH)2D
7. Vitamin D Biology TABLE 1
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activating a n d r e p r e s s i n g Vitamin D R e s p o n s e E l e m e n t s (VDREs)
Activating VDREs
VDRM
RXR
Rat osteocalcin
GGGTGA
atg
AGGACA
Human osteocalcin
GGGTGA
acg
GGGGCA AGGTTC
Mouse osteopontin
AGGTTC
acg
Avian oN[33 Integrin
GAGGCA
gaa
GGGAGA
Rat 24 hydroxylase (i)
AGGTGA
gtg
AGGGCG
(ii)
GGTTCA
gcg
GGTGCG
Repressing VD REs
Human PTH (antisense strand)
ATCTCAACTATAGGTTCAAAGCAGCACATA
Avian PTH
TGAGGGTCAGGAGGGTGTGCCTGCAGG
Human PTHrP
TAAAGTGCTATAGATTCATATTTGGTTTAT
RXR, half-site binding retinoid X receptor; VDR, half-site binding vitamin D receptor.
hormone induces the expression of the gene encoding the key effector of its catabolic breakdown, 25-hydroxyvitamin D-24-hydroxylase (C YP24). The CYP24 enzyme is also a mixed-function oxidase cytochrome P450 molecule. It is localized to the mitochondrial membrane and catalyzes the addition of a hydroxyl group on carbon 24 of the vitamin D secosteroid backbone. It can utilize several substrates: 25(OH)D to produce 24,25-dihydroxyvitamin D [24,25(OH)zD]; 1,25(OH)zD to produce 1,24,25-trihydroxyvitamin D [1,24,25(OH)3D], and even l~-hydroxyvitamin D [1 (OH)D] to generate l oL,24-dihydroxyvitamin D [1,24 (OH)zD]. The 24,25(OH)2D metabolite is the most abundant dihydroxylated metabolite in the circulation. Its ease of detection helped in the analysis of the 24hydroxylase enzyme, long before 1,25(OH)zD and the 1oL-hydroxylase were identified. The enzyme was purified to homogeneity from rat kidney mitochondria [69] and the purified protein was used to raise antibodies [70], permitting cloning of the cDNA [30]. This allowed cloning of the gene from various species [71-73], analysis and characterization of the promoter control elements, and production of the recombinant protein. In the mid-to late-1970s, the 24-hydroxylation step was shown to be induced by 1,25(OH)zD [74,75]. Since the product of this reaction, 1,24,25(OH)3D, was 10 times less active than the 1,25(OH)zD substrate [74], investigators reasoned that the 24-hydroxylation reaction was perhaps the first step in an inactivation process. It was also discovered that the 24-hydroxylated metabolites, 24,25(OH)zD and 1,24,25(OH)3D, could be further converted to different metabolic products with 24-oxo and/or 23-hydroxy groups [76-78]. Studies using perfused rat kidney led to the identification of additional metabolites--a 23-alcohol [79] and a 23-acid [80,81]. These metabolites had not been previously identified in
vitro. Calcitroic acid, the l~-hydroxylated 23-acid, was shown to be the main biliary excretory form of 1,25 (OH)zD [82]. With most metabolites identified, investigators deduced that 24-hydroxylation initiates the C 24 oxidation pathway that leads to 1,25(OH)2D degradation [80,81]. This pathway comprises five enzymatic steps that involve successive hydroxylation/oxidation reactions at C 24 and C 23, followed by cleavage of the secosteroid at the C23/C 24 bond and subsequent oxidation of the cleaved product to calcitroic acid [80,81]. The 1,25(OH)2D-inducible 24-hydroxylation and calcitroic acid production was observed in several cell lines from kidney, bone, intestine, skin, and breast [83-85], demonstrating that the C 24 oxidation catabolic pathway can be induced in a number of vitamin D target cells. The recombinant CYP24 protein, when associated with its electron-transport cofactors NADPH-ferredoxin reductase and ferredoxin, has been shown to perform multiple steps in the C 24 oxidation pathway. This includes 23-hydroxylation, dehydrogenation of the 24-hydroxyl group, and side chain cleavage [86,87]. The hypothesis that the main role of the C 24 oxidation pathway is attenuation of the 1,25(OH)zD biological signal inside target cells was tested in vitro using cytochrome P450 inhibitors. Blocking P450 activity by treatment of cells with ketoconazole inhibits catabolism and results in 1,25(OH)zD accumulation and extended hormone action [88]. This hypothesis was also tested and confirmed in vivo by engineering cyp24-deficient mice. Animals homozygous for the cyp24 mutation cannot effectively clear 1,25(OH)zD from their circulation [89]. The phenotype of the cyp24 knockout mice is described in detail later. The 1,25(OH)zD hormone induces CYP24 through a genomic mechanism; several groups have studied the promoter region of the CYP24 gene from various species and demonstrated that it contains functional VDREs. As
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previously mentioned and further detailed later, 1,25 (OH)2D binds the VDR, a class II nuclear hormone receptor that heterodimerizes with the retinoid X receptor (RXR) to bind DNA at consensus VDREs and regulate gene transcription [90]. In the rat cyp24 promoter region, two DR3-type VDREs (direct repeat hexanucleotide spaced by 3 bps) have been extensively characterized [91]. The two elements, separated by 94 bps within the proximal promoter region ( - 136/- 150 and -244/-258) [72,92-95], cooperate synergistically [96]. Additional transcriptional regulatory factors cooperate with the VDR/RXR dimer to regulate cyp24 expression levels. The ras-activated, phosphorylated Ets transcription factors form a trimeric complex with VDR and RXR to maximally induce cyp24 transcription [97]. On the other hand, the unliganded VDR was shown to interact with the N-CoR corepressor variant RIP13A1 to suppress basal cyp24 expression in the absence of 1,25(OH)2D [98]. Thus, in target cells that express the VDR, the receptor is involved in both basal promoter repression and ligand-inducible expression of the enzyme that leads to the inactivation of the 1,25(OH)2D hormone. The calciotropic peptide hormone PTH also regulates CYP24 expression, and its effects vary according to the target cell. In cells from the convoluted tubules of the kidney, the main site of 1,25(OH)2D synthesis, PTH inhibits CYP24 expression [99]. This is hypothesized to allow for maximal 1,25(OH)2D production with minimal concomitant catabolism. Due to the lack of PTH receptors in intestinal cells, CYP24 gene expression is not affected by PTH in the intestine. In osteoblasts, however, PTH synergizes with 1,25(OH)2D to stimulate CYP24 gene transcription [100,101]. The rationale for this synergy remains unclear. It could reflect tissue-specific requirements for 1,25(OH)2D catabolism. On the other hand, increased bone cell expression of CYP24 due to the synergism between PTH and 1,25(OH)2D could lead to augmented local production of 24-hydroxylated vitamin D metabolites, which have been hypothesized to be involved in cartilage development and fracture repair [102,103]. The putative role of vitamin D metabolites distinct from 1,25(OH)2D is discussed later.
MECHANISM OF ACTION The VDR is a member of the family of nuclear hormone receptors. This ligand-activated transcription factor belongs to a subfamily that also includes the triiodothyronine, retinoid X, and retinoic acid receptors. The VDR has been shown to mediate the effects of 1,25dihydroxyvitamin D by binding this ligand, heterodimerizing with the RXR, and interacting with DNA
sequences on target genes, thereby regulating their transcription. Nuclear receptor coactivators have been shown to contribute to the transcriptional effects of the liganded V D R - R X R heterodimer. Recent investigations suggest that the VDR may have ligand-independent actions; however, these effects are less well characterized. Vitamin D R e c e p t o r The biological effects of 1,25-dihydroxyvitamin D are thought to be mediated by a nuclear receptor, the VDR [90]. The VDR belongs to the subfamily of nuclear hormone receptors, which also includes the retinoic acid receptors, RXRs, and thyroid hormone receptors. Unlike these other members of the subfamily, which have more than one characterized isoform (oL, [3, and y), only one nuclear VDR has been isolated. Like other members of the nuclear receptor superfamily, the principal domains of the VDR are those involved in DNA binding, hormone binding, dimerization, and transcriptional activation (Fig. 2). These domains are evolutionarily conserved, with 75% identity between the human [104] and Xenopus [105] VDRs. The DNA-binding domain is more highly conserved, with more than 90% conservation of amino acid sequence across species (Fig. 2) [105]. Unlike most nuclear hormone receptors that contain a substantial amino-terminal transactivation domain (AF1), the human VDR has a very short region N terminal to the two zinc fingers that comprise the DNA-binding domain. Sequences within the DNA-binding domain (residues 24-90) have been shown to be responsible for DNA binding as well as nuclear localization, and they have also been shown to contribute to heterodimerization. The region of the VDR carboxy terminal to the DNA-binding domain contains residues involved in hormone binding, heterodimerization, transactivation, and interactions with nuclear receptor coactivators [90]. The affinity of the VDR for 1,25-dihydroxyvitamin D is approximately three orders of magnitude higher than
AF1
DBD
HR
HBD
AF2
I!i111111 iiiiiiii.iiiiii!iiiiiiiiiiiiiii!iiiiii!{ii3iiiiiiii{ii!ii!iii!ii!iil
Species Rat 95% Mouse 100% Chicken 48% Xenopus 62%
99% 99% 97% 93%
75% 74% 44% 45%
91% 90% 83% 76%
100% 100% 100% 75%
FIGURE 2 Evolutionaryconservation of functional domains in the vitamin D receptor (VDR). A schematicrepresentation of the VDR is shown with its principal domains-- AF1 and AF2 activation domains, DNA-binding domain (DBD), heterodimerizationdomain (HR), and hormone-binding domain (HBD). Indicated below is the evolutionary conservation of the amino acid sequence of the various domains between species.
7. VitaminD Biology that for 25-hydroxyvitamin D. Hormone binding by the receptor results in phosphorylation and promotes heterodimerization, high-affinity binding to DNA response elements, and the recruitment of nuclear receptor coactivators. A amphipathic e~-helical region at the C terminus (residues 416-422), referred to as the AF2 region, has been shown to be critical for transactivation as well as for recruitment of nuclear receptor coactivators. The transcriptional activity of the VDR is modulated by posttranslational modifications as well as by association with nuclear receptor coactivators. Like other members of the nuclear receptor superfamily, the VDR undergoes rapid phosphorylation. Phospho-amino acid analysis has shown that the predominant phosphorylated residue is serine. Both in vitro and in vivo studies have demonstrated that serine 208 is an important site of VDR phosphorylation. This residue is flanked by a casein kinase II recognition site and, in fact, purified casein kinase II has been shown to phosphorylate the human VDR at this residue [106]. Phosphorylation at Ser2~ has been shown to be the principal site of hormone-dependent VDR phosphorylation [107]. Although phosphorylation of this residue does not alter ligand binding [106], nuclear localization, or binding to VDREs, it results in a dose-dependent enhancement of 1,25-dihydroxyvitamin D-mediated transactivation of reporter genes [108], suggesting that it results in an enhanced interaction with nuclear receptor coactivators or with the basal transcription apparatus. In contrast, the rapid protein kinase A-mediated phosphorylation of the VDR is neither mediated by nor dependent on ligand. Protein kinase A-mediated phosphorylation of residues in the central region of the receptor attenuates, rather than enhances, transcriptional activity [109]. Protein kinase C has also been shown to phosphorylate the VDR. Attenuation of ligandmediated transactivation is also seen with protein kinase C-mediated VDR phosphorylation at ser 51 [110], correlating with impaired binding of the VDR to its DNA response elements [111]. The C-terminal region of the vitamin D receptor has been shown to interact with the basal transcription apparatus by directly contacting the transcription factor, TF IIB [112]. Although this interaction contributes to transactivation by the VDR, the observation that it occurs in a ligand-independent fashion suggests that it is only one of a series of protein-protein interactions that ultimately lead to the modulation of gene expression by the VDR. Nuclear R e c e p t o r Coactivators Nuclear receptor coactivators provide a critical link between ligand-activated nuclear receptors and the basal
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transcription machinery. The best characterized nuclear receptor coactivators belong to the SRC/pl60 (steroid receptor coactivator) family [113,114]. The members of this family interact with nuclear receptors via an oL-helical LXXLL motif that binds to an ~-helical region in the distal C-terminal region (416-422) of the VDR. It is postulated that ligand binding by the VDR results in a conformational change, exposing residues C terminal to the DNA-binding domain (224-246,255-278, and 416422), which in turn bind SRC-1. In fact, mutagenesis of these sequences results in impaired transactivation in the setting of normal hormone binding and heterodimerization with RXR [115]. SRC-1 has also been shown to enhance the ligand-dependent interaction between Smad-3, a protein involved in the transforming growth factor-13 signaling pathway, and the liganded VDR, suggesting that nuclear coactivators play a key role in modulating the VDR-mediated transcriptional effects of growth factors [116]. In addition to interacting with general transcription factors, including TF IIB and TATA-binding protein [117], SRC-1 family members have been shown to have histone acetyl transferase activity [118]. This latter enzymatic activity is thought to increase access to the general transcriptional apparatus by remodeling chromatin, thereby modifying the repressive effects of nucleosomes on gene expression. SRC/pl60 family members form complexes with CBP/p300, which also provides histone acetyl transferase activity [119]. Although CBP/p300 binds nuclear receptors with low affinity, its interactions with SRC-1 family members link CBP/p300, nuclear receptors, and other proteins that mediate gene regulation by nuclear receptors. NCoA-62 (also known as SKIP) is a coactivator that was cloned based on its interactions with the VDR. The mechanism by which NCoA-62 performs its transcriptional regulatory function is distinct from that of the SRC family members. Notable in this respect, it interacts with unliganded receptors and does not require the AF2 domain. NCoA-62 also has an independent transactivation domain in its C-terminal region and a distinct central region, which mediates its interactions with nuclear receptors [120]. A multimeric complex, isolated based on its affinity to interact with the hormone-binding domain of the VDR, DRIP (D receptor interacting proteins), is thought to play a crucial role in direct recruitment of the transcriptional machinery. This complex is capable of enhancing VDR-mediated transactivation in a cell-free system [120] and is essential for ligand-dependent transactivation [121]. Binding of liganded VDR to the DRIP complex enhances binding to RNA polymerase II, suggesting that this is a critical mechanism by which the liganded VDR mediates transcriptional activation [122].
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However, several components of the DRIP complex are also present in the activation complexes purified using nonnuclear receptor transactivators, such as Spl, suggesting that they may be universally required for transcriptional activation. Corepressors (NcoR and SMRT) have also been shown to interact with nuclear hormone receptors, and their actions are thought to be responsible for the transcription-repressing activities of unliganded receptors for triiodothyronine and retinoic acid. These corepressors stabilize chromatin by deacetylation of histones. However, they have not been shown to interact with the VDR and their contribution to VDR-mediated transcriptional repression is yet to be determined.
Vitamin D R e s p o n s e Elements a n d Target G e n e s The VDR has been shown to bind to direct repeat hexameric response elements, separated by three bases, on target genes that are upregulated by 1,25-dihydroxy vitamin D [123,124]. These response elements have been identified in several genes, including the osteocalcin [125,126], osteopontin [127], e~v[33 integrin [128,129], and vitamin D 24-hydroxylase genes [130,131]. Vitamin D responsiveness has also been demonstrated for the calbindin D 9K and 28K genes. Although vitamin D response elements have been characterized in both of these genes [132,133], 1,25-dihydroxyvitamin D does not induce the expression of calbindin D 9K in all cells that express this transcript [134,135]. Interestingly, transgenic experiments have demonstrated that the 1,25-dihydroxyvitamin D responsiveness of the 5' regulatory region of calbindin D 9K is not dependent on the characterized vitamin D response element but rather requires other DNA sequences, including the consensus-binding motif for the homeodomain gene Cdx2 [136]. The VDR has also been shown to bind to direct repeat response elements separated by four or five bases and to palindromic sequences, in addition to being capable of binding to response elements as a homodimer. However, the effect of these interactions on 1,25-dihydroxyvitamin D-mediated regulation of endogenous genes and the requirement for additional factors to stabilize the D N A protein interactions have not been definitively resolved. In the case of the human c-fos promoter, Nuclear Factor 1 has been shown to be involved in the V D R - R X R complex that interacts with a DNA sequence consisting of three direct repeats separated by seven bases [137]. In an analogous fashion, perhaps binding of the VDR to "nontraditional" response elements requires stabiliza-
tion of the DNA-protein interactions by recruitment of other nuclear proteins. Transcriptional repression mediated by the VDR is less well understood. The VDR has been shown to interact with DNA sequences in the PTH gene that are homologous to the motifs required for transcriptional induction by this receptor [138,139]. The mechanism by which these sequences lead to 1,25-dihydroxvitamin Dmediated transcriptional repression, rather than induction, has not been clarified. It is also uncertain whether this transcriptional repression is due to the use of alternate heterodimerization partners [140] or to recruitment of a different set of nuclear receptor coactivators or corepressors. The human PTH-related peptide negative VDRE has also been shown to bind the VDR but not RXR. Transcriptional repression mediated by this negative VDRE is dependent on interactions with the VDR and Ku antigen [141]. The sequences responsible for 1,25-dihydroxyvitamin D-mediated transcriptional repression of the vitamin D loL-hydroxylase gene bind a bHLH transcription factor [142]. Interaction of the VDR with these sequences is thought to result in transcriptional repression of the vitamin D l oL-hydroxylase gene by transcriptional interference. It is thought that the binding of the VDR to these sequences impairs binding of the bHLH protein, which in turn leads to decreased transcription of the l a-hydroxylase gene. In vitro data suggest that the unliganded VDR can repress basal transcription and that the unliganded VDR/RXR heterodimer causes VDR-mediated repression of basal transcription on classic vitamin D response elements [143]. The in vivo significance of these observations has not been established, but the existence of ligand-independent VDR actions is supported by the observation that mice and humans lacking functional VDRs develop alopecia [144], whereas the absence of ligand does not result in this phenotype [145]. Nonclassical Effects Several actions of 1,25-dihydroxyvitamin D occur too rapidly to be mediated by transcriptional mechanisms. These effects involve rapid activation of second messengers [146,147] and activation of voltage-dependent calcium channels [146-149]. Also, actions of naturally occurring vitamin D metabolites that bind the nuclear VDR with very low affinity have been shown to have specific biological effects, notably the effect of 24hydroxylated metabolites on growth plate chondrocyte maturation [150,151]. Studies in mice lacking the nuclear VDR are required to determine if these actions are mediated by a unique receptor and whether this alternative receptor is membrane associated or nuclear.
7. Vitamin D Biology
ROLE OF VITAMIN D IN CALCIUM HOMEOSTASIS Physiological Actions of 1~,25-Dihydroxyvitamin D Animal models and studies of humans with dietary vitamin D deficiency have contributed significantly to our understanding of the role of 1,25-dihydroxyvitamin D in mineral ion homeostasis [152-155]. These studies have demonstrated that 1,25-dihydroxyvitamin D plays a critical role in the intestinal absorption of calcium. Vitamin D-deficient animals develop hypocalcemia, hypophosphatemia, hyperparathyroidism, rickets, and osteomalacia [152]. In vitro studies have demonstrated that 1,25-dihydroxyvitamin D has an antiproliferative effect on parathyroid cells [156]. Furthermore, 1,25dihydroxyvitamin D has been shown both in vitro [157,158] and in vivo [159] to repress PTH gene transcription. These data provide much of the rationale for preventing and treating uremic hyperparathyroidism with pharmacological doses of vitamin D metabolites [160]. Numerous studies have examined the skeletal actions of 1,25-dihydroxyvitamin D. Disorganized growth plates are observed in rickets associated with vitamin D deficiency as well as with hypophosphatemia. Pathologically, these growth plates are characterized by an expansion in the layer of hypertrophic chondrocytes [152], although it is unclear whether this is due to a specific chondrocyte maturation defect, increased proliferation of these cells, or impaired apoptosis of the hypertrophic chondrocytes. Healing of the growth plate is seen with normalization of calcium and phosphorus levels in vitamin D-deficient animals and children, suggesting that impaired mineral ion homeostasis contributes to the rachitic changes [161-164]. Osteoblasts express vitamin D receptors, and 1,25dihydroxyvitamin D has been shown to play a critical role in the regulation of several bone matrix proteins. 1,25-Dihydroxyvitamin D has been shown to downregulate the transcription of the Otl(I) collagen gene in osteoblasts [165] and to be a potent transcriptional activator of the genes encoding osteocalcin and osteopontin [127]. However, experiments performed in vitamin D-deficient rats have failed to show significant differences in the bone content of several matrix proteins, including osteocalcin [166]. Studies performed both in vitamin Ddeficient rats [167] and in humans [163] with vitamin D receptor mutations have demonstrated that calcium and phosphorus repletion can mineralize osteomalacic lesions in the presence of impaired 1,25-dihydroxyvitamin D action, suggesting that this steroid hormone is not required for mineralization.
201
1,25-Dihydroxyvitamin D has been shown to play an important role in stimulating the differentiation of osteoclasts from monocyte-macrophage stem cell precursors in vitro. Mature osteoclasts do not express vitamin D receptors, and it has been demonstrated that monocyte-macrophage precursors isolated from vitamin D receptor null mice are capable of differentiating into multinucleated osteoclasts [168]. This is consistent with the effect of 1,25-dihydroxyvitamin D and parathyroid hormone on inducing the synthesis of RANK ligand (osteoclast differentiating factor) by the osteoblast. This membrane-bound factor then interacts with the RANK receptor on osteoclast precursors, leading to the differentiation of mature osteoclasts. RANK ligand synthesis is also induced by PTH, and in the absence of the genomic actions of 1,25-dihydroxyvitamin D it is believed that PTH-stimulated production of this factor is sufficient to sustain osteoclast differentiation. It is clear that 1,25-dihydroxyvitamin D plays a critical role in intestinal calcium absorption. Two calcium channels, expressed in the intestine, have recently been cloned--ECaC [169] and IcaC [170]. The relative roles of each of these channels in promoting intestinal calcium transport and the degree to which their synthesis and activity are modulated by 1,25-dihydroxyvitamin D remain to be clarified. However, 1,25-dihydroxyvitamin D has been shown to be a potent inducer of the gene encoding calbindin D 9K, a protein that binds calcium ions and is thought to play a role in transcellular calcium transport. Studies in vitamin D-deficient animals have demonstrated that calbindin D 9K levels are increased early and increase dramatically after injection of vitamin D metabolites, and they correlate with an increase in active transport of calcium across the intestine [171]. Vitamin D has been shown to have effects not related to mineral ion homeostasis. Consistent with these observations, the receptor for 1,25-dihydroxyvitamin D is expressed in many tissues, including skin, breast, prostate, pancreas, colon, muscle, and immune cells. 1,25-Dihydroxyvitamin D has been shown to induce the differentiation and slow the proliferation of both hematopoietic tumor cells [172] and keratinocytes [173-175]. Furthermore, 1,25-dihydroxyvitamin D analogs have been shown to have therapeutic potential in the treatment of psoriasis [176]. In addition to its immunomodulatory effects, 1,25-dihydroxyvitamin D has an antiproliferative effect on malignant cells, including breast [177,178] and prostate cancer cells [179], and numerous studies have suggested that vitamin D metabolites may protect against the development of several malignancies including colon cancer.
202.
Ren6 St.-Arnaud and Marie B. Demay
Animal M o d e l s
cyp27 Knockout CYP27 is the sole cloned cytochrome P450 able to hydroxylate vitamin D at position 25 [180,181]. The enzyme is also important for bile acid biosynthesis and regulation of cholesterol homeostasis [11,12]. Mutations in the gene cause cerebrotendinous xanthomatosis (CTX), a lipid storage disorder leading to premature atherosclerosis and progressive neurological dysfunction [7,8]. Mice deficient for cyp27 have been engineered [6]. Homozygous cyp27 -/- animals have larger livers and larger adrenals [182], decreased synthesis and excretion of bile acids [6], and hypertriglyceridemia [182]. The increased formation of 25-hydroxylated bile alcohols and cholestanol observed in CTX patients was not observed in cyp27 -/- mice, and no CTX-related pathological abnormalities were evident [6]. It remains unclear whether the cyp27-deficient mouse is a valid animal model for CTX. Interestingly, cyp27 null mice had normal serum concentrations of 1,25(OH)2D and slightly elevated levels of 25(OH)D in their blood [6]. These results strengthen the hypothesis that a distinct vitamin D 25-hydroxylase enzyme is involved in vitamin D metabolism. DBP Knockout
The bulk of 25(OH)D circulates bound to the carrier protein DBP, also known as the group-specific component of serum (Go-globulin). DBP shows higher binding affinity for 25(OH)D but can also bind 1,25(OH)2D and the parental vitamin D, albeit with 10-fold reduced affinity [183]. The role of DBP in vitamin D biology remained unclear until a line of mice deficient in DBP was generated. When fed a normal, vitamin D-replete diet, DBP -/mice had significantly reduced serum levels of 25(OH)D and 1,25(OH)2D but were otherwise normal [184]. The effect of the mutation was more evident when the mice were challenged by feeding them a vitamin D-deficient diet. Under these conditions, the DBP-deficient mice became hypocalcemic and hypophosphatemic, and they developed secondary hyperparathyroidism. These biochemical changes were accompanied by the typical bone changes associated with vitamin D deficiency~increased unmineralized bone (osteoid) and increased osteoblastic activity [184]. Thus, the absence of DBP leads to increased sensitivity to dietary vitamin D deprivation. With respect to vitamin D homeostasis, DBP -/- mice had a significantly decreased half-life of 25(OH)D in the circulation and increased liver uptake of vitamin D [184]. Urinary excretion of 25(OH)D was higher in DBP null mice [184]. DBP-deficient animals were less susceptible to the toxic effects of hypervitaminosis D, exhibiting re-
duced nephrocalcinosis and reduced hypercalcemia when compared with wild-type littermates [184]. This observation correlates with the profile of serum clearance of vitamin D measured in DBP null mice. Overall, analysis of the DBP -/- phenotype is consistent with a role for DBP in maintaining stable serum stores of vitamin D metabolites and influencing its bioavailability and activation. Megalin Knockout
The key role played by DBP in vitamin D metabolite activation was further confirmed by the analysis of surviving megalin knockout mice. Megalin is a multifunctional clearance receptor involved in the uptake of ligands from the luminal site into the proximal tubular cells of the kidney [185,186]. It binds numerous molecules, including vitamin-binding proteins, hormones, lipoproteins, proteases, and protease inhibitors [185,186]. Megalin is expressed in the neuroepithelium and in proximal tubular cells of the kidney; mice deficient for megalin die soon after birth from a developmental defect of the forebrain [187]. The severity of the phenotype differs between animals, however, and 1 in 50 of the megalin -/- mice survive to adulthood. Analysis of the phenotype of these survivors identified a key endocytic pathway involved in renal uptake and activation of 25 (OH)D [188]. Kidneys from megalin-deficient mice have decreased components of the endocytic apparatus, suggesting deficient uptake of filtered macromolecules [187]. Analysis of the urine from surviving adult megalin -/- mice revealed several excreted low-molecular-weight proteins that are usually reabsorbed in normal mice. Amino acid sequence analysis of the major protein band from knockout urine matched the sequence of DBP [188]. This suggested that megalin normally binds DBP and mediates its reabsorption from the urine. Indeed, it was demonstrated that megalin is the renal DBP receptor and that it mediates tubular uptake of unliganded DBP as well as 25(OH)DDBP complexes [188]. Moreover, perfused rat kidney experiments showed that blocking megalin activity in the tubules with a specific antagonist prevented conversion of 25(OH)D to 1,25(OH)2D [188]. These results suggest that filtered and reabsorbed DBP-bound 25 (OH)D serves as a substrate for the renal 1oL-hydroxylase enzyme and that loss of megalin activity prevents delivery of the substrate to tubular cells. Since approximately 90% of circulating 25(OH)D is bound to DBP [189], the massive excretion of DBP in the urine of megalin -/- mice leads to vitamin D deficiency and bone defects in megalin null mice [188]. The proposed model for the role of megalin in vitamin D homeostasis involves endocytosis of excreted 25(OH)D-DBP complexes into the proximal tubular kidney cells via megalin. Delivery of the endocy-
7. Vitamin D Biology
tosed complexes to lysosomes leads to DBP degradation and release of 25(OH)D, which is hydroxylated to 1,25 (OH)2D and resecreted into the circulation. These studies have elucidated a key step in vitamin D metabolism that was previously unrecognized.
cyp24 Knockout The CYP24 enzyme initiates the C 24 oxidation pathway, a succession of hydroxylation/oxidation reactions at carbons 24 and 23 that lead to 1,25(OH)2D inactivation [80,81]. The role of the CYP24 enzyme in the catabolism of 1,25(OH)2D was demonstrated in tissue culture [88]. The cyp24 gene has been inactivated in mice to examine the role of the CYP24 enzyme in vitamin D homeostasis in vivo [89]. Fifty percent of cyp24 -/- mice die before 3 weeks of age [89,190]. Analysis of macrophage function ruled out impaired responses to infection as the cause for postnatal death. The perinatal lethality is most likely a consequence of hypercalcemia secondary to hypervitaminosis D since the inactivation of the cyp24 gene in mice impaired the ability of the animals to clear 1,25(OH)2D. Bolus and chronic 1,25(OH)2D administration resulted in a marked elevation in serum 1,25(OH)2D levels in the mutant animals [89]. Chronic 1,25(OH)2D administration in cyp24 -/- mutants resulted in histological changes consistent with hypervitaminosis D in the kidney: cortical tubular dilation, necrotic debris, and mineralization (nephrocalcinosis). The inability to regulate 1,25(OH)2D and calcium homeostasis presumably leads to fatal hypercalcemia. Indeed, extremely high levels of circulating 1,25(OH)2D and calcium were measured in runted animals that died before weaning [89]. Since half of the mutant progeny appear unaffected by the cyp24 deficiency, these animals most likely use alternate means of regulating vitamin D homeostasis. We have measured clearance and metabolism of labeled 1,25(OH)2D in cyp24 -/- survivors and heterozygote controls. These experiments have shown that cyp24 null mice have impaired clearance of 1,25(OH)2D. Surprisingly, cyp24 -/- mice appear to lack not only 24-hydroxylated metabolites but also 1,25(OH)2D-26,23-1actone, supporting the view that the CYP24 enzyme is also responsible for hydroxylating 1,25(OH)2D at C 23 and C 26 [87,191]. These surprising findings suggest that the cyp24 null survivors adapt to the impaired vitamin D catabolism not by using an alternative catabolic route but by limiting the synthesis of the active compound. The survival of some cyp24 -/- mutant animals to adulthood has also allowed experiments designed to address the effect of perturbing vitamin D metabolism during development. Bone development is abnormal in homozygous mutants born from homozygous females
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[89,192]. Histological examination of the bones from these animals revealed an accumulation of osteoid at sites of intramembranous ossification [89,192]. No significant disruption of growth plate organization was noted. Control heterozygote littermates showed normal bone structure. Two major hypotheses can be formulated to account for the phenotype of the CYP24-deficient embryos from CYP24 mutant mice: (i) Perturbation of 1,25(OH)2D catabolism through the inactivation of the C 24 oxidation pathway affects bone development, and (ii) metabolites of vitamin D hydroxylated at position 24 are essential for bone development. To differentiate between these possibilities, the cyp24-deficient animals were bred to mice carrying an inactivating mutation of the vitamin D receptor gene [193]. If elevated 1,25(OH)2D levels, acting through the vitamin D receptor, were responsible for the observed phenotype, then mice lacking the receptor and the cyp24 gene should not show the aberrant intramembranous bone development. Using this elegant genetic strategy, incontrovertible evidence was obtained that expression of the VDR is required for the impaired mineralization phenotype of the cyp24-deficient animals: Double-mutant homozygotes (cyp24 -/- and VDR -/-) showed normal intramembranous bone formation at all sites examined [89]. This demonstrates that elevated 1,25 (OH)2D levels during gestation affect mineralization and suggests that impaired vitamin D metabolism during development perturbs bone formation.
la-Hydroxylase (cyp27B1)Knockout The 1et-hydroxylase enzyme (CYP27B 1) converts 25 (OH)D to 1,25(OH)2D, the biologically active metabolite of vitamin D. Mutations in the CYP27B1 gene cause PDDR, a rare autosomal disease characterized by growth retardation, failure to thrive, rickets, and osteomalacia [194,195] (see Chapter 24). Biochemical analysis of serum from affected patients reveals hypocalcemia, secondary hyperparathyroidism, and undetectable levels of 1,25(OH)2D3. An animal model of PDDR by targeted inactivation of the la-hydroxylase gene in mice has been generated [42]. The strategy, based on the Cre-Lox methodology, allows tissue-specific inactivation of the targeted gene. The technology utilizes the Cre recombinase from the P1 bacteriophage. This enzyme catalyzes recombination between two 34-bp recognition elements, called loxP sites, causing excision of the intervening sequences. Transgenic mice lines expressing Cre under the control of tissuespecific promoters are crossed with mice harboring loxP sites to achieve excision at will [196]. This strategy also allows whole-body knockouts from the same targeting event by transfecting a CMV-Cre expression vector in
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targeted ES cells and selecting for clones that have recombined between the loxP sites that flank the targeted exon. This conditional 1a-hydroxylase allele provides an invaluable genetic tool to analyze the putative autocrine/ paracrine roles that have been hypothesized for 1,25 (OH)eD in various cell types, such as osteoblasts, chondrocytes, macrophages, or keratinocytes. The l a-hydroxylase gene was cloned from a 129 SV mouse genomic library to construct a targeting vector in which exon 8, encoding the heme-binding domain [35], was flanked by a 5' loxP recognition site and by a 3' loxP-neo-loxP selection cassette. Homologous recombination at the l oL-hydroxylase locus followed by transient transfection of the embryonic stem cells with the Cre recombinase generated a targeted allele in which exon 8 was deleted, thus engineering a conventional knockout allele [42]. The engineered mutation was transmitted to the progeny with the expected Mendelian ratio (Table 2). Heterozygous animals had no discernable phenotype and were fertile. Homozygous mutant animals were phenotypically normal at birth but exhibited retarded growth as measured by weight gain from 3 to 8 weeks of age and femur length at 8 weeks (Fig. 3). Serum analysis of homozygous mutant animals confirmed that they were hypocalcemic (Fig. 4) and hypophosphatemic and had hyperparathyroidism and undetectable circulating levels of 1,25(OH)2D3 [42]. From a biochemical standpoint, the only difference between the l a-hydroxylase -/- mice and patients with P D D R is that patients with P D D R have normal serum levels of 25(OH)D [197-199] and 24,25(OH)2D [200], whereas elevated levels of 25(OH)D and very low levels of 24,25(OH)2D are seen in 1a-hydroxylase mutant mice [42]. The 1,25(OH)2D hormone is the main in vivo regulator of the expression of the CYP24 enzyme that catalyzes the synthesis of 24,25(OH)gD [201]. Therefore, low circulating 24,25(OH)2D levels and undetectable CYP24 expression in l a-hydroxylase -/- mice are anticipated. The discrepancy with the human disease remains to be explained but could result from species differences. This observed inhibition of CYP24 expression in mice, combined with the targeted ablation of the l oL-hydroxylase
TABLE 2
t h e I a - O H a s e null allele g e t s t r a n s m i t t e d with t h e expected Mendelian frequency
Number of births 584
+/+
+/-
-/-
154 (26.4%)
289 (49.5%)
141 (24.1%)
Adult heterozygotes males and females were intercrossed and the progeny genotyped by Southern blotting of tail DNA.
Femur length
(mm)
17.5
I
A
E E
.c -.01 e~
I
eeo
15.0
-ODOIDIDD-
GO
12.5
&& &A& &AA A&
-i|h- -ooo--AAA10.0
7.5
+1+
+/-
I
-/-
3 weeks
age:
+/8
-I-
weeks
FIGURE 3 Reduced growth of 25-hydroxyvitamin D-loL-hydroxylase (la-OHase) mouse mutants. Animals from all genotypes (+/+, + / - , and - / - ) and + / - and - / - littermates were sacrificed at weaning and at 8 months of age, respectively. Femurs were collected and measured with a caliper. There was no significant difference between femur length at 3 weeks of age (weaning), but l oL- OHase -/- animals ( - / - ) exhibited reduced growth rates and had significantly shorter femurs at 8 weeks compared to heterozygote ( + / - ) littermate controls.
Hypocalcemia in l(x-OHase-/- mice 3.25 - -
I
I
0
3.00 - E E ,_.., 2 . 7 5 - -
9
tl:i
O.Q, 2 . 5 0 E L
9
--A~-A~-AXA-AAA
2.252.00
VVV VV
+I+
+I-
"/"
**p
Genotype
FIGURE 4 Serum concentrations of total calcium in 25-hydroxyvitamin D- 1oL-hydroxylase(1oL-OHase) mutant mice. Pups were sacrificed by exsanguination under anesthesia at 3 weeks of age and genotyped by Southern blotting of tail DNA. Total calcium was measured from serum samples of wild-type (+/+), heterozygote (+/-), and homozygote mutant ( - / - ) animals using an automated Monarch analyzer. The horizontal bars indicate the mean for each population. **p < 0.01 by analysis of variance. The mean value for - / - mice is at the lower end of the normal range for this strain.
enzyme, leads to a metabolic block in mutant animals and an accumulation of the unprocessed 25(OH)D substrate [42]. Histological analysis of the bones from 3-week-old mutant animals confirmed the evidence of rickets [42]. At the age of 8 weeks, femurs from l c~-OHase-ablated mice presented a severe disorganization in the architecture of the growth plate and marked osteomalacia [42]. The X-ray features of the bones from 1oL-hydroxylase - / animals also matched the clinical manifestations of PDDR. Contact radiography of femurs from mutant
7. VitaminD Biology animals revealed diffuse osteopenia (hypomineralization) and rachitic metaphyseal changes [202]. Interestingly, the expression of vitamin D-dependent genes, such as osteopontin and osteocalcin, was not changed in bone tissue from 1oL-hydroxylase mutant mice [42]. The treatment of choice for PDDR is long-term replacement therapy with 1,25(OH)2D3 [195,197,203]. Recent results reveal that treatment of l~-hydroxylasedeficient mice with 1,25(OH)2D for 5 weeks corrects the hypocalcemia and secondary hyperparathyroidism. Bone histology and histomorphometry confirmed that rickets and osteomalacia were cured. The biomechanical properties of the bone tissue (load bearing, deformation, and stiffness) were also normalized by the rescue treatment [249]. Other rescue regimens, such as treatment with I~(OH)D [204] or feeding with a highcalcium, high-lactose diet [205], are currently being tested. Local production of 1,25(OH)2D could play an important autocrine or paracrine role in the differentiation or function of osteoblasts, chondrocytes, keratinocytes, and macrophages [102,206,207]. Crosses between transgenic mouse lines expressing the Cre recombinase under the control of the Otl(I) collagen gene promoter (Col ICre, expressed in osteoblasts) or the OLl(II) collagen promoter (Col II-Cre, expressed in chondrocytes) and mice harboring the conditional 1oL-hydroxylase allele will provide a powerful tool to address the physiological role of local production of 1,25(OH)2D in the context of normocalcemic animals. VDR Knockout
Although studies in vitamin D-deficient animals and in vitro studies using cell culture models clarified many of
the actions of vitamin D metabolites, there remained several unanswered questions concerning the principal actions of 1,25-dihydroxyvitamin D in vivo. To address these questions, four laboratories have generated mice with targeted ablation of the vitamin D receptor. As anticipated, these mice demonstrate several of the phenotypic abnormalities present in humans with vitamin D receptor mutations [193,208]. The pups are born phenotypically normal and are indistinguishable from their wild-type and heterozygous littermates. At the end of the third week of life, the mice develop hyperparathyroidism, presumably secondary to impaired intestinal calcium absorption. This hyperparathyroidism is accompanied by hypophosphatemia and hypocalcemia. The timing of the development of impaired intestinal calcium absorption is consistent with studies performed in rat pups, which demonstrated that intestinal calcium absorption is 1,25-dihydroxyvitamin D independent in the first 3 weeks of life [209]. Thereafter, it gradually be-
Z05
comes hormone dependent, and by 7 weeks of life a 1,25-dihydroxyvitamin D-dependent active transport mechanism is largely responsible for the active transport of calcium from the intestinal lumen. Associated with an elevation in circulating PTH levels is the gradual onset of parathyroid hyperplasia due to increased parathyroid cellular proliferation. There is a marked increase in proliferating nuclear antigen (PCNA) staining in the parathyroid glands of the VDR null mice by 35 days of age, associated with an increase in parathyroid glandular volume [205]. By 70 days of age, the circulating immunoreactive PTH levels in the VDRnull mice are elevated 16-fold and the parathyroid glandular volume is 10 times that of wild-type control littermates. Interestingly, prevention of abnormal mineral ion homeostasis in the VDR null mice prevents the development of secondary hyperparathyroidism and parathyroid glandular hyperplasia [205]. These data demonstrate that in the presence of normocalcemia, the genomic actions of 1,25-dihydroxyvitamin D are not required for parathyroid homeostasis. Whether these actions play an in vivo role in attenuating parathyroid cellular proliferation and PTH gene transcription in hypocalcemic states is not clear. However, these data suggest that hypocalcemia is the pathophysiological basis for hyperparathyroidism when the action of 1,25dihydroxyvitamin D is impaired, rather than lack of hormone. Analogous to humans with VDR receptor mutations and to vitamin D deficiency during growth, the VDR null mice develop severe rickets and osteomalacia. Expansion of the hypertrophic chondrocyte layer of the growth plate is evident as early as 21 days, 2 days after the development of secondary hyperparathyroidism. The absence of a growth plate phenotype prior to the development of impaired mineral ion homeostasis suggests that these rachitic changes are a direct consequence of hypocalcemia, hypophosphatemia, or secondary hyperparathyroidism rather than impaired genomic actions of 1,25-dihydroxyvitamin D. Confirming this hypothesis is the observation that prevention of impaired mineral ion homeostasis prevents the growth plate abnormalities in the VDR null mice [205]. Once hypocalcemia and hypophosphatemia have resulted in growth plate abnormalities, restoration of normal mineral ion levels is able to attenuate the progression of rachitic changes; however, skeletal remodeling is unable to restore a normal growth plate. Impaired mineralization of newly synthesized osteoid (osteomalacia) is a prominent feature in the VDR null mice [162]. By 70 days of age, there is a 30-fold increase in osteoid volume that leads to a 5-fold increase in bone volume. Despite this increase in bone volume, biomechanical analyses have demonstrated a marked decrease in
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the strength of the bones from the VDR null mice, consistent with the increased fracture risk of patients with osteomalacia [210-212]. Analysis of the cellular content of the bone of the VDR null mice demonstrates a 2-fold increase in osteoblast number, presumably due to the acceleration of bone turnover in response to profound secondary hyperparathyroidism. Paradoxically, the number of osteoclasts is not significantly affected, despite the elevation of immunoreactive PTH levels. In vitro studies have demonstrated that the absence of the VDR in the osteoclast precursors does not impair differentiation of these cells to mature osteoclasts. However, the absence of the VDR in marrow stromal cells (osteoblast precursors) leads to impaired osteoclastogenesis in response to 1,25-dihydroxyvitamin D but not to PTH [168]. These in vitro data suggest that there should be a compensatory increase in osteoclast number in the presence of secondary hyperparathyroidism. Two additional variables may explain the inappropriately normal osteoclast number in the VDR null mice with secondary hyperparathyroidism. First, the osteoclast Otv~3 integrin gene is highly induced by 1,25-dihydroxyvitamin D [128,129]. Second, osteoclasts are unable to resorb osteoid. Prevention of abnormal mineral ion homeostasis prevents the development of osteomalacia [162]. Furthermore, the increased bone volume in the hypocalcemic VDR null mice is not apparent in the normocalcemic counterparts, suggesting that the increase in matrix formation is due to the PTH-stimulated acceleration in bone turnover and the impaired resorption of unmineralized matrix by osteoclasts. VDR null mice with normal mineral ion homeostasis have a normal mineral apposition rate, confirming that the VDR is not required for mineralization of newly formed osteoid [162]. The biomechanical properties of the bones from the normocalcemic VDR null mice are indistinguishable from those of control littermates, as is the cellular content of the bone [162]. These data demonstrate that the genomic actions of the VDR are not required for skeletal homeostasis. The dramatic phenotypic rescue by normalization of mineral ion homeostasis in the VDR null mice confirms that the intestine is one of the critical targets of receptormediated actions of 1,25-dihydroxyvitamin D. Studies in the VDR null mice demonstrated a dramatic impairment in intestinal calcium absorption. This was associated with a reduction in mRNA levels for two calcium channels, ECaC and CAT1, which are thought to play a critical role in the regulation of calcium entry into the enterocyte [213]. Levels of calbindin D 9K mRNA were also dramatically reduced in the duodenum of the VDR null mice [213,214]; however, the level of the plasma membrane ATPase was not affected by VDR status [213]. These data suggest that expression of the channels that regulate calcium entry into the enterocyte and that
of calbindin D 9K, which is thought to play a role in intracellular calcium transfer, are the two principal mechanisms by which the nuclear VDR acts to promote intestinal calcium transport. A striking feature of both humans and mice with VDR mutations is the presence of alopecia. Alopecia totalis is not a feature of vitamin D deficiency [215]; however, there have been no cases of vitamin D deficiency reported with undetectable circulating levels of 1,25-dihydroxyvitamin D. Both mice and humans with VDR mutations are born with hair, suggesting that hair morphogenesis does not require a functional VDR. However, clinically, the VDR null mice begin to develop alopecia by the fourth week of life [193,208]. This process is not prevented by normalization of mineral ion levels, suggesting that it is truly VDR dependent [205]. Because 1,25-dihydroxyvitamin D has been shown to play an important role in inhibiting proliferation and promoting differentiation of neonatal keratinocytes [173], studies were undertaken to determine whether the absence of the VDR led to increased keratinocyte proliferation and impaired differentiation. Studies using neonatal keratinocytes isolated from the VDR null mice demonstrated that the proliferation rate and the acquisition of selected markers of keratinocyte differentiation were indistinguishable from those of keratinocytes isolated from control littermates [216]. These data suggested that either the defect leading to alopecia did not lie in the keratinocyte or that the actions of the VDR required for prevention of alopecia were only required after the morphogenetic period. Hair follicle morphogenesis in mice beings on Embryonic Day 14.5 and ends on Day 14 of life, the end of the first hair cycle. Although in control mice depilation in the third week of life results in initiation of a new hair cycle, with hair regrowth beginning 1 week later, the VDR null mice do not regrow hair in response to this stimulus [216]. Hair reconstitution assays were performed to determine whether the defect in initiation of postmorphogenetic hair cycles was due to the absence of the VDR in the mesenchymal (dermal papilla cell) or epidermal (keratinocyte) component of the hair follicle. These assays demonstrated that VDR expression in the keratinocyte was sufficient to prevent the defect in postmorphogenic hair cycling [145]. These studies led to in vivo analyses, which demonstrated that targeting expression of a VDR transgene to the keratinocytes of VDR null mice prevented the defect in the hair cycle. The question as to how the VDR acts to maintain a normal hair cycle remains unanswered. The observation that wild-type mice with undetectable circulating levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D do not develop alopecia [145] suggests that these actions of the VDR may be ligand independent. Mice with keratinocyte-specific ablation of RXRoL also demonstrate
7. Vitamin D Biology
impairment of postmorphogenetic hair cycling, suggesting that a functional VDR-RXR heterodimer is required for normal skin homeostasis. The keratinocyte-ablated RXRoL mice have a more profound skin phenotype than the VDR null mice, including keratinocyte hyperproliferation and impaired keratinocyte differentiation [217], suggesting that RXRoL interacts with another partner to mediate these latter homeostatic effects. Other nontraditional VDR target tissues studied in the VDR null mice include the immune system [218]. 1,25-Dihydroxyvitamin D has been shown to prevent the development of autoimmune diseases [219] and to delay graft rejection in experimental models. 1,25Dihydroxyvitamin D has also been shown to regulate the expression of several key genes involved in immunomodulation, including interleukin-1, interleukin-12, interferon-~/and granulocyte macrophage colony-stimulating factor. Vitamin D-deficient animals have been shown to have defects in macrophage and neutrophil chemotactic ability as well as impaired cell-mediated immunity. However, the contribution of impaired genomic actions of 1,25-dihydroxyvitamin D to the immune defects in vitamin D deficiency is not certain, in view of the hypothesis that ligand-bound vitamin D-binding protein has significant immunomodulatory effects. Specific studies of dendritic cell function in VDR null mice demonstrated that there was an increase in mature dendritic cells, consistent with the known effect of 1,25dihydroxyvitamin D in inhibiting dendritic cell maturation [220]. Interestingly, the increase in mature dendritic cells in the receptor-deficient animals was seen only in lymph nodes and not in the spleen. Other studies in VDR null mice demonstrated normal leukocyte and lymphocyte subset composition [221]. Splenocyte proliferation in response to numerous stimuli was notable only for a minor decrease in anti-CD3 stimulation. Despite mildly impaired macrophage chemotaxis, phagocytosis and killing by these cells was normal, as was in vivo graft rejection. Perhaps the most intriguing observation was the protection of the VDR null mice from low-dose streptozotocin-induced diabetes mellitus, a response that was reversed by correction of mineral ion homeostasis [221]. These data suggest that the immunomodulatory effects of 1,25-dihydroxyvitamin D are not dependent on the nuclear receptor and that at least some of the in vivo immune phenotype of vitamin D deficiency is a consequence of impaired mineral ion homeostasis. Vitamin D M e t a b o l i t e s The 1,25(OH)2D hormone is the biologically active metabolite of vitamin D involved in the control of calcium homeostasis. More than 37 vitamin D metabolites have been characterized [222]; although most are cata-
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bolic products, it has been proposed that some of these metabolites may exert distinct biological effects. The putative bioactivity of the most abundant dihydroxylated vitamin D metabolite, 24,25(OH)2D, remains controversial. An extensive literature demonstrates that CYP24 is expressed in growth plate chondrocytes and that cells from the growth plate respond to 24,25(OH)aD in a cell maturation-dependent manner [102]. Most of these studies were performed using the in vitro rat costochondral primary culture system. Dissection of the tissue allows isolation of cells from different regions of the growth plate. Each region represents a different maturation stage along the chondrocytic differentiation pathway. In this model system, the less differentiated cells of the resting zone, also called the reserve zone, respond to 24,25(OH)2D. The more mature cells of the growth zone, including the prehypertrophic and hypertrophic compartments, respond primarily to 1,25 (OH)2D. Interestingly, treatment of resting zone chondrocytes with 24,25(OH)2D induces a change in maturation state [223], supporting the hypothesis that 24,25 (OH)2D plays a role in cartilage development. The maturation stage-dependent responses of chondrocytes to the vitamin D metabolites include both genomic and nongenomic effects [102]. The 24,25(OH)2D metabolite was also shown to be important for development in vitamin D-deficient chick embryos [224] and to be essential for egg hatchability [225,226]. The pharmacological activity of 24,25 (OH)2D on bone mass has been studied using treatment of vitamin D-replete rats, rabbits, and dogs with high doses of the metabolite [227]. Another aspect of bone biology in which investigators have sought to identify a role for 24,25(OH)2D is fracture repair. The circulating levels of 24,25(OH)2D increase during fracture repair in chicks due to an increase in renal 24-OHase activity [228]. When the effect of various vitamin D metabolites on the mechanical properties of healed bones was tested, treatment with 1,25(OH)2D3 alone resulted in poor healing [103]. However, the strength of healed bones in animals fed 24,25(OH)2D3 in combination with 1,25 (OH)2D3 was equivalent to that measured in a control population fed 25-hydroxyvitamin D3 [103]. These results support a role of physiological concentrations of 24,25(OH)aD as an essential vitamin D metabolite for fracture repair. It is likely that 24,25(OH)2D acts through receptor-mediated signaling, and preliminary evidence suggests the presence of a nonnuclear membrane receptor for 24,25(OH)2D in the chick tibial fracture-healing callus [229,230]. The controversial nature of the putative biological activity of 24,25(OH)2D is due to convincing experimental results that contradict the results of previously cited studies. For many investigators in the field, the
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production of 24,25(OH)2D is meant to inactivate circulating 25(OH)D and thus regulate synthesis of 1,25 (OH)2D. Thus, 24,25(OH)2D is considered a catabolite of 25(OH)D. Support for this theory originally came from experiments utilizing analogs of vitamin D fluorinated at position 24 (thus preventing further hydroxylation at this position). When these analogs were used as the sole source of vitamin D, they produced the same biological responses as those resulting from 25(OH)D with respect to intestinal calcium transport, mobilization of calcium from bone, and mineralization of vitamin Ddeficient bone [231]. These results have been cited repeatedly to substantiate the hypothesis that 24-hydroxylation does not play a significant role in vitamin D function, bone growth, or development. Results obtained while characterizing the phenotype of cyp24-deficient mice support the notion that vitamin D metabolites hydroxylated at position 24, including 24,25(OH)zD, exhibit little bioactivity. Although cyp24 -/- mice show impaired 1,25 (OH)2D homeostasis due to the absence of the C 24 oxidation catabolic pathway, they develop normally when born from heterozygous crosses, ruling out a major role for 24,25(OH)zD during development [89]. When cyp24 -/ - pups were born from cyp24 -/- parents, mineralization of intramembranous bone was impaired [89]. Crossing the cyp24-mutated strain to VDR-ablated mice [193] provided incontrovertible genetic evidence that the abnormal mineralization phenotype was dependent on expression of the VDR and resulted from elevated 1,25(OH)2D and not from the absence of 24,25(OH)zD [89]. Is it possible that 24,25(OH)2D exerts biological activity in specific tissues and that genetic redundancy prevented the identification of this activity in cyp24 null mice? Using cyp24-ablated mice as a source of costochondral chondrocytes would confirm or refute the importance of local production of vitamin D metabolites in the maturation of chondrocytic cells in this culture system [232]. Similarly, the cyp24 -/- mice provide a unique model system to test the physiological relevance of cyp24 expression and 24,25(OH)2D during fracture repair. The CYP24 enzyme acts on 1,25(OH)2D to produce 1,24,25(OH)3D, the initial reactant in the C 24 oxidation pathway that leads to metabolite inactivation [80,81]. Treatment of ovariectomized rats with 1,24,25(OH)3D increased bone mass and serum calcium and reduced bone resorption [233]. The second enzymatic step in the C 24 oxidation pathway is the oxidation of 1,24,25 (OH)3D to 1,25(OH)2-24-oxo-D, a metabolite that showed activity in bone resorption assays and antiproliferation assays [234,235]. Hydroxylation of 1,25(OH)2D o n C 26 eventually leads to formation of 1,25(OH)zD-26,23-1actone [236]. This metabolite affects intestinal calcium transport and can modulate bone resorption [237,238].
The conversion of 1,25(OH)2D into 3-epi-1,25 (OH)2D is characterized by a switch of the hydroxyl group orientation on C 3 from the [3 to the oL position. The enzyme responsible for this epimerization is unknown. The 3-epimerization pathway is only detected in differentiated Caco-2 cells and not in proliferating, undifferentiated cells [239]. This observation suggests that 3-epimerization is a tightly regulated metabolic pathway and supports the hypothesis that 3-epi-1,25 (OH)zD may constitute a biologically active metabolite contributing to the diverse responses of target cells to the vitamin D endocrine system. Production of 3-epi-1,25 (OH)2D was further detected in bovine parathyroid cells [240], human keratinocytes [241], and rat osteosarcoma cells [242], but not in perfused rat kidney or human promyelocytic leukemia cells [243]. The 3-epi-1,25 (OH)zD compound was also detected as a circulating metabolite in rats [244]. Biological activity associated with the 3-epi-l,25(OH)zD metabolite includes suppression of PTH secretion in bovine parathyroid cells [240] and induction of keratinocyte differentiation [245]. Despite lower affinity for the VDR [240,246], all experimental evidence indicates that any bioactivity of the 3-epi-l,25(OH)2D metabolite is mediated through the VDR and not a distinct receptor [247,248]. In summary, although some vitamin D metabolites have been shown to exhibit bioactivity, they are less active than 1,25(OH)zD and seem to act through the classical VDR, raising doubts as to their importance under physiological conditions. The identification of putative specific receptors [229,230] and the further characterization of the novel genetic models with targeted deletions in vitamin D metabolic enzymes or effector pathways will help to establish the physiological role of these metabolites. SUMMARY AND PERSPECTIVES Molecular genetics-based research has allowed major progress in our understanding of vitamin D metabolism and function. For example, the engineering of VDRdeficient mice confirmed the major physiological role of 1,25(OH)2D in the regulation of calcium absorption from the gut and the regulation of mineral homeostasis. The same strain of mutant animals identified the hair follicle keratinocyte as a critical target tissue; these studies suggest ligand-independent roles of the VDR, which will undoubtedly be the focus of intensive research in the future. The VDR-ablated mice also served as a useful tool to allow the cloning of the l oL-hydroxylase cDNA. Analysis of the negative VDRE from the l~hydroxylase gene will identify new mechanisms of vitamin D-controlled gene expression. Mice deficient in
7. Vitamin D Biology
1a-hydroxylase activity have been engineered and represent a valid animal model for PDDR. The conditional ablation strategy used to engineer these animals will elucidate the hypothesized autocrine/paracrine roles of 1,25(OH)2D in target tissues such as bone, cartilage, skin, and macrophages. Yet another strain of knockout mice, the megalin-deficient animals, permitted the identification and characterization of a major metabolic pathway for vitamin D: megalin-dependent renal uptake and activation of 25(OH)D. The observation that cyp27 -/- mice have normal vitamin D homeostasis suggests that a distinct cytochrome P450 enzyme could be involved in the 25-hydroxylation of vitamin D under physiological conditions; the information contained in the human genome database could help to identify such an enzyme. Finally, studies using the animal models developed to date and additional molecular biological analysis will help clarify unresolved aspects of vitamin D biology, including whether a nonnuclear 1,25(OH)2D receptor exists or whether other receptors with high affinity for alternative vitamin D metabolites play physiological roles in vitamin D action.
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177. Colston, K. W., Mackay, A. G., James, S. Y., Binderup, L., Chanders, S., and Coombes, R. C. (1992). EB 1089: A new vitamin D analogue that inhibits the growth of breast cancer cells in vivo and in vitro. Biochem. Pharmacol. 44, 2273-2280. 178. Colston, K. W., Perks, C. M., Xie, S. P., and Holly, J. M. (1998). Growth inhibition of both MCF-7 and Hs578T human breast cancer cell lines by vitamin D analogues is associated with increased expression of insulin-like growth factor binding protein-3. J. Mol. Endocrinol. 20, 157-162. 179. Feldman, D., Skowronski, R. J., and Peehl, D. M. (1996). Vitamin D and prostate cancer. Adv. Exp. Med. Biol. 375, 53-63. 180. Usui, E., Noshiro, M., and Okuda, K. (1990). Molecular cloning of cDNA for vitamin D3 25-hydroxylase from rat liver mitochondria. F E B S Lett. 262, 135-138. 181. Su, P., Rennert, H., Shayiq, R. M., et al. (1990). A cDNA encoding a rat mitochondrial cytochrome P450 catalyzing both the 26hydroxylation of cholesterol and 25-hydroxylation of vitamin D3: Gonadotropic regulation of the cognate mRNA in ovaries. D N A Cell. Biol. 9, 657-667. 182. Repa, J. J., Lund, E. G., Horton, J. D., et al. (2000). Disruption of the stero127-hydroxylase gene in mice results in hepatomegaly and hypertriglyceridemia. Reversal by cholic acid feeding. J. Biol. Chem. 275, 39685-39692. 183. Cooke, N. E., and Haddad, J. G. (1989). Vitamin D binding protein (Gc-globulin). Endocr. Rev. 10, 294-307. 184. Safadi, F. F., Thornton, P., Magiera, H., et al. (1999). Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein. J. Clin. Invest. 103, 239-251. 185. Verroust, P. J., and Kozyraki, R. (2001). The roles of cubilin and megalin, two multiligand receptors, in proximal tubule function: Possible implication in the progression of renal disease. Curr. Opin. Nephrol. Hypertension 10, 33-38. 186. Barth, J. L., and Argraves, W. S. (2001). Cubilin and megalin: Partners in lipoprotein and vitamin metabolism. Trends Cardiovasc. Med. 11, 26-31. 187. Willnow, T. E., Hilpert, J., Armstrong, S. A., et al. (1996). Defective forebrain development in mice lacking gp330/megalin. Proc. Natl. Acad. Sci. USA 93, 8460-8464. 188. Nykjaer, A., Dragun, D., Walther, D., et al. (1999). An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3. Cell 96, 507-515. 189. Bikle, D. D., Gee, E., Halloran, B., Kowalski, M. A., Ryzen, E., and Haddad, J. G. (1986). Assessment of the free fraction of 25hydroxyvitamin D in serum and its regulation by albumin and the vitamin D-binding protein. J. Clin. Endocrinol. Metab. 63, 954-959. 190. St.-Arnaud, R. (1999). Targeted inactivation of vitamin D hydroxylases in mice. Bone 25, 127-129. 191. Miyamoto, Y., Shinki, T., Yamamoto, K., et al. (1997). 1Alpha,25-dihydroxyvitamin D3-24-hydroxylase (CYP24) hydroxylates the carbon at the end of the side chain (C-26) of the C-24-fluorinated analog of lalpha,25-dihydroxyvitamin D3. J. Biol. Chem. 272, 14115-14119. 192. St.-Arnaud, R., and Glorieux, F. H. (1997). Vitamin D and bone development. In Vitamin D (D. Feldman, F. H. Glorieux, and J. W. Pike, Eds.), pp. 293-303. Academic Press, San Diego. 193. Li, Y. C., Pirro, A. E., Amling, M., et al. (1997). Targeted ablation of the vitamin D receptor: An animal model of vitamin D-dependent rickets type II with alopecia. Proc. Natl. Acad. Sci. USA 94, 9831-9835. 194. Miller, W. L., and Portale, A. A. (1999). Genetic causes of rickets. Curr. Opin. Pediatr. 11, 333-339. 195. St.-Arnaud, R., and Glorieux, F. H. (2000). Hereditary defects in vitamin D metabolism and action. In Endocrinology (L. J. DeG-
7. Vitamin D Biology
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expression in mice lacking the vitamin D receptor. Endocrinology 139, 847-851. Pavlovitch, J. H., Galoppin, L., Rizk, M., Didierjean, L., and Balsan, S. (1984). Alterations in rat epidermis provoked by chronic vitamin D deficiency. Am. J. Physiol. 247, E228-E233. Sakai, Y., and Demay, M. B. (2000). Evaluation of keratinocyte proliferation and differentiation in vitamin D receptor knockout mice. Endocrinology 141, 2043-2049. Li, M., Chiba, H., Warot, X., et al. (2001). RXR-alpha ablation in skin keratinocytes results in alopecia and epidermal alterations. Development 128, 675-688. Hewison, M. (1992). Vitamin D and the immune system. J. Endocrinol. 132, 173-175. Mathieu, C., Waer, M., Laureys, J., Rutgeerts, O., and Bouillon, R. (1994). Prevention of autoimmune diabetes in NOD mice by 1,25 dihydroxyvitamin D3. Diabetologia 37, 552-558. Griffin, M., Lutz, W., Phan, V., La, B., and McKean, D. (2001). Dendritic cell modulation by 1,25-dihydroxyvitamin D and its analogs: A vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vivo and in vitro. Proc. Natl. Acad. Sci. USA 98, 6800-6805. Mathieu, C., Van Etten, E., Gysemans, C., et al. (2001). In vitro and in vivo analysis of the immune system of vitamin D receptor knockout mice. J. Bone Miner. Res. 16, 2057-2065. Bouillon, R., Okamura, W. H., and Norman, A. W. (1995). Structure-function relationships in the vitamin D endocrine system. Endocr. Rev. 16, 200-257. Schwartz, Z, Dean, D., Walton, J., Brooks, B., and Boyan, B. (1995). Treatment of resting zone chondrocytes with 24,25dihydroxyvitamin D3 [24,25-(OH)2D3] induces differentiation into a 1,25-(OH)2D3-responsive phenotype characteristic of growth zone chondrocytes. Endocrinology 136, 402-411. Sunde, M., Turk, C., and DeLuca, H. (1978). The essentiality of vitamin D metabolites for embryonic chick development. Science 200, 1067-1069. Norman, A., Leathers, V., and Bishop, J. (1983). Normal egg hatchability requires the simultaneous administration to the hen of l e~,25-dihydroxycholecalciferol and 24R,25-dihydroxycholecalciferol. J. Nutr. 113, 2505-2515. Henry, H. L., and Norman, A. W. (1978). Vitamin D: Two dihydroxylated metabolites are required for normal chicken egg hatchability. Science 201, 835-837. Ono, T., Tanaka, H., Yamate, T., Nagai, Y., Nakamura, T., and Seino, Y. (1996). 24R,25-dihydroxyvitamin D3 promotes bone formation without causing excessive resorption in hypophosphatemic mice. Endocrinology 137, 2633-2637. Seo, E. G., and Norman, A. W. (1997). Three-fold induction of renal 25-hydroxyvitamin D3-24-hydroxylase activity and increased serum 24,25-dihydroxyvitamin D3 levels are correlated with the healing process after chick tibial fracture. J. Bone Miner. Res. 12, 598-606. Seo, E. G., Kato, A., and Norman, A. W. (1996). Evidence for a 24R,25(OH)2-vitamin D3 receptor/binding protein in a membrane fraction isolated from a chick tibial fracture-healing callus. Biochem. Biophys. Res. Commun. 225, 203-208. Kato, A., Seo, E. G., Einhorn, T. A., Bishop, J. E., and Norman, A. W. (1998). Studies on 24R,25-dihydroxyvitamin D3: Evidence for a nonnuclear membrane receptor in the chick tibial fracture-healing callus. Bone 23, 141-146. Parfitt, A. M., Mathews, C. H., Brommage, R., Jarnagin, K., and DeLuca, H. F. (1984). Calcitriol but no other metabolite of vitamin D is essential for normal bone growth and development in the rat. J. Clin. Invest. 73, 576-586.
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232. Schwartz, Z., Brooks, B., Swain, L., Del Toro, F., Norman, A., and Boyan, B. (1992). Production of 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 by growth zone and resting zone chondrocytes is dependent on cell maturation and is regulated by hormones and growth factors. Endocrinology 130, 2495-2504. 233. Erben, R. G., Bante, U., Birner, H., and Stangassinger, M. (1997). Prophylactic effects of 1,24,25-trihydroxyvitamin D3 on ovariectomy-induced cancellous bone loss in the rat. Calcif. Tissue Int. 60, 434- 440. 234. Campbell, M. J., Reddy, G. S., and Koeffler, H. P. (1997). Vitamin D3 analogs and their 24-oxo metabolites equally inhibit clonal proliferation of a variety of cancer cells but have differing molecular effects. J. Cell. Biochem. 66, 413-425. 235. Stern, P. H., Rappaport, M. S., Mayer, E., and Norman, A. W. (1984). 24-Oxo and 26,23-1actone metabolites of 1,25-dihydroxyvitamin D3 have direct bone-resorbing activity. Arch. Biochem. Biophys. 230, 424-429. 236. Jones, G. (1997). Analog metabolism. In Vitamin D (D. Feldman, F. Glorieux, and J. Pike, Eds.), pp. 973-994. Academic Press, San Diego. 237. Ishizuka, S., Ishimoto, S., and Norman, A. W. (1984). Biological activity assessment of 1 alpha,25-dihydroxyvitamin D3-26,23-1actone in the rat. J. Steroid Biochem. 20, 611-615. 238. Ishizuka, S., and Norman, A. W. (1986). The difference of biological activity among four diastereoisomers of 1 alpha,25-dihydroxycholecalciferol-26,23-1actone. J. Steroid Biochem. 25, 505-510. 239. Bischof, M. G., Siu-Caldera, M. L., Weiskopf, A., et al. (1998). Differentiation-related pathways of 1 alpha,25-dihydroxycholecalciferol metabolism in human colon adenocarcinoma-derived Caco-2 cells: Production of 1 alpha,25-dihydroxy-3epi-cholecalciferol. Exp. Cell Res. 241, 194-201. 240. Brown, A. J., Ritter, C., Slatopolsky, E., Muralidharan, K. R., Okamura, W. H., and Reddy, G. S. (1999). 1Alpha,25-dihydroxy3-epi-vitamin D3, a natural metabolite of lalpha,25-dihydroxyvitamin D3, is a potent suppressor of parathyroid hormone secretion. J. Cell. Biochem. 73, 106-113. 241. Astecker, N., Reddy, G. S., Herzig, G., Vorisek, G., and Schuster, I. (2000). 1Alpha,25-dihydroxy-3-epi-vitamin D3, a physiological
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metabolite of 1alpha,25-dihydroxyvitamin D3: Its production and metabolism in primary human keratinocytes. Mol. Cell. Endocrinol. 170, 91-101. Siu-Caldera, M. L., Sekimoto, H., Weiskopf, A., et al. (1999). Production of lalpha,25-dihydroxy-3-epi-vitamin D3 in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8): Existence of the C-3 epimerization pathway in ROS 17/2.8 cells in which the C-24 oxidation pathway is not expressed. Bone 24, 457-463. Rao, D. S., Campbell, M. J., Koeffler, H. P., et al. (2001). Metabolism of 1alpha,25-dihydroxyvitamin D(3) in human promyelocytic leukemia (HL-60) cells: In vitro biological activities of the natural metabolites of lalpha,25-dihydroxyvitamin D(3) produced in HL-60 cells. Steroids 66, 423-431. Sekimoto, H., Siu-Caldera, M. L., Weiskopf, A., et al. (1999). 1Alpha,25-dihydroxy-3-epi-vitamin D3: In vivo metabolite of 1alpha,25-dihydroxyvitamin D3 in rats. F E B S Lett. 448, 278-282. Schuster, I., Astecker, N., Egger, H., et al. (1997). Vitamin Dmetabolism in human keratinocytes and biological role of products. In Vitamin D: Chemistry, Biology and Clinical Applications o f the Steroid Hormone (A. Norman, R. Bouillon, and M. Thomasset, Eds.), pp. 551-558. Univ. of California Press, Riverside. Norman, A., Bouillon, R., Farach-Carson, M., et al. (1993). Demonstration that l13,25-dihydroxyvitamin D3 is an antagonist of the nongenomic but not genomic biological responses and biological profile of the three A-ring diastereomers of 1a,25-dihydroxyvitamin D3. J. Biol. Chem. 268, 20022-20030. Harant, H., Spinner, D., Reddy, G. S., and Lindley, I. J. (2000). Natural metabolites of lalpha,25-dihydroxyvitamin D(3) retain biologic activity mediated through the vitamin D receptor. J. Cell. Biochem. 78, 112-120. Messerlian, S., Gao, X., and St.-Arnaud, R. (2000). The 3-epi-and 24-oxo-derivatives of lalpha,25 dihydroxyvitamin D(3) stimulate transcription through the vitamin D receptor. J. Steroid Biochem. Mol. Biol. 72, 29-34. Dardenne, O., Prud'homme, J., Hacking, S. A., Glorieux, F. H., and St-Arnaud, R. (2003). Rescue of the pseudo vitamin D deficiency rickets phenotype of CYP27B 1-deficient-mice by treatment with 1,25-dihydroxyvitamin D3: biochemical, histomorphometric, and biomechanical analyses. J. Bone Miner. Res. 18, in press.
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Other Factors Controlling Bone Growth and Development Calcitonin, CGRP, Osteostatin, Amylin, and Adrenomedullin JILLIANCORNISH* and THOMAS JOHN MARTIN t *Department of Medicine, University of Auckland, Auckland, New Zealand tSt. Vincent's Institute of Medical Research, Melbourne, Australia
INTRODUCTION
mammals, Copp and colleagues [1] discovered calcitonin. When they perfused the thyroparathyroid axis in dogs and sheep, they obtained evidence for the secretion, in response to a high calcium stimulus, of a factor that rapidly lowered the blood calcium. They called it calcitonin and suggested that it was produced by the parathyroid gland. This exciting discovery was quickly confirmed, but calcitonin was found to be a thyroid hormone in mammals. When Hirsch et al. [2] parathyroidectomized rats by cautery, they noted that these animals underwent a more rapid and profound decline in calcium than animals whose parathyroids were surgically removed. They then showed that acid extracts of rat thyroid caused a lowering of calcium when injected into young rats and concluded that the cautery was releasing a hypocalcemic factor that they called thyrocalcitonin. MacIntyre's group, using thyroparathyroid perfusions in dogs and goats, established the thyroid origin of the hypocalcemic agent [3]. It then became apparent that calcitonin and thyrocalcitonin are identical. The accepted usage became calcitonin, describing a new hormone of thyroid gland origin and probably important in calcium homeostasis. Calcitonin arises from the C cells of mammalian thyroid, with its secretion dependent on the prevailing serum calcium level [4]. Although the dominant site of production of calcitonin in mammals is the thyroid C cell, the distribution of these cells throughout the thyroid gland varies considerably among mammalian species, and there is evidence that in some animals calcitoninproducing cells might be found in other parts of the neck, including the thymus. In fish and most birds, calcitonin is produced by the ultimobranchial glands [5]. Whereas in
The continuous balance between bone formation and resorption must be maintained with the two processes equal if bone mass and architecture are to be maintained. In early development and in the young mammal before maturity, the rate of these processes is much greater than that after cessation of growth. Circulating hormones are important in contributing to the fine control necessary to achieve and maintain balance, but insights of the past few decades indicate that locally generated cytokines, growth factors, and peptides are crucial in influencing these processes. These paracrine influences are not exclusive of endocrine effects because there are many interactions between the circulating hormones and locally generated factors, the understanding of which would provide a better appreciation of the cellular and molecular basis of bone remodeling and could therefore be valuable in approaches to new therapies. In this chapter, we consider the actions of the hormones calcitonin and amylin and those of a number of proteins produced locally in bone, either in bone cells or nerves: parathyroid hormone-related protein (PTHrP), calcitonin gene-related peptide (CGRP), and adrenomedullin.
CALCITONIN In the course of experiments seeking to find some factor in addition to parathyroid hormone (PTH) that might contribute to the tight control of serum calcium in
PediatricBone
217
Copyright 2003, Elsevier Science (USA). All rights reserved.
2.18
lillian Cornish and Thomas John Martin
mammalian development the ultimobranchial bodies fuse with the posterior lobes of the developing thyroid to become the C cells, in submammalian vertebrates these bodies remain separate, and the ultimobranchial glands constitute a separate endocrine system [6]. The calcitonins of ultimobranchial origin are highly potent in their actions on mammalian targets [7], even though the physiological significance of calcitonin in fish and birds remains uncertain [8]. The serum and thyroid concentrations of calcitonin increase markedly with age in the rat, and this is associated with substantial increases in thyroid content of calcitonin m R N A [9]. In normal rats subjected to acute calcium stimulation in vivo, thyroid calcitonin mRNA was increased. Evidence suggests that calcium can stimulate both the synthesis and secretion of calcitonin by thyroid C cells. Although calcitonin secretion has been studied extensively in patients with medullary carcinoma of the thyroid, who have clearly assayable hormone levels, it has been difficult to establish the circulating hormone level in normal human subjects. Using the most sensitive and specific assays available, the level of calcitonin in normal human blood appears to be less than 10 pg/ml [8]. The first evidence of the mechanism of action of calcitonin was obtained by organ culture of bone in vitro that showed that calcitonin inhibited bone resorption [10]. The inhibition of resorption appeared to be explained by a direct action on osteoclasts. Calcitonin treatment of resorbing bone in vitro resulted in rapid loss of osteoclast ruffled borders and decreased release of lysosomal enzymes. In vivo evidence was also consistent with an inhibitory action on bone resorption. Thus, calcitonin infused into rats led to an immediate reduction in the rate of excretion of hydroxyproline, consistent with inhibition of breakdown of bone collagen [11]. Further-
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more, kinetic studies in rats led to similar conclusions, with no evidence to suggest any increase in the active uptake of calcium by bone [12]. For example, when calcitonin was infused into rats that had been injected 12 hr previously with radiolabeled 45Ca, hormone treatment lowered plasma calcium without affecting plasma 45Ca levels (Fig. 1). Under these experimental conditions, the disappearance of radioactivity from the plasma reflected uptake of 45Ca by the skeleton. The failure of calcitonin to influence this reflects an action of the hormone to prevent calcium efflux from bone and is not consistent with active stimulation of calcium uptake by bone. Studies of the actions of hormones on isolated bone cell populations established that calcitonin acts directly on osteoclasts, with receptor autoradiography establishing osteoclasts as the only discernible bone cell targets [13]. Consistent with this are observations of its actions in organ culture, especially the demonstration that calcitonin-treated osteoclasts in cultured mouse calvaria rapidly lose their ruffled borders. A similar in vivo observation of loss of ruffled border in osteoclasts has been made in patients with Paget's disease, in whom bone biopsies were taken before and 30 min after an injection of calcitonin [14]. In the same clinical study, calcitonin was noted to decrease the number of osteoclasts in addition to altering their ultrastructure. Studies using isolated osteoclast preparations indicate a direct effect of calcitonin on the osteoclast, in which the hormone rapidly inhibits the activity of osteoclasts. In further experiments, it was also noted that although isolated osteoclasts remained quiescent in calcitonin as long as the hormone was present, they regained activity when osteoblasts were added to the culture [15]. This escape of osteoclasts from inhibition by calcitonin took place at a rate proportional to the number of osteoblasts
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FIGURE 1 Effect of calcitonin infusion on plasma calcium, radioactivity, and specific activity in control (o) and in calcitonin-treated rats (.). 45Ca was injected 12 hr before beginning the 4-hr infusion of calcitonin or control solution [from Robinson, C. J., Martin, T. J., Matthews, E. W., et al. (1967). Mode of action of thyrocalcitonin. J. Endocrinol. 39, 71-77. Reproduced by permission of the Society for Endocrinology].
8. Other Factors Controlling Bone Growth and Development
with which they were in contact. Calcitonin reduced the cytoplasmic spreading of isolated osteoclasts in a dosedependent manner [15]. PTH had no effect unless osteoblasts were cocultivated with the osteoclasts, in which case the addition of PTH resulted in a marked increase in cytoplasmic spreading of osteoclasts. It cannot be assumed that these phenomena reflect the responses of cells in bone in vivo, but this work provided for the first time some useful direct observations of actions of hormones on isolated bone cell preparations containing osteoclasts. The molecular mechanisms by which calcitonin decreases osteoclast function have yet to be fully defined. The rapid effects of the hormone may occur as a result of actions on a cytoskeletal function of osteoclasts, after initial events involving generation of intracellular second messengers. With the development of improved methods of studying isolated osteoclasts, it has been possible to establish that mammalian osteoclasts possess abundant, specific, high-affinity receptors for calcitonin and that calcitonin stimulates cAMP formation in a sensitive and dose-dependent manner as well as increasing intracellular calcium levels [13]. Although calcitonin inhibits bone resorption, it has been found in organ cultures that calcitonin inhibition is followed by "escape," which is defined as an increase in resorption in bones stimulated by a resorptive agent despite the continued presence of concentrations of calcitonin that initially were maximally inhibitory [16]. Furthermore, rats treated chronically with calcitonin become refractory to the hypocalcemic action of the peptide [17]. Data from in vitro experiments suggested that escape was due to a change in responsiveness of the bones rather than a loss of activity of the hormone. Calcitonin-induced homologous desensitization has been studied directly in osteoclasts, with results showing that treatment with the hormone rapidly leads to receptor loss, related to diminution in m R N A for the calcitonin receptor [18]. The mechanism of calcitonin-induced receptor m R N A loss appeared to be due principally to destabilization of receptor m R N A [19]. The 3' untranslated region of the mouse and rat calcitonin receptor m R N A contain four A U U U A motifs as well as other A/U-rich domains and a large number of poly-U regions. Such motifs, commonly found in cytokines and oncogenes, function as signals for rapid m R N A inactivation. We have noted that the calcitonin receptor behaves in a manner similar to the [32-adrenergic receptor. In the latter case, A/U-rich elements in the 3' untranslated region have been shown to bind to a number of cytosolic proteins, some of which have been characterized and which probably accelerate m R N A degradation. An additional interesting aspect regarding calcitonininduced receptor regulation is that glucocorticoid treat-
2_19
ment substantially prevented calcitonin receptor loss [19]. Glucocorticoid treatment was shown by nuclear run-on analysis to increase transcription of the calcitonin receptor gene. It is worth noting that clinical evidence suggests that glucocorticoids, given together with calcitonin, might prevent to some extent calcitonin-induced resistance to its own action [20]. A novel recent finding is that Katl antigen, a unique cell surface antigen on rat osteoclasts, provokes a marked stimulation of osteoclast formation in the presence of calcitonin but not in its absence [21]. It does so even in the presence of OPG, and its production in response to calcitonin could be related to the mechanism of calcitonin-induced resistance. Our concept of the physiological role of calcitonin is that it is an inhibitor of bone resorption whose function is to prevent bone loss at times of stress on calcium conservation, including pregnancy, lactation, and growth. When calcitonin was discovered, it seemed to provide the necessary explanation for the tight control of serum calcium, but events proved otherwise. Concepts of the role of bone in maintaining extracellular fluid calcium relied on observations made in the young, growing rat, in which it was clear that if accretion continued at the same rate and resorption was inhibited, the result would be a lowering of plasma calcium. The younger the animal, the more rapid the bone resorption rate. It would therefore be expected that the calcium-lowering effect of calcitonin should be greater in younger than in older animals. This was indeed the case in the rat, in which it was noted that in the biological assay of calcitonin, which depends on the calcium-lowering effect of the hormone, the response became less marked with increasing age of the animals [22] (Fig. 2). It should be noted, however, that the ability of calcitonin to counteract the effects of a calcium load was not impaired in older animals, at least in the rat [22]. This observation has not been explained and has not been extended to other species. In normal adult human subjects, even quite large doses of calcitonin have little effect on serum calcium levels. In those subjects in whom bone turnover is increased (e.g., in thyrotoxicosis and Paget's disease), calcitonin treatment acutely inhibits bone resorption and lowers the serum calcium [23]. Given that the acute effect of calcitonin on serum calcium is related to the prevailing rate of bone resorption, it is not surprising that calcitonin has little or no effect on calcium in the mature animal or human subject since the process of bone resorption is slow in maturity. It may be that the role of calcitonin in bone throughout life is that of a regulator of the bone resorptive process, whatever the overall rate of the latter. In the young or during pathological states of increased bone resorption in
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maturity (e.g., Paget's disease and thyrotoxicosis), calcitonin inhibition of bone resorption can lower the serum calcium level, and there may even be a calcium homeostatic role for endogenous calcitonin in these circumstances. In a normal adult animal, however, when bone turnover is slow, even though calcitonin does not lower serum calcium, the physiological function of calcitonin in maturity may nevertheless be to regulate the bone resorptive process, in either a continuous or intermittent manner [8]. It follows that calcitonin should not necessarily be regarded as a calcium-regulating hormone in maturity but may yet be shown to be such in stages of rapid growth (e.g., in the young or in states of increased bone turnover). It is nevertheless important that bone resorption be regulated, and calcitonin could be capable of carrying out this function by a direct action on bone. Such a role might become more important in circumstances in which skeletal loss particularly needs to be prevented (e.g., in pregnancy and lactation). Evidence in support of such an important physiological role for endogenous calcitonin was provided by experiments showing that cancellous bone loss in thyroparathyroidectomized rats treated with PTH was greater than that in similarly treated sham-operated controls [24].
CGRP, a 37-amino acid peptide, has approximately 20% homology with calcitonin, and they have in common at the amino terminus a six-amino acid ring structure created by a disulfide bond as well as an amide group at the carboxyl terminus (Fig. 3). CGRP1 is generated by alternative processing of mRNA from the calcitonin gene, located on the short arm of chromosome 11. This gene has six exons, the first four of which produce mRNA for the precursor of calcitonin, preprocalcitonin. An alternative mRNA for the CGRP precursor, preproCGRP, is formed from exons 1-3,5, and 6. The alternative splicing of the calcitonin mRNA is tissue specific so that the predominant mRNA produced in the thyroid is that of calcitonin, whereas in the nervous system it is CGRP-1. A second form of CGRP, CGRP2, differs from CGRP by only three amino acids in the human and one amino acid in the rat. CGRP-2 is a product of a separate gene, also on the short arm of chromosome 11 [25]. CGRP is distributed throughout the nervous system and is one of the most abundant neuropeptides, with some data showing that CGRP circulates at concentrations of approximately 1 pmol/liter, levels that are increased by sex hormone replacement therapy in postmenopausal women. In the bone microenvironment, it is likely that CGRP concentrations are higher as a result of the local release of CGRP from nerve terminals. CGRPcontaining nerves develop when defects are created surgically in bone or following fractures. It is possible that CGRP aids bone growth through direct effects on osteoblast function. CGRP is a potent vasodilator, and because of the intimate association of the nerves with the blood vessels, CGRP may also have a role in regulating blood flow to sites of bone healing or growth. Soon after the discovery of CGRP [25] and its common origin with calcitonin, investigation of its effects on bone resorption was performed. CGRP lowers circulating calcium concentrations when the peptide is injected into intact animals [26]. In organ culture, CGRP was also demonstrated to lower radiolabeled 45Ca release from prelabeled neonatal mouse calvaria. The peptide inhibited both basal and stimulated bone resorption, but the half maximally effective concentration of CGRP was 500-fold higher than that of calcitonin [27]. A similar pattern of activity is seen with disaggregated isolated osteoclasts, where CGRP directly inhibits the activity of these cells. CGRP inhibits cell motility, probably via cAMP production; however, the osteoclast retraction seen with calcitonin is not produced by CGRP. CGRP inhibits the formation of TRAP-staining
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222
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mononuclear cells with similar potency (Fig. 4) and also inhibits the subsequent fusion of these cells to form multinucleated osteoclasts [28]. In in vivo bone models, when injected over the calvaria of adult mice, CGRP did not significantly inhibit bone resorption [29], and it has been shown that incomplete suppression of postovariectomy increases in bone resorption in rats with CGRP treatment [30]. In all studies illustrating the calcitonin-like ability of CGRP to inhibit bone resorption, whether in vitro or in vivo, the efficacy of CGRP is much less than that of calcitonin, indicating that it is unlikely that CGRP contributes to normal skeletal physiology through this mechanism. Rather, it reflects relatively weak interactions of CGRP with the calcitonin receptor on osteoclasts. While CGRP was being investigated as a calcitonin analog in bone, data began to emerge showing direct effects on osteoblasts to increase cAMP formation, in contrast to the lack of effect of calcitonin [31]. Subsequently, CGRP has been shown to have proliferative effects on primary cultures of osteoblasts, although at appreciably higher concentrations than those for amylin [29] (Fig. 5). Other studies indicated that CGRP activates an additional second messenger pathway by increasing
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intracellular calcium concentration [32,33]. CGRP may also act on preosteoblasts [34], influencing their development from precursor cells [35]. Bernard and Shih [36] demonstrated that CGRP stimulated the increase of bone colonies produced in bone marrow cultures. It is possible that CGRP regulates the function of both precursor cells and mature osteoblasts by modulating the production of cytokines and growth factors, such as increasing interleukin-6, insulin-like growth factor-1 (IGF-1), and IGF-2 [37,38]. The effects of CGRP on osteoblast function in vivo are Conflicting. Local injection of CGRP over calvaria in adult mice demonstrated no changes in osteoblast indices [29], and no change in bone formation rates in ovariectomized rats treated with CGRP has also been observed [30]. However, in mice with osteoblasts overexpressing CGRP, increases in bone formation indices, bone volume, and bone density were observed [39]. The possibility that these relatively weak effects of CGRP on osteoblast function may be mediated by receptors that have a higher affinity for amylin than for CGRP was recently proposed [40]. Amylin, another member of the calcitonin family that is mitogenic to osteoblasts, is described in detail later. In cultures of fetal rat osteoblasts, treatment with amylin increased cell number and thymidine and phenylalanine incorporation at 100-fold lower concentrations than those of CGRP, and amylin's maximal effects were significantly greater than those of CGRP. The two peptides were not additive in their effects. It was concluded from receptor antagonist experiments that amylin and CGRP probably act through a common receptor to stimulate osteoblast growth, and that this receptor has a higher affinity for amylin than for CGRP (Table 1). There is much capacity for cross-reactivity among the calcitonin/amylin family ligands and their seven-
8. Other Factors Controlling Bone Growth and Development TABLE 1 The c o m m o n receptor for Amylin and CGRP on osteoblasts as a higher affinity for amylin than for CGRR
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transmembrane G protein-coupled receptors. CGRP and amylin may interact with the calcitonin receptor on the osteoclast but with a much lower affinity, and, as discussed previously, they are therefore unlikely to contribute physiologically in this way. Uncertainty surrounds the identity of all the receptors for this family, including CGRP. Recent reports on nonosseous tissue indicate that receptor phenotypes for this family of peptides arise from an interaction between either the calcitonin receptor or the calcitonin receptor-like receptor (CRLR) gene products and one of the receptor activitymodifying proteins (RAMPs). RAMPs are novel proteins that may be present in many cell types, located intracellularly, and that transport receptors to the cell surface, where they become expressed as single transmembrane proteins altering the cell surface expression and phenotype of the associated receptor [41]. Receptors for CGRP are induced when CRLR couples with RAMP-1 [41,42]. Further studies are necessary to determine whether the CRLR or any of the RAMPs are present in osteoclasts. There is clearly a different receptor on osteoblasts that mediates the anabolic effects of CGRP and amylin on bone since calcitonin does not share these effects [29], unlike the antiresorptive effects of these peptides [43]. PARATHYROID HORMONERELATED PROTEIN PTHrP was discovered from studies of the mechanisms by which certain cancers cause hypercalcemia by acting on bone and kidney in a manner similar to the actions of PTH [44]. The similarities in amino acid sequence in the amino-terminal regions of PTH and PTHrP explain their actions through a shared G protein-coupled receptor, PTHR1. These structural similarities suggest that the two are most likely related by ancient gene duplication events. Many tumors produce PTHrP, and it has been identified in a variety of normal tissues of fetal and adult origin. It is readily measured in the circulation of patients
223
with several types of cancer-related hypercalcemia, but N-terminal PTHrP has never convincingly been assayed in plasma of normal human subjects [45]. Thus, PTHrP behaves as a hormone in these patients whose cancers produce it in excess. PTHrP can also be considered a hormone in the fetus, in which it is responsible for promoting calcium transport across the placenta from mother to fetus [46], and in lactation, in which PTHrP circulates [47] but the purpose of this has yet to be defined. The physiological roles of PTHrP in the mammal appear to relate to its function as a paracrine effector. PTHrP mRNA or protein have been detected in the following human tissues: adrenal, bone, brain, heart, intestine, kidney, liver, lung, mammary gland, ovary, parathyroid, placenta, prostate, skeletal, muscle, skin, spleen, stomach, testis, thymus, urinary bladder, uterus, and vascular smooth muscle [48]. As a result of a combination of physiological and genetic experiments, it has become clear that PTHrP plays an important role in many different tissues, both in development and in maturity. Examples are its regulation of epithelial-mesenchymal signalling during the development of mammary glands and hair follicles and its contribution to relaxation of uterine and vascular smooth muscle. The status of PTHrP as a paracrine or autocrine factor has been the subject of several reviews [49,50]. The discovery of PTHrP production in bone [51] raised the intriguing possibility that it has important local actions in bone, not only mimicking the hormone, PTH, but also possibly producing other effects through actions of different domains of the molecule. Its role in endochondral bone formation has been studied extensively. Targeted disruption of the genes for PTHrP or the common PTH/PTHrP receptor (PTHR1) in mice resulted in death in the perinatal period with gross skeletal abnormalities consistent with chondrodysplasia [52]. Histological studies of tibiae from homozygous PTHrP null mutants showed significant shortening of epiphyseal growth plates as a result of a markedly reduced number of proliferating chondrocytes, distortion of the orderly columnar arrays of hypertrophic chondrocytes by clusters of nonhypertrophic chondrocytes, alteration in the orderly program of cell death normally occurring in the metaphyseal region, and aberrant differentiation of periosteal progenitor cells. These results suggest a central role of PTHrP in fetal endochondral bone formation through its actions in maintaining a pool of proliferating chondrocytes, inhibition of terminal chondrocyte differentiation, retardation of cartilage matrix mineralization, and differentiation of periosteal mesenchymal precursors into cells of the chondrocytic or osteoblastic lineages. It appears that PTHrP might also be involved in intramembranous bone formation. In an experimental
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lillian Cornish and Thomas John Martin
model of this process in the rabbit, bone formation begins with the transformation of primitive mesenchymal cells of marrow into trabecular bone without any cartilage intermediate, and the synchronized appearance of hemopoietic marrow elements follows the onset of matrix mineralization. Cells of the osteoblast lineage consistently express PTHrP m R N A and protein throughout the bone formation sequence, With prominent production by cuboidal, actively synthesizing osteoblasts and weaker expression in lining cells on the mineralized trabeculae [53]. These observations, together with those of Suda et al. [51] and Amizuka et al. [54], support a role for PTHrP in the differentiation of mesenchymal precursors to the osteogenic lineage. From genetic experiments, heterozygous PTHrP null mice were shown to be phenotypically normal at birth, but by 3 months of age they were osteopenic as a result of a reduction in trabecular bone volume, and an increase in adipocytes was observed in the bone marrow [54]. The absolute requirement of PTHrP for normal bone integrity not only in the fetus but also postnatally, together with the finding that PTHrP haploinsufficiency is associated with less bone and with the preferential differentiation of stromal mesenchymal cells into adipocytes rather than osteocytes, indicates that PTHrP is a critical molecule in development and maintenance of bone. It is accepted that PTHrP mimics the actions of PTH through PTHR1 linked to adenylate cyclase activation. Thus, PTHrP promotes bone resorption in vitro and in vivo by acting directly on bone to increase production and activity of osteoclasts and on kidney to restrict calcium excretion [55]. Similarly, the anabolic effect of PTH is reproduced by PTHrP [56]. The ability of PTH (and PTHrP) to promote bone formation is dependent on the hormone being administered intermittently in a way that yields blood level peaks, which are not maintained. In this circumstance, processes are initiated in bone that result in anabolic effects, presumably as a result of activation of genes responding specifically to a rapid increase in PTH or PTHrP. On the other hand, if PTH or PTHrP is infused, or administered in such a way that elevated plasma levels are maintained, the dominant effect is stimulation of osteoclast formation and bone resorption, to the extent that these override any anabolic response. Recent in vivo studies in the rat support this view. Infusion of PTH into rats caused a robust and sustained increase in R A N K L and decrease in OPG production in bone as well as rapid depletion of matrix stores of OPG, all of which preceded hypercalcemia and enhanced osteoclast formation. Also in these conditions, sustained elevated levels of PTH resulted in decreased expression of genes associated with the bone formation phenotype of the osteoblast [57], including cbfal, osteocalcin,
bone sialoprotein, and type 1 collagen. These observations were in contrast to the findings with repeated single injections of PTH that, although they triggered a rapid but transient increase in the RANKL:OPG ratio, resulted in increased bone formation and enhanced expression of the genes associated with bone formation [58]. Much remains to be learned about the cellular and molecular events that determine whether the actions of PTH and PTHrP are resorptive or anabolic. It needs to be determined whether the anabolic response is predominantly the result of enhanced differentiation of existing osteoblast precursors, inhibition of apoptosis, or a combination of the two, or whether specific genes, such as cbfal, mediate the PTH/PTHrP effect. There may also be some relatively simple lessons and hypotheses that can be developed from the existing information. A central role for PTHrP in bone development and growth is suggested by the results of mouse genetic experiments [52], including the important observation that PTHrP haplosufficient mice are osteopenic [54]. In what ways can PTHrP as a paracrine/autocrine factor in bone contribute? The pharmacologic effects of intermittent versus sustained PTH/PTHrP treatment are striking and very different. If the behavior of osteoblasts in response to stimulation through PTHR1 requires this type of variation in delivery of the relevant ligand, it is doubtful that PTH, as a circulating peptide hormone, can achieve this. On the other hand, this role could be filled by local production of PTHrP, regulated in turn by hormones, cytokines, and/or neural transmission. In the case of PTHrP, there is also the possibility that biological activities within the remainder of the molecule could influence local events, either through independent processes or by modifying actions through the PTH 1R. Investigations of the actions of various PTHrP preparations on resorption by isolated osteoclasts have led to identification of a portion of the C-terminus region of PTHrP as an inhibitor of resorption (osteostatin) [59]. The physiological significance of this finding is uncertain. Although some attempts to establish this effect of C-terminus PTHrP in bone organ culture have failed [60], this may be due to a peptide stability issue; for example, when osteostatin is added more frequently (approximately 16 hr. intervals) to the culture system, bone resorption is inhibited (Cornish et al., unpublished data). The osteostatin sequence PTHrP(107-111), as well as PTHrP(107-139), has been shown to inhibit bone resorption in vivo either by systemic injection in ovariectomized rats [61] or by injection over the calvaria in intact mice [62]. There are a number of reports of biological activity of the osteostatin peptides on osteoblasts. Seitz
8. Other Factors Controlling Bone Growth and Development et al. [63] demonstrated the stimulation of cAMP by PTHrP(107-139) in three osteoblast cell preparations. Gagnon et al. [64] showed that PTHrP(107-111) stimulates protein kinase C activity in ROS 17/2 cells. In studies of the effects on osteoblast proliferation, PTHrP(107-139) and PTHrP(107-111) have been shown to promote a dose-dependent increase in cell number and thymidine and phenylalanine incorporation in primary neonatal rat osteoblasts [65]. In contrast, Esbrit and colleagues [66,67] demonstrated that the peptides reduced osteoblast proliferation in the osteoblastic osteosarcoma cell line UMR-106 and in human osteoblast-like cells. The finding that the effects are opposite in the primary rat cultures of osteoblasts to those seen in the transformed rat cell line is similar to the findings with both transforming growth factor-[3 and leukemia inhibitory factor, both of which stimulate the proliferation of authentic rodent osteoblasts in vitro but reduce that of transformed osteoblastic cells [68]. It is relevant that each of these factors stimulates bone formation when assessed in vivo [69,70]. The findings in the primary osteoblast cultures might be the better predictor of a factor's effect on osteoblast proliferation in vivo. In the human cells, effects were apparent after 6 days of incubation. The human osteoblasts were derived from patients with osteoarthritis, a condition in which substantial concentrations of PTHrP have been demonstrated in synovial fluid [71]. Thus, osteoblasts from this source might respond differently to PTHrP as a result of the previous prolonged exposure to high concentrations of this peptide. Whether C-terminus PTHrP is ultimately a stimulator or inhibitor of osteoblast growth needs to be determined in vivo. Limited data are available indicating that the peptide is a stimulator of bone formation. Rouffet et al. [61] studied the in vivo effects of the pentapeptide in ovariectomized rats. Although it was suggested that this peptide had positive effects on bone mass, there were inconsistencies in the data that make it difficult to assess. When injected locally above the calvaria of normal adult male mice, PTHrP(107-139) also resulted in an upward trend of mineralized bone area. The positive effects on bone result primarily from a marked inhibition of bone resorption. There was a much smaller reduction in bone formation indices; therefore, these findings do not necessarily rule out a positive effect of the peptide on bone formation in vivo and may merely reflect that it was overwhelmed by the coupling of resorption and formation. Thus, a decline in bone resorption results in a decline in bone formation, which may completely obscure any smaller direct effect on bone formation. In addition, Barengolts et al. [72] provided a preliminary report that C-terminus PTHrP, when injected into male mice for 7 days by a miniosmotic pump, increased bone formation two- or threefold.
225
AMYLIN Amylin was isolated from pancreatic amyloid deposits of patients with insulinoma or diabetes mellitis [73]. Amylin is cosecreted with insulin from the pancreatic beta cell and it appears that insulin and amylin genes share transcriptional regulators [74]. Amylin, like CGRP, is a 37-amino acid peptide, the genes for which have a common ancestral origin (Fig. 3). Amylin has 43% sequence identity with CGRP1, 49% with CGRP2, and 13% with calcitonin in humans. Human amylin appears to be produced from a single gene on the short arm of chromosome 12 consisting of three exons and, like calcitonin and CGRP, it is synthesized as a precursor molecule, preproamylin. Amylin is produced principally in the pancreas, but it also has been detected in tissues of the gastrointestinal tract, lung, dorsal route ganglion, hypothalamus, and neuroendocrine tumors [75]. Amylin production from a human osteoblast-like cell line has been reported [76], raising the possibility of amylin production locally within the bone microenvironment. However, we have been unable to confirm the presence of amylin m R N A in primary rat osteoblasts (Naot et al., unpublished data). Circulating amylin levels are 1-5 pmol/liter, increasing to 10-20 pmol/liter following a meal. Levels are also higher in obese subjects and those with type II diabetes [77], although it appears that amylin levels are decreased by leptin [78]. Amylin may contribute to the relationship between body mass and bone density. Body mass, particularly fat mass, is a major determinant of bone density in women [79]. Since both insulin and amylin are hypersecreted in obesity and since both may potentially act directly or indirectly to increase bone mass, they may contribute significantly to this relationship. Indeed, circulating insulin levels are directly related to bone density in normal postmenopausal women [80], and since amylin is cosecreted with insulin it seems likely that a similar relationship for this peptide exists. In type I diabetic patients, amylin and insulin circulate at low levels, and bone density is also reduced. We and others have demonstrated that amylin [29,81,82] and insulin [83] stimulate bone growth in vitro and in vivo. Soon after the isolation of amylin and the recognition of its similarity to calcitonin, its effects on bone resorption were investigated. As with calcitonin and CGRP, amylin lowers plasma calcium levels when injected into man and rats, although it is several orders of magnitude less potent than calcitonin [84]. Amylin inhibits osteoclast motility by increasing intracellular cAMP concentration [85]. Osteoclast development is also reduced by amylin, inhibiting both mononuclear osteoclast-like cell
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Jillian Cornish a n d T h o m a s John Martin
formation and fusion of these cells. In this system, calcitonin has a greater potency and amylin is more active at lower concentrations than C G R P [28] (Fig. 4). In organ culture, fetal rat long bone, or neonatal mouse calvaria cultures, amylin at nanomolar concentrations reduces both basal and stimulated bone resorption, and cAMP production is also increased [86-88]. Amylin may regulate bone resorption at physiological concentrations because this protein has a propensity to adhere to the surfaces of laboratory plasticware, suggesting that the actual concentrations of amylin in vitro may be one or two orders of magnitude less than the amount added to the media [89]. This activity is dependent on the presence of the C-terminus amide group and only occurs with the intact molecule. In contrast, amylin fragments [amylin (1-8) and -(8-37)] are active on osteoblasts [90]. The effect of amylin on resorption in vivo has been studied histomorphometrically in several different models. In a local administration model in which amylin was introduced over the calvaria of the adult mice, bone resorption was reduced by 60-70% [29]. Very similar changes in resorption indices were seen following systemic administration of amylin to adult mice for 1 month [91], whereas amylin(1-8) is without effect on resorption in this systemic model [92]. In ovariectomized rats, intact amylin reduces urinary excretion of deoxypyridinoline and reduces bone loss [82] (Fig. 6). In contrast, an earlier experiment in rats [93] showed only a nonsignificant trend toward reduced resorption, and Borm et al. [94] found no change in resorption markers in 23 diabetic patients who received the amylin analog pramlintide for 1 year. The latter study needs to be interpreted with caution since it used an amylin analog of unknown activity on bone and it was uncontrolled. The binding of amylin to osteoblast-like cells was demonstrated soon after the discovery of the peptide [95]. However, only recently was amylin demonstrated to stimulate the proliferation of fetal rat osteoblasts at concentrations as low as 10 -11 M (i.e., periphysiological concentrations) [29] (Fig. 5). The maximal effects of amylin on cell proliferation in this culture system are similar to those of other osteoblast mitogens [40], suggesting that these effects might be of physiological significance. Similar proliferative effects have been shown in human osteoblasts by Villa et al. [96] and by us (unpublished data). The effects of amylin on cAMP concentrations in osteoblasts are modest (in comparison with PTH), and recent work indicates that amylin activates mitogen-activated protein kinase in primary neonatal rat osteoblasts [97]. Amylin, calcitonin, and C G R P have been demonstrated to activate protein kinase C in human osteoblast-like primary cells [98]. The effects of amylin, CGRP, and PTH on the development of mineralized bone nodules in long-term osteoblast cultures have
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been compared, and amylin has been found to be a more potent agent than PTH or C G R P [81]. Structure-activity relationship studies demonstrated that amylin(1-8) also
227
8. Other Factors Controlling Bone Growth and Development
stimulates osteoblast proliferation, although its halfmaximal effect of concentration is 10-fold higher than that of the intact peptide (i.e., 3.4 • 10 -1~ M). This peptide also stimulates thymidine incorporation in neonatal mouse calvaria [99]. A number of in vivo studies on amylin's effects on bone have been performed. In rats, amylin increased cortical endosteal osteoblast numbers [100]. In adult mice, amylin administered locally over the calvaria for 5 days resulted in a 2- to 4-fold increase in the histomorphometric indices of osteoblast activity [29]. Similarly, daily systemic administration of amylin over 1 month to adult mice resulted in 30-100% increases in these indices [91]. Ovariectomized rats treated systemically with amylin demonstrated increases in serum osteocalcin concentrations [82] (Fig. 6). Daily systemic administration of amylin(1-8) to sexually mature male mice for 4 weeks almost doubled the histomorphometric indices of osteoblast activity [92]. Thus, a number of studies have found evidence of an anabolic action of amylin and its amino terminus in osteoblasts. Amylin and its fragments affect bone volume when administered in in vivo models. Amylin administration, at a dose of 4 • 10 - 9 moles per injection, significantly increased bone formation, reduced resorption, and led to a substantial increase in mineralized bone area [29]. At equimolar doses, CGRP in the same model produced no significant effects in the mineralized bone area, and calcitonin caused a small, nonsignificant increase. Similarly, in a systemic model in which amylin was administered to adult male mice at a dose of 10 p.g/day for 1 month, increases in formation and decreases in resorption led to a striking increase (70%) in trabecular bone volume in the tibia [91]. The cortical thickness was also significantly increased in the tibial shafts. Amylin also increased the tibial length and growth plate thickness, implying that the chondrocyte is also an amylin target cell. Indeed, amylin is proliferative to primary cultures of canine chondrocytes, increasing thymidine incorporation and cell numbers after treatment for 24 hr at concentrations of 10-11 M and higher (Cornish et al., submitted for publication). In another study, systemically administered amylin to male rats demonstrated small increases in bone mass in the absence of histomorphometric or biochemical indices of bone turnover [93]. Two recent studies of systemic administration of amylin to ovariectomized rats and diabetic rats, respectively, demonstrated significant increases in bone mineral density [82,101]. Bone volume changes were also demonstrated with amylin(1-8) in mice when administered systemically for 1 month at a dose of 2.2 i~g/day. There was an increase in formation and no change in resorption, resulting in a trabecular volume increase of 36% in the tibia (approximately half that seen for the amylin intact molecule), and a 3-point bending test for bone
strength resulted in displacement to fracture increases. In a separate experiment utilizing dynamic histomorphometry with bone-seeking fluorochrome labels, local injection of amylin(1-8) over the calvaria of male mice resulted in increases in bone formation and mineral apposition rates [92]. The effect was dose dependent from 0.4 to 40 nM and greater than that of an equimolar dose of hPTH(1-34) (Fig. 7). As discussed for CGRP, there is considerable crossreactivity within the members of this family of peptides and their receptors in bone tissue. In nonosseous tissue, an amylin receptor is created when the calcitonin receptor (CTR2) interacts with RAMP- 1 or -3 [102,103]. However, the primary osteoblasts do not exhibit CTR2 [104]; thus, the receptor through which amylin acts on the osteoblast has not been determined. What role amylin may play in normal bone metabolism and bone pathology remains to be determined. It has been hypothesized that amylin secretion following a meal directs the absorbed calcium and protein from the meal into new bone synthesis by increasing bone growth at a 100-]
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FIGURE 7 The effect of rat amylin(1-8) and human PTH(1-34) on the extent of the fluorochrome double-labeled surface and mineral apposition rate in mice calvariae. Mice were treated with local subcutaneous injections over the central calvariae for 5 consecutive days and sacrificed 10 days later [from Cornish et al. (2000). Systemic administration of a novel octapeptide, amylin(1-8), increases bone volume in male mice. Am. J. Physiol. (Endocrinol. Metab.) 279, E730-E735. Reproduced by permission of the American Physiological Society]. *Significantly different from control, p<0.04. ** significantly different from control, p<0.001. The groups receiving 40nM amylin and 40nM PTH were also significantly different from each other, p<0.02.
22.8
lillian Cornish and Thomas John Martin
time when the substrates are available [84,95]. Amylin may also contribute to the relationship between body mass and bone density. The effects of amylin are to increase bone formation and reduce bone resorption; as such, it is an attractive candidate for the treatment of generalized bone loss of osteoporosis and to promote healing of local bone effects. Amylin's efficacy has been demonstrated in several different animal models, although research must be performed to determine which fragments or analogs are the most promising candidates.
ADRENOMEDULLIN Adrenomedullin is a 52-amino acid peptide that is related to amylin and also has some homology to both calcitonin and CGRP. Adrenomedullin differs from the other peptides in that it has a linear amino-terminal extension consisting of 15 amino acids in humans and 13 in rats. All have in common the 6-amino acid ring structure at the amino terminus created by a disulfide bond, and the C termini are amidated (Fig. 3). Adrenomedullin was first identified in a phaeochromocytoma [106] and has since been found to be present not only in the normal adrenal medulla but also in many other tissues [107]. Adrenomedullin has been demonstrated to be expressed at high levels in the osteoblasts and chrondrocytes of mouse and rat embryos [108]. We recently showed by immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), and Northern blot analysis that adrenomedullin is also expressed in primary osteoblasts isolated from fetal rats [104]. Similarly, we have demonstrated the presence of adrenomedullin in human chondrocytes cultured from articular cartilage explants Cornish, et al. (2003). AdrenomeduUin-a regulator of bone formation. (Reg. Pep. 112) 79-860. The peptide has been shown to be produced in a macrophagemonocyte cell line [109], leading to speculation that adrenomedullin may be produced by the osteoclast. Adrenomedullin, like CGRP, is a potent vasodilator and it acts directly on the renal, cerebral, mesenteric, pulmonary, and systemic circulations, including the vascular supply of the skeleton [110]. Adrenomedullin circulates at picomolar concentrations in both rats and man [107,111]. An increasing number of conditions have been identified in which there are major perturbations of circulating adrenomedullin concentrations, including acute sepsis [112], hyperthyroidism [113], and pregnancy, during which high peptide concentrations have been reported in umbilical cord plasma and amniotic fluid [114]. Adrenomedullin, like amylin, is a potent osteoblast mitogen, increasing cell numbers and thymidine incorporation into DNA at physiological concentrations of
10-12 M and higher [115] (Fig. 5). Structure-activity relationship studies revealed that the activity on osteoblasts is preserved in peptides as short as adrenomedullin (27-52), suggesting that the disulfide bond and the ring structure created by it are not necessary. In contrast, in vascular smooth muscle, loss of the ring structure in the adrenomedullin molecule, resulting from amino-terminal truncation or removal of the disulfide bond, eliminates specific binding and cAMP formation [116]. This suggests that the receptor mediating the effects of adrenomedullin in osteoblasts differs from that in vascular smooth muscle. The activity of the short fragments is also surprising when comparison is made with amylin. When the ring structure is removed from amylin, the resulting peptide [amylin(8-37)] has no agonist activity on osteoblasts but rather is an antagonist. The intact molecules of both adrenomedullin and amylin are similar mitogens in terms of maximal effect, although adrenomedullin produces detectable growth stimulation at lower concentrations than amylin. Recent work indicates that adrenomedullin, like amylin, activates mitogen-activated protein kinase in osteoblasts [97]. Similar to CGRP, the proliferative effects of adrenomeduUin on osteoblasts are also blocked by amylin antagonists such as amylin(8-37), again suggesting involvement of the same receptor. Recently, we showed that the proliferative effects of adrenomedullin and amylin are dependent on the presence of the IGF-1 receptor [97,117], although neither of these peptides appears to compete for binding to this receptor, implying a less direct mechanism for its involvement. As discussed for CGRP, uncertainty surrounds the identities of the receptors for this family of peptides in nonbone cells. An adrenomedullin receptor, L1, was identified in 1995 [118], but recent work has cast doubt on the significance of this receptor [41]. McLatchie et al. [41] suggest that the adrenomedullin receptor, similar to the CGRP receptor, is formed by the CRLR in combination with RAMP molecules. CRLR in association with RAMP-1 constitutes the CGRP receptor, whereas the adrenomedullin receptor is produced by the interaction of RAMP-2 and RAMP-3 with CRLR. Other groups have confirmed that these CRLR-based receptors account for most of the specific binding of adrenomedullin and CGRP in a variety of rat tissues [42]. Recently, we demonstrated by RT-PCR and Northern blot analysis that primary rat osteoblasts and UMR-106.06 osteoblast-like cells express the mRNAs for all three RAMPs, CRLR, and the putative adrenomedullin L1 receptor [104] (Fig. 8). These results have been confirmed in MC3T3.E1 osteoblast-like cells [119]. We have also shown that the primary osteoblastic cells bind [125I]adrenomedullin with high affinity, and analysis of competitive binding data suggested the existence of two types of binding sites for adrenomedullin on primary
8. Other Factors Controlling Bone Growth and Development
2.2.9
FIGLIRE 8 Expression of genes for adrenomedullin (ADM) and its putative receptors in primary osteoblasts and UMR-106-06 cells. RT-PCR was carried out using specific primary pairs for the different cDNAs. The RT-PCR products were resolved on a 1% agarose gel except for RAMP3, which was resolved on a 2% gel. All the amplified cDNA fragments were extracted from the gels and their DNA sequence determined. Lanes 1-6 and 13, PCR products from primary osteoblasts; lanes 7-12 and 14, RT-PCR products from UMR-106-06 cells; lanes 1 and 7, ADM; lanes 2 and 8, L1 (ADMR); lanes 3 and 9, RAMP1; lanes 4 and 10, RAMP2; lanes 5 and 11, CRLR; lanes 6 and 12, CTR; lanes 13 and 14, RAMP3; L, 100-bp DNA ladder.
osteoblasts [104]. In all findings establishing the importance of CRLR in association with RAMPs as defining the receptors of this peptide family, the end point measured is a cAMP response, which may not be the second messenger mediating the action of these peptides on osteoblast proliferation (Cornish, Callon, and Reid, unpublished data). Therefore, the relevance of these findings to osteoblast biology remains to be determined. Neither the CGRP nor the adrenomedullin receptor shows significant cross-reactivity with the other ligand or with amylin, as is demonstrated in osteoblast proliferation experiments. Furthermore, the structure-activity relationship for the action of adrenomedullin on the receptor of McLatchie et al. [41] is quite different from that which we found in osteoblasts. These workers found that adrenomedullin (13-52) is the smallest active fragment, whereas we found full activity in adrenomedullin(27-52). Adrenomedullin, unlike the other members of this family, does not inhibit bone resorption as measured by 45Ca release in neonatal mouse calvarial organ cultures [115]. In addition, there is no reduction in bone resorption indices when adrenomedullin is administered either locally or systemically to live animals [120]. The in vivo effects of adrenomedullin were recently studied. Considering that adrenomedullin may be acting in an autocrine-paracrine manner, we used an in vivo model in which the local effects of factors on bone histomorphometry could be determined. In this model, the
peptide was administered directly above the calvaria of adult male mice, and adrenomedullin was seen to increase indices of bone formation to a similar extent to amylin (i. e., three or four times that of control). However, there was no effect on bone resorption. Adrenomedullin(27-52), which is an osteoblast mitogen in vitro (but no longer vasoactive), produced similar effects when injected systemically. In this model, 20 injections of 8.1 txg of the adrenomedullin fragment were administered over a 4week period. This produced increases in the indices of osteoblast activity, osteoid perimeter, and osteoblast perimeter. Osteoclast perimeter was not affected. There was a 21% increase in cortical width and a 45% increase in trabecular bone volume in animals treated with adrenomedullin(27-52). Assessment of bone strength by 3-point bending of the humerus showed that both the maximal force and the displacement to the point of failure were increased in the animals treated with adrenomedullin(2752) [120]. It is concluded that adrenomedullin(27-52) acts as an anabolic agent on bone. These findings may be relevant to the normal regulation of bone mass and to the design of agents for the treatment of osteoporosis.
References 1. Copp, D. H., Cameron, E. C., Cheney, B. A., et al. (1962). Evidence for calcitonin--A new hormone from the parathyroid that lowers blood calcium. Endocrinology 70, 638-649.
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2. Hirsch, P. F., Voelkel, E. F., and Munson, P. L. (1964). Thyrocalcitonin. Hypocalcemic hypophosphatemic principle of the thyroid gland. Science 146, 412-414. 3. Foster, G. V., Baghdiantz, A., Kumar, M. A., et al. (1964). Thyroid origin of calcitonin. Nature 202, 1303-1305. 4. Foster, G. V., Maclntyre, I., and Pearse, A. G. E. (1965). Calcitonin production and the mitochondrion-rich cells of the dog thyroid. Nature 203, 1029-1031. 5. Pearse, A. G. E., and Carvalheira, A. F. (1967). Cytochemical evidence for an ultimobranchial origin of rodent thyroid C cells. Nature 214, 929-931. 6. Perez Cano, R., Galan Galan, F., Girgis, S. I., et al. (1981). A human calcitonin-like molecule in the ultimo-branchial body of the amphibia (Rana pipiens). Experentia 37, 1116-1117. 7. Niall, H. D., Keutmann, H. T., Copp, D. H., et al. (1969). Amino acid sequence of salmon ultimobranchail calcitonin. Proc. Natl. Acad. Sci. USA 64, 771-778. 8. Martin, T. J., Findlay, M. M., Moseley, J. M., and Sexton, P. M. (1998). Calcitonin. In Metabolic Bone Disease and Clinically Related Disorders (L. V. Avioli and S. M. Krane, Eds.), 3rd ed., pp. 95-121. Academic Press, San Diego. 9. Jacobs, J. W., Simpson, E., Penschow, J., et al. (1983). Characterization and localization of calcitonin mRNA in rat thyroid. Endocrinology 113, 1616-1622. 10. Friedman, J., and Raisz, L. G. (1967). Thyrocalcitonin: Inhibitor of bone resorption in tissue culture. Science 150, 1465-1467. 11. Martin, T. J., Robinson, C. J., and Maclntyre, I. (1966). The mode of action of thyrocalcitonin. Lancet 1, 900-902. 12. Robinson, C. J., Martin, T. J., Matthews, E. W., et al. (1967). Mode of action of thyrocalcitonin. J. Endocrinol. 39, 71-77. 13. Nicholson, G. C., Moseley, J. M., Sexton, P. M., et al. (1986). Abundant calcitonin receptors in isolated rat osteoclasts: Biochemical and autoradiographic characterization. J. Clin. Invest. 78, 355-361.' 14. Singer, F. R., Melvin, K. E. W., and Mills, B. G. (1976). Acute effect of calcitonin on osteoclasts in man. Clin. Endocrinol. 5, 333S-340S. 15. Chambers, T. J. (1982). Osteoblasts release osteoclasts from calcitonin-induced quiescence. J. Cell Sci. 57, 147-160. 16. Raisz, L. G., Wener, J. A., Trummei, C. L., et al. (1972). Induction, inhibition and escape as phenomena in bone resorption. Excerpta Med. Int. Congr. 243, 446-453. 17. Messer, H. H., and Copp, D. H. (1974). Changes in response to calcitonin following prolonged administration to intact rats. Proc. Soc. Exp. Biol. Med. 146, 643-647. 18. Wada. S., Martin, T. J., and Findlay, D. M. (1995). Homologous regulation of the calcitonin receptor in mouse osteoclast-like cells and human breast cancer T47D cells. Endocrinology 136, 2611-2621. 19. Wada, S., Udagawa, N., Akatsu, T., Nagata, N., Martin, T. J., and Findlay, D. M. (1997). Regulation by calcitonin and glucocorticoids of calcitonin receptor gene expression in mouse osteoclasts. Endocrinology 138, 521-529. 20. Binstock, M. L., and Mundy, G. R. (1988). Effect ofcalcitonin and glucocorticoids in combination on the hypercalcemia of malignancy. Ann. Int. Med. 93, 269-272. 21. Kukita, T., Kukita, A., Watanabe, T., and Iijima, T. (2001). Osteoclast differentiation antigen, distinct from receptor activator of nuclear factor kappaB, is involved in osteoclastogenesis under calcitonin-regulated conditions. Endocrinology 170, 175-183. 22. Cooper, C. W., Hirsch, P. F., Toverud, S. V., et al. (1967). An improved method for the biological assay of calcitonin. Endocrinology 81, 610-617.
23. Martin, T. J., and Melick, R. A. (1969). The acute effects of porcine calcitonin in man. Aust. Ann. Med. 18, 258-263. 24. Yamomoto, M., Seedov, J. G., Rodan, G. A., et al. (1995). Endogenous calcitonin attenuates parathyroid hormone-induced cancellous bone loss in the rat. Endocrinology 136, 788-795. 25. Amara, S. G., Jonas, V., Rosenfeld, M. G., Ong, E. S., and Evans, R. M. (1982). Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 298, 240-244. 26. Tippins, J. R., Morris, H. R., Panico, M., Etienne, T., Bevis, P., Girgis, S., Maclntyre, I., Azria, M., and Attinger, M. (1984). The myotropic and plasma calcium-modulating effects of calcitonin gene-related peptide (CGRP). Neuropeptides 4, 425-434. 27. Yamamoto, I., Kitamura, N., Aoki, J., Shigeno, C., Hino, M., Asonuma, K., Torizuka, K., Fujii, N., Otaka, A., and Yajima, H. (1986). Human calcitonin gene-related peptide possesses weak inhibitory potency of bone resorption in vitro. Calcif. Tissue Int. 38, 339-341. 28. Cornish, J., Callon, K. E., Bava, U., Kamona, S. A., Cooper, G. J. S., and Reid, I. R. (2001). Effects of calcitonin, amylin, and calcitonin gene-related peptide on osteoclast development. Bone 29, 162-168. 29. Cornish, J., Callon, K. E., Cooper, G. J., and Reid, I. R. (1995). Amylin stimulates osteoblast proliferation and increases mineralized bone volume in adult mice. Biochem. Biophys. Res. Commun. 207, 133-139. 30. Valentijn, K., Gutow, A. P., Troiano, N., Gundberg, C., Gilligan, J. P., and Vignery, A. (1997). Effects of calcitonin gene-related peptide on bone turnover in ovariectomized rats. Bone 21, 269-274. 31. Michelangeli, V. P., Findlay, D. M., Fletcher, A., and Martin, T. J. (1986). Calcitonin gene-related peptide (CGRP)acts independently of calcitonin on cyclic AMP formation in clonal osteogenic sarcoma cells (UMR 106-01). Calcif. Tissue Int. 39, 44-48. 32. Gupta, A., Schwiening, C. J., and Boron, W. F. (1994). Effects of CGRP, forskolin, PMA, and ionomycin on pH(i) dependence of Na-H exchange in UMR-106 cells. Am. J. Physiol. 266, C1083-C1092. 33. Kawase, T., and Burns, D. M. (1998). Calcitonin gene-related peptide stimulates potassium efflux through adenosine triphosphate-sensitive potassium channels and produces membrane hyperpolarization in osteoblastic UMR-106 cells. Endocrinology 139, 3492-3502. 34. Thiebaud, D., Akatsu, T., Yamashita, T., Suda, T., Noda, T., Martin, R. E., Fletcher, A. E., and Martin, T. J. (1991). Structure-activity relationships in calcitonin gene-related peptide: Cyclic AMP response in a preosteoblast cell line (KS-4). J. Bone Miner. Res. 6, 1137-1142. 35. Mullins, M. W., Ciallella, J., Rangnekar, V., and McGillis, J. P. (1993). Characterization of a calcitonin gene-related peptide (CGRP) receptor on mouse bone marrow cells. Regul. Pept. 49, 65-72. 36. Bernard, G. W., and Shih, C. (1990). The osteogenic stimulating effect of neuroactive calcitonin gene-related peptide. Peptides 11, 625-632. 37. Sakagami, Y., Girasole, G., Yu, X. P., Boswell, H. S., and Manolagas, S. C. (1993). Stimulation of interleukin-6 production by either calcitonin gene-related peptide or parathyroid hormone in two phenotypically distinct bone marrow-derived murine stromal cell lines. J. Bone Miner. Res. 8, 811-816. 38. Vignery, A., and McCarthy, T. L. (1996). The neuropeptide calcitonin gene-related peptide stimulates insulin-like growth factor I production by primary fetal rat osteoblasts. Bone 18, 331-335. 39. Ballica, R., Valentijn, K., Khachatryan, A., Guerder, S., Kapadia, S., Gundberg, C., Gilligan, J., Flavell, R. A., and Vignery, A.
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expression of PTHrP during endochondral bone formation in mouse and intramembranous bone formation in an in vivo rabbit model. Bone 21, 385-391. Amizuka, N., Karaplis, A. C., Henderson, J. E., Warshawsky, H., Lipman, M. L., Matsuki, Y., Ejiri, S., Tanaka, M., Izume, N., Ozawa, H., and Goltzman, D. (1996). Hapl insufficiency of parathyroid hormone-related peptide (PTHrP) results in abnormal postnatal bone development. Dev. Biol. 175, 166-176. Kemp, B. E., Moseley, J. M., Rodda, C. P., Ebeling, P. R., Wettenhall, R. E. H., Stapleton, D., Diefenbach-Jagger, H., Ure, F., Michelangeli, V. P., Hollis, A., Simmons, H. A., Raisz, L. G., and Martin, T. J. (1987). Parathyroid hormone-related protein of malignancy: Active synthetic fragments. Science 238, 1568-1570. Hock, J. M., Fonseca, J., Gunness-Hey, M., Kemp, B. E., and Martin, T. J. (1989). Comparison of the anabolic effects of synthetic parathyroid hormone-related protein (PTHrP) 1-34 and PTH 1-34 on bone in rats. Endocrinology 125, 2022-2027. Ma, Y. L., Cain, R. L., Halladay, D. L., Yang, X., Zeng, Q., Miles, R. R., Chandrasekhar, S., Martin, T. J., and Onyia, J. E. (2001). Catabolic effects of continuous human PTH(1-38) in vivo is associated with sustained stimulation of RANKL and inhibition of osteoprotegerin and gene-associated bone formation. Endocrinology 142, 4047-4054. Onjia, J. E., Miles, R. R., Xang, X., Halladay, D. L., Hale, J., Glasebrook, A., McClure, D., Seno, G., Churgay, L., Chandrasekhar, S., and Martin, T. J. (2000). In vivo demonstration that human parathyroid hormone 1-38 inhibits the expression of osteoprotegerin in bone with the kinetics of an immediate early gene. J. Bone Miner. Res. 15, 863-871. Fenton, A. J., Kemp, B. E., Hammonds, R. G., Jr., Mitchelhill, K., Moseley, J. M., Martin, T. J., and Nicholson, G. C. (1991). A potent inhibitor of osteoclastic bone resorption within a highly conserved pentapeptide region of parathyroid hormone-related protein, PTHrP[107-111]. Endocrinology 129, 3424-3426. Sone, T., Kohno, H., Kikuchi, H., Ikeda, T., Kasai, R., Kikuchi, Y., Takeuchi, R., Konishi, J., and Shigeno, C. (1992). Human parathyroid hormone-related peptide-(107-111) does not inhibit bone resorption in neonatal mouse calvariae. Endocrinology 131, 2742-2746. Rouffet, J., Coxam, V., Gaumet, N., and Barlet, J. P. (1994). Preserved bone mass in ovariectomized rats treated with parathyroid-hormone-related peptide (1-34) and (107-111) fragments. Reprod. Nutr. Dev. 34, 473-481. Cornish, J., Callon, K. E., Nicholson, G. C., and Reid, I. R. (1997) Parathyroid hormone related-peptide-(107-139) inhibits bone resorption in vivo. Endocrinology 138, 1299-1304. Seitz, P. K., Zhang, R. W., Simmons, D. J., and Cooper, C. W. (1995). Effects of C-terminal parathyroid hormone-related peptide on osteoblasts. Miner. Electrolyte Metab. 21, 180-183. Gagnon, L., Jouishomme, H., Whitfield, J. F., Durkin, J. P., MacLean, S., Neugebauer, W., Willick, G., Roxon, R. H., and Chakravarthy, B. (1993). Protein kinase C-activating domains of parathyroid hormone-related protein. J. Bone Miner. Res. 8, 497-503. Cornish, J., Callon, K. E., Lin, C. Q. X., Xiao, L., Moseley, J. M., and Reid, I. R. (1999). Stimulation of osteoblast proliferation by C-terminal fragments of parathyroid hormone-related protein. J. Bone Miner. Res. 14, 915-922. Valin, A., Garcia-Ocana, A., de Miguel, F., Sarasa, J. L., and Esbrit, P. (1997). Antiproliferative effect of the C-terminal fragments of parathyroid hormone-related protein, PTHrP(107-111) and (107-139), on osteoblastic osteosarcoma cells. J. Cell. PhysioL 170, 209-215.
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67. Martinez, M. E., Garciaocana, A., Sanchez, M., Medina, S., Delcampo, T., Valin, A., Sanchezcabezudo, M. J., and Esbrit, P. (1997). C-terminal parathyroid hormone-related protein inhibits proliferation and differentiation of human osteoblast-like cells. J. Bone Miner. Res. 12, 778-785. 68. Lowe, C., Cornish, J., Callon, K., Martin, T. J., and Reid, I. R. (1991). Regulation of osteoblast proliferation by leukemia inhibitory factor. J. Bone Miner. Res. 6, 1277-1283. 69. Mackie, E. J., and Treschel, U. (1990). Stimulation of bone formation in vivo by transforming growth factor beta: Remodelling of woven bone and lack of inhibition by indomethacin. Bone 11, 295-300. 70. Cornish, J., Callon, K., King, A., Edgar, S., and Reid, I. R. (1993). The effect of leukemia inhibitory factor on bone in vivo. Endocrinology 132, 1359-1366. 71. Kohno, H., Shigeno, C., Kasai, R., Akiyama, H., Iida, H., Tsuboyama, T., Sato, K., Konishi, J., and Nakamura, T. (1997). Synovial fluids from patients with osteoarthritis and rheumatoid arthritis contain high levels of parathyroid hormone-related peptide. J. Bone Miner. Res. 12, 847-854. 72. Barengolts, E. I., Lathon, P. V., and Kukreja, S. C. (1996). Effect of various parathyroid hormone-related protein fragments on bone resorption and formation in vivo. J. Bone Miner. Res. 11 (Suppl. 1), $202. 73. Cooper, G. J. S., Willis, A. C., Clark, A., Turner, R. C., Sim, R. B., and Reid, K. B. M. (1987). Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. Proc. Natl. Acad. Sci. USA 84, 8628. 74. German, M. S., Moss, L. G., Wang, J., and Rutter, W. J. (1992). The insulin and islet amyloid polypeptide genes contain similar cellspecific promoter elements that bind identical beta-cell complexes. Mol. Cell. Biol. 12, 1777-1788. 75. Cooper, G. J. S. (2000). Amylin: Physiology and pathophysiology. The endocrine pancreas and regulation of metabolism. In The Handbook o f Physiology (L. S. Jefferson and A. D. Cherrington, Eds.). American Physiological Society/Oxford Univ. Press, New York. 76. Gilbey, S. G., Ghatei, M. A., Bretherton-Watt, D., Zaidi, M., Jones, P. M., Perera, T., Beacham, J., Girgis, S., and Bloom, S. R. (1991). Islet amyloid polypeptide: Production by an osteoblast cell line and possible role as a paracrine regulator of osteoclast function in man. Clin. Sci. 81, 803-808. 77. Butler, P. C., Chou, J., Carter, W. B., Wang, Y. N., Bu, B. H., Chang, D., Chang, J. W., and Rizza, R. A. (1990). Effects of meal ingestion on plasma amylin concentration in NIDDM and nondiabetic humans. Diabetes 39, 752-756. 78. Karlsson, E., Stridsberg, M., and Sandler, S. (1998). Leptin regulation of islet amyloid polypeptide secretion from mouse pancreatic islets. Biochem. Pharmacol. 56, 1339-1346. 79. Reid, I. R., Ames, R., Evans, M. C., Sharpe, S., Gamble, G., France, J. T., Lim, T. M. T., and Cundy, T. F. (1992a). Determinants of total body and regional bone mineral density in normal postmenopausal women--A key role for fat mass. J. Clin. Endocrinol. Metab. 75, 45-51. 80. Reid, I. R., Plank, L. D., and Evans, M. C. (1992b). Fat mass is an important determinant of whole body bone density in premenopausal women but not in men. J. Clin. Endocrinol. Metab. 75, 779-782. 81. Burns, M. D., and Kawase, T. (1997). Calcitonin gene-related peptide, amylin, or parathyroid hormone stimulate (in vitro) biomineralization. J. Bone Miner. Res. 12 (Suppl. 1), $324. 82. Horcajada-Molteni, M. N., Davicco, M. J., Lebecque, P., Coxam, V., Young, A. A., and Barlet, J. P. (2000). Amylin inhibits ovariectomy-induced bone loss in rats. J. Endocrinol. 165, 663--668.
83. Cornish, J., Callon, K. E., and Reid, I. R. (1996). Insulin increases histomorphometric indices of bone formation in vivo. Calcif. Tissue Int. 59, 492-495. 84. Maclntyre, I. (1989). Amylinamide, bone conservation, and pancreatic beta cells. Lancet 1, 1026-1027. 85. Owan, I., and Ibaraki, K. (1994). The role of calcitonin generelated peptide (CGRP) in macrophages: The presence of functional receptors and effects on proliferation and differentiation into osteoclast-like cells. J. Bone Miner Res. 24, 151-164. 86. Pietschmann, P., Farsoudi, K. H., Hoffmann, O., Klaushofer, K., Horandner, H., and Peterlik, M. (1993). Inhibitory effect of amylin on basal and parathyroid hormone-stimulated bone resorption in cultured neonatal mouse calvaria. Bone 14, 167-172. 87. Tamura, T., Miyaura, C., Owan, I., and Suda, T. (1992). Mechanism of action of amylin in bone. J. Cell. Physiol. 153, 6-14. 88. Cornish, J., Callon, K., Cooper, G. J. S., and Reid, I. R. (1994). The effect of amylin in neonatal mouse calvaria. Bone Miner. 25 (Suppl. 1), $41. 89. Young, A. A., Gedulin, B., Wolfelopez, D., Greene, H. E., Rink, T. J., and Cooper, G. J. S. (1992). Amylin and insulin in rat soleus muscle--Dose responses for cosecreted noncompetitive antagonists. Am. J. Physiol. 263, E274-E281. 90. Cornish, J., Callon, K., Lin, C.-Q., Xiao, C. L., Mulvey, T. B., Coy, D. H., Cooper, G. J. S., and Reid, I. R. (1998). Dissociation of the effects of amylin on osteoblast proliferation and bone resorption. Am. J. Physiol. 274, E827-E833. 91. Cornish, J., Callon, K. E., King, A. R., Cooper, G. J. S., and Reid, I. R. (1998). Systemic administration of amylin increases bone mass, linear growth, and adiposity in adult male mice. Am. J. Physiol. Endocrinol. Metab. 38, E694-E699. 92. Cornish, J., Callon, K. E., Gasser, J. A., Bava, U., Gardiner, E. M., Coy, D. H., Cooper, G. J. S., and Reid, I. R. (2000). Systemic administration of a novel octapeptide, amylin- (1-8), increases bone volume in male mice. Am. J. Physiol. 279, E730-E735. 93. Romero, D. F., Bryer, H. P., Rucinski, B., Isserow, J. A., Buchinsky, F. J., Cvetkovic, M., Liu, C. C., and Epstein, S. (1995). Amylin increases bone volume but cannot ameliorate diabetic osteopenia. Calcif. Tissue Int. 56, 54-61. 94. Borm, A. K., Klevesath, M. S., Borcea, V., Kasperk, C., Seibel, M. J., Wahl, P., Ziegler, R., and Nawroth, P. P. (1999). The effect of pramlintide (amylin analogue) treatment on bone metabolism and bone density in patients with type 1 diabetes mellitus. Horm. Metab. Res. 31, 472-475. 95. Datta, H. K., Rafter, P. W., Ohri, S. K., Maclntyre, I., and Wimalawansa, S. J. (1990). Amylin-amide competes with CGRP binding sites on osteoblast-like osteosarcoma cells. J. Bone Miner. Res. 5 (Suppl. 2), $229. 96. Villa, I., Rubinacci, A., Ravasi, F., Ferrara, A. F., and Guidobono, F. (1997). Effects of amylin on human osteoblast-like cells. Peptides 18, 537-540. 97. Grey, A. B., Cornish, J., Callon, K. E., Lin, C., Gilmour, R. S., and Reid, I. R. (1999). Amylin and adrenomedullin signalling to MAP kinase involves the IGF-1 receptor. J. Bone Miner. Res. 14 (Suppl. 1), $461. 98. Villa, I., Dal Fiume, C., Pagani, F., Rubinacci, S., and Guidobono, F. (2001). Calcitonin-related peptides activate protein kinase C in human osteoblast-like cells. Bone 28 (Suppl. 5), S133. 99. Cornish, J., CaUon, K. E., Lin, C. Q. X., Xiao, C. L., Mulvey, T. B., Cooper, G. J. S., and Reid, I. R. (1999). Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes. Am. J. Physiol. Endocrinol. Metab. 277, E779-E783. 100. Jacobs, T. W., Takizawa, M., Liu, C. C., Berlin, J. A., Katz, I. A., Stein, B., Joffe, I. I., Cooper, G. J. S., and Epstein, S. (1992).
8. Other Factors Controlling Bone Growth and Development Amylin stimulates bone cell turnover in vivo in normal and diabetic rats. J. Bone Miner. Res. 7 (Suppl. 1), $226. 101. Horcajada-Molteni, M. N., Chanteranne, B., Lebecque, P., Davicco, M. J., Coxam, V., Young, A., and Barlet, J. P. (2001). Amylin and bone metabolism in streptozotocin-induced diabetic rats. J. Bone Miner. Res. 16, 958-965. 102. Muff, R., Buhlmann, N., Fischer, J. A., and Born, W. (1999). Amylin receptor is revealed following co-transfection of a calcitonin receptor with receptor activity modifying proteins-l or-3. Endocrinology 140, 2924-2927. 103. Christopoulos, G., Perry, K. J., Morris, M., Tilakaratne, N., Gao, Y. Y., Fraser, N. J., Main, M. J., Foord, S. M., and Sexton, P. M. (1999). Multiple amylin receptors arise from receptor activitymodifying protein interaction with the calcitonin receptor gene product. Mol. Pharmacol. 56, 235-242. 104. Naot, D., Callon, K. E., Cooper, G. J. S., Reid, I. R., and Cornish, J. (2001). A potential role for adrenomedullin as a local regulator of bone growth. Endocrinology 142, 1849-1857. 105. Zaidi, M., Shankar, V. S., Huang, C. L. H., Pazianas, M., and Bloom, S. R. (1993). Amylin in bone conservation--Current evidence and hypothetical considerations. Trends Endocrinol. Metab. 4, 255-259. 106. Kitamura, K., Kangawa, K., Kawamoto, M., Ichiki, Y., Nakamura, S., Matsuo, H., and Eto, T. (1993). Adrenomedullin: A novel hypotensive peptide isolated from human pheochromocytoma. Biochem. Biophys. Res. Commun. 192, 553-560. 107. Hinson, J. P., Kapas, S., and Smith, D. M. (2000). Adrenomedullin, a multifunctional regulatory peptide. Endocrine Rev. 21, 138-167. 108. Montuenga, L. M., Martinez, A., Miller, M. J., Unsworth, E. J., and Cuttitta, F. (1997). Expression of adrenomedullin and its receptor during embryogenesis suggests autocrine or paracrine modes of action. Endocrinology 138, 440-451. 109. Kubo, A., Minamino, N., Isumi, Y., Katafuchi, T., Kangawa, K., Dohi, K., and Matsuo, H. (1998). Production of adrenomedullin in macrophage cell line and peritoneal macrophage. J. Biol. Chem. 273 (27), 16730--16738. 110. Kato, T., Bishop, A. T., Wood, M. B., and Adams, M. L. (1996). Effect of human adrenomedullin on vascular resistance of the canine tibia. J. Orthop. Res. 14, 329-333.
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111. Kitamura, K., Ichiki, Y., Tanaka, M., Kawamoto, M., Emura, J., Sakakibara, S., Kangawa, K., Matsuo, H., and Eto, T. (1994). Immunoreactive adrenomedullin in human plasma. FEBS. Lett. 341, 288-290. 112. Hirata, Y., Mitaka, C., Sato, K., Nagura, T., Tsunoda, Y., Amaha, K., and Marumo, F. (1996). Increased circulating adrenomedullin, a novel vasodilatory peptide, in sepsis. J. Clin. Endocrinol. Metab. 81, 1449-1453. 113. Taniyama, M., Kitamura, K., Ban, Y., Eto, T., and Katagiri, T. (1996). Elevated plasma adrenomedullin level in hyperthyroidism. Eur. J. Clin. Invest. 26, 454-456. 114. Di Iorio, R., Marinoni, E., Scavo, D., Letizia, C., and Cosmi, E. V. (1997). Adrenomedullin in pregnancy. Lancet 349, 328. 115. Cornish, J., Callon, K. E., Coy, D. H., Jiang, N. Y., Xiao, L. Q., Cooper, G. J. S., and Reid, I. R. (1997). AdrenomeduUin is a potent stimulator of osteoblastic activity in vitro and in vivo. Am. J. Physiol. Endocrinol. Metab. 36, E1113-El 120. 116. Eguchi, S., Hirata, Y., Iwasaki, H., Sato, K., Watanabe, T. X., Inui, T., Nakajima, K., Sakakibara, S., and Marumo, F. (1994). Structure-activity relationship of adrenomeduUin, a novel vasodilatory peptide, in cultured rat vascular smooth muscle cells. Endocrinology 135, 2454-2458. 117. Cornish, J., Callon, K. E., Grey, A. B., Balchin, L. M., Cooper, G. J. S., and Reid, I. R. (1999d). The proliferative effects of amylin and adrenomedullin on osteoblasts--An important role for the IGF-1 receptor. J. Bone Miner. Res. 14 (Suppl. 1), $338. 118. Kapas, S., and Clark, A. J. L. (1995). Identification of an orphan receptor gene as a type 1 calcitonin gene-related peptide receptor. Biochem. Biophys. Res. Commun. 217, 832-838. 119. Diampaka, E., Denne, M. A., de Vernejoul, M. C., and Cressent, M. (2001). Adrenomedullin and adrenomeduUin receptor mRNA expression in osteoblast-like cells. Dexamethasone effect. Bone 28 (Suppl. 5), S 146. 120. Cornish, J., Callon, K. E., Bava, U., Coy, D. H., Mulvey, T. B., Murray, M. A. F., Cooper, G. J. S., and Reid, I. R. (2001). Systemic administration of adrenomedullin(27-52) increases bone volume and strength in male mice. J. Endocrinol. 170, 251-257.
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J9 Peak Bone Mass and Its Regulation JEAN-PHILIPPE BONJOUR, THIERRY CHEVALLEY, SERGE FERRARI, and RENE RIZZOLI Department of lnternal Medicine, Division of Bone Diseases, Worm Health Organization Collaborating Center for Osteoporosis and Bone Diseases, University Hospital, Geneva, Switzerland
reported before puberty [1]. There is no evidence of a gender difference in bone mass at birth. Likewise, the volumetric BMD appears to also be similar between female and male newborns [9]. The absence of a substantial sex difference in bone mass is maintained until the onset of pubertal maturation [9-11]. During puberty, the gender difference in bone mass becomes expressed. This difference appears to be mainly due to a more prolonged bone maturation period in males than in females, with a larger increase in bone size and cortical thickness [10]. Bone size is much more affected during puberty than the volumetric BMD [1,2,10]. There is no significant sex difference in the volumetric trabecular density at the end of pubertal maturation [12-17]. During puberty, the accumulation rate in areal BMD at both the lumbar spine and the femoral neck increases four to sixfold during a 3- and 4-year period in females and males, respectively [18]. The change in bone mass accumulation rate is less marked in long-bone diaphysis [18]. There is an asynchrony between the increase in standing height and the growth of bone mineral mass during pubertal maturation [10,18,19]. This phenomenon may be responsible for the occurrence of a transient fragility that may contribute to the higher incidence of fractures known to occur when the dissociation between the rate of statural growth and mineral mass accrual is maximal [20,21].
DEFINITION AND IMPORTANCE OF PEAK BONE MASS Peak bone mass (PBM) corresponds to the amount of bony tissue present at the end of skeletal maturation [1]. It is a major determinant of the risk of fractures, such as those observed at the radial, vertebral, or femoral sites in osteoporotic patients. From epidemiological studies it can be assumed that a 10% increase (i.e., by approximately one standard deviation) in PBM in the female population decreases the risk of fracture by 50%, hence the interest of exploring ways to increase PBM in the primary prevention of osteoporosis. Bone mineral accumulation from infancy to postpuberty is a complex process. It can now be better appreciated due to the availability of dual X-ray absorptiometry (DXA) and quantitative computed tomography (QCT), which are noninvasive techniques that precisely measure areal or volumetric bone mineral density (BMD) at several sites of the skeleton [1-8]. These techniques also allow one to measure part of the change in the macroarchitecture or geometry of the bones, which along with the mineral mass strongly influence the resistance to mechanical strain. This chapter summarizes some of the knowledge that has accrued during the past few years on the characteristics of normal bone mass development from infancy to the end of skeleton maturation.
Time of P e a k B o n e M a s s A t t a i n m e n t In adolescent females, bone mass gain declined rapidly after menarche and was not statistically significant 2 years later [18]. In adolescent males, the gain in BMD/ bone mineral content (BMC) was particularly high from 13 to 17 years and markedly declined thereafter, although it remained significant between 17 and 20 years in both L2-L4 BMD/BMC and midfemoral shaft BMD
CHARACTERISTICS OF PEAK BONE MASS A C Q U I S I T I O N Bone Mass Development No substantial gender differences in bone mass of both the axial and appendicular skeleton have been
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Jean-Philippe Bonjouret aL
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[18]. In contrast, no significant increase was observed for femoral neck BMD. In subjects who reached pubertal stage P5 and grew less than 1 cm/year, a significant bone mass gain was still present in males but not in females. This suggests the existence of an important sex difference in the magnitude and/or duration of the so-called consolidation phenomenon that contributes to the PBM value. Observations made using QCT technology also indicate that the maximal volumetric BMD of the lumbar vertebral body is achieved soon after menarche since no difference was observed between the mean values of 16and 30-year-old subjects [22]. This is in keeping with numerous observations indicating that bone mass does not significantly increase from the third to the fifth decade [13,15,16,23-36]. Nevertheless, a few studies suggest that bone mass acquisition may be substantial during the third and fourth decades. In any case, most of the data do not support the concept that bone mass at any skeletal site, in both genders, in all races, and in any geographical area throughout the world continues to substantially accumulate until the fourth decade. In other words, PBM reached in the mid-30s does not seem to be a constant phenomenon of human physiology. Note that the external frame of the bones can become larger during adult life. This phenomenon has been documented by measuring the external diameter of several bones by radiogrammetry [37-40]. It may be the consequence of increased endosteal bone resorption with enlargement in the internal diameter. This would result in a transient reduction in cortical thickness, leading to augmentation in mechanical load on the remaining bony tissue. As a response to this increased load, the number and/or activity of osteogenic cells on the periosteal side of the cortex would be enhanced, giving rise to compensatory growth with enlargement of the external diameter. Thus, such a modeling phenomenon would be a response to bone loss, tending to compensate the reduction in the mechanical resistance [41]. Peak Bone M a s s Variance At the beginning of the third decade, there is large variability in the normal values of areal BMD in axial and appendicular skeleton [1]. This large variance, which is observed at sites particularly susceptible to osteoporotic fractures such as the lumbar spine and femoral neck, is only slightly reduced after correction for standing height and does not appear to substantially increase during adult life [42]. The height-independent broad variance in bone mass that is present before puberty appears to increase further during pubertal maturation at sites such as the lumbar spine and femoral neck [10,18]. Note
that in young healthy adults the biological variance in lumbar spine BMC is four or five times higher than that of standing height. It is also important to note that the variance in standing height does not increase during puberty [19].
CALCIUM-PHOSPHATE METABOLISM DURING GROWTH Endocrine a n d Transport Functions Several physiological functions influence bone accumulation during growth. Animal studies have identified physiological mechanisms that sustain increased bone mineral demand in relation to variations in growth velocity. In this context, two adaptive mechanisms affecting calcium-phosphate metabolism appear to be particularly important, namely the increase in the plasma concentration of 1,25-dihydroxyvitamin D3 (calcitriol) and the stimulation of the renal tubular reabsorption of inorganic phosphate (Pi). The elevation in the production and plasma level of calcitriol enhances the capacity of the intestinal epithelium to absorb both calcium and Pi. The increase in the tubular reabsorption of Pi results in an increase in its extracellular concentration. Without these two concerted adaptive responses, growth and mineralization cannot be optimal. Note that the increase in the tubular Pi reabsorption is not mediated by an increase in the renal production or the plasma level of calcitriol [43]. Analysis of cross-sectional studies suggests that these two adaptive mechanisms could be essential for coping with the increased bone mineral demand during the pubertal growth spurt. An increase in plasma calcitriol concentrations has been reported during pubertal maturation [44]. Both the pattern of this response and its consequence for intestinal calcium absorptive capacity in relation to pubertal bone mass acquisition are difficult to document since they require a time-integrated estimate of the controlling elements (calcitriol and intestinal calcium absorption). A tight relationship exists between the tubular reabsorption of Pi, plasma Pi level, and growth velocity in children [45]. An increase in plasma Pi during puberty has been reported [46,47]. Precise quantitation of the relationship between changes in the regulatory component of tubular Pi reabsorption and plasma concentration of Pi and bone mass gain during puberty remains to be done. However, similar to the calcitriol-intestinal calcium absorption regulatory pathway, a correct evaluation would require a timeintegrated assessment of the changes in tubular reabsorption and plasma concentration of Pi during the period of accelerated bone mass gain.
9. Peak Bone Mass and Its Regulation The mechanism underlying the parallel increase in calcitriol and tubular reabsorption of Pi has been clarified. In fact, experimental studies indicate that one factor, insulin-like growth factor-1 (IGF-1), could be responsible for stimulation of both calcitriol production and tubular Pi reabsorption (TmPi/GFR) in relation to the increased calcium and Pi demand associated with bone growth [10,48]. In humans, the plasma level of IGF-1 increases transiently during pubertal maturation and reaches a peak during midpuberty; thus, it occurs at an earlier age in females than in males [49]. The role of IGF-1 in calcium-phosphate metabolism during pubertal maturation in relation to essential nutrients for bone growth is illustrated in Fig. 1. The increases in plasma levels of IGF-1, calcitriol, and Pi are correlated with increases in indices of the bone appositional rate, such as alkaline phosphatase [50-53] and osteocalcin [52-55]. Note that the plasma concentrations of gonadal sex hormones, as well as those of adrenal androgens (dehydroepiandrosterone and androstenedione), which increase before and during pubertal maturation, do not seem to accord with the accelerated bone mass gain [56-58]. Whether differences in the adaptive responses that control calcium and phosphate homeostasis play a role in the increased variance in lumbar spine or femoral neck BMD/BMC remains to be explored. The interaction between the growth hormone-IGF-1 axis
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and sex steroids is quite complex [53]. The effect of these interactions on the increases in bone size and mass during pubertal maturation, independent of their influence on the rate and duration of longitudinal growth, remains largely unknown. Other potential players are the constituents of the PHEX complex [59] and FGF23 [60]. Bone Biochemical Markers during Puberty The interpretation of the changes in bone biochemical markers during growth is more complex than that for adulthood, particularly for the markers of bone resorption [53]. The plasma concentrations of the bone formation markers are highest when the velocity of bone mineral accrual is maximal, suggesting that the two phenomena are related. The high urinary excretion of bone resorption markers, such as collagen pyridinium crosslinks, observed during childhood decreases after the growth spurt and reaches adult values at the end of pubertal maturation (i.e., 15 or 16 years of age in females and 17 or 18 years of age in males) [53]. This probably reflects the decrease in resorption rate associated with the reduction and arrest in longitudinal bone growth. In a longitudinal study of pubertal girls, bone turnover markers (osteolcalcin, bone-specific alkaline phosphatase, and collagen pyridium cross-links) were modestly
FIGURE 1 Role of IGF-1 in calcium-phosphate metabolism during childhood and pubertal maturation in relation with essential nutrients for bone growth. During the pubertal bone growth spurt there is an increase in circulating IGF-1. The hepatic production oflGF-1 is under the positive influence of GH and essential amino acids (aa). IGF-1 stimulatesbone growth. At the kidneylevel, IGF-1 increasesboth the 1,25-dihydroxyvitaminD (1,25D) conversion from 25-hydroxyvitaminD (25-D) and the maximal tubular reabsorption of Pi(TmPi). By this dual renal action, IGF-1 favors a positive calcium and phosphate balance as required by the increased bone mineral accrual. Heredity, sex hormones, mechanical forces, and risk factors can either positively or negatively influence the bone response to IGF-1. See text for further details.
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related to statural height gain, but they were not predictive of gains in either total bone mineral content or density as assessed by DXA [61].
DETERMINANTS OF BONE MASS GAIN Many factors, more or less independently, are supposed to influence bone mass accumulation during growth, including heredity, sex, dietary components (calcium and proteins), endocrine factors (sex steroids, calcitriol, and IGF-1), mechanical forces (physical activity and body weight), and exposure to other risk factors [16,62-66]. Quantitatively, the most prominent determinant appears to be genetically related. Genetic D e t e r m i n a n t As previously mentioned, variability in BMD/BMC at the level of the lumbar spine and the proximal femur, unrelated to changes in statural height, increases during pubertal maturation. The contribution of heredity, compared to that of the environment, to this increased bone mass variability has not been clearly elucidated. Genetic factors account for a large percentage of the population variability in BMD among age- and sex-matched normal individuals [62]. Daughters of osteoporotic women have a low BMD [67], and BMD was decreased among relatives of 38 middle-aged men with severe idiopathic osteoporosis [68]. Two human models have been used to investigate the proportion of BMD variance across the population that is due to genetic factors, known as heritability [69]. In the twin model, within-pairs correlations for BMD are compared between monozygotic (MZ) twins, who share 100% of their genes, and dizygotic twins, who have 50% of their genes in common. Stronger correlation coefficients among adult MZ compared to DZ twins are indicative of the genetic influence on PBM, accounting for as much as 80% of lumbar spine and proximal femur BMD variance [62]. Lean and fat mass are also genetically determined [70]. Indeed, it appears that 80 and 65% of variance of lean and fat mass, respectively, are attributable to genetic factors. However, genetic factors affecting lean and fat mass have only little influence on lumbar spine or femoral neck BMD. These results differ from previous results indicating indirect genetic effects on bone mass occurring through the determination of lean body mass [71]. Parent-offspring comparisons have also shown significant relationships for BMD, although heritability estimates are lower (approximately 60%) than those in the twin model [72]. The magnitude of direct genetic
effects on PBM as evaluated in both human models may be overestimated due to similarities in environmental covariates [64,73]. We investigated correlations for BMC, areal and volumetric BMD, and bone area in the lumbar spine and femur (neck, trochanter, and diaphysis) in premenopausal women and in their prepubertal daughters [74]. Regressions were adjusted for height, weight, and calcium intake to minimize the impact of indirect genetic effects as well as dietary influences on bone mineral mass resemblance among relatives. The results indicated that despite great disparity in the maturity of the various constituents of bone mass before puberty with respect to peak adult values, heredity by maternal descent was detectable at all skeletal sites and affected virtually all bone mass constituents, including bone size and volumetric mineral density. Moreover, when daughters' bone values were reevaluated 2 years later, after puberty had begun and bone mineral mass had considerably increased, measurements were highly correlated with prepubertal values and mother-daughter correlations remained unchanged. Thus, most of this variance is due to genetic factors that are expressed before puberty, with subsequent tracking of bone mass constituent through the phase of rapid pubertal growth until PBM is achieved. Interestingly, it appears that male-to-male and male-to-female inheritance of bone mass may differ substantially [73]. Therefore, it might be erroneous to extrapolate genetic influences on bone mineral mass identified in women to the male population, which has not been investigated. In contrast to the clear heritability of PBM, the proportion of variance in bone turnover that is dependent on genetic factors, as assessed in this model by various markers of bone formation and resorption, appears to be small [75]. Hence, PBM is very likely determined by numerous gene products implicated in both bone modeling and remodeling. Taking into account the biological complexity of bone development, a large array of genes are probably involved in the determination of PBM at the various sites of the skeleton. There are several genes in which polymorphisms have been associated with differences in some bone variables, including osteoporotic fractures [76-79]. Polymorphisms of more than 20 candidate genes have been studied in relation to bone variables pertaining to the risk of osteoporosis and fragility fractures, including those coding for Receptors: Vitamin D (VDR), parathyroid hormone (PTH-1R), calcitonin, calcium, androgen, glucocorticoid, estrogen (ERe0, and tumor necrosis factor (TNF-R2) Hormones and cytokines: PTH, IGF-1, interleukin (IL)-6, TNF-ot, IL-1 receptor antagonist, and transforming growth factor-J3
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Enzymes: Aromatase (CYP19), methylenetetrahydrofolate reductase, and collagenase Bone matrix proteins." oL1 chain of type I collagen (CollA1), a2 chain of type I collagen (CollA2), osteocalcin (OC), oL2-HS glycoprotein, and bone morphogenetic protein-4 [76-79] However, for any of the genes in which polymorphisms have been associated with bone mass or fragility fracture, the functional significance of the genotype difference remains to be unequivocally demonstrated. Other polymorphisms of genes coding for molecules, having so far neither a structural nor a functional role in bone, have been found to be associated with BMD, such as apoprotein E [77,78]. As an alternative approach to the search for genetic linkage between candidate genes and bone mass, genomewide scanning in large kindreds with a clearly defined skeletal phenotype has localized quantitative trait loci (QTL) linked to bone mass. Some of the genomic regions with QTL appear to be more related to bone size than to bone volumetric density [76-79]. Recent findings strongly suggest that a gene coding for low-density lipoprotein receptor-related protein-5 (LRP5) could play a role in bone formation. Mutation in the LRP5 gene has been found in patients with human osteoporosis-pseudoglioma syndrome [80]. Interestingly Lrp5deficient mice develop an osteoporosis resulting in decreased osteoblast proliferation and function [81]. A gain-of-function mutation in LRP5 is associated with an autosomal dominant high bone mass [82,83]. Recent observations indicate that genomic variability in LRP5 is associated with differences in vertebral bone mass and size in healthy children, adolescents, and young adults [84]. It has been suggested that age and gene-environment and gene-gene interactions may explain the inconsistent relationships between bone mineral mass and 3' and 5' VDR genotypes [85-98]. Thus, significant BMD differences between 3~BsmI VDR genotypes were detected in children [99,100] but were absent in premenopausal women from the same genetic background [100]. Moreover, the latter study found that BMD in prepubertal girls was increased at several skeletal sites in Bb and BB subjects in response to calcium supplements, whereas it was unaffected in bb girls, who showed a trend for spontaneously higher BMD accumulation on their usual calcium diet [100,101]. Accordingly, a model taking into account the early influence of 3~ VDR polymorphisms, calcium intake, and puberty on BMD gain has been proposed to explain the relation between these genotypes and PBM [101]. Interestingly, several investigators have noted significantly lower height among women and men with the 3p VDR BB compared to Bb or bb genotypes
[100,102-105]. Considering the relationship between body size and bone size, as well as the influence of calcium intake on both body height and bone area during growth [105], it is tempting to speculate that 3' VDR alleles together with environmental calcium have an indirect and complex influence on PBM by regulating skeletal growth. Altogether, these observations indicate a possible physiological mechanism for the relationship between VDR gene polymorphisms and bone mass and emphasize the methodological limitations of earlier studies focusing on the association between VDR genotypes and BMD regardless of age and environmental factors. Moreover, other potential gene-environmental interactions, such as those involving physical exercise [106], as well as gene-gene interactions might further modulate the relationship between VDR gene polymorphisms and bone mass, such as an interaction between VDR and ER gene polymorphisms. In summary, 3~and 5~VDR alleles are possibly weak determinants of BMD, their effects being easily confounded by the influence of many other genes and environmental factors. Hence, VDR gene polymorphisms alone are not clinically useful genetic markers of PBM but could be a significant factor in explaining some of the variability observed in the population. Physical Activity Responsiveness to either an increase or a decrease in mechanical strain is probably greater in growing than adult bones [107-109]. Hence, the concept of public health programs aimed at increasing physical activity among healthy children and adolescents in order to maximize PBM has been promoted. Several recent reports of children or adolescents involved in competitive sports or ballet dancing indicate that intense exercise is associated with an increase in bone mass accrual in weight-bearing skeletal sites [110-117]. The question arises whether this increase in BMD/BMC resulting from intense exercise is translated into greater bone strength. A recent cross-sectional study in an elite male tennis player using peripheral QCT and side-to-side arm comparison indicated that the increase in BMC reflected increased bone size that was associated with an augmentation in an index of bone strength. In contrast, no change in either cortical or trabecular volumetric BMD was observed [118]. Whether the same type of beneficial structural change for bone strength is observed at other skeletal sites, such as vertebral bodies and proximal femur, in response to different kinds of intense exercise during childhood and adolescence remains to be documented. In terms of general public health, observations made in elite athletes cannot be the basis of recommendations for the general population since most individuals do not perform intense exercise. Much more
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relevant is information on the effect of moderate exercise on bone mass acquisition. Some [65,119,120] but not all [121-125] cross-sectional studies have found a slightly positive association between physical activity and bone mass values in children and adolescents. However, the positive association found cross-sectionally was not confirmed by observational longitudinal studies relating bone mass gain to physical activity [123,126]. Measurements of the duration, intensity, and type of physical activity that are based on recall are not very precise, particularly in children. Therefore, it is possible that negative findings could be ascribed to poor validity in the methods used to estimate physical activity. Recent controlled prospective studies carried out in prepubertal girls [127] and boys [128] indicate that exercise programs undertaken in schools, and considered on the average as moderate, can increase bone mass acquisition. These studies indicate that the growing skeleton is certainly sensitive to exercise and that prepuberty is an opportune time for implementing physical education programs consisting of various moderate weight-bearing exercises. Nevertheless, it remains uncertain to what extent the greater areal BMD gain in response to moderate and readily accessible weight-bearing exercise is associated with a commensurate increase in bone strength [128]. The magnitude of benefit in terms of bone strength depends on the nature of the structural change. An effect consisting primarily of an increased periosteal apposition and consecutive diameter will confer greater mechanical resistance than a response limited to the endosteal apposition rate leading essentially to a reduction in the endocortical diameter. Further studies examining the effects of mechanical loading components, such as the magnitude and frequency of various types of exercise, on the mass and geometry of bones in children and adolescents are necessary [129]. Furthermore, interaction between physical activity and the intake of nutrients, such as calcium, phosphate, and proteins, needs to be documented. A recent report suggested that the positive effect of physical activity on bone mineral mass gain is dependent on a high calcium intake [130]. Studies of elite adult athletes strongly indicate that increased bone mass gains resulting from intense physical activity during childhood and adolescence are maintained after training attenuates or even completely ceases [113,115,131-133]. Finally, the question of whether the increased PBM induced by physical exercise will be maintained into old age and confer a reduction in fracture rate remains unanswered. A recent cross-sectional study of retired elite Australian soccer players indicated that this may not be the case [134]. However, lack of information on the PBM values of these men does not allow firm conclusions to be drawn. In fact, another report indicated that high PBM attained by physical
activity in male hockey and soccer players during growth was associated with fewer fragility fractures in adult retired players (mean age, 69 years) [168] Nutritional Factors Puberty is considered to be a period of major behavioral changes and alterations in lifestyle. It is also assumed that important modifications in food habits occur during pubertal maturation, particularly in affluent societies. However, there is still a lack of quantitative and qualitative information regarding the evolution of both micro- and macronutrient intakes in relation to pubertal maturation. At the individual level, the extent to which variations in the intake of some nutrients in healthy, apparently well-nourished children and adolescents can affect bone mass accumulation, particularly at sites susceptible to osteoporotic fractures, has received increasing attention during the past 10 years. Most studies have focused on the intake of calcium. However, other nutrients such as protein should also be considered. Calcium
It is usually accepted that increasing calcium intake during childhood and adolescence is associated with a greater bone mass gain and thereby a higher PBM [135,136]. However, a survey of the literature on the relationship between dietary calcium and bone mass indicates that some [119,125,137-141] but not all studies [8,120,123,124,126,142] have found a positive correlation between these two variables. As with physical activity, several sets of cross-sectional and longitudinal data, including our unpublished results on dietary calcium intake and bone mass accrual in female and male subjects ages 9-19 years, are compatible with a two-threshold model. On one side of the normal range one can conceive of the existence of a low threshold, set at a total calcium intake of approximately 400-500 mg/day, below which a positive relationship can be found. Within this low range, the positive effect of calcium would be explained merely by its role as a necessary substrate for bone mass accrual. On the other side of the normal range, there would be a high threshold, set at approximately 1600 mg/day, above which the calcium intake through another mechanism could exert a slightly positive influence on bone mass accrual. In addition, the levels of the two thresholds could vary according to the stage of pubertal maturation. In our cross-sectional study [10], a significant positive relationship between total calcium intake as determined by two 5-day diaries was found in females in the pubertal subgroup P l-P4 but not in the P5 subgroup. Furthermore, in a longitudinal study [18], when results were analyzed taking into account the influence of age and
9. Peak Bone Mass and its Regulation
pubertal maturation, the relationship between the absolute values of calcium intake and the gain in BMD z score suggested that calcium may be more important before rather than during pubertal maturation [143]. Several intervention studies have been carried out on children and adolescents [105,144-148]. Overall, these indicate greater bone mineral mass gain in children and adolescents receiving calcium supplementation over periods varying from 12 to 36 months. The benefit of supplemental calcium is greater in the appendicular than in the axial skeleton [105,145]. Thus, in prepubertal children, calcium supplementation is more effective on cortical appendicular bone (radial and femoral diaphysis) than on axial trabecular-rich bone (lumbar spine) or on the hip (femoral neck and trochanter) [105,145]. The skeleton appears to be more responsive to calcium supplementation before the onset of pubertal maturation [145]. As intuitively expected, this benefit may be substantial in children with a relatively low calcium intake [105]. In 8-year-old prepubertal girls with a spontaneously low calcium intake, increasing the calcium intake from approximately 700 to 1400 mg augmented the mean gain in areal BMD of six skeletal sites by 58% compared to the placebo group after 1 year of supplementation [105]. This difference corresponds to a gain of 0.24 SD. If sustained over a period of 4 years, such an increase in calcium intake could augment mean areal BMD by 1 SD. Thus, milk calcium supplementation could modify the bone growth trajectory and thereby increase PBM. In this regard, it is interesting that an intervention influencing calcium-phosphate metabolism and limited to the first year of life may also modify the trajectory of bone mass accrual. In fact, a 400 IU/day vitamin D supplementation given to infants for an average of 1 year was associated with a significant increase in areal BMD measured at the ages of 7-9 years [149]. The areal BMD difference between the vitamin Dsupplemented and nonsupplemented groups was particularly significant at the femoral neck, trochanter, and radial metaphysis [149]. These observations are compatible with the "programming" concept, according to which environmental stimuli during critical periods of early development can provoke long-lasting modifications in structure and function [150,151]. Another aspect to consider is that the type of supplemented calcium salt may modulate the nature of the bone response. Thus, the response to administration of a calcium-phosphate salt from milk extract appears to differ from those of other calcium supplements. Indeed, the positive effect on areal BMD was associated with an increase in the projected bone area at several sites of the skeleton and a slight increase in statural height [105]. However, a recent report indicates that calcium carbonate supplementation can also increase standing
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height and bone mineral mass in male adolescents aged 16-18 years [152]. This type of response was not observed when calcium was given as citrate maltate salts [145,146], carbonate alone [147], or carbonate combined with gluconate lactate [148]. Interestingly, it was similar to the response to whole milk supplementation [153]. However, in the study by Cadogan et al. [153], the positive effect on bone size could be ascribed to other nutrients contained in whole milk, whereas in the study by Bonjour et al. [105] the tested calcium-enriched foods had the same energy, lipid, and protein content as those given to the placebo group. It is important to consider whether or not the gain resulting from the intervention will be lost after discontinuation of calcium supplementation. The answer to this question remains uncertain. It could depend on the type of bone response observed, which could differ according to the type of the supplemented calcium salt. As mentioned previously, with milk calcium-phosphate salt [105], the increase in areal BMD was associated with an increase in bone size. One year after discontinuing the intervention, differences in the gain in areal BMD and in the size of some bones were still detectable, at the limit of statistical significance [105,154]. We observed that this difference was still present 3 1/2 years after discontinuation of the supplementation [155]. These results need additional confirmation by long-term follow-up of the cohort, ideally until PBM has been attained, as well as by other prospective studies. Nevertheless, they apparently differ from results obtained with other calcium salt supplements [145,156]. In fact, calcium given in other forms to pre- or peripubertal girls does not appear to modify bone size [145-148] or to induce a persistent effect after the intervention is stopped [157,158]. This comparative analysis suggests that the positive effects observed on the areal BMD or BMC gain with citrate maltate salts [145,146] or carbonate alone [147] could be primarily related to an increment in the volumetric density resulting from an inhibition of bone remodeling. Despite a positive effect on mean areal BMD gain, there is still wide interindividual variability in the response to calcium supplementation. As discussed previously, it is possible that part of the variability in the bone gain response to calcium supplementation could be related to the VDR gene polymorphisms [100]. Nutrients Other Than Calcium
Among nutrients other than calcium, various experimental and clinical observations indicate the existence of a relationship between the level of protein intake and calcium-phosphate metabolism or bone mass or even osteoporotic fracture risk [159,160]. Nevertheless, any long-term influence of dietary protein on bone mineral
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metabolism and skeletal mass has been difficult to identify. Apparently contradictory information suggests that either a deficient or an excessive protein supply could negatively affect the balance of calcium and the amount of bony tissue contained in the skeleton [159,160]. Despite these uncertainties, multiple animal and human studies indicate that low protein intake per se could be particularly detrimental for both the acquisition of bone mass and the conservation of bone integrity with aging. During growth, undernutrition, including an inadequate supply of energy and protein, can severely impair bone development. Studies in experimental animals indicate that isolated protein deficiency leads to reduced bone mass and strength without histomorphometric evidence of osteomalacia [159,160]. Thus, inadequate supply of protein appears to play a central role in the pathogenesis of the delayed skeletal growth and reduced bone mass observed in undernourished children. Low protein intake could be detrimental to skeletal integrity by lowering the production of IGF-1. Indeed, the hepatic production and plasma concentration of this growth factor, which exerts several positive effects on the skeleton, are influenced by dietary protein [161]. Protein restriction has been shown to reduce circulating IGF-1 by inducing resistance to the hepatic action of growth hormone [162]. In addition, protein restriction appears to decrease the anabolic action of IGF-1 on some target cells. In this regard, it is important to note that growing rats maintained on a low-protein diet failed to restore growth when IGF-1 was administered at doses sufficient to normalize its plasma concentrations. Variations in the production of IGF-1 could explain some of the changes in bone and calcium-phosphate metabolism that have been observed in relation to intake of dietary protein. Indeed, the plasma level of IGF-1 is closely related to the growth rate of the organism. In humans, circulating IGF-1, of which the major source is the liver, increases progressively from 1 year of age to peak values during puberty. As described previously, this factor appears to play a key role in calcium-phosphate metabolism during growth by stimulating two kidney processes~Pi transport and the production of calcitriol [66,163]. IGF-1 is considered essential for bone longitudinal growth because it stimulates proliferation and differentiation of chondrocytes in the epiphyseal plate [164]. It also plays a role in trabecular and cortical bone formation. In experimental animals, administration of IGF1 also positively affects bone mass [165], increasing the external diameter of long bone, probably by enhancing the process of periosteal apposition. Therefore, during adolescence a relative deficiency in IGF-1 or resistance to its action possibly due to an inadequate protein supply may result not only in a reduction in skeletal longitudinal
growth but also in impairment of widthwise or crosssectional bone development. In well-nourished children and adolescents, the question arises whether variations in protein intake within the normal range can influence skeletal growth and thereby modulate the genetic potential in PBM attainment. There is a positive relationship between protein intake, as assessed by two 5-day dietary diaries with weighing of most food intakes [160,166], and bone mass gain during pubertal maturation [160]. Since both bone mass and protein intake increase in both sexes during adolescence, it is not surprising that there is a positive correlation between these two variables. However, we found that the correlation remained statistically significant even after correcting for the influence of either age or pubertal stage. The association between bone mass gain and protein intake was observed in both sexes at the lumbar spine, the proximal femur, and the femoral midshaft. The association appeared to be particularly significant from pubertal stage P2 to stage P4. However, these results should not be interpreted as evidence of a causal relationship between protein intake and bone mass gain. Indeed, it is quite possible that protein intake, which overall was related to the amount of ingested calories in our cohort, is to a large extent determined by growth requirements during childhood and adolescence. Recent data from our laboratory suggest that in prepubertal boys the response to calcium supplementation can be influenced by spontaneous protein intake [167]. As is the case for other nutrients such as calcium, only prospective interventional studies will establish whether variations in protein intake within the range recorded in our Western well-nourished population can affect bone mass accumulation during growth. Such prospective intervention studies should delineate the crucial years during which modifications in nutrition would be particularly effective for bone mass accumulation in children and adolescents. This kind of information is of importance in order to make credible and accurate recommendations for osteoporosis prevention programs aimed at maximizing PBM. CONCLUSIONS PBM is an important determinant of osteoporotic fracture risk; hence the interest in exploring ways to increase PBM in the primary prevention of osteoporosis. Bone mineral mass accumulation from infancy to postpuberty is a complex process implicating the interrelated actions of genetic, endocrine, mechanical, and nutritional factors. It can now be better evaluated due to the availability of noninvasive techniques, such as DXA and
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QCT, that precisely measure areal or volumetric BMD at various skeletal sites. From birth until PBM is attained, the increase in mass and strength is essentially due to an increase in bone size, with volumetric BMD changing very little during growth. Therefore, clinically the best estimate of bone strength is areal BMD rather than volumetric BMD, which does not take into account the size of the bone. It is estimated that in women an increase in PBM of 10% (i.e., approximately 1 SD) could decrease the risk of fragility fracture by 50%. Like standing height in any individual, bone mineral mass during growth follows a trajectory corresponding to a given percentile or standard deviation from the mean. Nevertheless, this trajectory can be influenced by environmental factors. Most important in the context of primary prevention of adult osteoporosis is that prospective randomized controlled trials strongly suggest that increasing the calcium intake or mechanical loading can shift the age-bone mass trajectory upward. Prepuberty appears to be an opportune time to obtain substantial benefit from increasing either calcium intake or physical activity. Further studies should demonstrate that the changes observed remain substantial at the end of the second decade and thus translate into a greater PBM. In this long-term evaluation of the consequences of modifying the environment, it will be of critical importance to assess whether any change in densitometric and morphometric bone variables observed at PBM confers a greater resistance to mechanical strain.
Acknowledgments This work was supported by the Swiss National Science Foundation (Grants 32-49757.96, 32-58880.99, and 32-58962.99), Nestec, Cerin, Novartis, and Institut Candia.
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144. Matkovic, V., Fontana, D., Tominac, C., Goel, P., and Chesnut, C. H., III (1990). Factors that influence peak bone mass formation: A study of calcium balance and the inheritance of bone mass in adolescent females. Am. J. Clin. Nutr. 52, 878-888. 145. Johnston, C. C., Miller, J. Z., Slemenda, C. W., Reister, T. K., Hui, S., Christian, J. C., and Peacock, M. (1992). Calcium supplementation and increases in bone mineral density in children. N. Engl. J. Med. 327, 82-87. 146. Lloyd, T., Andon, M. B., Rollings, N., Martel, J. K., Landis, J. R., Demers, L. M., Eggli, D. F., Kieselhorst, K., and Kulin, H. E. (1993). Calcium supplementation and bone mineral density in adolescent girls. J. Am. Med. Assoc. 270, 841-844. 147. Lee, W. T. K., Leung, S. S. F., Wang, S. H., Xu, Y. C., Zeng, W. P., Lau, J., Oppenheimer, S. J., and Cheng, J. C. (1994). Double-blind, controlled calcium supplementation and bone mineral accretion in children accustomed to a low-calcium diet. Am. J. Clin. Nutr. 60, 744-750. 148. Nowson, C. A., Green, R. M., Hopper, J. L., Sherwin A., J., Young, D., Kaymakci, B., Guest, C. S., Smid, M., Larkins, R. G., and Wark, J. D. (1997). A co-twin study of the effect of calcium supplementation on bone density during adolescence. Osteoporos. Int. 7, 219-225. 149. Zamora, S. A., Rizzoli, R., Belli, D. C., Slosman, D. O., and Bonjour, J. P. (1999). Vitamin D supplementation during infancy is associated with higher bone mineral mass in prepubertal girls. J. Clin. Endocrinol. Metab. 84, 4541-4544. 150. Barker, D. J. (1995). Intrauterine programming of adult disease. Mol. Med. Today 1, 418-423. 151. Cooper, C., Fall, C., Egger, P., Hobbs, R., Eastell, R., and Barker, D. (1997). Growth in infancy and bone mass in later life. Ann. Rheum. Dis. 56, 17-21. 152. Prentice, A., Stear, S. J., Ginty, F., Jones, S. C., Mills, L., and Cole, T. J. (2002). Calcium supplementation increases height and bone mass in 16-18 year old boys. J. Bone Miner. Res. 17 (Suppl. 1), $397. 153. Cadogan, J., Eastell, R., Jones, N., and Barker, M. E. (1997). Milk intake and bone mineral acquisition in adolescent girls: Randomised, controlled intervention trial. Br. Med. J. 315, 1255-1260. 154. Ammann, P., Rizzoli, R., Slosman, D., and Bonjour, J. P. (1997). Calcium supplements during childhood positively influence determinants of femoral neck bone strength. J. Bone Miner. Res. 12 (Suppl. 1), S 172. 155. Bonjour, J. P., Chevalley, T., Ammann, P., Slosman, D., and Rizzoli, R. (2001). The increase in bone mineral mass gain associated with calcium supplementation in prepubertal girls is observed more than 3.5 years after intervention discontinuation. Lancet, in press. 156. Rizzoli, R., and Bonjour, J. P. (1999). Determinants of peak bone mass and mechanisms of bone loss. Osteoporos. Int. 9 (Suppl. 2), S17-$23. 157. Slemenda, C. W., Peacock, M., Hui, S., Zhou, L., and Johnston, C. C. (1997). Reduced rates of skeletal remodeling are associated with increased bone mineral density during the development of peak skeletal mass. J. Bone Miner. Res. 12, 676-682. 158. Lee, W. T., Leung, S. S., Leung, D. M., and Cheng, J. C. (1996). A follow-up study on the effects of calcium-supplement withdrawal and puberty on bone acquisition of children. Am. J. Clin. Nutr. 64, 71-77. 159. Orwoll, E. S. (1992). The effects of dietary protein insufficiency and excess on skeletal health. Bone 13, 343-350. 160. Bonjour, J. P., and Rizzoli, R. (1995). Inadequate protein intake and osteoporosis possible involvement of the IGF system. In
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Challenges of Modern Medicine (P. Burckhardt and R. P. Heaney, Eds.), Ares Serono Symposia, Vol. 7, pp. 399-406. 161. Isley, W. L., Underwood, L. E., and Clemmons, D. R. (1983). Dietary components that regulate serum somatomedin-C concentrations in humans. J. Clin. Invest. 71, 175-182. 162. Thissen, J. P., Triest, S., Maes, M., Underwood, L. E., and Ketelslegers, J. M. (1990). The decreased plasma concentrations of insulin-like growth factor-I in protein-restricted rats is not due to decreased number of growth hormone receptors on isolated hepatocytes. J. Endocrinol. 124, 159-165. 163. Caverzasio, J., Montessuit, C., and Bonjour, J. P. (1990). Stimulatory effect of insulin-like growth factor-1 on renal Pi transport and plasma 1,25-dihydroxyvitamin D3. Endocrinology 127, 453-459.
164. Froesch, E. R., Schmid, C., Schwander, J., and Zapf, J. (1985). Actions of insulin-like growth factors. Annu. Rev. Physiol. 47, 443-467. 165. Ammann, P., Rizzoli, R., Muller, K., Slosman, D., and Bonjour, J. P. (1993). IGF-I and pamidronate increase bone mineral density in ovariectomized adult rats. Am. J. Physiol. 265, E770-E776. 166. Clavien, H., Theintz, G., Rizzoli, R., and Bonjour, J. P. (1996). Does puberty alter dietary habits in adolescents living in a western society? J. Adolesc. Health 19, 68-75. 167. Chevalley, T., Ferrari, S., Hans, D., Slosman, D., Fueg, M., Bonjour, J. P., and Rizzoli, R. (2002). Protein intake modulates the effect of calcium supplementation on bone mass gain in prepubertal boys. J. Bone Miner. Res. 17 (Suppl 1), S172. 168. Gustavsson (2002). J. Bone Miner. Res. 17 (Suppl. 1), $397.
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Pregnancy and Lactation ANN PRENTICE Medical Research Council Human Nutrition Research, Elsie Widdowson Laboratory, Fulbourn Road, Cambridge, United Kingdom
INTRODUCTION
content of this mineral. The proportion of calcium in fetal ash increases during early gestation and reaches a plateau of approximately 27% (g/g) by 4 months [2]. Substantial skeletal growth occurs from midgestation and maximal fetal calcium accretion occurs during the third trimester. Quantitatively, fetal calcium accretion increases from approximately 50 mg/day at 20 weeks of gestation to 330 mg/ day at 35 weeks [4]. For the third trimester of pregnancy, 200 mg/day is considered a typical calcium accretion rate. After delivery, skeletal growth and calcium accretion continue at a slower pace. A typical child accretes approximately 140 mg/day of calcium during the first year of life, with the rate being higher in the first months and slowing progressively with age [3,5]. The flux of calcium from mother to child across the placenta and via breast milk needs to be sufficient to match this accretion rate and, in the child, to meet any additional requirements imposed by gastrointestinal absorption and obligatory losses in urine, feces, and sweat. Breast milk calcium secretion averages approximately 200 mg/day at peak lactation but can be as high as 400 mg/day in some individuals [6]. Similar estimates can be made for the fluxes of the other primary bone-forming mineralsmphosphorus, magnesium, and zinc [3]. A newborn baby contains approximately 16 g of phosphorus, of which approximately 80% is contained in the skeleton. A typical whole-body phosphorus accretion rate in the first year of life is 70 mg/day. The magnesium content at birth is approximately 750mg, of which 60% is in the skeleton, and a typical whole-body accretion rate in infancy is 3 mg/day. For zinc, the whole-body content is 50mg at birth, of which 30% is in the skeleton, and the accretion rate in infancy of 0.4 mg/day [7]. Breast milk secretion during full lactation averages approximately 120,25, and 1.6 mg/ day for phosphorus, magnesium, and zinc, respectively, but can be higher in some individuals.
The fluxes of the primary bone-forming minerals, calcium, phosphorus, magnesium, and zinc, that occur between mother and offspring during pregnancy and lactation place considerable demands on maternal mineral economy. There are several possible biological strategies for meeting these extra requirements, including increased food consumption, elevated gastrointestinal absorption efficiencies, decreased mineral excretion, and mobilization of tissue stores. This chapter presents a review of the evidence on the extent to which these strategies apply in the human situation, the mechanisms by which they occur, the limitations imposed by maternal diet, and the possible consequences for the growth of the infant and bone health of the mother. It also discusses the importance of maternal vitamin D status in the mineral metabolism of the mother and infant. The evidence suggests that pregnancy and lactation are associated with physiological adaptive changes that are independent of maternal mineral supply, within the range of normal dietary intakes. These processes appear to provide the minerals necessary for fetal growth and breast milk production without requiring an increase in maternal dietary intake or compromising maternal bone health in the long term. More research is needed to define the limitations of these processes in women with marginal mineral intakes and poor vitamin D status.
MINERAL FLUXES FROM M O T H E R TO OFFSPRING At birth, the skeleton contains approximately 20-30 g calcium [1-3]. This represents 98 or 99% of the total body
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These mineral fluxes from mother to child represent a significant proportion of the mineral intakes of the mother, especially for calcium [8]. Intakes of these minerals vary in different areas of the world and range widely between individuals, but average intakes for women are of the order of 300-1000 mg/day calcium, 1000 mg/day phosphorus, 250 mg/day magnesium, and 10 mg/day zinc [9,10]. There are several possible biological strategies for meeting the extra demands that pregnancy and lactation make on the mineral economy of the mother, including increases in food consumption, elevated gastrointestinal absorption efficiencies, decreased mineral excretion, and mobilization of tissue stores. The extent to which these strategies apply in the human situation, the mechanisms by which they occur, the limitations imposed by maternal diet, and the possible consequences for the growth of the baby and bone health of the mother are discussed next.
PREGNANCY Mineral M e t a b o l i s m and Bone Biochemistry Human pregnancy is associated with major changes in mineral and bone metabolism (Table 1). These changes occur from early in gestation, in advance of the mineral requirement for skeletal development of the fetus. Calcium
During pregnancy, intestinal calcium absorption efficiency and urinary calcium excretion approximately double compared to the nonpregnant state (Fig. 1) [1117]. The elevation in calcium absorption is associated with increased intestinal expression of calbindin9K-D, a vitamin D-dependent calcium-binding protein [18,19]. The increased concentration of this calcium-binding protein
TABLE 1
T 1,25 (OH)2D 1" PTHrP 1" Prolactin 1" Placental lactogen
l
1" intestinal Ca** absorption 1" Ca-release from bone 1" Ca-uptake into bone
1" urinary Ca excretion
Ca transport to fetus
FIGURE 1 Schematic representation of the changes in calcium and bone metabolism that occur during pregnancy.
is mediated by 1,25-dihydroxyvitamin D, which is elevated in pregnancy. This acts on the enterocyte nucleus to increase transcription coupled with posttranslational effects that reduce degradation [18]. 1,25Dihydroxyvitamin D also acts at the enterocyte brush border to open the voltage-gated calcium channels (Fig. 2). The increase in urinary calcium excretion is largely due to the combined effects of the increases in glomerular filtration rate and calcium absorption. Fasting calcium excretion, corrected for creatinine clearance, is normal or decreased [13,20-22]. Measured calcium balance in the later stages of pregnancy is generally positive and retention approximates to that required for fetal growth [23]. The proportion of serum calcium circulating in the ionized form is increased in pregnancy. Serum ionized calcium concentration is unchanged or decreases slightly but remains within a narrow physiological range
Calcium and bone metabolic changes in human pregnancy, c o m p a r e d to the non-pregnant, non-lactating state
Calcium absorption Urinary calcium excretion, daily
T T
Serum 1,25 (OH)2vitamin D (flee and bound) Serum parathyroid hormone (intact)
T ~-~1 T
Fasting urinary calcium, creatinine corrected
~
Serum parathyroid hormone-related protein
Serum calcium (ionised) Serum calcium (total)
~(~)
Nephrogenous cyclic AMP Serum calcitonin
T
Tubular phosphate reabsorption
~
Urinary phosphate excretion
T
Bone resorption histology and markers* Bone formation markers (except Oc)**
T T
Serum phosphate
~l
Osteocalcin (intact)
1
*urinary collagen cross-links, telopeptides, hydroxyproline, serum tartrate-resistant acid phosphatase **serum bone alkaline phosphatase and procollagen peptides
10. Pregnancy and Lactation
Gut lumen
r-]
Maternal enterocyte ,,
,. . . . . . .
r
,,
Maternal circulation
~Ca2+ CaBP-D9K
251
tion and are not considered to represent alterations in maternal magnesium status [10]. Lymphocyte magnesium concentration is unchanged or decreased in pregnancy [29,30]. Urinary magnesium excretion is elevated in late pregnancy [15]. There is evidence that zinc absorption and urinary zinc excretion are increased, although individual responses are highly variable [32,33].
Bone Turnover 2+
FIGURE 2 Calcium transfer across maternal enterocyte by CaBPD9k, vitamin D-dependent calcium-binding protein (reproduced with permission from Hosking [18]).
throughout. The concentration of total serum calcium declines to a greater extent, with a slight increase toward the end of gestation. The decrease in total serum calcium reflects the change in serum albumin concentration associated with the increased intravascular fluid volume of pregnancy and the resulting hemodilution [17,24].
Phosphorus, Magnesium, and Zinc The patterns of change in the metabolism of the other bone-forming minerals largely parallel that of calcium, with decreases in serum concentrations coupled with increases in intestinal absorption and urinary excretion. There is no evidence for increased renal conservation of any of these minerals. Phosphorus balance becomes increasingly positive (i.e., absorption exceeds excretion) as gestation advances [11], and net absorption is higher in pregnant women compared with nonpregnant women [25]. Some studies have indicated a decrease in serum phosphorus concentration [13,15] and the renal phosphate threshold in the second and third trimesters of pregnancy, with a corresponding increase in urinary phosphate excretion [13]. The data are inconsistent, and other studies have shown no changes in phosphate metabolism [15,20,26]. The data on magnesium absorption and retention in pregnant women are very limited and inconclusive [10]. Ionized and total serum magnesium concentrations decrease with increasing gestational age [27-30] and are lower in pregnant women than preconception [15] or compared to nonpregnant controls [31]. These effects parallel the changes in serum proteins due to hemodilu-
Bone resorption increases during pregnancy. This has been indicated both histologically [34] and biochemically by urinary markers, such as collagen cross links, telopeptides, and hydroxyproline, and serum markers such as tartrate-resistant acid phosphatase [14-16,35]. After an initial decrease, increases are also noted in biochemical indices of bone formation, such as serum bone alkaline phosphatase and procollagen peptides, to levels higher than those observed prepregnancy [14-16, 35-38]. However, serum osteocalcin concentration, a commonly used marker of bone formation, is decreased relative to preconception levels [14,15], although its concentration in late gestation is higher than in early pregnancy [14-16,39]. Increases in bone turnover indices are observed in early gestation. Their levels increase by 50-200% during pregnancy [14-16,36]. The increases in markers of bone resorption occur before those of bone formation, indicating a substantial uncoupling of bone remodeling [16,40]. A study of women with multifetal pregnancies demonstrated that selective fetal reduction reduced circulating concentrations of the cross-linked carboxy (C)terminal telopeptide of type-1 collagen (ICTP), a marker of bone resorption, without corresponding changes in the C-terminal propeptide of type I pro-collagen (PICP), an index of bone formation [35]. This suggests that factors derived from the fetoplacental unit are involved in the stimulation of maternal bone turnover, primarily via an effect on bone resorption. There are problems in interpreting changes in biochemical indices during pregnancy because of the effects of hemodilution, alterations in creatinine excretion and renal clearance, and the contribution to and metabolism of these markers by the products of conception [19]. The disparity between the indices of bone formation, for example, may be due to the degradation or uptake of osteocalcin by the placenta [37,41]. Measurements of an osteocalcin metabolite (Ocf), adjusted for alterations in creatinine clearance, have indicated that despite the low measurable concentrations of the intact protein, osteocalcin production is not decreased in pregnancy [16]. Total alkaline phosphatase is not a useful index of bone formation in pregnancy because of the contribution from the placental isoform [19]. The increase in resorption
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markers may partly reflect a contribution from the turnover of the fetal skeleton. However, a study of the ratio of oL to 13 isomers of the C-terminal telopeptide of type 1 collagen (CTX), a bone resorption marker, suggested that the fetal contribution to maternal CTX excretion is small, amounting to less than 10% of oL-CTX and only 2% of 13-CTX [161.
Calciotropic Hormones The concentration of serum 1,25-dihydroxyvitamin D is increased throughout pregnancy [13-15,42]. This elevation occurs in both the free and protein-bound forms [20,43]. Until late in pregnancy, the increase in 1,25dihydroxyvitamin D parallels an increase in the concentration of vitamin D-binding protein [44,45]. The mechanisms underlying the increase in 1,25-dihydroxyvitamin D are unclear but may involve placental or fetal synthesis of the hormone from maternal 25-hydroxyvitamin D, upregulation of maternal renal 1-c~-hydoxylase by a variety of pregnancy-associated hormones, or an alteration in the balance between the production of 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D [17,19,46,47]. It is likely that the production of 1,25dihydroxyvitamin D by the maternal kidneys plays the greater role because low serum concentrations of this hormone have been reported from an anephric individual during pregnancy [19]. There is evidence of placental transfer of 25-hydroxyvitamin D, but the quantities are thought to be small and considered unlikely to compromise the vitamin D status of pregnant women [10,48]. Although serum 1,25-dihydroxyvitamin D is raised, there is no evidence of an increase in intact parathyroid hormone (PTH) concentration in pregnancy [13,17,20,26,49], and it may be decreased [14,15,50]. Early studies reported high concentrations of PTH during pregnancy, but these have had to be reinterpreted in light of research conducted after the advent of a sensitive two-site immunoassay specific for the intact molecule of PTH [17]. The elevated concentrations reported in the earlier studies are likely explained by the detection of multiple fragments of PTH, many of which are biologically inactive. Although increases in PTH production and turnover cannot be discounted, it appears that human pregnancy is not associated with an increase in PTH bioactivity [17,51]. This is supported by normal nephrogenous cyclic adenosine monophosphate (NcAMP) production, a marker of PTH-like bioactivity [13,26,49]. Consequently, the view of pregnancy as a period of physiological hyperparathyroidism driven by the fetal demand for calcium [52] is no longer regarded as tenable [13]. Increased concentrations of parathyroid hormonerelated protein (PTHrP) are detected in the maternal
circulation during pregnancy [49,53], probably originating from fetal, placental, or mammary tissues [17]. PTHrP, or more specifically its amino-terminal fragments, has close homology with the N-terminal 1-34 amino acid sequence of PTH. It has the ability to activate the PTH/PTHrP receptor [54] and, consequently, has PTH-like characteristics. It stimulates renal 1-t~-hydoxylase activity and NcAMP production, thereby promoting 1,25-dihydroxyvitamin D synthesis and calcium reabsorption [18]. In addition, N-terminal PTHrP promotes bone resorption via the classical PTH/PTHrP receptor, although the C-terminal fragment PTHrP(107139) inhibits osteoclastic bone resorption through a different receptor [54]. The role of PTHrP during pregnancy is unclear, however, but its presence may account, at least in part, for the increase in 1,25-dihydroxyvitamin D, which occurs even though intact PTH concentrations are reduced. Nonpregnant women administered PTHrP (1-36) subcutaneously have elevated 1,25-dihydroxyvitamin D levels, urinary calcium excretion, and NcAMP production with no alteration in serum PTH or calcium concentrations [55]ma response that resembles some of the biochemical changes of pregnancy. The response of calcitonin (CT) to pregnancy appears to be highly variable [56], with some studies reporting elevations [17,31,57] and others reporting no changes [14,46,56]. Increases in circulating CT have been observed in thyroidectomized women during pregnancy, probably as a result of CT synthesis by mammary and placental tissues [17]. The physiological function of CT is not fully understood, although a role in protecting the maternal skeleton from resorption during pregnancy has been proposed [58], and it may promote renal calcium excretion [19]. Many other hormones, growth factors, and cytokines are elevated in the maternal circulation during pregnancy that could stimulate or drive the observed changes in calcium absorption, bone turnover, and 1,25-dihydroxyvitamin D synthesis. These include prolactin, estrogen, progesterone, placental lactogen, placental growth hormone, tumor necrosis factor-a, and insulin-like growth factor-1 (IGF-1) [16,17,38]. Their relative contributions to mineral and bone metabolism in human pregnancy, and the interactions between their effects, have yet to be established.
Maternal Skeleton Physiological Changes Biochemical data suggest that human pregnancy is accompanied by alterations in the uptake and release of calcium and other minerals from the maternal skeleton. Whether the balance between storage and mobilization is
10. Pregnancy and Lactation
sufficient to result in an overall increase or decrease in bone mineral content is not clear. Changes in bone mineral content have been reported in pregnancy, but the response differs between individual women and between skeletal sites. No consistent pattern has emerged and the debate continues regarding the extent to which bone loss or gain is a physiological accompaniment to human pregnancy. Direct assessments of skeletal changes during pregnancy are restricted by the fact that the most sensitive methods for the measurement of bone mineral content are based on the attenuation of ionizing radiation, such as dual- and single-energy X-ray absorptiometry. Although the radiation dose incurred with modern instruments is low and similar to that received from background radiation, these techniques are avoided for measurements of the axial skeleton in pregnant women. As a consequence, investigations using these techniques are limited to estimating the integrated skeletal response over the whole of pregnancy by measuring bone mineral status before conception and after delivery. To date, relatively few such prospective studies have been undertaken, and these have involved only small numbers of individuals [14-16,40,59-63]. Increases in bone mineral content in the total body and at cortical bone sites have been observed in some studies [16] but not in others [14]. Decreases in bone mineral content have been seen in skeletal regions rich in trabecular bone, such as the spine and hip [16,40,59], whereas other studies have found no change in these regions [14,63]. In the case of women entering pregnancy during or after a period of extended lactation, substantial increases have been reported [60,61]. Measurements at peripheral skeletal sites, using absorptiometry or bone ultrasonography, have been used to investigate the pattern of skeletal response during pregnancy [15,20,36,64-68]. Many of these studies are difficult to interpret because the initial measurements were made when the women were already pregnant rather than before conception and, as is now well recognized, major changes in bone metabolism occur in the early stages of pregnancy [34]. Decreases in bone mineral content over the course of pregnancy have been noted in ultradistal scans of the forearm, a region rich in trabecular bone [40,66,67], but generally not at more proximal appendicular sites and not in all studies [15,20]. Ultrasound studies of the os calcis and phalanges have reported decreases in the measured variables in the later stages of pregnancy [36,68]. Although speed of sound and broadband ultrasound attenuation, measured by bone ultrasonography, are regarded as indices of bone mineral density, the validity of this assumption for pregnant women is not known, particularly in the presence of peripheral edema.
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The reasons for the variability in maternal skeletal response to pregnancy have yet to be determined. It is possible that the changes in bone mineral content are governed by a variety of influences, such as the mother's age or parity and her nutritional or endocrinological status prior to or after conception. For example, in studies of women who conceived during or soon after a period of extended lactation, increases in bone mineral content occurred during pregnancy that were similar to those required for the recovery of lactational bone losses [60,61]. However, those women who conceived after recovery of lactational bone loss had taken place showed little further change in bone mineral content by the end of the subsequent pregnancy. Similarly, slim pregnant women (body mass index < 22) exhibited significant increases in bone mineral content at the femoral neck and Ward's triangle that were independent of weight gain in pregnancy and were not observed in larger pregnant women or size-matched controls [62].
Osteoporosis of Pregnancy Fragility fractures due to osteoporosis can occur in pregnancy, although the incidence is rare [22,69,70]. The condition often involves the spine or hip, is more common in the first pregnancy, and usually resolves spontaneously a few months postpartum [22]. Osteoporosis of pregnancy is generally either idiopathic or secondary to treatment with corticosteroids, magnesium, or warfarin [69-71]. Some studies have suggested that pregnancy may unmask rather than cause low bone mineral density and that fractures result from alterations in posture or load bearing [22,72]. However, fractures associated with pregnancy can occur in the absence of low bone mineral density [73]. There are no data to suggest that osteoporosis of pregnancy is either an exaggerated metabolic response to pregnancy or a consequence of dietary deficiencies. Therefore, the fact that osteoporosis can occur in pregnant women cannot be taken as evidence either that bone mineral loss is a necessary corollary of normal pregnancy or that the condition can be prevented by alterations in diet and lifestyle.
Effect of Pregnancy on Later Bone Health The peak bone mass a women achieves as a young adult, before the onset of bone loss, is a predictor of her risk of fragility fracture after menopause. As a consequence, if the changes in bone mineral content associated with pregnancy are of sufficient magnitude to increase or decrease the mother's bone mineral status in the long term, pregnancy could alter the woman's risk of osteoporosis later in life. Retrospective studies of older women that have investigated the possible impact of pregnancy
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on osteoporosis risk have produced conflicting results [8,74]. The disparities may be due to the difficulties of defining reproductive history adequately, separating the effects of pregnancy and lactation, and controlling for possible confounders such as socioeconomic factors and body size. In addition, there appears to be considerable interindividual variability in the response to pregnancy. In general, however, evidence from retrospective studies indicates that parity is either associated with higher bone mineral content and reduced fracture risk [75-81] or has no effect [82-87]. There is no evidence that women who have become pregnant but miscarried have altered bone mineral status [88]. These data suggest that pregnancy is not a risk factor for osteoporosis and may be protective. This conclusion has to be viewed with caution because nulliparous women may have hormonal or metabolic characteristics that reduce their ability to conceive or to have successful pregnancies and that, independently, place them at greater risk of osteoporosis in later life [74]. The potential influence of individual differences in hormonal milieu has been suggested by a recent report of an interaction between parity and oral contraceptive use on hip fracture risk [79]. In this study, parity was associated with a reduction in hip fracture incidence among the population as a whole but with an increase in fracture incidence among women with a history of oral contraception [79].
Influence of M a t e r n a l Diet a n d Nutritional S t a t u s on Mineral M e t a b o l i s m of t h e P r e g n a n t M o t h e r a n d Skeletal D e v e l o p m e n t of t h e Fetus Calcium
There is only limited information about the impact of dietary calcium intake on maternal calcium and bone metabolism in pregnancy, and the studies that are available generally involve well-nourished women with a moderate to high calcium intake. Pregnant women exhibit an exaggerated calcemic response to an acute calcium load compared to nonpregnant controls [13,89] but experience similar increases in urinary excretion and decreases in bone resorption [89]. These data provide further evidence that pregnancy is a state of physiological hyperabsorption [13]. It is therefore unlikely that the observed biochemical changes imply an inadequacy of maternal dietary supply to meet the fetal demands for calcium. In support of this view, pregnancy-associated changes in calcium and bone metabolism are observed in women with a high calcium intake and in those who consume calcium supplements [15,90]. Few investigations have been conducted on women with a low calcium intake. A cross-sectional study of Malay women reported lower urinary calcium excretion and higher serum concentra-
tions of intact PTH in late pregnancy compared to early pregnancy [91]. This pattern of biochemical response differs from that reported from populations with higher customary calcium intakes and may indicate that PTHinduced renal conservation of calcium occurs in situations in which maternal calcium intake is low. Balance studies conducted in Indian women show that they achieve similar calcium retention as that of women in other countries despite their lower plane of calcium nutrition [23,92]. There is also little evidence to suggest that maternal calcium intake modifies the integrated skeletal response to pregnancy. In a study of women consuming an average of 1200mg/day of calcium, the change in femoral bone mineral content across pregnancy was not affected by calcium intake [62]. Conversely, larger decreases in phalangeal ultrasonographic bone propagation velocity were noted in pregnant women with a calcium intake less than 1000 mg/day compared with those with a higher intake [64]. However, the consumption of calcium supplements by pregnant Indian women with a customary calcium intake of approximately 300 mg/day did not lead to differences in radiographic bone density of the hand compared to women who did not receive the supplements [93]. An increase in serum lead concentration has been observed in pregnant women. It has been suggested that lead may be released from the skeleton because of the elevated bone turnover of pregnancy [94-96]. The increase in lead concentration is less in women with a high calcium intake and in those who take calcium supplements. It has yet to be established whether this is because a higher calcium intake alters the amount of skeletal lead released or decreases intestinal lead absorption [97a]. Taken together, the limited data indicate that physiological adaptations occur during human pregnancy that provide an adequate supply of calcium to meet fetal demands for bone growth and mineralization and that are not dependent on maternal dietary intake. However, whether fetal bone growth and development can be adequately supported by a very low maternal intake of calcium has still to be established. In an early study using radiographic densitometry, calcium supplementation of pregnant Indian mothers with an intake of approximately 300mg/day of calcium resulted in higher neonatal bone density compared with that of infants of control mothers, with no effect on birth weight or length [93]. This finding must be replicated with more sensitive and reliable absorptiometric techniques. A possible effect of maternal calcium intake on fetal mineralization has also been suggested from a study of United States women taking part in a calcium supplementation trial during pregnancy (2 g/d calcium or placebo from 22 weeks gestation). For women with a low customary calcium intake (<600md/d), calcium supplementation was associated with greater total bone mineral content of the
10. Pregnancy and Lactation
neonate, but there was no significant difference in infant size. No supplement effect was noted for women with higher calcium intakes.
Phosphorus, Magnesium, and Zinc No studies have investigated the effect of dietary phosphorus on phosphorus economy in pregnancy [10]. A number of studies have examined the impact of magnesium supplementation on aspects of pregnancy outcome, including preterm delivery and preeclampsia. These have shown no effect on fetal bone growth and intrauterine growth retardation [10]. Long-term use of intravenous magnesium sulfate treatment for preterm labor and preeclampsia has been associated with neonatal hypermagnesemia and bone abnormalities [98,99]. Although poor maternal zinc status, as indicated by depleted leukocyte concentrations, has been associated with fetal growth retardation in some studies, zinc supplementation of healthy pregnant mothers has not been shown to have an effect on infant birth weight, crown-heel length, or head circumference [100]. It is probable that, in healthy women, metabolic adaptation during pregnancy ensures an adequate transfer of zinc to the fetus [9].
Vitamin D Status Vitamin D deficiency during pregnancy is associated with congenital rickets and craniotabes in the newborn [48] and with the development of rickets in infancy, especially when the child is exclusively breast-fed [101,102]. Pregnant women who receive regular sunlight exposure during the summer months are not at risk of vitamin D deficiency, but women who wear concealing clothes, are housebound, or for other reasons do not receive adequate sunlight exposure are at risk unless their diet provides sufficient vitamin D [10,103]. There is evidence that poor maternal vitamin D status, at a level higher than that associated with rickets in the child, can affect fetal and infant skeletal growth and ossification, tooth enamel formation, and calcium handling [48,104,105]. Newborns of mothers at risk of vitamin D insufficiency given a vitamin D supplement during pregnancy have higher serum calcium concentrations than those of mothers who have not been supplemented [104,106,107]. In a Chinese study, the appearance of ossification centers was more likely to be delayed in infants from the north than from the south of the country and in those with a low cord serum 25-hydroxyvitamin D concentration [107]. Seasonal variations in maternal vitamin D status are reflected in neonatal bone mineral content. In a study from Korea, where women are at risk of vitamin D deficiency in the winter, lower total body bone mineral content and higher bone turnover were observed in winter-born neonates [108]. Conversely, in an American
255
study, neonates born in the winter had higher bone mineral content and lower bone turnover than those born in the summer [109,110]. The differences in these findings may indicate that there is a vulnerable period to vitamin D insufficiency in early gestation [48] or that there is greater use of vitamin D supplements by American women in winter [108]. Vitamin D supplementation of British Asian mothers, who are at increased risk of vitamin D deficiency, resulted in smaller fontanelles and a trend to higher birth weight in their newborns compared to controls [106]. The effects of maternal vitamin D status on infant growth appear to be long-lasting. In a follow-up of the British Asian study, the children born to the vitamin D-supplemented mothers were heavier and longer at 1 year than those born to control mothers [111].
General Nutrition Maternal undernutrition, in a general sense, has a major impact on fetal growth and birth weight, and hence on skeletal mass. With respect to bone formation, an adequate supply of protein and energy is required for collagen matrix synthesis, in addition to provision of the elements required for bone mineralization [112]. Poor nutrition during pregnancy may reduce neonatal bone density as well as size [112,113]. A detailed discussion of the relationship between maternal nutrition and fetal growth, and the extent and limitations of adaptive mechanisms, is beyond the scope of this chapter, but nutritional interventions aimed at preventing or treating impaired fetal growth have recently been subjected to systematic analysis [114]. Nutritional supplementation of undernourished pregnant mothers in developing countries can reduce the incidence of low-birth-weight infants and produce increases in skeletal length but not head circumference [115]. Size at birth and in infancy predicts adult bone mineral mass, probably through tracking of skeletal size through childhood [116,117]. This suggests that the nutritional environment in utero and early life, in addition to influencing the risk of adult vascular disease and diabetes [118], may be an important modulating factor of skeletal growth in childhood and of bone health in old age.
LACTATION Mineral M e t a b o l i s m a n d Bone Biochemistry Major changes in mineral and bone metabolism occur for several months postpartum (Figs. 3 and 4). The pattern of change is dependent on whether the mother chooses to breast-feed and, if lactation is initiated, on the pattern of breast-feeding adopted by the mother. Likely lactation-associated variables include the duration of
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Suckling
Estrogen repletion
T osteoblast activity
,I, GnRH
~
1
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1' PTHrP ,1, serum Ca ++ 1" bone resorption
1' PTH
1" 1,25 (OH)2D
1" blood Ca ++ ,1, PTH
$ Ca ++ uptake by mammary tissue
1" renal Ca ++ reabsorption
1' intestinal Ca ++ absorption
FIGURE 4 Schematic representation of the proposed mechanism that increases bone mineral after weaning (reproduced with permission from Kalkwarf [129]).
milk Calcium
FIGURE 3 Schematicrepresentation of the proposed mechanism by which the infant regulates calcium release from maternal bone and provision to the mammary gland for milk production (reproduced with permission from Kalkwarf[129]).
exclusive breast-feeding, the timing of introduction of other foods to the infant (as a complement to breastfeeding), breast-feed frequency (which will vary with stage of lactation), the initiation of replacement of breast-feeding by other meals (when breast-feeding frequency is reduced), and the timing of cessation of breast-feeding. The term weaning is ambiguous because it is used variously to describe the process of introducing the infant to solid foods, the transition period between introducing solid foods and the cessation of breastfeeding, and the cessation of breast-feeding. The main metabolic and biochemical differences for breast-feeding mothers during full lactation (first 3-6 months) and during the periods before and after cessation of breast-feeding are summarized in Tables 2 and 3. Interpretation is made difficult by the paucity of studies in which markers of mineral metabolism and bone biochemistry in lactation are compared to prepregnant levels within the same individual. In general, comparisons are made either with women of similar age and characteristics who have not recently been pregnant or to mothers at the same stage postpartum who are not lactating. Data using these two comparative groups are
shown in Tables 2 and 3. It should be appreciated that because nonlactating women may experience alterations in mineral and bone metabolism postpartum, changes in lactating women cannot be attributed solely to a response to lactation because they may be a consequence of having recently been pregnant. In addition, because nonlactating women may experience changes as a result of not initiating breast-feeding or stopping breast-feeding early, in addition to no longer being pregnant, comparisons between lactating and nonlactating postpartum women can obscure or exaggerate the response to lactation. Calcium
Calcium absorption and urinary calcium excretion decline from the high levels of pregnancy to prepregnancy levels after delivery [12,14,119,120]. The decrease in urinary calcium output partly reflects the reduction in glomerular filtration rate after parturition [19]. Further decreases in total urinary calcium output, and reductions in fasting calcium excretion, have been reported for breast-feeding women [14,17,31,32,119,121-123]. This finding has not been observed in all studies, particularly those that have made comparisons with nonlactating mothers at the same stage postpartum [15,124-127]. Serum ionized and total calcium concentrations are raised in lactation compared to late pregnancy and normal controls [14,17,90,123], whereas studies comparing
257
10. Pregnancy and Lactation TABLE 2
Calcium and bone metabolic changes in human lactation during 3-6 months of full breast-feeding, NP
Calcium absorption Urinary calcium excretion, daily Fasting urinary calcium, creatinine corrected Serum calcium (ionised) Serum calcium (total) Tubular phosphate reabsorption Urinary phosphate excretion Serum phosphate
~ ~ ~ 1 T'---' T~ T ~ T
NL
nk '---' ~ 1 T T
Serum 1,25 (OH)2vitamin D (free and bound) Serum parathyroid hormone (intact) Serum parathyroid hormone-related protein Nephrogenous cyclic AMP Serum calcitonin Bone resorption markers* Bone formation markers (not osteocalcin)** Osteocalcin (intact)
NP
NL
~1 ~l T T~ T~ T T ~
T ~-~ T T T T T
Data for lactating women compared to not-recently pregnant women (NP) and to post-pregnant mothers who did not lactate (NL) *urinary collagen cross-links, telopeptides, hydroxyproline, serum tartrate-resistant acid phosphatase * * serum bone alkaline phosphatase and procollagen peptides nk=not known
TABLE 3
Calcium and bone metabolic changes in human lactation during the later stages or after the cessation of breast-feeding. NP
Calcium absorption Urinary calcium excretion, daily Fasting urinary calcium, creatinine corrected Serum calcium (ionised) Serum calcium (total) Tubular phosphate reabsorption Urinary phosphate excretion Serum phosphate
NL T
~l ~
nk nk
T ~ ~1
Serum 1,25 (OH)2 vitamin D Serum parathyroid hormone (intact) Serum parathyroid hormone-related protein Serum calcitonin Nephrogenous cyclic AMP Bone resorption markers* Bone formation markers (except Oc)** Osteocalcin
NP
NL
T T~ nd ~ ~ ~ ~ ~
T T nd
T T T
Data for lactating women compared to not-recently pregnant women (NP) and to post-pregnant mothers who did not lactate (NL) *urinary collagen cross-links, telopeptides, hydroxyproline, serum tartrate-resistant acid phosphatase * * serum bone alkaline phosphatase and procollagen peptides nd = not detectable, nk = not known
lactating and nonlactating m o t h e r s at the same stage p o s t p a r t u m have reported no differences [124,126]. Studies c o m p a r i n g serum calcium in lactation and prepregnancy show either an elevation or no difference [14,15]. T a k e n together, the data suggest that the first 3-6 m o n t h s of breast-feeding are, or can be, associated with increased renal conservation of calcium but not with increased calcium absorption. The period immediately following the cessation of breast-feeding and, for w o m e n who breast-feed for 6 12 m o n t h s or more, the later m o n t h s of lactation m a y be a time of recovery when the changes in calcium and bone m e t a b o l i s m are relaxed and calcium retention is increased. Some studies have reported decreases in calcium excretion in w o m e n after breast-feeding has stopped
[15,123]. Others indicate that urinary calcium o u t p u t increases during long lactation and after the cessation of breast-feeding to p r e p r e g n a n c y levels or to levels typical of w o m e n of the same age who have not recently been p r e g n a n t [14,121,128]. A study c o m p a r i n g w o m e n in the period after breast-feeding to mothers at the same stage p o s t p a r t u m but who had not breast-fed found no differences in urinary calcium excretion or serum calcium concentration [126]. Increased calcium absorption efficiency has been observed 2 or 3 m o n t h s after the cessation of breast-feeding [120] but not in all studies [14,15] and not 6 m o n t h s or longer after breast-feeding has stopped [119]. The return of ovarian function m a y complicate the findings, although the picture is not clear-cut [129]. The r e s u m p t i o n of m e n s t r u a t i o n has been
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associated with decreased urinary calcium excretion [14,123] and higher calcium absorption efficiency [120] but not in all studies [14,126]. Use of oral contraceptives containing ethinyl estradiol by lactating women has been associated with reduced urinary calcium excretion [121]. Interpretation of changes in calcium metabolism during and after human lactation may be further complicated by alterations in dietary intake of calcium and other nutrients during and after lactation, and, in many studies, by small subject numbers.
Phosphorus, Magnesium, and Zinc Lactating women have greater tubular reabsorption, reduced urinary excretion of phosphate, and elevated serum phosphate compared with women who have not recently been pregnant [123,125,128]. Compared to nonlactating mothers at the same stage postpartum, breastfeeding women have similar or raised serum phosphate concentrations [10,124,126] and increased urinary phosphate excretion [124]. Tubular phosphate reabsorption is decreased [126] or unchanged [124]. In the later stages of lactation and after the cessation of breast-feeding, serum and urinary phosphate levels normalize [123,126,128] and may be reduced compared to prepregancy, with an increase in tubular phosphate reabsorption [15]. The picture for phosphorus therefore resembles that of calcium in that lactation is marked by renal conservation with normal or raised serum concentrations, with no evidence of changes in intestinal absorption. Similarly, urinary magnesium excretion appears to be reduced [130] or unaffected by lactation [15,121]. The picture is similar for zinc in that lactating women have been reported to excrete less zinc than nonlactating mothers and women who have not recently been pregnant [121], but not in a longitudinal study comparing zinc excretion in lactation with that in preconception [32]. However, in contrast to calcium and the other minerals, zinc absorption efficiency has been shown to increase markedly in early lactation compared to preconception levels [32].
Bone Turnover Biochemical markers of bone turnover are elevated in the first months of lactation compared to those of nonlactating mothers and to controls who were not recently pregnant [123,127,131]. Indices of bone resorption (collagen cross-links, N-telopeptide, and hydroxyproline) and bone formation (osteocalcin, PICP, and bone alkaline phosphatase) have been studied. Some changes are evident after delivery even in women who do not breastfeed [124,126]. Longitudinal studies suggest that bone turnover in early lactation is similar to or greater than
that at the end of pregnancy and is higher than that of prepregnancy [14,15]. Measured osteocalcin concentration is at variance with other markers because although it is elevated in lactation compared to nonlactating controls, this represents an increase fromthe low concentrations observed in pregnancy to levels similar to those prepregnancy [14,15,36,39]. The duration of lactation influences the patterns of change of these markers, which occur longer and are more pronounced in those who breast-feed for a longer time [36,131]. There is evidence of an asynchrony in the patterns of change between resorption and formation in the postpartum period, with the peak of resorption preceding that of formation by several weeks [126,128,132]. Such a pattern would allow for the release of mineral from bone, followed by its restitution after a period of time [128]. The concentrations of bone turnover markers decline after 6-12 months, even in women who continue to breast-feed for 18 months or longer [128]. The levels also decline when lactation stops, but differences have been observed for several weeks after the cessation of breast-feeding between mothers who lactated and those at the same stage postpartum who did not [126,131].
Calciotropic Hormones Serum PTH and 1,25-dihydroxyvitamin D concentrations are not elevated in lactating women compared to preconception concentrations in the same individual or to levels in women who have not recently been pregnant, and they may be slightly depressed [14,15,43,119,128,133]. Similar or higher concentrations of 1,25-dihydroxyvitamin D have been reported in lactating women compared to nonlactating mothers [120,124,133-136]. PTH is similar or lower in lactating women than in nonlactating women [126,133]. Raised serum CT concentrations in early lactation have been reported in some studies [31,128] but not in others [14,124,137]. Elevated or normal NcAMP production indicates enhanced PTH-like activity during lactation in the face of normal or lowered PTH concentrations [123,126]. Mothers who are breast-feeding twins have elevated concentrations of PTH, 1,25-dihydroxyvitamin D, and calcitonin compared to women nursing singletons [138]. In general, however, postpartum changes in the three calciotropic hormones do not correlate with the response to lactation as indicated by bone turnover markers, bone mineral content, or breast milk calcium concentration [128,133,137]. The later stages of lactation and the period following the cessation of breast-feeding have been associated with increased serum PTH and 1,25-dihydroxyvitamin D concentrations [15,123,128], although this finding is not consistent [133,136] and no changes are seen in serum CT
10. Pregnancy and Lactation
concentration [14,124]. Increases in PTH and 1,25-dihydroxyvitamin D have been reported by 6 months of lactation, with 1,25-dihydroxyvitamin D levels exceeding those of nonlactating mothers by this stage postpartum [126]. Other studies show no difference by breast-feeding status [133]. No difference in NcAMP production has been noted between women who have recently stopped breast-feeding and either nonlactating controls or women who have not recently been pregnant [123,126]. It therefore appears that the changes in calcium and bone metabolism observed in lactation are not driven by the classical PTH-vitamin D endocrine system and that other hormonal mechanisms must be involved in regulating calcium homeorhesis. There is evidence that PTH and 1,25-dihydroxyvitamin D may continue to play a role in the homeostatic regulation of serum calcium [126], although further research is needed. There is also evidence that PTH and 1,25-dihydroxyvitamin D may play a role during the period of recovery toward the end of lactation and after breast-feeding stops or in situations in which the demand for breast milk production is particularly high. However, it is clear that, as with pregnancy, current knowledge does not support the concept that human lactation is a period of physiological hyperparathyroidism [139]. PTHrP is regarded as a prime candidate for the role of principal regulator of calcium and bone metabolism in lactation [125,140,141]. It is produced by the lactating mammary gland, possibly under the influence of prolactin, and is secreted into breast milk and released in significant amounts into the maternal bloodstream [142,143]. PTHrP has PTH-like activity in the kidney, where it stimulates renal calcium conservation, increases urinary phosphorus excretion, and elevates NcAMP production [126]. Serum PTHrP concentration is higher in lactating women than in nonlactating postpartum women [133]. The possibility that PTHrP derived from the mammary gland plays a key role in calcium metabolism during lactation is supported by a clinical case report of a woman with PTH deficiency whose requirement for supplemental calcium and 1,25-dihydroxyvitamin D abated during lactation, a circumstance that was attributed to her elevated concentration of PTHrP [144]. In addition, women with pseudohypoparathyroidism, who are resistant to the amino-terminal actions of PTH and PTHrP, reportedly do not show the expected lactational changes in calcium metabolism [19]. However, evidence regarding the importance of PTHrP in lactation is inconsistent. Subcutaneous administration of PTHrP(1-36) to nonpregnant, nonlactating women elevates serum 1,25-dihydroxyvitamin D concentration, increases urinary phosphate and calcium excretion, and decreases serum phosphate [55]. This pattern of changes does not resemble the metabolic response to
259
lactation. One study of lactating women demonstrated that higher PTHrP concentrations were associated with higher prolactin concentrations, lower estradiol concentrations, and greater bone mineral changes, with no correlation with calciotropic hormone concentrations [133,140]. In contrast, serum PTHrP concentration has been shown to decrease over time in all postpartum women and becomes virtually undetectable after approximately 6 months, even in those who continue to breast-feed [133]. In addition, an earlier study found no correlations during established lactation between PTHrP and other biochemical indices or bone mineral changes [132] but demonstrated an inverse correlation with PTH during the initiation of lactation (2 or 3 days postpartum) [132]. However, the biology of PTHrP is complex, and it exists as a family of closely related peptides, all originating from the PTHrP gene but each with its own distinct physiological functions [54]. It is likely that investigations of PTHrP in relation to lactation have been limited by the assay systems used, and more studies are needed. Lactation is associated with changes in many other hormones and factors, and these may be involved in regulating the changes in maternal calcium and bone metabolism. For example, elevated prolactin and low estradiol levels are characteristic of the early stages of lactation and both have recognized effects on calcium and bone metabolism. However, the concentrations of prolactin and estrodiol tend to normalize as lactation progresses [133], and there is little evidence of synchronization between the skeletal response and the pattern of changes in these hormones. There is evidence that breastfeeding mothers who have resumed menstruation, and therefore have normalized estrogen levels, have higher serum 1,25-dihydroxyvitamin D concentrations than those who have not [126], but this has not been observed in all studies [120]. In addition, estrogen deficiency in nonpregnant, nonlactating women of reproductive age resulting from GnRH agonist therapy causes increased calcium excretion with suppression of PTH and 1,25dihydroxyvitamin D, a pattern that does not resemble the metabolic response to lactation [17,19]. It is likely that the observed changes seen in lactation are due to the combined effects of many different factors.
Effect on t h e M a t e r n a l S k e l e t o n
Physiological Changes Human lactation is accompanied by significant decreases in maternal bone mineral content during the first 3-6 months, as shown by several prospective, longitudinal studies [66,122-124,127,145-150]. The reductions are particularly pronounced in the axial skeleton,
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where average decreases of 3-5% have been observed at the spine and hip (Fig. 5). Changes in appendicular sites are less marked or absent. Whole-body bone mineral content is generally decreased by approximately 0.5-1%. For a typical woman, this represents a mobilization of approximately 5-10g of calcium, which, when averaged over a 3-month period, equates to approximately 50-100mg/day. The corresponding data for the release of phosphorus, assuming a Ca:P ratio in adult bone of 2.3:1 g/g [3], are approximately 2-4 g overall and 20-40 mg/day averaged over 3 months. These rates of change are remarkably rapid, given that menopausal bone loss is typically 1-3% per year. The magnitude and duration of the skeletal response are greater in women who breast-feed longer [140,146] and who produce larger volumes of breast milk [151]. The effects are attenuated or do not occur in mothers who do not breast-feed [146,148]. On an individual basis, the bone changes are highly variable, with some women losing up to 10% at the spine and with others experiencing little alteration in bone mineral content, even when exclusively breast-feeding for several months [6]. The reasons for this interindividual variability are not known. Predictors that have been identified in some studies, but not all, include maternal height [151], age, and parity [152]. Lactation-associated bone loss is recovered during late lactation and after breast-feeding stops [122,145,146]. For women who conceive during lactation, increases in bone mineral are observed during the following pregnancy [60]. At most skeletal sites, women have a higher bone mineral status after breast-feeding has stopped for at least 2 or 3 months than immediately after delivery [146,149]. Exceptions occur at the femoral neck and wrist, where bone mineral status tends to be lower after
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the cessation of breast-feeding than immediately postpartum [146]. However, similar changes at these sites are observed in mothers who do not breast-feed, and there is no evidence that duration of lactation, or lactation itself, is a determinant of a mother's bone mineral status after lactation [146,149]. There has been considerable debate about whether the trigger for the recovery of bone mineral is cessation of breast-feeding or the return of ovarian function. However, the strong interrelation between length of lactation and duration of amenhorrea makes it difficult to examine their influence independently. It is possible that neither factor is directly involved but provides information about some aspect of lactation behavior, such as suckling frequency or intensity [146]. This leads to difficulties in interpreting studies of long-term bone changes because of differences in the timing of the final measurement. This has been defined variously relative to the cessation of breast-feeding, the onset of regular menstruation, or delivery, with no control of the other variables [14,63,66,145,146,150,153,154]. In a study in which all women fully breast-fed for 6 months and stopped breast-feeding soon afterwards, those with an early return of menses had smaller decreases in bone mineral content from the spine by 6 months of lactation but gained less afterward [149]. By 18 months, there was no difference between these women and those mothers with a later return of menses. This adds to the evidence that there are different patterns of bone loss and gain depending on a number of reproductive and lactationassociated factors. Osteoporosis in the Postpartum Period In osteoporosis of pregnancy, symptoms often develop during the postpartum period and are more common among women who are breast-feeding [155]. In one study, more than half the patients had onset of symptoms after delivery and approximately three-fourths breast-fed [155]. A combination of increased plasma PTHrP concentration and a low bone mineral density has been implicated in postpartum-onset osteoporosis of pregnancy [156], but case reports have not addressed whether the patients had an exaggerated response compared to healthy mothers who had breast-fed for a similar amount of time. As for pregnancy-onset osteoporosis, there is no evidence that these effects are influenced by dietary calcium intake or other environmental factors.
Weeks post-partum
FIGURE 5 Effect of lactation on change in maternal bone mineral content (BMC) in British women. Data represent the percentage change in BMC from 0.5 months postpartum (mean + SE) at 3,6, and 12 months postpartum for 38 womenwho breast-fed exclusivelyfor at least 2 months (reproduced with permissionfrom Prentice et al. [6]).
Effect of Lactation on Later Bone Health There is conflicting evidence about the possible effects of lactation on the later bone health of the mother. Retrospective studies of pre- and postmenopausal women
10. Pregnancy and Lactation
have associated lactation history and duration of breastfeeding with increased bone mineral [75,157,158], with decreased bone mineral [159-161], or with no effect [75,78,154,162,163]. No association has been observed between lactation history and risk of spinal deformity [85]. Older women who breast-fed when younger appear to be at lower risk of hip fracture than women who had children but did not breast-feed, with the protective effect increasing with duration of lactation [84,86,164]. The lack of consistent definitions and the failure to adequately control for confounding factors, such as obesity and estrogen use, make interpretation of these studies difficult [6,74]. The term lactation encompasses a range of breast-feeding behaviors that differ in the duration of exclusive and partial breast-feeding, the number of breast-feeds given per day, the time at which other foods are introduced, the extent to which they are used, and the lactational performance of the mother. There are marked social class differentials in breast-feeding incidence and in body size that may confound the associations between bone mineral measurements and lactation history [6,83,165]. In addition, few studies have investigated the possibility that pregnancy and lactation may only pose a risk for later osteoporosis in women with a low intake of calcium or with other potentially adverse diet and lifestyle characteristics. In studies that have attempted to explore such interactions, no effects of low calcium intake have been identified [166]. However, women with a low customary calcium intake in developing countries who have many children and long lactation periods are not at increased risk of low bone mineral status or osteoporotic fractures in later life compared with women in Western countries [167-169]. S e c r e t i o n of Minerals in Breast Milk Calcium is present in breast milk predominantly (6070%) in a diffusible form, either as ionized calcium or associated with small inorganic ions such as citrate and phosphate [170]. The remaining 30-40% is associated with casein and whey proteins, such as cx-lactalbumin and serum albumin. This contrasts with bovine milk, in which 60-70~ of calcium is associated with casein. This is due in part to the fact that the caseins of human milk (which are 75~ [3- and 25% K-casein) bind less calcium per molecule than the caseins of bovine milk (which are 50% c~-, 37.5% [3-, and 12.5% K-casein). Phosphorus is a constituent of phospholipids and caseins, and it is also present as ionic phosphate and orthophosphate. Magnesium is mainly found as the free ion or bound to citrate and phosphate, with a small proportion associated with lipids and membranes [170]. The compartmentation of zinc in human milk is unclear, but at least 25% occurs in the fat layer. Because some breast milk calcium, phosphorus,
261
magnesium, and zinc is associated with the fat layer, particularly in stored samples, it is essential to use whole milk for the measurement of breast milk concentrations [171] and, in the case of zinc, to take precautions to collect samples containing a representative amount of fat. The calcium concentration of breast milk remains relatively constant during the first 6-12 weeks of lactation but declines progressively thereafter [6,172,173]. There are geographical variations in breast milk calcium concentration. Average values at 2 or 3 months of lactation range from 300mg/liter in regions of the United States and Europe to 200 mg/liter in areas of Africa and Asia [6,174]. Regional differences of similar magnitude have also been observed within the same country [6]. In addition, individual women differ in their breast milk calcium concentration, and these differences are maintained throughout the lactation period [6]. Typically, there is a twofold range of calcium concentrations between women from the same community measured at the same stage of lactation; this variation is threefold when women are compared across regions. The total amount of calcium transferred from the mother to her breast-fed infant depends on both the calcium concentration and the quantity of breast milk consumed; there is no relationship between the two [6,173,175]. Since both the calcium concentration and the volume of breast milk produced differ between women, there is a large variation in the amount of calcium secreted into breast milk on a daily basis. Quantitative measurements of breast milk calcium intake in healthy, exclusively breast-fed infants from the United Kingdom and The Gambia resulted in a range of 79-450 mg/day [6]. Breast milk concentrations of phosphorus, magnesium, and zinc also decline as lactation progresses [176178]. The following are typical concentrations at 3 months of lactation: phosphorus, 150mg/liter; magnesium, 30 mg/liter; and zinc, 2 mg/liter [174]. These correspond to intakes of these minerals by a child consuming 0.8 liters of breast milk per day of 120,25, and 1.6 mg/ day, respectively. In Western women, the ratio of breast milk calcium to phosphorus is approximately 2:1 mg/mg, and this remains relatively constant throughout the lactation period [177]. Studies in Africa suggest that women from developing countries may have a lower ratio of calcium to phosphorus, which declines further during lactation [177,178]. O t h e r M o d u l a t o r s of Bone Growth in Breast Milk The breast milk of well-nourished mothers contains all the nutrients required to support infant growth for at least the first 6 months of life [179]. In addition, breast milk contains hormones and growth factors that may
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promote bone development. Examples include PTH, CT, thyroid hormones, IGF, and transforming growth factor [179]. Breast-fed infants have higher circulating concentrations of osteocalcin than formula-fed infants, which suggests that they have higher rates of bone formation [180]. Influence of M a t e r n a l Diet a n d Nutritional Status on Mineral M e t a b o l i s m a n d Breast Milk P r o d u c t i o n of t h e Lactating M o t h e r Calcium
There is no evidence that maternal calcium intake modifies the biochemical response to lactation. Lactating women have an exaggerated reduction in urinary hydroxyproline excretion and decreased calciuric response to an acute oral calcium load but similar calcemic response compared to nonpregnant, nonlactating control women [89]. However, there is no indication that this pattern differs depending on the mother's calcium intake. Randomized, controlled intervention studies, conducted in women with high and low customary calcium intakes, have shown no effects of an increased calcium supply on bone turnover markers, serum mineral concentrations, fractional calcium absorption, or renal calcium handling [120,122,126,128,147,181]. Calcium supplementation produced a similar decrease in PTH and 1,25-dihydroxyvitamin D concentrations in lactating and nonlactating American mothers [126], whereas no effect on calciotropic hormone levels was seen in Gambian women who had elevated concentrations [128]. In a small study, breast milk PTHrP
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;
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Supplementn-30 Placebon--30
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Weeks of lactation FIGURE 6 Effect of calcium supplement on the breast milk calcium concentration of Gambian women. Values represent mean + SEM at different weeks of lactation for women in the supplemented and placebo groups (n = 30 per group). There were no significant differences (reproduced with permission from Prentice et al. [122]).
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concentration was not altered by calcium supplementation, suggesting that maternal calcium intake does not influence the production of this hormone in the mammary gland [143]. The pattern of bone loss and gain that accompanies lactation is also independent of the current calcium intake of the mother [6,182]. Lactating women with high customary calcium intakes exhibit the typical changes in bone mineral, as do those who consume calcium supplements [66,147,149,150]. Randomized, placebo-controlled studies have demonstrated little effect of calcium supplementation on the magnitude of bone changes during and after lactation [122,147,148,183]. Calcium supplementation produces a small increase in bone mineral status [148,149], but this is seen in both lactating and nonlactating women [148] and is likely a bone remodeling transient effect [149,184]. Most epidemiological studies have found no correlation between lactational bone changes and maternal calcium intake [66,145,146,150,151]. Where associations have been reported [124,185], these have been in bone mineral status and not in the magnitude of the lactational response, and they probably reflect the interrelationships between bone mineral density, body size, and dietary intake [165]. Adolescent mothers may be an exception because a high calcium intake was associated with an attenuated bone response in teenage American mothers compared with those with lower intakes [186]. However, no differences have been observed between teenage and adult lactating Gambian women in their response to calcium supplements, despite their very low customary calcium intake [122,128]. Similarly, the amount of calcium transferred into breast milk does not appear to depend on the calcium intake of the breast-feeding mother. Although breast milk calcium concentrations tend to be lower in countries in which the customary diet is low in calcium, and some observational studies have reported significant associations between maternal calcium intake and breast milk concentration, recent evidence indicates that calcium intake during lactation does not influence breast milk calcium secretion [6]. In particular, no changes in breast milk calcium concentration were observed in two randomized, controlled calcium supplementation studies of lactating mothers (Figs. 6 and 7) [122,148,187], even in women with a very low calcium intake (300mg/day) [122]. It is possible that the calcium intake of the mother duringpregnancy may predetermine breast milk calcium concentration in the subsequent lactation [188,189]. This would provide a link between the epidemiological data and the results of intervention studies. This hypothesis requires formal testing, and studies are under way. However, no association has been found between breast milk calcium concentration and infant bone growth at 3 months [6].
10. Pregnancy and Lactation 40
7_63
40
A
O'}
E 30 E ._m
30
20"
20
10'
10
O
E t~
0
1.5
3 16 12 Months postpartum
18
0
0.5
3 6 Months postpartum
FIGURE 7 Effectof calcium supplements on breast milk calcium concentration. (A) Gambian women consuming 283 mg calcium/daysupplemented with either 714 mg calcium/day(n = 30) or placebo (n = 30) from 0.5 to 12 months postpartum [122]. Milk samples were obtained from all participants at each time point. (B) American women consuming 720 mg calcium/daysupplemented with either 1000mg calcium/day(S) or placebo (P) from 0.5 to 6 months postpartum [148]. Milk samples were obtained from a subset of participants as follows: 0.5 months, S = 8 and P = 9; 3 months, S = 9 and P = 11; 6 months, S = 16 and P -- 16. Data are mean + SE. Solid bars, calcium group; open bars, placebo group (reproduced with permission from Prentice [187]).
Phosphorus, Magnesium, and Zinc Compared to calcium, there have been few studies of the effect of the maternal diet on the metabolism or breast milk concentrations of phosphorus, magnesium, and zinc. However, there is little evidence that dietary intakes of these minerals influence breast milk composition [190,191]. Zinc supplementation may slightly slow the decline in breast milk zinc concentration that occurs as lactation progresses [191], but the significance of this is not known [190].
than by maternal nutritional status during lactation [10,48]. Whether this applies when the infant has limited exposure to sunlight of the correct wavelengths is not known. Breast-fed black and Asian infants born in temperate climates are at risk of vitamin D-deficiency rickets [48,193,194]. This may be related to poor vitamin D status of the mothers during pregnancy and to lower vitamin D concentrations in breast milk, in addition to poor sunshine exposure in the infant [195].
General Nutrition Vitamin D There is no evidence that maternal vitamin D requirements are greater during lactation than at any other period [48]. It is possible that maternal vitamin D deficiency could impact on the breast milk concentrations of calcium and vitamin D, and lactating women are advised to ensure their diet contains 5-101xg/day vitamin D [10,103]. Breast milk, however, contains relatively small amounts of vitamin D and its metabolites. Concentrations of 25-hydroxyvitamin D in breast milk parallel the levels in the maternal circulation but do not influence the vitamin D status of the infant except when the mother consumes high doses of supplemental vitamin D [10,48]. In addition, maternal vitamin D status in the normal range does not influence breast milk calcium concentration [192]. The current consensus is that infant vitamin D status is influenced more by the vitamin D status of the mother during pregnancy and by the infant's sunshine exposure
In the past, it was commonly believed that undernourished mothers had impaired lactational performance in both the quantity and the quality of their breast milk. This view is not supported by evidence from recent studies, and human lactation appears to be robust in the face of poor maternal nutrition, unless there is severe malnutrition [179,196,197]. The exceptions are the concentrations of some micronutrients that are altered by maternal diet and nutritional status. Notable examples are the long-chain fatty acids, water-soluble vitamins such as riboflavin and vitamin C, and some trace elements [179]. The fat-soluble vitamins A, E, and K, as well as D, are less influenced by diet because of the buffering action of body stores and carrier proteins, although highdose supplements can result in elevated breast milk concentrations, potentially to toxic levels [190]. It is well recognized that breast-fed children have different rates of growth during infancy than those fed formula milk, and that the pattern of growth depends on
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the approach adopted to infant feeding [198-200]. Different patterns of growth in childhood may have longterm effects on skeletal size and bone health in adult life [116,201]. However, there is little evidence that maternal nutrition during lactation impacts on the growth and bone development of the breast-fed child, except in severe deficiency. SUMMARY This chapter presented a review of the evidence of the mechanisms that facilitate and support the transfer of minerals between mother and child during pregnancy and lactation, and it discussed the relationships between mineral fluxes, maternal nutrition, and skeletal growth of the offspring. The evidence suggests that human pregnancy and lactation are associated with physiological adaptive changes that are independent of maternal mineral supply, within the range of normal dietary intakes. These processes appear to provide the minerals necessary for fetal growth and breast milk production without requiring an increase in maternal dietary intake or compromising maternal bone health in the long term. More research is needed to define the limitations of these processes in women with marginal mineral intakes and poor vitamin D status. References 1. Widdowson, E. M., and Dickerson, J. W. T. (1964). Chemical composition of the body. In Mineral Metabolism. An Advanced Treatise (C. L. Comar and F. Bronner, Eds.), Vol. 2. Academic Press, New York. 2. Givens, M. H. (1933). The chemical composition of the human fetus. J. Biol. Chem. 102, 7-17. 3. Prentice, A., and Bates, C. J. (1994). Adequacy of dietary mineral supply for human bone growth and mineralisation. Eur. J. Clin. Nutr. 48S, 161-177. 4. Forbes, G. B. (1976). Calcium accumulation by the human fetus. Pediatrics 57, 976-977. 5. Fomon, S. J. (1974). Infant Nutrition. Saunders, Philadelphia. 6. Prentice, A. (1999). Lactation and bone development: Implications for the calcium requirements of infants and lactating mothers. In Nutrition and Bone Development (R. C. Tsang and J.-P. Bonjour, Eds.), pp. 127-145. Vevey/Lippincott-Raven, New York. 7. Krebs, N. F., and Hambidge, K. M. (1986). Zinc requirements and zinc intakes of breast-fed infants. Am. J. Clin. Nutr. 45, 288-292. 8. Prentice, A. (1994). Maternal calcium requirements during pregnancy and lactation. Am. J. Clin. Nutr. 59S, 477-483. 9. Department of Health (1991). Dietary reference values for food energy and nutrients for the United Kingdom. Report on Health and Social Subjects, No. 41. Her Majesty's Stationery Office, London. 10. Institute of Medicine Food and Nutrition Board (1997). Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. National Academy Press, Washington, DC.
11. Heaney, R. P., and Skillman, T. G. (1971). Calcium metabolism in normal human pregnancy. J. Clin. Endocrinol. Metab. 33, 661-670. 12. Kent, G. N., Price, R. I., and Gutteridge, D. H. (1991). The efficiency of intestinal calcium absorption is increased in late pregnancy but not in established lactation. Calcif. Tissue Int. 48, 293-295. 13. Gertner, J. M., Coustan, D. R., Kliger, A. S., Mallette, L. E., and Ravin, N. (1986). Pregnancy as a state of physiologic absorptive hypercalciuria. Am. J. Med. 81, 451-456. 14. Ritchie, L. D., Fung, E. B., Halloran, B. P., Tumlund, J. R., Van Loan, M. D., Cann, C. E., and King, J. C. (1998). A longitudinal study of calcium homeostasis during human pregnancy and lactation and after resumption of menses. Am. J. Clin. Nutr. 67, 693-701. 15. Cross, N. A., Hillman, L. S., Allen, A. H., Krause, G. F., and Vieira, N. E. (1995). Calcium homeostasis and bone metabolism during pregnancy, lactation, and postweaning: A longitudinal study. Am. J. Clin. Nutr. 61, 514-523. 16. Naylor, K. E., Iqbal, P., Fraser, R. B., and Eastell, R. (2000). The effect of pregnancy on bone density and bone turnover. J. Bone Miner. Res. 15, 129-137. 17. Kovacs, C. S., and Kronenberg, H. M. (1997). Maternal-fetal calcium and bone metabolism during pregnancy, puerperium, and lactation. Endocrine Rev. 18, 832-872. 18. Hosking, D. J. (1996). Calcium homeostasis in pregnancy. Clin. Endocrinol. 45, 1-6. 19. Kovacs, C. S. (2001). Calcium and bone metabolism in pregnancy and lactation. J. Clin. Endocrinol. Metab. 86, 2344-2348. 20. Kent, G. N., Price, R. I., Gutteridge, D. H., Allen, J. R., Rosman, K. J., Smith, M., Bhagat, C. I., Wilson, S. G., and Retallack, R. W. (1993). Effect of pregnancy and lactation on maternal bone mass and calcium metabolism. Osteoporosis Int. Suppl. 1, $44-$47. 21. Howarth, A. T., Morgan, D. B., and Payne, R. B. (1977). Urinary excretion of calcium in late pregnancy and its relation to creatinine clearance. Am. J. Obstet. Gynecol. 129, 499-502. 22. Mestman, J. H. (1998). Parathyroid disorders of pregnancy. Sem. Perinatol. 22, 485-496. 23. Paterson, C. R. (1978). Calcium requirements in man: A critical review. Postgrad. Med. J. 54, 244-248. 24. Pitkin, R. M., and Gebhardt, M. P. (1977). Serum calcium concentrations in human pregnancy. Am. J. Obstet. Gynecol. 127, 775-778. 25. Wilkinson, R. (1976). Absorption and calcium, phosphorus and magnesium. In Calcium, Phosphate and Magnesium Metabolism (B. E. C. Nordin, Ed.), pp. 36-112. Churchill Livingstone, Edinburgh, UK. 26. Gillette, M. E., Insogna, K. L., Lewis, A. M., and Baran, D. T. (1982). Influence of pregnancy on immunoreactive parathyroid hormone levels. Calcif. Tissue Int. 34, 9-12. 27. Standley, C. A., Whitty, J. E., Mason, B. A., and Cotton, D. B. (1997). Serum ionized magnesium levels in normal and preeclamptic gestation. Obstet. Gynecol. 89, 24-27. 28. Handwerker, S. M., Altura, B. T., and Altura, B. M. (1996). Serum ionized magnesium and other electrolytes in the antenatal period of human pregnancy. J. Am. Coll. Nutr. 15, 36-43. 29. Bardicef, M., Bardicef, O., Sorokin, Y., Altura, B. M., Altura, B. T., Cotton, D. B., and Resnick, L. M. (1995). Extracellular and intracellular magnesium depletion in pregnancy and gestational diabetes. Am. J. Obstet. Gynecol. 172, 1009-1013. 30. Seydoux, J., Girardin, E., Paunier, L., and Beguin, F. (1992). Serum and intracellular magnesium during normal pregnancy and in patients with pre-eclampsia. Br. J. Obstet. Gynaecol. 99, 207-211.
10. Pregnancy and Lactation 31. Dahlman, T., Sjoberg, H. E., and Bucht, E. (1994). Calcium homeostasis in normal pregnancy and puerperium. Acta Obstet. Gynecol. Scand. 73, 393-398. 32. King, J. C. (2001). Effect of reproduction on the bioavailability of calcium, zinc and selenium. J. Nutr. 131, 1355S-1358S. 33. King, J. C. (2000). Determinants of maternal zinc status during pregnancy. Am. J. Clin. Nutr. 71, 1334S-1343S. 34. Purdie, D. W., Aaron, J. E., and Selby, P. (1988). Bone histology and mineral homeostasis in human pregnancy. Br. J. Obstet. Gynaecol. 95, 849-854. 35. Ogueh, O., Khastgir, G., Abbas, A., Jones, J., Nicolaides, K. H., Studd, J. W., Alaghband-Zadeh, J., and Johnson, M. R. (2000). The feto-placental unit stimulates the pregnancy-associated increase in maternal bone metabolism. Hum. Reprod. 15, 1834-1837. 36. Yamaga, A., Taga, M., Minaguchi, H., and Sato, K. (1996). Changes in bone mass as determined by ultrasound and biochemical markers of bone turnover during pregnancy and puerperium: A longitudinal study. J. Clin. Endocrinol. Metab. 81, 752-756. 37. Rodin, A., Duncan, A., Quartero, H. W. P., Pistofidis, G., Mashiter, G., Whitaker, K., Crook, D., Stevenson, J. C., Chapman, M. G., and Fogelman, L. (1989). Serum concentrations of alkaline phosphatase isoenzymes and osteocalcin in normal pregnancy. J. Clin. Endocrinol. Metab. 68, 1123-1127. 38. Haruna, M., and Fukuoka, H. (1996). Metabolic turnover of bone during pregnancy and puerperium. Bull. Phys. Fitness Res. Institute 91, 109-115. 39. Cole, D. E. C., Gundberg, C. M., Stirk, L. J., Atkinson, S. A., Hanley, D. A., Ayer, L. M., and Baldwin, L. S. (1987). Changing osteocalcin concentrations during pregnancy and lactation: Implications for maternal mineral metabolism. J. Clin. Endocrinol. Metab. 65, 290-294. 40. Black, A. J., Topping, J., Durham, B., Farquharson, R. G., and Fraser, W. D. (2000). A detailed assessment of alterations in bone turnover, calcium homeostasis, and bone density in normal pregnancy. J. Bone Miner. Res. 15, 557-563. 41. Salle, B. L., Delvin, E. E., Lapillonne, A., Bishop, N. J., and Glorieux, F. H. (2000). Perinatal metabolism of vitamin D. Am. J. Clin. Nutr. 71, 1317S-1324S. 42. Kumar, R., Cohen, W. R., Silva, P., and Epstein, F. H. (1979). Elevated 1,25-dihydroxyvitamin D plasma levels in normal human pregnancy and lactation. J. Clin. Invest. 63, 342-344. 43. Wilson, S. G., Retallack, R. W., Kent, J. C., Worth, G. K., and Gutteridge, D. H. (1990). Serum free 1,25-dihydroxyvitamin D and the free 1,25-dihydroxyvitamin D index during a longitudinal study of human pregnancy and lactation. Clin. Endocrinol. 32, 613-622. 44. Bouillon, R., Van Assche, F. A., Van Baelen, H., Heyns, W., and De Moor, P. (1981). Influence of the vitamin D-binding protein on the serum concentration of 1,25-dihydroxyvitamin D3. Significance of the free 1,25-dihydroxyvitamin D3 concentration. J. Clin. Invest. 67, 589-596. 45. Bilke, D. D., Gee, E., Halloran, B., and Haddad, J. G. (1984). Free 1,25-dihydroxyvitamin D levels in serum from normal subjects, pregnant subjects, and subjects with liver disease. J. Clin. Invest. 74, 1966-1971. 46. Hillman, L. S., Slatopolsky, E., and Haddad, J. G. (1978). Perinatal vitamin D metabolism. IV. Maternal and cord serum 24,25-dihydroxyvitamin D concentrations. J. Clin. Endocrinol. Metab. 47, 1073-1077. 47. Zerwekh, J. E., and Breslau, N. A. (1986). Human placental production of 1oL,25-dihydroxyvitamin D3: Biochemical characterization and production in normal subjects and patients with pseudohypoparathyroidism. J. Clin. Endocrinol. Metab. 62, 192-196.
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48. Specker, B. L. (1994). Do North American women need supplemental vitamin D during pregnancy or lactation? Am. J. Clin. Nutr. 59 (Suppl.), 484S-491S. 49. Gallacher, S. J., Fraser, W. D., Owens, O. J., Dryburgh, F. J., Logue, F. C., Jenkins, A., Kennedy, J., and Boyle, I. T. (1994). Changes in calciotrophic hormones and biochemical markers of bone turnover in normal human pregnancy. Eur. J. Endocrinol. 131, 369-374. 50. Seely, E. W., Brown, E. M., DeMaggio, D. M., Weldon, D. K., and Graves, S. W. (1997). A prospective study of calciotropic hormones in pregnancy and post partum: Reciprocal changes in serum intact parathyroid hormone and 1,25-dihydroxyvitamin D. Am. J. Obstet. Gynecol. 176, 214-217. 51. Davis, O. K., Hawkins, D. S., Rubin, L. P., Posillico, J. T., Brown, E. M., and Schiff, I. (1988). Serum parathyroid hormone (PTH) in pregnant women determined by an immunoradiometric assay for intact PTH. J. Clin. Endocrinol. Metab 67, 850-852. 52. Cushard, W. G., Creditor, M. A., Canterbury, J. M., and Reiss, E. (1972). Physiologic hyperthyroidism in pregnancy. J. Clin. Endocrinol. Metab. 34, 767-771. 53. Ardawi, M. S. M., Nasrat, H. A. N., and BA'Aqueel, H. S. (1997). Calcium-regulating hormones and parathyroid hormone-related peptide in normal human pregnancy and postpartum: A longitudinal study. Eur. J. Endocrinol. 137, 402-409. 54. Wysolmerski, J. J., and Stewart, A. F. (1998). The physiology of parathyroid hormone-related protein: An emerging role as a developmental factor. Annu. Rev. Physiol. 60, 431-460. 55. Henry, J. G., Mitnick, M., Dann, P. R., and Stewart, A. F. (1997). Parathyroid hormone-related protein-(1-36) is biologically active when administered subcutaneously to humans. J. Clin. Endocrinol. Metab. 82, 900-906. 56. Pitkin, R. M., Reynolds, W. A., Williams, G. A., and Hargis, G. K. (1979). Calcium metabolism in normal pregnancy: A longitudinal study. Am. J. Obstet. Gynecol. 133, 781-790. 57. Whitehead, M., Lane, G., Osyth, Y., Campbell, S., Abeyasekera, G., Hillyard, C. J., Maclntyre, I., Phang, K. G., and Stevenson, J. C. (1981). Interrelations of calcium-regulating hormones during normal pregnancy. Br. Med. J. 283, 10-12. 58. Stevenson, J. C., Hillyard, C. J., and Maclntyre, I. (1979). A physiological role for calcitonin: Protection of the maternal skeleton. Lancet 2, 769-771. 59. Drinkwater, B. L., and Chesnut, C. H. (1993). Bone density changes during pregnancy and lactation in active women: A longitudinal study. Bone Miner. 14, 153-160. 60. Laskey, M. A., and Prentice, A. (1997). Effect of pregnancy on recovery of lactational bone loss. Lancet 349, 1518-1519. 61. Sowers, M. F., Randolph, J., Shapiro, B., and Jannausch, M. (1995). A prospective study of bone density and pregnancy after an extended period of lactation with bone loss. Obstet. Gynecol. 85, 285-298. 62. Sowers, M., Crutchfield, M., Jannausch, M., Updike, S., and Corton, G. (1991). A prospective evaluation of bone mineral change in pregnancy. Obstet. Gynecol. 77, 841-845. 63. Holmberg-Marttila, D., Sievanen, H., and Tuimala, R. (1999). Changes in bone mineral density during pregnancy and postpartum: Prospective data on five women. Osteoporosis Int. 10, 41-46. 64. Aguado, F., Revilla, M., Hernandez, E. R., Menendez, M., CortezPrieto, J., Villa, L. F., and Rico, H. (1998). Ultrasonographic bone velocity in pregnancy: A longitudinal study. Am. J. Obstet. Gynecol. 178, 1016-1021. 65. Christiansen, C., Rodbro, P., and Heinild, B. (1976). Unchanged total body calcium in normal human pregnancy. Acta Obstet. Gynecol. Scand. 55, 141-142.
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10. Pregnancy and Lactation 169. Aspray, T. J., Prentice, A., Cole, T. J., Sawo, Y., Reeve, J., and Francis, R. M. (1996). Low bone mineral content is common but osteoporotic fractures are rare in elderly rural Gambian women. J. Bone Miner. Res. 11, 1018-1024. 170. Neville, M. C., Zhang, P., and Allen, J. C. (1995). Minerals, ions, and trace elements in milk. A. Ionic internations in milk. In Handbook o f Milk Composition (R. G. Jensen, Ed.), pp. 577-592. Academic Press, San Diego. 171. Laskey, M. A., Dibba, B., and Prentice, A. (1991). A semiautomated micromethod for the determination of calcium and phosphorus in human milk. Ann. Clin. Biochem. 28, 49-54. 172. Vaughan, L. A., Weber, C. W., and Kemberling, S. R. (1979). Longitudinal changes in the mineral content of human milk. Am. J. Clin. Nutr. 32, 2301-2306. 173. Laskey, M. A., Prentice, A., Shaw, J., Zachou, T., and Ceesay, S. M. (1990). Breast-milk calcium concentrations during prolonged lactation in British and rural Gambian mothers. Acta Paediatr. Scand. 79, 507-512. 174. Prentice, A. (1995). Regional variations in the composition of human milk. In Handbook o f Milk Composition (R. G. Jensen, Ed.), pp. 115-221. Academic Press, San Diego. 175. Laskey, M. A., Jarjou, L., Dibba, B., and Prentice, A. (1997). Does maternal calcium intake influence the calcium nutrition of the breast-fed baby? Proc. Nutr. Soc. 56, 1A-6A. 176. Casey, C. E., Smith, A., and Zhang, P. (1995). Minerals, ions and trace elements in milk. C. Microminerals in human and animal milks. In Handbook o f Milk Composition (R. G. Jensen, Ed.), pp. 622-674. Academic Press, San Diego. 177. Laskey, M. A., Dibba, B., and Prentice, A. (1991). Low ratios of calcium to phosphorus in the breast-milk of rural Gambian mothers. Acta Paediatr. Scand. 80, 250-251. 178. Prentice, A., and Barclay, D. V. (1991). Breast-milk calcium and phosphorus concentrations of mothers in rural Zaire. Eur. J. Clin. Nutr. 45, 611-617. 179. Prentice, A. (1997). The constituents of human milk. Food Nutr. Bull. 17, 305-315. 180. Michaelsen, K. F., Johansen, J. S., Samuelson, G., Price, P. A., Christiansen, C., and Skakkebaek, N. E. (1992). Serum bone Gla protein (BGP, osteocalcin) in infants: Values positively correlated with human milk intake. In Mechanisms Regulating Lactation and Infant Nutrient Utilization (M. F. Picciano and B. Lonnerdal, Eds.), Contemporary Issues in Clinical Nutrition, Vol. 15., pp. 419-423. Wiley-Liss, New York. 181. Fairweather-Tait, S. J., Prentice, A., Heumann, K. G., Jarjou, L. M. A., Stirling, D. M., Wharf, S. G., and Turnlund, J. R. (1995). Effect of calcium supplements and stage of lactation on the efficiency of absorption of calcium by lactating women accustomed to low calcium intakes. Am. J. Clin. Nutr. 62, 1188-1192. 182. Prentice, A. (1997). Calcium supplementation during breastfeeding. N. Engl. J. Med. 337, 558-559. 183. Kent, G. N., Price, R. I., Gutteridge, D. H., May, K. D., Allen, J. R., Smith, M., Evans, D. V., and Bhagat, C. I. (1995). Site-specific reduction in bone loss by calcium supplements in normal lactation. Osteoporosis Int. 5, 315(A). 184. Parfitt, A. M. (1980). Morphologic basis of bone mineral measurements: Transient and steady state effects of treatment in osteoporosis. Miner. Electrolyte Metab. 4, 273-287.
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185. Morales, A., Tud-Tud Hans, L., Herber, M., Taylor, A. K., and Baylink, D. J. (1995). Lactation is associated with an increase in spinal bone density. J. Bone Miner. Res. 8, S 156. 186. Chan, G. M., McMurry, M., Westover, K., Engelbert-Fenton, K., and Thomas, M. R. (1987). Effects of increased dietary calcium intake upon the calcium and bone mineral status of lactating adolescent and adult women. Am. J. Clin. Nutr. 46, 319-323. 187. Prentice, A. (1998). Calcium requirements of breast-feeding mothers. Nutr. Rev. 56, 124-127. 188. Prentice, A., Dibba, B., Jarjou, L. M. A., Laskey, M. A., and Paul, A. A. (1994). Is breast-milk calcium concentration influenced by calcium intake during pregnancy? Lancet 344, 411-412. 189. Ortega, R. M., Martinez, R. M., Quintas, M. E., LopezSobaler, A. M., and Andrews, P. (1998). Calcium levels in maternal milk: Relationships with calcium intake during the third trimester of pregnancy. Br. J. Nutr. 79, 501-507. 190. Bates, C. J., and Prentice, A. (1996). Vitamins, minerals and essential trace elements. In Drugs and Human Lactation (P. Bennett, Ed.), 2nd ed., pp. 533-607. Elsevier, Amsterdam. 191. Krebs, N. F., Reidinger, C. J., Hartley, S., Robertson, A. D., and Hambidge, K. M. (1995). Zinc supplementation during lactation: Effects on maternal status and milk zinc concentrations. Am. J. Clin. Nutr. 61, 1030-1036. 192. Prentice, A., Yan, L., Jarjou, L. M. A., Dibba, B., Laskey, M. A., and Fairweather-Tait, S. (1997). Vitamin D status does not influence the breast-milk calcium concentration of lactating mothers accustomed to a low calcium intake. Acta Paediatr. 86, 1006-1008. 193. Mughal, M. Z., Salama, H., Greenaway, T., Laing, I., and Mawer, E. B. (1999). Lesson of the week: Florid rickets associated with prolonged breast-feeding without vitamin D supplementation. Br. Med. J. 318, 1417. 194. Kreiter, S. R., Schwartz, R. P., Kirkman, H. N., Charlton, P. A., Calikoglu, A. S., and Davenport, M. L. (2000). Nutritional rickets in African American breast-fed infants. J. Pediatr. 137, 153-157. 195. Specker, B. L., Tsang, R. C., and Hollis, B. W. (1985). Effect of race and diet on human milk vitamin D and 25-hydroxyvitamin D. Am. J. Dis. Child 139, 1134-1137. 196. Prentice, A. M., Paul, A. A., Prentice, A., Black, A. E., Cole, T. J., and Whitehead, R. G. (1986). Cross-cultural differences in lactational performance. In Human Lactation 2: Maternal and Environmental Factors (M. Hamosh and A. S. Goldman, Eds.), pp. 13-44. Plenum, New York. 197. Prentice, A. M., and Prentice, A. (1995). Evolutionary and environmental influences on human lactation. Proc. Nutr. Soc. 54, 391-400. 198. Whitehead, R. G., and Paul, A. A. (1984). Growth charts and the assessment of infant feeding practices in the Western world and in developing countries. Early Hum. Dev. 9, 187-207. 199. Michaelsen, K. F., Larsen, P. S., Thomsen, B. L., and Samuelson, G. (1994). The Copenhagen Cohort Study on Infant Nutrition and Growth: Breast milk intake, human milk macronturient content and influencing factors. Am. J. Clin. Nutr. 59, 600-611. 200. Prentice, A. (1991). Breast feeding and the older infant. Acta Paediatr. Scand.Suppl. 374, 78-88. 201. Cooper, C., Eriksson, J. G., Forsen, T., and Osmond, C. (2001). Maternal height, childhood growth and risk of hip fracture in later life: A longitudinal study. Osteoporosis Int. 12, 623-629.
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Fetal Mineral H o m e o s t a s i s CHRISTOPHER S. KOVACS Faculty of Medicine--Endocrinology, Memorial University of Newfoundland, Health Sciences Centre, St. John "s, Newfoundland, Canada
remaining 5 days of gestation [2]. The fetal lamb accretes 75 g of calcium by term, but most of the maternal-fetal transfer occurs late in gestation at the rate of 3 g of calcium per day [3]. In order to attain the required amount of calcium and regulate the fetal calcium level, the fetus makes use of the placenta, kidneys, bone, and intestine. The studies reviewed herein demonstrate that the fetal-placental unit functions relatively independently of the mother, such that it is capable of mineralizing the fetal skeleton and maintaining normal blood calcium, even in the presence of significant maternal hypocalcemia and vitamin D deficiency. In addition, this chapter shows that PTH-related protein (PTHrP) is a major regulator of placental calcium transport, whereas PTHrP and PTH act in concert to regulate the blood calcium and control skeletal mineralization. Due to obvious limitations in obtaining data from human fetuses, human regulation of fetal mineral homeostasis and placental calcium transport must be largely inferred from data obtained from studies in sheep, goats, pigs, rats, and mice. It is obvious that some of the observations in other animals may not apply to humans. With respect to inferences about placental function, it must be emphasized that mice and rats have hemochorial placentas that are structurally and functionally very similar to that of humans [4-7]. In contrast, the epitheliochorial placentas of sheep, goats, and pigs differ significantly in structure from the human hemochorial placenta and may therefore be functionally different as well [6]. This chapter reviews the existing data on fetal mineral homeostasis as extrapolated from human and animal data, including older studies involving surgically manipulated fetuses and more recent studies of genetically
FETAL ADAPTIVE GOALS As discussed in other chapters, much of normal calcium and bone homeostasis in adult humans and animals can be explained by the interactions of parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25-D), calcitonin, and the sex steroids. Deficiency of PTH causes hypocalcemia, which can lead to seizures, cardiac arrhythmia, and death. Vitamin D deficiency is associated with loss of calcium and phosphate from the skeleton, leading to severely weakened bones (osteomalacia). Chronic deficiency of estrogen increases the loss of calcium from the skeleton, leading to thinner, more fragile bones (osteoporosis). Based on knowledge of the roles that these hormones play, many current therapies for osteoporosis and other metabolic bone diseases are based on these hormones (or analogs) administered in pharmacological doses. In contrast to the adult, comparatively little is known about how calcium and bone homeostasis is regulated in the fetus. It is evident that fetal calcium and bone metabolism has been uniquely adapted to meet the specific needs of this developmental period, including the requirement to provide sufficient calcium (and other minerals) to fully mineralize the skeleton and the requirement to maintain an extracellular level of calcium (and other minerals) that is physiologically appropriate for fetal tissues (i.e., for cell membrane stability, blood coagulation, etc.). A human fetus typically accumulates 21 g of calcium by term, and 80% of this calcium is accumulated in the third trimester, necessitating an average daily transfer of 200-300mg calcium [1]. Similarly, the fetal rat accretes less than 0.5 mg of calcium in the first 17 days of gestation and approximately 12 mg of calcium in the
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Copyright 2003, Elsevier Science (USA). All rights reserved.
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Christopher S. Kovacs
[19,20,166] Deletion of Hoxa3 gene which results in abnormalities in tissues deriving from the 3 rd and 4 th pharyngeal arches, including absence of parathyroids and thymus. Circulating PTH is absent. Lethal at birth.
Hoxa3-null
Gcm2-null [192] Deletion of gene Gcm2 which results in absence of parathyroids but normal thymus. Thymus has hyperplastic parathyroid tissue that produces near-normal circulating PTH levels. Not lethal. Pth-null
[190] Deletion of gene encoding PTH. Non-lethal.
[11,44] Deletion of gene encoding PTHrP, resulting in severe chondrodysplasia and other abnormalities. Lethal at birth.
Pthrp-null
[11,120] Deletion of gene encoding PTH/PTHrP receptor (also known as PTH 1 receptor), resulting in absence of biological effects of amino-terminal PTH and PTHrP. Lethal between mid-gestation and birth, depending upon the genetic background. Pthrl-null
Cast-null [42,43] Deletion of the gene encoding the calcium-sensing receptor which results in fetal hyperparathyroidism. Lethal 2-3 weeks postnatal; heterozygotes are hypercalcemic but live normal life spans.
O-null [103,106] Deletion of gene encoding calcitonin and calcitonin gene-related peptide. Non-lethal; adults are fertile and live normal life spans.
centration is ionized, in the fetus approximately 80% of calcium is ionized [10]; only a small fraction is bound to albumin. Consequently, acute changes in pH have minimal effects on the ionized calcium level, at least in fetal mice (Fig. 1) [11]. Due to technical limitations in being able to obtain adequate blood samples from fetuses of earlier gestational ages, it is not known how early in gestation the fetal blood calcium begins to exceed the maternal calcium concentration. In humans, fetal hypercalcemia was documented at 15-20 weeks of gestation (by fetoscopy) [12] and at delivery of preterm singleton and twin pregnancies (mean gestational age of 33 weeks) [13]. In sheep, fetal hypercalcemia has been detected as early as Day 35 of gestation [14,15]. In fetal rats, there is
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Vdr-null [32,33]Deletion of the gene encoding the vitamin D receptor resulting in a murine form of vitamin D dependent rickets type II. Nonlethal, but results in significant hypocalcemia, osteomalacia and reduced fertility in adults.
engineered mice that lack calcitropic hormones or receptors. Earlier models (surgical parathyroidectomy and experimental vitamin D deficiency) provided the first insights into the unique regulation of fetal mineral homeostasis, whereas the use of genetically engineered mice has enabled the study of fetal models that cannot be created by surgical or pharmacological techniques, such as fetal mice that are completely devoid of PTHrP. To avoid confusion, this chapter emphasizes the species from which the data have been obtained (human, sheep, rat, mouse, etc.). The key mouse models discussed here are listed and briefly described in Table 1. "Unpublished data" refers to unpublished data from the author's research laboratory unless otherwise specified.
MINERALS AND CALCITROPIC HORMONES
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Calcium In all mammals that have been studied (humans, rodents, sheep, cattle, horses, monkeys, and pigs), the fetal blood calcium (both total and ionized) is maintained at a higher level than in the maternal circulation [8]. This elevation is mainly due to an increase in the ionized calcium level [9,10]. In contrast to the adult, in which approximately 45% of the circulating calcium con-
FIGURE 1 Whole blood pH (A) and ionized calcium (B) as determined in normal mouse fetuses removed sequentially from the uterus over 5 min. The littermate number indicates the order in which fetuses were removed. Although the fetal pH declines quickly (A), the ionized calcium increases only modestly (B). This is consistent with previous observations (discussed in text) that more than 80% of the blood calcium is ionized in fetuses; therefore, the ionized calcium is minimally affected by the action of H + to release calcium bound to albumin (Reproduced with permission from Kovacs et al. [11]. Copyright 1996 National Academy of Sciences, USA).
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1 1. Fetal Mineral H o m e o s t a s i s
a progressive increase in total and ionized calcium during the last week of gestation, corresponding to the time of a progressive decline in fetal pH [16-18]. In the author's laboratory, the increase in ionized calcium is present in mouse fetuses on Embryonic Day 15, the earliest time point from which sufficient blood samples can be obtained for assay (unpublished data). The physiological importance of fetal hypercalcemia is not known. Maintaining the fetal calcium concentration at least at the same level as the maternal calcium concentration (but not necessarily above it) appears to be important for ensuring adequate mineralization of the fetal skeleton. Fetal survival to the end of gestation appears unaffected by hypocalcemia, as observed in studies of Pthrp-null fetuses that have a blood calcium equal to the maternal calcium concentration [11], in Hoxa3-null fetuses that have more profound hypocalcemia [19], and in Hoxa3/Pthrp double mutants that have the lowest calcium concentration [20]. Normally, there is a sharp decline in the blood calcium to below the normal adult calcium level in the first 12 hr after birth, followed by an increase to the adult calcium concentration during the succeeding day [8]. Therefore, the high blood calcium level at birth may simply serve to protect against more profound neonatal hypocalcemia and tetany occurring immediately after birth. The normal decline in blood calcium is approximately 20% in humans [21-23] and 40% in rodents [17,24]. However, the postnatal decline in blood calcium is not a consistent finding in all mammals: Neonatal lambs do not have a decrease in blood calcium but maintain a higher blood calcium concentration for weeks to months after birth before it declines to the normal adult calcium concentration [25]. The fetus has a remarkable ability to maintain a higher level of blood calcium despite chronic, severe maternal hypocalcemia [8]. This includes, as observed in rats, severe maternal hypocalcemia due to a calcium-restricted diet [26], vitamin D deficiency [27-29], or thyroparathyroidectomy [30,31]. Murine fetuses have a similar, remarkable ability to maintain normal fetal hypercalcemia despite profound hypocalcemia in their mothers. Pregnant mice that are homozygous for loss of the vitamin D receptor gene (Vdr-null) have severe hypocalcemia and are prone to tetany as adults [32], but the ionized calcium level of all the pups of these Vdr-null mothers is unchanged [33]. Acute alterations in the maternal blood calcium of rodents and primates (by calcium, 1,25-D, calcitonin, PTH, or EDTA infusions) also have minimal or no effect on the fetal blood calcium level [34-38]. A decline in the fetal blood calcium after maternal parathyroidectomy in rats has also been reported, suggesting that the fetus may not always be able to maintain normal blood calcium when the mother is hypocalcemic [39-41]. In these studies, the fetal blood calcium was
normal between Days 12 and 17 of gestation but declined during the period of rapid fetal skeletal calcium accretion. Therefore, the ability of the fetal rat to set its blood calcium may break down during the time of rapid accretion of calcium by the skeleton if the mother has been parathyroidectomized. In adults, the calcium-sensing receptor (CaSR), which directly regulates PTH secretion, normally sets the blood calcium level. Evidence from studies of murine fetuses indicates that the CaSR has a role in normal fetal calcium metabolism, but it may not be directly responsible for setting the normal high fetal blood calcium level. Using genetically engineered mice that lack the CaSR gene (Casr) [42], it is clear that the murine fetus sets its blood calcium level independently of the ambient maternal calcium level [43]. Thus, a wild-type fetus will have the same blood calcium level regardless of whether its mother is normocalcemic (wild-type) or hypercalcemic (heterozygous for Casr ablation) (Fig. 2). The role of the CaSR in fetal calcium homeostasis is discussed in more detail later. The normal high fetal blood calcium may not be determined directly by the CaSR but by other factors, such as PTHrP and PTH. As noted previously, Pthrp-null fetuses [44] have lower blood calcium than normal, equal to that of the mother [11] (Fig. 3). PTHrP is evidently required to maintain the higher blood calcium level that is normally present in the fetus; in the absence of PTHrP, the blood calcium declines to a level equal to that normally set by the CaSR and PTH in the adult. PTH is also important for maintaining the fetal blood calcium since in the absence of PTH (Hoxa3-null), the fetal blood calcium declines well below the maternal calcium concentration (Fig. 4) [19]. Since such Hoxa3-null fetuses have normal circulating PTHrP concentrations [19], it is evident that PTH plays a greater role than PTHrP in maintaining the fetal calcium concentration. In summary, mammalian fetuses have higher levels of blood calcium than their mothers from early in gestation, mainly due to an increase in the ionized calcium level. The fetus establishes a particular fetal blood calcium level irrespective and independently of the ambient maternal blood calcium level. This ability persists in the presence of significant maternal hypocalcemia of various causes but may be impaired during the time of rapid accretion of calcium by the skeleton. The physiological importance of fetal hypercalcemia and the means by which it is maintained are not known.
Phosphate Serum phosphate levels are higher than maternal levels in humans [22,23,45,46], rats [18,47,48], mice (unpublished data), sheep [26], and pigs [49,50]. As with the
274
Christopher S. Kovacs
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FIGURE 2 Ionized calcium in late-gestation fetal mice obtained from Casr +/- females (a) and wild-type females (b) that have been mated with Casr +/- male mice. The fetal blood calcium of wild-type (WT) and Casr +/- ( + / - ) fetuses is the same regardless of the mother's genotype and blood calcium level. Dashed lines indicate the respective mean maternal ionized calcium for Casr +/- (a) and wild-type females (b). The number of observations for each genotype is indicated in parentheses (Reproduced with permission from Kovacs et al. [43]. Copyright 1998 American Society of Clinical Investigation).
serum calcium, the serum phosphorus declines sharply in the first few hours after birth, at least in rats [24]. The high serum phosphate level in fetuses has been interpreted to mean that phosphate is actively transported across the placenta, but the regulators of phosphate transport are unknown [51]. Both PTHrP and PTH are required to regulate the serum phosphate level because the serum phosphate level is increased in Pthrp-null and Hoxa3-null fetuses [19] (unpublished data). PTHrP and PTH do not stimulate placental transport of phosphate in sheep [52]. 1,25-D has been suggested to play a role in placental phosphate transfer [53]; however, in Vdr-null fetuses the serum phosphate levels and skeletal mineral content are normal [33]. Magnesium Fetal magnesium metabolism has not been extensively studied. In humans, a study of maternal and cord blood in 115 near-term deliveries found that the fetal magnesium concentration is significantly increased over the maternal magnesium concentration, although the difference (0.05 mmol/liter) was more modest than that normally observed between maternal and fetal calcium concentrations (0.30mmol/liter) [23]. Most studies are consistent with this result, finding that the fetal magnesium level is equal to or at most modestly different (i.e., modestly higher or lower) from the maternal magnesium concentration [22,23,54]. Fetal sheep appear to have modestly decreased magnesium levels compared to their mothers [26,55], although some have found both the total and ionized magnesium levels to be slightly increased in fetal sheep compared to their mothers (A. D. Care, un-
published data). In rats, the magnesium level is not significantly different than the neonatal (and presumably adult) magnesium level [24]; the same is true for fetal mice (unpublished data). Studies in sheep suggest that the fetus is able to maintain plasma magnesium concentrations independently of the maternal magnesium concentration [56]. Although the absence of PTHrP in the Pthrp-null fetuses does not affect serum magnesium concentrations (unpublished data), the absence of PTH (Hoxa3-null fetuses) results in a magnesium concentration that is significantly lower than the maternal magnesium concentration [19] and a significantly reduced magnesium content of the fetal skeleton [20]. The absence of calcitonin (Ct-null fetuses) was associated with a selective, stepwise decrease in the serum magnesium concentration of Ct +land Ct-null fetuses (but no alteration in calcium, phosphate, or other calcitropic hormone levels), suggesting that calcitonin may selectively regulate the serum magnesium concentration [57]. Parathyroid H o r m o n e In fetal humans and other animals, immunoreactive PTH blood levels have been found to be undetectable or very low (i.e., <0.5 pmol/liter) with respect to the maternal PTH level near the end of gestation [22,45,58-72]. Little information is available on PTH levels earlier in gestation. One study in fetal rats found that the PTH level declined in the last several days of gestation as the serum ionized calcium increased [16], whereas two crosssectional studies in preterm humans found that the fetal PTH level was not lower than the maternal PTH level [13,70]. Thus, it is uncertain whether PTH levels are
275
1 1. FetalMineral Homeostasis A
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-i-
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i
o
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+/-
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FIGURE 4 Hoxa3-null fetuses (-/-) have a blood calcium reduced below the maternal calcium concentration (line). The number of observations for each genotype is indicated in parentheses (adapted with permission from Kovacs et al. [19]. Copyright 2001 American Society of Clinical Investigation).
0.4 0.3
Iz LLI i a
0
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ci LuE N 1.3 Z O 1.2
B
i~ m
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-0.2
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FIGURE 3 Pthrp-nullfetuses (HOM) have lower blood calcium (A) and zero maternal-fetal calcium gradient (B) compared to their siblings (WT and HET). *p < < 0.001 vs WT or HET. The number of observations is indicated in parentheses (reproduced with permission from Kovacs et al. [11]. Copyright 1996National Academy of Sciences, USA).
suppressed throughout gestation after the formation of the parathyroids or only in late gestation. Although P T H levels are low in late gestation, it is clear that the fetal parathyroids are fully capable of synthesizing PTH. Parathyroids of rats and sheep contain P T H m R N A [73,74], and in humans PTH immunoreactivity is present in fetal parathyroid glands as early as 10 weeks of gestation [75]. P T H detected in the fetal blood likely derives from fetal sources alone. Several studies have demonstrated that intact P T H does not cross the placenta of nonhuman primates, sheep, and rodents [38,76,77]; fetal mice lacking parathyroids have undetectable PTH levels despite near-normal PTH levels in their mothers [19]. Therefore, P T H probably does not cross the human placenta as well. Studies in Casr-null fetal mice and their siblings indicate that in the wild type, P T H may be suppressed
by a normal CaSR in response to the increased fetal blood calcium [43]. In the absence of P T H r P (Pthrpnull fetuses), the circulating P T H level increases [20] and serves to maintain the fetal blood calcium at the level that is normally set by the actions of CaSR to regulate PTH in adult mice [11]. However, it is clear that maintenance of a normal blood calcium level is critically dependent on the normal low P T H level, and that lack of P T H has a greater impact on fetal blood calcium levels than lack of PTHrP. As noted previously, fetal mice lacking parathyroids and P T H (Hoxa3-null fetuses) have very low blood calcium levels, despite the presence of normal circulating levels of P T H r P (Fig. 4) [19]. The relevance of P T H to placental calcium transfer and skeletal mineralization is discussed later. The fetal PTH level responds to the maternal calcium concentration and is suppressed in response to maternal hypercalcemia. As observed in Casr knockout mice, the fetal P T H levels of Casr +/- and Casr-null fetuses obtained from hypercalcemic Casr +/- mothers are lower than the PTH levels of fetuses of identical genotype that have been obtained from normocalcemic, wild-type mothers (Fig. 5) [43].
1,25-Dihydroxyvitamin
D and Vitamin
D
Circulating 1,25-D levels are generally lower than the maternal level in late gestation of humans and rodents [59,78-80] (unpublished data) and pigs [81] but have been reported to be higher than the maternal 1,25-D concentration in fetal sheep [81]. 1,25-D does not readily cross the placenta, as observed in rats [82]; consequently, circulating levels of 1,25-D in the fetus are largely if not completely derived from fetal sources. The fetal kidneys and placenta possess the 10~-hydroxylase enzyme and
276
Christopher S. Kovacs
)<0.001
I
100
100 80
80 E
60
E
v
-1I---
v
-1I--
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+/(10)
40
-/(4)
0
p
//
//
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+/(6)
I
20
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Q.
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FIGURE 5 In the Casr knockout model, ablation of the CaSR results in a stepwise increase in PTH concentrations in the fetal circulation (a). Fetuses obtained from hypercalcemic Casr +/- mothers (a) have lower PTH levels than fetuses of identical genotype obtained from normocalcemic, wild-type mothers (b). The number of observations for each genotype is indicated in parentheses (reproduced with permission from Kovacs et al. [43]. Copyright 1998 American Society of Clinical Investigation).
convert 25-hydroxyvitamin D to the active form (1,25-D) [83,84]. The contribution of the fetal kidneys must be significant since fetal nephrectomy reduced the fetal 1,25-D levels in sheep and rats [85,86]. Also, in humans, umbilical artery levels of 1,25-D are higher than umbilical venous levels, confirming the contribution of the fetal kidneys [80]. 25-Hydroxyvitamin D levels have been found to be approximately equal to maternal levels in humans [79,80]; this is not surprising since 25-hydroxyvitamin D readily crosses the placenta, as observed in rats [87]. Levels of 24,25-dihydroxyvitamin D correlate with but are typically lower than maternal levels at term in humans [13,63,78]. The low circulating levels of 1,25-D in the fetus may be a response to the high serum phosphate and suppressed PTH levels in late gestation. The 10~-hydroxylase is responsive to PTH in utero, as evidenced by study of Casr +/- and Casr-null fetuses, which have a stepwise increase in PTH levels (Fig. 5a) and 1,25-D levels (Fig. 6) [43]. However, the high 1,25-D levels attained in Casr-null fetuses remain lower than that normally observed in adult wild-type mice (unpublished data). Maternal vitamin D deficiency reduces fertility and litter size in the rat [27,88], and the absence of the vitamin D receptor in mice (Vdr-null) similarly reduces fertility and litter size [33] (unpublished data). In contrast to these effects in the mother, evidence from several animal models indicates that 1,25-D is not necessary for normal calcium and bone homeostasis in the fetus. In pregnant rats, sheep, and pigs that were hypocalcemic due to experimentally induced severe vitamin D deficiency, the fetuses maintained completely normal blood calcium and phosphate levels and had fully mineralized skeletons at
160 -
f
p<0.02
I
c:l
,-- 140 E
~, 12o X
R loo t-
LID
~
80 60
WT
+/-
+/-
(5)
(11)
(5)
FIGURE 6 In the Casr knockout model, heterozygous ( + / - ) and homozygous ( - / - ) ablation of the CaSR results in a stepwise increase in 1,25-D concentrations in the fetal circulation. The number of observations for each genotype is indicated in parentheses (reproduced with permission from Kovacs et al. [43]. Copyright 1998 American Society of Clinical Investigation).
term, as determined by total weight, ash weight, and calcium content of femurs [26-29,89]. The interpretation of each of these studies was limited by the possibility that low levels of vitamin D might have reached the fetus. Definitive evidence that 1,25-D is not needed for normal fetal calcium and bone homeostasis derived from the 10~-hydroxylase-deficient Hannover pig model [49] and the vitamin D receptor ablation model in mice [32]. In the Hannover pig model, fetuses of homozygous 1,25-D-deficient sows also maintained completely normal blood calcium and phosphate levels and fully mineralized their skeletons by birth [49]. In the vitamin
2_77
1 1. Fetal Mineral H o m e o s t a s i s
D receptor ablation model (Vdr-null), fetal mice have normal calcium, magnesium, and phosphate levels and fully mineralized skeletons as determined quantitatively by analysis (atomic absorption spectroscopy) of the ashed skeletal residue [33] (unpublished data). It is only after weaning that Vdr-null neonates develop hypocalcemia and rickets [32], indicating that the VDR is also not required in the early neonatal period before intestinal calcium absorption becomes an active process. Additional evidence that 1,25-D is not required was demonstrated when fetal nephrectomy in rats did not affect fetal blood calcium or phosphate levels when measured 48 hr later, even though fetal 1,25-D levels had declined [86]. Finally, although the expression of the vitamin D-dependent calcium-binding proteins (calbindin-D9k and calbindin-Dzsk) is critically dependent on the presence of 1,25-D in the adult, fetuses of vitamin D-deficient rats and Vdr-null fetal mice have normal levels of calbindin-D9k and calbindin-D28k in placenta, intestine, and other tissues [33,89-91] (unpublished data). Some data from humans lend support to the observation that 1,25-D is not needed for normal fetal calcium and bone metabolism. At term, the cord blood calcium and skeletal mineralization are completely normal in the offspring of vitamin D-deficient mothers [92-94]. It is only in the first or second week after birth that hypocalcemia develops; skeletal demineralization and other rachitic changes are typically not detectable until 1 or 2 months of age [8]. These observations of a minimal effect of vitamin D deficiency on fetal calcium and skeletal metabolism do not mean that 1,25-D is inactive or has no role in fetal life. In rats, the VDR appears on Day 13 of gestation in the mesenchyme that will subsequently condense to form the skeletal tissues, and by Day 17 of gestation it is expressed in proliferating and hypertrophic chondrocytes and in osteoblasts of limb buds and the vertebral column [95]. The widespread expression of VDR early in fetal skeletal development suggests an important role for its ligand in fetal bone development, but evidence for this postulated role has not been found. Further studies manipulated the 1,25-D level in fetal animals to test the role of this hormone. Infusion of antibody to 1,25-D decreased the ovine fetal blood calcium level [85]. 1,25-D given to pregnant guinea pigs in pharmacological doses increased fetal calcium and phosphate levels [96]. Bilateral nephrectomy in fetal sheep resulted in reduced ionized and total calcium and increased phosphate and PTH levels; these changes could be reversed by administration of 1,25-D to the fetus [53]. Since these changes could be attributable to uremia and not loss of the renal l~-hydroxylase enzyme, additional fetuses underwent bilateral ureteral sectioning alone. This
surgical procedure allowed urine to drain into the fetal peritoneal cavity while retaining functional kidneys in situ. In these fetuses, ureteral sectioning had no effect on fetal calcium or calcitropic hormone levels. Thus, at least in the absence of normal renal function, 1,25-D may have a substantial influence on fetal mineral ion homeostasis. In summary, evidence from several (but not all) animal models indicates that deficiency of 1,25-D and the absence of VDR do not impair fetal skeletal formation and calcification nor the ability of the fetus to maintain a normal blood calcium. Pharmacological doses of 1,25-D have been demonstrated to affect fetal calcium metabolism. Although these data suggest a limited role for physiological levels of 1,25-D in the fetus, fetal production of 1,25-D and VDR mandates a continued search for fetal roles for 1,25-D. 25-Hydroxyvitamin D readily crosses the placenta and can be 1cz-hydroxylated by the fetal kidneys. However, 1,25-D does not cross the placenta, and fetal blood levels of 1,25-D are generally low in humans and other animals that have been studied (except sheep). Calcitonin Immunoreactive calcitonin can be detected in human fetal thyroid glands from as early as Week 15 of gestation [97], and fetal calcitonin levels are maintained at higher levels than maternal [24,46,59,62,80,98-101] (unpublished data). Maternal calcitonin cannot cross the placenta, as observed in rats [102] and in fetuses in which the calcitonin gene has been ablated [57,103] (unpublished data). The increased fetal levels of calcitonin are thought to reflect increased synthesis, but the metabolism and clearance of calcitonin have not been studied in fetal animals. Several acute experimental perturbations suggest a role for calcitonin in fetal calcium homeostasis. Infusion of calcitonin antiserum to fetal rats on Day 21.5 of gestation slightly increased the fetal blood calcium 1 hr later [104], whereas fetal injection of calcitonin caused hypocalcemia and hypophosphatemia [47]. However, fetal thyroidectomy with subsequent thyroxine replacement did not affect the fetal blood calcium in sheep, indicating that fetal thyroidal C cells alone may not affect the regulation of the blood calcium level [105]. Recently, examination of the calcitonin/calcitonin gene-related peptide gene ablation model [106] suggested that calcitonin is not required for normal fetal calcium homeostasis. Fetal ionized calcium levels were normal in Ct-null fetuses obtained from heterozygous and null mothers, and the calcium content of their skeletons was normal [103]. Serum magnesium levels were reduced stepwise in heterozygous and null siblings, and the
2,78
Christopher S. Kovacs
magnesium content of null skeletons was reduced, suggesting that calcitonin might play a selective role in the regulation of fetal magnesium homeostasis [57].
not either. PTHrP(1-86) did not cross the placentas of sheep and goats [112]; however, the possibility that smaller, biologically active fragments of PTHrP might cross the placenta has not been evaluated. PTHrP is produced in many sites throughout the developing embryo and fetus, including the fetal parathyroid glands (although this is uncertain) [74,113], skeletal growth plate [114,115], trophoblast cells of the placenta [73,113,116], intraplacental yolk sac [116], amnion [117,118], chorion [118], umbilical cord [119], and many other organs. All these sites may contribute to the circulating level of PTHrP in the fetus and may thereby be relevant to fetal calcium and bone metabolism. Since venous umbilical PTHrP levels were higher than umbilical arterial levels in pigs, the placenta may be an important source of systemically circulating PTHrP in the fetus [108]. Due to local production of PTHrP by the umbilical cord [119], the level of PTHrP in cord blood might not accurately reflect the systemic level of PTHrP, but this has not been tested. However, the importance of the placenta as a source of PTHrP in the fetal circulation is further supported by the following observations from two gene ablation models. In Hoxa3-null fetuses, the absence of the fetal parathyroids did not alter circulating PTHrP(1-34) levels (Fig. 8) or the expression of PTHrP m R N A and protein in the placenta [19]. In PTH/PTHrP receptor-null fetuses (Pthrl-null) [120] that have 11-fold increased circulating PTHrP levels (Fig. 8), placental expression of PTHrP (mRNA and protein) is increased (Fig. 9) [19]. PTHrP has multiple possible roles during embryonic and fetal development [110]. Pthrp-null fetuses have abnormalities of chondrocyte differentiation [44] and
Parathyroid H o r m o n e - R e l a t e d Protein Studies of PTH bioactivity in human umbilical cord blood (as determined by an in vitro cytochemical bioassay) found high PTH-like bioactivity, whereas immunoreactive PTH was simultaneously found to be undetectable or low [58,67,107]. Subsequently, it has been recognized that human cord blood PTHrP levels are significantly higher than maternal levels at term [65,72]. When both PTH(1-84) and PTHrP(1-86) were simultaneously measured by two-site immunoradiometric assays, human cord blood PTHrP levels were 24 pmol/liter, up to 15-fold higher than the levels of PTH (0.2-0.5 pmol/liter) [64-66]. It has yet to be definitively confirmed that PTHrP accounts for the high PTH-like bioactivity in human cord blood; however, studies in fetal pigs [108] and sheep [25,74] found that the levels of PTHrP and PTH-like bioactivity were tightly correlated in late gestation and the neonatal period. PTHrP is a prohormone that is processed into separate circulating fragments, each of which may have different functional roles and receptors (Fig. 7) [109-111]. Although the structures of these fragments have been deduced from studies of tumor cell lines transfected with the PTHrP gene, it has yet to be determined which of these fragments normally circulate in fetal life. Full-length PTHrP has twice the molecular weight of PTH; since PTH cannot cross the placenta, PTHrP probably does
RR KR
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FIGURE 7 PTHrP is a prohormone (top) that is cleaved into at least three circulating fragments (bottom), including a PTH-like N-terminal fragment that is indistinguishable from PTH in its ability to activate the PTH/ PTHrP receptor, a midmolecular fragment that encompasses amino acids 38-94 and is unable to activate the PTH/PTHrP receptor, and a C-terminal fragment osteostatin (reproducedwith permission from Wu et al. [111]. Copyright 1996American Societyfor Biochemistryand Molecular Biology).
279
1 1. Fetal Mineral Homeostasis P
._1
7.0-
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6.0-
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(9)
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"1I.n 2.0
30.0 -1I-.0.. 20.0
1.0
10.0
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(6)
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i
i
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Hoxa3 mice
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-I-
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,
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FIGURE 8
Circulating concentrations of PTHrP in fetuses are unaffected by ablation of the parathyroids in Hoxa3-null fetuses ( - / - ) compared to their siblings (A), whereas the concentration of PTHrP is increased 11-fold in Pthrl-null fetuses ( - / - ) that lack the PTH/PTHrP receptor (B). The solid line indicates the plasma PTHrP concentration in Pthrp-null fetuses from a similar genetic background and likely represents the detection limit of the assay under these experimental conditions. The number of observations for each genotype is indicated in parentheses (reproduced with permission from Kovacs et al. [19]. Copyright 2001 American Society of Clinical Investigation).
FIGURE 9 Respective bright field and dark field images of placenta (Embryonic Day 18.5) from wild-type (A and B) and Pthrl-null (C and D) fetuses, probed with a 35S-labeled antisense probe, demonstrated diffusely increased expression of PTHrP mRNA in the Pthrl-null placenta and yolk sac. Arrows indicate the outer rim of the placenta; arrowheads indicate the visceral (extraplacental) yolk sac. Scale bar = 501~m (reproduced with permission from Kovacs et al. [19]. Copyright 2001 American Society of Clinical Investigation).
null fetuses have increased PTH levels [20] but remain modestly hypocalcemic, it is clear that PTH cannot fully make up for lack of PTHrP. In sheep, fetal parathyroidectomy causes hypocalcemia that can be reversed by PTH or PTHrP infusion [105,122,123]. Since PTH normally circulates at low or undetectable levels in the fetus near term, it is possible that the hypocalcemic effect of fetal parathyroidectomy is at least partly due to the loss of PTHrP produced by the parathyroids. However, since the absence of the parathyroids in Hoxa3-null fetuses caused hypocalcemia (Fig. 4) and absent PTH, but no change in circulating PTHrP levels (Fig. 8), the fetal parathyroids may not contribute much PTHrP to the circulation. Whether the fetal parathyroids produce PTHrP at all remains uncertain. The unique role of PTHrP in stimulating placental calcium transport is discussed later. In summary, PTHrP is produced by diverse fetal tissues and circulates in fetal blood at levels higher than adult levels. PTHrP appears to regulate the fetal blood calcium as well as fetal-placental calcium transport.
Other Hormones and Factors aberrant breast development [121]. PTHrP may also be an important regulator of the fetal blood calcium. PTHrP levels correlate with the fetal ionized calcium levels in pigs [108]. In genetically engineered mice, homozygous ablation of Pthrp results in a fetal blood calcium no higher than that of the mother (Fig. 3) [11], confirming that PTHrP plays an: important role in fetal blood calcium regulation. Furthermore, since Pthrp-
The sex steroids (estradiol and testosterone) play an important role in regulating skeletal mineral content and bone turnover in the adult; deficiency of either hormone leads to loss of bone mineral content and osteoporosis. The role (if any) of the sex steroids in fetal skeletal development and mineral accretion is unknown, largely because the relevant analyses have not been performed. Analysis of estrogen receptor alpha and beta knockout
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Christopher S. Kovacs
mice has demonstrated effects on skeletal metabolism of adult mice, but no analyses have been performed on calcium homeostasis or the skeletal status of these mice during the fetal stage [124-126]. At the first postnatal measurement (3 weeks of age), there is no difference in such skeletal parameters as total weight, femur length, and bone density [126]; therefore, it is likely that no difference is present in these parameters at the end of gestation. However, additional study is needed to confirm whether fetal calcium and bone metabolism is unaltered by the absence of estrogen receptors. Recently, three new family members of the tumor necrosis factor ligand and receptor signaling system have been identified that play a critical role in the regulation of bone resorption: receptor activator of NF-KB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG). The role that this system plays in fetal bone metabolism and mineral accretion is not known. In the adult mouse, deletion of RANKL results in an osteopetrotic skeleton and lack of tooth eruption but normal levels of calcium, phosphate, and alkaline phosphatase [127]. The effect of deletion of RANKL on the fetal skeleton has not been characterized; the mice have radiographic evidence of osteopetrosis by 2 days after birth but normal growth until after weaning [127]. Deletion of RANK leads to mice that also appear normal at birth, but by 3 weeks of age they show evidence of stunted growth, osteopetrosis, lack of tooth eruption, hypocalcemia, hypophosphatemia, and secondary hyperparathyroidism [128]. Deletion of OPG causes an opposite phenotype in that adult mice develop early, severe osteoporosis and arterial calcifications [129,130]; however, it has not been determined if fetal skeletal development and calcium metabolism are affected. Deletion of c-Abl protein, a non-receptor tyrosine kinase, has been demonstrated to cause an osteoporotic phenotype in adult mice characterized by reduced trabecular and cortical bone volume, dysfunctional osteoblasts, and a decreased rate of mineral apposition [131]. The fetal skeletal phenotype has not been characterized apart from noting that the null mice have a normal birth weight; runting and osteoporosis have not been noted until after weaning. The serum ionized calcium concentration in fetuses differs markedly among different commercially available strains of normal mice; similarly, the ionized calcium concentration of adults differs markedly among normal strains [11] (unpublished data). Normal peak bone density or skeletal calcium content also differ markedly among different strains of normal adult mice [132]. These observations make it clear that other (as yet unidentified) genes (or combinations of genes) must regulate aspects of calcium and bone metabolism during fetal development and adults in order to account for these differences.
PLACENTAL CALCIUM TRANSPORT The fetus and placenta must obtain sufficient calcium to mineralize the skeleton and maintain an extracellular level of calcium that is physiologically appropriate for fetal tissues (i.e., for cell membrane stability, blood coagulation, etc.). As stated previously, the bulk of placental calcium transfer occurs late in gestation, such that 80% occurs in the third trimester in humans [1], whereas 96% occurs in the last 5 days of gestation in the rat [2]. The rapidity of calcium transfer is likely similar in mice, which have a gestation period of 19 days compared to 22 days in the rat. Active transport of calcium across the placenta is necessary in order for the fetal calcium requirement to be met [133,134]. The mechanisms by which active calcium exchange occurs across the placenta are not well understood. The site of active transport is likely the fetus-facing basement membrane of the syncytiotrophoblast cells in the human and the trophoblast cells and the basal surface of the endoderm of the intraplacental yolk sac in rodents [116,135,136]. Analogous to calcium transfer across the intestinal mucosa, it has been theorized that calcium diffuses into calcium-transporting cells through maternal-facing basement membranes, is carried across these cells by calcium-binding proteins, and is actively extruded at the fetal-facing basement membranes by Ca2+-ATPase [133]. Not all of the calcium is transported through active (ATP-dependent) means; some calcium may pass from maternal to fetal circulations by vesicular transport, paracellular transport, and simple diffusion. Replacement of the placenta of sheep by a semipermeable membrane results in a complete reversal of the calcium gradient between fetus and dam [9]. Attempts to quantitate the active, transcellular transfer of calcium as a percentage of the total forward (maternal ---, fetal) flow have resulted in estimates of one-third of the total flow in human placentas studied in vitro [137] and twothirds of the total flow in sheep placentas studied in vitro (A. D. Care, unpublished data). Calbindin-D9k is involved in the transfer of calcium across the kidney and intestine [138] and is thought to play a similar role in calcium transfer across the placenta. It is expressed in the rodent placenta as early as Day 10 of gestation [139]. The placental expression (mRNA and protein) of calbindin-D9k increases up to 135-fold during the last 7 days of gestation in the rat [140-142] and mice [143]. The expression of the CaZ+-ATPase increases 2fold during the same interval [140,144]. These observations are consistent with the theory that calbindin-D9k and CaZ+-ATPase are required for maternal-fetal calcium transfer in late gestation [116,133,136]. The activity of CaZ+-ATPase can be inhibited by dinitrophenol,
11. Fetal Mineral Homeostasis
ouabain, quercetin, and antibody to the human erythrocyte plasma membrane calcium pump [133,136]. Transplacental transport of calcium is generally considered to be a one-way processmthat is,, fetal-to-maternal flow of calcium is typically less than 1% of the forward (maternalto-fetal) flow [145,146]. However, backflux of calcium is technically difficult to measure accurately, and there is evidence that backflux may actually be more significant than previously considered. In rhesus monkeys, backflux was reported to be 80% of the forward flow [146]. In fetal mice lacking the PTH/PTHRP receptor (Pthrl-null) [120], placental calcium transfer is increased to 150% of the wild-type value [11]. Since the blood calcium, amniotic calcium level, and skeletal calcium content are all low in Pthrl-null fetuses [19], backflux of calcium must be increased in order to explain why the increased forward flow is not accompanied by increased calcium content in blood, amniotic fluid, and skeleton. Some of the discrepancies in the published data about backflux may represent true species differences or methodological differences. When active transport of calcium begins in gestation has not been determined due to technical difficulties involved in studying placental physiology early in gestation. However, active transport of calcium must be under way by the third trimester in humans and the last 5 days of gestation in rats, which is the time of rapid skeletal mineralization and peak fetal calcium requirement. Studies in fetal lambs have shown that a transplacental gradient in calcium is present from Day 90 of gestation [55]. The regulation of this placental calcium transfer is only partly understood, but it likely involves both maternal and fetal influences. M a t e r n a l Regulation Maternal hormones might influence fetal-placental calcium transport by increasing or decreasing the ambient maternal calcium level and by direct effects on the placenta. However, the published literature indicates that (at least in animal models) a normal rate of maternalto-fetal calcium transfer can usually be maintained despite the presence of maternal hypocalcemia or hormone deficiencies. Whether the same is true for human pregnancies is less certain. In pregnant sheep, maternal hypocalcemia due to parathyroidectomy or dietary calcium restriction did not affect the rate of fetal-placental calcium transfer as directly assessed in placental perfusion experiments [122,147]. Similarly, in fetal mice lacking the vitamin D receptor (Vdr-null), placental calcium transfer was normal even though the Vdr-null mothers were severely hypocalcemic [33] (unpublished data). However, a "normal" rate of maternal-fetal calcium transfer does not necessarily imply that the fetus is unaffected by the
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maternal hypocalcemia. The fetal-placental unit must be working harder to extract the normal amount of calcium required from maternal blood that has a lower calcium concentration than normal. In support of this hypothesis, it has been observed that a maternal calcium infusion caused a marked, acute increase in the blood calcium of fetuses from parathyroidectomized rats but had no effect on the fetuses of normal rats [148]. Such an upregulation in placental calcium transfer is necessary to compensate for the low ambient maternal blood calcium. Furthermore, in some instances of severe maternal hypocalcemia in humans, it is evident that the fetal response to this situation can have adverse effects on fetal calcium homeostasis and skeletal mineralization. In addition to the lack of effect of maternal parathyroidectomy, vitamin D deficiency, and ablation of the vitamin D receptor on fetal-placental calcium transfer, it has been noted that maternal lack of calcitonin does not impair maternal-fetal calcium transfer. Fetal mice lacking the calcitonin/calcitonin gene-related peptide gene (Ct-null) survive to adulthood and are fertile [106], but placental calcium transfer and fetal mineral accretion are not affected by the mother's genotype [57,103] (unpublished data). Several additional studies have examined only indirectly the effect of other maternal hormone deficiencies on placental calcium transfer. In these studies, net fetal accumulation of calcium at term was used as an index of placental calcium transfer during pregnancy in vitamin D-deficient rats [89]; thyroidectomized, thyroxinesupplemented (calcitonin-deficient) sheep [149,150]; and sheep that received daily administration of prolactin and/ or bromocriptine [151]. Since placental calcium transport was not directly assessed in these studies, conclusions cannot be drawn about the effect of maternal hyperprolactinemia and prolactin deficiency on placental calcium transport. The inferences from these studies about the effect of maternal vitamin D deficiency and calcitonin deficiency have since been confirmed by studies on Vdrnull and Ct-null mice, as described previously. Fetal Regulation Different techniques have been used to study placental calcium transfer in mammals. It is important to distinguish the techniques used because each has different limitations. The main techniques include placental perfusion in fetal lambs [122] and rats [152] and placental transfer in intact fetal mice [11]. In the placental perfusion technique used in lambs (Fig. 10), the fetus is removed and the placenta is perfused in situ with fetal blood or a blood substitute via the umbilical vessels in a semiclosed system in which the flow rate and perfusion pressure are kept constant. After 2 or 3 hr of baseline perfusion, test
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Christopher S. Kovacs
FIGLIRE 10 Placental perfusion studies in fetal lambs and rats. The fetus is removed from the uterus, and the umbilical vessels are connected to a semiclosed circuit to enable perfusion of the placenta in situ. Test peptides are administered on the arterial side of the circuit, and changes in maternal-fetal calcium transfer are detected by measurements on the venous side of the circuit. The reservoir contains autologous fetal blood or a blood substitute.
peptides are injected into the arterial circuit (placental inflow), and the calcium concentration in the venous circuit (placental effluent) is continuously monitored. In fetal rats, a similar artificial perfusion of the placenta in situ is done after removal of the fetus, and the clearance of 45Ca relative to 51Cr-EDTA is used as a measure of the rate of placental calcium transfer [153]. In the placental transfer experiments used in intact fetal mice, an intracardiac injection o f 45Ca and 51Cr-EDTA is given to the pregnant mother (Fig. 11A) [11]. 45Ca is actively transferred across the placenta, whereas 51Cr-EDTA crosses the placenta by passive diffusion only; 51CrEDTA serves to control for blood flow differences among placentas of a litter. The amount of 45Ca radioactivity present in each fetus of the litter (corrected for the rate of diffusion) is determined after 5 min as a measure of the rate of placental calcium transfer. To test the effect of hormones and drugs to regulate the rate of placental calcium transfer in fetal mice, intraabdominal injections of test peptides or diluent are administered to the intact fetuses in utero prior to performing the placental perfusion experiments (Fig. 11B). In addition to these main techniques used in lambs, rats, and mice, others have injected 45Ca into the maternal circulation of pregnant ewes and have used the amount of 45Ca activity present in a fetus after several days as an index of the rate of placental calcium transfer [149,154]. However, since 45Ca may cycle among several compartments within the fetus (skeleton, extracellular fluid, blood, urine, and amniotic fluid) and return to the maternal circulation, the amount of aSca present in a
FIGURE 1 1 Placental calcium transfer studies in fetal mice. The pregnant mother receives an intracardiac injection of isotopes [45Caand 51Cr-EDTA] (A). After 5 min, the fetuses are removed from the uterus, and the fetal radioactivity is individually measured. To test the effect of hormones and drugs to regulate the rate of placental calcium transfer, a laparotomy is performed and selected fetuses are given an intraabdominal injection of the test substance or diluent (B). Following a predetermined interval of 60 min or longer, the placental calcium transfer experiment proceeds with the intracardiac injection of isotope administered to the mother as in A.
fetus after several days is, at best, an indirect measure of the rate of placental calcium transfer. Fetal PTH, 1,25-D, calcitonin, and PTHrP have been studied to varying degrees regarding their potential roles
1 1. Fetal Mineral H o m e o s t a s i s
in regulating placental calcium transfer. A possible role for 1,25-D in fetal-placental calcium transport has been implied by the observation that VDRs are present in the placentas of humans, rats, mice, and sheep [116,155158]. Despite the presence of VDR in placenta, a crucial role for 1,25-D appears doubtful. The evidence in favor of a role for 1,25-D is the observation that prior nephrectomy of fetal sheep reduced calcium transfer in the placental perfusion model, and this effect could be partly restored by administering 1,25-D [133]. Also, pharmacological doses of 1,25-D or l~-cholecalciferol increased calcium transfer in placental perfusion models in rats, guinea pigs, and sheep [96,159,160]; these findings do not necessarily mean that 1,25-D is important at the physiological low levels that are normally present in the fetus. The evidence against a role for 1,25-D in regulating placental calcium transfer derives from Vdr-null fetal mice, which have a normal rate of placental calcium transfer and no alteration in calcitropic hormone levels that have been measured [33] (unpublished data). Since the absence of VDR does not impair placental calcium transfer, it is likely that 1,25-D is not required for the normal regulation of placental calcium transfer. Furthermore, Vdr-null placentas have normal expression of calbindin-D9k [33] (unpublished data), as do placentas obtained from rat models of vitamin D deficiency [89,91]. Therefore, although the expression of calbindin-D9k in the adult intestine is critically dependent on sufficiency of vitamin D and 1,25-D, expression does not require 1,25-D during fetal development. A role for fetal calcitonin in regulating placental calcium transfer is also doubtful. The only evidence in favor of a role for calcitonin came from studies in intact fetal sheep, in which administration of pharmacological doses of calcitonin was found to reduce the PTHrPmediated increases in the apparent rate of calcium transfer [149,154]. However, in these studies placental calcium transfer was not directly measured; the assay was the skeletal ash weight and mineral content several days after the initiation of treatment with calcitonin. In contrast, fetal thyroidectomy with subsequent thyroxine replacement (a surgical model of "calcitonin deficiency") did not alter placental calcium transfer in sheep, indicating that loss of the physiological amount of fetal calcitonin did not perturb the placental calcium pump [105]. Recent studies in Ct-null fetal mice indicate conclusively that the complete absence of calcitonin and calcitonin gene-related peptide does not impair placental calcium transfer or net calcium accretion by the skeleton [57,103] (unpublished data). The parathyroid glands have been extensively studied, although only recently has it been recognized that the actions of the parathyroids in fetal life may not be through PTH alone. The evidence regarding PTH and PTHrP is
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discussed next; the conflicting evidence regarding the parathyroid glands is also discussed here and later. The contribution of the parathyroids was studied in fetal lambs by performing a fetal thyroparathyroidectomy and administering thyroid hormone; 7-10 days after the surgical procedure, the fetuses were removed from the uterus, and the placentas were artificially perfused in situ as described previously [105,122]. In fetal rats, decapitation was performed to approximate a thyroparathyroidectomy, and 2 days later these fetuses were subsequently removed to allow perfusion of the placenta in situ [153]. Both thyroparathyroidectomy in fetal lambs and decapitation in fetal rats resulted in lower fetal blood calcium. When these surgically altered fetuses were removed so that the placentas could be artificially perfused in situ, active transport of calcium across these experimentally perfused placentas was found to be reduced [105,122,153]. These findings suggest that the parathyroid glands play a critical role in maintaining the fetal blood calcium and the active transport of calcium across the placenta. Infusion of autologous blood from fetuses with intact parathyroid glands restored calcium transport across the perfused placentas of thyroparathyroidectomized fetal lambs [105]. However, PTH failed to restore the active transport of calcium in fetal lambs under these conditions, suggesting that the parathyroids might produce a factor other than PTH to regulate placental calcium transfer [ 123,161]. The initial evidence that the factor might be PTHrP also came from the same studies of thyroparathyroidectomized fetal lambs. In these studies, synthetic PTHrP molecules of amino acid lengths 1-141,1-86, and 67-86 were found to stimulate placental calcium transport in the experimentally perfused placentas [123,161-163]. These results suggested that PTHrP, perhaps produced by the parathyroid glands, stimulates active transport of calcium across the placenta. In contrast, PTHrP(1-34), which contains only the PTH-like amino terminal, failed (like PTH) to stimulate calcium transport in this model. Studies in genetically engineered mice support the hypothesis that PTHrP stimulates placental calcium transport. A reduction in blood calcium to the maternal level (Fig. 3) and reduced placental transfer of calcium (Fig. 12) have been found in Pthrp-null fetal mice [11]. The placental transfer of calcium was acutely increased in the homozygous fetuses by treatment with PTHrP (1-86) (Fig. 13A) or PTHrP(67-86) but not by PTHrP (1-34) (Fig. 13B) or intact PTH [11]. The studies in fetal lambs and Pthrp-null fetuses both demonstrated that midmolecular forms of PTHrP (amino acids 67-86) stimulated placental calcium transfer, and that amino-terminal forms of PTHrP and PTH did not. Recently, additional studies in fetal lambs demonstrated that PTHrP(38-94), considered to be the true
284
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midmolecular form of PTHrP, also stimulated placental calcium transfer (Fig. 14) [111]. These findings indicate that fetal PTHrP regulates placental calcium transfer through a receptor (as yet uncloned) that is distinct
from the known PTH/PTHrP receptor (also known as the PTH1 receptor). The reason for this conclusion is 2fold. First, the PTH/PTHrP receptor is activated equally by amino-terminal fragments of PTH and PTHrP, but both peptides failed to stimulate placental calcium transfer in Pthrp-null fetuses and thyroparathyroidectomized fetal lambs. Second, PTHrP(67-86) and-(38-94) (the midmolecules of PTHrP) stimulated placental calcium transfer, but these fragments do not bind to or activate the known PTH/PTHrP receptor. This hypothesis was further supported by studies in Pthrl-null fetuses that lack the PTH/PTHrP receptor. These fetuses are also hypocalcemic, but placental calcium transfer is increased to 150% of the wild-type sibling value, perhaps due to upregulation of the PTHrP midmolecule and its effect (Fig. 15) [11]. Although a radioimmunoassay for midmolecular PTHrP was not available to confirm this, an immunoradiometric assay confirmed that the PTHrP(1-34) level was elevated 11-fold in Pthrl-null fetuses compared to their normal littermates (Fig. 8) [19]. Since the amino-terminal form of PTHrP is upregulated in Pthrl-null fetuses, the midmolecular forms may be as well. Midmolecular PTHrP would be able to stimulate placental calcium transfer in Pthrl-null fetuses since only the amino-terminal receptor is absent. These data on the effects of PTHrP, and lack of effect of PTH, in parathyroidectomized fetal lambs and Pthrpnull fetal mice are not supported by the following observations in fetal rats. As previously stated, fetal decapitation resulted in a decline in placental calcium transfer when the placentas were artificially perfused in situ, and in such decapitated fetuses placental calcium transfer was stimulated slightly (but not to normal) by PTH [153]. PTH was without effect in placentas obtained from intact fetuses. The finding that PTH had an effect in decapitated fetuses may reflect a true species difference or methodological differences (the cruder method and consequences of decapitation in rats compared to thyroparathyroidectomy in lambs or deletion of the PTHrP gene in mice). In additional experiments, PTHrP(1-34) and -(67-86) both failed to stimulate placental calcium transfer in perfused placentas obtained from intact fetal rats [164]. This latter result is consistent with the finding that wild-type fetal mice failed to stimulate placental calcium transfer in response to PTHrP(1-86) and PTHrP(67-86) injections, whereas Pthrp-null fetuses did respond (Fig. 13A) [11]. It may be that placental calcium transfer is maximally stimulated by physiological levels of PTHrP in normal fetal rats and mice, and that the circulating level of PTHrP must be reduced in order to demonstrate an effect from the pharmacological administration of PTHrP. The experimental findings in surgically altered lambs and in Pthrp-null fetuses are consistent in demonstrating
11. Fetal Mineral Homeostasis A: PRHrP 1-86 Injections
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a role for PTHrP in placental calcium transfer. The studies in lambs further indicated that the fetal parathyroids are an important regulator of placental calcium transfer, and they suggested that the fetal parathyroids might produce PTHrP. No measurements of PTHrP levels were made in these fetal lambs, and thus a reduction in the circulating PTHrP level as a consequence of parathyroidectomy has not been confirmed. The studies
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FIGURE 15 Placental calcium transfer is upregulated in Pthrl-null fetuses (HOM) compared to the littermates. *p < 0.005 vs HET. The number of observations is indicated in parentheses (reproduced with permission from Kovacs et al. [11]. Copyright 1996 National Academy of Sciences, USA).
on the Pthrp-null fetuses do not address the relative importance of parathyroid-derived PTHrP since PTHrP is absent from all tissues of Pthrp-null fetuses. It may well be that PTHrP derived from the placenta (or other fetal tissues) regulates placental calcium transfer. Recently, the question of the source of PTHrP has been readdressed by the study of genetic ablation models in fetal mice. Hoxa3-null fetuses lack parathyroid glands and PTH, among other abnormalities in tissues deriving from the third pharyngeal arch (athymia, thyroid hypoplasia, etc.) [165,166]. Such aparathyroid
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Christopher S. Kovacs
fetuses are hypocalcemic (Fig. 4) but have a normal rate of placental calcium transfer compared to that of their wild-type and heterozygous siblings (Fig. 16) [19]. Furthermore, the plasma PTHrP level is unaltered between wild-type and Hoxa3-null fetuses (Fig. 8A) [19]. Thus, both fetal lambs and Hoxa3-null fetuses have hypocalcemia as a consequence of the loss of parathyroids; in fetal lambs placental calcium transfer is reduced, whereas in Hoxa3-null fetuses placental calcium transfer is normal. The discrepancy between the results obtained in fetal lambs versus Hoxa3-null fetuses might be due to the nature of the models that were studied. The sheep models examined the effects of a surgical thyroparathyroidectomy in the absence of the fetus and fetal regulation, whereas the mouse model examined the effects of the absence of parathyroid glands throughout fetal development. In the former model, acute consequences of the surgical procedure and the absence of the fetus might have influenced the results, whereas in the latter model compensatory changes in response to the complete absence of parathyroids may have mitigated any changes in placental calcium transfer rates that would otherwise have been seen. To further resolve the role of the parathyroids in controlling placental calcium transfer, it is necessary to study additional models of selectively disrupted parathyroid function. The question of the parathyroids as an important source of PTHrP has also been addressed through the examination of the Pthrl-null fetus, which, as stated previously, has an increased rate of placental calcium transfer (Fig. 15) and an 11-fold increase in the circulating PTHrP concentration (Fig. 8B) [11,19]. Analysis of total RNA by Northern blot determined that PTHrP mRNA was upregulated in placenta and liver but not in the neck region that contains the parathyroids [19]. This result suggests that it is doubtful that the fetal parathyroids contribute 150.0 o t~
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significantly to the circulating PTHrP level, at least in mice. Molecular M e c h a n i s m s Although it has been established that calcium is actively transported across the placenta [133,134], little is known about how or in which cells the transport occurs. As stated previously, the site of active transport is likely in the trophoblast cells of human and other primate placentas and in the trophoblasts and the intraplacental yolk sac in rodents [116,135,136]. These cells are in closest proximity to the maternal circulation. Calbindin-D9k and Ca2+-ATPase (the putative "calcium pump") are required for maternal-fetal calcium transfer in late gestation [116,133,136]. Examination of the localization of calcitropic genes and proteins within the placentas of humans and other mammals provides some insight into the likely sites at which calcium might be transferred across the placenta. It should be noted that rodent placentas contain a unique structure called the intraplacental yolk sac that, as the name implies, consists of part of the primitive yolk sac that later becomes incorporated into the placenta [167]. The primitive yolk sac participates in nutrient exchange between the fetal and maternal circulations prior to the formation of the placenta [167,168]. Like the yolk sac from which it derives, the intraplacental yolk sac is a bilayered membrane, consisting of tall columnar cells on the visceral or endothelial side overlying fetal vessels and smaller parietal or cuboidal cells on the epithelial side that overlie a thick basement membrane (Reichert's membrane) and the maternal blood spaces. These two layers of the intraplacental yolk sac are separated by a potential space (sinus of D(ival) that communicates with the yolk sac cavity and, thereby, the uterine lumen. Given its anatomical position between fetal vessels and maternal blood spaces at the fetal pole of the placenta, it is well situated for exchange of substances between mother and fetus. The intraplacental yolk sac is found exclusively in rodent placentas (rat, mouse, gerbil, hamster, etc.) [168-170]; a corresponding structure has not been described in human or other primate placentas. The localization of calbindin-D9k differs significantly among species. Although highly expressed in trophoblasts of human placentas, in placentas of mice and rats it is not the trophoblasts but the intraplacental yolk sac cells that express the highest levels (mRNA and protein) of calbindin-D9K [116,141-143,171]. This localization of calbindin-D9k is specific to rodent placentas; in epitheliochorial placentas of sheep, cows, and goats, calbindin-D9k is mainly concentrated not in the placentome but in interplacentomal trophoblast cells (i.e., the flat intercotyledonary trophoblasts) [172-174].
1 1. Fetal Mineral H o m e o s t a s i s
Calbindin-D9k mRNA levels increase progressively during pregnancy in the interplacentomal epithelium but show no change during pregnancy in the placentome, which has the greater surface area for maternal-fetal exchange [173]. Similarly, although trophoblasts contain abundant CaZ+-ATPase in humans, in mice and rats the intraplacental yolk sac contains the most intense expression of this enzyme (mRNA and protein) [116,133,136]. In the epitheliochorial placentas of sheep, the placentome and the interplacentomal trophoblast epithelium have relatively equal expression of CaZ+-ATPase [173]. In human placenta, several studies have determined that PTHrP mRNA and peptide are widely expressed in syncytiotrophoblasts, cytotrophoblasts, amnion, decidua, and myometrium; the highest levels of PTHrP may be in the amnion [117,175,176]. PTHrP is expressed in giant trophoblasts of rat placenta between Embryonic Days (EDs) 7.5 and 13.5 [73,177]; in mouse placenta, PTHrP is expressed most intensely in columnar cells of the intraplacental yolk sac as well as in giant trophoblasts and spongiotrophoblasts [116]. In human placenta, low to undetectable levels of PTH/ PTHrP receptor mRNA were found in amnion, whereas abundant receptor mRNA was found in chorion decidua [176]. Using reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry, the human receptor was found to be present in smooth muscle cells and endothelium of the chorionic plate vessels, the syncytiotrophoblasts and the capillary endothelium of the chorionic villi, and myometrium [178]. In mice, the PTH/ PTHrP receptor is expressed in the preimplantation embryo [179] and later in the parietal and visceral extraembryonic endoderm of the postimplantation embryo from ED 7.5 through ED 10.5 [180]. The expression of PTH/PTHrP receptor (mRNA and protein) continues intensely in that part of the parietal and visceral extraembryonic endoderm that becomes the intraplacental yolk sac (especially the parietal cells thereof); the receptor was not detected in murine trophoblasts [116]. CaSR mRNA has been reported (by Northern blot analysis and RT-PCR) to be present in cytotrophoblasts of human term placenta [181]. In the mouse, using Casrnull placentas as controls, it is evident that CaSR mRNA and protein are expressed in trophoblasts and intraplacental yolk sac, with the most intense CaSR mRNA expression observed in the parietal cells of the intraplacental yolk sac [43,116]. The intraplacental localization of VDR in human placenta has not been described, although VDR has been isolated and purified from human placental membranes [157]. VDR had been localized in rat placenta and yolk sac by autoradiographic localization of radiolabeled 1,25-D [158]. Also, immunohistochemical study
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of rat placenta established a low prevalence of VDRimmunopositive cells in columnar cells of the intraplacental yolk sac, with no obvious staining in the remaining cells of the placenta [155]. Recently, using Vdr-null placentas as negative controls, expression of VDR in mouse placenta was confirmed to be confined to the intraplacental yolk sac and most intensely in columnar cells of this structure [116]. Evidence that human or rodent placenta express calcitonin was inconclusive, partly due to problems with antibody specificity [182,183]. Recently, Ct-null placentas have been used as negative controls to confirm that trophoblasts and intraplacental yolk sac diffusely express calcitonin (mRNA and protein) [116]. The presence of calcitonin receptor in human placentas was deduced by radiolabeled calcitonin binding studies [184] and has been localized to both fetus-facing and maternal-facing plasma membranes of syncytiotrophoblasts [185]. In mice, calcitonin receptor is expressed by trophoblasts and intraplacental yolk sac, but it is most intensely expressed in the extraplacental or visceral yolk sac [116]. Recent studies in murine placentas have determined that many calcitropic hormones and receptors (calbindin-D9k, CaZ+-ATPase, PTHrP, PTH/PTHrP receptor, CaSR, and VDR) are highly expressed in the intraplacental yolk sac compared to trophoblasts [116]. Therefore, this structure has calcium-sensing capability and expresses many of the factors that would be expected to regulate maternal-fetal calcium transfer. The intraplacental yolk sac is situated between maternal and fetal vessels within rodent placenta and communicates directly with the uterine lumen. This structure may enable the calcium demands of short-gestation, large-litter rodents to be met by providing an alternative route of calcium transfer in addition to that across the trophoblasts. Such a route may involve calcium secretion by uterine epithelial cells (analogous to the process of eggshell calcification in egg-laying mammals) into the uterine lumen and then to the intraplacental yolk sac and also direct exchange of calcium between maternal and fetal circulations across the sinus of Dfival. Due to the small size of the intraplacental yolk sac, it has not been technically possible to cannulate the structure and directly measure the movement of calcium and other solutes across it. As indirect evidence that the intraplacental yolk sac is required for maternal-fetal calcium exchange, the reduced placental calcium transfer of Pthrp-null fetuses is accompanied by a reduction in the amount of intraplacental yolk sac [116]. Also in Pthrp-null fetuses, the expression (mRNA and protein) of calbindin-D9K is significantly reduced in the intraplacental yolk sac but not in the trophoblasts [116]. There is no abnormality in intraplacental yolk sac structure and calcitropic gene or
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protein expression in Pthrl-null placentas that have an increased rate of placental calcium transfer [116]. These observations are compatible with the hypothesis that the intraplacental yolk sac plays a key role in placental calcium transfer of rodents. Fetuses lacking the gene encoding platelet-derived growth factor receptor alpha (Pdgfra-null) have no intraplacental yolk sac and all die conspicuously between midgestation and birth [186]; whether there is any abnormality in placental calcium transfer or skeletal mineralization of such fetuses has not been determined. In contrast to rodents, the epitheliochorial placentas of ruminants (sheep, goats, cows, and pigs) have calbindin-D9K expression most concentrated in the interplacentomal region, a structure that makes up approximately 2% of the volume of the mature placenta. It has been postulated that this structure may be an important site of calcium transfer between mother and fetus in ruminants [187]. Since lambs and rodents transfer proportionately more calcium but in a much shorter time frame than humans, these structures may have evolved to help meet the peak fetal calcium demands. In summary, human trophoblasts express various calcitropic factors (including calcium-binding proteins, Ca2+-ATPase, PTHrP, and CaSR) and maternal-fetal calcium exchange likely occurs across fetal-facing basement membranes of trophoblasts. In rodents, the highest expression of these same calcitropic factors is in the intraplacental yolk sac; this structure may play a key role in maternal-fetal calcium exchange of rodents. The interplacentomal cells may have a similar function in epitheliochorial placentas of ruminants. These findings emphasize that conclusions drawn from studies of placental function in rodents and ruminants are not necessarily applicable to human fetuses. PLACENTAL TRANSPORT OF MAGNESIUM AND PHOSPHATE Although less well studied, it is evident that magnesium and phosphate are also actively transported across the placenta against a concentration gradient [9,163]. Midmolecular fragments of PTHrP stimulated magnesium transport across in situ perfused placentas of fetal lambs [161,163]. In contrast, N-terminal and midmolecular forms of PTHrP failed to acutely affect the maternofetal clearance of magnesium in the perfused rat placenta [164]. With respect to phosphate transport, PTHrP and PTH do not stimulate placental transport of phosphate in sheep [52]; the regulators of phosphate transport are unknown [51].
FETAL PARATHYROIDS As noted previously, fetal parathyroids may contribute to calcium homeostasis by secretion of both PTH and PTHrP, unlike in the adult, in whom the parathyroids secrete PTH alone. However, the evidence that fetal parathyroids make PTHrP is not conclusive. Studies in parathyroids of fetal sheep and calves have noted immunoreactivity (by immunohistochemistry and Western blot) to both midmolecular and N-terminal fragments of PTHrP in the parathyroids [74,113]. Together with the finding of reduced PTHrP-responsive placental calcium transport in parathyroidectomized fetal lambs, it has been postulated that the parathyroids regulate placental calcium transport through the production of PTHrP. However, a detailed examination of normal fetal rat parathyroids found no detectable PTHrP mRNA by in situ hybridization or RT-PCR and no detectable PTHrP by immunohistochemistry [188]. The recent observation that lack of parathyroids in Hoxa3-null mice does not perturb the circulating PTHrP concentration (Fig. 8) or the rate of placental calcium transfer (Fig. 16) also casts doubt on the importance of parathyroids for producing PTHrP and regulating placental calcium transfer [19]. Furthermore, the observation that placenta and liver, but not the neck region, show upregulation of PTHrP mRNA in Pthrlnull fetuses also suggests that the fetal parathyroids may not be a dominant source of PTHrP in the circulation of fetal mice [19]. These latter studies underscore the need for further clarification of the postulated existence and role of parathyroid-produced PTHrP in the species that have been studied. The evidence is consistent across species that intact parathyroid glands are required for maintenance of a normal fetal calcium level, as demonstrated in studies of fetal lambs (thyroparathyroidectomy), rats (decapitation), and mice (loss of parathyroids through ablation of Hoxa3). Recent studies of genetically engineered mice have demonstrated that the role of PTH and PTHrP is additive, and that the parathyroids (and PTH) have a greater impact on blood calcium regulation than PTHrP [19,20]. The Pthrp and Hoxa3 gene deletions were placed into the same colony, such that Pthrp-null, Hoxa3-null, and Hoxa3/Pthrp double mutants would be present within the same litter. In this controlled situation, single mutant Pthrp-null fetuses had a modestly reduced blood calcium (equal to the maternal calcium concentration), single mutant Hoxa3-null fetuses had a markedly reduced blood calcium (below the maternal calcium concentration), and Hoxa3/Pthrp double mutants had the lowest blood calcium (Fig. 17) [20]. Thus, lack of PTH had a greater impact on the fetal blood calcium than lack
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FIGURE 1 7
Serum ionized calcium in wild-type (WT), single mutant Hoxa3-null, single mutant Pthrp-null, and Hoxa3/Pthrp double mutant fetuses. For clarity, the other five possible genotypes have been omitted. The upper dashed line indicates the mean maternal ionized calcium level in this genetic background (equal to Pthrp-null), whereas the lower dashed line indicates the mean ionized calcium level in Pthrlnull fetuses in a similar genetic background (reproduced with permission from Kovacs et al. [20]. Copyright 2001 The Endocrine Society).
of PTHrP; the combined loss of both PTH and PTHrP resulted in the lowest blood calcium level. Fetal parathyroids are also required for normal regulation of serum magnesium and phosphate concentrations, as observed in thyroparathyroidectomized fetal lambs that had a reduced serum magnesium concentration [161] and an increase in the serum phosphate level [15,189]. Similarly, the absence of PTH and parathyroids in Hoxa3-null fetuses also causes hypomagnesemia and hyperphosphatemia [19]. The absence of PTHrP alone causes hyperphosphatemia, but the serum magnesium concentration is unaltered [19] (unpublished data). Although a Pth-null model has been published, the effects of lack of PTH on fetal calcium, phosphate, or magnesium concentrations have not been reported [190]. As discussed previously, there is consistent evidence that PTHrP regulates placental calcium transfer but conflicting evidence between sheep and mouse models that parathyroids are required for this process. The fetal parathyroids are required for normal accretion of mineral by the fetal skeleton, as discussed in more detail later.
289
blocked by simultaneous deletion of the PTH/PTHrP receptor (Casr/Pthrl double knockout), then ablation of the CaSR does not affect the fetal blood calcium level [43]. These observations confirmed that the effects of CaSR ablation in fetal life are mediated by PTH. Postnatally, homozygous ablation of the CaSR results in severe hyperparathyroidism and hypercalcemia [42], analogous to the human condition of neonatal severe primary hyperparathyroidism. However, in fetal life the serum calcium of Cast-null fetuses is no higher than that of the heterozygous siblings (Fig. 2) [43], indicating that some aspect of the intrauterine environment prevents Casr-null fetuses from achieving a higher blood calcium level. It appears that the CaSR functions in fetal life (as it does in the adult) to regulate PTH secretion, and that the CaSR does not regulate PTHrP. The fetal serum calcium is maintained at a higher level than that of the mother partly through the action of PTHrP; the CaSR suppresses PTH in response to this calcium level (Fig. 18A). In the absence of PTHrP (Pthrp-null), the fetal serum calcium is maintained at the normal adult level through the actions of the CaSR, which stimulates fetal PTH (Fig. 18B) [11,20]. In the absence of PTH (Hoxanull), the serum calcium decreases sharply because the CaSR has no ability to stimulate PTHrP. Ablation of the CaSR not only increases the fetal serum calcium (Fig. 2) but also decreases the rate of transfer of calcium across the placenta (Fig. 19) [43]. As noted previously, the CaSR is expressed in murine placenta [116]. The reduction in placental calcium transfer may be a consequence of the loss of calcium-sensing capability within the placenta; alternatively, it is possible that downregulation of calcium transfer occurs in response to the elevated serum calcium concentration or
CALCIUM-SENSING RECEPTOR Ablation of the CaSR (as in Casr+/- and Casr-null fetuses) increases both the fetal blood calcium (Fig. 2) and the circulating PTH level (Fig. 5) above the corresponding wild-type values [43]. If the effect of PTH is
FIGURE 1 8 Fetal blood calcium regulation. (A) Normal high fetal calcium level, which is dependent on PTHrP, activates the parathyroid CaSR, and PTH is suppressed. (B) In the absence of PTHrP, fetal calcium declines to a level that is set by the parathyroid CaSR; PTH is stimulated to maintain the ionized calcium at the normal adult level (maternal).
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mally present in mice but have parathyroid tissue in the thymus that produces relatively normal amounts of PTH [192]. In contrast, H o x a 3 - n u l l mice lack parathyroids and thymus, and they completely lack PTH [19]. Whether thymic PTH contributes to fetal calcium metabolism has not been determined. Rats and mice normally have two parathyroid glands, in contrast to humans, who normally have four (and occasionally more). Whether thymic parathyroid tissue in mice is the evolutionary equivalent of the lower parathyroids of humans has not been determined.
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FETAL KIDNEYS AND AMNIOTIC FLUID b
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FIGURE 19 Placentalcalcium transfer at 5 min is decreased by ablation of Cast in fetuses of Cast +/- mothers (a) and infetuses of normal mothers (b). The number of observations is indicated in parentheses (reproduced with permission from Kovacs et al. [43]. Copyright 1998 American Societyof Clinical Investigation).
the elevated PTH concentrations that occur in these null mice. In addition to increasing the blood calcium and increasing PTH secretion, ablation of the CaSR is expected to decrease renal calcium clearance as it does in the adult [42]. However, Casr +/- and Casr-null fetal mice have increased amniotic fluid levels, suggesting that renal calcium excretion is increased in proportion to the increased serum calcium concentration [43]. The discrepancy between adult and fetal effects of CaSR ablation on renal calcium handling may be explained by the observation that the kidneys express very low levels of CaSR mRNA until Postnatal Day 1 [191].
THYMUS PTH is not solely produced in the parathyroids; G c m 2 - n u l l mice lack the two parathyroids that are nor-
Fetal kidneys may partially regulate fetal calcium homeostasis by adjusting the relative reabsorption and excretion of calcium, magnesium, and phosphate by the renal tubules in response to the filtered load and other factors, such as PTHrP and/or PTH. Evidence for a contribution of the fetal kidneys includes the observation that thyroparathyroidectomy in fetal lambs resulted in an increase in fractional excretion of calcium by the fetal kidneys and reduced phosphate excretion [189,193]. These effects were reversed by treatment with aminoterminal fragments of either PTH or PTHrP [189,193], resulting in hypercalcemia, hypocalciuria, hypophosphatemia, and hyperphosphaturia. Thus, the hypocalcemia of thyroparathyroidectomized fetal lambs may be a consequence of several factors, including the reduction in placental calcium transfer and the loss of the effects of parathyroid-derived PTH and PTHrP on the renal tubules and skeleton. In contrast to these results in fetal lambs, the absence of parathyroids in H o x a 3 - n u l l fetuses, and the absence of the PTH/PTHrP receptor in P t h r l - n u l l fetuses, was accompanied by a marked reduction in the amniotic fluid calcium content, which is an indirect measure of the renal calcium handling [19] (unpublished data). Since both H o x a 3 - n u l l and P t h r l - n u l l fetuses have a very low circulating calcium concentration, the renal filtered load of calcium is likely very low and may account for the low amniotic fluid calcium content. It has not been technically possible to directly measure the fractional renal excretion of calcium in fetal mice; thus, it cannot be determined whether the results from lambs and mice are in disagreement. The fetal kidneys may also participate by synthesizing 1,25-D. Evidence includes the observation that nephrectomy in fetal lambs resulted in hypocalcemia, hyperphosphatemia, and reduced placental calcium transfer [53,133]. These effects were attributed to loss of renal production of 1,25-D because nephrectomy caused a
11. Fetal Mineral Homeostasis
60% decrease in plasma 1,25-D levels. However, it is not possible to exclude the possibility that other consequences of nephrectomy (loss of renal filtering function and surgical effects) influenced the results. Given that the levels of 1,25-D in the fetus are normally low (except in sheep, as discussed earlier), and that the absence of VDR in fetal mice does not impair fetal calcium homeostasis or placental calcium transfer [33], it appears likely that renal production of 1,25-D is relatively unimportant for fetal calcium homeostasis in most animals. Compared to that in the adult, renal calcium handling in fetal life may be less important for the regulation of calcium homeostasis because calcium excreted by the kidneys is not permanently lost to the fetus. Fetal urine is the major source of fluid and solute in the amniotic fluid, whereas fetal swallowing is likely the major pathway for clearance of amniotic fluid and is a pathway by which excreted calcium can be made available again to the fetus [194]. Other sources may contribute to the amniotic fluid, including secretions from the respiratory tract and exchange of fluid and/or solute across the fetal skin, fetal membranes (amnion, chorion, and chorionic plate); and umbilical cord [194]. Nevertheless, amniotic fluid represents a pathway by which excreted calcium may be recirculated to the fetus.
FETAL SKELETON The skeleton must undergo substantial growth and be sufficiently mineralized by the end of gestation to support the organism. As in the adult, the fetal skeleton participates in the regulation of calcium homeostasis; calcium accreted by the fetal skeleton may be subsequently resorbed to help maintain the concentration of calcium in the blood. Several lines of evidence indicate that skeletal calcium may be mobilized in response to reduced transfer of calcium from mother to fetus. Maternal hypocalcemia due to thyroparathyroidectomy or calcitonin infusion increased the basal level of bone resorption in subsequently cultured fetal rat bones [195, 196]. These effects were blocked by prior fetal decapitation, which is thought to mimic the effects of thyroparathyroidectomy [195,196]; thus, fetal hyperparathyroidism mobilized calcium from the skeleton. Furthermore, the fetal parathyroid glands enlarge in response to maternal hypocalcemia in the rat [197,198], and fetal femur length and mineral ash content are subsequently reduced [39]. Several recent observations in genetically engineered mice also support a role for the skeleton in fetal calcium homeostasis. The ionized calcium of PTH/PTHrP receptor-ablated fetal mice (Pthrlnull) is lower than that of Pthrp-null fetal mice, despite
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the fact that placental calcium transport is supranormal in Pthrl-null fetuses and subnormal in Pthrp-null fetuses [11]. Lack of bone responsiveness to the amino-terminal portion of PTH and PTHrP may well contribute to the hypocalcemia in mice without PTH/PTHrP receptors. Placement of a constitutively active PTH/PTHrP receptor into the growth plates of Pthrp-null fetuses not only reverses the chondrodysplasia [199] but also results in a higher fetal blood calcium level (unpublished data). Casr-null fetuses have a higher ionized calcium than normal, and this is maintained at least in part through increased PTH-stimulated bone resorption [43]. The skeletal calcium and magnesium contents of Casr-null skeletons are significantly reduced compared to those of their siblings, and bone resorption is increased as determined by measurement of the amniotic fluid concentration of deoxypyridinoline [43] (unpublished observations). Intact fetal parathyroid glands may be needed for normal skeletal mineral accretion since thyroparathyroidectomy in fetal lambs caused decreased skeletal calcium content and rachitic changes [200,201]. These effects could be partly reversed or prevented by fetal calcium and phosphate infusions; thus, much of the effect of fetal parathyroidectomy was caused by a decrease in blood levels of calcium and phosphate [201]. Therefore, functioning fetal parathyroids (PTH and/or PTHrP) may be required for normal fetal bone mineralization. Results from recent examination of the skeletons of the aparathyroid Hoxa3-null fetuses are consistent with observations in fetal lambs since despite a normal rate of placental calcium transfer, the skeletons of Hoxa3-null fetuses accrete less calcium and magnesium by the end of gestation (Fig. 20) [20]. Further comparative study of Pthrp-null, Pthrl-null, and Hoxa3-null fetuses has clarified the relative role of PTH and PTHrP in regulation of the development and 1203
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FIGURE 20 Deletion of parathyroids (Hoxa3-null) results in a significant decrease in the calcium accreted by the fetal skeleton as determined by quantitative assessment by atomic absorption spectroscopy on the ashed fetuses. The magnesium content and total skeletal weight are also significantly reduced (not shown) (adapted with permission from Kovacs et al. [20]. Copyright 2001 The Endocrine Society).
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mineralization of the fetal skeleton. PTHrP produced locally in the growth plate directs the development of the cartilaginous scaffold that is later broken down and transformed into endochondral bone [202], whereas PTH controls the mineralization of bone through its contribution to maintaining the fetal blood calcium and magnesium [20]. In the absence of PTHrP, a severe chondrodysplasia results [44], but the fetal skeleton is fully mineralized [20]. In the absence of parathyroids and PTH (Hoxa3-null), endochondral bone forms normally but is significantly undermineralized [20]. Since blood calcium and magnesium were also significantly reduced in Hoxa3-null fetuses, lack of PTH action on bone may have affected the maintenance of blood calcium and magnesium. That is, by impairing the amount of mineral presented to the skeletal surface and to osteoblasts, lack of PTH thereby impaired mineral accretion by the skeleton. When both PTH and PTHrP are deleted (Hoxa3/ Pthrp double mutants), the typical Pthrp-null chondrodysplasia results but the skeleton is smaller and contains less mineral [20]. Similarly, in the absence of the PTH/ PTHrP receptor, Pthrl-null skeletons are significantly undermineralized [20]. Therefore, functioning fetal parathyroids are required for normal mineralization of the skeleton; the specific contribution may be through PTH alone. Whether the contribution is through direct actions of PTH on osteoblasts, or whether it is indirect through the actions of PTH to maintain the fetal blood calcium, remains to be clarified. Apart from undermineralization of the skeleton, the lengths of the long bones and the growth plates of the Hoxa3-null were normal at both the gross and microscopic level, and the expression of several osteoblast and osteoclast-specific genes was unaltered by loss of parathyroids and PTH [20]. In other words, loss of PTH did not appear to affect the development of the cartilaginous scaffold or the bone matrix that replaced it, but loss of PTH did impair the final mineralization of the bone matrix. It is therefore unlikely that abnormal osteoblast function can explain the reduced mineralization of Hoxa3-null bones. However, it is clear that the PTH1 receptor influences osteoblast function and the normal regulation of osteopontin, osteocalcin, and interstitial collagenase in the fetal growth plate since Pthrl-null growth plates show a defect in osteoblast function and reduced expression of osteopontin, osteocalcin, and interstitial collagenase [203,204]. Since PTHrP is produced locally in the growth plate and periosteum, it is likely the ligand that normally acts on the PTH 1 receptor to regulate these genes. On the other hand, since the expression of osteopontin, osteocalcin, and interstitial collagenase is not reduced in the Pthrp-null fetuses [203] and there is no evidence of impaired osteoblast function [204], PTH may be able to penetrate the relatively avas-
cular growth plate and compensate for the absence of PTHrP. The elevated PTH levels observed in the Pthrpnull fetus are compatible with this observation [20]. Therefore, osteopontin, osteocalcin, and interstitial collagenase may be downregulated in the Pthrl-null because neither PTH nor PTHrP can act in the absence of the PTH 1 receptor; these genes are not downregulated by the absence of PTH or PTHrP alone. Since only the Pthrlnull shows evidence of impaired osteoblast function [204], but both the Hoxa3-null and the Pthrl-null show a similar degree of reduced mineralization [20], the undermineralization of both null phenotypes may be due to the reduced availability of mineral presented to the osteoblast surface (i.e., the reduced blood calcium and magnesium level in both phenotypes); the availability of mineral is dependent on the action of PTH. The recently reported Pth-null mice also have undermineralized skeletons, but they differ from the phenotype of Hoxa3-null mice in that the long bones of the Pth-null mice are modestly shortened, and there is evidence of reduced osteoblast number and function from studies that were not performed on Hoxa3-null mice [190]. The Pth-null and Hoxa3-null models need to be compared within the same genetic background in order to determine which aspects of the respective phenotypes are due to loss of PTH and which might be due to other confounding effects (e.g., aparathyroid and athymic in Hoxa3-null, marked parathyroid hyperplasia in Pth-null, and lower blood calcium in the C57BL6 background of Pth-null mice versus higher blood calcium in the Black Swiss background of Hoxa3-null mice). In summary, normal mineralization of the fetal skeleton requires intact fetal parathyroid glands and adequate delivery of calcium to the fetal circulation. Although both PTH and PTHrP are involved, PTH plays a more critical role in ensuring full mineralization of the skeleton before term.
MATERNAL SKELETON There is evidence that the maternal skeleton may accrete mineral early in gestation in preparation for the peak fetal demand later in pregnancy, that the maternal skeleton contributes to the calcium ultimately accreted by the fetal skeleton, and that there is a small net decline in maternal mineral content by term [8,205]. This is in contrast to lactation, in which there is normally a 5-10% decline in bone density in humans [8,205] and a 30% or greater decrease in maternal skeletal mineral content in rats that may be worsened if the mother is calcium deficient [206,207] or rendered deficient in calcitonin by thyroidectomy [39,208-211]. The finding that lack of
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calcitonin worsened the loss of mineral from the skeleton prompted the hypothesis that calcitonin normally protects the maternal skeleton from excessive resorption during pregnancy and lactation. The decline in bone mineral content that occurs during pregnancy and especially lactation is normally reversed after weaning, but not in vitamin D-deficient rats [212]. The effect of pregnancy on maternal bone mineral content has not been extensively examined, and it has not been examined at all in the currently available gene ablation models.
FETAL RESPONSE TO MATERNAL HYPERPARATHYROIDISM In humans, maternal primary hyperparathyroidism has been associated with an alarming rate of adverse fetal outcomes, including a 30% rate of spontaneous abortion or stillbirth [213,214] and a 50% rate of tetany and a 25% rate of death in the neonatal period [213,215]. These adverse fetal and neonatal outcomes are thought to result from suppression of the fetal parathyroid glands; suppression may occasionally be prolonged for months [216,217]. PTH cannot cross the placenta [19,38,76,77]; therefore, fetal parathyroid suppression is thought to result from increased net calcium flux across the placenta to the fetus, facilitated by the maternal hypercalcemia. The fetal parathyroid glands may also be suppressed when the mother has hypercalcemia due to familial hypocalciuric hypercalcemia [218-221]. Evidence from animal models confirms that acute elevations in the maternal serum calcium cause an increase in the fetal serum calcium and a decrease in fetal PTH level [222]. Chronic elevation of the maternal serum calcium in C a s r +/- mice (the equivalent of familial hypocalciuric hypercalcemia in humans) results in suppression of the fetal PTH level compared to that of fetuses obtained from wild-type sibling mothers (Fig. 2) [43]. Because of the alarming rate of adverse fetal and neonatal outcomes, surgical correction of primary hyperparathyroidism during the second trimester has been almost universally recommended [214,223-225]. Several case series have found elective surgery to be well tolerated and to dramatically reduce the rate of adverse events compared to the results of earlier cases [223,224,226,227]. However, many women in the early cases were symptomatic and had nephrocalcinosis or renal insufficiency. The early case reports may also have reflected reporting bias of adverse fetal and neonatal outcomes. Whether the milder, asymptomatic form of primary hyperparathyroidism commonly seen today has the same risk of adverse fetal or neonatal outcomes has not been determined. In several case reports, mild
elevations in maternal serum calcium were followed without operative intervention, and no adverse fetal or neonatal outcome occurred [228,229].
FETAL RESPONSE TO MATERNAL HYPOPARATHYROIDISM Maternal hypoparathyroidism in human pregnancy has been associated with the development of intrauterine, fetal hyperparathyroidism. This condition is characterized by fetal parathyroid gland hyperplasia, generalized skeletal demineralization, subperiosteal bone resorption, bowing of the long bones, osteitis fibrosa cystica, rib and limb fractures, and low birth weight [230-234]. Spontaneous abortion, stillbirth, and neonatal death have also been associated with this condition [235-237]. Similar skeletal findings have been reported in the fetuses and neonates of women with pseudohypoparathyroidism [238,239], renal tubular acidosis [240], and chronic renal failure [241]. Although these skeletal changes have been interpreted to indicate fetal hyperparathyroidism, no serum measurements of intact PTH (or PTHrP) have been reported for this condition, and the serum calcium level has generally been reported to be normal. These changes in human skeletons differ from what has been found in animal models of maternal hypocalcemia, in which the fetal skeleton and the blood calcium are generally normal.
INTEGRATED FETAL CALCIUM HOMEOSTASIS The evidence discussed in the preceding sections suggests the summary models discussed in the following sections. Calcium S o u r c e s The main flux of calcium (and other minerals) is across the placenta and into fetal bone, but calcium is also made available to the fetal circulation through several routes (Fig. 21). Some calcium filtered by the kidneys is reabsorbed into the circulation. Calcium excreted by the kidneys into the urine and amniotic fluid may be swallowed and absorbed by the intestine. Calcium is also resorbed from the developing skeleton to maintain the circulating calcium concentration. Some calcium also returns to the maternal circulation (backflux). The maternal skeleton is a critical source of mineral (in addition to maternal dietary intake), and the maternal skeleton is
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FIGURE 21 Calcium sources in fetal life. The main flux of calcium is across the placenta and into fetal bone. Some calcium returns to the maternal circulation (backflux). Calcium filtered by the kidneys is partly reabsorbed into the circulation; calcium excreted by the kidneys into the urine and amniotic fluid may be swallowed and absorbed by the intestine. Calcium is also resorbed from the developing skeleton to maintain the circulating calcium concentration.
FIGURE 2 2 Fetal blood calcium regulation. PTH has a more dominant effect on fetal blood calcium regulation than PTHrP, with blood calcium represented schematically as a thermometer (light gray, contribution of PTH; dark gray, contribution of PTHrP). In the absence of PTHrP, the blood calcium declines to the maternal level. In the absence of PTH (Hoxa3-null that has absent PTH but normal circulating PTHrP levels), the blood calcium declines well below the maternal calcium concentration. In the absence of both PTHrP and PTH (Hoxa3/Pthrp double mutant), the blood calcium declines even further than in the absence of PTH alone. Whether the parathyroids normally contribute PTHrP to the circulation has not been ruled out by these observations.
compromised in maternal dietary deficiency states in order to provide for the fetus. Blood Calcium Regulation Fetal blood calcium is set at a level higher than maternal blood calcium through the actions of PTHrP and PTH acting in concert (among other potential factors) (Fig. 22). Although the parathyroid CaSR appears to respond appropriately to this increased level of calcium by suppressing PTH, the low level of PTH is critically required for maintaining a normal blood calcium and normal mineral accretion by the skeleton. 1,25-D synthesis and secretion, in turn, are suppressed due to the effects of low PTH and high blood calcium and phosphate. The parathyroids may play a central role by producing PTH and PTHrP, or they may produce only PTH while PTHrP is produced by the placenta and other fetal tissues. It is well appreciated that the PTH1 receptor mediates actions of two ligands, PTH and PTHrP. These ligands, both present in the fetal circulation, independently and additively regulate the fetal blood calcium, with PTH
having the greater effect. Neither ligand can make up for the absence of the other: If one ligand is missing, the blood calcium is reduced, and if both ligands are missing, the blood calcium is reduced even further. How one receptor can simultaneously and independently mediate the actions of these two ligands in the circulation is not clear. The contribution of PTHrP to the fetal blood calcium may not be through the PTH1 receptor at all, but perhaps only through the actions of midmolecular PTHrP to regulate placental transfer of calcium (a process that has been shown to be independent of the PTH1 receptor). Thus, PTH may contribute to the blood calcium through actions on the PTH1 receptor in classic target tissues (kidney and bone), whereas PTHrP might contribute through placental calcium transfer and actions on other (non-PTH) receptors. The normal elevation of the fetal blood calcium above the maternal calcium concentration was historically considered the first evidence that placental calcium transfer was largely an active process. However, the fetal blood calcium level is not simply determined by the rate of
11. FetalMineral Homeostasis placental calcium transfer since this is normal in Hoxa3null and increased in Pthrl-null, but both null phenotypes have significantly reduced blood calcium levels [11,19]. Also, Casr-null fetuses have reduced placental calcium transfer but markedly increased blood calcium levels [43]. P l a c e n t a l C a l c i u m Transfer Placental calcium transfer is regulated by P T H r P but not by PTH (Fig. 23). Although the exact tissue source(s) of P T H r P that is relevant for placental calcium transfer remains uncertain, the placenta is one proven source of P T H r P that is likely involved~ in calcium transfer. Whether the parathyroids produce P T H r P or not is uncertain; in contrast to fetal lambs, experiments in Hoxa3null mice indicate that the absence of parathyroids does not impair placental calcium transfer [19]. Skeletal Mineralization PTH and P T H r P have separate roles with respect to skeletal development and mineralization (Fig. 24). PTH normally acts systemically (i.e., outside of bone) to direct the mineralization of the bone matrix by maintaining the blood calcium at the adult level and possibly by direct
Thyroid and parathyroids FIGURE 23 Placentalcalcium transfer is regulated by PTHrP but not by PTH. Although the exact tissue source(s) of PTHrP that is relevant for placental calcium transfer remains uncertain, the placenta is one proven source of PTHrP that is likely involved in calcium transfer. Whether the parathyroids produce PTHrP or not is uncertain; experiments in Hoxa3-null mice indicate that the absence of parathyroids does not impair placental calcium transfer.
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actions on osteoblasts within the bone matrix. PTH is capable of directing certain aspects of endochondral bone development in the absence of P T H r P (e.g., regulation of expression of osteocalcin, osteopontin, and interstitial collagenase within the growth plate) [20]. In contrast, P T H r P acts both locally within the growth plate to direct endochondral bone development and outside of bone to affect skeletal development and mineralization by contributing to the regulation of the blood calcium and placental calcium transfer. PTH has the more critical role in maintaining skeletal mineral accretion. The rate of placental calcium transfer has been historically considered to be the rate-limiting step for skeletal mineral accretion. However, it has been shown that this is not the rate-limiting step for skeletal mineralization since the accretion of mineral was reduced in the presence of normal placental calcium transfer (Hoxa3-null) and increased placental calcium transfer (Pthrl-null) [20]. Furthermore, Pthrp-null fetuses showed normal skeletal mineral content in the presence of reduced placental calcium transfer and a modestly reduced blood calcium [20]. The rate-limiting step for skeletal mineralization appears to be the blood calcium level, which in turn is largely determined by PTH. The level of blood calcium achieved in the Pthrp-null (i.e., the normal adult
FIGURE 24 Schematicmodel of the relative contribution of PTH and PTHrP to endochondral bone formation and skeletal mineralization. PTHrP is produced within the cartilaginous growth plate and directs the development of this scaffold, which will later be broken down and replaced by bone. In the absence of PTHrP (Pthrp-nuU) a severe chondrodysplasia results but the skeleton is normally mineralized. PTH reaches the skeleton systemicallyfrom the parathyroids and directs the accretion of mineral by the developing bone matrix. In the absence of PTH (Hoxa3-null), the bones form normallybut are severely undermineralized.
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level of blood calcium) is sufficient to allow normal skeletal accretion of mineral, whereas lower levels of blood calcium (Hoxa3-null, Pthrl-nuU, and Hoxa3/Pthrp double mutant) impair the rate of mineral accretion.
Acknowledgments My research endeavors have been supported in large part in the form of a fellowship, a scholarship, and operating grants from the Canadian Institutes for Health Research (formerly Medical Research Council of Canada), in addition to support from Memorial University of Newfoundland. I am most grateful for the support and advice of Dr. Henry M. Kronenberg, who supervised my postdoctoral fellowship (and acted as midwife to the birth of my interest in calcium metabolism during the reproductive periods) at Harvard Medical School and Massachusetts General Hospital. I acknowledge my collaborators (Drs. Marie Demay, James Friel, Robert Gagel, Andrew Karaplis, Gerard Karsenty, Nancy Manley, Jack Martin, Jane Moseley, Ernestina Schipani, and Peter Wookey), who kindly permitted me to make use of their models, assays, and antibodies. I thank Dr. Anthony D. Care f o r reviewing the manuscript and permitting me to cite his unpublished data; Dr. John Harnett for his enthusiastic support as chair of medicine at Memorial University of Newfoundland; my graduate students Mandy L. Woodland and Kirsten R. McDonald, whose work is cited herein; and my research assistants Neva J. Fudge and Linda L. Chafe. Finally, I acknowledge my wife, Dr. Susan MacDonald, and our children, Caileigh and Jamieson, without whose support and patience none of this would have been possible.
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Noninvasive Techniques for Bone Mass Measurement STEFANO MORA,* LAURA BACHRACH, t AND VICENTE GILSANZ* 9Laboratory of Pediatric Endocrinology, Scientific Institute H San Raffaele, Milan, ltaly f Division of Endocrinology, Stanford University School of Medicine, Stanford, California 9Childrens Hospital Los Angeles, Department of Radiology University of Southern California School of Medicine, Los Angeles, California
Each is more sensitive than conventional radiography for detecting deficits in bone mineral. This chapter discusses the pediatric use of dual-energy X-ray absorptiometry (DXA), quantitative computed tomography (QCT), peripheral QCT (pQCT), quantitative ultrasound (QUS), magnetic resonance imaging (MRI), and X-ray radiogrammetry. These techniques can be compared using several criteria, including speed of measurement, radiation exposure, cost, and regions of the skeleton that can be scanned. These techniques also vary in accuracy (the difference between bone measurement and ash weight) and precision (the reproducibility of repeated measurements). For clinical purposes, precision is of greater importance than accuracyl QCT and MRI can distinguish trabecular from cortical bone, whereas DXA provides only a composite measurement of both compartments. Finally, there are differences in the availability of normative pediatric data to be used as reference values when interpreting scans performed for clinical purposes.
INDICATIONS FOR BONE MASS MEASUREMENTS The foundation for lifetime bone health is established during childhood and adolescence [1]. Peak bone mass is achieved during the third decade, serving as the bone bank for the remainder of life. Acquiring a robust "account" counteracts the inevitable bone loss due to aging, menopause, and other factors and delays or prevents osteoporosis. Puberty is a particularly critical period for mineral accrual since the amount of bone mineral gained in healthy youth equals the amount lost throughout the remainder of life [2]. As recognition of the importance of early bone health has increased, so has the demand for bone mineral assessments in children and adolescents. Researchers have sought a means to define the tempo and magnitude of bone mineral accrual and the factors modulating peak bone mass in healthy youth. With this information, investigators hope to develop interventions that will optimize mineral accrual to prevent osteoporosis. For clinicians, densitometry is viewed as a means of screening the bone health of young patients with myriad chronic diseases associated with low bone mass and fractures early in life [3]. The goal of mass assessments in this setting is to identify patients with skeletal fragility and to monitor response to therapy. A variety of noninvasive methods are available to assess the peripheral, central, or entire skeleton [4].
PediatricBone
CHALLENGES TO INTERPRETING BONE MASS MEASUREMENTS IN C H I L D H O O D AND ADOLESCENCE Bone mass measurements have proven to be powerful predictors of bone fragility in adults [5]. The correlation between bone density and fracture risk is sufficiently
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strong that the World Health Organization has established diagnostic criteria for osteoporosis based on bone mineral density (BMD) criteria alone. Osteopenia (low bone mass) is defined as a BMD between 1 and 2.5 standard deviations (SDs) below the mean for healthy young adults; osteoporosis is defined by a BMD SD score lower than -2.5. The risk of bone fractures is estimated to double with each SD that BMD falls below the mean. Some studies in children have also observed a correlation between bone mass or area and fracture risk [6,7], but the data are not sufficient to establish a "fracture threshold" such as that developed in adults. This may well reflect the difficulties in accurately interpreting bone mineral measurements in growing children as well as the important influence of factors other than bone mass in determining bone strength. The interpretation of bone mineral measurements is far more complex in children than in adults [8-10]. Unlike adults, whose bone dimensions are stable with time, children and adolescents are very much moving targets. As discussed in previous chapters, the process of bone mineral accrual throughout childhood and adolescence involves changes in bone size, geometry, and mineral content. These processes evolve at varying rates in different regions of the skeleton, with appendicular growth preceding spinal mineral acquisition [11]. Furthermore, within a given region of interest, trabecular and cortical compartments respond variably to sex steroids, calcium intake, and mechanical loading. The tempo of mineral accrual is more closely linked to pubertal and skeletal maturation than to chronologic age, and these processes vary with gender and ethnicity [12,13]. To accurately interpret bone mineral measurements, the influence of bone size and maturation must be considered. One additional challenge in interpreting pediatric densitometry data is the lack of pediatric reference data. Although several recent publications have provided normative data for DXA and QCT, data are limited for some skeletal sites and age groups. Fewer data are available for newer techniques, such as pQCT and ultrasound. Whole bone structural strength is determined not only by the material properties (bone mass and stiffness) but also by the geometry of the bone [14]. Bone size and the distribution of bone mineral within the bone influence the resistance of bone to fracture. Given two bones of equal material properties, the larger bone will be less likely to fracture. Similarly, resistance of bone to torsion increases with the distance of bone mass from the center of the bone. Currently available noninvasive techniques vary considerably in their ability to quantify the true density, geometry, and size of bone. In some cases, mathematical models have been created to estimate volumetric density [15] or key geometric parameters such as the
moment of inertia [16] using existing data derived from the scans. The assessment techniques also vary in the extent to which they distinguish between trabecular and cortical compartments.
NONINVASIVE MEASUREMENT TECHNIQUES Radiogrammetry and Radiographic A b s o r p t i o m e t r y Radiogrammetry was first described in 1960 for the measurement of bone to assess osteoporosis. Since that time, several scores obtained using conventional radiographs and a standard ruler have been published for diverse skeletal sites (femur, hand, and spine) [17]. The dimensions of tubular bones are delineated as follows: the outside diameter (D); the inside diameter (d) (Fig. 1); the cortical index, which is the ratio between these measurements ( D - d / D ) ; and the combined cortical thickness (D - d) [18-20]. Changes in cortical index and cortical thickness have been studied in several diseases. Defects of total bone formation or decreases in subperiosteal width can be differentiated from excess endosteal surface resorption or increases in medullary cavity width [21]. Current applications of radiogrammetry have taken advantage of advancing technologies in the computer sciences and computerized image processing to provide automated measurements of bone dimensions [22]. A second method for obtaining bone measurements using plain radiographs is radiographic absorptiometry. In this technique, the X-ray absorption of the bone is compared to that of a standard reference. Early descriptions of this technique used an aluminum wedge to assess
FIGURE 1 Radiogrammetrymeasurements in a tubular bone. The inner (d) and outer (D) diameters are measured.
12. Noninvasive Techniques for Bone Mass Measurement
the radiographic optical density of a tooth [23] and an ivory wedge to assess bone [24]. Recently, radiographic absorptiometry has been computerized. Some software even allows for the correction of differences in the degree of radiographic exposure or soft tissue thickness [25]. In one approach, two posteroanterior radiographs of the hand with an aluminum reference wedge placed near the bone are obtained at two different exposure settings [26-29] (Fig. 2). The image is captured electronically, and bone mineral "density" is calculated in arbitrary units using the aluminum reference wedge as a calibration material. The integral bone (cortical and cancellous bone together) is evaluated at sites such as the phalanges and/or the radius. Precision of measurements ranges from 0.68% for radiogrammetry [22] to approximately 1% for radiographic absorptiometry [30]. Studies on the accuracy of these techniques are only available for radiographic absorptiometry, which demonstrates an excellent correlation between ash weight and the bone mineral content values expressed in arbitrary units [30,31]. Both methods are derived from radiographs and therefore imply radiation exposure. Skin dose varies from 1 to 5mSv.
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However, the effective dose, which depends not only on the absorbed radiation but also on the size of the irradiated volumes, the relative sensitivity of the irradiated organs, and the radiation energies [32], has been reported to be as low as 1 pSv [22,31]. Several studies in healthy children have been performed with these methods. Radiogrammetry has been used to study the effect of calcium intake on bone mass in adolescent girls [33] and to assess determinants of bone mass in preadolescent girls [34]. In the latter study, skeletal age was found to be the major determinant of bone mass measured at the second metacarpal. A large study in young children (aged 6.8-10.7 years) showed that bone mineral content measured by radiographic absorptiometry increased with skeletal age and that girls had higher BMD at the diaphyseal and metaphyseal sites than boys [35]. In contrast, another study showed no gender differences in children and small increases in BMD until the age of 11.5 years. Thereafter, bone density increased markedly in both boys and girls [36]. Recently, normative data for radiographic absorptiometry have been published using skeletal age groups rather than chronological ages [37]. In general, the data indicate that BMD remains fairly constant until skeletal age 12 years in boys and 10 years in girls, when it markedly increases. In addition, there are no gender differences, except at bone ages 11-14 years, when girls showed higher BMD values. Dual-Energy X-Ray A b s o r p t i o m e t r y
FIGURE 2 Radiographic absorptiometry. Hand radiographs are taken and a reference aluminum standard is placed closed to the index finger.
DXA has become the most commonly employed technique worldwide for the assessment of bone mineral content [38]. Bone mineral measurements by DXA rely on the attenuation (absorption) of energy that occurs as the X-ray beam scans across the region of interest [39,40]. Two energy settings are used to optimize the separation of mineralized and soft tissue components in the area analyzed. The low-energy photons penetrate only the soft tissue surrounding the bone, whereas the highenergy photons penetrate both the soft tissue and the bone (Fig. 3). A detector located above the X-ray tube measures the exiting photons from the site scanned and a computer subtracts the low-energy values from the highenergy measurements. With the use of calibration materials, the difference in relative attenuation is calculated and expressed as bone mineral content (BMC) in grams. Manufacturers of DXA equipment have various approaches to calibration. One scanner, which derives the two-photon energies by rapid switching of the X-ray tube potential, employs a synchronized rotating filter that includes a sector of hydroxyapatite to provide internal calibration on a pixel-to-pixel basis. Other scanners use a constant potential X-ray source and absorption edge
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FIGURE 4 (A) Representation of the pencil beam technique for DXA bone mineral measurements. (B) The fan beam technique.
FIGURE 3 Schematicrepresentation of the dual-energytechnique.
filtration to split the spectrum into two parts with different effective energies. They rely on a very stable X-ray source and daily checks with an external standard. Because of the different calibration methods, the results obtained with one DXA device are not directly comparable with those obtained from others. Several attempts have been made to cross-calibrate the different instruments and to derive mathematical formulae to convert the results from one scanner to another, but they have not been successful. BMC values are frequently divided by the projected area of the bones analyzed, and the resulting measurements are conventionally referred to as BMD and expressed as grams per cubic centimeter. DXA values, however, do not represent a volumetric density measurement and are therefore referred to as areal density measurements. Additionally, projection techniques, including DXA, cannot independently assess cancellous and cortical bone, and the resulting values reflect the sum of both components (integral bone measurements). Bone measurements using DXA have been obtained in the lumbar spine, femurs, forearms, and whole body. The initial DXA scanners used a collimated X-ray beam (pencil beam), which has recently been replaced by a fan beam (Fig. 4). Precision of DXA BMD measurements has been extensively studied both in vitro and in vivo and shows a high degree of reproducibility [41-44]. Results vary depending on the site measured: Lumbar spine errors have been reported to be as low as 0.7-1.7%, the forearm varies between 0.9 and 1.9%, total body values
are 0.7%, and the femur has the highest coefficient of variation at 1.3-2.6%. Studies assessing the accuracy of DXA measurements have yielded conflicting results. Some reports showed high accuracy in vitro by scanning anthropomorphic phantoms or by comparing BMC values obtained from excised bones with the dry or ash content [43,45,46]. Other in vivo studies demonstrated that accuracy at each skeletal site depends greatly on the soft tissue surrounding the bone [44]. In general, accuracy errors less than 6 or 7% of BMD measurements have been reported for posteroanterior spine, forearm, and femur [44]. Radiation exposure from DXA exams is minimal (Table 1), as indicated by dosimetry studies in adults [47-49] and in pediatric subjects [50]. Effective dose depends on the X-ray energy used and on the irradiated organs. In particular, irradiation of reproductive tissues increases the effective dose value. As shown in Table 1, the effective dose for the anteroposterior spine scan is less than 1 gSv when using standard procedures, but it is as low as 0.2 gSv when using a pediatric scan mode [50]. Similarly, the effective dose for total body scans varies depending on the scanning mode used; it has been calculated that the real effective dose for children weighing up to 35 kg is approximately 0.02 gSv [50]. Normative data for DXA values in pediatrics are available in the literature and are included in most DXA software packages (Fig. 5). However, there is substantial variation in the normative data published by manufacturers of DXA equipment; therefore, caution is advised when attempting to utilize published normal values. Table 2 summarizes the studies performed using different DXA techniques grouped by manufacturer. Although leading DXA manufacturers have proposed standardization for BMD measurements of the lumbar spine [51],
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Young Adu!t ~ % T 38 42 39 31 40 40 37 40 37 35
-7.21 -7.23 -7.60 -8.59 -7.19 -7.29 -7.66 -7.44 -7.83 -18.08
=====================
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Age Matched ~ % Z 50 63 59 47 62 61 57 5! 56 53
-2.96 -2.98 -3.35 -4.34 -2.94 -3.04 -3.41 -3.19 -3.58 -3.83
Example of a DXA report.
such changes have not been included in the standard software of the commercial devices. Numerous studies have been published on bone mass measurements obtained using DXA in healthy children of all ages [12,52-93], including newborns [60-62,93] and infants [60,61,65,72]. In general, BMD and BMC values measured at different skeletal sites increased from infancy to adulthood [12,52,53,55-57,59,63-66,68-72,74,75,78, 85,86,88,90,92]. The relationship between age and lumbar spine BMD shows a remarkable increase during
childhood followed by a greater increase during the pubertal period that ends in the third decade of life [12,52,53,57,59,64-73,75,78,85]. Similar relationships with growth can be observed for the femoral neck, the femoral shaft [12,53,57,59,64,66-68,70,71], and the entire skeleton [63,66,68,70,79,81,88,92]. Opinions regarding the possible relationship between radial bone measurements and age differ; some studies show an age-related increase in radial bone values [59,68,69,89], whereas others show no such relationship [75,80].
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TABLE 1 Valuesof skin and effective doses for irradiation from different sources (adapted from refs. 47-50) Skin Dose (ttGy)
Effective Dose (ttSv)
Radiogrammetry Radiographic Absorptiometry
100-500
1
DXA Posteroanterior lumbar spine 5 year-old1 10 year-old1 Proximal femur Total Body Pediatric medium~ Pediatric large1
35 6 6 60 18 0.12 0.12
0.12-0.5 0.282 0.201 0.14-1.4 4.6 0.029 0.021
QCT bone mineral measurement Vertebrae Femur
3000 1500
30 3
pQCT bone mineral measurement (forearm)
300
10
Chest X-ray
500
50
Type of Irradiation
Natural background radiation per month
200
1 Examinations performed using specificpediatric scan modes.
Studies assessing the possible association between pubertal development, as determined by the method of Tanner, and DXA bone measurements indicate that BMD and/or BMC increase markedly between Tanner stages II and IV or V in both girls and boys [12,52-56, 65,67-70,73,75,79,80,84,85,88,89,92]. In some cases, Tanner stage was considered to be more closely related to bone measurements than chronological age [55,79,80], although one study failed to observe this relationship [75] and two studies suggested that skeletal age was a better predictor than Tanner stage [74,75]. Gender differences have been reported in some [53,57,65,66,68-70,79,81,92] but not all studies [52,63, 66,79,85]. The majority of studies indicate that by the age of 16 or 17 years, boys have greater BMD than girls [53,68,70,92]. However, one study showed that girls have higher BMD values at the femoral neck [20], and another showed sex differences only at the lumbar spine and not in the whole skeleton [79]. Most DXA studies have examined Caucasian children and adolescents; few have reported data from children of Asian [59,71,76,78,79,84,85,91], African [72,78,79,82-84, 91], or Hispanic [78,82,84] origin. Although there is general agreement on the absence of racial differences between Caucasian and Hispanic children and adolescents
[82,84], studies examining other racial differences in bone mass have yielded conflicting results. Some studies found no difference between bone measurements in Caucasian and African children [55,72], and some showed children of African origin to have higher bone mass BMD values at most skeletal sites [78,82-84,91]. In contrast, when compared with Caucasian children, Asian children have been reported as having either comparable [84,91] or lower [40] DXA values, the latter being attributed to differences in bone size [76]. Quantitative Computed Tomography Computed tomography introduced the technique of digital imaging to diagnostic radiology. The transverse anatomic sections afforded by this digital technique provide a three-dimensional image unobscured by overlying structures. The data displayed as the CT image are actually a representation of attenuation values or CT numbers of the object scanned. These numerical values are stored in digital form and are accessible for future study. The use of this digital information to provide quantitative information has been called QCT. QCT is an established technique for measuring BMD in the axial and appendicular skeleton [94,95]. The average scanning time is less than 10 min, and examinations are performed using a commercial CT scanner and a bone mineral reference standard to calibrate each scan. The procedures for bone mineral measurements of axial and appendicular bone differ slightly. Based on a lateral localizer image, or scoutview, single 1 0 - m m - t h i c k sections are obtained through the midplane of each vertebra (usually two or three vertebrae of T12-L4) using a low-dose technique, with the gantry angled parallel to the vertebral end plates. A region of interest (ROI) is then positioned in the interior portion of trabecular bone of the vertebral body for analysis (Fig. 6) [94,95]. In some approaches, this ROI may be positioned automatically [96,97]. The CT density of the selected area of interest is measured in Hounsfield units (HUs); an HU is a unit of measurement based on an arbitrary scale of 1000 positive values ranging from 0 (the value of water) to 1000 (the attenuation of compact bone) and 1000 negative values, where -1000 corresponds to the attenuation coefficient of air. Conversion to grams per centimeter 3 is performed by using linear regression to relate the HU number of the vertebral bone to that of the compartments of the calibration standard. In addition to measurements of bone density, QCT offers the possibility of measuring the height and the cross-sectional area of each vertebral body [98]. The heights of the anterior, middle, and posterior portions of the vertebral bodies are measured on lateral scoutview, whereas the area of the vertebral body is calculated
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12. Noninvasive Techniques for Bone Mass Measurement TABLE 2
Reference data on bone mineral measurement in children grouped according to DXA manufacturer.
Hologic
Lunar
Glastre et al. 1990 (52) Bonjour et al. 1991 (53) Sentipal et al. 1991 (54) Southard et al. 1991 (55) Katzman et al 1991 (56) Lloyd et al. 1992 (58) Tsukuhara et al. 1992 (59) Braillon et al. 1992 (60) Venkataraman et al. (61) Theinz et al. 1992 (12) Salle et al. 1992 (62) Faulkner et al. 1993 (63) Ruiz et al. 1995 (67) Moreira-Andr6s et al. (69) Faulkner et al. 1996 (70) Takahashi et al. 1996 (71) Rupich et al. 1996 (72) Lonzer et al. 1996 (73) Bhudhikanok et al. 1996 (76) Moro et al. 1996 (78) Molgaard et al. 1997 (81) Ellis KJ. 1997 (82) Nelson et al. 1997 (83) Wang et al. 1997 (84) Mckay et al. 1998 (86) Molgaard et al. 1998 (88) Bailey et al. 1999 (90) Godfrey et al. 2001 (93)
Kr6ger et al. 1992 (57) Kr6ger et al. 1993 (64) del Rio et al. 1994 (65) Lu et al. 1994 (66) Ilich et al. 1996 (75) Boot et al. 1997 (79) Maynard et al. 1998 (87) Horlik et al. 2000 (91) Nguyen et al. 2001 (92)
on C T scans corresponding to a R O I in each vertebral body and excluding structures behind the m o s t anterior margin of the spinal canal. The recent application of Q C T to assess the appendicular skeleton has significantly i m p r o v e d the ability to measure cortical bone. Three bone p a r a m e t e r s can be m e a s u r e d by Q C T in the appendicular skeleton: the cross-sectional area (cm 2) of the bone, the cortical bone area (cm2), and the cortical bone density (Fig. 7) [99,100]. A p p e n d i c u l a r bone m e a s u r e m e n t s are usually p e r f o r m e d at the midshaft of the femur. The scanning site is located by physical examination, and C T m e a s u r e m e n t s are obtained f r o m single 1.5-mm-thick imaging scan at the m i d p o r t i o n of the distance between the greater trochanter and the lateral condyle [99]. The cortical bone density is m e a s u r e d using a calibration device, which is placed
Norland
Zanchetta et al. 1995 (68) Plotkin et al. 1996 (77) Uusi-Rasi et al. 1997 (80) Cheng et al. 1998 (85)
Others
Sabatier et al. 1996 (74) Bonofiglio et al. 1999 (89)
under the thigh. The procedure for calculation of bone density is similar to that described for the vertebrae. The outer and inner boundaries of the cortex are identified by special software at the place of the m a x i m u m slope of the femoral profile t h r o u g h the bone. The area within the outer cortical shell represents the cross-sectional area, whereas the area between the outer and inner shells represents the cortical bone area. The m e a n C T n u m b e r s of pixels within the inner and outer cortical shells provide the average density of bone [99]. As stated earlier, Q C T m e a s u r e m e n t s of bone are able to separately assess the density of cortical and trabecular bones. Moreover, the scan section has a definite thickness, which varies according to the region examined. Bone density m e a s u r e m e n t s are expressed as grams per cubic centimeter and are therefore true volumetric
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measurements. However, some limitations are present in CT bone measurements. The CT image is formed by
FIGURE 6 Vertebral tomogram for QCT bone density assessment. The reference calibration phantom is scanned simultaneously and is placed under the body of the child.
al.
thousands of pixels, which are small squares that have a different optical density according to the tissue they represent. The smaller the pixel size, the better the resolution of the image. Unfortunately, the size of the trabeculae in cancellous bone is frequently smaller than the pixel size; therefore, not only bone but also marrow arerepresented. The optical density will change according to the amount of prevalent tissue within the area of the pixel. This phenomenon is defined as volume averaging and is a potential problem when measuring trabecular bone or an extremely porous cortical bone. In children, QCT measurements at the midshaft of the femur reflect the material density of the bone (the amount of collagen and mineral in a given volume of bone) because the relative lack of porosity and the thickness of the cortex circumvent volume-averaging error [99]. The minimum thickness necessary for an accurate density evaluation of cortical bone by QCT is 2-2.5 mm; below this threshold, QCT values decline in a linear way relative to width. Above this thickness, the measured pixel represents the combination of the attenuation coefficients defined by the densities and concentrations of osteoid and mineral. Although the nonmineral fraction may contribute to minor fluctuations in measurements of cortical bone density,
FIGURE 7 (A) QCT imageof the midshaft of the femur. (B) The mean CT numbers of the pixelswithin the inner and outer cortical shells provide the average density of bone. The area within the outer cortical shell represents the cross-sectional area (C), whereas the area between the outer and inner shells represents the cortical bone area (D). The small scale bar is the reference calibration phantom.
12. Noninvasive Techniques for Bone Mass Measurement
QCT numbers are primarily based on the calcified bone fraction, which has a high attenuation coefficient [100]. These measurements are analogous to in vitro determinations of the intrinsic mineral density of bone, which are commonly expressed as the ash weight per unit volume of bone [101]. In children, the material density of cortical bone in the appendicular skeleton measured by QCT remains fairly constant [99]. Commercially available calibration devices differ in the composition of materials used and are designed for adult subjects. Special calibration reference phantoms have therefore been developed for children to provide precise measurements, even in the very young [102]. The precision of QCT bone measurement is good in children. Coefficients of variation for determination of cancellous bone density, vertebral body height, and vertebral cross-sectional area have been calculated as 1.5,1.3, and 0.8%, respectively [103,104]. The coefficients of variation for repeated QCT measurements of cortical bone density, cortical bone area, and the cross-sectional area of the femur range between 0.6 and 1.5% [105]. Radiation exposure is related to the technique employed and can be as low as 1500 ~tGy localized to the ROI in the appendicular or axial skeleton. The effective dose of radiation varies from 3 to 30 gSV (Table 1), and these data include radiation associated with screening digital radiographs used to localize the site of measurement [47]. This amount of radiation is far lower than that associated with other CT imaging procedures, accounting for the wide range of published data on the radiation dose associated with CT measurements. It is also less than that of many other commonly used radiographic diagnostic tests. Therefore, bone measurement determinations using QCT, like those using DXA, do not expose children to amounts of ionizing radiation that deviate from the amount that constitutes part of their normal life experience. Analysis of BMD and bone size by CT requires the availability of a CT scanner and a proper monitored setting. Recently, smaller, portable, and less expensive QCT devices have been introduced [106]. These scanners are not designed for axial skeleton examination, but they assess the bones of the appendicular skeleton: for this reason, the technique has been called peripheral QCT. pQCT measurements are performed at the forearm and at femur with a small CT apparatus equipped with a low-energy X-ray tube. Calibration of the system is not achieved simultaneously at each scan, but it is done periodically with a specifically designed phantom. A scoutview is performed to localize the measurement site, and usually a single 2-mm-thick tomographic slice is taken (Fig. 8). The pixel size for the newer pQCT devices is as small as 0.4mm. Scanning time ranges from 2 or 3 min in smaller children to 4 or 5 min in adults.
FIGURE 8
31 1
Example of a pQCT scan image.
Similar to QCT examinations, several bone measurements are performed with pQCT. Cross-sectional area (CSA) is calculated after detecting the outer bone contour at a threshold of 280 g/cm 3. Total BMD (vBMDtot) is defined as the mean density of the total cross section, whereas trabecular BMD (vBMDtrab) is determined as the mean density of the 4% central area of the bone's cross section. The pQCT system also provides a parameter defined as cortical + subcortical BMD (vBMDcort), which represents the mean density in the 55% peripheral bone area. pQCT bone measurements show good precision. The CV values for CSA, vBMDtot, vBMDtrab, and vBMDcort measurements are 1.4,1.5,0.8, and 1.1%, respectively [106]. Accuracy of pQCT measurements, evaluated with a specifically designed phantom [107], showed low errors for vBMDtot and vBMDtrabparameters (2.6 and 1.9%, respectively) and higher errors for CSA and vBMDcort variables (10 and 14%, respectively) [ 106]. The radiation dose measured at the skin level for a single pQCT scan is 0.3 mSv, corresponding to an effective dose of approximately 0.01 mSv (Table 1). In healthy children, studies using QCT showed that pubertal development plays a crucial role in the increase of cancellous bone density during growth [108-111]. In both sexes, a remarkable increase in BMD measurements is observed after the onset of puberty (Tanner stage Ii), reaching a peak at approximately the time of cessation of longitudinal growth and epiphyseal closure [112]. Interestingly, different results have been obtained with pQCT. No age-dependent changes of vBMDtrab have been observed in girls [106,113], whereas a slight increase has been documented in boys at Tanner stage III [106]. In children, the material density of cortical bone in the appendicular skeleton when measured by QCT remains fairly constant (2.00 + 0.065 g/cm 3) throughout childhood and adolescence [99,111]. Similarly, cortical bone
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density values measured by pQCT remain stable between 6 and 15 years of age and show moderate increase after age 15 to reach adult levels [106]. In contrast, age-related increases have been reported in a study in which cortical bone density was evaluated at the distal radius by pQCT [113]. QCT studies have also shown a greater increase in vertebral cancellous bone density in African American girls than in Caucasian girls during the later stages of puberty [108,109]. Other studies using QCT analyzed the influence of gender on the amount of bone that is gained during childhood and adolescence [104,105]. QCT has established that the lower vertebral bone mass of females, compared with that of males, results from early gender differences in the size of the bones rather than differences in cancellous bone density [98,104]. Even after accounting for differences in body size, the cross-sectional area of the vertebral body is approximately 20% smaller in girls than in boys [104]. In contrast to these findings, the cross-sectional area at the midshaft of the femur does not differ between boys and girls matched for age and anthropometric parameters [105]. QCT values for the size and the amount of bone in the appendicular skeleton, however, correlate strongly with all anthropometric indices, suggesting that weightbearing and mechanical stresses are the major determinants of the increases in size and volume of cortical bone during growth [110]. Moreover, evidence suggests that insulin-like growth factor-1 is a major determinant of bone dimensions, but it does not influence the material density of cortical bone [114]. Bone dimensions measured at the distal radius by pQCT increase markedly during growth. CSA doubles between 6 and 15 years of age in both sexes, and it does not show major changes after age 15 [106]. In contrast, cortical thickness increased little between 6 and 13 years in girls and 6 and 15 years in boys and showed rapid changes in both sexes up to 18 years of age [115]. Quantitative Ultrasound The use of ultrasound as a medical diagnostic procedure began soon after the end of World War II, expanded greatly in the 1950s, and is currently the second most widely used diagnostic imaging modality. In 1984, Langton et al. [116] theorized that the properties of ultrasound waves might make it suitable for the clinical assessment of bone status; since then, many studies have been conducted both in vitro and in vivo to validate this technique. When applying ultrasound measurements for the study of bone, the results are shown as pure number or graphs rather than images. Hence, this technique is referred to as QUS. Ultrasound is a mechanical wave with frequencies extending from 20 kHz to 100 MHz. Its energy is trans-
mitted through a medium (bone) and can be quantified by appropriate receivers. As the mechanical energy of the ultrasound wave interacts with the bone, the cortex and the trabecular network vibrate, altering the shape, intensity, and speed of the wave. The first generation of QUS systems characterized the bone tissue using two relevant parameters: the speed of sound (SOS) and the attenuation of the signal [broadband ultrasound attenuation (BUA)]. The velocity of an ultrasound wave depends on the properties of the medium through which it is propagating and its mode of propagation [119]. The complex nature of bone makes it difficult to model the relationship between the mechanical properties of bone and the ultrasound velocity. Nevertheless, under simplified conditions it can be represented by the following equation: SOS - (E/p) l/a, where E is the modulus of elasticity (a measure of resistance to deformation), and p is the physical density of bone. The SOS through bone is obtained by dividing the distance traversed by the transit time and is expressed in meters per second. When sound propagates through a material, some of its energy is transferred to the tissue--a phenomenon called attenuation. Attenuation of ultrasound beams occurs as the result of diffraction (beam spreading), scattering, and absorption. The latter is the transfer of the ultrasound energy to the tissue (generating heat), whereas scattering is the re-emission of waves in all directions by the internal structures of the medium. The amount of scattering depends on a number of factors, including the structure, the specific acoustic properties of the medium, and the wavelength of the ultrasound signal used [120]. In ultrasound measurements performed in vivo, it is not possible to separate absorption from scattering, resulting in measurement of total attenuation. Nevertheless, in a study of bone parameters, it is claimed that the bone architecture is responsible for the scattering mechanism [121]. The specific roles of scattering and absorption in determining the overall attenuation are not completely known. In some studies, the frequency dependence of attenuation observed in cancellous bone has been attributed mainly to absorption [120,121]. Conversely, in other studies, energy losses by scattering in the internal structure of cancellous bone or friction at the bone marrow interfaces have been considered the main cause of attenuation [122-124]. Attenuation strongly depends on the frequency of the ultrasound wave employed. With the use of a lowfrequency range (200-600 kHz), attenuation is almost a linear function of frequency. In QUS, attenuation is measured over a low-frequency range, and the slope of the regression line is the BUA value, given in decibels per megahertz. Some QUS devices provide a combined measurement called stiffness or quantitative ultrasound index. This
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parameter is mathematically calculated from both SOS and BUA values, and it is claimed to simplify the interpretation of the two single parameters. Newer-generation QUS devices and ongoing research on the properties of ultrasound waves for studying bone have introduced new or modified ultrasound parameters. The observation that the amplitude of the ultrasound signal decreases with the increase in bone porosity led to the identification of an amplitude-related measurement of SOS. This new parameter (AD-SOS) is able to magnify the differences in SOS as measured in diverse bone statuses (i.e., normal or osteoporotic bone) [125,126]. AD-SOS is expressed in meters per seconds. In other QUS devices, ultrasound velocity is measured as apparent velocity of ultrasound (AVU), which differs from true velocity by approximately 100 m/sec. The difference between the measured and the true velocity occurs because the detection algorithm was designed to respond to a prominent, easily recognizable portion of a signature waveform, ignoring the first part of the signal. This is done to obtain a more precise measurement of arrival time of the ultrasonic pulse [127,128]. The systematic study of the morphology of the received ultrasound signal enabled the identification of an association between specific ultrasound parameters and the physical characteristics of bone (mechanical resistance, structure, elasticity, and fragility). One of these parameters has been defined as the bone transmission time (BTT) or time frame, which represents the time interval between the first received signal and the speed value of 1570 m/sec, expressed in microseconds [129]. Ultrasound equipment consists of two transducers, a transmitter and a receiver, that are placed on opposite sites of the bone of interest (Figs. 9 and 10). The ultrasound wave produced by the transmitter crosses the
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bone and is received by the second transducer. All of the devices that study the crossing of the skeletal segment by ultrasound require that the bone surface is homogeneous and level and that the surfaces on which the signal enters and exits are parallel. Moreover, soft tissue thickness should be minimal because it affects the velocity and attenuation of the signal. As in all other ultrasound applications, to enhance the transmission of the ultrasound wave, a coupling medium is used between the transducers and the skin. In general, QUS systems use water or ultrasound gel as coupling substances (Fig. 9). Because the ultrasound wave crosses the bone being examined, the information pertains to both cortical and cancellous portions. QUS devices that combine the transmitter and the receiver in one probe have recently been introduced. These systems measure the ultrasound velocity longitudinally along the selected skeletal site (Fig. 10), giving information only on the bone cortical shell [130,131]. The first QUS devices measured velocity and attenuation in the heel, and for years ultrasound investigations concentrated on the calcaneus. The number
FIGURE 10 Single-pointQUS system for measurements of ultrasound parameters at the finger phalanxes using gel coupling.
FIGURE 9 Schematicrepresentation of fixed single-point transmission devices employing different coupling substances. (Left) A water bath-based foot placement; (right) coupling by means of ultrasound gel.
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F I G U R E 1 1 Assessment of u l t r a s o u n d velocity parameters of cortical shaft using online transducers. Coupling is obtained with ultrasound gel.
TABLE 3 Reproducibility of ultrasound measurements at different skeletal sites. Site
CV%
9 C V % in children r
Calcaneus BUA
0 . 8 % - 2.5%
1 . 4 % - 5.4%
SOS
0.2% - 0.6%
0.14% - 0.64%
Patella
1.5 %
0.49%
Tibia
0.2% - 0.7%
0.43%
Radius
0.4 - 0.8%
Phalanges
0.5% - 1.1%
0.55%
of skeletal sites chosen for QUS studies has gradually expanded to include both short bones (calcaneus and patella) and long bones (hand phalanges, tibia, and radius). The values of reproducibility for the various Skeletal sites are shown in Table 3. CV values obtained in children are comparable to those computed in adult patients. As for adults, BUA measurements in the heel usually show poorer precision than SOS. Precision is affected not only by the skeletal site chosen but also by the QUS device, the coupling medium, and repositioning errors. When using a water bath as the coupling medium, factors that may affect precision include immersion time of foot, water depth, and water temperature. Foot positioning is critical for the QUS measurements of the heel because of the inhomogeneity of the calcaneus. Rotation of the foot about the axis of the leg significantly affects BUA measurements [132]. Improved precision is obtained with the newer devices (using coupled or in-line transducers) by rotating the probe around the long bone and by averaging the readings of several scans (Fig. 11). As stated earlier, ultrasound examination of the bone does not assess a single quantitative parameter such as bone mineral content, but QUS measurements combine several data, including information on the structure of
bone. Therefore, for QUS bone measurement it is not possible to assess the accuracy of each method, given the lack of appropriate correlate for which to control. QUS devices have several advantages compared to X-ray-based techniques in terms of cost and health risks. QUS equipment is smaller in size and usually mobile. On average, the cost does not exceed half the cost of a DXA scanner, and maintenance is less expensive. Obviously, the absence of ionizing radiation makes the method suitable for use even in small subjects and critically ill patients. The use of QUS equipment does not require a controlled environment or specific authorization by health or radiation safety authorities. There are few studies assessing the possible relations between QUS parameters and age or pubertal status, and comparison of their results is complicated by the heterogeneous skeletal sites measured and by the use of different QUS devices within the same site [133-145]. The majority of studies evaluating BUA values measured at the calcaneus show an increase with age [134,137-139,142]. The increase in BUA was accelerated during adolescence in some studies [138] and constant in others [139,142], whereas no further increase was reported in late adolescence [142]. In a recent study, no substantial age-related changes were observed [144]; additionally, heel QUS parameters did not correlate with skeletal age. Different results have been found for SOS calcaneal measurements. The first published study demonstrated an age-related increase of SOS values that peaked at puberty and plateaued thereafter [133]. Similarly, SOS values at the heel have been found to increase with age [134,138, 142]. Nevertheless, two studies (one performed in a very large sample of approximately 1800 healthy children and adolescents) found that calcaneal SOS values remained constant during growth in both sexes [139,144]. When healthy subjects were grouped according to their stage of sexual development, Tanner stage was found to be a better predictor of QUS parameters than chronological age when measured at the calcaneus [144]. In general, no relevant sex differences were observed in calcaneal QUS measurements. In some studies, sex differences were present only during the adolescence period because of the different tempo of onset of puberty in the two sexes [135,140,141,143]. Few studies have documented changes in QUS parameters at the patella [133,135,140]. SOS values increased with age, reaching a peak at 20-25 years of age [133]. When AVU was measured in another study, changes related to pubertal development were observed [135]. In particular, AVU values increased from Tanner stage I to stage V, and sex differences were present only at stages II and III. The same authors examined a subgroup of subjects 3 years after the first study [140]. This longitudinal investigation showed that the patella AVU
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rate of change was greater at ages 10.5-13.5 in girls and between 14.5 and 17.5 years in boys. Moreover, AVU values increased significantly with increasing level of Tanner stage in both sexes. The amplitude-dependent speed of sound measured in the phalanxes in a very large number of healthy subjects was found to increase progressively from 3 to 21 years of age [143]. The authors observed a remarkable increase of AD-SOS between ages 15 and 16. When the QUS data were analyzed according to pubertal development, significant increases in AD-SOS values were found between Tanner stages III and V in boys and stages II and IV in girls. The same QUS device was used to study a group of term newborns and to compare the results with those obtained from a group of preterm infants [146]. Because the phalanxes of infants are much smaller than the width of the calipers, the authors adapted the instrument to study the humerus of these subjects. Preterm infants showed significantly lower SOS and BTT values than infants born at term. Measurements of SOS at the cortical shaft of the tibia in a large sample of healthy children and adolescents showed a strong age dependence [141]. SOS values increased progressively in boys and girls, reaching a plateau in girls after age 17. Significant increases were noted at Tanner stage II in girls and boys. The use of a unique probe for the measurements of SOS in the cortical shaft enables the study of this ultrasound parameter even in small subjects. Probes of appropriate size may be used for the measurement of QUS parameters in the long bones of newborns and very small infants born before term (S. Mora and R. Rovelli, unpublished observation). One study compared the results obtained with two different QUS devices that measured SOS at the calcaneus and the tibia [144]. The authors did not find a relationship between the two measurements, showing that QUS measurements are site specific. Comparison between QUS and other techniques for bone mass measurement has been performed in three studies [136,138,145]. In a small sample of healthy youth, total body BMD values were found to correlate with BUA measurements obtained at the calcaneus [136]. In another study, DXA measurements obtained from the spine, the femoral neck, and the whole skeleton showed a relationship with SOS and BUA values obtained at the os calcis [138]. Finally, a moderate direct correlation was found between tibial SOS values and BMD measured at the midphalanxes by radiogrammetry [145]. Magnetic Resonance Magnetic resonance offers unique qualitative capabilities to noninvasively evaluate the muscoloskeletal system, including assessment of compositional changes
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in the bone marrow and structural changes in osseous tissue. This technique has profoundly changed medical imaging in general by the application of high magnetic fields, transmission of radiofrequency (RF) waves, and detection of RF signals from excited hydrogen protons. Recently, the capabilities of MR have been explored in the study of osteoporosis. The most widely used application of MR in the diagnosis of osteoporosis does not rely on the quantitation of bone mass, but in the detection of osteoporotic bone fractures [147-149]. However, MR has also been proposed as a technique for providing information related to bone density as well as structure. Direct MR measurement of mineralized elements has been relatively unsuccessful because bone tissue is a solid material that has low mobile proton density. Therefore, quantitative information on cortical bone is limited to the thickness of the cortex and the amount of cortical bone in a given volume. Trabecular bone can be assessed indirectly by measuring the surrounding bone marrow. This method is based on evidence that near the boundary of two physical phases of different magnetic susceptibility (trabecular bone and bone marrow), the magnetic susceptibility of trabecular bone is much smaller than that of the bone marrow. This causes spatial inhomogeneities in the static magnetic field, the magnitude of which depends on the number of bone/marrow boundaries, the surface size of trabecular structures, and the field strength. The magnetic quality of the protons within the field inhomogeneities does not contribute to the measurement of field strength, causing a decrease in T2 relaxation times. Wider gaps between trabecular structures cause magnetically more homogeneous areas. The effect of these inhomogeneities principally used for measurement is dephasing of the transverse magnetization, leading to a decrease in the T2* relaxation times and a decrease in signal intensity for gradient echo images. Shortening of relaxation time becomes greater with an increase in the concentration of trabecular bone in the surrounding homogeneous marrow tissue. Thus, in normal dense trabecular network, T2* shortening should be more pronounced than in rarefied osteoporotic trabeculae. The relationship between bone density and T2* relaxation time has been studied in vitro as well as in vivo [150-156]. Recently, the relationships between bone mineral measurements obtained with standardized techniques (i.e., DXA, QCT, and QUS) and T2* relaxation time have been extensively studied [157-161]. MR microscopy may be an additional MR-based technique to study trabecular microarchitecture in a quantitative manner both in vitro and in vivo. In vitro, using small RF surface coils in high-field scanners, MR microscopy can be performed at resolutions sufficient to discriminate individual bone trabeculae [162,163]. The
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resolution in vivo is lower, but images have been obtained at resolutions of 78 x 78 x 300 Ftm in the phalanges [164], 156 x 156 x 700 gm in the distal radius [165], and 200 x 234 x 1000gm in the calcaneus [166]. However, the appearance of the image in MR depends on several factors other than image resolution. The pulse sequence used to obtain the image (whether a spin echo or gradient echo), the echo time, and the magnetic field strength are all important factors that may modify the trabecular dimensions depicted in MRI [167]. Repeatability and accuracy have been assessed, as has the possibility of using MR to monitor changes occurring after heart surgery [168,169]. Another approach has been proposed for the evaluation of bone trabeculation [170-172]. This technique uses phase images of a gradient echo sequence. In phase images, the gray level of the pixel represents the phase of the magnetization in the voxel. By measuring the variance of the mean phase per voxel in a ROI of a phase image, information on the phase distribution can be obtained. In vivo studies on the distal femur and the distal radius [171,172] have shown that the distribution of phases is related to the amount of bone as well as structure parameters, such as trabecular number, thickness, spacing, and fractal dimension [173]. Unfortunately, this technique shows a strong dependence on the imaging parameters. Ongoing in vitro and in vivo research is focused on solving this problem [174,175]. MR is a potential tool for assessing trabeular bone structure in vivo. It is a noninvasive, nonionizing technique and it can provide three-dimensional images in arbitrary orientations. Moreover, it can depict trabecular structure. Furthermore, it provides a platform for monitoring in vivo structural changes of trabecular bone; therefore, it may help in understanding the pathophysiologic changes of various diseases. However, high costs and technical problems limit the use of MR as a competitive technique for bone measurements.
DATA INTERPRETATI O N The ideal densitometer for pediatric use would assess true volumetric BMD, bone size, and trabecular and cortical compartments at all skeletal sites. The method would accomplish all this with speed, precision, low cost, and little or no exposure to ionizing radiation. A large set of normative values from a representative population of healthy youth would be available for comparison. Finally, densitometry results would be predictive of clinical outcome such as low trauma fracture. Since none of the currently available techniques fit this tall order, the choice of methodology depends on the clinical or research
question to be addressed. Awareness of the complexities of pediatric bone densitometry will enhance the value of this diagnostic tool while reducing the likelihood of misdiagnosis of skeletal fragility states in the growing child. Limitations and A d v a n t a g e s of Available Techniques All currently available techniques for assessing bone mass in children have advantages and limitations. Comparison between these techniques, which are totally dissimilar in the way they acquire data, is difficult; often, judgment regarding their value has been at least partially subjective. In this section, a synthesis of advantages and limitations intrinsic in each technique is presented, emphasizing the potential biases that might affect bone measurements and, when possible, the solutions proposed to overcome such problems. Radiogrammetry and radiographic absorptiometry are popular techniques for measurements of bone because of their ease of use, low cost, and the ubiquitous availability of X-ray equipment. These techniques can be used for the assessment of bone status where more sophisticated devices are unavailable, and the analysis of the radiographs can be performed off site, thereby reducing additional costs. The main limitation of radiogrammetry and radiographic absorptiometry for bone measurements is that the values are mainly obtained from the long bones in the hand; because of the great variability in manual dexterity among humans, they do not reflect mineralization at other skeletal sites. Bone mass measurements obtained with DXA in children have the advantages of low cost, accuracy, and low radiation exposure. Theoretically, this technique would be most suitable for longitudinal monitoring of bone mineral changes, especially in chronic diseases. However, several limitations should be stressed with reference to the use of DXA in growing individuals, when major changes in body composition, body size, and skeletal mass occur. First, DXA is a projection technique, and its measurements are based on the two-dimensional assessment of a three-dimensional structure. Thus, DXA values are a function of three skeletal variables: the size of the bone, the volume of the bone examined, and its mineral density [15]. Because bones with equal mineral density but different dimensions give different results with DXA [15], the influence of bone size on these measurements needs to be accounted for. Unfortunately, corrections of BMC data obtained by dividing the mineral content by the surface area of the bone examined [bone area (BA)] are not sufficient and are prone to error. Several authors have therefore tried to develop mathematical models that account for the dimensions of the bone~that is,
12. Noninvasive Techniques for Bone Mass Measurement
the cross-sectional area of the vertebra is shaped like a cube [15,176,177], a cylinder with a circular base [57,64, 177, 178], or a cylinder with an elliptic base area [179, 180]. Similar closed formulas have been proposed for the femur and the midradius [57,177,178]. Other approaches for correcting the influence of bone geometry on DXA values take into account the height of the child [77]. In one technique, all DXA BA measurements are multiplied by the height of the subject, producing a new variable called corrected BMD (= BMC/BA x height) and expressed as grams per centimeter 3. A more complex approach was presented by a British group that studied the relationship between BMC and projected BA and developed the following equation: BMD = BMC/ BA h, where A is the exponent to which BA is raised to remove its influence on BMC [181]. The value of A changes with anthropometric measurements and/or pubertal stage. Similarly, an intricate but effective approach to solve the problem of body and bone size-dependent measurements has been proposed by a Danish group [81]. After having analyzed numerous whole body DXA scans, this group developed percentile curves for BA and age, BMC and age, BA and height, and BMC and BA. DXA results for each individual are plotted on height for age, BA for height, and BMC for BA curves. The three steps correspond to different causes of reduced bone mass, such as short bones, narrow bones, and low-density bones. Lastly, when comparing different groups of subjects, a simple approach is that of using multivariate analyses, where BMC is the dependent variable, and BA, anthropometric measurements, age, and gender are the confounding variables [182]. This approach does not make assumptions about correction factors, and it allows for simultaneous correction of different variables, but it is not applicable to single measurements. Second, another source of error for DXA bone mineral analysis is the use of inappropriate software for both the acquisition and the subsequent analysis of the data. The standard software from most DXA manufacturers has been designed for adults, although several manufacturers have developed software to be used in pediatrics. The use of pediatric software has improved the precision and accuracy of examinations in small subjects, but discrepancies have been noted when comparing the adult software to the pediatric software [183,184]. These differences may affect longitudinal studies of skeletal growth and the compilation of reference data from infancy through adulthood. Pediatric software usually requires longer scanning time, making cooperation from young children difficult and resulting in motion artifacts, which may markedly alter DXA values [185]. Third, inaccuracies of DXA measurements can result from the nonhomogeneous distribution of soft tissues
31 7
adjacent to the bone being analyzed. Because corrections for soft tissue are based on the assumption of a homogeneous fat distribution around the bone, changes in DXA values are observed if fat is distributed inhomogeneously around the bone. Inhomogeneous fat distribution may influence DXA measurements by as much as 10% [186]. Although this is not of concern when studying subjects whose weight and body composition remain fairly constant, longitudinal DXA values in children may be subject to considerable error and measurements may reflect the changes in body size and composition occurring during growth rather than true changes in bone density. Fourth, interpretation of DXA measurements of the total body should take into account the finding that BMD values for the skull explain most of the variance of total body BMD values [187]. Because head BMD is poorly related to age, height, or weight, the inclusion of this variable in the calculation of total body BMD contributes to the low predictive value of age and anthropometric measurements for DXA total body measurements. Lastly, as previously described, newer DXA scanners utilize a fan beam approach to greatly reduce scanning time. Fan beam X-rays produce a distortion of the image of the bone scanned, which is inversely proportional to the distance between the bone examined and the detector and to the size of the bone. When fan beam densitometers are used in small subjects, the chance of greater distortion is enhanced and increases the probability of errors in the bone measurements. The ability of QCT to assess both the volume and the density of bone in the axial and appendicular skeletons, without influence from body or skeletal size, is the major advantage of this modality when used in children. Unfortunately, the cost and inaccessibility of CT scanners have markedly limited its use for bone measurements. In addition, although with low settings CT exams can be performed with a very low level of radiation exposure, the dose is higher than that associated with DXA. CT scanners are expensive, large, nonportable machines that require costly maintenance and considerable technological expertise for proper function. Moreover, this equipment is usually located in the radiology department and is under constant clinical demand, resulting in a lack of accessibility. These disadvantages have been partially overcome by the recent development of smaller, mobile, and less expensive peripheral QCT scanners. Precision of trabecular bone QCT measurements is greatly affected by volume averaging. Cancellous bone exists as a three-dimensional lattice of plates and columns (trabeculae). The trabeculae divide the interior volume of the bone into intercommunicating pores, which are filled with a variable mixture of red and yellow marrow [188]. Because of the relatively small size of the trabeculae when compared to the pixel, the CT unit of
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measurement, QCT values for cancellous bone density reflect not only the amount of mineralized bone and osteoid but also the amount of marrow per pixel [189]. Similar limitations apply to in vitro determinations of the volumetric density of trabecular bone that are obtained by washing the marrow from the pores of a specimen of cancellous bone, weighing it, and dividing the weight by the volume of the specimen, including the pores [188]. Both QCT and in vitro bone density determinations of cancellous bone are directly proportional to the bone volume fraction and inversely proportional to the porosity of the bone. The relatively large coefficient of variation for values of cancellous bone density reflects the considerable variations in the dimensions of the pores throughout the vertebral body. It should also be noted that the precision and accuracy of QCT for bone mineral determinations depend on the percentage of fat in the marrow. Because the marrow of children contains less fat than the marrow of older subjects, the precision of cancellous measurements by QCT in children is considerably better than that in adults [103]. It should also be noted that when assessing the metaphyseal regions of the long bones, trabecular bone measurements are influenced by cortical bone thickness due to beam-hardening effects or photon scattering. This error is especially prominent in pQCT evaluations of the radius. Previous studies indicate that if the effect of scatter of the X-ray beam is corrected adequately, the accuracy error can be reduced to less than 4% [190,191]. Finally, because diverse calibration standards give different results, it is important that serial evaluations employ the same reference phantom [192]. Advantages for using QUS in children include its low cost, ease of use, and the absence of ionizing radiation. The small dimensions of many QUS devices enhance the portability of the instruments, making this technique ideal for screening large groups of subjects. However, several issues should be considered in the applicability of this method for obtaining bone measurements. Despite extensive research, the question regarding what is really measured by QUS remains unanswered. Although SOS signals are greatly influenced by the material density of bone, BUA depends on many structural parameters that contribute to scattering and attenuation of sound waves. For this reason, it has been suggested that BUA measurements reflect bone structure. However, multiple authors have not been able to extrapolate this assumption to in vivo studies [193,194]. Additionally, QUS measurements seem to be related to bone size [195, 196], and the significant increases of QUS values noted during childhood may be related more to changes in skeletal size than to changes in bone structure or density. Technique also affects QUS measurements. Foot positioning is considered to an important source of error in
BUA assessment of the calcaneus, resulting in nonhomogeneous spatial distribution of calcaneal trabeculae. This is especially important in pediatric subjects because reproducibility of foot positioning in the very young is difficult, limiting the value of QUS in pediatric longitudinal studies. Like BUA, SOS measurements are affected by the position of the transducers along the bone examined. In an attempt to overcome these limitations, a rigid protocol for positioning should be followed, regardless of the site of measurement and the QUS device used. QUS measurements are commonly performed at skeletal locations where the interference of soft tissue is minimal, such as the os calcis, the patella, and the phalanxes. Unfortunately, it is not known how representative the determinations at these skeletal sites are for the entire skeleton. This potential problem may be overcome with the newer QUS devices that are able to measure SOS at various skeletal sites, including the radius and the tibia [198]. Nonetheless, bone measurements by X-ray-based devices known to predict fracture risk have correlated poorly with those obtained with QUS [197]. N e e d for E n h a n c e d R e f e r e n c e D a t a for All M e t h o d s Comparisons with appropriate pediatric bone mineral reference data are essential to accurately describe the clinical impact of a disease on bone development; to monitor changes of BMC, BMD, or QUS variables; and to identify patients for treatment protocols. Misclassification of a bone mineral measurement may lead to unnecessary testing, parental anxiety, alteration in treatment of underlying disease, initiation of needless treatment, or the absence of intervention. The use of appropriate reference data has been stressed by a survey that compared the results of DXA examinations with different normative data sets [199]. Great variation in classification of children with various diseases was observed, and the need for race- and sexspecific data sets has been enforced. The classification of osteoporosis or osteopenia in adults is based on the results of t or z scores, obtained respectively in young adult populations and age-matched individuals. The use of z scores for children is not recommended for bone mineral measurements that are influenced by the size of the bones. However, an interesting approach was recently proposedmthe use of z scores derived from a prediction model that takes into consideration the age, height, and ethnicity of the subjects [200]. Because there are important differences among the sites of measurement, calibration, and software applications in devices belonging to the same group of bone measurement technique, it is advisable to create normative data sets specific for each instrument and for differ-
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ent populations. Although this is of pivotal importance for optimal comparison, it represents only one issue of a more complex problem. In fact, bone variables are measured in a population of growing individuals, who show a wide spectrum of growth velocity due to individual physiological variations and to disease interference. Therefore, chronological age- and sex-matched normal values are not sufficient to correctly classify bone measurements. For a correct comparison of individual measurements, the following variables should be determined: weight and height, skeletal age, and pubertal stage. Whenever one of these variables is advanced or delayed compared to reference data, the comparison with norms should be corrected and chronological age-matching should not be employed. The ultimate test of any measurement of bone mass is its ability to predict clinical bone fragility. In adults, BMD has been shown to be a very significant correlate of fracture. However, the risk of fracture for children whose bone densities decline 1 or more SDs below the mean for age, bone age, or body size has not been established. The challenges for pediatric bone research are to refine noninvasive tools for bone mass measurements; to enrich the normative data from healthy youth; and to optimize the methods used to adjust for bone size, maturity, and geometry.
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ll3[ A s s e s s m e n t of Maturation Bone Age and Pubertal Assessment NOF_L CAMERON Human Biology Research Centre, Department of Human Sciences, Loughborough University, Loughborough, Leicestershire, United Kingdom
BACKGROUND
INITIAL C O N S I D E R A T I O N S
The process of maturation is continuous throughout life: It begins at conception and ends at death. This chapter focuses on the assessment of the process of maturation from birth to childhood--that is, the part of the total process that is intimately linked to physical growth. It is therefore important to differentiate between "growth" and "maturation." Bogin [1] defines the former as "a quantitative increase in size or mass," such as increases in height or weight. Development or maturation, on the other hand, is defined as "a progression of changes, either quantitative or qualitative, that lead from an undifferentiated or immature state to a highly organized, specialized, and mature state." The end point of maturation, within the context of growth, is the attainment of adulthood, which I define as a functionally mature individual. Functional maturation, in a biological context, implies the ability to successfully procreate and raise offspring who themselves will successfully procreate. In addition to the obvious functional necessities of sperm and ova production, reproductive success within any mammalian society is also dependent on a variety of morphological characteristics, such as size and shape [2,3]. The too short or too tall, and the too fat or too thin, are unlikely to achieve the same reproductive success as those within an acceptable range of height and weight values that are dependent on the norms in a particular society. Thus, in its broadest context, maturation and growth are intimately related and both must reach functional and structural end points that provide the opportunity for successful procreation.
In order to understand how maturation can be assessed, it is important to first appreciate that maturation is not linked to time in a chronological sense. In other words, 1 year of chronological time is not equivalent to 1 year of maturational "time." This is perhaps best illustrated in Fig. 1, in which three boys and three girls of precisely the same chronological ages demonstrate dramatically different degrees of maturity as evidenced by the appearance of secondary sexual characteristics. In addition, they exhibit changes in the proportion and distribution of subcutaneous fat, and the development of the skeleton and musculature, that result in sexually dimorphic body shapes in adulthood. Although individuals have passed through the same chronological time span, they have done so at very different rates of maturation. Second, maturation is most often assessed by the identification of maturity indicators. Such indicators are discrete events or stages recognizable within the continuous changes that occur during the process of maturation. Thus, the indicators that identify changes in the radiographic appearance of the radial epiphysis or changes in breast or pubic hair development divide the continuous changes that occur during skeletal and sexual maturation into discrete stages. Third, there is variability of maturation within the individual. For instance, although skeletal and secondary sexual maturation are associated, they are not correlated so significantly that one can categorically associate a particular stage of sexual maturation with a
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No~! Cameron Fifth, there is clear sexual dimorphism within human growth and maturation such that females tend to be advanced relative to males at any particular chronological age. In Fig. 1, for instance, the females are aged exactly 12.75 years and the males 14.75 years, but their levels of secondary sexual development are similar. Sixth, maturation is not related to size except in very general terms; a small human is likely to be a child and thus less mature than a large human, who is more likely to be an adult. As the ages of the two individuals become similar, the distinction between size and maturity narrows and disappears such that within a group of similar maturity there will be a range of sizes and within a group of similar size there will be a range of maturity levels. Thus, when maturation is assessed, size must be controlled for or excluded from the assessment method. These six considerationsmthe relationship of maturity to time, the quantification of the continuous process of maturation by using discrete events, the relative independence of different processes of maturation within the individual, the appreciation of uneven maturation, sexual dimorphism, and the lack of a relationship between maturity and size--have governed the development of techniques for the assessment of maturation.
The Concept of Time Acheson [6] elegantly described the problem of time within the development of skeletal maturity assessment methods as follows: FIGURE 1 Three normal boys aged 14.75 years and three normal girls aged 12.75 years demonstrating dramatically different stages of maturation [reproduced with permission from Tanner, J. M. (1975). Growth and endocrinology of the adolescent. In Endocrine and Genetic Diseases of Childhood (L. Gardner, Ed.), 2nd ed. Saunders, Philadelphia].
Because maturation is distinct from growth it merits a distinct scale of measurement; indeed the whole basis of the medical and scientific interest it attracts is that it does not proceed at the same rate in the various members of a random group of healthy children. The corollary of this is that the unit of measurement, "the skeletal year," does not have the same meaning for any two healthy children, nor even.., does a skeletal year necessarily have the same meaning for two bones in a single healthy child. (p. 471)
particular skeletal "age" [4,5]. In the closest association, skeletal age with menarcheal age, it is possible to state that a girl with a skeletal age less than 12 years is unlikely to have experienced menarche and that one with a skeletal age of 15 years is likely to be postmenarcheal. However, it cannot be stated with any real degree of confidence that the association of these two maturational processes is closer than that. Fourth, within a particular maturational process, such as sexual maturation, it is apparent that different structures (e.g., genitalia and pubic hair) will not necessarily be at precisely the same level of maturity. Similarly, within a particular anatomical region, such as the hand and wrist, all bones will not be at precisely the same stage of maturation. Thus, there is a process of uneven maturation.
The core problem is the use of an age scale to represent maturity. This fails at the extreme because no particular age can be associated with full maturity, and it fails prior to full maturity because of the lack of a constant relationship between maturity and time both between and within the sexes. Thus, when using the Greulich-Pyle atlas technique for skeletal maturity [7], one is using the final standards for males and females that correspond to an age of 18 years but that in fact represent full maturity or the maturity found in any individual who has achieved total epiphyseal fusion regardless of his or her actual chronological age. Acheson [8,9], and Tanner and colleagues [10-13] overcame this problem in the assessment of skeletal maturity by moving away from an age-based method and
13. Assessment of Maturation developing the bone-specific scoring techniques in which numerical scores are assigned to each bone rather than a bone age. Acheson's early attempt, which became known as the Oxford method, simply gave scores of 1,2,3, etc. to each stage. However, this scoring method did not account for the fact that the differences between scales were not equivalent; the difference between stage 1 and stage 2 was not necessarily equivalent in terms of the advancement of maturity to the difference between stages 2 and 3. Tanner's basic principle was that the development of each single bone, within a selected area, reflected the single process of maturation. Ideally, the scores from each bone in a particular area should be the same and the common score would be the individual's maturity. However, such scores would not be the same because of the large gaps between successive events in a single bone. Thus, the scoring process would need to minimize the overall disagreement between different bones, which is measured by the sum of squares of deviations of bone scores about the mean score. In order to avoid what Tanner described as the "trivial solution" of perfect agreement by giving the same scores to each stage, the scores were constrained on a scale of 0 to 100 (i.e., each bone starts at 1 and matures at 100). In essence, each maturity indicator is rated on a maturity scale from 0% maturity to 100% maturity. Without dwelling on the mathematics, which are given in detail by Tanner and colleagues [12,13], the principle is an important one and should be applied to any new system of assessing maturity. In addition, the bone-specific scoring approach can be applied to an appropriate sample of radiographs from any population to derive maturity norms. The principle of scoring maturity indicators was applied to the assessment of dental maturity by Demirijan et al. [14], but it has not been applied to other attempts at maturational assessment such as for secondary sexual development. This apparent neglect may be due to the fact that we still use the staging system originally developed by Nicholson and Hanley [15] and modified by Tanner [16]. Only five stages are used within any particular area, and these are often difficult to assess accurately. Also, secondary sexual development occurs over a relatively short period of time (e.g., between 10 and 17 years in girls)compared to the birth-to-adulthood temporal basis of skeletal maturity. Thus, there are fewer maturity indicators within a short period of time and the application of a scoring technique has seemed inappropriate. Maturity
the identification of maturity indicators, skeletal maturity was assessed by the number of ossification centers method in which a count was made, either from the hand and wrist [19,20] or from a skeletal survey of each child [21], of the number of centers that were present or absent in the total skeleton. Alternatively, planimetry was used to assess the total amount of bony tissue apparent in radiographs [22-24]. The former method failed because of a lack of appreciation of the fact that the order of appearance of ossification centers is largely under genetic control [25]. The latter method failed because only the carpus was used, which, as now known, is not representative of overall maturity. Todd identified determinators of maturity within the changing radiological appearance of long and short bones. Greulich and Pyle [7] later defined these maturity indicators as "those features of individual bones that can be seen in the roentgenogram.., and which, because they tend to occur regularly and in a definitive and irreversible order, mark their progress towards maturity." Although this concept of maturity indicators was primarily applied to the skeleton, it is apparent that such indicators are also visible in other aspects of maturation. Figure 2 illustrates the maturity indicators for the developing radius identified independently by two groups of researchers--Greulich and Pyle (GP) [7] and Tanner, Whitehouse, and Healy (TW) [10]. Both groups examined the development of the radius apparent in radiographs of the left hand and wrist of children from birth to adult maturity. The former group identified 11 indicators, whereas Tanner et al. described 8. It is apparent, however, that visually at least, the indicators of Tanner and colleagues are not dramatically different from those of Greulich and Pyle. Indeed, it is very important that these indicators are similar. If the two groups of researchers had arrived at very different maturity indicators, within I
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the same skeletal area, then each group would have been identifying different aspects of maturation and this would have cast doubt on our ability to recognize the process of maturation. The similarity of the indicators defined by these two independent research groups illustrates that they were recognizing the same process and using similar criteria to measure its progression. Regardless of the particular maturational process under investigation, the identification of maturity indicators is fundamental to quantifying the process and determining measures of individual and population variation. Maturity indicators must conform to certain prerequisites if they are to be useful, however. They must possess the quality of universality in that they must be present in all normal children of both sexes, and they must appear sequentially, and in the same sequence, in all children. Roche and colleagues added to these criteria while developing the Fels hand-wrist method [26,27]. They tested their maturity indicators for discrimination (the ability of a maturity indicator to distinguish between children of the same chronological age), reliability (good inter- and intraobserver reliability), validity (the ability to reflect genuine maturational change; i.e., they should reflect a continuous process of maturation rather than a discontinuous process), and completeness (prevalence in the population).
Maturational Variation Maturational variation covers two aspects: the variation of maturation within a process and the variation of maturation between processes. The former aspect may be observed within sexual maturation from the data published by Marshall and Tanner [4,5] on British children. They illustrated variation by investigating the percentage of girls or boys in any particular stage of development of one indicator of maturation when they entered a particular stage of another indicator. For instance, 84% of girls were in at least stage 2 of breast development when they entered stage 2 of pubic hair development. In other words, they did not enter pubertal maturation in both breast and pubic hair development simultaneously. Breast development for the vast majority was the first stage of puberty, followed by pubic hair development. Similarly, 39% of girls were already adult for breast development when they became adult for pubic hair development. A similar pattern of variation was observed in males, with 99% of boys starting genitalia development prior to pubic hair development. This variation is critical in that it requires any modification of the method to allow for intraindividual variation. Within clinical situations, for instance, the difficulties in accurately rating the various stages of breast, genitalia, or pubic hair development
within the Tanner 5-point classification have resulted in combination of the stages into a 3- or 4-point "pubertal" staging technique. Thus, variations within individuals between the different aspects of secondary sexual development are impossible to quantify, and in terms of investigating variability in maturation, the pubertal staging technique loses significant sensitivity. The variation of maturation between different aspects of maturity presents difficulties with regard to applying a general maturational level to the individual. For example, entry into the early stages of puberty is not associated with any particular level of skeletal maturity except in the broadest sense. The only exception to this rule, with regard to skeletal and sexual maturation, is menarcheal age, in which skeletal age and chronological age are associated at a level of 0.35 and in which menarche tends to occur at a skeletal age of 12.5-14.0 "years" regardless of chronological age. However, it may be that this apparent lack of association between skeletal maturity and sexual maturity is actually due to our inability to assess sexual maturation as accurately as we can assess skeletal maturity. The maturity indicators for secondary sexual development are far less easily identified, and the apparent changes between adjacent stages are not easily observed.
Sexual Dimorphism Ideally, any method that assesses maturity should be able to assess the same process of maturation in both males and females. Although this criterion is true for skeletal maturity assessment methods and also for dental maturation and perhaps methods that might be developed from mathematical models of the pattern of human growth, it is not true of all aspects of secondary sexual development. In skeletal and dental maturity assessments, sexual dimorphism is accounted for by gender-specific scores for each bone or tooth and in the latter by identifying equivalent functional processes in the different sexes. However, the interpretation of maturation, or the meaning of the attainment of a particular level of maturity, may be different within the sexes. Spermarche and menarche, for instance, are often thought to be equivalent stages of maturation in males and females, but their position within the pattern of growth is quite different; thus, their association with other aspects of maturation also differs. Menarche occurs following peak height velocity and toward the latter part of secondary sexual development (i.e., in breast stages 3-5) [4]. Relatively sparse data on spermarche indicate that it occurs at approximately 14 years in boys, in the early or middle part of the adolescent growth spurt and thus indicative of an earlier stage of pubertal maturation.
13. Assessment of Maturation
Maturity and Size The early methods of assessing skeletal maturity by planimetry used the reasoning that size and maturity were closely related. It is now clearly recognized that, except in very general terms, size does not play a part in the assessment of maturation. However, size does play a role in assessment as a maturity indicator as a ratio measure. For example, the maturity indicator for stage D in the radius of the TW II/III system is the fact that the epiphysis is half or more the width of the metaphysis (i.e., the size is relative to another structure within the same area). However, except for such a ratio situation, the only maturity assessment method that uses a quantitative indicator of maturity is testicular volume; 4 ml represents the initiation of pubertal development and 12 ml midpuberty. This is not to say that there is no variation in testicular volume. Like all aspects of growth and development, variability is an inherent aspect of testicular growth. However, clinicians use the previous measures as indicators of normal testicular growth and the initial and middle stages of pubertal development.
329
west of the 1930s, a socially advantaged group later described by Greulich and Pyle as above average in economic and educational status. From each chronological age group the films were arrayed, concentrating on one bone at a time, in order of increasing maturity. The film exhibiting the modal maturity for the age group was selected and the maturity indicators of that particular bone were described. The appearance of these indicators was taken as typical for a healthy child of a particular age and sex. Having described these indicators for each bone and each age, the series was reexamined to identify radiographs showing, for every bone, the modal maturity for each age and sex. Each of these standards was assigned a skeletal age determined by the age of the children on whom the standard was based, and it is these standards that appeared in the atlas. Continuing Todd's work, William Greulich, Idell Pyle, and Normand Hoerr published a variety of atlases between 1950 and 1969 to describe the skeletal maturation of the hand and wrist, knee, and foot and ankle [7,29-31]. The atlas for the hand and wrist is the best known and is referred to universally as the Greulich-Pyle atlas.
M E T H O D S OF ASSESSMENT
Bone-Specific Scoring Techniques Skeletal D e v e l o p m e n t Three techniques are most commonly used in clinical situations to estimate skeletal maturity; the atlas technique of Greulich and Pyle [7], the Tanner-Whitehouse bone-specific scoring technique [10-13], and the Fels hand-wrist method [26,27]. All use the left hand and wrist to estimate a skeletal age or bone age, but the latter two are different both in concept and in method from the former.
Atlas Techniques The atlas technique has its origins in the pioneering work of Todd, who published an atlas of skeletal maturity in 1937 [17]. A "skiagraphic" atlas showing the development of the bones of the hand and wrist had, in fact, been published in London in 1898 by John Poland [28]. This contained anatomical descriptions of the development of each bone and a series of"roentgens" of children (mostly boys) from 12 months to 17 years of age. As a system of skeletal maturity assessment, its appearance was an isolated event until Todd's atlas. Todd based his atlas on the hand-wrist radiographs of 1000 children from the Brush Foundation Study of Human Growth and Development, which started in 1929 in Cleveland, Ohio. Children were only admitted to the study by the application of a pediatrician and were thus, in the Mid-
Bone-specific techniques were developed in an attempt to overcome the two main disadvantages of the atlas techniques--the concept of the :evenly maturing skeleton and the difficulty of using age in a system measuring maturity. Acceptance of the evenly maturing skeleton was compulsory if one used the atlas method of comparing the radiograph with standard plates. This acceptance decreased the significance of individual variation within the bones of the hand-wrist. Similarly, the acceptance of age from the standards implied the acceptance of a chronological time series.
Oxford Method Acheson, working on radiographs from approximately 500 preschool children in Oxford, England, devised a scoring system for the hand and wrist and knee that permitted maturation to be rated on a scale that did not require direct consideration of the size of the bone and was independent of the age of the child [6]. In essence, he identified maturity indicators and assigned scores ranging from 0, when no center was present, to 1 for the initial appearance, 2 for a clear shape, and so on until full maturity. By summing the scores for each bone, he arrived at a bone maturity score. He decided that this total maturity score should bear a linear relationship to age and thus used a weighting system to achieve this end. The system, like all subsequent bone-specific systems, depended on the
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assignment of a number or score to each maturity indicator or a combination of maturity indicators. However, Acheson's scores were arbitrary: They were not weighted in the statistical sense because "the decision as to what did, and what did not, constitute a maturity indicator was somewhat arbitrary in any case." The problem of this technique, called the Oxford method by Acheson, is that it does not deal with the problem of dysmaturity in that similar total scores from different individuals may be the result of the maturity of different bones. Even though the Oxford method fell short of an acceptable technique, it did allow Acheson to investigate the nature of maturity indicators. The fundamental flaws in the Oxford method, however, led Tanner and colleagues to develop their techniques a few years later. Tanner-Whitehouse M e t h o d
In 1959 and 1962, Tanner and Whitehouse, working in England, published their first attempt at a bone-specific scoring system. This was known as TW1 but was later revised and published as TW2 [10-12] and recently as TW3 [13]. The basic rationale was that the development of each single bone reflected a single process that they defined as maturation. Scores could be assigned to the presence of particular maturity indicators within the developing bones. Ideally, each of the n scores from each of the bones in a particular individual should be the same. This common score, with suitable standardization, would be the individual's maturity. To develop a practical technique, a variety of modifications to this rationale had to be made. In addition, Tanner and colleagues were highly critical of the method and how it operated in practicemhow well it served the pediatric and research communities for which it was intended. Their monitoring of the system promoted the various modifications that resulted in TW2 and TW3. The underlying rationale of the Tanner-Whitehouse techniques was based on dissatisfaction with a maturity system based on chronological age and thus the need to define a maturity scale that does not refer directly to age. The result of such a system would be that in any particular population the relationship between maturity and age could be studied and maturity standards, similar to height or weight standards, could be produced. Concentrating on the bones of the hand and wrist, they defined a series of eight maturity indicators for each bone and nine for the radius. (As with the Oxford method, the sesamoid bones were ignored.) These maturity indicators were then evaluated, not in relation to chronological age but in relation to their appearance within the full passage of each specific bone from immaturity to maturity. Thus, for example, it was possible to say that a particular indicator on the lunate first appeared at 13% maturity and that a process of fusion in the first metacarpal
started at 85% maturity. In addition, Tanner and colleagues were of the opinion that the metacarpals and phalanges, being greater in number than the carpal bones, would weight the final scores in favor of the long bones; therefore, they omitted rays 2 and 4 from the final calculations. Furthermore, they weighted the scores so that half of the mature score derived from the carpal bones and half from the long and short bones. The scores were so proportioned that the final mature score totaled 1000 points. Five thousand radiographs of normal British children were then rated using this technique to develop population standards that related bone maturity scores to chronological ages. The resulting curve of bone maturity score against age was sigmoid, demonstrating a nonlinear relationship between skeletal maturity and chronological age. There were three problems with TW1. First, some of the maturity indicators involved the assessment of size relations between bones that may be altered by pathological conditions, and thus TW1 violates the requirement of universality in the selection of maturity indicators. Second, by constraining the number of maturity indicators to eight, Tanner et al. weakened their system by ignoring the fact that some bones may exhibit greater or fewer maturity indicators than the eight required by the TW1 system. Third, the contribution of the carpus to 50% of total maturity presents a problem in terms of the repeatability of assessing maturity indicators (i.e., the carpus is less reliable) and because the carpus is not known to play a major role in growth in height or in epiphyseal fusion. Tanner and colleagues took cognizance of these criticisms in their development of the TWII system, which was in general use in Europe for 20 years. They did not change the maturity indicators, but they changed the scores assigned to the individual bones to allow the calculation of a bone maturity score based on the radius, ulna, and short bones (RUS) only or the carpal bones (CARPAL) only in addition to the full 20-bone score [TW2(20)]. The mathematical rationale for the TW systems is of considerable importance. The problem with the Oxford technique was that assigning scores of 1,2,3, etc. to the appearance of maturity indicators does not allow for the fact that changes from one maturational level to another may be very different in different bones. Tanner and colleagues believed that the development of each bone reflects primarily a single process defined as maturation, and that the scores from each of the bones in a particular individual should, with suitable standardization, be the same and this common score should be the individual's maturity. In practice, the scores of the various bones are not identical, with one of the most important reasons being the large gaps between successive events in a single
13. A s s e s s m e n t of Maturation
bone. Tanner et al. therefore defined the scores in such a way as to minimize the overall disagreement between the different bones. First, the disagreement in a particular individual is measured by the sum of squares of deviations of his or her bone scores about their mean value. Second, the scores are constrained to avoid the solution in which perfect agreement is reached by giving the same score to every stage. Table 1 illustrates this procedure. Two rival systems of scores are illustrated, labeled L and M, as are the stages for three bones in a particular individual. Regardless of whether system L or M is used, the resulting mean value is 9. The disagreement between the bones, however, is greater for system L than for system M when measured by the sum of squares of deviations about the mean (146 for system L and 42 for system M). Tanner et al. [10] generalized this by using all the bones and all the stages and by adding the total disagreement sum of squares over all members of a large standardized group. The system producing the overall minimum sum of squares of deviations is the preferred one. (The mathematical basis of the system is complex but may be studied in the second and third editions of the Tanner-Whitehouse technique [12,13] or in the paper by Healy and Goldstein [32].) The TW2 skeletal maturity system thus addressed the disadvantages of both the Greulich-Pyle atlas method and the Oxford method. It allowed an assessment of skeletal maturity that was age independent, and because of the three systems available from a single rating
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[TW2 (20), RUS, and CARPAL] it allowed considerable flexibility in both the assessment and the monitoring of skeletal maturity. Tanner and colleagues have published an updated method known as TW3 [13]. It was published approximately 20 years after the second edition of TW2 was published and, like all systems within growth research that rely on source samples from a particular historical time, Tanner and colleagues were acutely aware of the secular trend. This trend is almost universal and has been a recognized aspect of generational differences in human growth for many years. The secular trend affects growth both in overall size and in maturity such that size becomes larger and maturational events occur earlier with each succeeding generation. Thus, the rate of skeletal maturation will also have advanced and bone-specific scoring techniques should reflect or allow for this advancement. In addition, some important conceptual advances have occurred in the past 20 years, such as the fact that it is now widely recognized that standards and references are not the same. Standards are now viewed as being prescriptive and are based on desirable growth of groups of healthy children living in optimal environments (i.e., disease free and environmentally ideal). References are descriptive and are based on the growth of children living in normal environments in which they experience normal levels of infectious diseases and are not protected from environmental insult (i.e., growth "as is"). The source samples from which the reference charts within TW3 are constructed are not composed of children with optimal growth living in optimal environments. They therefore reflect a process of normal growth and should be called references. There are four major differences between TW2 and TW3. The most important, however, is the fact that the descriptions and manual ratings of the stages of the bones have not been altered. They remain the same so that previous ratings and calculations of bone maturity scores in TW2 are still valid for TW3. However, the TW2(20) bone score was abolished because it was believed that the mixture of the carpal maturity scores with the RUS maturity sores was not of major value. Skeletal maturity of the carpal bones in isolation is problematical in most situations. They appear to give different information about the process of maturity. The RUS bones are certainly more useful both in terms of reflecting general skeletal maturity and in the prediction of adult height. Second, the source samples for the reference charts have been updated so that they now reflect the norms for more recent samples of children from Europe and North America. Thus, the conversion to bone age also changes, particularly from approximately 10 years onwards. The third and fourth changes relate to the height prediction technique rather than the assessment of
3 32
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Roche- Wainer-Th&sen Technique
skeletal maturity per se. RUS bone score is now used rather than bone age in the prediction equations, and the source sample has been improved by using more appropriate data from the Zurich longitudinal growth study. The new maturity score, called EA90 (to reflect the European and American sources) or TW3, is based on data from samples of children in Europe, North America, and Japan assessed in the 1970s-1990s. These included Belgian data (21,174 boys and 10,000 girls) from the Leuven growth study, Spanish children ( N - - 2000 with more than 5000 radiographs) from the Bilbao study, Japanese children (N = 1000) from Tokyo, Italian children (950 boys and 880 girls) from Genoa, Argentinean data from the early 1970s, and data from Project Heartbeat from approximately 1000 normal European American children in Texas [13]. The new EA90 bone age values were chosen to match this scoring system but mainly concentrated on the Belgian, Spanish, and American samples. The differences in RUS scores between TW2 and TW3 for both sexes are shown in Fig. 3. At preadolescent ages the scores for boys vary little between the systems. After 9 years of age in boys and from approximately 5 years in girls, the differences increase quite dramatically so that, for instance, a boy scoring 405 would have a TW2 bone age of 13 years and a TW3 bone age of 11.7 years. This difference is consistent during adolescence, reflecting the relative advancement of the EA90 sample compared to the TW2 sample.
60 40
In 1975, Roche, Wainer, and Thissen published a technique to estimate the skeletal maturity of the knee[33]. Roche in particular was critical of the hand-wrist techniques because the bones of the hand and wrist exhibit few maturational changes over the age ranges of 11 to 15 in boys and 9 to 13.5 in girls [34]. In addition, the usefulness of the hand-wrist techniques was limited at early ages when few centers were visible and in later ages when some areas (e.g., the carpus) reached their adult maturity levels prior to others. He chose the knee as an area for assessment because he believed that the area investigated should be closely related to the reason for assessment: Maturity of the knee relates closely to growth in height. Thus, when one is dealing with growth disorders or height prediction, the knee should give a more appropriate estimation of skeletal maturity, but this may not actually be the case. Roche changed his opinion in the following decade, and with his colleagues Cameron Chumlea and David Thissen he produced a hand-wrist scoring technique in 1988 known as the Fels hand-wrist method [26,27].
Fels Hand- Wrist Technique The theoretical basis for the Fels hand-wrist method is little different from that of the earlier TannerWhitehouse methods. Roche and colleagues went through the laborious process of identifying suitable
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13. A s s e s s m e n t of Maturation
maturity indicators from 13,823 serial radiographs of children from the Fels Longitudinal Growth Study. The radiographs were taken between 1932 and 1972 and thus may appear to be dated and susceptible to the problems of secular change. From a possible 130 maturity indicators taken from the literature, 98 were selected that conformed to the criteria of universality, discriminative ability, reliability, validity, and completeness. In addition to graded indicators, Roche and colleagues used metric ratios of lengths of radius, ulna, metacarpals, and phalanges. Roche et al. [26,27] maintain that the Fels method differs from previous methods in terms of the observations made, the chronological ages at which assessments are possible, the maturity indicators, the statistical methods, and scale of maturity. In order to translate the ratings of the bones into a skeletal age, specific computer software (FELShw) is required [26]. The data-entry forms reflect the fact that the method can use ratings from the radius, ulna, all carpal bones, and, like the TW systems, the phalanges of rays I, III, and V. The output is an estimated skeletal age and an estimated standard error to provide an indication of the confidence of the estimated age. The Greulich-Pyle atlas technique and the TW2 and Fels scoring systems are the result of attempts to quantify skeletal maturation from different theoretical standpoints. The TW2 and Fels systems gained much from the study of the disadvantages of the Greulich-Pyle system and attempted to overcome them. There are advantages to both systems depending on the context within which they are used, but in order to decide on the most appropriate system the clinician or research worker must have at least a "nodding acquaintance" with their theoretical bases and indeed their practical use and the reliability to be expected from each system.
RELIABILITY Tanner et al. [13] provide a detailed description of the comparative reliability of the different systems. Experienced raters tend to obtain 95% confidence limits of +0.5 to +0.6 "years" based on two standard errors of measurement [13]. Thissen [35] provides examples that suggest a greater error when using the Fels hand-wrist technique; however, as for the reliability of TW methods, this may be because of the chronological age of the specific example chosen. Reliability tends to be greater or lesser depending on chronological age because at certain ages minor changes in maturity indicators can result in major changes in skeletal age. Thus, a miss-rating of one of these indicators will result in an inflated standard error of measurement from a test-retest reliability study.
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COMPARABILITY OF THE ATLAS AND BONE-SPECIFIC METHODS In a recent comprehensive comparison of the atlas and bone-specific techniques, Aicardi and colleagues [36] compared the Greulich-Pyle, Tanner-Whitehouse, and Fels methods using a sample of 589 children (250 girls and 339 boys) aged 2 to 15 years. These Italian children from Genoa were admitted to a clinical pediatrics department for investigation of growth, obesity, and acute diseases. Although the Italian boys were generally delayed in relation to all methods, bone age was closer to chronological age using the RWT knee method rather than the hand-wrist methods, with an average deviation o f - 0 . 1 1 years. The Fels hand-wrist method had an average deviation o f - 0 . 3 2 years, the Tanner-Whitehouse RUS -0.35 years, and the Greulich-Pyle method -0.61 years. Conversely, girls were generally advanced, with equivalent values between chronological and skeletal age of 0.06 (RWT), 0.18 (Fels), 0.23 (TW-RUS), and -0.04 years (GP). Naturally, such studies are rare because of the generally uncommon situation in which children have both hand-wrist and knee radiographs taken during the course of a hospital entry. Aicardi et al. concluded that further comparisons are required to decide whether hand-wrist or knee methods are most useful in clinical settings, particularly when there is a concern for growth potential. Tanner and colleagues include a discussion on population differences in skeletal maturity in the most recent TW3 method [13] and good historical data are provided in Eveleth and Tanner's review of growth globally [37,38]. The major point is that differences between countries and indeed within countries are to be expected because skeletal maturity, like all aspects of maturation, reflects the interaction of both genetic and environmental forces. Although ideally reference values should be developed for each relevant population, in the absence of such developments it seems reasonable to suggest that the method of choice will depend on the proximity of the child under investigation to the source sample of the particular method, the availability of appropriate radiographs of the hand-wrist and/or knee, and, in the case of the Fels hand-wrist method, the availability of the appropriate software. Given the presence of secular changes in the appearance of maturity indicators and therefore in the degree of advancement or delay of the child/sample, it seems sensible to use the most recent methods and those that expose the child to the lowest radiation dosage. Thus, the Tanner-Whitehouse and Fels hand-wrist methods are preferred over the Greulich-Pyle and the RWT knee techniques. The TannerWhitehouse technique is most often used in Europe and
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the Fels hand-wrist technique in North America, but this situation probably reflects marketing and familiarity rather than scientific considerations.
SECONDARY SEXUAL DEVELOPMENT Secondary sexual development is assessed using maturity indicators that provide discrete stages of development within the continuous process of maturation. The most widely accepted assessment scale is the Tanner Scale or the Tanner Staging Technique. It was developed by Tanner [16] and was based on the work of Reynolds and Wines [39] and Nicholson and Hanley [15]. Tanner classified the processes of breast development in girls, genitalia development in boys, and pubic hair development in both sexes into five stages and axillary hair development in both sexes into three stages. The usual terminology is to describe breast development in stages B1-B5, genitalia development in stages G1-G5, pubic hair development in stages PH1-PH5, and axillary hair development in stages A1-A3. The following descriptions of the stages of development of the breasts, genitalia, and pubic hair are found in Tanner [16] and Marshall and Tanner [4,5] and are provided here to accompany the illustrations of secondary sexual development (Figs. 4-6): Breast development Stage 1: Preadolescent--elevation of papilla only. Stage 2: Breast bud stage--elevation of breast and papilla as small mound; enlargement of areolar diameter. Stage 3: Further enlargement and elevation of breast and areola, with no separation of their contours. Stage 4: Projection of areola and papilla to form a secondary mound above the level of the breast. Stage 5: Mature stage--projection of papilla only, due to recession of the areola to the general contour of the breast. Genitalia development Stage 1: PreadolescentDtestes, scrotum, and penis approximately the same size and proportion as in early childhood. Stage 2: Enlargement of scrotum and testes--the skin of the scrotum reddens and changes in texture. There is little or no enlargement of the penis at this stage. Stage 3: Enlargement of penismthis occurs first mainly in length; further growth of testes and scrotum. Stage 4: Increased size of the penis with growth in breadth and development of glans; further enlargement of testes and scrotum; increased darkening of scrotal skin.
FIGURE 4 Pubichair developmentin boysand girlsaccordingto the Tanner staging technique [reproduced with permission from Tanner, J. M. (1962). Growth at Adolescence, 2nd ed. Blackwell,Oxford].
Stage 5: Genitalia adult in size and shape. Pubic hair development Stage 1: Preadolescent--the vellus over the pubes is not further developed than that over the abdominal wall (i.e., no pubic hair). Stage 2: Sparse growth of long, slightly pigmented downy hair, straight or only slightly curled, appearing mainly at the base of the penis or along the labia. Stage 3: Considerably darker, coarser, and more curled. The hair spreads sparsely over the junction of the pubes. Stage 4: Hair now resembles adult hair in type, but the area covered by it is still considerably smaller than in the adult. There is no spread to the medial surface of the thighs. Stage 5: Adult in quantity and type, with distribution of the horizontal or classically feminine pattern; spread to the medial surface of the thighs but not up the linea alba or elsewhere above the base of the inverse triangle.
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FIGURE 5 Genitaliadevelopment in boys according to the Tanner staging technique [reproduced with permission from Tanner, J. M. (1962). Growth at Adolescence, 2nd ed. Blackwell,Oxford].
Clinical Evaluations The assessment of secondary sexual development is a standard clinical procedure for which the full Tanner Scale is used. There are some practical problems with the Tanner stages, however, in that the unequivocal observation of each stage is often dependent on having longitudinal observations. In most situations, outside the clinical setting, the observations are cross sectional. This practical difficulty led to the amalgamation of some of the stages to create pubertal stages. These pubertal stages are either on a 3- or 4-point scale and combine breast/genitalia development with pubic hair development [40,41]. In the 3-point technique, for instance, stage P1 represents the prepubertal state (B1/G1 and PH1), and stages P2 (B2-B4/G2-G4 and PH2-PH4) and P3 (B5/G5 and PH5) represent the midpubertal and postpubertal states, respectively. All indicators of maturational change between the prepubertal and postpubertal extremes have been combined into the P2 stage. Assessing breast/genitalia development with pubic hair development is obviously much easier than assessing these maturity indicators separately but inevitably leads to a lack of sensitivity in the interpretation of the timing and duration of the different stages of pubertal development. Indeed, the intrasubject variation in the synchronous appearance of pubic hair and breast/genitalia stages, illustrated in British children by Marshall and
FIGURE 6 Breast development in girls according to the Tanner staging technique [reproduced with permission from Tanner, J. M. (1962). Growth at Adolescence, 2nd ed. Blackwell,Oxford].
Tanner [4,5], suggests that it may be misleading to expect stage synchronization in as many as 50% of normal children.
S e l f - A s s e s s m e n t of Pubertal S t a t u s The assessment of secondary sexual characteristics is, to some extent, an invasive procedure in that it invades the privacy of the child or adolescent involved. Thus, such assessments of normal children who participate in growth studies, as opposed to those being clinically assessed, are problematical from both ethical and subject compliance standpoints. In order to overcome this problem, the procedure of self-assessment has been developed and validated in a number of studies.
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The self-assessment procedure requires the child to enter a well-lit cubicle or other area of privacy in which are provided pictorial representations of the Tanner scales and suitably positioned mirrors on the wall(s). The pictures may be either photographs or line drawings, as long as the contents are clear. To each picture of each stage is appended an explanation of what the stage represents. The participant is instructed to remove whatever clothing is necessary in order for him or her to properly observe his or her pubic hair/genitalia or pubic hair/ breast development in the mirrors. The participant then writes on paper his or her stage of development and seals the paper in an envelope on which is marked the study identity number of the participant. The envelope is either left in the cubicle or handed to the observer on leaving the cubicle. The results of validation studies vary greatly depending on the age of the participants (e.g., early or late adolescence) [42], gender [42,43], the setting in which assessments are performed (e.g., school or clinic) [44,45], ethnicity [46], and whether the participants represent a distinct diagnostic group such as cystic fibrosis [47] or anorexia nervosa [48] or are socially disadvantaged [49]. Younger, less developed children tend to overestimate their development, and older, more developed children tend to underestimate. Boys have been found to overestimate their development, whereas girls' estimates have been more consistent with those of experts [43]. The amount of attention given to explaining the required procedure appears to be of major importance. Thus, excellent rating agreement between physicians and adolescents has been found in clinical settings, with ~: coefficients between 0.66 and 0.91 [45,50,51], but there has been less agreement in school settings (~c = 0.35-0.42; correlations = 0.25-0.52) [44,45]. Improved agreement in clinical settings probably reflects the more controlled environment in these settingsas opposed to a school setting. The main reason for low correlations and thus poor validity in any setting with any group of participants likely concerns the amount of explanation that is provided to the child. When the participant has been the subject of a clinical trial, and the scientist/clinician has spent considerable time and effort ensuring that the child is completely appraised of what he or she has to do, validity is high. Less effort in explaining procedures leads to lower validity. The observer should explain the procedure thoroughly to the participant using appropriate (nonscientific) language and invite questions to ensure that the participant fully understands the procedure. Only when the observer is sure that understanding is total should the child be allowed to follow the procedure. Randomized reliability assessment by the observer is an ideal but
requires a full and carefully considered argument to obtain the appropriate ethical permission. Age at M e n a r c h e Age at menarche is usually determined in one of three ways: status quo, retrospectively, or prospectively. Status quo techniques require girls to respond to the question, "Do you have menstrual cycles (periods)?" The resulting data from a sample of girls will produce a classical dose-response sigmoid curve that may be used to graphically define an average age at menarche. More commonly, the data are analyzed using logit or probit analysis to determine the mean or median age at menarche and the parameters of the distribution, such as the standard error of the mean or the standard deviation. Retrospective techniques require the participants to respond to the question, "When did you have your first period?" Most adolescents can remember to within 1 month, and some to the day, when this event occurred. Others may be prompted to remember by reference to whether the event occurred during summer or winter, whether they were at school or on vacation, and so on. One interesting result of such retrospective analyses is that there appears to be a negative association between the age of the women being questioned and the age at which they report menarche: The older the women, the younger they believe they were when menarche occurred. Such results have been found in both developed and developing countries and cast doubt about the reliability of retrospective methods beyond the teenage and early adult years. The prospective method is normally only used in longitudinal monitoring situations, such as repeated clinic visits or longitudinal research studies. This method requires the teenager to be seen at regular intervals (usually every 3 months) and to be asked on each occasion whether or not she has started her period. As soon as the response is positive, an actual date on which menarche occurred can be easily obtained. There is little doubt that the prospective method is the most accurate in estimating menarcheal age, but it has the disadvantage of requiring repeated contact with the subjects. This is seldom possible, except in clinical situations; thus, status quo and retrospective methods are usually the techniques of choice. Status quo techniques that rely on logit or probit analysis require large sample sizes because the analysis requires the data to be grouped according to age classes. With few subjects, broader age ranges are required, such as whole or half years, with a consequent loss of precision in the mean or median value. Retrospective methods result in parametric descriptive statistics but have the problem of accuracy of recalled ages when this particular event occurred.
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Secondary Sexual Events in Boys Although status quo, prospective, and retrospective methods may easily obtain age at menarche, assessments of secondary sexual development in boys are complicated by the lack of a similar clearly discernible maturational event. Attempts to obtain information on the age at which the voice breaks or on spermarche are complicated by the amount of time necessary for the voice to consistently remain in a lower register and the logistical complications involved in the assessment of spermarche. Testicular volume, using the Prader orchidometer [52], is commonly the only measure of male secondary sexual development outside the rating scales previously mentioned, although other measurement techniques have been described to estimate testicular volume [53]. The detection of spermatozoa in the urine has been proposed as a quick, noninvasive method to assess the functional state of the maturing gonad and may be useful as a screening technique in population studies [54-59]. Its use, however, may be limited because longitudinal [58,60] and cross-sectional [57] studies have shown that spermaturia is a discontinuous phenomenon.
13.
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1. Bogin, B. (1988). Patterns of Human Growth. Cambridge Univ. Press, Cambridge, UK. 2. Tovee, M. J., Maisey, D. S., Emery, L., and Cornelissen, P. L. (1999). Visual clues to female physical attractiveness. Proc. R. Soc. London Ser. B 266, 211-218. 3. Zaadstra, B. M., Seidell, J. C., Vannord, P. A. H., Tevelde, E. R., Habbema, J. D. F., and Vrieswijk, B. (1993).Fat and female fecundity-Prospective study of the effect of body fat distribution on conception rates. Br. Med. J. 306, 484-487. 4. Marshall, W. A., and Tanner, J. M. (1969). Variations in the pattern of pubertal changes in girls. Arch. Dis. ChiM. 44, 291-303. 5. Marshall, W. A., and Tanner, J. M. (1970). Variations in the pattern of pubertal changes in boys. Arch. Dis. Child. 45, 13-23. 6. Acheson, R. M. (1966). Maturation of the skeleton. In Human Development (F. Falkner, Ed.), pp. 465-502. Saunders, Philadelphia. 7. Greulich, W. W., and Pyle, S. I. (1959). Radiographic Atlas of the Skeletal Development of the Hand and Wrist, 2nd ed. Stanford Univ. Press, Palo Alto, CA. 8. Acheson, R. M. (1954). A method of assessing skeletal maturity from radiographs. J. Anst. (London) 88, 498-508. 9. Acheson, R. M. (1957). The Oxford method of assessing skeletal maturity. Clin. Orthop. 10, 19-39. 10. Tanner, J. M., Whitehouse, R. H., and Healy, M. J. R. (1962). A New System for Estimating the Maturity of the Hand and Wrist, with Standards Derived from 2600 Health British Children. Part II. The Scoring System. International Children's Centre, Paris. 11. Tanner, J. M., Whitehouse, R. H., Marshall, W. A., Healy, M. J. R., and Goldstein, H. (1975). Assessment of Skeletal Maturity and Prediction of Adult Height. Academic Press, London. 12. Tanner, J. M., Whitehouse, R. H., Cameron, N., Marshall, W. A., Healy, M. J. R., and Goldstein, H. (1983). Assessment of Skeletal
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Maturity and Prediction of Adult Height, 2nd ed. Academic Press, London. Tanner, J. M., Healy, M. J. R., Goldstein, H., and Cameron, N. (2001). Assessment of Skeletal Maturity and Prediction of Adult Height (TW3 Method), 3rd ed. Academic Press, London. Demirjian, A., Goldstein, H., and Tanner, J. M. (1973). A new system of dental age assessment. Hum. Biol. 45, 211-227. Nicholson, A. B., and Hanley, C. (1952). Indices of physiological maturity. ChiM Dev. 24, 3-38. Tanner, J. M. (1962). Growth at Adolescence, 2nd ed. Blackwell, Oxford. Todd, T. W. (1937). Atlas of Skeletal Maturation. Part I: The Hand. Mosby, St. Louis. Hellman, M. (1928). Ossification of epiphyseal cartilages in the hand. Am. J. Phys. Anthrop. 11, 223-257. Rotch, T. M. (1909). A study of the development of the bones in childhood by the roentgen method, with the view of establishing a developmental index for the grading of and the protection of early life. Trans. Am. Assoc. Phys. 24, 603-630. Bardeen, C. R. (1921). The relation of ossification to physiological development. J. Radiol. 2, 1-8. Sontag, L. W., and Lipford, J. (1943). The effect of illness and other factors on appearance pattern of skeletal epiphyses. J. Pediatr. 23, 391-409. Lowell, F., and Woodrow, H. (1922). Some data on anatomical age and its relation to intelligence. Pedagog. Sem. 29, 1-15. Carter, T. M. (1926). Technique and devices in radiographic study of the wrist bones of children. J. Ed. Psychol. 17, 237-247. Flory, C. D. (1936). Osseous development in the hand as an index of skeletal development. Monogr. Soc. Res. Child. Dev. 1, 3. Pryor, J. W. (1907). The hereditary nature of variation in the ossification of bones. Anat. Rec. 1, 84-88. Roche, A. F., Chumlea, W. C., and Thissen, D. (1988). Assessing the Skeletal Maturity of the Hand-Wrist: Fels Method. Thomas, Springfield, IL. Chumlea, W. C., Roche, A. F., and Thissen, A. F. (1989). The FELS method for assessing the skeletal maturity of the hand-wrist. Am. J. Hum. Biol. 1, 175-183. Poland, J. G. (1898). Skiagraphic Atlas Showing the Development of the Bones of the Wrist and Hand. For the Use of Students and Others. Smith, Elder, London. Greulich, W. W., and Pyle, S. I. (1950). Radiographic Atlas of the Skeletal Development of the Hand and Wrist. Stanford Univ. Press, Palo Alto, CA. Pyle, S. I., and Hoerr, N. L. (1955). A Radiographic Standard of Reference for the Growing Knee. Thomas, Springfield, IL. Pyle, S. I., and Hoerr, N. L. (1955). A Radiographic Standard of Reference for the Growing Knee, 2nd ed. Thomas, Springfield, IL. Healy, M. J. R., and Goldstein, H. (1976). An approach to the scaling of categorized attributes. Biometrika 63, 219-229. Roche, A. F., Wainer, H., and Thissen, D. (1975). Skeletal Maturity." the Knee Joint as a Biological Indicator. Plenum, London. Roche, A. F. (1970). Associations between rates of maturation of the bones of the hand-wrist. Am. J. Phys. Anthrop. 33, 341-348. Thissen, D. (1989). Statistical estimation of skeletal maturity. Am. J. Hum. Biol. 1, 185-192. Aicardi, G., Vignolo, M., Milani, S., Naselli, A., Magliano, P., and Garzia, P. (2000). Assessment of skeletal maturity of the hand-wrist and knee: A comparison of methods. Am. J. Hum. Biol. 12, 610-615. Eveleth, P. B., and Tanner, J. M. (1976). Worldwide Variations in Human Growth. Cambridge Univ. Press, Cambridge, UK.
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38. Eveleth, P. B., and Tanner, J. M. (1990). Worldwide Variations in Human Growth, 2nd ed. Cambridge Univ. Press, Cambridge, UK. 39. Reynolds, E. L., and Wines, J. V. (1948). Physical changes associated with adolescence in boys. Am. J. Dis. Child. 75, 329-350. 40. Kulin, B. E., Bwibo, N., Mutie, D., and Santner, S. J. (1982). The effect of chronic childhood malnutrition on pubertal growth and development. Am. J. Clin. Nutr. 36, 527-536. 41. Chaning-Pearce, S. M., and Solomon, L. (1986). A longitudinal study of height and weight in black and white Johannesburg children. S. Afr. Med. J. 70, 743-746. 42. Varona-Lopez, W., Guillemot, M., Spyckerelle, Y., and Deschamps, J. P. (1988). Self assessment of the stages of sex maturation in male adolescents. Pediatrie 43, 245-249. 43. Sarni, P., de Toni, T., and Gastaldi, R. (1993). Validity of selfassessment of pubertal maturation in early adolescents. Minerva Pediatr. 45, 397-400. 44. Wu, W. H., Lee, C. H., and Wu, C. L. (1993). Self-assessment and physician's assessment of sexual maturation in adolescents in Taipei. Chung Hua Min Kuo Hsiao ErhKo I Hsueh Hui Tsa Chih 34, 125-131. 45. Schlossberger, N. M., Turner, R. A., and Irwin, C. E., Jr. (1992). Validity of self-report of pubertal maturation in early adolescents. J. Adolesc. Health 13, 109-113. 46. Hergenroeder, A. C., Hill, R. B., Wong, W. W., Sangi-Haghpeykar, H., and Taylor, W. (1999). Validity of self-assessment of pubertal maturation in African American and European American adolescents. J. Adolesc. Health 24, 201-205. 47. Boas, S. R., Falsetti, D., Murphy, T. D., and Orenstein, D. M. (1995). Validity of self-assessment of sexual maturation in adolescent male patients with cystic fibrosis. J. Adolesc. Health 17, 42-45. 48. Hick, K. M., and Kutzman, D. K. (1999). Self-assessment of sexual maturation in adolescent females with anorexia nervosa. J. Adolesc. Health 24, 206-211.
49. Hardoff, D., and Tamir, A. (1993). Self-assessment of pubertal maturation in socially disadvantaged learning-disabled adolescents. J. Adolesc. Health 14, 398-400. 50. Duke, P. M., Litt, I. F., and Gross, R. T. (1980). Adolescents' selfassessment of sexual maturation. Pediatrics 66, 918-920. 51. Brooks-Gunn, J., Warren, M. P., Russo, J., and Gargiulo, J. (1987). Validity of self-report measures of girls' pubertal status. ChiM Dev. 58, 829-841. 52. Prader, A. (1966). Testicular size: Assessment and clinical importance. Triangle 7, 240. 53. Daniel, W. A., Feinstein, R. A., Howard-Pebbles, P., and Baxley, W. D. (1982). Testicular volume of adolescents. J. Pediatr. 101, 1010-1012. 54. Schaefer, F., Marr, J., Seidel, C., Tilgen, W., and Scharer, K. (1990). Assessment of gonadal maturation by evaluation of spermaturia. Arch. Dis. Child. 65, 1205-1207. 55. Baldwin, B. (1928). The determination of sex maturation in boys by a laboratory method. J. Comp. Psychol. 8, 39-43. 56. Richardson, D., and Short, R. Time of onset of sperm production in boys. J. Biosoc. Sci. 5, 1525. 57. Hirsch, M., Shemesh, J., and Modan, M. (1979). Emission of spermatozoa: Age of onset. Int. J. Androl. 2, 289-298. 58. Nielson, C. T., Skakkebaek, N. S., and Richardson, D. W. (1986). Onset of the release of spermatozoa (spermarche) in boys in relation to age, testicular growth, pubic hair, and height. J. Clin. Endocrinol. Metab. 62, 532-535. 59. Kulin, H. E., Frontera, M. E., Demers, L. D., Bartholomew, M. J., and Lloyd, T. A. (1989). The onset of sperm production in pubertal boys. Am. J. Dis. Child. 143, 190-193. 60. Hirsch, M., Lunenfeld, B., Modan, M., Oradia, J., and Shemesh, J. (1985). Spermarche--The age of onset of sperm emission. J. Adolesc. Health Care 6, 35-39.
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]14] Biochemical Markers of Bone M e t a b o l i s m ECKHARD SCHONAU* and FRANK RAUCH t *University Children "s Hospital Cologne, Germany t Genetics Unit, Shriners Hospital for Children, Montreal Quebec, Canada
INTRODUCTION
W h a t is B o n e M e t a b o l i s m ? Before dealing with individual indicators of bone metabolism, it may be of interest to consider what an ideal bone marker reflects. Bone metabolism in children is the sum of three different physiologic mechanisms: bone elongation, increase in bone circumference, and bone remodeling. The longitudinal growth of most bones occurs by endochondral bone formation, which basically involves two steps. First, cartilage tissue is added to the growth zones of a bone (the growth plates). Second, this cartilaginous scaffold is transformed into bone tissue in the adjacent metaphyses (Fig. 2). This second step involves the degradation of most of the cartilage matrix and the rapid secretion and mineralization of woven bone matrix adjacent to the remaining columns of chondrocytes. The resulting primary spongiosa is successively removed and replaced by mature secondary spongiosa, which no longer contains cartilaginous remnants. The conversion of primary into secondary spongiosa is often referred to as remodeling [3]. However, little is known about this process, which is likely different from the remodeling of mature bone, particularly because the turnover of primary spongiosa is far more rapid than that of mature bone. Increase in bone circumference occurs by a process that Frost [35] termed bone modeling. Bone modeling involves the presence of active osteoclasts and osteoblasts on opposite sides of a given piece of bone. During growth in width of long bone diaphyses or vertebral bodies, osteoblasts are typically located on the outer (periosteal) surface of a bone cortex, where they deposit bone matrix that is later mineralized (Fig. 3). Thereby, the outer circumference of a long bone or a vertebral body is gradually increased. At the same time, osteoclasts
Biochemical markers of bone metabolism are compounds that are released from bone tissue into the circulation and can be quantified in serum or urine samples (Fig. 1). The past 15 years have seen a dramatic increase in the number of commercially available bone metabolism markers. Although primarily designed for diagnosis and follow-up of metabolic bone diseases in adults, these are employed in pediatrics as well. In this chapter, we focus on issues of interest when these makers are used in the pediatric setting.
FIGURE 1 Markers of bone formation and resorption. Reproduced with permission from Sch6nau, E., Rauch, F., Blum, W. F. (2001). Prediction of growth response to GH treatment: A reference guide. Abingdon, United Kingdom: TMG Healthcare Communications Ltd.
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FIGURE 2 Longitudinal bone growth occurs by endochondral ossification. Growth plate chondrocytes proliferate and hypertrophy while secreting cartilage matrix, which is subsequently mineralized. In the metaphysis, this mineralized cartilage is gradually replaced by mature bone. Reproduced with permission from Sch6nau, E., Rauch, F., Blum, W. F. (2001). Prediction of growth response to GH treatment: A reference guide. Abingdon, United Kingdom: TMG Healthcare Communications Ltd.
FIGURE 3 Bone growth in width by modeling of the bone cortex. Osteoclasts and osteoblasts are located on different sides of the cortex and are active without interruption. The direction of movement of the cortex is indicated by the arrows.
located on the inner (endocortical) surface of the cortex resorb bone, thus increasing the size of the marrow cavity. Because osteoblasts are active without interruption during bone modeling, rapid increases in the amount of bone tissue can occur. During this process, osteoclasts usually remove less bone tissue than is deposited by osteoblasts. As a result, modeling usually leads to a net increase in the amount of bone tissue.
The bone tissue that is created either by endochondral ossification or by modeling is continuously turned over in a process that Frost [36] called remodeling. Remodeling consists of successive cycles of bone resorption and formation on the same bone surface (Fig. 4). The basic features of this process are identical for trabecular and cortical bone [89]. A group of osteoclasts removes a small quantity ("packet") of bone tissue, which after a reversal phase is replaced by a team of osteoblasts. The entire group of cells involved in this process is called the remodeling unit or basic multicellular unit. The fact that osteoblast activity is linked to previous osteoclast action is referred to as coupling [89]. The difference between the amount of bone removed and the amount added during one remodeling cycle is called remodeling balance. In young adults, the remodeling balance is typically near zero. Consequently, the amount of bone remains largely unchanged. The remodeling process renews the bone tissue and thereby prevents an accumulation of tissue damage. The concepts of coupling and remodeling balance are frequently confused in reports on biochemical markers of bone metabolism. For example, when biochemical markers indicate that more bone is lost than is formed, many authors conclude that formation and resorption are "uncoupled." However, there is no need to always postulate such a drastic derangement in the
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Biochemical Considerations
FIGURE 4 Bone maintenance by remodeling of cortical and trabecular bone. (a) Intracortical remodeling. Osteoclasts in the cutting cone create a tunnel that subsequently is filled in by osteoblast, leaving only a narrow opening, the Haversian canal. The end result is a new osteon (osteonal remodeling). (b) Trabecular bone remodeling. Bone tissue is continuously turned over by remodeling units consisting of osteoclasts and osteoblasts. These units move parallel to the surface, as indicated by the arrows. A remodeling unit in trabecular bone corresponds to half of a remodeling unit in cortical bone (hemiosteonal remodeling). The amount of bone resorbed is similar to the amount of bone subsequently deposited (i.e., the remodeling balance is near zero). Therefore, only slow changes in bone shape and mass can be achieved with remodeling. Reproduced with permission from Sch6nau, E., Rauch, F., Blum, W. F. (2001). Prediction of growth response to GH treatment: A reference guide. Abingdon, United Kingdom: TMG Healthcare Communications Ltd.
The previous considerations assumed that measurable markers of bone cell activity are entirely specific for bone metabolism. However, the concentration of most bone markers in serum or urine does not just depend on the rate of production in bone and their subsequent release into the circulation. Similar to any other substance present in the bloodstream, serum levels of biochemical markers of bone turnover also depend on the rate of elimination. Elimination occurs either by metabolic degradation or by excretion via liver or kidneys, depending on which marker is considered, and impaired function of these organs can thus affect the circulating concentration of these markers. Furthermore, levels of urinary markers of bone turnover depend on the relative amount that is excreted into the urine. The concentration of urinary solutes also depends on water diuresis. To adjust for this factor, levels of urinary bone markers are usually normalized to urinary creatinine concentration, which increases analytical accuracy. However, if creatinine excretion is reduced, for example, due to decreased muscle mass [47], interpretation of urinary measurements may be different.
MARKERS OF BONE FORMATION Because new bone is formed by osteoblasts, bone formation markers reflect the activity of osteoblastics. The cellular origin of these markers is schematically represented in Fig. 5. Alkaline P h o s p h a t a s e
remodeling process to explain bone loss. Bone loss usually occurs during a continuous process, in which resorption and formation remain coupled but the overall remodeling balance is negative (i.e., the amount formed is less than the amount resorbed). In fact, it is not certain that complete uncoupling exists at all The relative contributions of endochondral bone formation, modeling, and remodeling to the total amount of bone turned over during a given time interval are unknown. It is therefore not possible to obtain separate information on the three bone metabolic activities by determining levels of bone markers in children and adolescents. This is an important difference to the situation in adults, in whom bone metabolic activity is basically limited to remodeling and all biochemical measurements reflecting the activity of the different bone cells can thus be regarded as remodeling markers.
Alkaline phosphatases (ALPs) are a group of enzymes that are present in many different tissues. The two major ALP isoforms in human serum, produced in bone and liver, are difficult to distinguish because they are both encoded by the tissue-nonspecific ALP gene; both isoforms differ only in their patterns of posttranslational glycosylation [13]. Many methods have been developed for the determination of bone-specific ALP, but specificity remains a problem for all these assays [99]. Interestingly, total bone-specific ALP can be further separated into isoforms that are specific for cortical and trabecular bone, respectively [73]. If confirmed in larger studies, it might thus be possible to obtain separate information on metabolism in trabecular and cortical bone. Although the physiological role of ALP in the skeleton is not entirely clear, it is obviously involved in the mineralization process. In fact, when tissue-nonspecific ALP is lacking (hypophosphatasia), severe mineralization
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FIGURE 5 Osteoblasticbone collagen formation. Reproduced with permission from Sch6nau, E., Rauch, F., Blum, W. F. (2001). Prediction of growth response to GH treatment: A reference guide. Abingdon, United Kingdom: TMG Healthcare CommunicationsLtd.
defects can occur [145]. The clearance of ALP from serum may be transiently impaired after viral infections in young children (transient hyperphosphatasia), leading to excessively high total ALP serum activity without any detectable consequences [119]. Osteocalcin Osteocalcin, or bone Gla (7-carboxyglutamic acidcontaining) protein, is a 5.8-kDa protein that is exclusively synthesized by osteoblasts [13]. The molecule is gamma-carboxylated intracellularly under the influence of vitamin K. It is secreted and incorporated into the organic bone matrix. Some newly synthesized osteocalcin escapes incorporation into matrix and reaches the bloodstream. Theoretically, osteocalcin should be the most accurate marker of osteoblast activity. However, diagnostic use of this molecule is hampered by its instability and by difficulties in distinguishing between the various molecular forms that are found in the circulation [75]. Intact osteocalcin is metabolized primarily by the kidneys and it is excreted into the urine, where several different osteocalcin fragments have been identified [76]. Since the function of osteocalcin is uncertain, it is difficult to know what aspect of bone formation is assessed by measuring serum osteocalcin concentration. Surprisingly, mice lacking osteocalcin have stronger bones than wild-type mice [31]. Procollagen Type I P r o p e p t i d e s Collagen type I is by far the most abundant protein synthesized by osteoblasts [13]. After secretion into the extracellular space, the procollagen type I C- and N-terminal propeptides (PICP and PINP) are cleaved off and are released into the circulation (Fig. 5). The remaining collagen molecule will undergo several pro-
cessing steps and finally be integrated into a collagen fibril. PICP is a soluble trimeric globular protein with a molecular weight of approximately 100 kDa. It is not excreted by glomerular filtration but is taken up through the mannose receptor by liver endothelial cells, and it is degraded intracellularly [126]. This uptake can be disturbed, resulting in very high serum levels of PICP [127]. PINP is also taken up by liver endothelial cells, but this occurs via the scavenger receptor [77]. Radioimmunoassays have been developed to measure both PICP and PINP in serum samples [78,79]. Although collagen type I is found in many different tissues, it is assumed that serum levels of these propeptides reflect primarily bone matrix synthesis because bone is the major organ of collagen type I synthesis [13]. The appeal of PICP and PINP as markers of bone formation is that they reflect the activity of a crucial and wellcharacterized step of bone formation, the synthesis of collagen type I. This facilitates the rational interpretation of results.
MARKERS OF BONE RESORPTION Most currently used indices of bone resorption arise during the posttranslational modification of bone type I collagen and are released upon bone matrix degradation during osteoclastic bone resorption (Fig. 6). The positions of these compounds within the intact collagen fibril are schematically represented in Fig. 7.
Hydroxyproline Hydroxyproline is the traditional marker of bone resorption. Hydroxyproline is a product of posttranslational hydroxylation of proline in the procollagen chain,
14. Biochemical Markers of Bone Metabolism
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FIGURE 6 Osteoclastic bone resorption. Osteoclasts seal off an area of bone and acidify the newly created extracellular lysosome. Bone mineral dissolves in this area and collagen type I molecules can then be digested by proteases. The large number of degradation products that arise enter the circulation and are excreted by the kidneys. Reproduced with permission from Sch6nau, E., Rauch, F., Blum, W. F. (2001). Prediction of growth response to GH treatment: A reference guide. Abingdon, United Kingdom: TMG Healthcare Communications Ltd.
FIGURE 7 Bone resorption markers derived from bone type I collagen. Reproduced with permission from Sch6nau, E., Rauch, F., Blum, W. F. (2001). Prediction of growth response to GH treatment: A reference guide. Abingdon, United Kingdom: TMG Healthcare Communications Ltd.
which is released when type I collagen is degraded [13]. Since collagen breakdown occurs during bone resorption, hydroxyproline is regarded as a marker of bone resorption. However, some newly synthesized collagen chains are degraded even before they are secreted by the osteoblast [63]; therefore, hydroxyproline is also influenced by osteoblast activity. In addition, type I collagen turnover in tissues other than bone and nutritional collagen intake also contributes to the circulating pool of hydroxyproline [13]. Hydroxyproline is mostly excreted unchanged via the kidneys but can also be metabolized
in the liver [13]. Due to these multiple confounding factors, urinary hydroxyproline is not an ideal indicator of bone resorption. For this reason, other bone resorption markers, notably type I collagen cross-linkbased compounds, have become more popular in recent years. Collagen Pyridinium Cross-Links Pyridinoline (PYD) and deoxypyridinoline (DPD) are generated from hydroxylysine and lysine during the
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extracellular maturation of the collagen fibril [33]. Pyridinium cross-links are interchain bonds that stabilize the collagen fibril (Fig. 7). PYD is the prevalent crosslink in bone, but it is also found in articular cartilage and other connective tissues [33]. DPD is thought to be more specific for bone because its extraosseous concentrations are comparatively lower [13]. In the serum of children, the fraction of free cross-links is approximately 16-18% and the remainder is peptide bound [18]. In urine, the free fraction is approximately 40% and the peptidebound forms represent approximately 60%. Excretion of urinary cross-links is thought to be a better marker of bone resorption than hydroxyproline because crosslinks are released only during collagen degradation, are not metabolized, and are independent of nutritional collagen intake [13]. Various immunoassays have been developed to quantify cross-links in one form or another [13], including assays using antibodies against the pyridinium structures [6,123] or against the cross-link containing C-terminal telopeptide of type I collagen (ICTP) [106]. Other assays also use antibodies against amino acid sequences within the collagen type I C- and N-terminal telopeptides (CTX and NTX, respectively), which do not contain the cross-link compounds [8,9]. For several years, ICTP was the only serum marker within this group, but recently serum assays for NTX and CTX have become available
[15,108]. Hydroxylysine Glycosides Similar to the pyridinium cross-links, hydroxylysine glycosides are derived from hydroxylysine (Hyl) residues of collagen chains. They arise during posttranslational modifications of the collagen chain within the endoplasmic reticulum [13]. Hydroxylysine can be glycosylated to galactosyl-hydroxylysine (Gal-Hyl) or to glucosylgalactosyl-hydroxylysine (Glc-Gal-Hyl) [120]. Neither of these compounds is entirely specific for bone collagen. However, the concentration of Glc-Gal-Hyl is higher in skin than in bone collagen, whereas the reverse is true for Gal-Hyl [120]. For this reason, Gal-Hyl is regarded as a more useful bone marker [98]. Like the other collagenbased resorption markers, Gal-Hyl and Glc-Gal-Hyl are released during collagen degradation and are not reutilized for new collagen formation (Fig. 7) [13,120]. Hydroxylysine glycosides have received less attention than hydroxyproline or pyridinium cross-links as markers of bone resorption in children and adolescents probably because they can only be determined by highperformance liquid chromatography. An immunoassay has recently been developed to quantify Gal-Hyl in urine
samples [66], but the clinical experience with this assay appears to be limited. Tartrate-Resistant Acid P h o s p h a t a s e Tartrate-resistant acid phosphatase (TRAcP) is special among markers of bone resorption in that it is an enzyme derived from the osteoclast that can be quantified in serum samples [13]. Assays to quantify TRAcP enzyme activity have been available for a long time [135]. In recent years, a variety of immunoassays have been developed [40,41].
BONE MARKERS DURING NORMAL DEVELOPMENT
Biological Variation Several markers of bone and collagen metabolism show circadian variations, usually with higher values at night than during the day [37,46,100,112]. In children, PICP varies by 10-20%, osteocalcin by 3060%, and Gal-Hyl by 30% over a 24-hr period, although bone ALP does not show significant circadian variation [137]. To minimize the effect of such variation when assessing children longitudinally, the time of day when samples are collected should be standardized. Within-individual, day-to-day variation of the urinary markers can also be considerable [74,100,124,136] but has not been reported for serum markers due to ethical constraints. Between-individual biological variation is wide for all markers at all ages. Single measurements therefore have limited value unless they are very aberrant. Prenatal Period Little is known about bone metabolism during normal intrauterine development. A number of studies attempted to gain insight into fetal bone metabolism by analyzing cord blood from babies born prematurely or from term newborns. Early studies focused on cord blood levels of osteocalcin, with variable results [17,29,68,82,93,122,125]. Osteocalcin cord blood levels were described as being lower [93] or much higher [17,29,68] than the mean value for healthy adults. However, studies are in agreement that osteocalcin levels are higher in fetal than in maternal blood, partly because maternal osteocalcin levels are very low at the end of pregnancy [17,29,93,107,125]. An increase in cord blood osteocalcin was described after 22-27 weeks of gestation
14. Biochemical Markers of Bone Metabolism
and a decrease thereafter [122]. In term newborns who were small for gestational age, osteocalcin cord blood concentrations were lower than those in newborns whose size was appropriate for gestational age [82]. In recent years, researchers have increasingly employed markers of collagen type I turnover. The concentration of PICP, PINP, and ICTP in cord blood decreases with gestational age [53,56,87,110,121]. There is no relationship between fetal and maternal bone marker levels at birth [17,147]. In all these studies, blood was obtained after birth, which obviously leaves open the possibility that results are influenced by the birth process or by the condition leading to premature birth. Recently, bone markers have also been quantified in surplus amniotic fluid obtained during amniocentesis. Levels of collagen turnover markers decreased with gestational age [43,58]. Interpretation of such data is difficult because it is unknown how these collagen markers enter amniotic fluid and how they are eliminated. Probably the most reliable information on fetal bone metabolism derives from studies that used material from fetal blood sampling by intrauterine cordocentesis. The few available data confirm that bone and collagen metabolism decreases from 18 weeks of gestation to birth. This pattern was found for total ALP activity [83] as well as for PICP and ICTP [88], reflecting similar changes in the rate of intrauterine weight gain [32]. Few data are available on the clinical utility of evaluating biochemical bone markers prenatally or at birth. Case reports have suggested that bone markers can help in the prenatal diagnosis of congenital bone disorders, such as hypophosphatasia and osteogenesis imperfecta [58,138]. Markers of collagen metabolism might be useful for studies that assess the effect of obstetric interventions, such as antenatal dexamethasone therapy, on fetal bone development [56,109]. In contrast, attempts to gain insight into the pathophysiology of bone metabolism in infants of diabetic mothers have been largely unsuccessful [30,43,87]. N e o n a t a l Period, Infancy, a n d C h i l d h o o d The immediate postnatal period is characterized by rapid adaptational changes in many organ systems. The skeleton has to adapt to the sudden interruption of placental supply of nutrition and hormones as well as to the transition from a mechanical environment in which movements occurred against the resistance of the uterine wall to an environment with unrestricted movement [101]. Probably the best studied aspect of early skeletal
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changes is the decrease in neonatal serum calcium levels after the placental supply of calcium is cut off, leading to a secondary increase in parathyroid hormone levels [62]. This increase occurs within hours after birth. Repeated measurements within the first few days of life are therefore necessary to study the effects on early postnatal bone metabolism adequately. In one of the few longitudinal studies on healthy term newborns, Loughead et al. [68] reported a dramatic decline in serum osteocalcin levels within 2 hr after birth and no further changes during the first 24 hr of life. This contrasts with the findings of Delmas et al. [29], who showed that osteocalcin levels during the first day of life are similar to cord blood levels and increase during the following 4 days. Urinary levels of hydroxyproline or NTX related to creatinine increase 50-80% during the first 48 hr of life [81,144], but it is unclear whether these findings reflect increasing collagen type I degradation or a decrease in creatinine excretion during the immediate postnatal period. Cross-sectional studies found decreasing PICP but stable ICTP levels during the first 24 hr of life [95,96]. There are insufficient data to draw firm conclusions about the immediate postnatal changes in bone metabolism. However, several studies are in agreement that bone formation decreases immediately after birth, whereas bone resorption remains similar or even increases. This scenario appears plausible when skeletal adaptation is considered from a nutritional (mobilization of calcium after interruption of placental supply and initially insufficient intake) or from a biomechanical standpoint (bone loss in a disuse situation). Once the early adaptational hurdles have been cleared and the phase of regular and rapid growth begins, an increase in bone metabolism is expected. The few available studies on term newborns confirm this expectation. Serum concentrations of ALP, osteocalcin, PICP, ICTP, as well as urinary hydroxyproline and cross-link to creatinine ratios increase after the first 3 days of life [2,23, 94-96,140,144]. In these studies, collagen markers PICP, ICTP, and urinary cross-links were maximal at approximately 4 weeks of age and decreased thereafter, whereas ALP activity had a plateau between 45-90 days and osteocalcin continued to increase until 3 months of age [94]. During the remainder of the first year of life all bone markers decrease rapidly, and this decline continues more slowly until 3 or 4 years of age. This pattern was first noted in a study on total serum ALP activity [14], and it is also observed with newer serum markers of bone metabolism [1,8,19,67,72,80,113,137,139,144]. After 4 years of age, serum bone marker levels usually
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products is offset by the concomitant increase in creatinine excretion. After pubertal stage 5, bone marker levels decrease to adult levels (Fig. 8) [98].
remain stable until puberty [14,17,27,57,137,139]. Urinary resorption markers related to creatinine tend to decrease after 4 years of age until puberty [1,8,50,102,144], but this may reflect increasing creatinine excretion rather than decreasing bone resorption [98].
BONE AND COLLAGEN MARKERS IN METABOLIC BONE DISEASES
PUBERTY All three mechanisms contributing to bone turnover in growing individuals (longitudinal growth, modeling, and remodeling) accelerate during puberty [38,90,134]. This pubertal growth spurt is reflected by a corresponding increase in many markers of bone metabolism. Since pubertal peak height velocity occurs earlier in girls but is lower than that in boys [134], the peak in bone marker levels is earlier and less marked in girls [7,14,17,44,57,113,137,146]. However, most studies on urinary resorption markers normalized to creatinine failed to detect a pubertal peak [7,74,100,102] because the increase in the excretion of collagen breakdown
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Interpretation a n d Problems of Bone Marker Results What is osteoblast activity? An ideal biochemical marker specifically reflects the activity of one of the two effector cell types in the skeletonmosteoblasts and osteoclasts. As such, they are classified as indicators of osteoblast activity or osteoclast activity. These terms are often used in the bone marker literature, but they are rarely defined. Here, we consider what determines the levels of markers of osteoblast activity. Analogous considerations apply to osteoclast activity.
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14. Biochemical Markers of Bone Metabolism
these components can be estimated by methods other than biochemical bone marker analysis. Total body bone mass can be determined by densitometric techniques, whereas the number of osteoblasts per unit bone surface area and osteoblast activity can be measured using bone histomorphometry. Bone surface-tobone mass ratio is closely related to a histomorphometrical parameter, the bone surface-to-bone volume ratio. It thus becomes possible to interpret bone marker results in a more detailed fashion.
Biochemical markers of bone formation provide systemic information. As such, they mirror the sum activity of all osteoblasts at all skeletal regions. Systemic osteoblast activity is determined by the number of osteoblasts in the body and by the average activity of single osteoblasts (Fig. 9). In the literature, bone formation markers are often interpreted as if they reflected only the activity level of osteoblasts, but the number of osteoblasts is probably a much more important determinant of whole body bone formation activity [89]. What determines the total number of osteoblasts in the skeleton? Osteoblasts are exclusively located on bone surfaces (periosteal, intracortical, endocortical, and trabecular). Therefore, total body osteoblast number is determined by two factors (Fig. 9):(i) the total skeletal bone surface area, corresponding to the areas of periosteal, intracortical, endocortical, and trabecular surfaces combined, and (ii) the average number of osteoblasts per unit bone surface area. Finally, total skeletal bone surface area can be regarded as the product of two factors--total body bone mass and the average bone surface per bone mass ratio (Fig. 9). The dependence of bone marker levels on bone mass is rarely considered, but it is obvious that a skeleton weighing 2 kg will produce twice as much bone marker molecules as a skeleton weighing 1 kg, if everything else is equal. In synthesis, systemic osteoblast activity can be broken down into four factors: total body bone mass, bone surface per unit bone mass, the number of osteoblasts per unit bone surface area, and mean osteoblast activity (Fig. 1). The advantage of breaking down whole body osteoblast activity into these factors is that each of
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Osteogenesis imperfecta is characterized by bone fragility and decreased bone mass. Detailed densitometric and bone histomorphometric analyses have been published [71,103]. It is thus possible to interpret bone marker findings in light of the system developed in the previous section. Serum levels of the bone formation marker PICP are typically low in patients with osteogenesis imperfecta [11,70,97]. Values average approximately 50% of the mean result in age-matched control subjects. At the same time, the urinary bone resorption parameter NTX related to creatinine is elevated by at least 50% compared to age-specific mean values [12]. These results seem to show that bone formation is decreased and bone resorption is increased in osteogenesis imperfecta, explaining why bone mass is low in osteogenesis imperfecta. Although attractive at first glance, this model is nevertheless implausible. A condition in which the amount of bone formed is decreased
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to half the normal value and the amount of bone resorbed is increased by 50% would lead to the rapid disappearance of the skeleton. For example, it can be calculated from histomorphometric data [103] that iliac bone trabeculae would be completely resorbed within 2 or 3 years. This does not happen, and in fact bone mass increases after birth even in children with severe osteogenesis imperfecta, although it occurs at a much slower than normal rate. This raises the question: What is wrong with these bone marker studies? As mentioned previously, it is incorrect to interpret serum bone marker levels simply as a mirror of single cell activity. This erroneous view, however, is the basis for the explanation that PICP is low in osteogenesis imperfecta because single osteoblasts produce only half the normal amount of bone. As discussed earlier, bone marker levels also depend on bone mass, bone surface-tomass ratio, and cell number-to-bone surface ratio. These parameters can only be determined by histomorphometry. Table 1 shows the relevant histomorphometric data for osteogenesis imperfecta type III; findings for type I and IV are similar [103]. As expected, osteoblasts of osteogenesis imperfecta patients form bone at only half the normal rate. However, the number of osteoblasts relative to bone surface area is very much increased, resulting in increased surface-based bone formation rates. The bone surface-to-mass ratio is also increased, but bone mass is very much decreased. The net result of these abnormalities is that bone formation rate on the level of the entire bone is low. In addition, low height
TABLE 14-1 B o n e f o r m a t i o n r a t e s in o s t e o g e n e s i s i m p e r f e c t a t y p e 111 o n i n c r e a s i n g levels of biological o r g a n i z a t i o n
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very high osteoblast number more than compensates low activity of single cell
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a high bone-surfaceto-mass ratio leads to even higher bone mass related bone formation rate
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extremely low bone mass leads to low bone size related bone formation rate
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velocity also contributes to low serum bone marker levels. Thus, the low serum levels of the bone formation marker PICP reflect low bone mass and slow bone growth rather than the weakness of individual osteoblasts. Why then is there an increase in urinary levels of the bone resorption marker NTX? It is often forgotten that urinary concentrations of bone resorption markers are related to creatinine to allow for differences in water diuresis. Urinary creatinine excretion reflects muscle mass [47], which is clearly decreased in osteogenesis imperfecta patients. Thus, high urinary NTX-to-creatinine ratios are likely to reflect low muscle mass rather than increased bone loss. What can be learned from this example? Biochemical markers of bone metabolism necessarily reflect a variety of skeletal properties other than bone turnover, including bone mass, bone surface-to-mass ratio, osteoblast or osteoclast number relative to bone surface area, and longitudinal growth rate. Conclusions that are based only on bone marker levels and do not take into account these properties of bone are suspect. This is especially true for attempts to elucidate the pathophysiology of bone disorders in cross-sectional studies that compare bone markers in a group of patients and healthy controls. Short-term longitudinal studies that analyze changes in biochemical bone markers after an intervention are less affected by these limitations. In this situation, changes in bone markers are more likely to directly reflect changes in bone metabolism because bone structure and mass can be assumed to remain similar during the study interval. Prematurity The postnatal changes in bone markers after premature birth are similar to those after birth at term if the clinical course is uncomplicated [23,29,84,121,141 ]. The best studied parameter is total ALP activity, which is typically approximately two or three times higher than adult levels at birth and increases further during the first 3-6 weeks of life [5,23]. Elevated levels during the first weeks of life indicate a problem with bone mineralization (rickets and osteomalacia) and are associated with growth delay that may persist until at least 12 years of age [34,69]. It is important that the ALP isoenzyme pattern in the neonatal period is different from that in later life [13,23]: A so-called fetal intestinal isoform, which is not detectable at birth, increases after the start of enteral feeding and peaks approximately 2 weeks later. The maximum level of the fetal intestinal isoform is negatively correlated with gestational age and is approximately 30-50% of total ALP levels in very premature infants [23]. Bonespecific ALP levels might therefore reflect bone metabolism better than total ALP in these infants.
14. Biochemical Markers of Bone Metabolism
Bone I n v o l v e m e n t in Acute a n d Chronic Illnesses Seriously ill children do not grow well and, not unexpectedly, levels of bone markers are low in such patients. This has been consistently demonstrated for osteocalcin, bone-specific ALP, PICP, PYD, and DPD in disease states as diverse as malnutrition, phenylketonuria, acute lymphatic leukemia, active rheumatic diseases, and severe burns [10,22,42,48,60,92,104,128]. Bone markers also reflect the acceleration of growth during the successful therapy of these disorders [60,104,128].
Corticosteroid Therapy Children with asthma treated with intravenous methyl-prednisolone showed a rapid decrease in PICP within 24-48 hr of initiating treatment [51]. In preterm infants with bronchopulmonary dysplasia who were treated with a daily dose of 500 gg/kg dexamethasone, marked decreases in PICP and ICTP were seen within 3 days of starting treatment, accompanied by growth arrest and weight loss [24]. During steroid weaning, all markers began to increase at a daily dose of approximately 200 gg/kg. After stopping dexamethasone, collagen markers and anthropometric indices rebounded in several infants. In children with inflammatory bowel disease, significantly lower PICP and height velocities were found among subjects receiving daily prednisone therapy than in those receiving alternate-day or no corticosteroid therapy [52]. Following surgery and withdrawal of corticosteroid therapy, PICP and height velocity increased within 2 months. During the induction treatment of children with acute lymphoblastic leukemia, high doses of oral prednisolone are administered along with other chemotherapeutic agents. This is associated with apparent growth arrest and a decrease in several markers of bone and collagen turnover [21,22]. After prednisolone is discontinued, longitudinal growth resumes and all markers increase. Similar phases of decrease and recovery of markers occurred during intensification cycles that included corticosteroids
[20]. These studies confirm the well-known fact that systemic glucocorticoid administration is associated with reduced longitudinal bone growth and overall bone turnover. They also demonstrate the ability to rebound rapidly once systemic glucocorticoid administration is stopped. The effect of inhaled steroids on bone development is less obvious than that of systemic steroids. A crosssectional study of asthmatic children did not detect an effect of inhaled beclomethasone on the bone formation
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markers bone ALP and osteocalcin [61]. In a more sensitive longitudinal study, PICP and osteocalcin, but not bone ALP, decreased within 1 month of starting treatment with inhaled beclomethasone [130]. One month of treatment with 800 fg of inhaled budesonide had a similar effect and resulted in significantly decreased levels of PICP, PINP, ICTP, PYD, DPD, and NTX [129]. Even lower dose budesonide (200 gg per day) led to detectable suppression of these collagen markers [45]. Thus, markers of collagen metabolism are sensitive indicators of the systemic effects of glucocorticoid treatment. The other markers of bone metabolism seem to yield more variable results. These longitudinal studies represent good examples of how biochemical markers can be used to obtain meaningful results. Bone markers were assessed before and after a short period of therapy. A panel of markers of bone formation and resorption were used to ensure that results indeed reflected bone metabolism. Growth Disorders a n d Growth Prediction All three mechanisms that characterize skeletal growth (endochondral bone formation, modeling, and remodeling) involve the formation and degradation of bone matrix. It is therefore not surprising that various biochemical parameters derived from bone and collagen metabolism show some correlation with longitudinal growth of children. Indeed, the association between growth and bone markers has been recognized for more than five decades [14]. Since that time, few authors using bone markers in pediatric studies have failed to comment on the potential utility of bone markers as indicators of growth. The open question is whether this association with growth can actually be used for diagnostic purposes. Predicting the response to growth hormone treatment could be one of the clinically most relevant situations for the use of growth indicators. The individual therapeutic effect on growth is quite variable and it would indeed be helpful to have a way to evaluate responsiveness after a short period of therapy. Currently, therapeutic success is typically assessed only after 1 year of therapy, and the dosage is increased when growth is not satisfactory. A sufficiently precise prognosis regarding whether the growth response will match expectations would allow for a faster dose adjustment. Many studies have examined markers of bone turnover in growth hormone-deficient children. In untreated patients, levels of osteocalcin [55,85,111,116], PICP [54,111], total and bone-specific ALP [85,137], ICTP [65], PYD, DPD [37], and galactosyl-hydroxylysine [100] are lower than in healthy control groups. However, there is considerable overlap and these markers cannot be used to diagnose growth hormone deficiency in individual patients.
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After treatment with growth hormone is started, levels of all markers of bone metabolism increase within 1-12 weeks [37,54,55,65,85,100,111,116,118,137,139]. Positive associations with growth velocity after 1 year of growth hormone treatment have been reported for the increase of PICP after 1 week [111], PYD and DPD after 1 month [131], and galactosyl-hydroxylysine and osteocalcin after 3 months [55,98,100] (Fig. 10). Fewer such studies have been performed in short children without growth hormone deficiency. In children with short stature due to intrauterine growth retardation, osteocalcin levels were normal before treatment and increased in a dose-dependent manner after 1 year of growth hormone therapy [28]. Crofton et al. [25,26] demonstrated that total and bone-specific ALP were indicators of height velocity in most children with idiopathic short stature receiving growth hormone treatment. Similar results were obtained using PICP and ICTP [143]. Thus, there is ample evidence that markers of bone metabolism reflect height velocity. Unfortunately, there is a large gap between a statistically significant association and a clinically relevant prediction, even if widely used statistical jargon suggests otherwise. For practical purposes, it is certainly not sufficient to show that there is a high correlation coefficient between a marker and height velocity. More important is the confidence 20 15
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interval of the prediction. The reality is that the confidence interval of the predictions based on a single bone marker typically encompasses the entire range from low to high height velocities. The limited utility of using a single bone marker for growth prediction is widely realized. Therefore, recent attempts to predict the success of growth hormone therapy used combinations of clinical and biochemical measures, including markers of bone and collagen metabolism [98,118] (Fig. 11). The accuracy of the prediction that can be achieved in this manner might be adequate for clinical use. However, these models require validation in larger groups of individuals before firm conclusions can be reached.
Renal Osteodystrophy Chronic renal failure in children is often associated with slow growth and other skeletal disorders, including secondary hyperparathyroidism, aluminum-related low turnover bone disease, osteomalacia, and adynamic osteopathy [49]. Although the distinction between these conditions may have important therapeutical implications, few studies have analyzed the value of bone markers ICTP [143]. Thus, there is ample evidence that markers of bone metabolism reflect height velocity. Unfortunately, there is a large gap between a statistically significant association and a clinically relevant prediction, even if widely used statistical jargon suggests otherwise. For practical purposes, it is certainly not sufficient to show that there is a high correlation coefficient between a metabolism in these patients because in conditions associated with slow growth, the relative input of the bone isoform to total ALP decreases. In fact, total ALP has been shown to be a poor predictor of bone histology in children undergoing peritoneal dialysis [114,115]. Renal
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FIGURE 1 1 Residuals of a prediction based on 3-month height velocity, relative bone age retardation before therapy, pretreatment IGF-1, and DPD measured after 1 month of therapy versus observed 12-month height velocity. Reproduced with permission from Schtinau, E., Rauch, F., Blum, W. F., (2001). Prediction of growth response to GH treatment: A reference guide. Abingdon: United Kingdom: TMG Healthcare Communications, Ltd.
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14. Biochemical Markers of Bone Metabolism
in bone histomorphometry in adults with predialytic renal failure [16].
bone disease might be an indication to determine bonespecific ALP, which has been shown to correlate with histomorphometrically determined bone formation rate in adults [142]. The value of other bone markers in renal osteodystrophy remains to be elucidated. Urinary markers and serum assays of small molecules that are eliminated via the kidneys (such as osteocalcin and ICTP) are probably not useful when renal clearance is impaired. In contrast, serum levels of PICP are independent of renal function [16] and have been found to reflect dynamic parameters
Rickets Vitamin D deficiency rickets is the classical metabolic bone disease in childhood, which today is relatively rare in Western countries. Elevated total ALP activity is a characteristic finding and is very useful in monitoring the effect of treatment [4,59] (Fig. 12). The value of other markers is less certain. Because the contribution of the
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FIGURE 12 Longitudinal variation of mean serum levels of alkaline phosphatase (A), osteocalcin (B), C-terminal propeptide of type I procollagen (PICP) (C), cross-linked C-terminal teleopeptide of type I collagen (ICTP) (D), and mean urinary excretion values of cross-linked N-teleopeptides of type I collagen (NTX) (E) in children with vitamin D deficiency rickets before (time point 0) and during vitamin D treatment. Duration of vitamin D treatment: 8 weeks, n = 14; 10 weeks, n -- 10; 12 weeks, n = 14; 14 weeks, n -- 1. The rectangular area represents mean + 2 SD of controls for each biochemical bone marker: a, p -- not significant; b, p < 0.05; c, p < 0.01; d, p < 0.001 in comparison to baseline values (time point 0). Reproduced, with permission, from Bone turnover in children with vitamin D deficiency rickets before and during treatment. Acta. Paediatr. (2000), 89, 513-518.
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liver isoform to total ALP activity is negligible in children with increased bone turnover, who do not simultaneously suffer from cholestatic liver disease [99], the determination of bone-specific ALP does not offer any advantage in rickets [105]. Osteocalcin levels do not show a consistent pattern in children with vitamin D or calcium deficiency rickets [39,64,86,117]. Levels of PICP and ICTP are mildly elevated before treatment [4,113,117]. E h l e r s - D a n l o s S y n d r o m e Type Vl Ehlers-Danlos syndrome type VI is a rare disorder affecting connective tissue in many organs [133] and is caused by a deficiency in lysyl-hydroxylase, an enzyme involved in the posttranslational modification of the procollagen molecule. Because PYD is synthesized from three hydroxylysine residues, whereas DPD is made up of one lysine and two hydroxylysine residues [33], lack of lysyl-hydroxylation leads to the preferential synthesis of DPD. This can be easily diagnosed in urine samples: The PYD:DPD ratio, which is approximately 4:1 in healthy subjects, is approximately 1:4 in individuals affected by Ehlers-Danlos syndrome type VI [91,!32].
CLINICAL AND RESEARCH VALUE OF BIOCHEMICAL MARKERS OF BONE METABOLISM As demonstrated in this chapter, a considerable amount of pediatric literature has accumulated on biochemical markers of bone and collagen metabolism. However, for most situations there is still no answer to the clinically most relevant question: Do the newer bone markers improve clinical management of bone disorders in children and adolescents? In clinical care, total serum activity of ALP remains the mainstay of diagnosis and follow-up of such conditions, and the other markers are largely dispensable. Currently, biochemical markers of bone and collagen metabolism are mostly used as research tools. Such research might benefit from a more critical approach to bone markers. It is tempting to simply take results of bone markers at face value, especially when the data appear to confirm a study's hypothesis. However, no marker exclusively reflects bone metabolism, and the many confounding factors should be considered to avoid misinterpretation. A classical example is the influence of muscle mass and metabolism on urinary resorption markers that are normalized to creatinine. It should also be acknowledged that little is known about the metabolic pathways of most markers in children and adolescents. How diseases and therapies affect these pathways is virtually
unexplored. These gaps in our knowledge call for caution in the interpretation of results. Even an ideal bone marker that would exclusively reflect bone metabolism can only provide information on the lump activity of bone growth in length, growth in width, and bone maintenance. A bone disorder or a therapy might have opposing effects on these mechanisms, and it is impossible to detect this on the basis of bone markers alone. The effect on growth in length could be partly accounted for by relating results of bone markers to height velocity. This would require that reference data be presented not only as a function of age but also as a function of longitudinal growth rate [98].
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FIGURE 13 The median levels of the serum markers of bone formation (top) and the levels of the markers of collagen degradation (bottom) in 14 children with asthma inhaling budesonide for 6 months (higher dose for 1 month and lower dose for 5 months) and nedocromil during 6 subsequent months (n = 12). Reproduced, with permission, from changes in bone markers in children with asthma during inhaled budesonide and nedocromil treatments. Acta. Paediatr. (1996), 85, 1176-1180.
14. Biochemical Markers of Bone Metabolism
The limitations of bone markers particularly affect the utility of cross-sectional studies. It is extremely difficult to gain useful insights on the pathophysiology of bone disorders by performing cross-sectional studies in a group of patients. In most of these situations, bone histomorphometric data are required for proper evaluation of bone metabolism. Nevertheless, biochemical bone markers can yield useful information in the pediatric context. Their advantage compared to bone histomorphometry is that they can be obtained in a noninvasive manner and thus can be determined repeatedly in short time intervals. As exemplified by Fig. 13 [129], the main strength of bone markers is to assess short-term changes following therapeutic interventions. In summary, bone markers are mostly research tools that can provide some preliminary insights in situations in which bone histomorphometric data are not available and cannot be obtained.
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[15I Bone Histomorphometry FRANK RAUCH Genetics Unit, Shriners Hospital for Children, Montreal, Quebec, Canada
INTRODUCTION
aim is to open this field to the nonspecialized reader with an interest in pediatric bone disorders. More detailed accounts of methodology can be found elsewhere [3,4]. Readers who wish to know more about the physiological and pathophysiological meaning of histomorphometric parameters can find information in A. M. Parfitt's writings [5-14].
Since Harold Frost pioneered bone histomorphometry in the early 1960s, this technique has been a key tool for studying bone metabolism and, to a lesser extent, bone mass and structure. Histomorphometry of undecalcified bone samples is a method to directly obtain quantitative information on bone. It allows the determination of the amount and distribution of bone tissue with unsurpassed resolution. When tetracycline labeling is performed prior to biopsy, bone histomorphometry offers the unique possibility to study bone cell function in vivo. At the same time, qualitative histologic assessment is possible, which allows the detection of subtle disorders of bone structure. Importantly for pediatric use, the growth process does not interfere with the measurements. Bone histomorphometry is also an excellent educational tool. The insight gleaned from studying bone tissue can be used to better understand results of indirect methods of bone analysis. For example, much of the current confusion surrounding the interpretation of bone densitometry data in children could certainly have been avoided if the tissue-level characteristics of bone were better known in the pediatric scientific community [1,2]. On the other hand, knowledge of bone tissue is crucial to put the disparate findings of molecular and cellular studies into perspective. Despite its many advantages over indirect methods of bone analysis, bone histomorphometry is dramatically underused in pediatrics. This might be partly due to the fact that it is an invasive technique. Bone histomorphometry is also time-consuming, labor intensive, and requires special equipment and expertise. This chapter summarizes the methodology of bone histomorphometry and highlights the tissue-level characteristics of normal and abnormal bone development. The
PediatricBone
METHODOLOGY B o n e Biopsy Clinical Procedure
Bone histomorphometry was first developed to study rib bone samples. This was soon abandoned because the ilium proved to be a much more convenient site for obtaining bone samples. In principle, histomorphometric analysis can be performed in any bone. In clinical pediatrics, however, the utility of samples from nonstandard sites is limited because reference data are only available for the ilium. Quantitative bone histomorphometry requires an intact biopsy specimen of good quality. This implies that the transiliac sample must be obtained under standardized conditions and with appropriate tools. It is essential that the sample is not fractured or crushed and contains two cortices separated by a trabecular compartment. These requirements are often quite difficult to meet in small or very osteopenic children. Therefore, the key to a meaningful histomorphometric analysis is that the sample be taken by a skilled and experienced operator. Bone specimens for histomorphometric evaluation are horizontal, full-thickness (transfixing) biopsies of the ilium from a site 2 cm posterior from the anterior superior iliac spine (Fig. 1). This bone is easily accessible, does
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Copyright 2003, Elsevier Science (USA). All rights reserved.
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not require extensive surgery, and is associated with few postoperative complications. Also, this is the only site for which pediatric histomorphometric reference data have been published [15]. It is important to note that horizontal transiliac samples are required for histomorphometric evaluation. Vertical samples (from the iliac crest downwards) cannot be used. The transiliac sample must be obtained at a site well below the iliac crest growth plate. Specimens containing growth cartilage do not allow for a reliable quantitative analysis because turnover is very high and cortical thickness is very low in the bone adjacent to the growth plate. The usual bone biopsy instrument is the Bordier trephine (Fig. 2). The core diameter of the trochar must be at least 5 mm and should preferably be 6 or 7 mm. Most children younger than 14 years of age require general anesthesia for the procedure. Local anesthesia can be sufficient for older adolescents. This procedure does not have side effects other than transient local discomfort. Patients are allowed to get out of bed after 3 hr and can usually be discharged on the same day.
FIGLIRE 1 procedure.
Anatomic location for the transiliac bone biopsy
Obviously, the operator's experience is an important factor in keeping intervention-related morbidity to a minimum. Another prerequisite for histomorphometric evaluation is bone labeling. Dynamic parameters of bone cell function can only be measured when the patient has received two courses of tetracycline label prior to biopsy. Tetracycline compounds form calcium chelate complexes that bind to bone surfaces. These complexes are buried within the bone at sites of active bone formation, whereas they redissociate from the other bone surfaces once serum tetracycline levels decrease. The tetracycline trapped at formation sites can then be visualized under fluorescent light (see color plate 1). Figure 3 shows the labeling schedule used at the Shriners Hospital Montreal. The tetracycline compound used is demeclocycline hydrochloride (Declomycin) at a dose of 15-20 mg/kg per day (maximum dosage, 900 mg per day). The daily amount is given orally in two doses. The drug is given for 2 days in both label courses. The two courses are separated by an interlabel time of 10 days. Bone biopsy is performed 4-6 days after the last administration of demeclocycline. Although children and adolescents generally tolerate tetracycline double labeling well, some side effects might be observed, such as allergic reactions, vomiting, and photosensitivity. Administering the drug after meals can diminish gastrointestinal side effects. It is important that these meals do not include milk or other dairy products because tetracycline complexes with calcium contained in the food and is not absorbed adequately. Sun exposure must be avoided while taking tetracyclines. Tetracycline use is generally not recommended for children younger than 9 years of age because discoloration of teeth may occur. However, the previously mentioned schedule appears to be safe in this respect. At the Shriners Hospital Montreal, it has been used for more than 300 biopsies in children younger than 9 years of age and tooth discoloration has never been observed.
Sample Processing
FIGLIRE 2
View of a 5-mm trephine for transiliac bone biopsy.
The biopsy sample should be placed into a fixative as soon as possible after the procedure. The fixation process aims at the preservation of bone tissue constituents by inactivating lysosomal enzymes. The choice of fixative and temperature at which the sample should be kept depends on the planned staining techniques. For routine histomorphometry, 70% ethanol or 10% buffered formalin at room temperature can be used. The duration of fixation should be at least 48 hr but should not exceed 10 days because the tetracycline labels are washed out when fixation is too long. Following
15. Bone Histomorphometry
Patient's N a m e : ..................................................... Date of b i r t h " . ................................... Dosage of demeclocycline: 15-20 mg / kg / d (per os). Maximum" 900 mg/ d. Declomycin (Lederle) is available in 150 nag or 300 rng tablets to be taken orally. Weight"
kg
Daily dose
mg
IMPORTANT.
Give the medication as indicated on the calendar below. If for any reason
you cannot follow the schedule, indicate all changes on the document and call . . . . . . . . . . . . . . . . . . .
Bring this document with you at the time of admission and sign that the patient received the medication as indicated on the schedule.
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Name of patient or legal guardian FIGURE 3
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Date
Schedule for prebiopsy tetracycline labeling.
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fixation, the specimen is 326 dehydrated in absolute ethanol and embedded in methylmethacrylate. Cutting mineralized bone requires a special microtome operated by a skilled technician. For each specimen, two to five series of undecalcified, 6- to 10-gm-thick consecutive sections should be cut at least 150 gm apart. The sections are then deplastified to allow for optimal staining. The most widely used staining methods for histomorphometric analysis are probably toluidine blue and Masson Goldner trichrome. The section that will be used for fluorescence microscopy is mounted unstained. An appropriately large sample area must be available to obtain representative measures. Therefore, at least two sections of a biopsy should be available for each type of analysis in order to obtain a measurable tissue area of 40-50 mm 2. Measurement Procedure
The actual histomorphometric analysis requires a high-quality microscope that is suitable for fluorescence microscopy. In the early years, histomorphometric measurements were performed by manual or point-counting techniques. These methods involved the use of a grid placed in the microscope eyepiece. This has been replaced by computerized systems that allow for automation of the analysis process. Whatever method is used, histomorphometric analysis is time-consuming because even the most advanced systems rely on the operator's judgment to correctly identify the individual histoanatomical components. Histomorphometric Measures Definitions
Reporting histomorphometric results was greatly facilitated by the introduction of a standardized and well-
FIGURE 4
defined terminology in 1987 [5]. Articles published before 1987 are often quite difficult to read because many authors used private nomenclature. An introductory overview of the most important histomorphometric terms is given here. More detailed information can be found in the 1987 terminology report [5]. In histomorphometry, bone is defined as bone matrix, whether mineralized or not. Unmineralized bone is called osteoid, whereas mineralized bone does not have a special designation. Bone and the associated soft tissue, such as bone marrow, are referred to as tissue. Osteoblasts are cells on bone surfaces that are producing and secreting bone matrix currently or with only temporary interruption. Flat cells of the osteoblast lineage that cover quiescent nonperiosteal bone surfaces are referred to as lining cells. The term osteoclast is restricted to cells that are currently in contact with a bone surface and are actively resorbing bone. A transiliac biopsy specimen consists of two cortices separated by a cancellous compartment (Fig. 4). The terms cancellous and trabecular are usually used interchangeably. The outer delimitation of the cortex is called periosteal surface, and the inner border is the endocortical surface (Fig. 4). Osteonal and Volkmann canals are lined with intracortical surfaces. The bone surfaces in the cancellous compartment are referred to as cancellous (or trabecular) surfaces. Intracortical, endocortical, and trabecular surfaces are in continuity and together form the endosteal surface or envelop. In the literature, there is confusion regarding the latter term because many authors use the term endosteal surface when in fact referring to the endocortical surface. Histomorphometric measurements are performed in two-dimensional sections. This may cause conceptual problems because bone is a three-dimensional organ. What is perceived and measured as an area in the
Schematic representation of a bone biopsy section. The different types of bone surfaces are indicated.
15. Bone Histomorphometry histologic section in fact reflects a volume. In order to highlight the three-dimensional nature of bone, threedimensional terminology is favored when reporting results. For example, the percentage of unmineralized bone is measured in the two-dimensional bone slice as osteoid area relative to total bone area. However, the result of this ratio is reported as osteoid volume per bone volume. This is done simply by convention, and it should not be mistaken as an actual three-dimensional measurement. Histomorphometric Parameters
Terminology for most histomorphometric parameters follows a standardized schema: source - measurement/ referent. Source refers to the type of bone that is measured (e.g., cancellous or cortical). Since analyses are often limited to cancellous bone, the source prefix is usually omitted, as long as there is no possibility of confusion. Measurement is the type of parameter that is determined. Histomorphometric data are usually not given as absolute values but are related to each other. This is what is meant by "referent." Most parameters are related to a surface area or a volume. Histomorphometric parameters can be classified into four categories (Table 1): Structural parameters, static bone formation parameters, dynamic formation parameters, and static bone resorption parameters. Dynamic parameters can only be determined when tetracycline labeling is performed prior to obtaining the biopsy. There are no dynamic parameters of bone resorption, which is one of the main shortcomings ofhistomorphometry.
363 TABLE 1 The most commonly used histomorphometric parameters
Parameter
Abbreviation
Structural Parameters
Core width Cortical width Cortical Porosity Bone Volume / Tissue Volume Trabecular Thickness Trabecular Number Trabecular Separation
C.Wi Ct.Wi Ct.Po BV/TV Tb.Th Tb.N Tb.Sp
Static Formation Parameters
Osteoid Thickness Osteoid Surface / Bone Surface Osteoid Volume / Bone Volume Osteoblast Surface / Bone Surface Osteoblast Surface / Osteoid Surface Wall Thickness
O.Th OS/BS OV/BV Ob.S/BS Ob.S/OS W.Th
Dynamic Formation Parameters
Mineralizing Surface / Bone Surface Mineralizing Surface / Osteoid Surface Mineral Apposition Rate Adjusted Apposition Rate Mineralization lag time Osteoid maturation time Bone Formation Rate / Bone Surface Bone Formation Rate / Bone Volume Activation Frequency Formation Period
Structural Parameters
Static Resorption Parameters
The overall size of an intact biopsy specimen is expressed as core width (C.Wi) (Fig. 5), which is the mean distance between the two periosteal surfaces of the sample. Cortical width (Ct.Wi) is determined as the mean distance between the periosteal and endocortical surfaces of each cortex. Usually, results from both cortices are combined. Determination of Ct.Wi is not as straightforward as the widespread use of this parameter in radiological techniques might suggest. Indeed, there is often a smooth transition from cortical to cancellous bone, and it is a matter of subjective judgment where the border between the two compartments is drawn. Bone volume per tissue volume (BV/TV) of trabecular bone is the combined volume of mineralized and unmineralized bone matrix relative to the total volume of the trabecular compartment (Fig. 5). BV/TV can also be measured in cortical bone, but many authors prefer to express cortical results as cortical porosity (Ct.Po), which is simply the complement of BV/TV in cortical bone (Ct.Po -- 100% - BV/TV). The two surface-to-volume ratios (BS/BV and BS/TV) are important for establishing
Eroded Surface / Bone Surface Osteoclast Surface / Bone Surface Number of Osteoclasts / Bone Perimeter
MS/BS MS/OS MAR Aj.AR Mlt Omt BFR/BS BFR/BV Ac.f FP ES/BS Oc.S/BS N.Oc/B.Pm
the link between bone surface-based cellular activity and the effect on the amount of bone. For example, the differences in turnover of cortical and cancellous bone mostly reflect differences in the bone surface-to-volume ratio, whereas the surface activity of bone cells is quite similar [6]. In trabecular bone, BV/TV can be schematically divided into two separate components: mean trabecular thickness (Tb.Th) and trabecular number (Tb.N). Tb.N is equivalent to the number of trabeculae that a line through the cancellous compartment would contact per millimeter length. The mean distance between two trabeculae, trabecular spacing (Tb.Sp), can be mathematically derived from Tb.Th and Tb.N.
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FIGURE 5 Basichistomorphometricparameters of bone structure.
Apart from these classic structural parameters, a set of indices have been developed to quantitatively describe the architecture of trabecular bone [16]. These approaches have not been applied to pediatric histomorphometry. Static Formation Parameters
Osteoid surface per bone surface (OS/BS) is the surface of all the osteoid seams in the cancellous compartment relative to the total surface of trabecular bone (Fig. 6). Osteoid thickness (O.Th) corresponds to the mean distance between the surface of the osteoid seam facing the bone marrow and the mineralization front (Fig. 6). Osteoid seams do not end abruptly, so some minimum width must be specified. Osteoid volume per bone volume (OV/BV) is the amount of unmineralized osteoid relative to the total amount of mineralized and unmineralized bone. This value is calculated from OS/BS and O.Th. Osteoblast surface per bone surface (Ob.S/BS) is a measure reflecting the area of the interface between osteoblasts and bone relative to the total bone surface. During their active life span, osteoblasts become con-
tinuously flatter and those that remain on the bone surface turn into lining cells. Thus, there is a continuum between fiat osteoblasts and lining cells, and it is necessary to indicate the criteria used to distinguish between the two cell types. Osteoblast surface per osteoid surface (Ob.S/OS) is the percentage of osteoid surface that is covered by osteoblasts. This value is calculated from Ob.S/BS and OS/BS. Wall thickness (W.Th) reflects the amount of bone that is created by the action of a single remodeling unit. It is defined as the mean thickness of the bone that has been laid down at a completed remodeling site (i.e., at locations that are covered by lining cells and where osteoid production has stopped) (Fig. 6). W.Th should not be confused with cortical thickness, with which is does not bear any relationship. The confusion can arise when cortices are inappropriately termed cortical walls, as is sometimes done in the radiological literature. W.Th is measured as the mean distance between the surface of a trabecula and the cement line. The cement line is created in the reversal phase of a remodeling cycle after the osteoclasts have left and before the osteoblasts have arrived (Fig. 6), and for this reason it is also called the reversal line. Reversal lines only appear during remodeling; thus, they represent a histological criterion to distinguish bone created by remodeling from bone made by modeling [6]. The reversal line is difficult to visualize unless special staining procedures are used; alternatively, the wall can be detected by the abrupt change in collagen fiber orientation between adjacent bone lamellae [5]. This is usually easily visible under polarized light (see color plate 2), Dynamic Formation Parameters
The dynamic formation parameters yield information on in vivo bone cell function. Therefore, these parameters are the key to understanding bone physiology, pathophysiology, and the effect of treatment on a tissue level. This underscores the importance of performing tetracycline labeling prior to biopsy.
FIGURE 6 Schematicrepresentation of a remodelingsite in trabecular bone. Osteoclastsin the front dig a trench across the bone surface, which is then refilled by a team of osteoblasts.
15. Bone Histomorphometry
Mineralizing surface per bone surface (MS/BS) represents the percentage of bone surface exhibiting mineralizing activity. This is usually measured as the total extent of tetracycline double label plus half the extent of single label. Mineralizing surface per osteoid surface (MS/OS) is the percentage of osteoid surface undergoing mineralization. This is equivalent to the fraction of the osteoid seam life span during which mineralization occurs [5]. MS/OS is calculated from MS/BS and OS/BS. Mineral apposition rate (MAR) is the distance between the midpoints of the two labels divided by the time between the midpoints of the labeling interval. This is one of the most important parameters because it reflects the activity of individual teams of osteoblasts. Adjusted apposition rate (Aj.AR) is calculated as the product of MAR and MS/BS. As such, it represents the mineral apposition rate averaged over the entire osteoid surface. In a steady state of pure remodeling activity and in the absence of osteomalacia, Aj.AR is the best estimate of the mean rate of osteoid apposition [6] because the rate of bone mineralization is identical to the rate of osteoid production under these conditions. Mineralization lag time (Mlt) is the average interval between the deposition and mineralization of matrix. Mineralization occurs much more rapidly at new formation sites with young osteoblasts than at locations where osteoblasts approach the end of their active careers. Mlt therefore represents a value that is averaged over the entire osteoid seam life span. Mlt is calculated as the ratio between O.Th and Aj.AR. Osteoid maturation time (Omt) is the mean time interval between matrix deposition and the onset of mineralization at a new bone forming site. Omt reflects what is happening at new formation sites when osteoblasts are still "young and dynamic.: Therefore, Omt is almost always shorter and never longer than Mlt. Omt is calculated as the ratio between O.Th and MAR. Bone formation rate per bone surface (BFR/BS) is the volume of mineralized bone formed per unit time and per unit bone surface. It is calculated as the product of MAR and MS/BS. Since bone metabolism occurs only on the surfaces of bone, expressing bone formation rate relative to bone surface is the most logical approach when hormonal effects on bone remodeling are considered [17]. Bone formation rate expressed relative to bone volume (BFR/BV) is equivalent to the bone turnover rate (i.e., it indicates the percentage of bone that is turned over per year). This determines the mean age of the bone tissue [18]. It is often mistakenly assumed that a higher bone formation rate is equivalent to a higher accumulation of bone. However, remodeling removes approximately as much bone as it forms, and bone formation rate does not give any information about the balance between the two
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processes. It is therefore more appropriate to interpret bone formation rate as an index of bone turnover rate rather than of bone gain. The formation period (FP) is the mean time required for building a new bone structural unit or osteon from the cement line back to the bone surface at a single location. FP is calculated as the ratio between W.Th and Aj.AR. Activation frequency (Ac.f) represents the probability that a new cycle of remodeling will be initiated at any point on the surface by the event of activation. Ac.f is the key indicator of remodeling activity. It is calculated as the ratio between BFR/BS and W.Th.
Static Resorption Parameters Eroded surface per bone surface (ES/BS) is defined as the percentage of bone surface presenting a scalloped or ragged appearance of the bone-bone marrow interface with or without the presence of osteoclasts. This is a controversial measure of bone resorption since there is a high degree of subjectivity in recognizing a surface as "ragged" [19]. In addition, many of the locations that are classified as presenting an eroded aspect are the result of aborted resorption attempts rather than of remodelinglinked osteoclast action [19]. Osteoclast surface per bone surface (Oc.S/BS) is the percentage of the bone surface that is in contact with osteoclasts. Apart from measuring the length of the osteoclast-bone surface interface, it is possible to determine the number of osteoclasts. Numbers without units are related to two-dimensional referents because the spatial relationships change with cell morphology, and consequently conversion into three-dimensional values is inappropriate [5]. The number of osteoclasts per bone perimeter (N.Oc/B.Pm) corresponds to the number of osteoclasts in contact with cancellous bone. It is expressed as the number per millimeter length of bone perimeter in a two-dimensional section.
Reproducibility of Histomorphometric Measures Only one study has evaluated the reproducibility of bone histomorphometric measures in children and adolescents [15]. Structural parameters showed a variability of 5-10%, whereas variations were highest for cellular parameters (20-30%). Reproducibility was best for two primary parameters of osteoblast team function~ mineral apposition rate and wall thickness (4 and 5%, respectively). The variability of repeated measurements was smaller in children than in adults [20,21], which may be explained by the higher bone turnover in children. This effect reduces the sampling error for parameters of bone formation and resorption.
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PEDIATRIC BONE HISTOMORPHOMETRY IN HEALTH AND DISEASE The purpose of this section is to highlight histologic and histomorphometric aspects of normal and abnormal bone development in children.
Normal Bone Development The use of bone histomorphometry in pediatrics was long hampered by the lack of normative data. This gap was filled by a recent publication that presented results from 58 individuals between 1.5 and 22.9 years of age who underwent surgery for reasons independent of ab-
TABLE 2
normalities in bone development and metabolism [15]. The results are shown in Table 2. Cortical width and cancellous bone volume increase significantly with age, with the latter due to an increase in trabecular thickness. Osteoid thickness does not vary significantly with age. Bone surface-based indicators of bone formation show an age-dependent decline, reflecting similar changes in activation frequency. Mineral apposition rate decreases continuously with age. Parameters of bone resorption do not vary significantly between age groups. In principle, these results can only be used for comparisons if the same methods are used for processing and analyzing the samples. However, some parameters are more likely to vary with methodology than others.
Reference d a t a (mean_+SD) for iliac b o n e h i s t o m o r p h o m e t r y from 1.5 t o 23 years.
Age (years)
1.5-6.9
7.0-10.9
11.0-13.9
14.0-16.9
17.0-22.9
Structural C.Wi (mm)
5.3+1.4
7.9+1.7
7.1+1.8
8.6+2.4
8.2+1.6
Ct.Wi (mm)
0.70+0.28
0.97+0.37
0.90+0.33
1.18+0.35
1.01+0.20
BV/TV (%)
17.7+2.6
22.4+4.2
24.4+4.3
25.7+5.3
27.8+4.5
Tb.Th (l~m)
101+11
129+17
148+23
157+22
153+24
Tb.N (/mm)
1.77+0.31
1.73+0.17
1.66+0.22
1.63+0.16
1.83+0.27
Tb.Sp (~tm)
481+112
453+62
464+78
461+70
404+77
6.9+1.2
Static Formation O.Th (l~m)
5.8+1.4
5.9+1.1
6.7+1.7
6.3+1.0
OS/BS (%)
34+7
29+13
22+8
26+8
17+5
OV/BV (%)
4.0+1.2
2.6+1.0
2.1+1.0
2.2+0.9
1.6+0.7
Ob.S/BS (%)
8.5+4.1
8.2+4.4
6.7+4.5
7.9+4.1
5.3+2.7
Ob.S/OS (%)
26+14
29+15
29+13
31+12
32+12
W.Th (lxm)
33.9+3.8
40.6+3.0
45.1 +6.9
44.4+3.2
41.1 +2.5
Dynamic Formation MS/BS (%)
12.5+4.5
14.9+4.5
11.7+5.0
12.5+3.4
7.9+2.7
MS/OS (%)
38+13
50+22
53+12
52+14
58+14
MAR (l.tm/d)
1.04+0.17
0.95+0.07
0.87+0.09
0.81+0.09
0.75+0.09
Aj.AR (lam/d)
0.40+0.16
0.47+0.18
0.46+0.10
0.42+0.11
0.43+0.12
Mlt (d)
16.7+6.4
14.1+4.3
14.5+3.0
15.3+3.6
17.3+6.5
Omt (d)
5.7+1.3
6.5+1.0
7.6+1.8
7.6+1.2
9.4+2.3
BFR/BS (lxm3/~tm2/y)
48+19
52+16
37+17
37+10
22+9
BFR/BV (%/y)
97+42
78+27
50+21
48+ 19
29+ 13
Ac.f (/y)
1.40+0.53
1.25+0.37
0.83+0.35
0.83+0.27
0.54+0.23
FP (d)
105+18
99+34
103+28
114+32
102+27
ES/BS (%)
14.8+4.4
17.0+6.0
14.9+5.6
18.0+5.7
18.0+6.1
Oc.S/BS (~
1.1+0.8
1.3+0.6
0.9+0.4
1.1+0.7
1.0+0.4
N.Oc/B.Pm (/mm)
0.35+0.23
0.36+0.16
0.29+0.14
0.34+0.22
0.31+0.14
Static Resorption
15. Bone Histomorphometry
Different staining and handling procedures probably least influence measures of bone structure. The results for osteoid thickness and osteoid surface extent depend on the cutoff threshold for osteoid width. Wall thickness depends on the type of staining and whether the cement line or the abrupt change in collagen fiber orientation is used to define a wall [6]. Cellular parameters depend on the degree of cellular preservation, which is influenced by sample fixation and staining technique. Mineralizing surface and mineral apposition rate vary with the labeling substance, labeling schedule, and dosage used [22]. What is measured as erosion surface depends on what the observer believes is a scalloped surface, which is a subjective interpretation [19]. Apart from the practical utility of these data as reference material, these results provided a unique possibility to gain insight into the cellular and structural features of normal human bone development [14]. The relative bone volume of cancellous iliac bone increases markedly between 2 and 20 years of age. As demonstrated in this study, this is entirely due to trabecular thickening, whereas there is no change in trabecular number. Trabecular thickening is due to remodeling with a positive balance, which is on the order of a few percent of the total amount of bone turned over. This means that the amount of bone deposited during a remodeling cycle is slightly higher than the amount of bone removed. Since the difference between resorption and formation is very small, a high remodeling activity is necessary to achieve trabecular thickening. Bone formation rate and activation frequency were very high in the youngest children in this study, decreased until approximately age 8 or 9 years, and then increased again to a pubertal peak. After the age of puberty, the values declined to the low adult ranges. Interestingly, wall thickness increased with age, whereas mineral apposition rate decreased at the same time. This indicates that the active osteoblast life span is much shorter in younger children than in adults. Regarding the outer bone dimensions and cortical bone, this study demonstrated that ileal bone development is characterized by an outward modeling drift [14,23]. Both cortices move through tissue space in parallel by reciprocal activities of their surfaces. The external cortex moves through periosteal apposition and endocortical resorption, whereas the internal cortex migrates laterally through periosteal resorption and endocortical apposition. The movement of the external cortex is considerably faster than that of the internal cortex, which leads to an increase in the size of the bone. At the same time, periosteal apposition is faster than endocortical resorption on the external cortex, whereas endocortical apposition is faster than periosteal resorption on the internal cortex. This accounts for the cortical
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thickening during the growth period. This mode of development also implies that a large proportion of ileal trabeculae is created by cancellization of the external cortex rather than by endochondral ossification, as had been previously believed. Also, due to trabecular compaction, the internal cortex contains material from what was formerly cancellous bone. The mode of ileal bone development indicates that this process cannot be determined by factors residing in the bone marrow. The two endocortical surfaces are in contact with the same bone marrow compartment but undergo different changes. These observations are more in line with the hypothesis that the cells on cortical surfaces respond to signals that emanate from the cortical bone. Thus, these findings are compatible with the idea that bone modeling and remodeling are governed by signals from osteocytes that are transmitted to the surfaces via the canalicular network [24,25].
Disorders of Bone Mineralization In the growing skeleton, mineralization occurs in two different types of tissue~growth plate cartilage and bone matrix. Deficient mineralization at the level of the growth plate is called rickets, and impaired mineralization of bone matrix is termed osteomalacia. By default, only osteomalacia can occur after growth plates have fused. Rickets is usually diagnosed clinically or radiographically, whereas osteomalacia is essentially a histologic diagnosis. Therefore, this chapter focuses on histomorphometric aspects of osteomalacia. The physiological process of mineralization represents the incorporation of mineral (calcium, phosphorous, and others) into organic bone matrix after it has been synthesized and deposited by osteoblasts [6,26]. Discussing mineralization disorders has become complicated in recent years by the widespread misuse of the term mineralization in the densitometric literature. Decreased bone mineralization is often said to be present when low bone mineral density is found. However, mineralization can only occur where bone matrix has been previously deposited. Most cases of low bone density are not due to a problem in the incorporation of mineral into matrix but rather reflect insufficient bone matrix production or increased matrix removal. Osteomalacia is a disorder of the physiologic process of mineralization (i.e., the incorporation of mineral into the organic bone matrix is disturbed) [12]. This leads to an accumulation of unmineralized bone matrix because osteoblasts continue to secrete osteoid for some time (see color plate 3). The aspect of tetracycline labels reflects the severity of the mineralization defect. In a mild mineralization disorder~ the proportion of tetracyclinelabeled osteoid is decreased and the distance between
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labels is decreased. In severe cases, the tetracycline labels may be blurred and can even be absent. In quantitative histomorphometric terms, osteomalacia is defined as the simultaneous occurrence of increased osteoid thickness and increased mineralization lag time. The criteria for abnormality in these parameters depend on the source of reference data. The usual cutoffs used for adults are 12.5 gm for osteoid thickness and 100 days for mineralization lag time [12]. These values are probably not appropriate for children because bone turnover is faster during growth. Following a widely used approach to separate "normal" from "abnormal," a value higher than two standard deviations above the mean in control subjects might be used to define an "increased result." On the basis of pediatric reference data [15], the cutoff values for osteoid thickness and mineralization lag time would be calculated as approximately 9 gm and 25 days, respectively. The diagnosis of osteomalacia can only be confirmed when both osteoid thickness and mineralization lag time are abnormally high. An isolated increase in osteoid thickness can be due to an increased osteoid production rate, whereas an isolated increase in mineralization lag time can be due to slow bone turnover. In addition to abnormally high osteoid thickness and mineralization lag time, osteomalacia is also characterized by reduced mineral apposition rate. However, this finding is not specific. Low mineral apposition rate can not only be caused by a defect in mineralization but also may result from a reduction in matrix deposition rate, as occurs in osteogenesis imperfecta and other osteopenic disorders. Calcipenic Disorders of Bone Minerafization In calcipenic forms of rickets and osteomalacia, hyperparathyroidism occurs in addition to the mineralization defect. This secondary hyperparathyroidism leads to increased bone turnover and deep osteoclastic resorption cavities during the early stages of the disease. As osteomalacia progresses, the mineralized bone surface is increasingly covered with thick osteoid seams and thus becomes inaccessible to osteoclasts. In such cases, tunneling resorption may be apparent. Paratrabecular fibrosis can be seen in severe forms of hyperparathyroid bone disease (see color plate 4).
Vitamin D Deficiency Vitamin D deficiency rickets/osteomalacia is very frequent in many areas of the world, but histomorphometric data from children with this condition have not been published. This is probably because the diagnosis can be confirmed with less invasive methods, and treatment is straightforward and leads to rapid improve-
ment. However, a histomorphometric study in rats contributed to the elucidation of the pathophysiology of osteomalacia in this disorder. In vitamin D-deficient rats, osteomalacia could be prevented by continuous calcium and phosphorus infusion [27]. This suggested that decreased availability of calcium and phosphorus may be the sole basis of the mineralization defect seen in vitamin D deficiency
Calcium Deficiency Calcium deficiency can cause histologic and histomorphometric abnormalities that follow the usual pattern of a calcipenic mineralization defect, as outlined earlier. In younger children, osteomalacia appears to predominate [28], whereas in teenagers histologic signs of hyperparathyroidism, overt osteomalacia, or a mixture of both can be found [29]. As described earlier, hyperparathyroid bone disease and osteomalacia may reflect different stages of the mineralization defect. Alternatively, variations in phosphorus intake may be responsible for the development of the two different histological defects. In fact, baboons fed a low-calcium and low-phosphorus diet develop osteomalacia, whereas baboons on a lowcalcium, high-phosphorus diet have features of hyperparathyroid bone disease [30]. The effects of low calcium intake can be exacerbated by concomitant fluorosis, as has been reported from South Africa and India [31,32]. In fluorosis, high cancellous bone volume is usually found in addition to osteomalacia [31]. Phosphopenic Disorders of Bone Minerafization Secondary hyperparathyroidism is typically absent in phosphopenic forms of osteomalacia. Consequently, increased bone turnover and deep erosion cavities are not usually seen in these disorders.
X-Linked Hypophosphatemic Rickets Children and adults with classical X-linked hypophosphatemic rickets show the typical features of osteomalacia [33-36]. There are very thick osteoid seams and a grossly increased mineralization lag time, both in trabecular and in cortical bone [35,36]. Despite low bone turnover rates, the osteoid surface extent (OS/BS) is increased because mineralization proceeds slowly at individual remodeling sites. Cancellous bone volume is high in most patients, but this includes a large amount ofunmineralized matrix. The mineralized fraction of cancellous bone is typically at the mean for age [35]. X-linked hypophosphatemic rickets can be distinguished histologically from other causes of osteomalacia because osteocytes are surrounded by a halo of unmineralized bone [37] (see color plate 5).
369
15. Bone Histomorphometry
With current standard treatment with phosphate and calcitriol, osteoid thickness and mineralization lag time decrease markedly [33,34,38]. However, in many patients these parameters do not normalize completely. Also, the periosteocytic lesions persist to a large extent despite adequate therapy [39].
Other Forms of Hypophosphatemic Rickets The histological features of non-X-linked hypophosphatemic rickets/osteomalacia have been studied in less detail. The histomorphometric findings in hereditary hypophosphatemic rickets with hypercalciuria resemble those of X-linked disease [40]. Only case reports are available on the bone tissue-level features of tumor-induced osteomalacia. Trabecular bone surfaces are almost completely covered with thick layers ofosteoid, and osteoblast activity is low. In one case, normalized osteoid thickness and vigorous bone formation activity were found 3 months after removal of the underlying tumor [41].
and cortical width during growth are determined by modeling processes [48], these observations suggested a modeling defect in OI. This is an important aspect of the disease because deficient bone modeling will result in smaller cross section and thinner cortices of long bones and thus reduced bone strength. In addition to diminished external size and cortical width, OI is also characterized by a low amount of cancellous bone, which is largely due to decreased trabecular number (Fig. 7). Low trabecular number can result from either increased loss or decreased production of trabeculae. There was no evidence that children with
Cancellous bone volume (BV/TV, %) 30 25 20 15 10
Osteopenic Disorders
5
Primary Osteopenic Disorders in Children and Adolescents
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Osteogenesis imperfecta (OI) has been classically divided into four clinical types [42]. Type I OI comprises patients with a mild presentation and a low normal or slightly reduced height, whereas type II OI is usually lethal in the perinatal period. Type III OI is the most severe form in children surviving the neonatal period. Patients who do not fit into one of these categories are usually classified as having type IV OI. Although OI is not rare, there arc surprisingly few studies on the bone tissue characteristics of OI. Early qualitative studies evaluated samples obtained at sites of deformity or fracture during surgical procedures so that results were obscured by injury or repair reactions [43]. Several studies of small series of OI patients showed decreased cancellous bone volume [44-46]. Cancellous bone turnover was increased in children with OI. These studies provided valuable preliminary information, but only a recently published study had a sufficiently large sample number to provide certainty regarding the histomorphometric features of OI [47]. Static and dynamic parameters were measured in iliac bone specimen from 70 children with types I, III, and IV OI between 1.5 and 13.5 years of age. Results were compared to those of 27 age-matched controls without metabolic bone disease. The external size of the biopsy core did not increase with age in OI patients, and cortical width was generally markedly below normal. Because external bone size
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FIGURE 7 Age variation of structural cancellous bone parameters in osteogenesis imperfecta (OI). The difference in cancellous bone volume between controls and OI patients increases with age due to insufficient (OI type I) or absent (OI type III and type IV) thickening of trabeculae. In contrast, there are no significant changes in trabecular number.
3
70
Frank Rauch
OI lose secondary trabeculae because trabecular number remained constant with age (Fig. 7). By exclusion, this suggested that fewer secondary trabeculae are produced. Trabeculae consist of lamellar bone, but lamellae tend to be thinner than those in healthy children (see color plate 6). Inadequate trabecular thickening in OI is caused by a defect in bone remodeling. In the control group, each remodeling cycle added 2.8~tm more bone than it resorbed. In OI type I, the positive balance was only 1.1 I~m and it was approximately 0 in types III and IV. The insufficient performance of the osteoblast team was the consequence of the fact that the amount of work achieved by an individual cell was decreased by approximately 50% (Fig. 8). This was only partly compensated by an increased number of osteoblasts per remodeling unit, as estimated from Ob.S/OS. Although the amount of bone turned over in individual remodeling cycles is decreased in OI, the number of remodeling cycles that occur on a given bone surface per unit time (Ac.f) is increased (Fig. 8). The cause of increased recruitment
FIGURE 8
of remodeling teams is not clear, but increased microdamage in the bone matrix due to impaired mechanical resistance is the likely cause. If so, increased remodeling in OI may be largely ineffective for improving the quality of the bone tissue because the newly deposited matrix harbors the same structural defect as the old matrix. This study showed that in OI a single genetic defect in the osteoblast interferes with multiple mechanisms that normally ensure adaptation of the skeleton to the increasing mechanical needs during growth.
OI Types Identified by Bone Histology and Histomorphometry: OI Types V and 111 Many OI patients present unusual clinical features. One of these is hyperplastic callus formation, which can appear spontaneously, following fracture, or with intramedullary rodding [49]. While studying bone sections from OI patients, it was realized that those with a history of hyperplastic callus formation also showed an abnormal pattern of lamellation under polarized light.
Bone formation abnormalities in OI on three levels of biological organization.
15. Bone Histomorphometry Lamellae were arranged in an irregular fashion and had a coarsened or even mesh-like appearance under polarized light. It was then noted that patients with this particular histological pattern also had distinctive features, including calcifications of the interosseous membrane at the forearm, hyperdense metaphyseal bands, and a lack of mutations in collagen type I. These observations led to the classification of this disease entity as OI type V [50]. Comparison of quantitative histomorphometric results in OI type V and controls revealed no difference in most bone surface-based indicators of bone formation and resorption. However, parameters reflecting bone formation activity in individual remodeling sites (MAR and Aj.AR) were clearly decreased. The rate of matrix deposition as estimated by adjusted apposition rate was less than half of the control value, and correspondingly osteoid seams were very thin. Thus, bone remodeling in type V OI is characterized by a normal rate of activation of remodeling units but an impaired bone formation within individual remodeling units. Bone histology and bone histomorphometry also allowed the identification of another subgroup of OI patients. These individuals initially had been diagnosed with OI type IV on clinical grounds. However, evaluation of iliac crest biopsy samples yielded surprising results. There was loss of the normal orientation of the lamellae and a "fish-scale" pattern under polarized light. A large amount of osteoid was present, and inspection under fluorescent light revealed poor or diffuse uptake of the tetracycline labels. These findings suggested that there was a defect in matrix mineralization in these patients. Quantitative histomorphometry revealed that cortical width and trabecular thickness were diminished in OI type VI, but that trabecular number was similar to that of healthy controls. Both osteoid thickness and surface were significantly increased in OI type VI patients compared to healthy controls, resulting in a grossly elevated osteoid volume. The mineral apposition rate and the adjusted apposition rate were decreased, and the mineralization lag time was significantly prolonged in OI type VI patients. As mentioned previously, increased osteoid thickness and a prolonged mineralization lag time are the defining elements of osteomalacia. It is interesting to compare the bone formation abnormalities in OI type VI and types I, III, and IV. Relative osteoid surface (OS/BS) is increased in all these disorders, but for different reasons. In OI types I, III, and IV, osteoid surface extent is elevated because remodeling activity is high and thus a large number of remodeling teams simultaneously work on the trabecular surface. However, osteoid thickness is normal because the process of mineralization is not impaired. In contrast, remodeling activity is not increased in OI type VI, but osteoid accumulates because mineralization is delayed.
3 71
Histological Phenocopy of OI Type I without Collagen Mutation: OI Type VII In a recently discovered kindred with a novel form of autosomal recessive OI, collagen mutations could be excluded [51]. There is a moderate to severe OI phenotype that is characterized by fractures at birth, bluish sclerae, early deformity of the lower extremities, coxa vara, osteopenia, and rhizomelia [52]. Similar to OI type I, bone size is small, cortical width is reduced, and cancellous bone volume is low in this form of OI. All bone surface-based parameters of bone formation and resorption (i.e., OS/ BS, Ob.S/BS, MS/BS, BFR/BS, Oc.S/BS, and ES/BS) are markedly increased, but the mineral apposition rate is decreased. These observations demonstrate that the tissue-level manifestations of OI can result from mutations in genes other than collagen type I.
Idiopathic Juvenile Osteoporosis Idiopathic juvenile osteoporosis (IJO) is a primary bone disorder with bone fragility and low bone mass. In contrast to OI, IJO is not a congenital disease and no genetic defect is known. It is often difficult to distinguish between IJO and mild forms of OI clinically, but a recent report shows that there are characteristic differences between the two disorders on the tissue level [53]. In contrast to OI, biopsy specimens from IJO patients are usually of normal size. Qualitatively, a lack of activity is usually noted in IJO, whereas there is hypercellularity in OI (see color plate 7). In quantitative histomorphometric terms, this translates into low activation frequency and low bone surface-based remodeling parameters in IJO and an increase in these values in OI. More detailed analysis of the bone formation defect in IJO revealed that the bone formation rate per unit bone surface (BFR/BS) is decreased to 38% of the value found in age-matched controls due to two abnormalities: Fewer osteoblast teams are recruited, and the individual team performs worse than normal. Reconstruction of the formation site showed that the osteoid apposition rate of IJO patients is already very much decreased during the first few days of osteoblast activity. This suggested that the osteoblast team is headed for a lower target from the start, with accordingly scaled down intermediate steps. In contrast to bone formation, no defect in bone resorption was detectable. These findings suggested a pathophysiologic model of IJO, in which insufficient production of cancellous bone creates weaknesses at locations where trabeculae are most needed to maintain bone stability (i.e., the metaphyses of long bones and vertebrae). Mechanical strain eventually exceeds the fracture threshold, and fractures at these sites ensue. Interestingly, no abnormalities were detected in intracortical remodeling activity [23]. Both structural
3 72,
Frank Rauch
parameters reflecting intracortical remodeling (cortical porosity and the diameter of osteonal canals) and bone surface-based metabolic parameters (OS/BS, Ob.S/BS, MS/BS, Oc.S/BS, ES/BS, and BFR/BS) were normal in IJO patients. Thus, it appears that the bone formation defect in IJO is limited to cancellous bone. Skeletal Dysplasias and S y n d r o m e s with Skeletal Involvement Many skeletal dysplasias are rare disorders often defined on the basis of radiological appearance. The gene defects underlying these conditions are elucidated with increasing speed. The missing link usually concerns how the molecular defect is functionally related to the phenotype. In order to fill this gap in our understanding, a quantitative description of the tissue-level characteristics of a disease would be helpful. However, only a few reports have included histomorphometric data on skeletal dysplasias.
Fibrous Dysplasia A large number of qualitative descriptions have been published concerning the histological appearance of fibrous bone dysplasia [54] (see color plate 8). However, quantitative histomorphometric evaluation of the lesion has been reported for only a few cases [55]. Trabecular surfaces were almost completely covered with osteoid; nevertheless, osteoclast numbers were relatively high. It is not known whether and how drug therapy with bisphosphonates change this picture.
Osteopetrosis Bone histology in autosomal recessive infantile osteopetrosis is characterized by a large amount of calcified cartilage and a normal or abnormally high number of osteoclasts [56,57] (see color plate 9). In contrast, the histological abnormalities of autosomal dominant (adult) osteopetrosis are mild. Cortical width and cancellous bone volume are only slightly increased, and bone formation and resorption indices show only subtle deviations from normal [58]. S e c o n d a r y Bone Disorders
Renal bone disease The skeletal effects of chronic renal failure in children and adolescents are difficult to study. The patient population is quite heterogeneous regarding underlying condition, age at onset, and progression of renal failure. In addition, treatment schedules for the various stages of
chronic renal failure change quickly and this may affect the type of skeletal involvement. Nevertheless, renal osteodystrophy is the pediatric bone disorder for which the largest number of histologic and histomorphometric studies have been performed. This important topic is treated in a separate chapter.
Secondary Osteopenic Disorders in Children and Adolescents Although osteopenia and bone fragility are major problems in children undergoing long-term corticosteroid treatment, little is known about the tissue-level features of this secondary bone disorder. Bone volume and bone formation rate were reportedly normal in four children with chronic steroids use [59]. Larger studies are required to adequately address this important topic. Bone mass can be low in children following severe burns. The tissue-level mechanisms responsible were investigated in one pediatric histomorphometric study [60]. Within a few weeks after severe burns, bone formation activity was below the detection limit. Seventeen out of 18 study subjects did not have any tetracycline uptake in cancellous bone. There was no indication of increased bone resorption in this study. It is unknown whether this type of reaction is specific for burn injuries or a general occurrence after major trauma in children.
INDICATIONS FOR BONE BIOPSY AND HISTOMORPHOMETRY IN PEDIATRIC BONE DISEASES Clinical Practice For diagnostic purposes, qualitative assessment of bone biopsies is usually sufficient in clinical practice. A bone biopsy can aid in correctly classifying various osteopenic and bone fragility disorders. It is often possible to distinguish IJO from OI. Also, severe forms of polyostotic fibrous dysplasia can be mistaken for OI on clinical grounds, whereas the correct diagnosis is readily apparent in the tissue section. The new forms of OI (types V and VI) can be recognized on the basis of their histological appearance. Bone biopsies are usually not necessary to correctly diagnose and treat the various forms of rickets. However, bone histology can be helpful when the clinical appearance is not typical and the response to treatment is not satisfactory. For example, X-linked hypophosphatemic rickets can be distinguished from other forms of hypophosphatemic rickets by the abundance of hypomineralized periosteocytic lesions. Bone histology is also
15. Bone Histomorphometry
useful in suspected mineralization disorders after growth plate fusion. In fact, suspected osteomalacia is one of the most frequent indications for bone biopsy in adults.
Clinical Studies Histomorphometric evaluation of bone biopsies should be performed whenever an experimental form of drug therapy is administered to children with bone diseases in whom an iliac bone sample can be safely obtained. Current noninvasive methods for studying the amount, distribution, and metabolism of bone are fraught with technical limitations and uncertainties regarding the interpretation of results. The availability ofhistomorphometric data allows one to make treatment recommendations on a firm and rational basis. In addition to characterizing the effects, bone biopsies are also important for detecting skeletal side effects of therapy. Thus, including bone histomorphometry in study protocols is crucial for documenting the efficacy of therapy as well as its safety.
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ll61 A Diagnostic Approach to Skeletal Dysplasias SHEILA UNGER*, ANDREA SUPERTI-FURGAt, and DAVID L. RIMOIN * 9Division of Clinical and Metabolic Genetics, Hospital for Sick Children, University of Toronto, Toronto, Canada t University of Lausanne, Division of Molecular Pediatrics, Centre Hospitalier Universitaire Vaudors, Lausanne, Switzerland ~Medical Genetics Birth Defects Center, Ceders-Sinai Health System and Department of Pediatrics and Medicine, UCLA School of Medicine, Los Angeles, California
INTRODUCTION
and outlines some of the more important radiographic findings.
The skeletal dysplasias are disorders characterized by developmental abnormalities of the skeleton. They form a large heterogeneous group and range in severity from precocious osteoarthropathy to perinatal lethality [1,2]. Disproportionate short stature is the most frequent clinical complication but is not uniformly present. There are more than 100 recognized forms of skeletal dysplasia, which can make determining a specific diagnosis difficult [1]. This process is further complicated by the rarity of the individual conditions. The establishment of a precise diagnosis is important for numerous reasons, including prediction of adult height, accurate recurrence risk, prenatal diagnosis in future pregnancies, and, most important, for proper clinical management. Once a skeletal dysplasia is suspected, clinical and radiographic indicators, along with more specific biochemical and molecular tests, are employed to determine the underlying diagnosis. This process starts with history gathering, including the prenatal and family history. This is followed by clinical examination with measurements and radiographs. It is important to obtain a full skeletal survey because the distribution of affected and unaffected areas is key to making a specific diagnosis [3]. Only after a limited differential diagnosis has been established should confirmatory molecular investigations be considered. When available, histological examination of cartilage is a useful diagnostic tool. This is especially important for those conditions that are lethal in the perinatal period. In these instances, the acquisition of as much information as possible, while the material is available, is critical. This chapter reviews this sequence of diagnostic steps
PediatricBone
BACKGROUND Each skeletal dysplasia is rare, but collectively the birth incidence is approximately 1/5000 [4,5]. The original classification of skeletal dysplasias was quite simplistic. Patients were categorized as either short trunked (Morquio syndrome) or short limbed (achondroplasia) [6] (Fig. 1). As awareness of these conditions grew, their numbers expanded to more than 200 and this gave rise to an unwieldy and complicated nomenclature [7]. In 1977, N. Butler made the prophetic statement that "in recent years, attempts to classify bone dysplasias have been more prolific than enduring" [8]. The advent of molecular testing allowed the grouping of some dysplasias into families. For example, the type II collagenopathies range from the perinatal lethal form (achondrogenesis type II) to precocious osteoarthritis [9]. Although grouping into molecularly related families has simplified the classification, the number of different genes involved is very large. There remain a large number of dysplasias without a known molecular defect that are grouped with others on the basis of a shared clinical or radiographic feature. The nomenclature continues to undergo revisions as new molecular genetic information becomes available [1]. The nomenclature has been renamed as a nosology to indicate that it represents a catalog of recognized skeletal dysplasias rather than a succinct grouping of the varied and numerous disorders (Table 1). A framework in which to classify the skeletal dysplasias on the basis of
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Copyright 2003, Elsevier Science (USA). All rights reserved.
3 76
Sheila Ungeret aL
FIGURE 1 Differencesin body proportions. (A) A boy with achondroplasia due to the less common FGFR3 mutation (G375C). The shortening is predominantly limb shortening with the proximal segments most affected (rhizomelia). (B) A child with Morquio syndrome (mucopolysaccharidosis type IVA). Although there is overall shortening, it is clear that the trunk is more severelyaffected.
their molecular defects has recently been developed [10,11] that groups the skeletal dysplasias by the basic function of the defective gene/gene product but does not delineate the biological pathway involved [10]. The spectrum of skeletal dysplasias ranges from perinatal lethal to individuals with normal stature and survival but early onset osteoarthrosis [1]. The approach to diagnosis varies between the lethal/semilethal disorders and those compatible with life; thus, they are reviewed separately. Most lethal skeletal dysplasias (and many nonlethal ones) can be identified on prenatal ultrasound. An attempt should be made to make a precise diagnosis during pregnancy, but this may be impossible until after pregnancy termination/delivery. However, under experienced eyes, a prenatal ultrasound distinction can usually be made between those disorders compatible with life and those lethal prenatally or during early postnatal life. Patients with a nonlethal skeletal dysplasia generally present to their physician for evaluation of short stature. It is sometimes unclear whether the cause of growth failure is systemic or skeletal. Renal, endocrine, and cardiac abnormalities might need to be ruled out. However, these conditions present with proportionate short stature, whereas
the dysplasias usually cause disproportionate short stature. Also, some genetic syndromes cause significant prenatal growth failure but should be easily distinguishable on the basis of associated features, such as developmental delay and dysmorphic facies, and by radiographs. In fact, a chapter in Smith's Recognizable Patterns of Human Malformation [12] is dedicated to disorders with "very small stature, not skeletal dysplasia." HISTORY AND PHYSICAL EXAMINATION i
When presented with a child with disproportionate short stature, a focused history can give invaluable clues as to the differential diagnosis. In genetics, this starts with prenatal history and includes length at birth. Many of the nonlethal dysplasias (e.g., achondroplasia) present with short stature at birth [13], whereas others (e. g., pseudoachondroplasia) present with a normal birth length with subsequent failure of linear growth [14]. Although the age at which growth failure is first noted for a specific skeletal dysplasia is variable, it tends to be
TABLE 16 International Nosology and Classification of Constitutional Disorders of Bone Osteochondrodysplasias Mode of Inheritance
Chromosome Locus
Gene
Gene Product
Thanatophoric dysplasia, Type I1
4~16.3
FGFR3
FGFR3
Achondroplasia
4p16.3
FGFR3
FGFR3
Hypochondroplasia
4p16.3
FGFR3
FGFR3
EVC
EVC
OMIM Syndrome
Comments
1. Achondroplasia group Thanatophoric dysplasia, Type I (includes San Diego Type)
Hypochondroplasia SADDAN (severe achondroplasia, developmental delay, acanthosis nigricans)
other AD
2. Severe Spondylodysplastic dysplasias Lethal platyspondylic skeletal dysplasias
SP
151210
(Torrance type, Luton type) Achondrogenesis type 1A Opsismodysplasia SMD Sedaghatian Type see also: Thanatophoric dysplasia Types 1\11 Achondrogenesis Types IBIII and Group 3. 3. Metatropic dysplasia group Fibrochondrogenesis
AR
Schneckenbecken dysplasia
AR
Metatropic dysplasia (various forms)
AD
4. Short-rib dysplasia (SRP) (with or without polydactyly) group SRP type 11111
AR
SRP type I1
AR
SRP type IV
AR
Asphyxiating thoracic dysplasia (Jeune)
AR
Chondroectodermal Dyplasia (Ellis-van Creveld dysplasia)
AR
Thoracolaryngopelvic dysplasia
AD
4p16
(continues)
TABLE 16 (continued) Mode of Inheritance
OMIM Syndrome
Comments
Chromosome Locus
Gene
Gene Product
5q32-q33
DTDST
5q32- q33
DTDST
Sul. transporter Sul. transporter
5q32- q33
DTDST
Sul. transporter
5q32-q33
DTDST
Sul. transporter
lp36.1
PLC (HSPG2)
Perlecan
5. Atelosteogenesis-Omodysplasia group Atelosteogenesis type I (includes "Boomerang dysplasia")
SP
Omodysplasia I (Maroteaux)
AD
Omodysplasia I1 (Borochowitz) Atelosteogenesis Type 111 de la Chapelle dysplasia 6. Diastrophic dysplasia group
Achondrogenesis 1B
AR
Atelosteogenesis type I1 Diastrophic dysplasia see also: Group 11
Autosomal Recessive MED
7. Dyssegmental dysplasia group Dyssegmental dysplasia, Silverman-Handmakertype
AR
Dyssegmental dysplasia, Rolland-Desbuquois type 8. Type I1 collagenopathies
AR
Achondrogenesis I1 (Langer- Saldino)
12q13.1-q13.3
COL2Al
Type I1 collagen
Hypochondrogenesis
12q13.1- q13.3
COL2A1
Type I1 collagen
Spondyloepiphyseal dysplasia (SED) congenita Spondyloepimetaphyseal dysplasia (SEMD) Strudwick type Kniest dysplasia
12q13.1-q13.3
COL2Al
Type I1 collagen
12q13.1-q13.3
COL2A1
Type I1 collagen
12q13.1-q13.3
COL2Al
Type I1 collagen
SED Namaqualand Type
12q13.1-q13.3
COL2A1
Type I1 collagen
SED with brachydactyly Mild SED with premature onset arthrosis
12q13.1-q13.3
COL2A1
Type I1 collagen
12q13.1- q13.3
COL2Al
Type I1 collagen
Stickler dysplasia Type I
12q13.1- q13.3
COL2Al
Type I1 collagen
lp21
COLl lAl
Type XI collagen
6p21.3
COLl lA2
Type XI collagen
6p21.3
COL1lA2
Type XI collagen
9. Type XI collagenopathies
Stickler dysplasia Type I1
heterogeneous with or without ocular involvement
Marshall syndrome
AD
Otospondylomegaepiphysealdysplasia (OSMED)
AR
Recessive haploinsuficiency mutations
Dominant mutations; also called WeissenbachZweymiiller or Stickler dysplasia without ocular involvement
Otospondylomegaepiphysealdysplasia (OSMED)
10. Other spondyloepi-(meta)-physeal [SE(M)D]dysplasias XLD
COLllA2
Type XI collagen
313400
Xp22.2-p22.1
SEDT
SEDLIN
208230
6q22-q23
WISP3
WNT 1-inducible signaling pathway protein 3
18q12
FLJ90130
FLJ90130
Wolcott-Rallison dysplasia
2p12
Immuno-osseous dysplasia (Schimke)
2q34-q36
EIF2AK3 SMARCALl
EIF2AK3 SMARCALl
lp36.1
PLC (HSPG2)
Perlecan
X-linked SED tarda
SEMD Handigodu Type Progressive pseudorheumatoid dysplasia
AD AR
Dyggve-Melchior-Clausen dysplasia
includes Burton dysplasia and Kyphomelic dysplasia; see also dyssegmental dysplasiaSilvermanHandmaker (see group 7)
Schwartz-Jampel syndrome
SEMD with joint laxity (SEMDJL) SEMD with dislocations (Hall) (leptodactylicType) Sponastrime dysplasia see also: Group 12
SEMD short limb - abnormal calcification Type SEMD Pakistani Type
see: opsismodysplasia Group 2 11. Multiple epiphyseal dysplasias & pseudoachondroplasia Pseudoachondroplasia
AD
see also: Groups 8/10
19p12-13.1
COMP
COMP
Multiple epiphyseal dysplasia (MED)
AD
see also: recessive MED in Group 6
19~13.1
COMP
COMP
(Fairbanks and Ribbing types)
Familial hip dysplasia (Beuke)
6q13
COL9Al
Type IX collagen
1~32.2-33
COL9A2
Type IX collagen
20q13.3
COL9A3
Type IX collagen
2p23-24
MATN3
matrilin 3
4q35 (continues)
TABLE 1 6 (continued) Mode of Inheritance
OMIM Syndrome
Comments
Chromosome Locus
Gene
Gene Product
12. Chondrodysplasia punctata (CDP) (stippled epiphyses group) Rhizomelic CDP Type 1
AR
PTS2 peroxisomal biogenesis receptor
Rhizomelic CDP Type 2
AR
1q42
DHPAT
DHAPAT
Rhizomelic CDP Type 3
AR
2q3 1
AGPS
Zellweger syndrome
AR
7q11.23
PEXl
ADHAPS Peroxin- 1
AR
8q21.1
PEX2
Peroxin-2
AR AR
6q23
PEX3
Peroxin-3
12~13.3
PEX5 (PXRI)
AR
6p21.1
Peroxin-5 Peroxin-6
AR
PEX6 PEXl2
Peroxin- 12
CDP Conradi-Hiinermann Type
XLD
17q11.2 Xpll
EBP
EBP
CDP X-linked recessive Type
XLR
Xp22.3
ARSE
Arylsulfatase E
EBP
EBP NAD(P)H steroid dehydrogenase like protein
(brachytelephalangic)
0
CDP Tibia-metacarpal Type CHILD (limb reduction icthyosis)
AD XLD
CHILD (limb reduction icthyosis)
XLD
Xpl 1 Xq28
Hydrops-ectopic calcification-motheatenHEM (Greenberg dysplasia)
AR
1q42
Dappled diaphyseal dysplasia 13. Metaphyseal dysplasias
AR
Jansen Type
AD
Schmid Type
AD
Cartilage-Hair-Hypoplasia (McKusick)
NSDHL LBR
Lamin B receptor
3p22-p21.1 6q21q22.3
PTHRl COLlOAl
PTHRPTHRP Type X collagen
AR
9p21-p12
RMRP
RNA subunit of RMRP RNA'ase
Metaphyseal dysplasia without hypotrichosis
AR
9p21-p12
RMRP
RNA subunit of RMRP RNA'ase
Metaphyseal anadysplasia (various types)
AD1 XLD
Metaphyseal dysplasia with pancreatic insufficiency and cyclic neutropenia (Shwachman Diamond)
AR
7pll-qll
SBDS
SBDS
Adenosine deaminase (ADA) deficiency
AR
20q- 13.11
ADA
Adenosine deaminase
Metaphyseal chondrodysplasia Spahr Type
AR
Acroscyphodysplasia (various types)
AR
14. Spondylometaphyseal dysplasias (SMD) Spondylometaphyseal dysplasia Kozlowski Type
AD
Spondylometaphyseal dysplasia (Sutcliffelcorner fracture Type) SMD with severe genu valgum (includes Schmidt and Algerian Types)
AD AD see also: SMD Sedaghatian Type (Group 2)
15. Brachyolmia spondylodysplasias Hobaek (includes Toledo Type) Maroteaux type Autosomal dominant type 16. Mesomelic dysplasias Dychondrosteosis (Leri-Weill)
Pseudo AD
127300
Langer type (homozygous dyschondrosteosis)
Pseudo AR
249700
Homozygous dominant
Xpter- p22.32
SHOX
Short stature homeobox protein
Xpter-p22.32
SHOX
Short stature homeobox protein
Nievergelt Type Kozlowski-Reardon Type Reinhardt-Pfeiffer Type Werner Type Robinow Type, dominant Robinow Type, recessive
Receptor tyrosine kinase-like orphan receptor 2
Mesomelic dysplasia with synostoses Mesomelic dysplasia Kantaputra Type Mesomelic dysplasia Verloes Type Mesomelic dysplasia Savarirayan Type 17. Acromelic dysplasias Acromicric dysplasia Geleophysic dysplasia Myhre dysplasia Weill-Marchesani dysplasia Trichorhinophalangeal dysplasia Types 11111
8q24.12
TRPSl
Trichorhinophalangeal dysplasia Type I1 (LangerGiedion)
8q24.11-q24.13
TRPSl EXTl (contiguous gene deletion) (continues)
TABLE 1 6 (continued) Mode of Inheritance
OMIM Syndrome
Brachydactyly type Al
AD
112500
Brachydactyly type A2
AD
112600
Comments
Chromosome Locus
Gene
Gene Product
2q35
112700
Brachydactyly type A3 Brachydactyly type B
AD
113000
9q22
ROR2
Receptor tyrosine kinase-like orphan receptor 2
Brachydactyly type C
AD
113100
2Oqll
CDMPl
cartilage derived morphogenic protein 1
20q13
GNAS l
guanine nucleotide-binding protein, a subunit
12~12.2-p11.2
HTNB
Camptodactyly arthropathy coxa vara pericarditis (CACP) 18. Acromesomelic dysplasias
1q25-31
PRG4
Acromesomelic dysplasia Type Maroteaw
9~13-p12
12q24 Brachydactyly type D Brachydactyly type E Pseudohypoparathyroidism (Albright Hereditary Osteodystrophy) Acrodysostosis Saldino-Mainzer dysplasia Brachydactyly-hypertension dysplasia (Bilginturan) Craniofacial conodysplasia Angel-shaped phalango-epiphysealdysplasia (ASPED)
Acromesomelic dysplasia Type Campailla-Martinelli Acromesomelic dysplasia Type FerrazIOhba Acromesomelic dysplasia Type Osebold Remondini 20q11.2
Grebe dysplasia Cranioectodermal dysplasia
AR
CDMPl
cartilage derived morphogenic protein 1
CBFAltRUNX2
core binding factor ctl-subunit
218330
19. Dysplasias with predominant membranous bone involvement Cleidocranial dysplasia
AD
119600
Yunis-Varon dysplasia Parietal foramina (isolated)
llp11.2
ALX4
Aristaless-like 4
Parietal foramina (isolated)
5q34- q35
MSX2
Muscle segment homeobox 2
20. Bent-bone dysplasia group Campomelic dysplasia Cumming syndrome Stiive-Wiedemann dysplasia see also Antley-Bixler syndrome 21. Multiple dislocations with dysplasias Larsen syndrome
AD
150250
Larsen-like syndromes (including La Reunion Island)
AR
245600
Desbuquois dysplasia
AR
251450
Pseudodiastrophic dysplasia
AR
264 180 see also: Group 10
22. Dysostosis multiplex group Mucopolysaccharidosis IH
AR
IDA
a-1-1duronidase
Mucopolysaccharidosis IS
AR
IDA
a- 1-1duronidase
Mucopolysaccharidosis I1
XLR
IDS
Iduronate-2-sulfatase
Mucopolysaccharidosis IIIA
HSS
Mucopolysaccharidosis IIIB
AR AR
Mucopolysaccharidosis IIIC
AR
Heparan sulfate sulfatase N-Ac-a-D-glucosaminidase Ac-Coa:a-glucosamine-Nacetyltransferase
W
0 Mucopolysaccharidosis IIID
12q14 16q24.3
GNS GALNS
N-Ac-glucosamine-6-sulfatase
Mucopolysaccharidosis IVA
3~21.33
GLBI
O-Galactosidase
Mucopolysaccharidosis VI
5q13.3
ARSB
Arylsulfatase B
Mucopolysaccharidosis VII
7q21.11
Fucosidosis
lp34
GUSB FUCA MAN MANB
Mucopolysaccharidosis IVB
See also: GMIGangliosidosis
a-Mannosidosis
19~13.2-q12
b-Mannosidosis
4q22-q25
Aspartylglucosaminuria
Galactosamine-6-sulfatase
P-Glucuronidase a-Fucosidase a-Mannosidase b-Mannosidase
AgA GLBl
Aspartylglucosaminidase
3p21.33
Sialidosis, several forms
6~21.3
NEU
a-Neuraminidase
Sialic acid storage disease
6q14-q15
SIASD
Galactosialidosis, several forms
20q13.1
PPGB
GMI Gangliosidosis, several forms
Multiple sulfatase deficiency
4q32-q33 See also: MPS IV B
!3-Galactosidase
b-Galactosidase protective protein Multiple sulfatases (continues)
TABLE 16 (continued) Mode of Inheritance
OMIM Syndrome
Chromosome Locus
Gene
Gene Product
Mucolipidosis I1
AR
252500
4q21-23
GNPTA
N-Ac-Glucosaminephosphotransferase
Mucolipidosis I11
AR
252600
4q21-23
GNPTA
N-Ac-Glucosaminephosphotansferase
Osteogenesis irnperfecta I (normal teeth)
COLl A1
Type I collagen
Osteogenesis Imperfecta I (normal teeth)
COLlA2
Osteogenesis irnperfecta I (opalescent teeth)
COLlA2
Type I collagen Type I collagen
Comments
see also: Groups 8,10,11,14 23. Osteodysplastic slender bone group Type I microcephalic osteodysplasticdysplasia Type I1 microcephalic osteodysplasticdysplasia Microcephalic osteodysplastic dysplasia (Saul Wilson). 24. Dysplasias with decreased bone density
COLlA2 Osteogenesis imperfecta I1
Osteogenesis imperfecta 111
COLlAl COLIA2
Type I collagen Type I collagen Type I collagen
COLl Al
Type I collagen
COLlAl
Type I collagen
COLlA2
Type I collagen
COLlA2
Type I collagen
Osteogenesis imperfectaIV (normal teeth)
COLlA2
Type I collagen
COLl Al
Type I collagen
Osteogenesis irnperfecta IV (opalescent teeth)
COLIA2
Type I collagen
COLl Al
Type I collagen
Osteogenesis Imperfecta V Osteogenesis Imperfecta VI Cole-Carpenter dysplasia Bruck dysplasia I Bruck dysplasia I1 Singleton-Mertondysplasia Osteopenia with radiolucent lesions of the mandible
Osteoporosis-pseudoglioma dysplasia
Low density lipoprotein receptor-related protein:
AR SP
231070 259750 24 1500 146300
lp36.1-p34
ALPL
alkaline phosphatase
Hypophosphatasia adult form
AR AD
lp36.1-p34
ALPL
alkaline phosphatase
Hypophosphatemic rickets
XLD
307800
Xp22.2-p22.1
PHEX
Phosphate regulating endopeptidase
AD
193100
12~13.3
FGF23
Fibroblast growth factor 23
Neonatal hyperparathyroidism
AR
239200
3q21- q24
CASR
Calcium-sensing receptor
Transient neonatal hyperparathyrodism with hypocalciuric hypercalcemia
AD
145980
3q21-q24
CASR
Calcium-sensingreceptor
1lq13.4- q13.5
TClRGl
16~13
CLCN7
vacuolar proton pump Chloride channel 7
8q22
CA2
carbonic anhydrase I1
Xq28
IKBKG (NEMO)
NF-kB signalling
lq21
CTSK
cathepsin K
Geroderma osteodysplasticum Idiopathic juvenile osteroporosis 25. Dysplasias with defective mineralization Hypophosphatasia-perinatal lethal and infantile forms
Some families not linked to this locus
26. Increased bone density without modification of bone shape Osteopetrosis Infantile form
AR AR
259700
With infantile neuroaxonal dysplasia
AR?
600329
Delayed forms Intermediate form (possibly heterogeneous)
AD
166600
AR
259710
With renal tubular acidosis (carbonic anhydrase I1 deficiency)
AR
259730
Dysosteosclerosis With ectodermal dysplasia and immune defect (OLEDAID)
AR
224300
XL
300301
Osteomesopyknosis Cranial osteosclerosis with bamboo hair (Netherton)
AD
166450
Pyknodysostosis
AR AR
256500 265800
Osteosclerosis Stanescu type
AD
122900
Osteopathia striata (isolated) Osteopathia striata with cranial sclerosis
SP ADIXL D?
166500
D? Melorheostosis Osteopoikilosis Mixed sclerosing bone dysplasia
SP AD
155950 166700
SP (continues)
TABLE 16
(continued) Mode of Inheritance
OMIM Syndrome
Diaphyseal dysplasia Camurati Engelmann
AD
131300
Diaphyseal dysplasia with anemia (Ghosal)
AR
231095
Craniodiaphyseal dysplasia
?AR
218300 122860
Comments
Chromosome Locus
Gene
Gene Product
19q13.1-13.3
TGFbl
transforming growth factor beta 1
27. Increased bone density with diaphyseal involvement
Lenz Majewski dysplasia
151050
Endosteal hyperostosis van Buchem type
Sclerostin Sclerostin
Sclerosteosis Worth type Sclero-osteo-cerebellar dysplasia Kenny Caffey dysplasia Type I Kenny Caffey dysplasia Type I1 Osteoectasia with hyperphosphatasia (Juvenile Paget disease) Diaphyseal medullary stenosis with bone malignancy Oculodentoosseous dysplasia
239000
8q24.2
AD
112250
9p2 1-p22
AR
257850
AD
164200
AD
190320
28. Increased bone density with metaphyseal involvement Pyle dysplasia AR
265900
Trichodentoosseous dysplasia
AR
TNFRSFl lB
Osteoprotegerin
Distal-less 3 protein
Craniometaphyseal dysplasia Severe type Mild type
5~15.2-p14.2
ANKH
Pyrophosphate channel
see also: Group 29 29. Craniotubular digital dysplasias Frontometaphyseal dysplasia
XLR
305620
Xq28
FLNA
Osteodysplasty, Melnick-Needles
XLD
309350
Xq28
FLNA
Filamin 1 Filamin 1
Xq28
FLNA
Filamin 1
Xq28
FLNA
Filamin 1
Precocious osteodysplasty (terHaar dysplasia)
AR
249420
Otopalatodigital syndrome Type I
XLD
311300
Otopalatodigital syndrome Type I1
XLR
304120 see also: Group 28
30. Neonatal severe osteosclerotic dysplasias Blomstrand dysplasia
AR
215045
Raine dysplasia
AR
259775
Prenatal onset Caffey disease
AD ?AR
114000
Astley-Kendall dysplasia
AR
PTHR l see also: Mucolipidosis I1
31. Disorganized development of cartilaginous and fibrous components of the skeleton Dysplasia epiphysealis hemimelica
SP
127800
Multiple cartilaginous exostoses
EXT 1
exostosin-1
EXT2
exostosin-2
GNAS 1
guanine nucleotide-binding protein, a subunit
Enchondromatosis, Ollier
I
Enchondromatosis with hemangiomata (Maffucci)
SP
166000
Spondyloenchondromatosis
AR
27 1550
Spondyloenchondromatosis with basal ganglia calcification
AR
Dysspondyloenchondromatosis Metachondromatosis Osteoglophonic dysplasia Enchondromatosis Carpotarsal osteochondromatosis Fibrous dysplasia (McCune-Albright and others)
174800
Jaffe Campanacci
SP mosaic SP
Fibrodysplasia ossificans progressiva
AD
135100
Cherubism
AD
118400
Cherubism with gingival fibromatosis
AR
135300
SH3 domain-binding protein 2
32. Osteolyses Multicentric-hands andfeet Multicentric carpal-tarsal osteolysis with and without nephropathy Shinohara carpal-tarsal osteolysis Winchester syndrome Torg syndrome (continues)
TABLE 16 (continued) Mode of Inheritance
OMIM Syndrome
AD
228600
AD
161200
Comments
Chromosome Locus
Gene
Gene Product
18q21.1- q22
TNFRSFl l A
RANK
LMXlB
LIM homeobox transcription factor 1
Distal phalanges Hadju-Cheney syndrome Mandibuloacral syndrome Diaphyses and metaphyses Familial expansile osteolysis Juvenile hyaline fibromatosis (includes systemic juvenile hyalinosis)
33. Patella dysplasias Nail patella dysplasia Scypho-patellar dysplasia
AD
Ischiopubic patellar dysplasia
AD
147891
AR
224690
Genitopatellar syndrome Ear patella short stature syndrome (Meier Gorlin)
16. A Diagnostic Approach to Skeletal Dysplasias
fairly constant and can be used in developing a differential diagnosis. Increasingly, both lethal and nonlethal skeletal dysplasias are being detected on prenatal ultrasound, and it is worthwhile to inquire if any ultrasounds were done during pregnancy and if any discrepancy was noted between fetal size and gestational dates [15]. Inquiry should be made for findings related to the skeletal system. Some of these are obvious, such as joint pain and scoliosis. Some skeletal dysplasias present with multiple congenital joint dislocations (e.g., atelosteogenesis type III) [16]. Other findings that the family might notice include ligamentous laxity or conversely progressive finger contractures. Fetal joint dislocations due to extreme laxity can present at birth as contractures due to failure of proper in utero movement [17]. It is also important to ascertain when growth failure was first noted. Sometimes, findings unrelated to the skeletal system can be most helpful in making the diagnosis, such as abnormal hair and susceptibility to infections in cartilage-hair hypoplasia (McKusick metaphyseal dysplasia) [18]. Unfortunately, these findings are by no means constant. Parents may not consider other manifestations relevant to the diagnosis and a history will not be offered unless specifically asked for. Conversely, the diagnosis of a specific skeletal dysplasia may also lead the physician to detect abnormalities that had not been apparent to the patient or the family, such as renal abnormalities in asphyxiating thoracic dysplasia (ATD or Jeune syndrome) [19]. Most skeletal dysplasias are associated with normal intellectual development. However, a developmental history should be taken because there are notable exceptions to this rule. For children with achondroplasia, there is a gross motor developmental delay in the first 2 years of life likely related to large head size and ligamentous laxity [20]. Specific learning disabilities have been reported in hypochondroplasia and achondroplasia, but their significance remains controversial [21]. Certainly, there is marked developmental delay in children with the syndrome known as severe achondroplasia with developmental delay, which is a related fibroblast growth factor receptor 3 disorder [22,23]. Dgyvve-Melchior-Clausen and dysosteosclerosis are both rare dysplasias associated with severe to profound mental retardation [24,25]. A detailed family history should also be taken. Obviously, if another family member has a skeletal dysplasia, this will be important in assessing the mode of inheritance. It is also important to note parental heights since it is possible that the child might simply have familial short stature. Frequently, there is no family history of dwarfism because many, if not most, of the skeletal dysplasias, including the most common (achondroplasia), are autosomal dominant but most often caused by new mutations rather than being inherited [26]. Certain dysplasias are
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more common among certain ethnic groups, such as cartilage-hair hypoplasia in the Amish [27] and spondyloepimetaphyseal dysplasia with joint laxity in the South African Afrikaner population [28]. On physical examination, various growth parameters must be precisely determined. It is important to note not only the height of the child but also the weight and head circumference. This can sometimes establish a pattern; for example, in children with achondroplasia, the head circumference is larger than normal but height is dramatically reduced compared to normal [13]. A simple method of determining proportions consists of measuring the lower segment (symphysis pubis to floor) and subtracting this figure from the total height to determine the upper segment and then calculating the upper segmentto-lower segment ratio. This ratio, along with the arm span-to-height ratio, is used to document whether the spine or limbs are more severely shortened. When there is limb shortening, it is helpful to classify it as rhizomelic (proximal), mesomelic (middle), or acromelic (distal) depending on which segment is most affected. Once a specific diagnosis has been established, it is useful to plot the child's growth against disorder-specific growth curves. Specialized growth curves have been developed for achondroplasia, pseudoachondroplasia, spondyloepiphyseal dysplasia congenita, and diastrophic dysplasia [13,29]. These curves are most helpful for achondroplasia and should be used more cautiously for the other disorders because they show much more allelic heterogeneity and thus much greater phenotypic variability. In addition, assessment of symmetry or asymmetry can indicate certain diagnoses (e.g., chondrodysplasia punctata Conradi-Hfinermann) [30] (Fig. 2). As in other genetic syndromes, ancillary signs can be helpful in securing the diagnosis; thus, a general physical examination is also recommended. These signs include such findings as congenital heart disease, polydactyly, and dystrophic nails in chondroectodermal dysplasia (Ellis-van Creveld syndrome) [31]. A single finding is never present in 100% of patients but if present can be instructive. A good example of this is the cystic ear swellings seen in children with diastrophic dysplasia, which are fairly specific for this disorder [32] (Fig. 3). In general, children with skeletal dysplasias do not show dysmorphic features of the head and neck, but one important feature is the Pierre-Robin sequence seen in the type II collagenopathies and campomelic dysplasia [9] (Fig. 4). DIAGNOSTIC IMAGING The number of clinical discriminators is far less than the number of skeletal dysplasias; thus, radiographs are necessary for diagnosis. A complete skeletal survey is
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FIGURE 3 (A) A young child with diastrophic dysplasia. Note the gross deformation of the helical contour of the ear by the underlying cystic swelling. Generally, these swellings are not present at birth but develop during the first year of life and can be quite useful in establishing the diagnosis. (B) Another clue to the diagnosis of diastrophic dysplasia is this deformity of the thumb. Note the absence of flexion creases at the phalangeal joints.
FIGLIRE 2 (A) A 21-year-old woman with chondrodysplasia punctata Conradi-Htinermann type CDP-CH. Her face and limbs show the asymmetry characteristic of this disorder. She presented at birth with hypoplasia of the right side of her body and icthyosis. She also had scoliosis, bilateral club feet, and laryngeal stenosis requiring surgical correction. On radiographs, there were multiple areas of stippling, particularly at the right knee and ankle. Her diagnosis was confirmed by plasma sterol analysis, which showed an increase in 8(9) cholestenol (analysis by Dr. Lisa Kratz and Dr. Richard Kelley). recommended because the demonstration of normal findings in a specific region (e.g., the hands) can be important in making a differential diagnosis. The genetic skeletal survey should include the following views: lateral skull, anteroposterior and lateral thoracic and lumbar spine, and separate lateral views of the cervical spine, thorax, pelvis with hips, long bones, hands, and feet [3]. An assessment of the size, structure, and shape of the individual bones should also be performed. The dysplasias are traditionally classified by the parts of the skeleton that are involved. The patterns may include any or all of the following: spondylo-, epiphyseal-, metaphyseal-, and diaphyseal abnormalities. Recognition of the area or areas involved helps to narrow the differential. Pseudoachondroplasia (PSACH) is a classic example of a
spondyloepimetaphyseal dysplasia. In childhood, children with PSACH have anterior beaking of their lumbar vertebrae, small irregular epiphyses, and metaphyseal flaring (Fig. 5). This pattern of features is specific to PSACH and sufficient for making the diagnosis [33]. This dysplasia also illustrates that radiographic features of a dysplasia are not static. As with most dysplasias, the diagnosis of PSACH is much more difficult using adult radiographs when the epiphyses have fused and the anterior beaking of the vertebrae is replaced by nonspecific platyspondyly. In addition to the pattern of skeletal abnormalities, the region of the skeleton that is affected can be used to narrow the differential diagnosis. For example, in cartilage-hair hypoplasia (CHH) (McKusick metaphyseal dysplasia), the metaphyses are abnormal with relative sparing of the epiphyses and spine, but not all metaphyses are equally affected. The knees are most compromised, with relative sparing of hips [34]. This pattern of affected regions helps to differentiate C H H from the other metaphyseal dysplasias and nutritional rickets [35]. The pattern is key to the diagnosis because few radiographic features are specific. One notable exception is the finding of iliac horns (Fig. 6) in nail-patella syndrome, which are essentially pathognomonic for the disorder [36]. Although it is a subjective assessment, the bone quality can also help to discriminate between various dysplasias. Dense bones are seen in several disorders, including osteopetrosis and pycnodysostosis [37,38] (Fig. 7). Osteopenia is seen in another group of disorders, including osteogenesis imperfecta and hypophosphatasia [39].
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FIGURE 4 (A) A newborn with campomelic dysplasia and typical craniofacial features. He has midface hypoplasia, protuberant eyes, and Pierre-Robin sequence (U-shaped cleft soft palate and micrognathia). (B) A 4-year-old girl with Stickler syndrome. She has high myopia and hearing loss (note hearing aids), in addition to the Pierre-Robin sequence. She has a proven type II collagenopathy with a 9 base pair deletion in exon 41.
Bone mineral density studies are available to quantify the impression of osteopenia, but care should be taken to use age-matched controls. The spine radiographs can reveal more than simple platyspondyly. In the newborn period, several disorders, including Kniest dysplasia and various forms of chondrodysplasia punctata, have multiple coronal clefts [40]. One of the more specific findings in the spine is the "double hump" seen in Dgyvve-Melchior-Clausen syndrome [24] (Fig. 8). Again, it is important to keep in mind the "fourth dimension" or the evolution of findings over time [41]. The humped vertebrae of spondyloepiphyseal dysplasia tarda will not be apparent until adolescence, and the abnormalities in the lumbar spine in sponastrime dysplasia change from platyspondyly with an anterior protrusion to biconcave deformities of the posterior portion of the vertebral bodies [42,43]. Abnormal findings have been recorded for every bone or anatomical region. The hands are worthy of special mention because of the variety of abnormal findings and their frequently critical role in establishing a diagnosis. Although bone age is not reliable for estimating potential adult height in a person with a skeletal dysplasia, it can be a useful indicator. Several skeletal dysplasias show retarded osseous maturation, whereas advanced carpal bone age has been reported in few, such as Desbuquois dysplasia [44]. Cone-shaped epiphyses are a cardinal finding that can help establish a limited differential diagnosis. Cone-shaped epiphysis refers to an epiphysis that is broader at the base than distally and is frequently associated with an indentation in the metaphysis, most often in the phalanges but occasionally in the metacarpals. Experts in this field can recognize 38 types of cones and certain types are specific for distinct disorders [45].
The classic example is type 12 cone epiphyses in the trichorhinophalangeal disorders [46] (Fig. 9). Brachydactyly can be the only radiographic abnormality in certain syndromes (multiple forms have been delineated) or seen as part of a more generalized dysplasia (e.g., Robinow's syndrome) [47]. Campomelia (bowed bones) should not be considered a specific indicator but rather as a starting point for generating a differential diagnosis. Campomelic dysplasia is named for the bowing seen classically in the femurs and tibiae and associated with an overlying skin dimple. However, the bowing is merely one of the radiographic criteria, and more specific and constant findings are actually seen in the chest, including hypoplastic/aplastic scapulae, hypoplastic thoracic vertebral pedicles, and 11 pairs of thin gracile ribs [48]. Several children with acampomelic campomelic dysplasia due to point mutations in SOX9 or chromosomal rearrangement have been reported [49,50] (Fig. 10). Campomelia is also seen in other dysplasias, such as Stuve-Wiedemann syndrome [51], and more commonly as a reflection of fractures/bone fragility in osteogenesis imperfecta [52]. Campomelia can also be seen in nonskeletal dysplastic conditions, such as Meckel-Gruber syndrome, presumably as a consequence of fetal hypokinesia [53]. Like campomelia, chondrodysplasia punctata is a radiographic sign and not a specific diagnostic entity [54]. The terms chondrodysplasia punctata, stippled epiphyses, and punctate epiphyses have been used interchangeably in the literature. Although this finding will help generate a differential diagnosis, it is seen in more than 20 disorders, including teratogen exposures, intrauterine infections, chromosomal abnormalities, and some metabolic diseases [55]. Punctate epiphyses
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FIGURE 6 Radiograph of the pelvis of an approximately 4-year-old girl who has nail-patella syndrome. She has short stature, dystrophic nails, and absent patellae. The radiograph shows bilateral iliac horns, which were asymptomatic.
FIGHRE 5 Radiographs of a 5-year-old girl with pseudoachondroplasia (PSACH). The lateral spine radiograph shows anterior beaking with central protrusion, which is typical of the disorder. At her knee, the epiphyses are small and dysplastic and the metaphyses are flared. In PSACH, the radiographic findings are sufficient and specific enough to allow for diagnosis.
disappear with age as the multiple calcified centers coalesce, reinforcing the need for an early and complete skeletal survey if a dysplasia is suggested.
FIGHRE 7 AP radiograph of the left hand of a 41/2-year-old boy with pycnodyostosis. Of note are the osteolysis seen in all the distal phalanges and the increased density of the bones, which are both typical of this disorder.
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FIGURE 10 AP radiograph of a newborn with campomelic dysplasia. Of note is the absence of vertebral pedicles (small arrowheads) in the thoracic spine (present in the lumbar spine) and 11 pairs of ribs. A nearly diagnostic and uniform feature is the hypoplastic scapulae (large arrowhead). Not seen here but often present are cervical kyphosis and cervical or thoracic scoliosis. FIGURE 8 Lateral radiograph of the lumbar spine of a teenage girl with Dgyvve-Melchior-Clausen syndrome. She presented with short stature, dysplastic hips, and developmental delay. The spine has a "double-hump" appearance with a central indentation. This is one the few skeletal dysplasias associated with developmental delay.
FIGURE 9 (A) Radiograph of the left hand of a 3-year-old boy with Langer-Giedion syndrome (trichorhinophalangeal syndrome type II). He presented with short stature, unusual facies, and severe developmental delay. There are multiple cone epiphyses particularly well seen in the middle phalanges (arrows) and exostoses at both the distal ulna and radius (arrowheads). (B) Radiograph of the knee demonstrates multiple exostoses at the distal femur and both tibiae and fibulas (arrows).
Radiographic views of the pelvis can also be important in the differential diagnosis. In a child with ATD, the neonatal manifestations are due to the small chest size, but this does not differentiate ATD from other disorders associated with short, horizontally oriented ribs, such as Barnes syndrome [56]. Although the pelvic abnormalities
FIGURE 1 1 The pelvic radiographic findings in asphyxiating thoracic dysplasia (ATD) are important diagnostic features. This radiograph shows the typical pelvis of ATD with narrow sacrosciatic notches and trident appearance of the acetabular roof (radiograph provided by Dr. Elke Schaefer).
are clinically silent, they are diagnostically important (Fig. 11). The pelvic abnormalities in some conditions, such as Schneckenbecken and baby rattle dysplasias, are so striking that they have been used in naming the conditions [57,58].
BIOCHEMICAL INVESTIGATIONS Biochemical investigations are not often useful but in certain instances can be invaluable. Classic examples of
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dysplasias diagnosed in this manner are the mucopolysacharidoses and mucolipidoses. Screening is done by quantitation of urine mucopolysaccharides and oligosaccharides and diagnosis is by specific enzyme assay on leukocytes or fibroblasts. These disorders have varying degrees of skeletal involvement but follow a pattern known as dysostosis multiplex [59]. The findings in the skull include J-shaped sella turcica and premature fusion of the cranial sutures. The vertebral bodies tend to be ovoid in shape and there can be ossification defects. The ossification defects are pronounced in Morquio syndrome and, along with the platyspondyly, result in the gibbus deformity, which is frequently the presenting sign of the disorder [60] (Fig. 12). There are also characteristic changes in the hands, including short proximally pointed metacarpals and bullet-shaped phalanges (Fig. 13). Wide ribs that narrow posteriorly are a frequent sign of dysostosis multiplex [59] (Fig. 14).
Recently, abnormalities in sterol metabolism have been recognized as causing several forms of chondrodysplasia punctata, including chondrodysplasia punctata Conradi-Hunermann and congenital hemidysplasia
FIGURE 13 Radiographof the left hand of a 10-month-oldboy with Hurler disease (0t-iduronidase deficiency). Of note is the marked proximal pointing of the metacarpals, resembling a sharpened pencil.
FIGURE 12 Lateralradiograph of a 41/2-year-oldboy with Morquio A (N-acetyl-galactosamine sulfatase deficiency). In the cervical spine, note the platyspondyly and hypoplastic dens. The lumbar spine is typical of severe dysostosismultiplex, with flattening and midanterior beaking. There is a kyphosis of approximately 15~
FIGURE 14 Chest radiograph of a 5-month-old girl with I cell disease who died at 7 months of age. Particularly noteworthy is the expansion of the ribs.
16. A Diagnostic Approach to Skeletal Dysplasias with icthyosis and limb defects [30,61]. Sterol analysis was useful to show that these were metabolically related disorders and is now used for confirmation of diagnosis [61]. Another example of biochemical analysis is the measurement of GNAS1 function in the diagnosis of Albright hereditary osteodystrophy [62]. Quantitative analysis of this protein's activity in the erythrocyte membrane has been used for diagnosis prior to gene discovery [63].
CARTILAGE HISTOLOGY Although not commonly used, histological assessment can be helpful and occasionally crucial to the diagnosis of skeletal dysplasias identified both prenatally and postnatally, especially if the molecular defect is unknown. The most useful bone from an autopsy is the femur because it offers bone tissue, cartilage tissue, and two large growth plates. Iliac crest biopsies from living patients can be quite useful. The following are useful criteria for the distinction and diagnosis of bone dysplasias: (i) Where is the primary abnormality--in bone tissue (e.g., osteogenesis imperfecta), in cartilage tissue (e.g., achondrogenesis l b and 2), or at the growth plate (thanatophoric dysplasia)? (ii) Is the extracellular matrix affected or is it microscopically normal? (iii) Are the
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chondrocytes morphologically normal or do they show changes in shape (e.g., spindle shaped as in fibrochondrogenesis or ballooned as in collagen 2 dysplasias)? (iv) Within the growth plate, are the relative widths of the columnar zone, the hypertrophic chondrocyte zone, and the provisional calcification zone correct? Routine hematoxylin and eosin staining is of limited value because of the poor affinity of cartilage matrix for these dyes. Whenever possible, Azan-Mallory staining or another trichrome staining method should be performed to visualize collagen fibers, and staining with a cationic azo dye (Alcian blue or toluidine blue) should be performed to visualize the anionic sulfated proteoglycans in the cartilage matrix. To obtain the best visualization of cellular and matrix components, specimens should not be decalcified and embedding should be done in a plastic such as methylmethacrylate rather than paraffin. Fibrochondrogenesis is a lethal (presumed autosomal recessive) disorder named for its unusual histological pattern [64]. Radiographically, there is a resemblance to lethal metatropic dysplasia, but microscopic evaluation of the growth plate revealed a very disturbed pattern compared to controls that is particular to fibrochondrogenesis [64,65]. The columnar zone is reduced in width in the thanatophoric dysplasia/achondroplasia group, whereas it can be markedly wider than normal in hypophosphatasia [66] (Fig. 15). The importance of cartilage
FIGURE 15 Examplesof architectural disturbances at the metaphyses. (Left) Metaphysis of a long bone of a fetus (28 weeks) with thanatophoric dysplasia type 1. The width of the proliferating, columnar chondrocyte zone (between the arrows) is dramatically reduced; column formation is barely recognizable. There is a dense fibroosseous band just proximal to the growth zone that correlates with a cupped appearance of the metaphysis on radiographs. (Right) Metaphysis of a long bone of a fetus (33 weeks)with hypophosphatasia. The defect in alkaline phosphatase activity impairs terminal differentiation of the proliferating chondrocytes to hypertrophic chondrocytes. Therefore, column formation is exuberant (some columns can be followed almost to the bottom of the figure). Magnification, approximately 20x. (see color plate.)
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FIGURE 16 Examples of different patterns of changes in chondrocytes and cartilage matrix in epiphyseal cartilage of fetuses with achondrogenesis type 1A (left) and type 1B (middle) and fibrochondrogenesis (right). (Left) The cartilage matrix in achondrogenesis type 1A is smooth and homogeneous and thus near normal. The chondrocytes have irregular sizes, and in some vacuolization of the cytoplasm can be recognized. Also, some chondrocytes display eosinophilic inclusions (which would show better after PAS staining). (Middle) The cartilage matrix in achondrogenesis type 1B does not have a smooth ground-glass pattern but shows instead coarse collagen fibers that tend to coalesce around the chondrocytes. Some of the chondrocytes show a limited pericellular (territorial) zone with some preservation of matrix. (Right) Fibrochondrogenesis. With this conventional hematoxylin and eosin staining, the main abnormality visible is the spindle-shaped (fibroblast-like) chondrocytes that tend to be grouped in nests separated by fibrous strands. (see color plate.)
histology is further demonstrated in the achondrogenesis group: Many of the distinctive radiographic features are not reliably detected in midgestation fetuses, but cartilage histology may allow for reliable distinction between type 1B (normal chondrocytes and rarefied matrix with coarse collagen fibers), type 1A (normal matrix and inclusions in chondrocytes), and type 2 (matrix dehiscence and vacuole formation and ballooned chondrocytes) [67,68] (Fig. 16). These data can be used to decide what confirmatory laboratory investigations should be obtained first. Although the role of careful histological examination for diagnostic purposes is undisputed, its contribution to suggesting possible pathogenetic mechanisms is controversial because it has been helpful in some cases (e.g., in linking dyssegrnental dysplasia to the perlecan gene by virtue of histologic analogies to a perlecan mouse knockout) but misleading in others [69]. For example, histochemical evidence suggesting a proteoglycan defect in achondrogenesis and diastrophic dysplasia has been present for many years, but the disorders were linked only after biochemical and molecular evidence of a common sulfation defect; histochemical data had long been interpreted as suggestive of a collagen 2 defect or a metabolic defect leading to cellular demise in diastrophic dysplasia.
The intermediate defect, atelosteogenesis 2, was separated from severe diastrophic dysplasia despite radiographic and histologic evidence of a close relationship between the two.
M O L E C U L A R BASIS As the molecular basis has become known for increasingly more skeletal dysplasias, mutation analysis has become an increasingly useful tool for confirmation of the clinical/radiographic diagnosis. The determination of a specific molecular diagnosis can have clinical implications for prognosis of the patient and for recurrence risk for the family. This is particularly important for those disorders that are inherited in an autosomal recessive manner or have significant germline mosaicism and that might be amenable to prenatal diagnosis. Knowledge of the gene defect also allows for the description of the complete spectrum of a disorder and the overlap of certain disorders. For example, it has been shown that recessive metaphyseal dysplasia without hypotrichosis is a variant of C H H and that hair anomalies and immunodefciency are not obligate features of C H H [70]. Similarly, molecular analysis has revealed that Ehlers-Danlos syndrome
16. A Diagnostic Approach to Skeletal Dysplasias type 7 is caused by splicing mutations in the type 1 collagen genes, thus explaining the phenotypic overlap between this disorder and osteogenesis imperfecta [71].
PRENATAL DETECTION OF SUSPECTED SKELETAL DYSPLASIA In recent years, ultrasonographic examination during pregnancy has become part of standard prenatal care, and measurements of the skull, abdomen, and femurs are a routine part of the exam. Currently, more than 80% of the lethal dysplasias are detected on prenatal ultrasound, and the nonlethal or variably lethal skeletal dysplasias are increasingly detected [72]. The most c o m m o n findings prompting suspicion of a skeletal dysplasia are short limbs for gestational age or polyhydramnios [73]. Once a skeletal dysplasia is suspected, the patient is referred to a tertiary care center for detailed anatomic screening. Although historically in utero radiographs were used to establish a diagnosis, in practice this has been abandoned due to its limitations and the advances in ultrasound technology [74]. Prenatally, it is most important to determine whether or not the fetus actually has a skeletal dysplasia and, if
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so, whether it is lethal because this will often play a role in pregnancy management. Perinatal lethality in skeletal dysplasias is usually secondary to restrictive lung disease as a consequence of a small bony thorax; thus, measurements of the thoracic circumference and the thoracic/abdominal ratio are the best indicators of lethality [75] (Fig. 17). Severely shortened limbs (micromelia) are a useful but indirect indicator of lethality and can sometimes be appreciated earlier in the pregnancy than small thoracic circumference [75]. However, not all short limbs are due to dysplasia, and intrauterine growth retardation can be mistaken for a skeletal dysplasia. This is important to recognize because it will affect prognosis for the current pregnancy and recurrence risk is dependent on the underlying cause of the growth failure is [76]. Prenatally, in addition to assessing the individual bones, such as by examining postnatal radiographs, it is necessary to assess the pattern of bony abnormalities. It is more difficult to judge radiographic features prenatally, but an examination of the skeleton and the various patterns seen in dysplasias can help formulate a reasonable differential diagnosis. Ultrasound visualization of a skull defect might lead to the diagnosis of osteogenesis imperfecta or hypophosphatasia. However,
FIGURE 17 (A) Ultrasound performed at 23.5 weeks due to suspicion of skeletal dysplasia. Mildly shortened femurs were noted at 12 weeks and repeat ultrasound at 19 weeks showed micromelia, small thorax, and marked midface hypoplasia. Based on the findings, the parents were counseled that the fetus had a lethal condition and that thanatophoric dysplasia (TD) was the likely diagnosis. This view of the fetus shows the narrow chest diameter compared to the abdomen. After termination of pregnancy, the diagnosis of type 1 was confirmed by radiographs and molecular analysis. (B) Radiograph of the fetus with TDI. This diagnosis was subsequently confirmed by molecular analysis, which showed the C742T mutation in the FGFR3 gene (typical of TDI). The radiographic findings are severely shortened limbs with trident positioning of the fingers and bowed femurs. In the thorax, there are H-shaped platyspondyly and short ribs. TD is broadly classified into two types. TD1 causes bent/ angulated femurs. TD2 is associated with cloverleaf skull caused by multiple craniosynostoses and relatively straight femurs.
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FIGURE 18 A fetus assessed for short limbs at 22 weeks of gestation. Of note, the head was relatively large and on profile had features of achondroplasia. Most noticeable was the prominent forehead and the depressed nasal bridge. (B) On inspection of the extremities, the diagnosis of achondroplasia was supported by the finding of trident hand. The diagnosis was confirmed postnatally.
a more detailed examination of the fetal skeleton might reveal absent clavicles and delayed ossification of the pubis and lead to the diagnosis of cleidocranial dysplasia [77]. Examination of the fetal head and neck might reveal other clues, such as the kleeblattschadel of thanatophoric dysplasia type II or the micrognathia of the type II collagenopathies [78] (Fig. 18). A detailed ultrasound examination of fetal structures and organs is recommended because ancillary ultrasound findings are helpful in forming the differential diagnosis. Findings include polyhydramnios, abnormal fetal positioning (e. g., club feet and contractures), and congenital heart defects [76]. Accurate prenatal diagnosis of skeletal dysplasias remains problematic. In order to ensure appropriate counseling, posttermination or postnatal examination should be done, including clinical exam/autopsy and radiographs. Unless a specific diagnosis is highly suspect, molecular testing should be reserved until after delivery or termination of pregnancy to avoid inaccurate prenatal diagnosis leading to "normal" molecular results and false reassurance of the expectant parents.
CONCLUSION In practice, the diagnosis of skeletal dysplasias is not difficult, but it remains complicated. It demands a familiarity with numerous rare conditions and good patternrecognition skills. The sequence of steps in this chapter provides a framework for establishing a differential diagnosis, but consultation with an expert in the field of
skeletal dysplasia is a key step in refining a suspected diagnosis. Despite advances in molecular medicine, the interpretation of skeletal radiographs is still essential for diagnosis. When the clinician has delineated the pattern of radiographic abnormalities and clinical features, it is possible to search the medical literature and radiographic atlases for a matching pattern. However, as the number of skeletal dysplasias that are molecularly defined increases, mutation analysis is becoming an increasingly more important method of confirming the suspected diagnosis of rare entities. Establishment of a precise and correct diagnosis is important for appropriate counseling regarding potential complications, expected adult height, and recurrence risk.
References 1. International Working Group on Constitutional Diseases of Bone (1998). International nomenclature and classification of the osteochondrodysplasias. Am. J. Med. Genet. 79, 376-382. 2. Ala-Kokko, L., Baldwin, C. T., Moskowitz, R. W., and Prockop, D. J. (1990). Single base mutation in the type II procollagen gene (COL2A1) as a cause of primary osteoarthritis associated with a mild chondrodysplasia. Proc. Natl. Acad. Sci. USA 87, 6565-6568. 3. Lachman, R. S. (1998). Radiologic and imaging assessment of the skeletal dysplasias. In Growth Disorders (C. J. H. Kelnar, M. O. Savage, H. F. Stirling, and P. Saenger, Eds.), pp. 251-264. Chapman & Hall, London. 4. Andersen, P. E., and Hauge, M. (1989). Congenital generalised bone dysplasias: A clinical, radiological, and epidemiological survey. J. Med. Genet. 26, 37-44. 5. Orioli, I. M., Castilla, E. E., and Barbosa-Neto, J. G. (1986). The birth prevalence rates for the skeletal dysplasias. J. Med. Genet. 23, 328-332.
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11__7__1 The Spectrum of Pediatric Osteoporosis Leanne M. Ward* and Francis H. Glorieux t *Department of Pediatrics, Division of Endocrinology and Metabolism University of Ottawa and The Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada fDepartments of Surgery, Pediatrics and Human Genetics, McGill University and the Shriners Hospital for Children, Montreal, Quebec, Canada
to aggressive medical therapy for chronic systemic disease, with improved long-term outcome for children. In addition, health care providers are identifying osteoporosis in childhood more frequently, through systematic monitoring of bone health in the first two decades of life. The purpose of this chapter is to provide an approach to the diagnosis and treatment of pediatric osteoporotic conditions described in the current literature.
ABSTRACT Osteoporosis is increasingly recognized as an important medical problem among pediatric patients with genetic disorders and chronic illnesses. The goal of this chapter is to provide a review of the scope of the problem in children and adolescents, highlighting the unique issues that arise from the assessment and treatment of osteoporosis in the growing patient. In order to understand the determinants of bone health and disease during the pediatric years, a "functional model" of bone development is proposed. An overview of the various forms of primary and secondary osteoporosis and of the existing evidence regarding preventive and treatment strategies is provided. An approach to the differentiation of child abuse from bone fragility conditions is also proposed. While the past decade has seen tremendous progress in the identification of pediatric osteoporosis, there is ongoing need for prospective, longitudinal studies to fully define the natural history of the various forms of childhood osteoporosis and the effects of intervention during the growing years.
DEFINITION AND DIAGNOSIS OF OSTEOPENIA/OSTEOPOROSIS IN PEDIATRIC PATIENTS Osteoporosis is defined as low bone mass and microarchitectural deterioration of bone tissue resulting in fragility (atraumatic) fractures. Osteopenia (literally = scarcity of bone) is the precursor of osteoporosis, and is simply defined as a reduction in bone mass for age. This term may be confused with osteomalacia, which is a mineralization defect of bone tissue. Both osteomalacia and osteoporosis are associated with low bone mass, but due to completely different mechanisms. In osteoporosis, there is a decreased amount of bone tissue (osteopenia) associated with atraumatic fractures (osteoporosis). Osteopenia and osteoporosis are caused either by insufficient deposition or increased resorption of organic bone matrix. The incorporation of calcium and phosphate into the bone matrix, however, is not usually affected. In osteomalacia, there is an accumulation of unmineralized bone matrix, as osteoblasts continue to secrete osteoid for some time despite impaired mineralization. In both osteoporosis and osteomalacia, bone mineral density (BMD, bone mass per volume of tissue) and bone
INTRODUCTION Osteoporosis is the most common metabolic bone disorder in adults, and remains a major health problem worldwide [1,2]. While previously considered a disease of the aging, there is increasing awareness that osteoporosis may affect children, either because of an intrinsic skeletal defect (primary osteoporosis) or as the result of other diseases or their treatment (secondary osteoporosis). There is a growing list of secondary osteoporoses, due
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mineral content (BMC) are all reduced, which has led to confusion among these definitions. Thus, to say that bones are poorly mineralized implies a mineralization defect, or osteomalacia. To say that there is a reduction in bone mass per volume of tissue, in the absence of a mineralization defect, defines osteopenia/osteoporosis. Various methods have been employed to assess bone health and detect osteopenia and osteoporosis in children and adolescents. These have included measurements of bone pain and mobility [3] as well as X-ray confirmation of fractures, bone densitometry, assessment of biochemical markers of bone metabolism, peripheral quantitative computed tomography (pQCT), quantitative ultrasound and bone histology/histomorphometry [3-7]. A more detailed discussion of noninvasive techniques for bone mass measurement is presented in chapter 12, bone turnover markers in chapter 14, and bone histomorphometry in chapter 15. The most widely used tool for the diagnosis of osteopenia and osteoporosis is dual energy X-ray absorptiometry (DXA). DXA is considered the preferred method for quantifying bone mass because of its precision, reproducibility, speed, and minimal exposure to radiation [8]. Its limitation is that it assesses areal bone mineral density (aBMD) and thus requires that bone size be taken into account (see below). Published pediatric normative data are available for Lunar [9-11], Hologic [1215] and Norland [16] instruments. While pQCT allows independent measurement of bone size and volumetric BMD (vBMD), it has more limited use in pediatrics because of fewer reference data for children [17,18] and the requirement that the patient must remain still for several minutes to complete the test. Quantitative ultrasound is appealing because of the low ionizing radiation dose, relative low cost and portability. As such, it is presently under study at various sites in children, but normative data are currently limited, [6,19,20] and its validity has yet to be established. The interpretation of DXA in children and adolescents poses unique challenges due to the changes in bone size and shape that occur during the growing years. Confusion has arisen from the fact that bone density by DXA is traditionally reported as bone mineral content (BMC, in grams) per cm 2 of mineralized bone tissue. This is a twodimensional measurement of a three-dimensional structure and represents the "areal" projection of bone in the coronal plane. The height and width of the scanned bone are known, but the depth is not because of technological limitations. As a result, a bigger bone will appear to have a greater density when it does not. The bigger bone holds more mineral, but this mineral is contained within the larger volume of the entire bone. Areal bone density (aBMD), then, suggests that a child's bone becomes significantly denser as the child grows, when it does not.
When aBMD is interpreted without considering bone size, the vBMD (g/cm 3) of a smaller child with smaller bones is underestimated while a tall child's vBMD is overestimated (and a diagnosis of osteoporosis may be missed). In adults, aBMD is a reasonable surrogate for vBMD, since BMC may change (decline) but bone size (at least its depth) is relatively stable. The failure of densitometric technology to account for bone size has led to the erroneous interpretation that there is a global increase in mineral density of the skeleton during the growing years. In actual fact, vBMD in long bones is independent of age, while modest gains in vBMD at the spine occur around the time of puberty [9]. In light of this problem, several approaches have been proposed which take bone size into account through estimation of vBMD [9,21,22]. In these models, BMC is divided by the estimated volume of bone in the region of interest. Molgaard et al. have suggested correcting total body BMC for height [23]. Despite these recommendations, there is no universally accepted method for correction of bone size in children. In addition, the patient's gender, pubertal stage (ie. skeletal age) and ethnicity must also be considered given the observed differences in these parameters. For example, males attain final height and peak bone mass later than females [24,25], the rate of bone mineral accrual accelerates at the time of puberty [26] and Caucasians have a lower bone mass than African-Americans [15,17,27]. Failure to consider gender, developmental stage and ethnicity in DXA interpretation of bone mass may lead to erroneous conclusions and misdiagnoses [28]. For adults, specific diagnostic criteria for osteopenia and osteoporosis based on bone densitometry have been developed. According to the World Health Organization definition [2], an aBMD measurement of 2.5 SD or more below the healthy young adult mean (the T-score) meets the criteria for osteoporosis. Osteoporosis is considered "established" when at least one documented fragility fracture is associated with the densitometric criteria for the disease. Osteopenia is said to exist when the T-score is situated between-1 and-2.5 SD. At the present time, there are no data to define osteopenia/osteoporosis in pediatric patients. Furthermore, it is inappropriate to use T-scores in young patients who have not yet achieved their peak bone mass. For children, if the diagnosis of osteoporosis were based on a purely statistical (Gaussian) distribution of BMD, then in densitometric terms it would be defined as a BMD of 2 SD or more below the mean value (the Z score) compared to age- and sex-matched healthy controls. According to this approach, osteopenia would be said to exist when the BMD Z score lies between - 1 and - 2 SD. Among adults, aBMD is considered a strong predictor of fracture risk [29,30], and a 1 SD reduction
17. The Spectrum of Pediatric Osteoporosis
in aBMD from the healthy adult mean corresponds to a 2- to 3-fold increased risk for fracture in postmenopausal women [31]. For children, however, the fracture threshold for BMD has not been clearly established. In a recent study, Goulding et al. [32] showed that for each 1 SD reduction in total body BMD, there was a doubling of risk for new fractures in young girls. Similar studies on large numbers of children are needed to determine whether the number of observed fractures in a given cohort exceeds the expected incidence of fractures for age and gender. Until more data is available, the clinical risk of a reduced bone density (either estimated vBMD or aBMD) cannot be clearly stated as for adults. Moreover, the fracture threshold in children is likely to vary at different skeletal sites, depending on the timing and duration of exposure to illness. This is because bone mineral accrual and bone growth proceed at different rates, at different skeletal sites [24,25]. Finally, it is possible that different illnesses will produce different fracture thresholds, a question that has not been addressed in the pediatric literature to date. Until further studies of bone density and fracture risk at different ages and skeletal sites are performed, osteopenia/osteoporosis should not be diagnosed in children based solely on the statistical distribution of BMD. Rather, osteoporosis should be clearly defined in functional terms as recently proposed [33]. In this way, osteoporosis will be said to exist when the skeleton has not been able to withstand its mechanical challenges (growth and muscle force) due to inadequate bone mass and/or structure, resulting in atraumatic fractures. This means that a child will not be labeled with osteoporosis unless there is a history of fractures that occur with minimal trauma. With such an approach, the term osteopenia will not be employed in the assessment of a child's bone health. If osteoporosis is to be defined on a non-densitometric basis, then what is the role of bone densitometry and how should it be interpreted? Densitometry should serve as a guide to monitor the patient's gain (or lack thereof) in estimated vBMD over time in response to therapy or in the context of evolving disease. It should be interpreted cautiously, in view of the patient's age, gender, size, pubertal stage and ethnicity, and correlation with more direct clinical indices of skeletal health such as fractures, bone pain, limb deformity and impaired mobility should be sought in all cases. Biochemical markers of bone metabolism may aid in the monitoring of patients receiving anti-resorptive medication for the treatment of osteoporosis, since markers of resorption are correlated inversely with bone area and spinal bone density [4]. Biochemical markers have not been useful, however, for the diagnosis of osteoporosis in children, due to the wide range of normal values and the need to adjust for pubertal stage. Iliac crest bone
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biopsies have proven useful for the histological characterization of different forms of osteogenesis imperfecta (OI) [34-36], and for the study ofbisphosphonate effect at the tissue level in children with OI [37]. Recently, pediatric normative data for iliac crest bone histology and histomorphometry have been published [7]. However, bone biopsies are invasive procedures and as such, their use is presently restricted to centres that specialize in the diagnosis and treatment of pediatric bone disease.
THE ROLE OF THE MECHANOSTAT IN THE PATHOGENESIS OF PEDIATRIC O S T E O P O R O S I S The importance of building the best possible skeleton during childhood and adolescence is increasingly recognized by physicians who care for the young and old alike, and prevention of osteoporosis has been heralded a pediatric responsibility [38,39]. Previous approaches to osteoporosis in childhood have emphasized the determinants of "peak bone mass" and their relationship to the subsequent risk of fractures in later life. Bone mass is easily quantified with current techniques and thus is frequently used as an index of skeletal health. Studies have shown that 25 percent of peak bone mass is accrued during the two-year period surrounding peak height velocity [40] and at least 90 percent of peak bone mass is obtained by the age of 18 years [40,41]. Furthermore, the amount of bone mineral accrued during the pubertal years is equal to the amount typically lost throughout adulthood [26]. Up to 80 percent of the variability in peak bone mass is genetic [42,43], but the achievement of one's genetic potential can be influenced by a number of risk and protective factors. Threats to bone health that are operative in childhood may significantly reduce the mineral deposit to the bone bank (termed "acquisitional osteopenia"). The site and severity of compromised bone growth and mineral accretion will be influenced by the timing and duration of exposure to insult, since growth and mineral accrual proceed at different rates for different skeletal sites [24,25]. For example, trabecular bone is acquired before cortical bone, and most of the appendicular growth is complete before puberty while spinal growth is enhanced under the influence of sex steroids [24,25]. On the other hand, protective factors such as exercise [44,45] and improved calcium intake [46,47] result in greater benefit to mineral acquisition when exposure occurs during the growing years. To date, the pediatric osteoporosis literature has focussed on the array of osteotrophic factors such as physical activity, nutrition, hormones, and molecular, environmental and genetic events, which are known to
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play individual roles in the attainment of peak bone mass (the "cumulative model of bone development") [48]. In a recent discussion on bone development, Rauch and Schoenau [48] suggested there are two problems with this approach. First, while the evidence suggests that childhood is an important period for bone mineral accrual and that peak bone mass may be correlated with subsequent skeletal health [29], a focus on the cumulative factors which affect mineral acquisition does not directly address the more clinically relevant question--what is the functional goal of bone development? These authors noted that the goal of bone development should not be to accumulate mineral and become as heavy as possible, since weight is not the most functionally important property of bone. Instead, it stands to reason that the ultimate goal of bone development is to maximize bone strength, so that bones are as strong and as stable as they need to be (to meet the mechanical demands in a given individua!). Optimizing bone mass through mineral accrual, then, is not likely to be the sole quest of bone development but rather one of the means by which bone strength is ultimately achieved. The other way in which bone strength and stability are achieved is through skeletal architectural design [49,50]. The second problem with this approach is that for the "cumulative model of bone development" (Figure 1a) to be true, the genes involved in the process must contain the entire blueprint for construction of the changing skeleton during the growing years. Rauch and Schoenau [48] note that while this is likely true for skeletal patterning during embryogenesis (which occurs in a soft tissue), it is more difficult to reconcile skeletal development with an assembly process that is orchestrated by gene products once mineralization has already occurred. Spatial information necessary for skeletal patterning is provided by morphogens, which are distributed within the soft tissue of the skeletal template and which effect change in the geometric shape of an organ tissue by concentration-dependent diffusion [51]. However, once the skeletal template has been constructed and mineralization has occurred, cellular communication through diffusion of coordinating molecules is not possible, as the cells are separated by mineralized tissue. It is difficult to explain, then, how bone would be able to both sense and respond to the changes required for growth and mineral accrual, when the genetic blueprint is a static entity. As such, Rauch and Schoenau [48] propose a "functional model for bone development" (Figure l b) based on Frost's mechanostat theory [52]. According to this theory of bone development, the genetic blueprint provides spatial information for development of the cartilaginous template of bone. Once this is complete and mineralization occurs, bone cell communication is effected through the mechanical requirements
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FIGURE I A: A cumulative model of bone development. B: A functional model of bone development based on the mechanostat theory. Bone development is regulated by the feedback loop between bone deformation and bone strength. During growth this homeostatic system is continually forced to adapt to external challenges. Reprinted with permission from Rauch et al. [48]. 9 Lippincott, Williams and Wilkins.
of the bone. This means, that when the mechanical challenges exerted on the bone exceed a certain limit (the setpoint of the mechanostat) and cause bone deformation or "strain," this is sensed by the mechanostat and bone is added at the precise location where tissue is needed to withstand the challenge. The primary mechanical challenges for the developing (post-embryonic) skeleton are increases in muscle force and in bone length. The osteotropic factors (genes, nutrition, hormones, physical activity) still play an important role in the mechanostat model, either directly through modification of the mechanostat's response threshold, or indirectly through alterations in bone length and/or muscle mass. Either way, the required bone strength (defined by the mechanical forcesbearing upon the bone) determines its mass and architecture. Thus, genetically determined linear bone growth and extrinsic muscular forces catapult skeletal development (ie. mineral accrual and architectural design) in order to achieve bone strength and stability. It is proposed that the effect of genes and their products on post-embryonic bone development is to regulate the size of bones and muscles ("the sizostat") and the set-point of the mechanostat [53]. This functional model of bone development shifts the focus of our approach to understanding osteoporosis in
17. The Spectrum of Pediatric Osteoporosis
pediatric patients. It encourages clinicians/researchers to consider whether the low bone mass in various disorders is the result of a decrease in bone length, an increase in the mechanostat set-point, or diminished muscle force applied to bone. Considering childhood osteoporosis in this light takes us beyond bone densitometric issues, which are made difficult by the constant change in bone size for pediatric patients. In this chapter, the functional model of bone development has directed the overall approach to osteoporosis in children, which may result from mainly mechanical factors (changes in muscle force/bone length) or primarily non-mechanical influences (which act directly on bone and may alter the mechanostat setpoint). In some cases, health and disease may affect more than one component of the mechanostat. For example, the hormonal milieu at the time of puberty enhances growth plate activity, increases muscle mass, and alters the mechanostat threshold in response to strain.
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TABLE 1
Differential Diagnosis of O s t e o p o r o s i s in Children and Adolescents ( m o s t c o m m o n c a u s e s , b a s e d on current pediatric literature)
I. Primary Osteoporosis I. Heritable Disorders of Connective Tissue
a. Osteogenesis Imperfecta b. Bruck Syndrome c. Osteoporosis Pseudoglioma Syndrome d. Ehlers-Danlos Syndrome e. Marfan Syndrome f. Homocystinuria 2. Idiopathic Juvenile Osteoporosis 2. Secondary Osteoporosis 1. Neuromuscular Disorders
a. Cerebral Palsy b. Duchenne Muscular Dystrophy c. Prolonged Immobilization 2. Chronic Illness
THE SCOPE OF THE PROBLEM
a. Leukemia b. Rheumatologic Disorders
Osteoporosis in childhood is usually suspected when a patient presents with frequent and/or low trauma fractures, chronic pain, or an incidental finding of "possible osteopenia" on plain X-rays. The skeletal health of children with known risk factors for the disease, such as neuromuscular disorders, chronic glucocorticoid use, endocrinopathy or poor nutrition may be evaluated even before clinical symptoms are present, unveiling the presence of low bone mass or subtle vertebral compression. In this chapter, the disease has been divided into "primary osteoporosis" resulting from an intrinsic skeletal defect, and "secondary osteoporosis" due to underlying illness or treatment. In some cases, the osteoporosis may be of mixed etiology, as in the case of OI exacerbated by immobilization due to fractures and chronic pain. An overview of the differential diagnosis of osteoporosis in children, based on current literature, is presented in Table 1.
c. Anorexia Nervosa d. Cystic Fibrosis e. Inflammatory Bowel Disease f. Other: primary biliary cirrhosis, cyanotic congenital heart disease, thalassemia, malabsorption syndromes, organ transplantation 3. Endocrine and Reproductive Disorders
a. Disorders of Puberty b. Turner Syndrome c. Growth Hormone Deficiency d. Hyperthyroidism e. Diabetes Mellitus f. Hyperprolactinemia g. Athletic Amenorrhea h. Glucocorticoid Excess 4. Iatrogens
a. Glucocorticoids b. Methotrexate c. Cyclosporine
Primary O s t e o p o r o s i s
d. Heparin e. Radiotherapy
Primary osteoporosis due to an intrinsic skeletal defect can be further divided into two main groups: the heritable disorders of connective tissue (including OI) and idiopathic juvenile osteoporosis (IJO). In general, the primary osteoporoses result from genetic defects that impact on bone development. In IJO, the underlying defect is unknown, however, given the lack of extraskeletal manifestations of the disease, it is presently classified as a primary osteoporosis. There is a growing number of disorders which are associated with primary
f. Medroxyprogesterone acetate g. GnRH agonists h. L-Thyroxine suppressive therapy 5. Inborn Errors of Metabolism
a. Lysinuric Protein Intolerance b. Glycogen Storage Disease c. Galactosemia d. Gaucher Disease
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osteoporosis and while the list is not exhaustive, the more commonly reported diagnoses are outlined in Table 2. A detailed discussion of all the primary osteoporoses being beyond the scope of this chapter, only the most frequently discussed causes will be presented here. Heritable Disorders of Connective Tissue
The heritable disorders of connective tissue represent a group of diseases wherein the underlying gene defect affects bone as well as other supporting tissues. The most widely studied heritable disorder of connective tissue in the pediatric osteology literature to date is OI. This likely reflects the frequency of the disease compared to other disorders of connective tissue, and the advances in treat-
TABLE 2
ment options for children with severe forms [3,54]. A detailed overview of the diagnosis and treatment of OI is presented in chapter 16.
Osteogenesis Imperfecta OI is a heritable disorder of bone where the hallmarks are bone fragility and low bone mass. The incidence of the disease is estimated at 1:15,000 live births [55]. Four different types are commonly distinguished on the basis of clinical features and disease severity [56]. Patients with OI type I have a mild phenotype with normal or nearnormal height and typically blue sclerae, while OI type II is usually lethal in the perinatal period. OI type III, known as progressive deforming OI, is the most severe form in children surviving the neonatal period. These
Major Causes of Primary Osteoporosis in Childhood and Adolescence
Disorder
Inheritance
Genetic/Underlying Defect
Osteogenesis Imperfecta Type I-IV
AD/AR
Mutations involving type I collagen (COL1A1/COL1A2 genes)
Type V
AD
Unknown
Type VI
Unknown
Unknown
AR
Unknown (localized to chromosome 3p)
Classical type (EDS I and II)
AD
Majority of patients show mutations in type V collagen (COL5A1/COL5A2 genes). Rare mutations in type I collagen (COL1A1 gene)
Hypermobility type (EDS III)
AD
Mutations in type III collagen (COL3A1 gene), though the majority of mutations in EDS III have not been defined
AD
Mutations in type III collagen (COL3A1 gene)
AD/AR
Mutations in lysyl hydroxylase (PLOD 1 gene)
Type VII Ehiers-Danlos Syndrome
Vascular type (EDS IV) Kyphoscoliosis type (EDS VI) Arthrochalasia type (EDS VIIa and VIIb)
AD
Mutations in COL1A1/COL1A2 genes alter the procollagen N-peptidase cleavage site of the alpha 1 and alpha 2 chains of type I collagen
Dermatosparaxis type (EDS VIIc)
AR
Mutations in procoUagen N-peptidase
Marfan Syndrome
AD
Mutations in FBNI gene, one of two genes encoding fibrillin-1. Fibrillin is the main structural component of elastin-associated cross-links
Homocysffnuria
AR
Most often due to a cystathionine b-synthetase defect, resulting in elevated plasma homocysteine levels. Hyperhomocysteinemia may damage fibrillin-1
Bruck Syndrome
AR
Mutation in bone-specific telopeptidyl lysyl hydroxylase, which interferes with collagen cross-link formation in bone (but not ligaments or cartilage)
Cuffs Laxa
AR
Lysyl oxidase deficiency (mutations in LOX gene) with abnormal synthesis of collagen and elastin cross-links
Other: Progeroid EDS, Periodontitis type, Fibronectin-deficient type, X-linked Type V)
AD
Mutations in the elastin (ELN) gene
Menkes and Occiptal Horn Syndromes
XLR
Mutation in the gene encoding Cu2+ transporting ATPase alpha-polypeptide with lysyl oxidase deficiency and aberrant synthesis of collagen cross-links
Osteoporosis Pseudoglioma Syndrome
AR
Congenital Contractural Arachnodactyly Idiopathic Juvenile Osteoporosis
AD/AR
Mutation in the LRP5 gene which is expressed in osteoblasts and associated with transduction of Wnt signaling Mutation in FBN2 encoding fibrillin-2, a structural protein in bone matrix Unknown
17. The Spectrum of Pediatric Osteoporosis
patients have a characteristic phenotype including extreme short stature, severe deformity of the spine, thoracic cage and extremities, white or blue sclerae and often a triangular facies. Patients with a moderate to severe form of the disease who do not fit one of the above descriptions are classified with OI type IV; as such, this group is extremely heterogeneous. The underlying genetic defect in a proportion of OI patients is attributable to mutations in the two genes encoding collagen type I at chains (COL1A1 and COL1A2); however, in many patients with moderate (type IV) OI, there are no detectable mutations [57]. Glorieux et al. [35] recently described a group of patients initially classified with OI type IV, who presented a discrete phenotype including hyperplastic callus formation, a dense metaphyseal band adjacent to the growth plate and calcification of the interosseous membrane. These patients were also unique histologically, with a coarsened, mesh-like lamellar pattern of the bone matrix under polarized light. Mutations in type I collagen were absent. In keeping with the numerical classification for OI forms, this entity was called "OI type V". The inheritance clearly followed an autosomal dominant pattern. Another novel phenotype which has emerged from the OI type IV group, named "OI type VI", features osteopenia and bone fragility due to a mineralization defect, in the absence of any abnormality in mineral metabolism [36]. On iliac crest bone biopsies, there was loss of the normal birefringent pattern of lamellar bone, often with a fish-scale appearance. The inheritance in OI type VI is presently not known, since none of the parents of the affected children possessed the phenotype and none of the patients, themselves, have had offspring. As for OI type V, the underlying genetic defect in OI type VI remains to be elucidated. The majority of OI forms are inherited in an autosomal dominant fashion, whereas autosomal recessive inheritance has been described in rare kindreds [58-65]. The most recent novel form of OI to be reported, called OI type VII, showed autosomal recessive inheritance in a consanguineous community from Northern Qu6bec [66]. Rhizomelia and coxa vara were striking clinical features, associated with slightly blue sclerae, normal dentition, and moderately severe long bone deformity. As for OI types V and VI, mutations in the genes encoding type I collagen were absent, and type I collagen protein studies were normal [67]. The disease was subsequently linked to the short arm of chromosome 3 [67], outside the loci for the type I collagen genes. Histomorphometric analyses of iliac crest bone biopsies revealed findings similar to OI type I, with decreased cortical width and trabecular number, increased bone turnover and preservation of the birefringent pattern of lamellar bone. These observations demonstrate that the tissue level manifestations of
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OI are not specific for defects in type I collagen, but may result from mutations in other genes. The diagnosis of OI, particularly for milder forms, may be difficult to distinguish from IJO and from physical abuse (further discussion of these two entities is present in ensuing sections of this chapter). The emergence of novel forms of OI in which type I collagen studies are normal emphasizes that the diagnosis must not rest solely on currently available genetic analyses. Bone fragility and low bone mass in conjunction with a family history of OI, blue sclerae, limb deformity or dentinogenesis imperfecta is suggestive of the diagnosis. However, when these features are absent, it may be difficult to say with certainty on clinical grounds whether a form of OI exists. Secondary forms of osteoporosis should be ruled out, leaving IJO the only other consideration. Bone biopsy may be helpful in distinguishing IJO from OI, if it is available. A recent study of the bone histomorphometric characteristics of children with OI sub-types I to IV showed abnormalities in the three mechanisms that normally produce an increase in bone mass throughout childhood [34]. Defects in modeling of external bone size and shape, in production of secondary trabeculae by endochondral ossification, and in thickening of secondary trabeculae by remodeling were evident in the 70 OI children studied [68]. Compared to children with IJO, cancellous bone volume was similarly decreased in OI but bone turnover was much lower in IJO (as evidenced by a reduction in activation frequency and bone surface-based remodeling parameters) [68]. The brittle mineralized matrix of OI bone has been hypothesized to impair sensing by the mechanostat, through interference with the fluid flow in canaliculi [34,69]. Previous modalities for the treatment of OI including fluoride, magnesium oxide, calcitonin, growth hormone and anabolic steroids [70,71] have been met with limited success and are not currently recommended [72]. However, the quality of life for children with severe OI has improved remarkably through the administration of cyclical intravenous pamidronate, a potent inhibitor of bone resorption [73], in conjunction with multi-disciplinary (surgical and rehabilitative) care. A detailed discussion of the approach to medical treatment of patients with OI is provided in Chapter 16. Bruck Syndrome
Bruck syndrome is a rare, autosomal recessive disorder, with nine kindreds reported worldwide [74]. The Bruck syndrome phenotype shares features with OI, including bone fragility, deformity of the spine and extremities, low bone mass, wormian bones, and blue or white sclerae [75,76]. Despite the shared OI features, Bruck syndrome is clearly distinguishable by the presence
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Leanne M. Ward and FrancisH. Glorieux
of congenital joint contractures and an absence of the typical alterations in type I collagen that are found in OI patients [77-79]. Nevertheless, an abnormality of the collagenous structure of bone had long been suspected in Bruck syndrome, and was recently elucidated [74]. Bank et al. [74] reported that Br.uck syndrome is due to a deficiency of a bone-specific telopeptide lysyl hydroxylase, responsible for the formation of type I collagen crosslinks in bone, but not in ligaments or cartilage. In the Bruck syndrome, lysine residues within the telopeptide region of type I collagen are underhydroxylated, leading to aberrant crosslinking, while the lysine residues within the triple helical domain are normally modified. These findings are consistent with the observation that crosslink patterns are tissue-specific and not related to a specific collagen protein. Through a mapping approach based on homozygosity by descent in a consanguineous Bruck syndrome kindred, Bank et al. [74] further showed localization of the disease to chromosome 17p12. As such, the investigators suggested this might represent the genetic region encoding the bone-specific telopeptide lysyl hydroxylase. Studies pertaining to the treatment of Bruck syndrome with anti-resorptive therapies have not been reported. Osteoporosis Pseudoglioma Syndrome
The osteoporosis pseudoglioma syndrome (OPPG) is another autosomal recessive condition with phenotypic features shared by patients with moderate to severe OI including reduced bone mass, short stature, and skeletal deformity. However, patients with OPPG can be distinguished from those with OI by the presence of congenital blindness. The ocular defect (which resembles a pseudoglioma), arises from persistent hyperplasia of the vitreous, which in turn is hypothesized to result from failure of the primary vitreal vasculature to undergo involution during embryogenesis [80], as would normally occur at the beginning of the second trimester. Recently, the Osteoporosis-Pseudoglioma Collaborative Group [80] demonstrated that homozygous (likely loss-of-function) mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene were responsible for the OPPG phenotype. Heterozygotes demonstrated reduced bone mass, while eyesight appeared to be preserved. The LRP5 molecule is expressed by developing and mature osteoblasts, and affects bone mineral accrual during Wnt-mediated osteoblastic proliferation and differentiation. The mutant LRP5 molecule reduces bone mass accrual in murine models, and children with OPPG demonstrate very thin cortices and few trabeculae on iliac crest samples [80]. The importance of LRP5 as a modulator of bone mass accrual is further substantiated by the recent observation that an activating mutation in the LRP5 gene results in an autosomal dominant High
Bone Mass Trait [81]. The ocular pathology associated with LRP5 mutations is hypothesized to result from transient expression of mutant LRP5 by cells within the vitreal vasculature, leading to failed involution of the vascular network. Children with OPPG may have severe osteoporosis with significant pain from vertebral and extremity fractures. As such, anti-resorptive therapies have been explored, and recently described [82]. Three children, ages 9 to 11 years, received either pamidronate or clodronate for the treatment of symptomatic vertebral compression fractures with subsequent improvement in pain, mobility and in the size of the vertebral bodies after two years of therapy. There were no new fractures during the treatment interval, growth and puberty proceeded normally, and the medications were well-tolerated. The authors concluded that bisphosphonate therapy may be justified in patients with OPPG who have symptomatic vertebral compression. Ehlers-Danlos Syndrome
Patients with Ehlers-Danlos syndrome (EDS), like those with OI, demonstrate an extremely heterogeneous phenotype. EDS patients have in common joint and skin hyperlaxity as well as easy bruising [83]. Other variable characteristics of the disease include recurrent joint dislocations, fragile (cigarette paper-like) scars, mitral and triscuspid valve prolapse, kyphoscoliosis, and fragility of the ocular, cardiovascular and gastrointestinal systems. In 1998, the classification of EDS was revised by Beighton et al. [84] such that the numerical system was replaced by descriptive terms. A full discussion of the various EDS sub-types is beyond the scope of this chapter. However, the major forms and their underlying genetic defects are presented in Table 2. Most of the EDS forms are due to a collagenopathy, resulting from mutations in the genes encoding collagen type V (classical EDS), collagen type III (hypermobility and vascular EDS) and collagen type I (arthrochalasia EDS), or in the enzymes necessary for pro-collagen cleavage (dermatosparaxis EDS) and post-translational modification (kyphoscoliosis EDS). The skeletal phenotype associated with EDS is variable and includes scoliosis, kyphosis, thoracic lordosis, lumbar platyspondyly, subluxation of the sternoclavicular joints, chest wall deformity, radio-ulnar synostosis, congenital hip dislocation and clubfoot [85]. There is a paucity of studies addressing the bone health of patients with the various forms of this disease, and previously osteoporosis was not considered a cardinal feature. However, a number of studies have found reductions in bone mass at either the spine [86], femoral neck [87], or both [88]. Dolan et al. evaluated 23 patients (mean age 38.5Y, SD15.5) compared to controls. Prior history of
17. The Spectrum of Pediatric Osteoporosis
fracture was 10 times more common in EDS with 86.9 percent of patients reporting a total of 47 low impact fractures, compared to 8.7 percent of controls. A significant reduction in BMD was found at the femoral neck and at the lumbar spine. In a more recent article, Carbone et al. [89] found no difference in BMD at the lumbar spine nor in biochemical markers of bone and mineral metabolism for 23 adult patients with EDS III (hypermobility and vascular EDS) compared to controls. EDS subjects did have a significantly reduced BMD at the femoral neck, but this difference compared to controis disappeared after adjustment for body height, weight and physical activity levels. The reasons for these discrepant findings may in part be due to the classification of patients. In the study by Carbone et al. [89], the patients were diagnosed with EDS III and thus were a more homogeneous group than those reported in prior studies [86-88]. Furthermore, patients with co-morbidities known to affect bone and mineral metabolism were excluded from this report. Regardless, the increased fracture rate reported by Dolan et al. [88] suggests abnormal bone strength and thus comprehensive studies of bone health in sufficient numbers of patients with the various forms of EDS are warranted before further conclusions can be drawn. M a r f a n Syndrome
Marfan syndrome (MFS) is a common autosomal dominant condition with variable skeletal, ocular and cardiovascular manifestations [90]. The disease has been shown in many families to result from mutations in the FBN1 gene (chromosome 15), one of two genes coding for the glycoprotein fibrillin [91,92]. Fibrillin is the main structural component of the elastin-associated microfibrils in the extra-cellular matrix [93], and its presence has been identified in bone [94]. It has been hypothesized that a defect in this molecule may alter the distribution of mechanically induced strain [95] or interfere with calcium binding [96]. Rarely, mutations in COL1A2 (one of the genes encoding type I collagen) have been described [97]. Osteoporosis is a reported feature among adults with MFS [94,98], and has been found in children with the disease as well [95]. Kohlmeier et al. [95] described 9 boys and girls (ages 9.9 to 17.5 years) with MFS who were tall for age but of normal weight. All were moderately active and had no history of atraumatic fractures. The BMD correlated with age, height and pubertal development as expected. Femoral neck aBMD was reduced, with a trend toward a decreased aBMD at the lumbar spine. These authors also found that 32 women with MFS had both axial and appendicular osteopenia, while Carter et al. noted only axial osteoporoSis among both sexes [94]. Tobias and colleagues [99] found that BMD was
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similar to the reference population at both the hip and the lumbar spine in post-menopausal women with MFS. The discrepancy among these findings in adults has been attributed to differences in the criteria for diagnosis of the syndrome. Criticisms of the 1986 Berlin nosology [100] include over-diagnosis of MFS among relatives. The 1996 revised nosology [101] now includes the potential for molecular studies in assisting the diagnosis and more stringent criteria for diagnosis in relatives of an affected individual. The subjects in the study by Carter et al. [94], then, may represent those with true MFS and as such, the assessment of the bone mineral status in these patients may be more accurate. The importance of exercise in the development of low bone mass in children with MFS has not been fully explored. Musculoskeletal pain and the recommendation to avoid contact sports because of risk of aortic rupture are likely to interfere with physical activity and thus reduce mechanical loading. These aspects have not been addressed formally, though Kohlmeier et al. did show that none of their subjects with MFS participated in vigorous or competitive sport [95]. 9At present, the fracture risk associated with a reduced BMD in MFS has not been determined. Fractures have been reported in 16/48 (33 percent) of adults with the syndrome [102]. However, the site and degree of trauma associated with these fractures was not indicated, and the fractures in these patients were attributed to the characteristic joint hypermobility of this disease. Kohlmeier et al. [95] noted that none of the adults sustained non-traumatic fractures, though 50 percent of premenopausal women and 12.5 percent of children sustained peripheral traumatic fractures. Further studies are required to determine the clinical significance of the reduced bone mass in children and adults with MFS. Homocyst&uria
Homocystinuria is an autosomal recessive connective tissue disease associated with mental delay, ectopia lentis, marfanoid habitus, osteoporosis and early onset thrombotic vascular disease. The occurrence of homocystine in the urine may result from a number of different genetic defects, the most common of which is a deficiency in cystathionine b-synthetase. This enzyme is important in the transsulfuration pathway, responsible for the conversion of methionine to cysteine and ultimately to sulfate. Homocysteine is an intermediate in the transsulfuration pathway, and defective cystathionine b-synthetase activity leads to an accumulation of methionine and homocysteine in plasma [103]. The multisystem toxicity of hyperhomocysteinemia is attributed to its spontaneous chemical reaction with many biologically important molecules, primarily proteins. Irreversible homocysteinylation of proteins leads
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to cumulative damage and progressive clinical manifestations. Krumdieck et al. [104] noted that the glycoprotein fibrillin-1 is particularly susceptible to homocysteine attack through homocysteinylation of the epidermal growth factor (EGF)-like domains of the molecule. Fibrillin-1, the main structural component of elastin microfibrils [105], is found in the medial layer of all elastic arteries, and in bone, cartilage, skin, and the suspensory ligament of the lens, all structures that are compromised in both MFS and homocystinuria. The recent mutations described in the fibrillin-1 gene giving rise to the MFS phenotype support the hypothesis that fibrillin-1 is an important target of homocysteinylation and that damage to its structure may be responsible for many of the abnormalities that severe hyperhomocysteinemia has in common with MFS, including osteoporosis. A number of skeletal manifestations have been demonstrated in homocystinuria, including scoliosis, arachnodactyly, enlarged carpal bones, pectus excavatum/ carinatum, limb deformity (bowing), humerus varus, joint contractures and pes planus/cavus. Osteoporosis was first noted on plain radiographs [103,106]. In a report of 26 patients, 25 were found to be osteoporotic on plain radiographs, and 19 showed vertebral compression fractures. Yap and Naughten [107] reported their 25 years of experience with 25 Irish homocystinuric patients (age 2.5 to 23 years) who were diagnosed through a national screening program. They found by radiological examination that osteoporosis was present in 33 percent of patients who were non-compliant with medical therapy. Osteoporosis was absent in all of the 18 compliant patients. A recent report of osteoporosis among 6 adult patients (ages 27 to 48 years) with a late diagnosis of homocystinuria showed significant reduction in BMD at the femoral neck [108], with even more profound reduction in the spine. These authors found that the osteoporosis was not more severe in the oldest patients compared to the younger ones. Taken together, these findings suggest that osteoporosis is a common feature of homocystinuria, that it is present at an early age, and that medical treatment may alter the severity of the disease. The treatment of patients with homocystinuria due to a defect in cystathionine b-synthetase includes vitamin B6, low methionine diet and recently, adjuvant therapy with betaine. This agent lowers plasma homocysteine through augmentation of homocysteine methylation to methionine [109]. At the present time, the clinical significance of the low bone mass and its relation to fractures risk in this disease are unknown.
Idiopathic Juvenile Osteoporosis Idiopathic juvenile osteoporosis (IJO) is a rare, selflimiting disorder first described in detail by Dent and
Friedman in 1965 [110]. Since then, only approximately 100 cases have been reported in the literature. The disease typically presents in previously healthy children during the 2-3 years prior to puberty [111,112], although the age at onset has been reported as early as 3 years [113]. There is no gender selection, and it does not appear to be heritable. Children report a gradual onset of pain in the back, hips, knees and feet, sometimes with difficulty walking [114]. Vertebral compression fractures are frequent and may significantly compromise the length of the upper body segment. Long bone fractures, usually of the metaphyses, may also be present. While the physical examination may be normal, kyphosis, scoliosis, pectus carinatum, long bone deformity and difficult ambulation may be evident. Radiological studies may show evidence of new, abnormal bone formed in the metaphyseal areas (neoosseous osteoporosis), appearing as a radiolucent, submetaphyseal band (Figure 2a and b). Long bones usually have normal length and cortical width, while wedgeshaped or biconcave vertebrae may be evident. Proszynska et al. showed that carboxyterminal pro-peptide of type I pro-collagen (PICP) levels were higher in patients with IJO compared to those with OI [115]. Other changes in biochemical markers of bone and mineral metabolism have been inconsistently reported in the literature [112,116], and to date, no specific laboratory hallmarks of the disorder have been identified. As such, IJO remains a diagnosis of exclusion, once other primary as well as secondary causes of osteoporosis have been considered. As previously discussed, differentiating IJO from a milder form of OI may be difficult. The discriminating features (which may or may not be present in any one patient) are presented in Table 3. The most remarkable feature of IJO is the spontaneous remission which occurs over 2 to 5 years [112], usually around the time of puberty. While this is the typical pattern, Smith et al. [112] reported that of 21 patients with the disease, three had persistent disability in adulthood. The underlying pathogenesis remains unclear. A recent histomorphometric study of iliac crest bone biopsies in nine patients with IJO showed a decreased cancellous bone volume and very low bone formation rates on cancellous surfaces. The results pointed to a disorder of osteoblast team performance, with a resulting defect in bone formation as evidenced by a 54 percent reduction in the activation frequency (the number of new osteoblast teams recruited per unit time) [68]. In a further study by Rauch et al. [117], the authors demonstrated through iliac crest histomorphometric studies that the disturbance in bone remodeling among children with IJO was limited to cancellous bone. In addition, the widths of the internal, but not external, cortices were decreased due to a decrease in the modeling
41 1
17. The Spectrum of Pediatric Osteoporosis
FIGURE 2a: Neo-osseous osteoporosis in a patient with IJO. b: Complete resolution of the sub-metaphyseal lesion nine months later, in the absence of specific medical therapy. TABLE 3
Idiopathic Juvenile O s t e o p o r o s i s (IJO) and O s t e o g e n e s i s Imperfecta (O!): Distinguishing Features
Feature
OI
IJO
Age at onset
Clinical features may manifest in utero, at birth, or during childhood/adolescence, depending upon disease severity
Clinical features usually manifest 2to 3 years prior to onset of puberty
Family history
Often positive
Negative
Duration
Lifelong
Usually remits after 2-5 years, during puberty
May improve but does not completely remit with puberty Skeletal manifestations
Frequent long bone fractures
Reduced upper:lower segment ratio
Metaphyseal fractures rare
Metaphyseal fractures common
Normal or short stature
Normal stature
Gracile bones
Neo-osseous osteoporosis*
Long bone deformity, thin cortices
Long bones straight, normal cortices
Wormian bones Thin ribs Hyperplastic callus formation (OI type V) Extra-skeletal manifestations
Blue sclerae
None
Dentinogenesis imperfecta Joint hyperlaxity Deafness, cardiac lesions Bone histolQgy
Genetic/molecular studies
Increased bone turnover
Decreased bone turnover
Lamellation normal in OI types I-IV
Lamellation normal
Abnormal lamellar pattern in OI types V,VI
Surface-specific remodeling and modeling abnormalities
Type I collagen abnormalities often detected in OI types I-IV, due to mutations in COL1A1/COL1A2
No known molecular defect
*considered pathognomonic of IJO (see Figure 2)
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activity on the endocortical surface of the external cortex. These authors concluded that the disturbed bone metabolism in IJO primarily affected bone surfaces that were in contact with bone marrow. These results suggest that the skeleton of IJO patients is unable to adapt to the increasing mechanical challenges that occur during growth and development. The association with the onset of puberty calls into question the role of sex hormones. However, no direct evidence for a hormonal effect has been found and further, very young patients with the disorder have also been reported [113]. Treatments for IJO are virtually impossible to assess because of the spontaneous improvement that usually occurs. In addition, the disease is often difficult to diagnose and very rare, making formal study of outcomes challenging. Saggese et al. [116] reported clinical improvement with calcitriol therapy, and Glorieux et al. [118] found an increase in trabecular bone volume and bone formation rate in patients treated with long-term sodium fluoride. Calcitonin therapy was attempted in one patient who had an elevated 1,25 dihydroxyvitamin D level, but this was not met with any clinical improvement [119]. It should be noted that some patients have demonstrated low circulating levels of 1,25 dihydroxyvitamin D [116]. Hoekman et al. [120] observed impressive results following two months of treatment with intravenous pamidronate in a 13 year old boy. A positive calcium balance quickly ensued after initiation of therapy and iliac crest bone biopsy taken at the beginning of treatment and then 2 months after therapy showed thin trabeculae pre-treatment with poor marrow cellularity followed by a marked increase in new osteoblasts after treatment. The role of spontaneous recovery in this patient's clinical improvement is of course unknown. Shaw et al. [121] found similar results, when 5 patients with pediatric osteoporosis, one of whom was diagnosed with IJO, were administered intravenous pamidronate. This patient (an 11 year old girl) presented with painful vertebral fractures, for which she received three treatments with intravenous pamidronate over one week's time. The onset of pain relief was also rapid, occurring within two weeks of the initial treatment. Until further studies and treatment guidelines are available for IJO, optimization of calcium and vitamin D intake is a reasonable recommendation. It is also advisable to protect the spine until recovery occurs, through avoidance of heavy back-packs and high risk physical activity. Physical activity without risk of trauma, however, should be encouraged through supervised physiotherapy programs. Finally, the use of bisphosphonates may be justified in select cases of IJO, when patients present with significant pain secondary to vertebral or limb fractures.
Secondary Osteoporosis Osteoporosis is considered "secondary" when it results from an underlying disorder or from the treatment of such a disorder. There is a growing list of secondary osteoporoses in children (see Table 1), due in part to improved long-term outcome for children because of advances in such areas as oncology, immunosuppressive therapy, rehabilitative care, and pediatric pharmacology. The identification of osteoporosis in children has also been furthered through systematic monitoring of skeletal health for those with known osteoporosis risk factors. The goal here is to provide an overview of the more commonly described causes of secondary osteoporosis in children.
Neuromuscular Disease
The influence of muscular strength on developing bone is well described [122], though to date this knowledge has received little attention in pediatric science and practice. As previously discussed in this chapter, an increase in muscle load applied to bone is one of two mechanical challenges (the other being bone growth) which brings about adaptational changes in bone mass and architecture. The ultimate goal of these adaptational responses is to fashion bones that are as strong as they need to be in order to withstand the mechanical challenges of growth and development that are exerted upon them. Contrary to popular belief, the ultimate goal of bone development is not to become as heavy as possible (not to accumulate bone mass). Rather, the aim of the developing skeleton is to become as strong and stable as necessary. The accumulation of bone mass is one of the means by which this goal is achieved. This concept, based on the mechanostat theory [52], is clearly demonstrated in children with neuromuscular disorders such as cerebral palsy, muscular dystrophy and any condition leading to chronic immobilization. With muscle disuse, either voluntarily through inactivity or involuntarily through neuromuscular disease/prolonged therapeutic recumbancy, there is a reduction in the mechanical challenges endured by bone, resulting in accelerated bone loss in adults, and failure to accrue bone mass in children. In this section the major causes of abnormal muscle force and its consequences will be presented. Cerebral Palsy
Cerebral palsy (CP) is defined as a nonprogressive disorder of posture, tone and/or movement that results from a static insult to the developing brain. The estimated prevalence is 2/1,000 [123]; thus, CP is a very common disorder. The precise etiology remains un-
17. The Spectrum of Pediatric Osteoporosis
known. It was once attributed to birth asphyxia, however, despite considerable advances in obstetric and neonatal care, the incidence remains unchanged. CP is associated with a number of clinical manifestations that include epilepsy, cognitive delay, and speech and sensory impairment. The disorder has been classified according to the predominant motor abnormality into a number of categories including spastic, dyskinetic, ataxic, hypotonic and mixed forms [123]. These features, combined with a description of the affected body distribution and functional status of the patient, provide a commonly used clinical classification for patients with CP. Spastic CP is the most common sub-type, with hemiand diplegia being frequent distributions. CP may give rise to a number of painful skeletal complications including scoliosis, joint subluxation and dislocation, and torsional bone deformities [124]. Fractures are also a problematic complication of the disease, occurring in 5-30 percent of CP children. Brunner et al. [125] surveyed 37 patients with CP who had sustained 54 fractures with minimal trauma and found that the majority were in the femoral shaft and supracondylar region. These authors proposed that long, fragile arms and stiffness due to contractures in the major joints, especially knees and hips, were the major contributors to the increased fracture rates in this disorder. In association with minimal trauma fractures, low bone mass is a characteristic feature of CP, as documented by a number of groups [126-129]. The pathogenesis of osteoporosis in CP is attributed in large part to muscle disuse from reduced mobilization and thus diminished muscle load upon the developing skeleton [130]. Using the mechanostat model, one might hypothesize that with a reduction in this critical mechanical challenge (muscle load), the skeletal response is to reduce bone mass accretion compared to motorically normal peers. As such, it is not the low bone mass per se, that is responsible for the fractures, but rather the abnormal relationship between mechanical forces (which are usually reduced but may be intermittently high in CP) and bone mass. For example, if a CP patient with low bone mass experiences ongoing (albeit reduced) mechanical challenges that are within the bone's capability to withstand, the patient is unlikely to fracture. On the other hand, if there are intermittent mechanical challenges that exceed the bone's ability to withstand them, then the patient's bones are likely to break. The intermittent mechanical challenges in CP that override the bone's ability to cope might occur, for example, with forceful muscle contractions during seizures and with occasional weight bearing/transfers. The degree of preserved ambulation (and thus muscle use) has been shown to correlate positively with bone mass in CP [126,128]. Similarly, Lin et al. [131] showed that the BMC in the affected limb of
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children with spastic hemiplegic CP was significantly reduced compared to the healthy limb. For the upper extremities, the reduction was 26.5 percent in the affected limb while in the lower extremities, BMC was reduced by 15.6 percent compared to the uninvolved limb. Lean muscle mass was reduced by 15 percent in the hemiparetic limbs. Chad et al. [132] found similar results in nutritionally adequate patients with CP. Bone mineral content, BMD and bone-mineral-free lean tissue were consistently lower in the non-independent ambulators compared to independent ambulators. In keeping with these studies is the intervention trial by Chad et al. [130] who proposed a programme of weight-bearing for children with CP. These authors noted that after 8 months of such activity, the femoral neck BMC and vBMD increased significantly compared to a control group. While ambulatory status appears to play the most significant role in determining CP patients' risk for osteoporosis, other factors are contributing determinants. Henderson et al. [133] found that after mobilization, nutritional status as determined by caloric intake, skin fold thickness and body-mass index was the second most important variable. Reduced calcium intake was an additional, though less significant, adverse factor. Serum vitamin D levels did not correlate with BMD results, however. In another report, Henderson et al. [134] examined Vitamin D levels in 125 noninstitutionalized children with various forms of CP and found that 25Hydroxyvitamin D (calcidiol) levels were significantly reduced compared to normal pediatric subjects. In contrast, 1,25 dihydroxyvitamin D (calcitriol) levels were normal in all but 2 percent of CP patients, and comparable to their healthy counterparts. For institutionalized children with CP, Bischof et al. [135] found that patients with long-bone fractures had more severe biochemical and radiographic evidence of rickets compared to CP children without fractures. The fractures were thus attributed to Vitamin D deficiency, possibly secondary to anticonvulsant use, thus compounding a lack of sunlight. The influence of anti-convulsant therapy on bone development has been the source of much debate in a variety of clinical conditions, including CP. Some investigators have provided evidence for an association between anticonvulsants and abnormal vitamin D metabolism [135,136], while others have not [134,137]. Recently, Rieger-Wettengl et al. [138] evaluated 39 children with isolated epilepsy receiving either carbamazepine or valproic acid using pQCT at the distal radius. These authors found that calcium and 25-hydroxyvitamin D levels were similar to controls. Trabecular vBMD was decreased in the patients receiving anti-convulsant medication while bone mass and grip strength were normal for age. Bone turnover, as assessed by deoxypyridinoline, was elevated. These authors concluded that a normal BMC despite
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reduced trabecular vBMD was due to a compensatory increase in cortical BMC. In the past, increases in bone turnover in this setting have been attributed to vitamin D deficiency and resulting osteomalacia [136]. However, markers of bone turnover remained elevated when Vitamin D supplementation was given to carbamazepinetreated patients [139], and it has since been suggested that the increased bone turnover is due to a direct effect of anti-convulsants on bone cells [140]. Similar studies have not been performed in children with CP; however, the study by Rieger-Wettengl et al. [138] calls into question the assumption that anti-convulsant therapy interferes with the absolute amount of bone mass that is accrued and with mineral metabolism. This issue requires attention in future studies. The diagnosis of osteoporosis in CP frequently comes to the clinician's attention after fractures have been sustained, or upon referral from an orthopedic surgeon. Quantification of osteoporosis in this population is hampered by the usual problem of bone density interpretation with DXA due to variation in bone size. DXA is also made difficult by the fact that some children with CP are unable to position properly for the measurement. Since CP patients often prefer lying on their side, Harcke et al. [141] tested the feasibility and accuracy of measuring BMD at four distal femur sub-regions in the lateral projection, with favourable results. Recurrent, minimal trauma fractures strongly suggest the diagnosis of osteoporosis, even in the absence of BMD measurements. Ancillary data should include biochemical measurements of bone and mineral markers, and the evaluation of the patient's calcium and vitamin D status. If the biochemistry is suggestive, plain x-rays should be obtained to ensure a diagnosis of rickets is not overlooked. The treatment of osteoporosis in CP has been ventured by few investigators. Jekovec-Vrhovsek et al. [142] treated 13 children with severe spastic quadriplegic CP receiving anti-convulsants with calcitriol 0.25 micrograms ~and calcium 500 mg/day for 9 months. BMD was measured by DXA and showed significant increases from baseline values compared to untreated controls with similar degrees of handicap. Shaw et al. [127] assessed the bone status of 9 non-ambulant children with CP, and found profound reductions in BMD despite adjustments for body weight. Vitamin D and parathyroid hormone levels showed no consistent abnormality while three patients had significant hypercalciuria. Three children with recurrent fractures received intravenous pamidronate for 12 to 18 months and demonstrated increases in bone density ranging from 20 to 40 percent. The drug was well tolerated in these three CP patients. Intravenous pamidronate was also used in a double-blind, placebo-controlled study of six pairs of non-ambulatory children with CP [143]. One member
of each pair randomly received intravenous saline placebo while the other received pamidronate. In the metaphyseal region of the distal femur, aBMD increased 89 percent + 21 (mean + SEM) at 18 months in the pamidronate group compared to 9 percent + 6 in the control group. Age-matched aBMD Z scores increased from -4.0 + 0.6 to -1.8 + 1.0 in the pamidronate group while there was no significant difference from baseline in the untreated group. Chad et al. [130] offered a nonpharmaceutical approach by prescribing a weight bearing physical activity programme to patients with CP and found significant increments in femoral neck BMC and vBMD, and in total proximal femur BMC compared to controls, after an 8 month trial. The skeletal health of children with CP represents an important area of future research given the burden of frequent fractures to patients and their families. The current literature suggests that a multi-disciplinary approach to prevention and treatment which includes optimization of nutrition, weight-bearing physical activity and medical intervention with anti-osteoporotic agents may lead to significant improvements in the lifestyle of patients with CP. Duchenne Muscular D y s t r o p h y
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder due to mutations in the dystrophin gene. The most distinctive feature of DMD is a progressive proximal muscular dystrophy with characteristic pseudohypertrophy of the calves. There is massive elevation of creatine kinase levels in the blood and myofiber degeneration with fibrosis and fatty infiltration on muscle biopsy. The onset of DMD is usually before 3 years of age, and the patient is chair-ridden by the early teens. Unfortunately, while DMD is being diagnosed earlier than in previous years, survival has not changed significantly and most patients with DMD still die in early adulthood [144]. The progressive loss in muscle function, particularly in the lower extremities, is associated with frequent fractures. Larson et al. [145] performed a longitudinal study of 41 boys with DMD and evaluated their bone density at the lumbar spine and proximal femur while ambulatory, and again when they were no longer walking. During the ambulatory phase, the aBMD at the lumbar spine was only slightly decreased (mean Z score -0.8). With loss of ambulation, the BMD fell significantly (mean Z score -1.7). In contrast, aBMD at the proximal femur was profoundly reduced even when gait was minimally affected (mean Z score -1.6) and then progressively fell to almost 4 SDS below the mean, compared to agematched controls (mean Z score -3.9). Though bone size was not taken into account, aBMD correlated with the site of pathologic fractures, suggesting the reductions
17. The Spectrum of Pediatric Osteoporosis
in BMD were associated with important clinical consequences. Eighteen of the boys (44 percent) sustained at least one fracture, and 66 percent of these involved the lower extremities. There were no compression fractures at the lumbar spine. Four of nine boys who were walking with aids/support at the time of fracture did not resume walking after the incident. Thus, the site of the most profound reduction in bone mass (the proximal femur) correlated with the site of greatest muscle weakness. Furthermore, frequent fractures resulted in premature loss of ambulation, raising the issue that fracture prevention in this group of patients is an important health issue despite the reduced longevity. A number of therapies have been studied in order to preserve muscle function in DMD, including prednisone [146], prednisolone [147], azathioprine [148], oxandrolone [149] and deflazacort [150]. With the exception of azathioprine, these therapies have proven to slow muscle deterioration and lengthen the ambulatory period to variable extents. The use of steroids has been limited in some patients because of weight gain, cataracts, impaired growth and behavioural changes; however, these effects have usually been milder with deflazacort [150]. Shortterm studies of deflazacort suggested that this agent may have a bone sparing effect in children [151,152]. However, Chabot et al. [153] reported on 46 boys with DMD who received deflazacort over a four year period, and found that 26/46 (52 percent) of the boys suffered 37 fracture events. Of the 37 fractures, 39 percent were vertebral compression fractures while the remainder were fractures of the long bones. Significant decrements in bone mass occurred over the study period. These results contrasted those of Larson et al. [145] who found an absence of vertebral fractures in DMD patients who did not receive steroids, and suggest that the osteotoxic effect of deflazacort appears to exacerbate the underlying predisposition for osteoporosis in boys with DMD and has a predilection for the spine. Whether antiosteoporotic agents such as bisphosphonates can prevent osteoporosis in patients with DMD has yet to be explored in the medical literature. Immobilization and L i m b Disuse
The immobilization and disuse osteoporosis provides researchers with a unique opportunity to study the skeleton's adaptation to reduced muscle load. A number of clinical conditions characterized by immobilization, ranging from complete motor paralysis to temporary therapeutic recumbancy, are associated with loss in muscle and bone mass. The results from studies of immobilization and limb disuse are remarkably concordant despite different methodologies and clearly demonstrate the importance of weight-bearing and muscular activity in regulating the skeleton's adaptational responses.
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The effect of voluntary bed rest for three months on healthy subjects was evaluated in a controlled study by Zerwekh et al. [154]. There was a rapid and sustained increase in bone resorption and a more subtle decrease in bone formation as reflected in bone histological and biochemical studies. Dramatic changes in muscle mass and decrements in bone mineral density have been observed following one month of bed rest [155]. Complete paralysis due to spinal cord injury represents the most extreme clinical case of immobilization, since neither muscle activity nor weight-bearing are present in body regions below the level of the lesion. Studies in older adolescents and adults have shown that most of the decrements in bone mass following spinal cord injury and paralysis occur within the first year after injury [156,157]. Bone loss begins immediately following injury with a mean rate of loss in the hip that is greater than 2 percent per month for the first half of the year, then 1 percent per month for the remainder of the year [158]. This significant rate of loss resolves during the third or fourth year post-injury, and there is little evidence of measurable loss afterward [159]. As predicted, lesser degrees of loss occur with incomplete paralysis, due to residual muscle function. With lower extremity paralysis, lumbar spine bone mass is not different from ambulatory controls, due to the muscle forces exerted on the spine during maintenance of an erect posture [159]. Pathologic fractures may be frequent in spinal cord injured patients [158], and most often occur at the distal femur, in the supracondylar region. Other conditions involving immobilization or limb disuse include congenital neuromuscular disorders as previously discussed, chronic diseases requiting prolonged hospitalizations, amputation (with subsequent immobilization of adjacent structures) and frozen shoulder. As expected, with regional disuse there are regional reductions in bone mass. For example, following above the knee amputation, a 28 percent reduction in femoral neck bone mineral has been documented [160]. Lumbar spine bone mineral loss may occur at a rate of 2 percent per week following scoliosis surgery plus 3-6 weeks of recumbancy [161], and a 50 percent reduction in bone mineral in the "frozen shoulder" has been documented compared to the contralateral limb [162]. While the data are somewhat inconsistent, studies suggest that complete restitution of bone mass following temporary immobilization may not always be possible despite resumption of normal physical activity [163]. This may be due in part to failure to participate in enough physical activity for adequate stimulation of mineral accretion. A number of therapies have been attempted to restore bone mass following immobilization and disuse injuries, including weight-bearing, electrical stimulation of muscle activity, and pharmacologic
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intervention (calcitonin, bisphosphonates) [158]. Passive loading through sitting or standing in a standing frame has shown no effect on bone mass in adult males with permanent spinal cord injuries [164]. Electrical stimulation of muscles to elicit active contractions has shown improvement in parameters of muscle function, but no study has been able to show a positive effect on bone mass [158]. Calcitonin monotherapy had a transient effect on hypercalcemia in acute spinal cord injury [165], while the use of bisphosphonates (clodronate and tiludronate) following spinal cord injury has shown promise when administered shortly after injury, during the phase of most rapid bone mineral loss [166,167]. The positive effect of muscle force on bone health has been illustrated by Montpetit, et al. [168] who showed that following treatment with pamidronate, pediatric patients with severe OI had a significant increase in muscle force as measured by grip strength. These authors hypothesized that the dramatic improvement in BMD with pamidronate was in part due to a direct drug effect on bone and in part due to the improved muscle load to bone that resulted from chronic pain relief and enhanced physical activity. As in severe OI, the duration of bisphosphonate therapy that is required for sustained improvement in the context of the immobilization osteopenias needs further study. Chronic Illness
With improved long-term outcome for children because of advances in diagnostic techniques and in pharmacologic/rehabilitative care, there has been increasing attention to the sequelae of chronic pediatric disease. A number of chronic disorders have been associated with bone morbidity in children, including leukemia and other cancers, rheumatologic disorders, inflammatory bowel disease and cystic fibrosis. Poor nutrition may play a central role in the abnormal skeletal status, as in the case of anorexia nervosa, or it may have a secondary, but nonetheless important, role in the pathogenesis. The more frequently reported chronic illnesses associated with adverse effects on skeletal health in youth are discussed in this section. Leukemia
Bone morbidity associated with acute lymphoblastic leukemia (ALL) and other childhood cancers has been a focus of attention in recent years [169]. ALL is the most common pediatric malignancy, with an overall survival rate now exceeding 70 percent [170]. As such, there is an increasing population of survivors who are at risk for long-term sequelae of childhood ALL. Musculoskeletal pain and gait abnormalities have been reported in one third of children with ALL at diagnosis, a sub-set of whom also demonstrate fractures [171]. Bone mass is usually within the normal range
compared to age-matched, healthy peers at the outset of the illness. Radiographs of painful regions show metaphyseal lucencies, sclerotic lesions and sites of periosteal reaction in many of the patients with bone pain at presentation. A number of mechanisms have been proposed for the skeletal morbidity in ALL observed at diagnosis, including infiltration of bone by leukemic cells, paraneoplastic factors, and disordered mineral metabolism [171]. Several groups have reported loss of bone mass during therapy for ALL [171-173]. The greatesl~ reductions in bone mass occur during the first 6 months of therapy, consistent with the effect of glucocorticoids on bone metabolism [174,175]. Compared to findings at diagnosis, a significant rise in fracture rates and gait abnormalities has been observed throughout therapy, with fractures occurring in over one third of patients [171]. Halton et al. [171] observed more frequent fractures in the lower extremities compared to the spine and upper extremity, and van der Sluis [175] found a fracture rate that was 6 times higher in ALL patients compared to healthy controls. Pubertal patients may be more susceptible to skeletal insult during treatment than younger children [171], and the magnitude of the bone mass decrement during therapy appears to be a stronger determinant of fracture risk than the absolute standard deviation score (SDS) value [175]. The adverse skeletal effects observed during treatment for ALL have been attributed to corticosteroids, methotrexate, cranial irradiation, poor nutrition, impaired mobility, and disordered mineral metabolism [171,175-177]. van der Sluis et al. [175] found reductions in biochemical markers of bone formation at diagnosis in pediatric ALL patients, while markers of bone resorption were normal at diagnosis but increased during treatment. The long-term effect of skeletal morbidity in ALL has been the subject of recent studies, as investigators have questioned whether the bone mass decrements and abnormal mineral metabolism at diagnosis and during treatment are sustained in later years [178]. Furthermore, these earlier studies of bone morbidity in ALL often included patients who had received cranial irradiation, which has been shown to be a risk factor for reduced bone mass [176]. Cranial irradiation is now used far less frequently as part of current ALL protocols. KadanLottick et al. [178] studied 75 survivors (11 to 82 months post-diagnosis, mean age at diagnosis 6.8 yrs) who were diagnosed with ALL between 1991 and 1997. Overall, the mean whole body aBMD Z score was normal (+0.22 + .96), and a significant positive correlation was found between whole body aBMD and years elapsed since the start of maintenance therapy, when adjustment for risk status/age category, history of cranial irradiation and total days hospitalized was carried out. Patients receiving maintenance therapy did have reduced bone
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mass, with an increased incidence of fractures. This study is in agreement with the report by van Der Sluis et al. [179] who showed that survivors of ALL who had received high dose methotrexate and dexamethasone but no cranial irradiation had normal bone mass measurements (total body and lumbar spine) at a mean of 10 years post-treatment. Therefore, based on current knowledge, it appears that the significant bone morbidity observed at diagnosis and during therapy for ALL may not be associated with long-term effects when cranial irradiation and prolonged hospitalizations are avoided. Survivors of ALL are at risk for a number of sequelae that may interfere with their quality of life, including hypothyroidism, hypogonadism and growth hormone insufficiency. To foster skeletal health during and following ALL treatment, surveillance for endocrinopathies should be undertaken and appropriate therapy instituted if deficiencies are documented. It is prudent to encourage weight-bearing physical activity and adequate nutrition, including calcium and vitamin D supplementation, once the diagnosis is made. More aggressive medical therapy with anti-osteoporotic agents such as bisphosphonates to prevent decrements in bone mass or fractures during therapy has not been systematically studied, though pamidronate has been used with success to treat leukemia-associated hypercalcemia [180,181]. Rheumatologic Disorders
Inflammatory diseases of childhood, including juvenile rheumatoid arthritis (JRA), systemic lupus erythematosis, and dermatomyositis, are well-known to be associated with compromised skeletal health. Atraumatic fractures may occur at an early age [182-184], and reductions in bone mass have been documented in a number of studies [182,185-189], though BMD measurements were adjusted for bone size in only one of them [182]. Of the pediatric rheumatologic conditions, bone morbidity has been most extensively studied in JRA. There are a number of factors that may adversely affect bone mass in pediatric JRA patients. Active arthritis may reduce bone mineral accrual around affected joints (periarticular osteopenia), and in skeletal sites far from the diseased joint as well. Reed et al. [189] measured cortical bone density by single photon absorptiometry in a group of 27 patients with JRA, with repeat measurements performed 36 months later. Only those patients whose disease became inactive over the study period had significant increases in BMD, and 89 percent of patients had a radial BMD more than 2 SD below the expected mean (without correction for bone size). In support of the hypothesis that disease activity impairs normal bone metabolism, negative correlations between bone mass and clinical measures of disease activity have been documented by others as well [190,191].
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In addition to inflammatory disease activity, a number of contributing factors may play a role in the bone morbidity observed in rheumatologic disorders, the most important of which is glucocorticoid use [192]. Glucocorticoids have a potent effect on skeletal metabolism, as discussed in section 4.2.4.a. Methotrexate [193,194] and cyclosporine [195] have also been associated with osteoporosis, though the precise osteotoxic mechanism is unclear. Weight-bearing activity is a strong stimulus for mineral accrual and microarchitectural adaptation, both of which enhance bone strength. Mobility may be compromised in pediatric rheumatologic conditions, depending upon disease severity. There are few studies that specifically address treatment of osteoporosis in juvenile rheumatologic disease. Calcium, vitamin D and 25-hydroxyvitamin D have been studied in pediatric patients with various rheumatologic conditions, without convincing results [196,197]. Shaw et al. [121] administered cyclical intravenous pamidronate to five pediatric patients with osteoporosis, one of whom had had JRA. Treatment with an anti-resorptive agent was initiated in this case because of severe back pain secondary to vertebral compression fractures. Pain relief was effected within one week of pamidronate therapy, and the lumbar aBMDZ score increased from -3.5 to -3.0 SDS within one year. Bianchi et al. [198] performed a study of daily alendronate (5 or 10mg) in 38 children with a variety of rheumatologic conditions compared to 38 untreated controls with the same disorders. BMD increased by a mean of 14 + 9.8 percent in the alendronate-treated group compared to 2.6 + 5 percent in the control patients. Low trauma fractures were documented in 20 percent of the patients prior to alendronate therapy, while no new fractures were sustained during the 12 month treatment period. As for other chronic diseases associated with osteoporosis, the therapeutic plan for children with rheumatologic conditions should identify and treat all risk factors for reduced bone mass, including disease activity, poor nutrition, impaired physical activity and delayed puberty. If corticosteroids are necessary, the minimum effective dose should be used and steroids should be discontinued as soon as possible, according to disease activity. For patients receiving medications with potent skeletal toxicity, such as glucocorticoids, it appears that anti-osteoporotic agents may be needed in order to prevent clinically significant decrements in bone mass and atraumatic fractures. Randomized, placebo-controlled trials of such agents in pediatric rheumatologic conditions are now needed. Anorexia Nervosa
Among teenage girls, anorexia nervosa (AN) is an increasingly common disorder with an estimated
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prevalence of 0.2 to 1.0 percent in Western countries [199,200]. AN is a psychiatric condition characterized by self-inflicted food restriction resulting in malnutrition and primary or secondary amenorrhea. Excessive physical activity in the face of apparent inanition is also characteristic of the disease. Reduced bone mass is a frequent complication of AN among female youth [201-203] and may be severe enough to result in fragility fractures and an increased fracture risk throughout life [204]. Bone density is reduced by more than 2.5 SD at the hip or the spine in 38 percent of women with AN and by more than 1.0 SD in 92 percent of AN patients [204]. Significant reductions in bone mass have been observed less than one year following diagnosis among teenage girls [201], highlighting the vulnerability of the peri-pubertal skeleton to the effects of malnutrition. That skeletal health is compromised in the majority of patients with AN is not surprising, given the timing of disease onset and the multitude of risk factors for osteoporosis in this condition. Up to 25 percent of girls with AN develop the disease before 13 years of age, with the peak incidence of disease onset occurring during midadolescence, prior to the attainment of peak bone mass. Caloric intake is severely restricted and thus dietary intake of calcium, vitamin D and protein is inadequate to meet the needs of the developing teenager. Hypogonadotrophic hypogonadism with estrogen and testosterone deficiency [205] are cardinal features of the disease resulting from starvation-induced hypothalamic dysfunction. Hypogonadism is not the only nutritionallymediated abnormality affecting bone trophic hormones, as hypercortisolemia [206] and reductions in circulating insulin-like growth factor-1 (IGF-1) [205] have also been documented. Low levels of dehydroepiandrostenedione (DHEA) or its sulfate have been reported in some [207-209] but not all [205] studies. Recently, Soyka et al. [205] studied 19 adolescent girls with AN compared to the same number of bone age-matched female controls in order to elucidate the potential mechanisms of osteoporosis in this condition. The authors found that lumbar anterioposterior BMD was more than 1 SD below the mean in 42 percent of patients and that a similar reduction in lateral spine BMD was present in 63 percent of patients, compared to healthy controls. The duration of the AN was the most significant predictor of spinal BMD. Estrogen and free testosterone levels were significantly decreased in the AN group. Similarly, bone formation markers, including IGF-1, were significantly reduced in AN while measures of bone resorption were comparable between the two groups. The primary correlate of bone formation in this study of adolescent AN was IGF-1. These authors suggested that reductions in circulating IGF-1 as well as gonadal steroid deficiencies
were critical factors in the development of bone morbidity in adolescents with AN. The first principle of osteoporosis treatment for patients with AN is restoration of a healthy weight. Increases in BMD associated with weight gain have been documented in a number of studies using both DXA [202,204,210,211] and QCT [212] techniques. However, not all studies have found improvement in BMD with increased weight [213,214]. Furthermore, studies of the potential for recovery following improved nutritional status and eumenorrhea have failed to demonstrate complete restitution of bone mass [204,211,214]. Thus, it appears that restoration of the normal nutritional and hormonal milieu may not be sufficient to overcome the deleterious effects of prolonged anorexia. The role of other therapeutic modalities to minimize or treat AN-associated bone morbidity has been the source of ongoing study and debate. Calcium and vitamin D intake does not appear to correlate with BMD nor prevent reductions in bone mass [201,213-216]. Bed rest is likely to lead to accelerated bone loss while excessive physical activity increases the risk of atraumatic fractures, poor weight gain and prolonged amenorrhea. Moderate physical activity may have a protective effect against reductions in bone mass [215], though this has not been consistently documented [201,214]. Even when these general measures to protect against reductions in bone mass are carried out, compromised skeletal health is likely to occur in AN patients. As such, a number of specific pharmacologic interventions have been explored. Patients with AN may have profound estrogen deficiency, yet estrogen replacement therapy has yielded conflicting results [211,215,217,218]. In a double-blinded, randomized controlled trial, Klibanski et al. [212] reported that spinal bone mass (measured by QCT) was no different in estrogen-treated subjects after 1.5 years of therapy compared to untreated controls. Estrogen therapy provided protection against further bone loss only for patients who were less than 70 percent of their ideal weight. On the other hand, Seeman et al. [215], in a retrospective cross-sectional study, found that patients receiving oral contraceptives had higher spinal aBMD than nonusers, although the BMD remained significantly reduced for age in both groups. In a large, cross-sectional study, Karlsson et al. [211] found that a substantial proportion of the deficit in bone mass in AN patients was due to smaller bone size. These authors also reported that estrogen replacement was associated with increased (though not normal) vBMD and bone size relative to untreated controls. These authors further noted that recovery from the illness was associated with the best outcome for vBMD and bone size. Therefore, while estrogen therapy may be of benefit to bone health in the severely malnourished patient, its use does not
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replace nutritional intervention. Furthermore, estrogen is ideally avoided in patients who have not yet reached final height, as estrogen therapy will hasten epiphyseal closure. Recent studies suggest that more aggressive medical therapy may be needed to treat severely malnourished patients with significant reductions in bone mass. In a prospective trial of nine months' duration, Grinspoon et al. [216] studied the effects of an anabolic (recombinant human IGF-1, rhIGF-1) and an anti-resorptive (the oral contraceptive pill, OCP) agent in severely malnourished AN patients. Patients were randomly assigned to one of four treatment groups and received either physiologic doses of rhIGF-1 (sub-cutaneously, twice daily), the OCP, these agents in combination, or rhIGF1 placebo without the OCP (Figure 4). A significant, modest increase in spinal aBMD was observed following rhIGF-1 therapy compared to placebo. In contrast, no effect of OCP on bone density was observed at any site. The greatest aBMD response was seen in the combined treatment group (rhIGF-1 and OCP) compared to untreated controls. RhIGF-1 therapy was associated with an increase in lean body mass, but not weight, and correlated with the change in spinal aBMD. These results suggested that the effects of rhIGF- 1 on bone density may be due to anabolic effects on body composition, and that an anabolic agent in conjunction with an-antiresorptive drug such as the OCP may confer the greatest benefit to severely malnourished patients. In a randomized trial, the effects of the anabolic agent, DHEA, compared to conventional hormone replacement therapy (ethinyl
FIGURE 4 Effectof recombinant human IGF-1 and oral contraceptive administration on bone density in females aged 18-38 years with anorexia nervosa. Percent change from baseline in AP spinal bone density. , p < 0.05 vs. control subjects. Results are mean + SEM. Reprinted with permission from Grinspoon et al. [216]. 9 The Endocrine Society.
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estradiol plus levonorgestrel) were recently assessed by Gordon et al. [219]. During the one year of treatment, hip and spinal aBMD were maintained to a similar extent, but there was no significant increase after accounting for weight gain. It remains to be determined whether potent anti-resorptive agents such as bisphosphonates, either alone or in combination with anabolic medications, will play a role in the therapeutic approach to patients with AN-associated bone morbidity.
Endocrine and Reproductive Disorders Disorders o f Puberty
Sex steroids are necessary for the growth spurt at puberty, completion of epiphyseal maturation and bone mineral accrual [24,25,220]. The effect of gonadal hormones on the developing skeleton is reflected in the fact that one third to one half of the adult bone mass is accrued in the pubertal years. During puberty, the marked increase in height and bone mass are temporally dissociated, with mineral accrual following increases in longitudinal growth [221]. Fournier et al. [221] found the greatest disparity in height and BMD gains during midpuberty, between 13 and 14 years of age for boys, and between 11 and 12 years of age for girls. These observations are consistent with the increased fracture rate that occurs around the same time [222]. These findings are also in line with the mechanostat theory, since the skeletal response cannot precede, but rather must follow, the mechanical challenge of growth. The lag between longitudinal growth and enhanced bone strength is thus exaggerated when longitudinal growth accelerates [223]. Because the timing of maximal increases in bone length and muscle mass vary at different musculoskeletal sites, it is not surprising that the increases in bone mass that occur at the time of puberty follow a region-specific pattern [24,25]. The mechanostat model also helps to understand the sexual dimorphism in skeletal development that occurs during the pubertal years. Periosteal apposition increases bone width in boys and girls during pre-pubertal growth. Estrogen exposure serves to lower the mechanostat setpoint at endosteal bone surfaces [224]. As such, when estrogen levels rise in puberty, these surfaces are resensitized to mechanical strain, leading to endocortical apposition at many skeletal sites and excess bone mineral accrual relative to muscle strength. Consequently, in the post-pubertal, pre-menopausal phase, girls and women have more bone relative to their mechanical needs than males [225,226]. It is hypothesized that this estrogen-dependent bone reservoir can be tapped during pregnancy and lactation, when the demands of the fetus and infant take their toll on the female skeleton [227]. In
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boys, puberty is associated with rising levels of testosterone, which increase muscle mass and force, stimulating adaptational responses that lead to increased bone crosssectional size. This occurs through accelerated periosteal apposition with less endocortical expansion, resulting in enlargement of the bone diameter, cortical thickening, and an increase in the medullary diameter [228]. These surface-specific changes place the cortical bone mass further from the neutral axis of the long bone in men than women, which confers more strength and stability than when bone is deposited by endocortical apposition near the neutral axis of long bones [229]. While early puberty hastens mineral accrual and bone growth, and a later puberty retards this process, whether early or late exposure to sex hormones ultimately influences peak bone mass remains controversial. In a retrospective study, Finkelstein et al. [230] reported significant reductions in aBMD at the spine and radius in 23 men with constitutional delay of puberty (CDP), evaluated at a mean age of 26 + 2 years, compared to men with a normally timed puberty. This deficit could not be attributed to delayed mineral accrual, since subjects were compared to younger controls with similar durations of sex steroid exposure. Furthermore, two years later, 18 of these men were re-evaluated and there were no further gains in bone mass [231]. In contrast, Bertelloni et al. [232] found no difference in estimated vBMD in men with a history of delayed puberty compared to controls with normal pubertal development. Upon recalculation of BMD data to evaluate the differences in estimated vBMD compared to controls, Finkelstein et al. found persistently low values at the radius and spine [233]. These authors proposed that the mean age of the controls in the study by Bertelloni et al. (19.3 + 1.3 years) may have contributed to the disparity in results, since these controls may not yet have reached their peak bone mass. Although short-term gains in radial aBMD have been found with exogenous testosterone in males with CDP, additional evidence suggests that such therapy improves neither final height [232] nor peak bone mass [232,234] significantly, compared to subjects without pubertal delay. In the absence of data to the contrary, it appears that the decision to treat CDP with testosterone rests upon patient preference, as skeletal health seems neither to be enhanced nor adversely affected by such intervention. The effect of CDP on bone mineral accrual in otherwise normal, healthy females has not been a focus of study. This likely reflects the fact that girls with CDP come to medical attention less frequently, and are not advised to receive exogenous estrogen, given the concern that hormonal therapy will accelerate epiphyseal fusion and thus compromise final height. In addition, there is less patient demand to treat girls, since the stigmata of
short stature and delayed puberty appears generally to be less profound in girls with CDP than in boys. Most studies of delayed puberty and skeletal health in girls have evaluated athletic amenorrhea or eating disorders, where additional risk factors for osteoporosis are present. It appears that women with later menarche have a slightly larger bone marrow cavity at the distal radius compared to those with an earlier menarche [235], and an inverse relationship between age at menarche and peak bone mass has also been observed [236]. The influence of these observations on fracture risk is unknown. Studies of hormonal intervention for hypogonadotropic hypogonadism in women have demonstrated only marginal benefit to bone mass, and benefit to the fracture threshold is undetermined [237]. Patients with hyperandrogenic amenorrhea appear to be spared from decrements in bone mass [238], possibly due to a protective effect of the hyperandrogenemia. Precocious puberty, most commonly diagnosed in otherwise healthy girls, results in accelerated growth, mineral accrual and skeletal maturation. Compared to age- and sex-matched peers, children with precocious puberty demonstrate increases in aBMD at the spine [239] and femoral neck [239,240], while whole body BMD has been normal [239]. Increased lumbar aBMD was attributed to advanced skeletal maturation and increased bone size. BMD corrected for skeletal age is normal [240] or reduced [239] and estimation of vBMD at the lumbar spine is normal for chronological age [239]. Gonadotropin-releasing hormones (GnRH) analogues are used to arrest further pubertal development and thus delay epiphyseal fusion in children with central precocious puberty. Longitudinal follow-up to nearfinal [175] and final [241] height in males and females [242] with precocious puberty treated with GnRH analogues have shown preservation of genetic height potential and peak bone mass. Furthermore, when children with precocious puberty do not receive a GnRH analogue, as for patients with the slowly progressing variant, peak bone mass does not appear to be adversely affected. Turner S y n d r o m e
The skeletal status of girls and women with Turner Syndrome (TS) has long been a subject of scrutiny and debate. Turner syndrome is associated with multiple skeletal abnormalities including short stature, Madelung's deformity, cubitus valgus, scoliosis and foreshortening of the fourth metacarpal. In addition, retrospective studies have documented an increased fracture risk in women and children with TS [243-246]. The etiology of this increased fracture incidence has been unclear, as attempts to quantify bone mass have led to conflicting results because of different methodologies and the need to consider the reduced bone size and delayed skeletal
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maturation that is typical of TS. Investigators have sought to account for the reduction in skeletal size by comparing TS patients with size- and weight-matched healthy controls [243,247] or by using mathematical estimates of vBMD [248], with normalization of bone mass or BMD values. However, Bechtold et al. [249] recently questioned whether these results were valid, because the assumption of normal bone shape (inherent in the estimation of vBMD) might be incorrect in TS. These authors evaluated 21 older teenage and young adult TS women (all of whom had received GH) by pQCT at the forearm (Figure 3) and found bone shape was abnormal, with a markedly decreased radial length, but the total cross-sectional area (CSA) at the distal radius was normal. Bone mass and density were reduced, due to a reduction in cortical width. Muscular CSA was normal. The relationship between muscle CSA and bone size was similar to healthy women, but TS women had less BMC and cortical CSA relative to muscle CSA compared to healthy post-pubertal patients. In fact, the muscle-bone relationship in TS patients was similar to healthy prepubertal girls, despite estrogen supplementation (started at a mean age of 13.7 + 1.2yr). These findings were consistent with a normal adaptation of external bone (ie. bone size) to muscle force, and a lack of adequate estrogen effect on the endocortical bone surface. The authors concluded that reduced bone strength and relatively high body weight (+0.8 SD) in TS patients might contribute to the increased fracture incidence. These observations are in line with a recent report by Carrascosa et al. [250] who noted bone mass was normal in TS when pubertal development was spontaneous as opposed to induced. To explain these findings, it has been proposed that estrogen supplementation for puberty induction does not sufficiently mimic the natural estrogen milieu, and/or that normal gonadal function and thus estrogen exposure is needed from infancy onwards for normal bone development. TS girls are often treated with rhGH to augment linear growth and with sex steroids to promote pubertal development. The effect of this therapy on bone mass has been difficult to discern for reasons already discussed. The observed gains in bone mineral are confounded by changes in bone size and maturation, and made difficult to assess because of abnormal bone shape. Studies assessing the influence of GH therapy have been inconsistent, with lower than expected BMD values in some [251] but not all [252,253] studies. Estrogen therapy, either in combination or alone, appears to augment bone mass [254,255], though it appears difficult to attain a normal peak bone mass despite long-term exogenous estrogen therapy [255,256]. The ideal age for initiation of exogenous therapy for optimization of bone mass is presently unknown. One
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study noted that initiation of estrogen before age 12 was associated with a higher radial BMC than later therapy [257], but bone size was not considered in the analysis and randomization to timing of treatment was not performed. Others have shown that early estrogen therapy is not justified on the basis of bone mineral status, when TS girls are treated with rhGH [252]. Further studies are needed to determine the optimal age at which to initiate estrogen therapy in order to minimize bone mass decrements, weighing this against the need to maximize final height. Prospective studies to assess the effect of hormonal therapy on fracture incidence in TS are also warranted. Growth Hormone Deficiency
Growth hormone (GH) and IGF-1, the anabolic effector of GH, are essential for normal bone growth and for the development of muscle mass [258,259]. Growth hormone deficiency (GHD), if not recognized and properly treated, results in extreme short stature, reduced lean body mass and poor muscle strength [260]. According to the mechanostat model, G H D has the potential to compromise two key mechanical challenges, bone growth and muscle force, which would normally stimulate accrual of bone mineral and adaptations in skeletal architectural design, for the purpose of fashioning strong and stable bones [261]. The effects of G H D on BMD have been challenging to discern because of the influence of reduced bone size and skeletal maturation on aBMD, which has led to conflicting results. Deficits in BMD have generally been persistent in both children [260,262] and adults [263], when bone size has been considered through estimation of vBMD. Stronger evidence for compromised skeletal health in G H D patients is the finding that fracture rates are higher among adults compared to the normal population. Wuster [264] analyzed results from a large pharmacoepidemiological survey of adults with GHD and found the prevalence of fractures in G H D adults was 2.7 times that in the control population. This fracture risk was independent of whether the patient had isolated G H D or combined pituitary hormone deficits. Similar epidemiological studies of fracture rates have not been performed in the pediatric population. The reductions in bone mass seen in GHD patients have been attributed to impairment in bone formation [265]. This conclusion is substantiated by the proposed mechanism of actions of GH in vitro, including a direct action of GH on osteoblasts to stimulate production of bone tissue factors, as well as enhanced intestinal calcium absorption, increased intestinal cell sensitivity to PTH, and increased renal hydroxylation of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D [258].
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FIGURE 3 Muscle-bone interrelationship at the proximal forearm in patients with TS (mean age 19.5 + 2.3 yr) compared to females at Tanner Stage 5 (left) and Stage 1 (fight). Reprinted with permission from Bechtold et al. [249]. 9 The Endocrine Society.
Recombinant human growth hormone (rhGH) has revolutionized the treatment of short stature due to GH deficiency. When rhGH is given six to seven days per
week at doses of 0.16-0.20 mg/kg/week (0.5-0.6 IU/kg/ week), bone mass decrements are minimized or eliminated altogether in growth hormone deficient children
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during short-term studies [260,266,267], and there is a positive effect on lean tissue mass. Higher doses do not appear to confer greater gains in mineral accrual [268]. There are inconsistent reports as to whether normal peak bone mass can be obtained and subsequently maintained during the adult years following treatment with rhGH for GHD during childhood [263,265,267,269]. In addition, from the small numbers of studies that are currently available, it appears difficult to completely normalize bone mass with GH therapy in GH deficient adults [270]. Placebo-controlled trials of 12-18 months' duration have failed to demonstrate improvements in bone mass [271], while short-term uncontrolled studies have suggested that GH treatment may increase bone mass by 0.SSD in adults [272]. Further studies are needed to evaluate whether therapy in GH deficient adolescents should be extended beyond attainment of final height, until peak bone mass is achieved, in order to maximize muscle strength and mineral accrual during this critical period. The effect of GH therapy on fracture incidence in adulthood is an important question, the answer to which will be instrumental in determining the direction of bone health trials for older teenagers and young adults with GH deficiency. Hyperthyroidism
The effect of thyrotoxicosis on bone and mineral metabolism has been well documented in the adult and pediatric literature [273-276]. Binding ofT3 to its nuclear receptors directly stimulates osteoblasts, and osteoblastic activity mediates T3 activation of osteoclasts, resulting in bone resorption [277,278]. Accelerated bone resorption increases serum levels of calcium and phosphate. Parathyroid hormone and 1,25-dihydroxyvitamin D are subsequently suppressed, which in turn reduces gastrointestinal absorption of calcium and phosphate and increases calciuria. Ultimately, thyroid hormone excess increases bone turnover in favour of net bone resorption, which may result in significant decrements in bone mass [273,274,279]. Biochemical markers of bone metabolism reflect the increased bone turnover, with elevated indices of bone formation (serum alkaline phosphatase, osteocalcin, propeptide of type I collagen) and resorption (collagen cross-links pyridinoline and deoxypyridinoline) [275]. The risk of osteoporosis increases with the duration and severity of untreated or uncontrolled thyrotoxicosis [276]. In children, both endogenous hyperthyroidism and excessive thyroid hormone replacement have been associated with accelerated resorption and decrements in bone mass [4,280]. Mora et al. [273] found, in a longitudinal study of 13 hyperthyroid girls, that at diagnosis an inverse correlation existed between serum free T4 levels and spinal and whole body bone density. Free T4 and T3
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levels correlated positively with urinary NTX (Nterminal telopeptide of type I collagen), consistent with the hyperresorptive state. Similar results were reported by Lucidarme et al. [274], who in addition noted a preferential loss of cortical compared to trabecular bone in 26 young patients with Graves' disease. Both Mora et al. (N=13) [273] and Lucidarme et al. (N=26) [274] reported full restitution of bone mass within 12 to 24 months following successful treatment with antithyroid medication. While recovery in these studies was rapidly achieved following induction of euthyroidism, the long-term effect of Graves' treatment with anti-thyroid medication on bone metabolism has not been studied prospectively in children. Concern has recently been raised in the pediatric thyroidology literature regarding the long-term efficacy of anti-thyroid medication for the pediatric patient [281]. Anti-thyroid medication is generally considered first-line therapy for children and adolescents with Graves' disease, while radioiodine ablation of the thyroid gland has been deemed a second line option despite proven efficacy. However, hyperthyroidism may not be adequately controlled in many pediatric patients on medical therapy because of side effects, failure to achieve remission, non-compliance, or relapse once medical therapy is discontinued. A history of hyperthyroidism is a known risk factor for osteoporosis later in life [282,283]. As such, the long-term effect of anti-thyroid medication on bone health needs further assessment to determine whether the encouraging short-term results described by Mora [273] and Lucidarme [274] are sustained and whether the current approach to Graves' treatment and subsequent bone health needs revision. High dose L-thyroxine therapy (120 micrograms/m2/ day) has been shown to reduce proximal forearm BMC in children and adolescents treated for endemic goiter, Hashimoto's thyroiditis or cancer [280]. Children with congenital hypothyroidism treated with high dose Lthyroxine in the first few months of life to rapidly restore euthyroidism have also been evaluated for skeletal effects of this therapy. Kooh et al. (N=20) [284] and Leger et al. (N=44) [285] did not detect any lasting adverse effects when congenital hypothyroid patients were studied at a mean age of 8 years [284,285]. On the other hand, Demeester-Mirkine et al. [286] evaluated the calcitonin and bone mineral status of 9 adults with hypothyroidism due to thyroid agenesis/dysgenesis. These authors found that post-calcium infusion calcitonin levels were lower in hypothyroid women compared to normal women and further, hypothyroid women demonstrated a 10 percent reduction in BMC at the radial diaphysis. These authors hypothesized that both calcitonin deficiency and thyroid hormone replacement at higher than physiologic doses could have played a role in the observed bone loss.
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Based on the current literature, it is advisable to avoid suppressive doses of L-thyroxine, except in cases of thyroid cancer where the benefits of therapy outweigh the risks. For patients with congenital hypothyroidism, initial high dose therapy is necessary to preserve cognitive potential [287] and does not appear to adversely affect skeletal health, but surveillance of bone mineral accrual may be prudent in patients with thyroid agenesis/ dysgenesis because of their known risk for calcitonin deficiency. The clinical significance of the reduced bone mass in adults with thyroid agenesis/dysgenesis is not known. Diabetes Mellitus
Attention to skeletal health among patients with type 1 diabetes mellitus (DM) has resulted in ongoing controversy [288-291]. Adult type 1 DM patients of both sexes show a reduction in BMD, more commonly at appendicular sites, and complications such as neuropathy and microangiopathy appear to worsen the observed deficits [288,292]. However, there is no convincing evidence of an increase in fragility fractures among adult type 1 DM patients who have reductions in bone mass. As such, the clinical relevance of the observed osteopenia is questionable. While some investigators have shown normal bone mineral density studies in pediatric patients with type 1 DM for up to 13 years' duration [289,293,294], others have demonstrated decrements at the lumbar spine or appendicular skeleton [295-298]. Cortical bone has been found to be more markedly affected in some cases [295] while in others, trabecular bone mass has been reduced [299]. Some studies have observed lower, bone mass for boys [296] or girls [290], and others have found no sex difference [295]. Different methodologies for estimating bone mass and failure to correct for bone maturation and size may have contributed to the disparity among results in children. Based on current studies, it appears that mild reductions in bone mass are a feature of type 1 DM in adults and possibly in children. The degree of bone mass deficit, when it is present, increases only marginally or not at all with time [290,297]. The clinical importance of osteopenia in type 1 DM remains in question, given that fractures are not reported more frequently in children or in adults with long-standing disease [288]. In an attempt to explain the reductions in bone mass that have been observed in some studies, correlation with features of type 1 DM and indices of bone health have been explored, with inconclusive results. Osteopenia may be present at diagnosis [291] and some studies have shown an association between subsequent glycemic control (as estimated by HbA1C) [292], while others have not [298]. The duration of type 1 DM has also been
inconsistently correlated with bone mass decrements [291,292,297]. The majority of studies suggest diabetic osteopenia results from impaired bone formation [300,301]. Guarneri et al. [301] showed reduced serum osteocalcin levels at the time of diagnosis in 31 children with diabetes. After 15 days of insulin therapy, the osteocalcin levels completely normalized. Osteocalcin levels were negatively correlated with glycosylated hemoglobin and positively correlated with the degree of metabolic acidosis. These authors hypothesized that during glycometabolic imbalance, there is a decrease in bone turnover that may contribute to diabetic osteopenia. Animal models have been consistent with reduced bone formation. Abnormal glycosylation of collagen, reduced GH and IGFs and decreased osteoblast recruitment have been hypothesized to be contributing factors in the pathogenesis [300]. Abnormalities in Vitamin D and its receptor have also been observed. The role of these factors in the development of diabetic osteopenia in adults, and the clinical significance of the bone mass deficits, remain to be determined. Hyperp r o lac t inem ia
Hyperprolactinemia is an infrequent diagnosis in childhood and adolescence and typically presents with amenorrhea and galactorrhea in girls and with arrested puberty in boys. In adolescence, hyperprolactinemia has been shown to cause deficits in bone mass that correlate inversely with the duration of hyperprolactinemia [302]. Short-term studies in women and teenage girls with hyperprolactinemia have shown that normalization of prolactin levels and resumption of menses do not necessarily lead to complete restitution of bone mass [302,303]. There are insufficient data to determine whether complete restitution of bone mass is possible over the long-term following normalization of prolactin levels and return of normal gonadal function [302,303]. Studies of the relative contributions of hyperprolactinemia-induced hypogonadism and the prolactin excess itself suggest that gonadal function is a more important determinant of bone mass decrements than hyperprolactinemia [304,305]. Hyperprolactinemia may occur in association with growth hormone deficiency, which adds further to the risk of reduced bone mass [306]. The skeletal status of pediatric patients with hyperprolactinemia should be closely monitored, and treatment to suppress prolactin and restore normal growth and gonadal function should be instituted as quickly as possible in order to minimize decrements in bone mass. The long-term fracture risk for patients with a history of hypogonadism secondary to hyperprolactinemia during childhood and adolescence is unknown.
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Iatrogens Glucocorticoids
The morbidity associated with glucocorticoids has been studied for over 70 years, since Harvey Cushing's first report of endogenous glucocorticoid excess [307]. While endogenous hypercortisolemia is a rare entity in children, exogenous corticosteroids are commonly prescribed for the treatment of numerous pediatric conditions such as autoimmune/inflammatory disease, asthma, leukemia and organ transplantation, to name a few. In adults, it has been estimated that up to 50 percent of patients on long-term (greater than 1 year) glucocorticoids have osteoporosis, and that a large number of these patients suffer from fragility fractures [308]. While precise incidence data on the frequency of steroid-induced skeletal morbidity in children with various diseases are not available, a number of studies attest to the potentially serious effects of steroids on bone health among pediatric patients [182,184,185]. The pathogenesis of glucocorticoid-induced osteoporosis (GOP) is complex, due to the multiplicity of corticosteroid effects on bone and mineral metabolism. Recent developments in cell biology suggest glucocorticoids inhibit osteoblast- and osteoclastogenesis, and promote apoptosis of osteoblasts and osteocytes [309]. It is proposed that reduced production of osteoclasts explains the observed reduction in bone turnover while apoptosis and decreased production of osteoblasts account for the decline in bone formation and trabecular width. It has been further suggested that the accumulation of apoptotic osteocytes may contribute to osteonecrosis, a known side effect of hypercortisolemia. Excess glucocorticoid also diminishes intestinal calcium absorption and renal tubular calcium reabsorption, resulting in a negative calcium balance. Muscle and growth plate are other targets of glucocorticoid excess. Long-term use may result in significant myopathy [310] and growth inhibition [311], thereby diminishing two key mechanical challenges that, in accordance with the mechanostat model, would normally foster bone strength. Bone loss in adults with GOP is biphasic. A precipitous drop in bone mass is observed in the first 6 to 12 months of therapy, followed by gradual but sustained loss in subsequent years [174,312]. Weinstein et al. [313] recently demonstrated that the early loss of bone with glucocorticoid excess is caused by extension of the life span of pre-existing osteoclasts. The rate of bone loss appears to be similar at the lumbar spine and femoral neck when measured by DXA [314,315], however, it has been suggested that trabecular bone is more sensitive to the deleterious effect of steroids than cortical bone [316]. As such, QCT or lateral spine DXA scans may be
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superior for assessment of bone mass in GOP than the more commonly used anterior-posterior spine and proximal femur DXA studies [316]. In adults, there is potential for restitution of bone mass following discontinuation of glucocorticoid therapy, and a concomitant reduction in fracture risk [317,318]. There are little data regarding the natural history of GOP in childhood and adolescence. Gafni et al. [319] recently hypothesized that temporary steroid use early in life would have little or no effect on adult bone mass because many areas of the young skeleton are replaced entirely through skeletal growth. These investigators demonstrated that tibial bone density in 5 week-old dexamethasone-treated rabbits was reduced following 5 weeks of treatment, as expected. Complete recovery through bone growth was observed by 16 weeks following the cessation of dexamethasone. If these data can be generalized to the pediatric skeleton, they suggest that temporary insults to bone mineral acquisition early in life may not adversely affect peak bone mass. On the other hand, drugs and/or disease which are operative long-term, or which adversely affect bone during the later growing years, may not be associated with complete recovery. Fractures have been reported in children receiving long-term glucocorticoids [169], but the overall fracture incidence is unknown. Patients with steroid-induced osteoporosis are heterogeneous, and thus the fracture incidence may vary depending upon the underlying disease and associated risk factors. In adults, it has been suggested that fracture susceptibility is higher in GOP than in involutional osteoporosis [320]. A six-fold increase in the risk of vertebral fracture was associated with a decrement of 1 SD or less in lumbar spine BMD among steroid-treated post-menopausal women with rheumatoid arthritis [321]. It has been proposed that alterations in bone quality independent of BMD may explain these observations. Glucocorticoid-induced bone loss appears to be dosedependent [182,184]. However, it remains unsettled whether low doses cause bone loss in all patients. An adult dose of 7.5 mg/day of prednisone or glucocorticoidequivalent has been proposed as the threshold d o s e - the dose above which most adults will lose a clinically significant amount of bone [322]. The concept of a threshold dose is controversial, however, as even low doses of glucocorticoids affect skeletal metabolism [323]. In adults with rheumatoid arthritis, a dose less than 5 mg/ day is relatively safe [324,325]. The lowest-dose threshold for children, if it exists, has not been determined. Alternate day steroid use does not appear to reduce the skeletal effect compared to daily administration [326]. Deflazacort, an oral steroid derivative, has a bone-sparing effect when compared to prednisone or methylprednisone in
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the short-term [151,152]. Whether this bone-sparing effect can be sustained with long-term administration remains to be determined. Inhaled glucocorticoids have fewer systemic, including skeletal, effects compared to oral or intravenous therapy [327-330], unless they are administered at high doses [331]. A comprehensive assessment of all risk factors for osteoporosis should be undertaken before initiating a treatment plan for pediatric patients with GOP. Variability in the skeletal effect of glucocorticoids may reflect genetic factors in determination of bone mass, differences in pharmacokinetics, or it may be the result of factors such as disease activity, mobility, nutrition, delayed puberty and/or treatment with other osteotrophic drugs. As a general principle for patients with steroid-responsive diseases, the minimally effective dose to treat disease activity should be prescribed, and topical or inhaled therapies should be offered when appropriate. It is also recommended to optimize nutrition and ensure the recommended daily intake of calcium and Vitamin D. Hypogonadism, malabsorption, and impaired mobility should also be addressed as part of the management plan. In the case of DXA evaluation of bone mineral density, correction for bone size and pubertal stage are critical as both sexual maturation and stature may be adversely affected by glucocorticoids. In our experience, these treatment measures are frequently inadequate and pediatric GOP patients often have persistent pain and vertebral fractures despite attempts to quell disease activity and restore pubertal development, nutrition and mobility. The treatment of pediatric GOP with anti-osteoporotic agents is virtually unchartered in the literature to date, with studies of calcitonin [332], alendronate [198], pamidronate [333], and growth hormone [334] restricted to isolated cases or small numbers of patients. Trials of rescue therapy (secondary prevention) in adults, once osteoporosis is established, have led to the study of calcium, vitamin D (calciferol), vitamin D analogues (calcitriol and alphacalcidiol), calcitonin, hormone replacement and bisphosphonates. Calcium and vitamin D have not been shown to be effective in reducing fracture rates among patients on long-term glucocorticoids [335]. Studies of sex hormones, vitamin D analogues and calcitonin have not been sufficiently powered to address fracture incidence, though BMD has been positively affected in a number of studies [177,336-339]. None of these agents appear to be as effective as bisphosphonates, where evidence of benefit has been more consistently documented [340-342]. Daily sub-cutaneous PTH, a novel anabolic therapy, has been shown to prevent osteoblast and osteocyte apoptosis [343], and preliminary results in the treatment of adults with GOP are promising [344]. Recent attention has also turned to primary prevention of adult GOP with
bisphosphonates, given the precipitous loss in bone mass which occurs during the first few months of glucocorticoid therapy. The co-prescription of a bisphosphonate at the time of glucocorticoid initiation appears to be an effective method for maintaining bone mass, at least in the short-term [345,346]. Given the documented morbidity associated with pediatric GOP, there is considerable need for prospective studies of fracture incidence as well as prevention and intervention trials.
Other Iatrogens Glucocorticoids have been the most extensively studied osteotoxic agents in pediatric osteoporosis. However, a number of other therapies also exert a negative skeletal effect, though in many cases the precise effect on bone and mineral metabolism is unclear. A list of these iatrogens and their proposed threats to bone health is provided in Table 4.
APPROACH TO PREVENTION AND INTERVENTION In keeping with the principles put forth by the mechanostat model, the fashioning of strong and stable bones during the critical years of growth and puberty is dependent upon two key factors: 1) the magnitude of the mechanical challenges (bone growth and muscle force) on bone and 2) the skeleton's ability to sense and respond to these challenges (via the mechanostat and its setpoint). Both of these mechanisms promote bone strength through bone mineral accrual and changes in the skeleton's architectural design. The mechanostat set-point may be lowered or raised by osteotrophic factors, depending on the functional needs of the skeleton. This we have seen with the estrogen effect on bone during female puberty (see section on disorders of puberty). Primary prevention of osteoporosis has been declared a pediatric responsibility, because of the enormous changes in skeletal architecture and mineral accrual that occur during the growing years. While mineral accrual and skeletal design are strongly determined by genetic influences, there are a number of modifiable determinants of skeletal health, to which the pediatric skeleton may be particularly sensitive. The most frequently identified, modifiable skeletal influences will be discussed here. Muscle force should be adequate to stimulate mineral accretion and changes in skeletal architectural design. A moderate amount of physical activity, especially weightbearing exercise, has a positive impact on mineral accrual and bone size [41,44,347]. However, there appears to be a threshold of physical activity above which there may be a negative effect on bone, particularly if frequent, intense
17. The Spectrum of Pediatric Osteoporosis TABLE 4 Iatrogen
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Frequently used iatrogenic agents associated with osteoporosis in the pediatric population Proposed osteotoxic effect (direct or indirect)
Glucocorticoids
Apoptosis of osteoblasts and osteocytes; decreased osteoblastogenesis; impaired intestinal calcium absorption and renal reabsorption Methotrexate Uncertain. Proposed mechanisms include impaired protein synthesis by osteoblasts, interference with vitamin C metabolism Cyclosporine Uncertain. High-turn over state with increased resorption has been observed Heparin Uncertain. Proposed mechanisms include inhibition of renal 1-alpha-hydroxylaseactivity with reductions in circulating levels of 1,25-dihydroxyvitaminD and concomitant increases in PTH; direct drug effect on bone tissue with increased resorption and decreased formation, affecting primarily cancellous bone R a d i o t Hormonal h e r (growth a hormone, p ycentral[or peripheral 3 7 7 ] avascular deficiencies hypogonadism), necrosis, muscle atrophy Depot medroxyprogesterone acetate Central hypogonadism GnRH analogues Central hypogonadism L-thyroxine suppressive therapy Osteoblast mediated T3 activation of osteoclasts, resulting in bone resorption Anti-convulsants Reduced trabecular bone, but compensatory increase in cortical bone, with preservation of the absolute bone mass
exercise is accompanied by low bone mass, poor nutrition or hypogonadism [348-350]. Endocrine abnormalities such as pathologically delayed puberty, growth hormone deficiency, hyperthyroidism, hyperprolactinemia and hypercortisolemia should be identified and treated in a timely fashion in order to restore the normal hormonal milieu for bone growth and development. Similarly, caloric intake should be adequate throughout the years, and particular attention to the nutritional status of young girls and teenagers should be paid in order to identify those with self-induced caloric restriction. The role of calcium and vitamin D intake in accrual of bone mass during the growing years has been the source of considerable study and merits particular attention here. In the absence of rickets and osteomalacia, the mineral content of bone per unit volume of matrix varies within narrow limits, and almost all dietary calcium is either excreted or retained in bone mineral. Bailey et al. [351], in a landmark study, showed that peak calcium accretion rates during puberty were about 350 mg in boys and 300mg in girls on a calcium diet between 1,1101,140mg/day. Furthermore, calcium retention efficiencies were high during this critical period (36.5 percent for boys and 29.6 percent for girls). Balance studies have shown that adolescents continue to increase calcium retention at dietary intakes above 1,500 mg/day [352]. As a result, dietary calcium recommendations for healthy adolescents have increased to 1,300mg/day [353]. Whether peak bone mass can be enhanced by calcium supplementation has been evaluated by a number of investigators. Studies have shown a positive effect of calcium supplementation on bone mass in children and
Reference
[309] [375] [195] [376]
[3781 [3791 [277,278,2801 [138]
adolescents [46,47,354,355], though one study suggested the effect was only operative if calcium was administered prior to menarche [46]. Whether the positive effect on bone mass can be maintained once supplementation is withdrawn is unclear. Nowson et al. [354] showed the greatest effect of calcium on B M D within the first 6 months of therapy. Thereafter, the differences were maintained, but did not increase, with continued supplementation. Lee et al. [355] showed the benefits of supplementation in Chinese children on a habitually low calcium diet disappeared 18 months following withdrawal, while Bonjour et al. [47] demonstrated a sustained effect for an additional 12 months following calcium therapy. Thus, it appears that calcium supplementation can enhance BMD among children and adolescents, at least in the short term. However, further studies are required to determine whether the increases in bone mass are sustained, whether they result in a higher peak bone mass, and whether children with chronic diseases have the same calcium requirements as healthy youth. For children with chronic systemic diseases, early identification and treatment of the underlying condition is paramount to skeletal health. Bone mass should be monitored at the time of presentation and throughout the growing years, with careful attention to bone size and skeletal maturation when interpreting D X A results. Osteotoxic drugs should be used sparingly if possible, and their use should be weighed against the need to control disease activity. However, often treatment of the chronic disease and institution of strategies to protect the skeleton as already discussed are not enough to
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prevent decrements in bone mass and atraumatic fractures. In such cases, therapy with anti-osteoporotic agents should be considered. The most extensively studied pharmacologic agents for the treatment of pediatric osteoporosis to date are anti-resorptive agents, and in particular, bisphosphonates. Bisphosphonates are synthetic analogues of pyrophosphate that inhibit bone resorption by inactivation of osteoclasts [356]. Although not yet approved by the FDA for their use in children, bisphosphonates have shown benefit to children with a variety of metabolic bone diseases, including OI [3,357], IJO [120,357], rheumatoid conditions [198,357], fibrous dysplasia [358], Gaucher's disease [359], hypercalcemia [360], familial hyperphosphatasia [361], mitochondrial myopathy [357] and fibrodysplasia ossificans progressiva [362]. As a class of drugs, they are well-tolerated both orally and intravenously [357]. The most extensively studied bisphosphonate in children with osteoporosis, intravenous pamidronate, has led to improved bone mass, reduced pain, enhanced mobility and a lower rate of fragility fractures in young patients with congenital and acquired osteoporotic conditions [3,54,121,357]. The largest study of bisphosphonate use in children to date, an uncontrolled study of 30 patients with severe OI who received intravenous cyclical pamidronate showed remarkable benefit to patients' quality of life. Common adverse effects observed in the majority of pediatric patients who have received intravenous pamidronate have been a transient low-grade fever and flu-like symptoms (known as the acute phase reaction) [363]. Oral administration occasionally has been associated with chemical esophagitis [364]. Bisphosphonates suppress bone resorption and turnover, leading to reduced levels of serum alkaline phosphatase, asymptomatic hypocalcemia/hypophosphatemia, and decreased production of collagen breakdown products. Anterior uveitis and scleritis are rare complications, and transient decreases in lymphocyte counts have also been observed [357]. Theoretical concerns about the effect of bisphosphonate therapy on the growing skeleton have not been confirmed after a decade of clinical and histological observation [357,365]. Zeitlin et al. [365] found that long-term, cyclical intravenous pamidronate therapy was associated with significant height gain in OI types I, III and IV. Irreversible effects on bone remodeling and loss of bone mineral density/increase in fractures following treatment have been sought but not observed [3,366-368]. When treatment is given before closure of the epiphyses, sclerotic lines appear at the distal metaphyses of long bones. Despite this finding, skeletal maturation proceeds at a normal rate [357]. The effect of bisphosphonates on fracture healing in young patients with osteoporosis remains under study.
These data strongly suggest that bisphosphonate therapy can be beneficial to young patients with osteoporosis for whom no other options are currently available. The clinical benefit observed thus far justifies further controlled studies in conditions such as OI and the more common secondary osteoporoses. For optimal therapy and for monitoring of very long-term effects in children, the authors feel that present treatment with bisphosphonares should be restricted to specialized centres with expertise in the treatment of pediatric metabolic bone diseases, and informed consent should be obtained prior to therapy. The safety and efficacy of new compounds on the horizon, such as PTH and vitamin D analogues, have not been determined in the pediatric population.
DIFFERENTIATING CHILD ABUSE FROM BONE FRAGILITY CONDITIONS Child abuse has been formally recognized as a clinical entity in the pediatric literature for approximately 50 years. Pediatricians, child protection specialists and metabolic bone experts may be asked to participate in the challenging task of evaluating a child with suspected abuse. This section will focus on aspects pertaining to the musculoskeletal assessment, as well as the features that may aid in distinguishing the abused child from a child with a bone fragility condition. While there is a great deal of medical literature pertaining to the differentiation of physical abuse from accidental trauma, there is little information available for distinguishing the abused child from a child with bone fragility. Therefore, much of the information presented here is based on the authors' own experiences. It is important to note that a child with bone fragility may also be abused, and thus the discovery of an organic disease does not necessarily dismiss a diagnosis of abuse. As a general principle, where there is doubt as to the mechanism of injury, error should be made on the side of caution to prevent an abused child from being placed back into a dangerous situation. Identifying a child with a bone fragility condition versus physical abuse requires knowledge of the disorders that predispose to low-trauma fractures. Any of the osteoporotic diseases discussed in this chapter may be associated with fractures. In our experience, however, the condition most frequently mistaken for abuse is OI. When the classic features of OI are present, such as long bone deformity, a triangular facies, dentinogenesis imperfecta, extreme short stature and strikingly blue sclerae, the diagnosis should be obvious. However, when these features are subtle or absent, as may be the case in OI types I, atypical OI type IV, and the newer OI
17. The Spectrum of Pediatric Osteoporosis
forms (types V-VII), the diagnosis may be overlooked and families are then subjected to the difficult process of an abuse assessment. This occurs not infrequently in patients with milder and novel forms of OI, particularly when there is no family history of the disease, or when there are undiagnosed family members. While disorders characterized by low bone mass make up the majority of bone fragility conditions, increased fragility may occur when bone mass is normal, or pathologically elevated (osteosclerosis). The vast majority of physically abused children are under 4 years of age [369]. Younger children are at increased risk for abuse due to the inability to verbally or physically resist harm, greater time spent in direct contact with caregivers, and greater need for assistance in daily activities. Children with special needs (mental or physical handicaps), born prematurely, or with difficult temperaments are more likely to be abused, and boys appear to be at increased risk compared to girls [370]. The abuser is a related caregiver in 90 percent of cases, a sibling in 1 percent, and an unrelated caregiver in the remainder [369]. Substance abuse, mental illness and previous history of abuse in childhood are risk factors for abuse by the caregiver. The family dynamic is an important factor in abuse risk, and where there is social isolation, financial stress, domestic violence, unwanted pregnancies, young caregivers and stressful life events, there is an increased risk for child abuse. Suspicion of abuse is usually based on a history that is not in keeping with physical findings or the child's developmental stage. For an abused child with normal skeletal strength, the injury will be more severe than suggested by the history. For the child with a bone fragility condition, the history is usually not consistent with the severity of the injury either, since fractures occur with minimal trauma. Thus, the severity of the injury is not a strong distinguishing feature. However, the distribution of the injuries is helpful as it may be discordant with the mechanism of the injury in an abused child, and should be concordant in the child with organic disease. The key is to obtain as much detail as possible in the history in order to illuminate any differences between the injuries and the explanation provided. A family history of bone fragility should be sought, since a parent may have undiagnosed OI. The physical examination should then be viewed in light of the historical details surrounding the incident. The physical signs of skeletal trauma (swelling, tenderness, deformity) are not universally present in cases of inflicted fracture, since healing of the fracture may have already begun, and signs of acute injury may have resolved. On the other hand, children with undiagnosed bone fragility are usually brought to medical attention by anxious parents immediately, or very soon after the inci-
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dent. Fractures in children who are non-ambulatory should raise suspicion, and fractures of certain bones are unusual in accidental injury and disease. These include the sternum, spinous processes and scapulae [371]. Skull fractures are also relatively infrequent in bone fragility conditions, where fractures of the extremities predominate. Fractured ribs are rare in accidental injury, but may occur in bone fragility conditions. Rib fractures characteristic of abuse are frequently multiple, bilateral and anterior or posterior (attributed to a squeezing injury) [372]. Vigorous pulmonary resuscitation has not been found to fracture ribs in normal children [373]. The presence of skin trauma, oral lesions, retinal hemorrhages, and abdominal/thoracic visceral injuries suggests physical abuse. On the other hand, joint and/or skin hyperlaxity, scoliosis, thoracic and long bone deformity, blue sclerae, dentinogenesis imperfecta, vertebral compression fractures, coxa vara/valga, protrusio acetetabulae, skull deformity (in the absence of contusions/fractures) and enlarged fontanelles point to organic pathology. Attention should also be paid to the overall status of the child, including growth and nutrition, psychomotor development, hydration and hygiene. A complete radiographic skeletal survey is mandatory in the assessment of possible abuse. The single view "babygram" is inadequate and unacceptable. The skeletal survey should include an AP and lateral skull (including cervical spine), supine AP and lateral chest, AP pelvis, AP and lateral upper and lower extremities (to detect subtle bowing not visible on physical examination), and lateral thoracolumbar spine. Abnormalities in bone quality or structure (such as osteosclerosis, long bone deformity, or thin cortices) may be detected. Long bone fractures in OI tend to be transverse, at times subperiosteal, and may or may not be in alignment. Spiral fractures are more typical of abuse, and the "corner" or "bucket-handle" fracture (also called the classic metaphyseal lesion or CML) is a frequent occurrence in abuse. The CML occurs as a result of indirect forces when the extremity is pulled, pushed, twisted or when the infant is shaken. A word of caution has come from a recent report where the CML was observed following serial casting for clubfoot [374]. CT or MRI may be indicated in infants with injuries consistent with shaken-baby syndrome (such as rib fractures and retinal hemorrhages), even in the face of a normal neurological examination. Wormian bones may be or may not be present in OI, and may be seen in other diseases unassociated with bone fragility (Prader-Willi and Down syndromes, hypothyroidism, hydrocephalus). As such, the presence of wormian bones does not rule in OI, nor does their absence eliminate the diagnosis. Additional tests are in accordance with the degree of suspicion for physical abuse versus organic disease.
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A coagulation profile is an essential part of the routine work-up. Biochemical parameters of bone and mineral metabolism are normal in OI, and generally are not helpful in the evaluation (unless hypocalcemia is suspected as the cause of the fractures). Bone densitometry by DXA may be more of a hindrance than help, for numerous reasons. First, normative data for children less than 5 years of age are not supplied by all densitometer manufacturers, and institution-specific data may not be available. Second, correction for bone size and skeletal age must be considered in the interpretation of these results, and compared to normative data. Third, while bone fragility conditions are usually associated with BMD values outside the normal range, bone mass may also be normal or near-normal. Patients with OI type I may have low-normal bone mass, and we have recently identified three patients with an OI-like phenotype but with normal bone mass, bone fragility and unique histology at the iliac crest (unpublished data). Both of these examples emphasize that normal bone mass does not rule out the possibility of a bone fragility condition. Finally, the use of bone densitometry in an abused child with co-morbid conditions which adversely affect bone mass (such as neuromuscular disease) is particularly limited. Similarly, the diagnosis of a bone fragility condition should not rest on genetic studies. While mutations in the only two genes presently known to cause OI (COL1A1/ COL1A2) are found in the majority of patients, a sub-set of patients with atypical OI type IV and types V-VII show no evidence of an abnormality in the type I collagen genes or their protein product. Furthermore, false negative results are possible in any genetic screening program, because of inherent technical limitations. Finally, children with suspected abuse are best evaluated by a multi-disciplinary child protection team. In some cases, it is not possible to definitively diagnose either abuse or an underlying bone fragility condition at the initial evaluation. In such cases, measures should be taken to ensure the child's safety until further investigations or observation can be carried out. If a child with suspected abuse and frequent fractures is removed from his/her home environment, such a child should have ongoing medical surveillance to ensure that fractures are not continuing, and that a diagnosis of bone fragility has not been missed.
SUMMARY AND FUTURE DIRECTIONS Thirty years ago, Charles Dent gave the keynote address at the First Conference on Clinical Aspects of
Metabolic Bone Disease at the Henry Ford Hospital [38]. He proposed that age-related osteoporosis could be "best guarded against by ensuring that you build up the best possible skeleton in childhood, when stresses and strains can be so clearly shown to produce a larger and stronger skeleton. How far it can be said that senile osteoporosis is a pediatric disease...needs further study". Most bone health advocates would now agree that Dent's hypothesis has revolutionized the approach to osteoporosis across the lifecycle. Over the past decade, increased attention to pediatric bone diseases has opened our eyes to the magnitude of bone morbidity in a growing list of genetic disorders and chronic illnesses of childhood. The mechanostat theory provides a framework to guide our approach to bone research and clinical practice based on the functional requirements of the skeletal systemmstrength and stability. When evaluating the skeletal health of youth, the most important questions to be asked are whether the bones are fragile, the frequency with which bones break in a given condition, the risk factors for bone fragility, and whether fractures can be minimized or prevented in the short- and long-term. A number of modifiable osteotrophic factors have been identified which impact on bone mass in childhood, but whether these factors have sustained beneficial effects on bone mass and whether they ultimately influence bone strength remain in question. Over the past decade, the pediatric bone literature has been flooded with studies of bone densitometry, but because of technical limitations (failure to account for bone size and shape), this has led to misinterpretation of the data and false conclusions. There is a need for further exploration of tools that provide precise measurement of bone mass, size and density in the growing patient, and to consider which indices of skeletal health best reflects skeletal strength. While the past decade has seen tremendous progress in the identification of pediatric osteoporosis, there is ongoing need for prospective longitudinal studies with sufficient numbers of patients to fully define the natural history of the various forms of childhood osteoporosis and the effect of intervention during the growing years. Whether the prevention and reversibility of decrements in bone mass and fractures can be achieved through basic measures such as nutrition, weight-bearing and treatment of co-morbid conditions remains in question. Randomized, controlled trials of bisphosphonates (and potentially other anti-osteoporotic agents) are needed in several conditions associated with significant bone morbidity, such as glucocorticoid-induced osteoporosis. Given the relatively small numbers of patients with osteoporosis in childhood, multi-center studies will be needed to address the questions that face the pediatric osteoporosis scientific community.
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17. The Spectrum of Pediatric Osteoporosis (1998). Alendronate for the prevention and treatment of glucocorticoid-induced osteoporosis. Glucocorticoid-Induced Osteoporosis Intervention Study Group. N Engl J Med 339, 292-299. 343. Jilka, R. L., Weinstein, R. S., Bellido, T., Roberson, P., Parfitt, A. M., and Manolagas, S. C. (1999). Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J Clin Invest 104, 439-446. 344. Lane, N. E., Sanchez, S., Modin, G. W., Genant, H. K., Pierini, E., and Arnaud, C. D. (2000). Bone mass continues to increase at the hip after parathyroid hormone treatment is discontinued in glucocorticoid-induced osteoporosis: results of a randomized controlled clinical trial. J Bone Miner Res 15, 944-951. 345. Gonnelli, S., Rottoli, P., Cepollaro, C., Pondrelli, C., Cappiello, V., Vagliasindi, M., and Gennari, C. (1997). Prevention of corticosteroid-induced osteoporosis with alendronate in sarcoid patients. Calcif Tissue Int 61, 382-385. 346. Boutsen, Y., Jamart, J., Esselinckx, W., and Devogelaer, J. P. (2001). Primary prevention of glucocorticoid-induced osteoporosis with intravenous pamidronate and calcium: a prospective controlled 1-year study comparing a single infusion, an infusion given once every 3 months, and calcium alone. J Bone Miner Res 16, 104-112. 347. Slemenda, C. W., Miller, J. Z., Hui, S. L., Reister, T. K., and Johnston, C. C., Jr. (1991). Role of physical activity in the development of skeletal mass in children. J Bone Miner Res 6, 1227-1233. 348. Myburgh, K. H., Bachrach, L. K., Lewis, B., Kent, K., and Marcus, R. (1993). Low bone mineral density at axial and appendicular sites in amenorrheic athletes. Med Sci Sports Exerc 25, 1197-1202. 349. Myburgh, K. H., Hutchins, J., Fataar, A. B., Hough, S. F., and Noakes, T. D. (1990). Low bone density is an etiologic factor for stress fractures in athletes. Ann Intern Med 113, 754-759. 350. Snow-Harter, C. M. (1994). Bone health and prevention of osteoporosis in active and athletic women. Clin Sports Med 13, 389-404. 351. Bailey, D. A., Martin, A. D., McKay, H. A., Whiting, S., and Mirwald, R. (2000). Calcium accretion in girls and boys during puberty: a longitudinal analysis. J Bone Miner Res 15, 2245-2250. 352. Jackman, L. A., Millane, S. S., Martin, B. R., Wood, O. B., McCabe, G. P., Peacock, M., and Weaver, C. M. (1997). Calcium retention in relation to calcium intake and postmenarcheal age in adolescent females. Am J Clin Nutr 66, 327-333. 353. Institute of Medicine. (1997). Dietary reference intakes for calcium, phosphorus, magnesium, Vitamin D, fluoride. Washington, D.C. National Academy Press. 354. Nowson, C. A., Green, R. M., Hopper, J. L., Sherwin, A. J., Young, D., Kaymakci, B., Guest, C. S., Smid, M., Larkins, R. G., and Wark, J. D. (1997). A co-twin study of the effect of calcium supplementation on bone density during adolescence. Osteoporos Int 7, 219-225. 355. Lee, W. T., Leung, S. S., Wang, S. H., Xu, Y. C., Zeng, W. P., Lau, J., Oppenheimer, S. J., and Cheng, J. C. (1994). Doubleblind, controlled calcium supplementation and bone mineral accretion in children accustomed to a low-calcium diet. Am J Clin Nutr 60, 744-750. 356. Rodan, G. A., and Reszka, A. A. (2002). Bisphosphonate mechanism of action. Curt Mol Med 2, 571-577. 357. Brumsen, C., Hamdy, N. A., and Papapoulos, S. E. (1997). Longterm effects of bisphosphonates on the growing skeleton. Studies of young patients with severe osteoporosis. Medicine (Baltimore) 76, 266-283.
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358. Chapurlat, R. D., Delmas, P. D., Liens, D., and Meunier, P. J. (1997). Long-term effects of intravenous pamidronate in fibrous dysplasia of bone. J Bone Miner Res 12, 1746-1752. 359. Samuel, R., Katz, K., Papapoulos, S. E., Yosipovitch, Z., Zaizov, R., and Liberman, U. A. (1994). Aminohydroxy propylidene bisphosphonate (APD) treatment improves the clinical skeletal manifestations of Gaucher's disease. Pediatrics 94, 385-389. 360. Sellers, E., Sharma, A., and Rodd, C. (1998). The use of pamidronate in three children with renal disease. Pediatr Nephrol 12, 778-781. 361. Cassinelli, H. R., Mautalen, C. A., Heinrich, J. J., Miglietta, A., and Bergada, C. (1992). Familial idiopathic hyperphosphatasia (FIH): response to long-term treatment with pamidronate (APD). Bone Miner 19, 175-184. 362. Rogers, J. G., Dorst, J. P., and Geho, W. B. (1977). Use and complications of high-dose disodium etidronate therapy in fibrodysplasia ossificans progressiva. J Pediatr 91, 1011-1014. 363. Glorieux, F. H. (2001). The use of bisphosphonates in children with osteogenesis imperfecta. J Pediatr Endocrinol Metab 14, 1491-1495. 364. de Groen, P. C., Lubbe, D. F., Hirsch, L. J., Daifotis, A., Stephenson, W., Freedholm, D., Pryor-Tillotson, S., Seleznick, M. J., Pinkas, H., and Wang, K. K. (1996). Esophagitis associated with the use of alendronate. N Engl J Med 335, 1016-1021. 365. Zeitlin, L., Rauch, F., Plotkin, H., and Glorieux, F. H. (2003). Height and Weight Development During Four Years of Therapy with Cyclical Intravenous Pamidronate in Children and Adolescents with Osteogenesis Imperfecta Types I, III and IV. Pediatrics in press. 366. Rossini, M., Gatti, D., Zamberlan, N., Braga, V., Dorizzi, R., and Adami, S. (1994). Long-term effects of a treatment course with oral alendronate of postmenopausal osteoporosis. J Bone Miner Res 9, 1833-1837. 367. Huaux, J. P., and Lokietek, W. (1988). Is APD a promising drug in the treatment of severe osteogenesis imperfecta? J Pediatr Orthop 8, 71-72. 368. Landman, J. O., Hamdy, N. A., Pauwels, E. K., and Papapoulos, S. E. (1995). Skeletal metabolism in patients with osteoporosis after discontinuation of long-term treatment with oral pamidronate. J Clin Endocrinol Metab 80, 3465-3468. 369. Marshall, W. N., Jr., Puls, T., and Davidson, C. (1988). New child abuse spectrum in an era of increased awareness. Am J Dis Child 142, 664-667. 370. Ammerman, R. T. (1990). Predisposing child factors. In: Children at Risk: an Evaluation of Factors Contributing to Child Abuse and Neglect Eds. R.T. Ammerman, M. Hersen, 199-221.
371. Worlock, P., Stower, M., and Barbor, P. (1986). Patterns of fractures in accidental and non-accidental injury in children: a comparative study. Br Med J (Clin Res Ed) 293, 100-102. 372. Cadzow, S. P., and Armstrong, K. L. (2000). Rib fractures in infants: red alert! The clinical features, investigations and child protection outcomes. J Paediatr Child Health 36, 322-326. 373. Feldman, K. W., and Brewer, D. K. (1984). Child abuse, cardiopulmonary resuscitation, and rib fractures. Pediatrics 73, 339-342. 374. Grayev, A. M., Boal, D. K., Wallach, D. M., and Segal, L. S. (2001). Metaphyseal fractures mimicking abuse during treatment for clubfoot. Pediatr Radiol 31, 559-563. 375. Jones, G., and Sambrook, P. N. (1994). Drug-induced disorders of bone metabolism. Incidence, management and avoidance. Drug S a f 10, 480-489.
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376. Mutoh, S., Takeshita, N., Yoshino, T., and Yamaguchi, I. (1993). Characterization of heparin-induced osteopenia in rats. Endocrinology 133, 2743-2748. 377. Ramuz, O., Bourhis, J., and Mornex, F. (1997). Late effects of radiations on mature and growing bone. Cancer Radiother 1, 801-809. 378. Cromer, B., and Harel, Z. (2000). Adolescents: at increased risk for osteoporosis? Clin Pediatr (Phila) 39, 565-574.
379. Yanovski, J. A., Rose, S. R., Municchi, G., Pescovitz, O. H., Hill, S. H., Cassorla, F. G., and Cutler, G. B. (2002). Luteinizing hormone-releasing hormone agonist-Induced delay of epiphyseal fusion prolongs the growth period and increases adult height of adolescents with short stature: results of a randomized, placebocontrolled trial. Pediatric Research 51, 742.
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[18I Osteogenesis Imperfecta HORACIO PLOTKIN*, DRAGAN PRIMORAC t, and DAVID ROWE ~ *Inherited Metabolic Diseases Section, Department of Pediatrics, University of Nebraska Medical Center and Children's Hospital, Omaha, Nebraska tLaboratory of Clinical and Forensic Genetics, Split University Hospital and School of Medicine, Split, Croatia CDepartment of Genetics and Developmental Biology, University of Conneticut Health Center, Farmington, Conneticut
INTRODUCTION
pyknodysostosis [9]. The back of the skull can be flat due to bone fragility and lack of head control in infants
During the past decade, the concept of osteogenesis imperfecta (OI) has changed from "a collagen disorder caused by mutations in the collagen genes, divided in four types, for which there is no medical treatment" to a fascinating group of heterogeneous conditions characterized by bone fragility, caused by numerous different mutations, with at least 10 different clinical forms and with effective symptomatic treatment and exciting prospects for gene therapy. The prevalence of OI is estimated to be 1 in 15,000-20,000 infants [1], but misdiagnosis is frequent because it is a heterogeneous condition. The prevalence appears to be the same throughout the world [2-5]. During the evolution of understanding of the disease, OI has served as the paradigm for heritable disease of connective tissue from which advances in molecular diagnosis, mode of inheritance, and new concepts of therapy have been applied. It should continue to play this pivotal role in the future. In the vast majority of cases, mutations within the C O L I A 1 or C O L I A 2 genes are responsible for the phenotype, although it is now recognized that mutations in other genetic loci can produce a similar clinical outcome (Table 18). A comprehensive list of the mutations within type I collagen genes resulting in OI [6] is maintained in the OI mutation database (http://www.le.ac.uk/genetics/collagen).
The hallmark of OI is brittle bones. All other characteristics of OI are variable, with heterogeneity even in different members of the same family [7]. Wormian bones are present in the skull in approximately 60% of cases [8] (Fig. 1), although they can be present in other conditions, such as progeria, cleidocranial dysplasia, Menkes syndrome, cutis laxa, Cheney syndrome, and
Pediatric Bone
FIGURE 1 Wormian bones are detached portions of the primary ossification centers in adjacent membranous bones. Theyare suggestive of osteogenesis imperfecta but not pathognomonic.
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CLASSIFICATION
FIGURE 2 Skull in a severe case of OI. Infants with severe OI have soft skulls that are easily flattened in the back because of the inability to support their heads.
with severe OI (Fig. 2). Affected children may suffer recurrent fractures resulting in pain and immobilization, particularly in preschool years. The long-term evolution of the disease is also a matter of concern, particularly in women after menopause. It is wellknown that the peak of bone mass accretion is attained during puberty [10,11]; therefore, it is in the early years of life when treatment efforts should be focused. These individuals have osteopenia due to the basic defect, often worsened by immobilization secondary to fractures or surgery, and decreased physical activity. They also have muscle weakness and ligament laxity. Fractures do not cease at puberty [12] and bone fragility persists throughout life. Chronic, unremitting bone pain may also be present. Thus, this is a complex life-long disease for which the pediatrician will play an essential role in developing a plan that optimizes the quality of life for patients.
The severity of OI ranges from mild forms with no deformity, normal stature, and few fractures to forms that are lethal in the perinatal period. OI was classified into two forms by Looser in 1906 [13]. He classified OI as congenita (Vrolik) or tarda (Lobstein) depending on the severity of presentation. Infants with OI congenita have multiple fractures in utero, whereas in individuals with OI tarda fractures occur at the time of birth or later. OI tarda has also been subdivided into gravis and levis [13]. This classification is no longer used because it understates the complexity of the disease. The first clinical classification of OI to reflect the spectrum of the disease severity was proposed by Sillence [4,14]. Although there is no consistency in the literature regarding the characteristics of the different types, and even though members of the same family (that should have the same OI type) may differ dramatically in severity and clinical presentation [15], the classification has received general acceptance. In the original report [4], Sillence, et al. classified 154 subjects into four groups. Common use has derived into the definition of "types." Group 1 included individuals with bone fragility, blue sclerae, and presenile deafness. The majority of the subjects in this group had their first fracture in the preschool period (in 5 patients, fractures were present at birth). This is an important issue when evaluating cases of suspected child abuse. Of note is that 50% of the subjects in this group were short for age by adult life. Head circumference was large for age. Inheritance was dominant in all cases. Group 2 included patients with lethal perinatal OI with radiographically crumpled ("accordion-like") femora and beaded ribs. In most cases, sclera was blue, but it was white in some. Group 3 patients had progressive deformity and pale blue sclerae at birth becoming normal at puberty.. Easy bruising was not considered common in these individuals. All cases in this group were sporadic. Group 4 patients had white sclerae, and inheritance was dominant. Clinical features were heterogeneous. As per Sillence et al., patients in this group may or may not have a history of fractures, skeletal defomities are variable, and all have osteoporosis and white sclera. They may have dentinogenesis imperfecta but no hearing loss. Paterson et al. [16] described 48 subjects who had white sclerae and dominant inheritance, providing a more extensive description of Sillence's type 4. They found that there is a wide range for age at first fracture and for total number of fractures. They note the differ-
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ent level of severity of members of the same family, including parents with mild manifestations who had children with severe phenotype. The authors classify the patients according to the color of the sclerae, although they mention that several subjects included in Sillence's type 4 had pale blue sclerae. Sillence made it clear that patients in group 4 may have blue sclerae at younger ages that fades as they grow [17]. On these bases, it may not be possible to differentiate group 1 and group 4 individuals at an early age. In clinical practice, scleral hue has very little significance in the diagnosis and classification of OI because blue sclera may occur in normal children and in several other diseases. The numeric classification of OI should be used with caution, and the clinical form and severity must be referred to in each individual case. Hanscom et al. [18] proposed classifying OI according to X-rays. They classified subjects into six groups (A-F), depending on bowing of long bones, the shape of vertebrae, cystic changes of metaphysis, and cortex of long bones and ribs. Unfortunately, some of the changes suggested for classification do not present until age 5 or 10 years, so young children cannot be classified. This approach seems to be realistic and could be developed in more detail. Others classified OI according to severity [12,19]. The mode of inheritance in OI is almost always dominant, and there is a high incidence of new mutations, regardless of the form of OI. Exceptions to this type of inheritance are Type VII OI [20] and osteoporosis pseudoglioma syndrome [21]. Also, in some families from South Africa and Ireland, a recessive pattern of inheritance has been demonstrated [22,23]. The possibility of a germ cell mosaicism [24] has been proposed to explain cases in families with healthy parents who have more than one child with OI [25,26]. These cases were previously thought to have been transmitted in a recessive fashion. It is considered that in at least 6% of cases of lethal OI, one of the parents is a carrier of a germ cell line mosaicism [27]. Thus, there is no clear genotype/phenotype correlate in individuals with OI, and classification should remain clinical. More types were added to the four described by Sillence, including osteoporosis with pseudoglioma [28,29], OI type VI (with mineralization defect) [30], OI with congenital joint contractures (Bruck's syndrome) [31,32], Rhizomielic OI [20], and OI with craniosynostosis and ocular proptosis (Cole-Carpenter syndrome) [33]. Following Rowe and Shapiro [34], we propose that patients should be described in reference to their severity, regardless of the type. Clinical forms described in the literature are summarized in Table 1.
TABLE 18
Recognized regions of mutations in patients with Osteogenesis Imperfecta
Mutation Location
3p22-24. 1
Gene
Phenotype
UNK
Rhizomielic OI (85) OP + pseudoglioma (88)
1lq12-13
LRP5 (87)
chromosome 17
UNK
OI + contractures (95)
17q21.3-q22
Pro-Collagen
Heterogeneous (6)
7q21.3-q22.1
Pro-Collagen
Heterogeneous (6)
TYPES OF Ol (MODIFIED FROM SlLLENCE, ET AL. [4,14] AND ROWE AND SHAPIRO [34]) N o n d e f o r m i n g Ol (Type 1) Mild Ol with Normal Stature
People with mild OI typically have normal stature and few fractures, mostly during the first years of life. They do not present with bowing of the long bones. This condition is transmitted with an autosomal dominant trait. Bone density can be very low, with little relationship to clinical severity. Typically, they are fully ambulatory. Fractures may be present during the first years of life, even at birth [35], but decrease dramatically after puberty. In some cases, the diagnosis is made after the disease is detected in an offspring, or it is an incidental finding after a fracture [36]. Therefore, it is very important to examine the parents of any child with OI in whom an aggravated form of osteoporosis may be the adult manifestation of the disease. Dentinogenesis imperfecta (DI) can be present, and it has been suggested that this is useful to distinguish two discrete forms of mild OI [37]. Early hypoacusia is typical of this form of OI. The cause is not clear. Some studies suggest that it is a neuronal syndrome [38], whereas others indicate it is a conductive abnormality [39]. Cardiovascular problems can be present in these patients, particularly aortic valvular disease [35]. The most common mutation (silenced allele) causing type I OI reduces the expression of otherwise normal type I collagen. Because of the two-to-one requirement for the formation of heterotrimeric collagen, the level of COL 1A1 expression directly influences the production of normal type I collagen molecules. Reduced output from a single COL1A1 allele will cause decreased production of heterotrimeric collagen. Thus, the degree that one of the COL1A1 alleles underperforms may be one of the determinanants of the severity of osteopenia in type I OI.
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The most frequent cause of diminished activity from a collagen gene is a mutation that introduces a premature stop codon in the collagen m R N A [40-42]. This type of mutation leads to rapid destruction of the RNA by a recently described cellular process called nonsense-mediated RNA decay [43-45]. This process appears to be an important mechanism for preventing a truncated protein from being expressed, thus saving the cell from producing proteins with unintended function. Mutations of these surveillance genes are incompatible with development [46]. A truncated 0~1(I) chain produced from a COL1A1 transcript in vitro helps to determine the presence and location of such a stop codon [47]. Otherwise, finding the mutation using a molecular approach is laborious and the mutation can be missed. A second cause of an underproducing COL1A1 allele is a mutation that leads to retention of an intron within the mature transcript. Although this is an uncommon cause of a type I OI, it has provided insight into the normal pathway for splicing a complex transcript such as collagen [48]. Other causes for diminished transcriptional activity from a collagen gene are extremely rare. Mutations within the 3t untranslated region affecting polyadenylation has been reported, and mutations in the 5t untranslated region are predicted to have a modified phenotype but have not been observed. Finally, a nonfunctional collagen gene can result from the synthesis of a procollagen chain that is unable to incorporate itself within the triple-helical molecule. Frameshift mutations within the terminal exon of either collagen gene have been identified that lead to the synthesis of a full-sized procollagen chains, which are rapidly degraded intracellularly when failing to incorporate into the collagen molecule [49].
D e f o r m i n g Ol
Lethal OI (Type II) In this form of OI, newborns do not survive the perinatal period. Death is caused by extreme fragility of the ribs and pulmonar hypoplasia [50] or by central nervous system malformations [51] or hemorrhage. Bone mineral density is severely decreased, and infants present with multiple intrauterine fractures (including skull, long bones, and vertebrae), beaded ribs, and severe deformity of the long bones [52]. Prenatal ultrasound may show shortened and broad limbs, with very low echogenecity and absent acoustic shadow [53], abnormal compressibility of the vault by the transducer, unusually good visualization of the orbits, increased visualization of arterial pulsations, increased through-transmission of the ultrasound beam due to extremely poor mineralization, and abnormally small thorax [54]. However, prenatal differ-
ential diagnosis between severe and lethal OI is not possible. Differential diagnosis includes chondrodysplasia punctata [55] and other forms of OI. In extremely severe cases, patients can be born dismembered [56]. They may have low birth weight, micro- or macrocephalus, and cataracts [57]. In the majority of cases, they are caused by autosomal dominant new mutations [27,58,59]. Unaffected parents may have more than one child with lethal OI due to germ cell line mosaicism [60]. There may be different clinical forms of lethal OI [61]. Virtually all of the mutations that cause the deforming forms of OI act in a dominant negative manner (i.e., the presence of the abnormal type I collage gene product causes the disease). The deleterious effect of the mutant collagen gene is a consequence of the three-dimensional structure of the collagen fibril that is dependent on the tight association of the Gly-X-Y amino acid triplet. A glycine substitution in the helical domain of the collagen ~1(I) chain is the most common mutation. Glycine is the smallest amino acid and must fit in a sterically restricted space in which the three chains of the triple helix join. Depending on the helical location of a mutation, disease severity can range from lethal to severely deforming and mildly deforming. The potential amino acid substitutions are cysteine, alanine, arginine, aspartic acid, cysteine, glutamic acid, serine, valine, and tryptophan. Substitution destabilizes the conformation of the collagen helix, although current biochemical analysis does not always predict clinical severity. Since the helix assembles from the C-terminal propeptide, a mutation in the Cterminal helical and propeptide region results in greater instability and more severe disease, whereas mutations in the midhelical domain tend to be less severe. However, mutations in the midhelical domain can have a severe phenotype, suggesting that subdomains within the helix are critical for functions other than contributing to an intact helical structure. Mutations located at the N-terminal domain of either chain can be extremely mild and are classified as type I OI. Maps relating mutation type and location to clinical phenotype are graphically presented in an interactive pdf format at http://www.le.ac. uk/genetics/collagen/. Other molecular mechanisms that result in a disrupted collagen helix include mutations in the consensus donor or acceptor site that can lead to exon skipping, and the production of a shortened helix [62]. Much less common are mutations that delete a portion of the gene and a number of inframe exons [63] or mutations that insert a segment of intron that remains inframe with the entire transcript [64]. Severe disease results from a dominant negative mutation in the type I collagen gene with the exception of a null mutation of the COL1A2 gene. Formation of the heterotrimeric collagen molecule requires that the
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~2(I) chain account for 50% of the available chains when the procollagen molecule is assembled. When this requirement is not met, because of either underproduction chain or overproduction of the 0~1(I) chain, of the 0r homotrimeric molecules are formed. The severity of disease depends on the balance between homotrimeric and heterotrimeric molecules within the bone matrix. This may explain the spectrum of disease severity ranging from type III OI type, when both COL1A2 alleles are affected, to measurable osteopenia and fragility in the heterozygous state [65,68] and an association with osteoporosis due to the spl polymorphic alteration in the COL1A1 gene. This variation in disease severity acts in a recessive manner or as a quantitative trait in which gene dosage contributes to the severity of bone disease.
Severe Ol with Triangular Face (Type III) Due to overdevelopment of the head and underdevelopment of the face bones, these patients have a characteristic triangular face. They also have short stature, severe deformities of the long bones, vertebral fractures, and scoliosis and chest deformities. Characteristically, they have marked elongation of the pedicles of the vertebrae in all cases and posterior rib angulation [67]. They are frequently wheelchair bound, although some are able to walk with canes or a walker. Prenatal diagnosis is sometimes possible using ultrasonography [68]. Long bones are severely deformed, and altered structure of the growth plates lead to a particular "popcorn" appearance of the metaphyses and epihpyses (Fig. 3).
Moderate Ol with Short Stature (Type IV) These individuals have short stature for age, bowing of long bones may be present, and frequently they also have vertebral fractures. Scoliosis and joint laxity may be present. Patients with moderate OI are generally ambulatory, although sometimes they need aids for ambulation. Interestingly, birth length appears to be normal in this form of OI (personal observation). As with mild OI, moderate OI has been subdivided into two forms: with and without DI [37].
O t h e r Clinical Forms of O!
Type V Ol Some patients with OI develop hyperplastic calluses in long bones that can appear spontaneously or follow fracture or intramedullary rodding [36] (Fig. 4a). These patients present with hard, painful, and warm swellings over bones that initially may suggest inflammation or even osteosarcoma. After a rapid growth period, the
FIGURE 3 "Popcorn" appearance of the epiphysis. Severe OI causes distortion of the growth plate, with zones of partially calcified cartilage and broadening of the epiphysis.
size and shape of the callus may remain stable for many years [70], unless a new fracture occurs at the same site. Microscopically, there is increased production of poorly organized extracellular matrix, which is incompletely mineralized [71]. The first description of hyperplastic callus formation in OI was made in 1908 [13]. A number of case reports have been published [13,70,72-77]. In a series of 60 patients, 10 (17%) developed hyperplastic callus before age 20 years [78]. In a follow-up of 334 patients with OI, we detected hyperplastic callus in 9 patients (2.6%) [data not published]. Familial occurrence of hyperplastic callus with an autosomal dominant pattern of inheritance has been described [19,36,77]. These calluses were in some cases associated with calcification of the interosseous membrane between radius and ulna and irregular collagen fibril diameter [13,70,79]. Magnetic resonance imaging of the hypercallus is not contributory in the differential diagnosis with osteosarcoma, but computed tomography shows a calcified rim of the lesion associated with the absence of cortical destruction that may be useful for ruling out malignancy [80]. It is important to note that although rare, osteosarcoma may develop in patients with OI [81,82]. The
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Inheritance appears to be autosomal dominant, with variable penetrance.
Ol with Mineralization Defect (Type VI) This is a rare form of OI [30], with a prevalence of approximately 6%, and it is undistinguishable from moderate to severe OI on a clinical basis. It is diagnosed by iliac crest bone biopsy, from which a mineralization defect affecting the bone matrix and sparing growth cartilage is evident. These patients have no DI or wormian bones. There are no radiological signs of growth plate involvement compatible with rickets, despite the mineralization defect. The pattern of inheritance is not clear, but the case of two siblings from healthy consanguineous parents has been described, suggesting gonadal mosaicism or a somatic recessive trait. There are no mutations of COL1A1 and COL1A2 genes, and collagen is normal.
Type VII Ol
FIGURE 4 OI with hypercallus formation. Individualswith OI with hypercallus formation develop redundant bony formations around fractures (a), which are sometimesconfused with osteosarcoma. These patients also present with ossificationof the interosseousmembrane of the forearm and the leg (b).
hypercallus may also be present in flat bones [83]. Gloriuex's group analyzed in depth a group of seven children with OI who presented with specific changes in the bone biopsy of the iliac crest [69]. Matrix lamellae were arranged in a mesh-like fashion, as opposed to a parallel arrangement that is seen in controls and in patients with other types of OI. Five patients also had hyperplastic callus formation in long bones, and all showed radiological signs of calcification of the interosseous membrane of the forearm. This determines a clinical sign: patients are unable to pronate and supinate the forearm. The membrane between the tibia and fibula may also present with abnormal calcification (Fig. 4b). The patients also had hyperdense metaphyseal bands in the metaphyses of long bones. The significance of these bands is unknown. None presented with blue sclerae or DI. Electron microscopy analysis of bone of patients with this form of OI showed failure of patches of bone to mineralize [84]. It was not possible to demonstrate any mutations in the collagen genes in this group of patients.
A rareform of OI was recently described in a First Nations community in Quebec [20]. The affected individuals have rhizomelia: shortness of humeri and femora. The phenotype is moderate to severe, with fractures at birth, early lower limb deformities, coxa vara, and osteopenia. Histomorphometrically, the bone in this form of OI is not different from that of type I OI. This type of OI is inherited in an autosomal recessive fashion, and the disease locus has been mapped to the short arm of chromosome 3 by linkage analysis [85]. This genomic location excludes COL1A1 and COL1A2 (respectively located in chromosomes 7q and 17q) as candidate genes. Direct sequencing has also excluded PTH/ PTHrP and TGF-[3 R II genes.
Osteoporosis-Pseudoglioma Syndrome This form of OI was first described in 1972 in three families [86]. Subsequently, the syndrome was described in a South African family of Indian stock [21]. Six members of this family had a severe form of OI and also blindness due to hyperplasia of the vitreous, corneal opacity, and secondary glaucoma. The pedigree was consistent with autosomal recessive inheritance. Bone involvement is mild to moderate. Cases have been observed in the United States and Canada that follow a similar inheritance mode (unpublished data). It has been speculated that ocular pathology results from failed regression of the primary vitreal vasculature during fetal growth [87]. The genetic defect was mapped to chromosome region 1 lq12-13 [88], and later it was shown that the defect is in the L R P 5 gene, which encodes for the low-density lipoprotein receptor-related protein 5 [87].
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It is a member of the Wnt signaling pathway, which has been extensively studied in flies and mice as a fundamental molecular pathway controlling early organogenesis including the skeleton. Ventricular septal defect was also seen in three affected siblings of a consanguineous family [89]. Two other forms of OI with ocular involvement have been described: one variant with optic atrophy, retinopathy, and severe psychomotor retardation [90] and another with microcephaly and cataracts [57].
Ol with Craniosynostosis and ocular proptosis(ColeCarpenter Syndrome) Two boys and a girl have been described with this particular form of OI [33,91]. All were normal at birth, but after several months they developed multiple metaphyseal fractures associated with low bone density in the entire skeleton and craniosynostosis, hydrocephalus, ocular proptosis, and facial dysmorphism. One of the patients also had hypercalciuria. Neurological development is normal in this form of OI. All patients were wheelchair bound at adult age, with very short stature, severe bone involvement (Fig. 5), and normal intellectual and neurological development (unpublished data).
Ol with Congenital Joint Contractures (Bruck Syndrome) This form of OI was first described by Bruck et al. in 1897 in an adult patient [92]. Patients with Bruck's syndrome are born with brittle bones, leading to multiple fractures and joint contractures and pterygia (arthrogryposis multiplex congenita) [31,32]. Wormian bones are present in the skull. It appears to be inherited in a recessive fashion [93,94]. In three patients studied, it was not possible to demonstrate any mutations in the COL1A1 and COL1A2 genes [32]. The basic defect in this syndrome was mapped to locus 17p12 (18 cM interval), and a defect in bone specific telopeptidyl hydroxylase has been identified[95]. This leads to underhydroxilated lysine residues within the telopeptides of collagen type I and, therefore, to aberrant cross-linking in bone but not in cartilage or ligaments. The lysine residues within the triple helix are normally modified, suggesting that collagen cross-linking is regulated primarily by tissue-specific enzymes that hydroxylate only telopeptide lysine residues but not those in the helical portion of the molecule [95].
DIFFERENTIAL DIAGNOSIS Frequently, family history, biochemical profile, and clinical features are sufficient for diagnosing OI. When
FIGURE 5 Severebony involvementin a patient with Cole-Carpenter syndrome. There is no identifiable bone in the midshaft of the humerus of this 17-year-old male with OI with craniosynostosis and ocular proptosis.
feasible, a bone biopsy with histomorphometric analysis is best for making the differential diagnosis of OI. Genetic testing may also be useful, although it is not always possible to find mutations in the COL1A1 and COL1A2 genes; therefore, the diagnosis of OI should not rely on genetic test results. Currently, two laboratories in the United States offer molecular diagnostic services based on DNA sequencing from peripheral blood or cultured fibroblasts (http://www.som.tulane.edu/ gene_therapy/ matrix/matrix_dna_diagnostics.shtml) or on collagen products from cultured cells (http://www.pathology. washington.edu/clinical/byers.html). Readers can inquire about laboratories in Europe offering diagnostic services through
[email protected] or
[email protected]. Premature infants are at risk of osteopenia. Eighty percent of bone mineralization in fetuses occurs during
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the third trimester [96]. Inadequate postnatal management of parenteral or enteral nutrition may also lead to osteopenia. Nonaccidental injury (NAI) is one of the most challenging differential diagnosis of OI [97]. Although the social history may be contributory and certain signs are suggestive of NAI [97], such as hand fractures in the nonambulant child, acromial fractures, fractures of the outer end of the clavicle, and spinal, posterior rib, and metaphysial fractures [98,99], diagnosis is often difficult [100]. Metaphyseal fractures may occur in children with OI but probably only in the presence of obvious bone disease with radiologically abnormal bones [101]. This is complicated by the fact that children with OI may also suffer NAI [102,103]. It is important to note that there are no pathognomonic radiological signs of NAI [104]. Idiopathic juvenile osteoporosis (IJO) is another difficult differential diagnosis. Although IJO is an acquired form of osteoporosis, it is often difficult to be certain that the bone problem was not present from birth, and it was not found because it was not severe enough to cause fractures of the long bones. It is possible to make a differential diagnosis of IJO and OI using histomorphometry: In IJO, there is a twofold decrease in cancellous bone formation, suggesting that there is a lower bone turnover compared with that of OI, with no evidence of increased bone resorption [105]. Hypohposphatasia can resemble OI clinically, but low levels of serum alkaline phosphatase activity and radiological characteristics make the diagnosis [106]. Other differential diagnoses include Cushing's disease, glucocorticoid induced osteoporosis, homocystinuria, lysinuric protein intolerance, glycogen storage disease, congenital indifference to pain, calcium deficiency, malabsorption, immobilization, anticonvulsant therapy, and acute lymphoblastic leukemia [107].
GENERAL CLINICAL FINDINGS Laboratory Markers of bone metabolism are difficult to interpret in children with OI. After a fracture, serum alkaline phosphatase may be elevated, especially in the case of patients with OI type V when there is hypercallus formation. The bone resorption marker type I collagen Ntelopeptide normalized to urinary creatinine (NTX/ uCr) is higher than the 50th age- and sex-specific percentile in 25 and 75% of patients with type I and III OI, respectively [108]. NTX/uCr is significantly higher in type III than in type I OI patients. However, serum creatinine is lower in patients with type III OI, and
serum creatinine is negatively correlated with NTX/ uCr. Differences in NTX/uCr between type I and type III OI are not significant after adjusting for serum creatinine. These findings suggest that the increased NTx/uCr in type III OI could be a consequence of decreased serum creatinine. Serum creatinine is a function of muscle mass in the absence of renal impairment. Therefore, higher NTX/uCr in type III OI may at least be partly due to the underdeveloped muscle system of these children [109]. In severely affected children, hypercalciuria may be present [110], but there is no compromise of renal function [111]. Kidney stones and nephrocalcinosis may also be present [8]. Neurological involvement Basilar invagination is an uncommon but potentially fatal complication of OI. The incidence of this complication in patients with OI is unknown. There is no gender predominancy for this complication [112]. Symptoms of basilar invagination in OI are headache (in approximately 76% of patients), lower cranial nerve palsy, dysphagia, hyperreflexia, quadriparesis, ataxia, nystagmus, and hearing loss. Patients can be asymptomatic and present with large, normal, or small head circumferences [113]. Sawin and Menezes [112] recommend ventral decompression followed by occipitocervical fusion with contoured loop instrumentation to prevent further squamooccipital infolding. The authors note that basilar invagination tends to progress despite fusion in 80% of cases, and that prolonged external orthotic immobilization may stabilize symptoms and halt further invagination [112]. One case of paraplegia occurring in an adolescent girl with OI after chiropractic manipulation has been reported [114]. Reflex sympathetic dystrophy has been described in adults with OI [115]. The cases described in the literature occurred in patients 26-59 years of age. The incidence of this condition in OI patients is not clear [116]. Other neurological manifestations of OI include benign communicating hydrocephalus; macrocephalus; cerebral atrophy [117], usually with no alteration of intellectual status; Dandy-Walker malformation; and idiopatic seizures. Abnormalities of the central nervous system were noted in autopsies of patients with the lethal form of OI, including perivenous microcalcifications, hippocampal malrotation, agyria, abnormal neuronal lamination, white matter gliosis, and migrational defects [118,119]. Hypoaccusia is present in approximately 50% of individuals with mild forms of OI, generally only after the third decade of life [120]. However, this problem is probably more prevalent than appreciated because of the lack of proper studies in children. King and Boblechko [13]
18. Osteogenesis lmperfecta suggested that the incidence of deafness is directly related to severity. The prevalence of hearing loss in OI appears to be between 20 and 60% [121,122]. With increasing age, the prevalence of hearing impairment in patients with OI may be approximately 100% [123]. Hearing loss may be due to otosclerosis [124,125], to middle and inner ear pathology [126,127], or a neuronal syndrome [38]. It has been suggested that there is a structural change in the mineral crystals of the ear bones from hydroxyapatite to brushite in patients with osteosclerosis [128]. It has also been recommended that children with OI undergo audiometry at 10 years of age and repeat the study every 3 years thereafter [129]. Stapedectomy has been performed in patients with OI with success [130-132]. Other otologic findings include lopped pinna, notching of the helix of the pinna, rosy flush of the medial wall of the middle ear, and vestibular abnormalities [127]. Cardiovascular involvement There are several published reports of congenital malformations of the heart in children with OI [8,133], but their incidence is probably not higher than that in the unaffected population. In a series of 58 children with OI, 4 (6.9%) had congenital cardiac malformations [134]. Aortic regurgitation was present in only 2% of patients in another series of affected individuals, whereas aortic root dilatation was present in 12.1% [133] Dilatation was mild [133,135]. The prevalence of mitral valve prolapse varies from 3.4% [134] to 6.9% [133] in published series, which is not different from the prevalence of mitral valve prolapse in the general population (4-8%) [136]. Others have found that the prevalence of mitral valve prolapse in OI is slightly higher (10%) than in the normal population [135]. These lesions are rarely clinically important [34]. Valve replacement has been performed successfully in patients with OI [137-139]. Epoetin-~ has been used to increase hematrocrit preoperatively in mitral valve replacement surgery because of the high risk of perioperative bleeding [140]. Ulnar artery aneurism has been reported in a patient with OI [141], which may be due to increased weakness of vessel walls that can also produce spontaneous carotidcavernous fistulas [142]. Renal involvement Hypercalciuria is a common finding in children with OI, being present in 36% of a series of 47 patients [110,111]. In 124 patients from 14 days to 18 years of age, studied at the Montreal Shriners Hospital, 24 (19%) had at least one episode of hypercalciuria, during a period of observation that ranged from 1 to 8 years,
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before receiving bisphosphonate treatment (unpublished data). This hypercalciuria did not affect renal function, concordant with what was previously described in one series of 12 hypercalciuric patients [111]. In a series of 58 patients, 4 patients developed kidney stones and 1 had papillary calcification without kidney stones. However, it was not clear if these patients were hypercalciuric [134]. One patient was described in the literature with chronic renal failure secondary to obstructive uropathy caused by bony pelvic outlet deformities [1431. Endocrine c h a n g e s Growth hormone (GH) deficiency is rare in patients with OI. Of 22 children with OI tested by Marini et al. [144], none fulfilled the standard criteria for GH deficiency. Children with OI may present with hypopituitarism [145]. Some patients with OI have a hypermetabolic state, typically reflected by excessive diaphoresis and associated with increased oxygen consumption and elevated thyroxine levels [146]. The cause of this hypermetabolic state is not known. For reasons that are unclear, women with OI have late menarche [147]. Respiratory p r o b l e m s Patients with OI may have respiratory complications secondary to kyphoscoliosis. Young patients with OI appear to have normal ventilation/perfusion rates, and restrictive complications are associated with spine deformities [148]. Pulmonary hypoplasia has been described in a newborn with lethal OI [53]. Studies of pulmonary function in patients with OI may show different results [148], and some patients may develop restrictive lung disease, leading to right ventricular failure. When hypoxemia was present, it was not severe, and hypercapnia was never observed [50]. Connective tissue alterations Individuals with OI have a tendency to bruise easily. This may be related to increased capillary fragility caused by the underlying collagen defect. Decreased platelet retention and reduced factor VIII R:Ag have also been described in individuals in OI [149]. Skin of people with OI is stiffer and less elastic than normal skin [150]. Muscle strength is reduced in moderate and severe forms of OI [151,152]. Joint hyperlaxity is common, especially in affected females [153], and it can lead to dislocation of hips and radial heads. Certain individuals with OI are prone to sprains. Flat feet are commonly seen in patients with OI. Hernias can be present [154].
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Constipation is common, which may be due to severe protusio acetabulae and pelvic deformation in children with severe OI [155]. Treatment of constipation is difficult and frequently frustrating.
Ocular changes Individuals with mild OI frequently demonstrate blue sclerae and premature arcus corneae. Arcus corneae juvenilis is an unusual eye finding commonly associated with hypercholesterolemia, but in a large series of patients it was not associated with other clinical or laboratory findings of hypercholesterolemia [17]. In subjects with mild OI there is a progressive arcus corneae that can first be seen in some patients in their late teens [156]. Contrary to what is commonly stated, scleral thickness is normal in OI type I, and the blue color is not a consequence of its transparency. The blue hue results from differential back-scattering of short wavelengths of light by the abnormal molecular organization of the matrix in the sclerae [17]. Scleral collagen fibrils are of normal diameter in OI type I, but there is an increase in electron-dense granular matrix material between collagen fibrils [157]. In other types of OI, the sclerae may be thin, scleral collagen fibrils are reduced in diameter, and the intercollagenous matrix is normal. On the other hand, corneal thickness is significantly reduced in OI type I [156], as in other types of OI. Teeth Some individuals with OI have DI (Fig. 6) [158]. Although the enamel of the teeth with DI is normal anatomically, it may not attach normally to the dentin [159]. The pulp chambers and root canals are completely or partially obliterated by abnormal dentin. The junctions between the crowns and roots are more constricted than normal [160]. The severity of DI is not related to the severity of skeletal involvement in the case of OI,
FIGURE 6 Dentinogenesis imperfecta (DI). Teeth of affected individuals appear transparent due to abnormal dentin. Enamel is normal. The severity of the DI has no relation to the severity of the skeletal involvement in the case of OI. (see color plate.)
and it may be present in patients with mild and severe forms of the condition. Severity may be different in affected members of the same family [161]. The primary dentition is always more affected than the permanent dentition. Radiographically, the teeth show bulbous crowns with a constriction at the coronal-radicular junction. The roots are shorter and more slender than normal. The pulpal spaces are narrow or obliterated [162]. Subjects with OI do not have an increased susceptibility to cavities and do not necessarily have more dental pain. There is no effective way to prevent the problems associated with teeth in persons with OI. One method for treating DI is to crown the teeth as they erupt. The back teeth are especially important to help guide the permanent teeth into place and for proper chewing throughout life. Malocclussion is a common finding in patients with OI, particularly class III (the cusp of the posterior mandibular teeth interdigitate a tooth or more ahead of their opposing maxillary counterparts [163]), and the prevalence is 60-80% [161,164]. This complication is more common than DI, with a prevalence of approximately 28% [164]. Patients may require surgical correction of the malocclussion [166]. Changes in the position of the basal bones also may require orthognatic surgery, which has been performed successfully in these patients [167,168]. Unerupted first and second molars are frequent in OI patients in permanent dentition, which is rare in the general population [164]. Other abnormalities include invaginations and hypodontia [169], which have no relation to the existence of DI. Dental treatment to help prevent dental fractures is available, such as ready-made crowns for primary dentition and tooth-colored crowns for permanent dentition [170]. Birth a n d a n e s t h e t i c c o m p l i c a t i o n s a n d life e x p e c t a n c y There is an increased incidence of breech presentation of OI fetus at term [171]. Recently, a retrospective study on the mode of delivery of children with OI concluded that cesarean delivery does not appear to decrease fracture rates at birth in infants with nonlethal forms of OI, nor does it prolong survival for those with lethal forms [171]. Patients with OI should be considered as high risk for anesthesia [172]. They are prone to fracture and may have neck and jaw deformities that will make intubation difficult, and sometimes severe thoracic deformities and kyphoscoliosis may cause restrictive problems [173]. Also, DI and valvular heart disease may increase the anesthetic risk of these patients. Children with OI may have hyperthermia during anesthesia, but this is
18. Osteogenesis lmperfecta usually not associated with muscle rigidity and rarely progresses to malignant hyperthermia (MH) [173,174], although a case of MH in OI has been described in the literature [175]. MH is a familiar disease, and patients with OI may also be affected, but prophylactic use of dantrolene in these patients is not warranted [174] because MH is considered to be a coincidental occurrence in patients with OI [176]. However, certain drugs should be avoided in patients with OI. Succinylcholine may cause fractures as a result of muscle fasciculation. Pancuronium bromide and atracurium are the muscle relaxants of choice [174]. Despite all these potential problems, life expectancy in subjects with nonlethal OI appears to be the same as that for the normal population [177], except in cases of severe OI with respiratory or neurological complications [178].
PATHOPHYSIOLOGY Collagen plays an essential role in forming an interactive network between the cells that make the extracellular matrix [179] and noncollagenous proteins that lead to proper mineralization of bone. Thus, it is not surprising that when the fundamental structure of the helix is disturbed by a mutation a complex series of secondary consequences will develop. The following discussion categorizes these consequences at increasing levels of tissue organization.
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mutant molecules [189], allowing for a substratum of relatively normal collagen fibers to accumulate. Other matrix proteins can modify the size and organization of otherwise normal type I collagen fibrils and can affect the mechanical properties of the collagen fibers [190,191 ]. For example, copolymerization of type V collagen within the type I collagen fibril influences the size and structure of the type I collagen fibril [192,193]. Another modifier of collagen fiber size is the incorporation of unprocessed type I procollagen producing another form of Ehlers-Danolos Syndrome (EDS) that can overlap with features of type I OI. The EDS-OI-like symptoms appear to result from impairing cleavage of the procollagen propeptide secondary to glycine substitution disruption in the N-terminal helical domain. A similar problem might be expected with a mutation affecting cleavage of the C-terminal propeptide [194]. Mutations in noncollagenous proteins such as decorin [195], fibromodulin [196,197], and microfibrillin [198] can affect the structure or organization of type I collagen fibers, indicating that physical interaction between the two components plays an important role in this process. It would not be surprising if the nonclassical forms of OI result from mutations in proteins that affect some of these binding interactions. Although the absolute amount and composition of hydroxyapatite within OI bone are probably not abnormal, the deformed crystal structure probably contributes to the overall weakened nature of the bone [199-204]. How the helix influences the interaction of noncollagenous proteins and mineral is not fully understood.
Formation of O s t e o i d a n d Mineralization
Function of t h e Ol o s t e o b l a s t
The impact of the glycine substitution on the structure of the collagen triple-helical structure has been demonstrated by X-ray diffraction [180], nuclear magnetic resonance, and circular dichroism [181-184]. The altered structure of the individual triple-helical molecule affects the subsequent formation of collagen fibrils that form from lateral association of individual collagen molecules. X-ray diffraction has shown small fibers with less welldefined lateral growth and more fiber disorganization in tissue obtained from OI subjects [185]. Transmission and scanning electron myography have shown that the periodicity of OI fibrils is normal but the fibrils are disorganized and have wide variation in fiber diameter [186]. Mutations that interrupt the helix decrease the thermal stability of procollagen molecules and render the molecules more susceptible to proteolytic attack by tissue proteases [187]. This may explain the observation that mutant collagen molecules are not uniformly distributed throughout matrix but are found on the surface of bone [188]. Tissue proteases probably select against the
The rough endoplasmic reticulum of OI fibroblasts and osteoblasts is grossly dilated [205] and the secretion of fully formed but mutant procollagen is impaired [206,207]. The role that the hsp47 chaperone protein plays in determining the trafficking of normal and mutant molecules within these cells is believed to be important in detecting the mutant collagen chains and eliciting a cellular mechanism to prevent their secretion [208]. In fact, gene knockout of the hsp47 protein is an embryonic lethal in which an abnormal type of collagen accumulates [209], suggesting that this chaperone protein plays an essential role in selecting for correctly assembled collagen molecules [210]. The retention of the mutant procollagen molecule also leads to posttranslational overmodification of the lysine residues in the helical domain that may further affect the quality of fibril formation. In vitro studies of osteoblasts derived from OI humans [209,210] or OIM mice [211] show diminished markers of osteoblastic differentiation, as well as a reduced rate of
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cell proliferation. If this property of the OI osteoblast persists in vivo, it may be a secondary contributor to the severity of bone disease. Not only is there an impairment in the quantity or quality of the matrix that is produced, but the number of differentiated osteoblasts capable of making a mineralized matrix may also be reduced. The mechanism for diminished osteoblast proliferation and differentiation could be a direct consequence of the retained procollagen molecules with the distended rough endoplasmic reticulum. It may reflect an indirect effect of the quality or quantity of the extracellular matrix made by the preosteoblastic cell that is necessary for osteoblast differentiation [306,307]. Possibly, the high rate of bone turnover characteristic of this disease may lead to exhaustion and/or premature senescence of stem cells capable of generating vigorous osteoblastic cells in vitro, which if present in intact bone will further contribute to the severity of the bone disease, particularly in elderly subjects with OI. M e t a b o l i c activity of Ol b o n e Intact bone is able to sense its mechanical environment and initiate a new round of bone removal or reformation when defective matrix, usually a microfracture, develops (Fig. 7). This fundamental principle of bone biology is continuously called on in OI because the matrix that is produced is defective and subject to microfracture. This situation is reflected in the histology of OI bone, which shows a state of ineffective high bone formation by increased numbers of osteoblasts and osteocytes [84] and an increased number of double-labeled surfaces of normal thickness [214]. In the case of type I OI, the amount of bone formed during a remodeling cycle is decreased compared to controls [214]. It is of note that the occurrence of nonunion fractures is increased in children with OI [215], which is probably related to the decreased bone formation mentioned previously. The level of bone matrix destruction in OI, although not obvious in histological studies, is revealed in the urinary excretion of bone collagen degradative products. Although the measurements are variable because of differences in growth rate and in the underlying mutation [216-218], the dramatic decrease in excretion of degradative products and subsequent increase in bone matrix accretion after bisphosphonate treatment attest to the contribution of osteoclastic activity to the pathogenesis of OI. Murine models of OI are particularly instructive in defining the pathophysiology of OI bone. The OIM mouse model is equivalent to severe nonlethal OI in humans. Analysis of osteoblastic activity in this model suggests that the osteoblast lineage is under constant stimulation to proliferate to build up sufficient numbers
of precursor cells that are then required to progress to full osteoblast differentiation [219]. The activated osteoblastic lineage can be demonstrated by measuring the content of collal m R N A in OI bone or the activity of a type I collagen promoter transgene that is sensitive to osteoblastic activity. In both cases, a high level of transcriptional activity for type I collagen can be demonstrated relative to normal bone. At the same time, the number of osteoclasts is greatly elevated, as is the excretion of collagen-derived cross-links. The net effect is an uncoupling between the signals transmitted from the bone matrix to the bone lineage, in which the bone cells do respond at the gene level but cannot deliver at the protein level. The lineage is already maximally stimulated in response to the activated osteoclastic pathways, but the new matrix that is produced does not improve the mechanical properties of the bone. By analogy, this form of OI can be viewed as a hemolytic anemia of bone. This concept is particularly important for understanding the growth retardation and enhanced fragility of bone during childhood (Fig. 8). It is the balance between matrix formation and resorption that determines bone strength in OI. During periods of rapid linear growth, the deficit between formation and resorption is maximal because bone turnover is enhanced beyond the level that is responsive to mechanical forces. Although normal bone has the reserve within the bone lineage to increase its rate of matrix formation, the OI bone lineage is already maximally stimulated so that it is during the period of linear growth that the deficit in net bone formation is most severe [219]. This may explain why fractures are so severe in the rapidly growing child. Growth retardation may also result from diminished bone formation at the collar region of the growth plate, where signaling between newly forming cortical bone and the proliferating chondrocyte has been demonstrated. With the completion of puberty and cessation of linear growth (the loss of proliferating chondrocytes), bone remodeling slows and a balance between bone formation and resorption becomes more favorable. Thus, puberty does not improve bone strength by stimulating the lineage but instead it stabilizes the skeleton and reduces the need for bone remodeling. When menopause reinstates a state of high bone resorption, the balance between formation and resorption again becomes unfavorable and fractures can return. The additional effect of a chronically stimulated osteoprogenitor lineage and gradual loss of proliferative or differentiation potential with advancing age could result in additional factors contributing to bone loss. Thus, one rationale for instituting antiresorptive therapy is to reduce the rate of bone turnover and prolong the ability of the osteoprogenitor lineage to generate productive osteoblasts into later adulthood.
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FIGURE 7 The bone-forming/resorbing unit and its relationship to OI. A remodeling cycle is initiated by osteoclasts removing old bone matrix followed by new bone matrix filling in the resorption pit. The osteoblast and osteoclast lineages are closely intertwined in this process such that bone mass is increased during childhood and maintained in adulthood. (A) The osteoblast lineage arises from a mesenchymal precursor cell and undergoes a series of proliferative and differentiation steps. (B) In normal bone, the activities of the two lineages are balanced. (C) In OI bone, the osteoclastic lineage is highly activated to remove defective matrix and the osteoblastic lineage responds in an attempt to replace the resorbed bone. However, the synthetic activity of the formation response is compromised and the new matrix that is produced is no better than that which was removed. Thus, OI bone is characterized by an increased number of bone-resorbing and -forming packets. The bone is more cellular because the rapid turnover precludes the time needed for late bone maturation and the formation of resting osteocytes.
T h e h e t e r o z y g o u s M o v 13 m o u s e is a m u r i n e m o d e l for mild nondeforming OI. Affected mice demonstrate h a l f o f n o r m a l levels o f collal m R N A as a c o n s e q u e n c e
o f i n a c t i v a t i o n b y a r e t r o v i r a l i n s e r t i o n a l event. T h e b o n e s s h o w d i m i n i s h e d c o r t i c a l t h i c k n e s s , w h i c h is c o n s i s t e n t w i t h h u m a n O I , a n d l o w levels o f p r o c o l l a g e n
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FIGURE 8 Bone formation and degradation in normal and OI bone. During the period of rapid growth, normal children have an accelerated rate of bone formation to make new matrix to support somatic growth and to replace the bone lost due to remodeling. Once the full skeletal mass is acquired, the rate of bone remodeling decreases, and the rate of growth decreases to match the remodeling rate. Thus, bone mass increases rapidly during somatic growth and peak volume is achieved in early adulthood. In OI, bone degradation is high due to the effort to remove defective matrix, and most of the bone-forming activity is expended to keep pace with the intrinsic rate of bone loss. The additional bone loss secondary to somatic growth is not compensated by a further increase in bone formation so that somatic mass does not increase during childhood. Thus, without the addition of bisphosphonates, bone mass increases and somatic growth occurs very slowly. Once puberty is attained and linear growth stops, the extra loss of bone matrix attributable to somatic growth is eliminated so that the bone formation effort can result in increased quantity and quality of the bone matrix. Thus, bone mass does increase after puberty and the fracture rate declines because the bone can be remodeled to become more structurally sound. With the loss of sex hormones and a return of a higher rate of bone degradation, the deficit in bone formation relative to bone loss will return.
propeptide in blood reflect the low output of the type I collagen-producing cells [220]. Histomorphometry does show increased osteoblast cellularity and bone-forming units, and dynamic histomorphometry suggests a decrease in osteoid seams [221]. Excessive osteoclastic activity does not appear to be present. In both mouse and man with type I OI, significant skeletal remodeling is apparent upon sexual maturation so that the mechanical properties of the bone are near normal [222,223]. Although further analysis of a murine model that is
healthy into adulthood is necessary, it appears that the deficit between bone formation and resorption in type I OI is much less than that in deforming forms of OI, particularly after the adult skeleton is established. Thus, it is during adulthood that a relatively normal bone matrix is accumulated and fractures are uncommon. Only during growth and menopause is this relationship unfavorable, again emphasizing the value of bisphosphonates for improving bone strength during these periods.
18. Osteogenesis Imperfecta THERAPY Until recently, treatment of OI focused on fracture management and surgical correction of deformity whenever possible. All medical therapies other than those directed at symptomatic pain relief had been ineffective [224], including vitamin C [225,226], sodium fluoride [12,227,228], magnesium [229,230], and anabolic steroids [231,232]. Early studies of the use of calcitonin for the treatment of OI appeared to show significant biochemical changes in patients with OI and a reduction in the number of fractures from pretreatment to treatment periods [233-235]. Other studies, however, showed that biochemical changes are not accompanied by significant clinical responses, and that patients may develop complications such as calcitonin dose-related hypomagnesemia [236,237]. The use of calcitonin treatment for OI has been abandoned. Antiresorptive a g e n t s Pamidronate is a second-generation bisphosphonate with a chemical structure based on pyrophosphate, the only naturally occurring inhibitor of bone resorption [238]. The exact mechanism of action of the bisphosphonates remains unclear, although effects on both osteoblasts [239,240] and osteoclasts [241] have been documented. There have been several case reports of treatment of children withOI with bisphosphonates [242,243,244,245, 313]. Glorieux and his group administered pamidronate by intermittent intravenous infusion for up to 9 years in more than 150 children with severe OI aged 2 months to 18 years. In the first publication [246], they studied 30 children over 3 years of age. Cyclical IV pamidronate resulted in sustained reduction in serum alkaline phosphatase concentrations and in the urinary excretion of calcium and type I collagen N-telopeptide. There was a mean annualized increase of 41.9 + 29.0% in bone mineral density, and the deviation of bone mineral density from normal, as indicated by the z score, improved from -5.3 + 1.2 to -3.4 + 1.5. The cortical width of the metacarpals increased significantly, and the increase in the size of the vertebral bodies suggested that new bone had formed. The mean incidence of radiologically confirmed fractures decreased. Treatment with pamidronate did not alter the rate of fracture healing, the growth rate, or the appearance of the growth plates. All children reported substantial relief of chronic pain and fatigue. In children younger than 3 years of age, the results were more remarkable. A group of nine patients severely affected with OI (types III and IV; mean age: 10.2 months at entry. Range: 2.6 to 20.7 months) received pamidro-
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nate treatment for 12 months [247]. The drug was administered intravenously in cycles of 3 consecutive days. Patients received doses ranging from 8.5 to 20.5 mg/kg/ year. This group was compared to a historical control group consisting of six age-matched, severely affected OI patients who had not received any treatment for OI but had followed the same multidisciplinary support program. Under cyclical pamidronate treatment, bone mineral density (BMD) increased 65-227% in 1 year. The z score increased significantly, whereas in the control group a significatn decrease in the BMD z score was observed. Vertebral coronal area increased in all treated patients but remained unchanged in the untreated group. In treated patients, the fracture rate was also significantly lower than in the control group. No adverse side effects were noted apart from the well-known acute phase reaction during the first infusion cycle. Signs of bone pain (e.g., crying while being handled) disappeared within days.Vertebral size increased in all treated children, as should be expected in growing individuals. In contrast, a decrease in vertebral size was noted in half of the untreated children, indicating that vertebral collapse had occurred in these patients. The youngest patient to start pamidronate treatment in the Montreal clinic was 14 days of age. The radiological and microscopic changes under treatment were striking (Figs. 9 and 10). Fracture incidence is a weak efficacy parameter in open therapeutic studies of OI patients because it can be influenced by external factors (e.g., mode of handling and mobility) and may also spontaneously decrease with age [8]. However, despite higher risk of injury due to increased mobility, a marked decrease in fracture rate was noted, suggesting a direct effect of therapy. Bisphosphonates, on the other hand, do not appear to interfere with fracture healing [248]. The disappearance of bone pain and decreased fracture incidence may have contributed to greater mobility [249]. Physical activity is an essential factor for the development of the skeletal system [250]. Thus, increased mobility may synergize with the direct inhibitory effect of pamidronate on bone resorption to increase bone mass. The effect of bisphosphonate therapy on growth was a matter of concern before the treatment was used in children. In animal studies, long-term treatment with bisphosphonates did not affect linear growth unless very high doses were administered [251]. In young patients, pamidronate did not have a detrimental effect on growth. Instead, the height Z score increased in all the patients who started treatment before 3 years of age [247]. In a larger group of patients, height Z-scores increased significantly in patients with type II OI and did not change in patients with type I and type IV OI after one year of pamidronate therapy. After four years of
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pamidronate therapy, mean height Z-scores increased significantly in children with type IV OI, while patients with type I and type III OI showed non-significant trends of increase [252]. The success of bisphosphonate treatment in patients with Paget disease of bone appears to be related to the unremitting osteoclastic activity characteristic of OI. The effect of the drug can be monitored by measuring parameters of bone resorption, such as urinary calcium excretion and the excretion of collagen breakdown by-products such as the collagen hydroxylysine glycpsides [253] and the collagen cross-links pyridinoline and deoxypyridinoline. Plasma alkaline phosphatase activity (as a measure of bone osteoblast activity) also decreases [254,255]. Clinical symptoms of bone pain and diaphoresis also correlate with the inhibitory effect of the drug on osteoclastic activity, suggesting that it is the process of high bone turnover and associated high
blood flow, not unlike a pagetic lesion, that underlies these symptoms. The most common side effects are a flu-like syndrome in 80% of the patients the first time they received treatment and, in some infants, a transient decrease in blood cell count that recovered to normal values in 48-72 hours. Delayed fracture healing may be present with chronic pamidronate use (personal observation). Patients taking alendronate have the theoretical risk of gastric discomfort or even severe burning of the esophagus if the drug is not taken properly. Histomorphometric studies [256] showed that biopsy size does not change significantly with pamidronate treatment in children with OI, but cortical width increases by about 90%, and cancellous bone volume increases by about 45%. This is due to higher trabecular number, whereas trabecular thickness remained stable. Indicators of cancellous bone remodeling decrease by 26 to 75%. These results suggest that
FIGURE 9 Radiological changes under bisphosphonate treatment. Progressive healing of vertebral fractures (a), increased length and cortical width of long bones (b), and reshaping of the head due to growth of the facial bones (c) are observed with the use of intravenous pamidronate in children with OI. Note the hyperdense bands in the metaphysis; each one corresponds to a treatment cycle.
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FIGURE 9
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in the growing skeleton pamidronate has a two-fold effect: Both bone resorption and formation are inhibited, but osteoclasts and osteoblasts are active on different surfaces (and are thus uncoupled) during modeling of cortical bone. This causes a selective targeting of resorption while continuing bone formation can increase cortical width. Importantly, there was no evidence for a mineralization defect in any of the 45 patients studied.
Biochemistry studies [108] showed that concentrations of ionized calcium drop and serum parathyroid hormone levels almost double after the first pamidronate infusion. At the same time, urinary excretion of the bone resorption marker type I collagen Ntelopeptide related to creatinine (uNTX/uCr) decreases by approximately 60-70%. Two to four months later, ionized calcium returns to pretreatment levels, and parathyroid
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FIGURE 10 Microscopic bone changes under bisphosphonate treatment in a 10-year-old boy with OI. Cortical width is significantly increased after 2 years of treatment with intravenous pamidronate, as seen in this pair of iliac crest biopsies stained with trichrome. The baseline biopsy is shown on the left. Bone biopsy courtesy of F. Rauch. (see color plate.)
hormone concentrations are still above baseline values in patients below two years of age. During four years of pamidronate therapy in 40 patients, ionized calcium levels remained stable, but parathyroid hormone levels increased by about 30%. However, no patient had a result that was more than 60% above the upper limit of the reference range, uNTX/uCr, expressed as a percentage of the age and sex-specific mean value in healthy children, decreased from a mean of 132 at baseline to a mean of 49 after four years of therapy. Therefore, serum calcium levels can decrease considerably during and after pamidronate infusions, requiring close monitoring, especially at the first infusion cycle. In long-term therapy, bone turnover is suppressed to levels that are lower than in healthy children. These treatments should always be given as part of a strictly controlled protocol. An adequated calcium and vitamin D intake is warranted, particularly in regions with low sun exposure. Daily vitamin D requirements are of 400 IU, and calcium requirements vary with age. Long-term effects of bisphosphonate treatment are not know. Therefore, this therapy should be administered under strict research protocols. Anabolic a g e n t s Growth hormone, insulin-like growth factor-l,and parathyroid hormone have the potential to increase bone mass. Except for a treatment protocol with GH in children with deforming OI, most experience with these
agents has been anecdotal. There are no large studies on the use of GH therapy in patients with OI. An increase of fracture rate during GH therapy has been reported [257,258]. In a controlled study comparing seven children with mild OI with seven children receiving no treatment, the fracture rate was not different between the groups [259]. Reported side effects of GH therapy are arthralgia, myalgia, carpal tunnel syndrome, pesudotumor cerebri, benign intracranial hypertension, slipped capital femoral epiphysis, and transient insulin resistance [260]. There are no data regarding final height in OI patients treated with GH. It has been suggested that GH should probably not be used as a first-line therapy in OI [261]. Like all children who are initially started on GH, OI children do experience an initial acceleration of growth rate [109]. Given the underlying physiological basis of OI, it would be surprising if an agent that furtherstimulates more bone turnover as part of its anabolic action has a longterm beneficial effect. The osteoblast lineage is already maximally stimulated and the addition of agents that enhance osteoclastic activity will only contribute to the deficit between formation and degradation. The transiently increasing growth rate that is seen in children with GH occurs because the growth plate is stimulated to proliferate. If the bone that contains the growth plate (collar region and primary spongiosa) is not more structurally sound than before the stimulus, damage to the growth plate might be anticipated. Potentially, the combination of GH and bisphosphonate might provide a compromise that is acceptable, and studies using this
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combination may be of interest. However, this is another therapeutic setting requiring animal experimentation for concept validation. Orthopedic management The surgical outcome of patients with OI has improved significantly with the introduction of bisphosphonate treatment. Patients can now have rodding surgery as early as 18 months of age without complications. The preferred technique is to rod one leg and wait at least 7 days before rodding the other leg. Using this technique, the need for a blood transfusion is minimized (F. Fassier, personal communication). For the femora, the extensible rods (Dubow-Bailey [262,263] or Fassier-Duval [264]) are preferred, and the number of osteotomies should be as small as possible. Patients should weight-bear as soon as possible, usually approximately 3 weeks after surgery. After rodding surgery, most previously nonambulatory patients with OI are able to walk [265]. The complication rate for Dubow-Bailey rods ranges from 63.5% [266] to 72% [267] and is approximately 50% for nonelongating rods. The reoperation rate is similar for both types of rods. The most common complication is rod migration, and infections, pseudoarthrosis, lack of elongation or overelongation of rods. Loosening of the terminal T piece may also occur [266]. A new elongating rod, called Fassier-Duval [264], opens interesting possibilities for the surgical treatment of patients with OI. This rod allows for the introduction of the whole device through the greater trochanter without the need to open the knee joint. Post-operative management is then greatly facilitated. For tibiae, due to the difficulty of opening the ankle joint, proximal insertion of rush rods is preferred. Patients should wear below-knee orthosis to protect the bone from fracturing, particularly after they have outgrown the rod, and to prevent bowing of the unprotected distal part of the tibiae. Patients with severe OI almost always have spinal deformities, with a prevalence that may be as high as 92% [19,268]. More than half of the cases of scoliosis are located in the thoracic region, and pectus carinatum and pectus excavatum are common associated findings [269]. These deformities increase with age [268,270], and bracing does not stop the progression of the curve [271]. Thoracic scoliosis of more than 60 ~ has severe adverse effects on pulmonary function in patients with OI [272]. Fusion with bank bone graft [273] and with Keil bone graft without instrumentation [274] has been used for the treatment of severe scoliosis in patients with OI, as has halo gravity traction and posterior spondylodesis with instrumentation [275]. The ideal surgical treatment of severe scoliosis in patients with OI has yet
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to be determined, and it remains a difficult procedure [276]. Occupational therapy and physiotherapy Physical activity is a key factor in the response of these patients to treatment and for the achievement of a better quality of life [277]. When a patient has a fracture and is immobilized for a certain period of time, the bone density declines dramatically [278]. The bisphosphonates will protect the patient from bone loss, but there may be little or no gain in the bone density for a while. It is important that physiotherapy be administered by professionals who have experience with patients with OI. Some evaluation tools have been validated for OI [279]. Exercises should be prescribed following a program designed specifically for each individual, encouraging parental participation and bonding. Water therapy is highly recommended for patients with OI. In three different groups of OI patients able to ambulate, it was found that preventable functional impairment is caused by shoulder joint and hand contractures and upper extremity weakness in children able to stand in braces; by hip flexion and plantar flexion contractures of the feet, shoulder joint contractures, and upper extremity weakness in patients able to ambulate short distances without braces; and by poor lower extremity joint alignment, impaired balance, and low endurance in children able to ambulate in the community without assistance [280]. The aim of these programs is to employ children in graded exercise regimes and foster their increased involvement in school and social situations. Results suggest that aggressive physical therapy and rehabilitation have a major role in the overall care of infants and children with OI [281]. Sitting devices should be designed to allow comfortable sitting positions as early as possible. Children develop tolerance for sitting position gradually by progressively decreasing the degree of tilt of the sitting devices. The goal is head control (J. Ruck-Gibis and K. Montpetit, personal communication). Psychosocial aspects are extremely important in the management of patients with OI. Issues regarding selfesteem, sexuality, and peer integration must be addressed to properly care for these patients, particularly during adolescence [282]. OI children have no intellectual deficits; therefore, they should be attending regular schools. The following rules should be followed to permit better school integration of children with OI: 9 School must be accessible for handicapped children. 9 The school should have an access ramp, accessible toilets, mobile tables and chairs, and a wheelchair in case of emergency. 9 The school should have an emergency evacuation plan adapted to handicapped children in case of fire.
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9 Some OI children lack balance and need better supervision in school yards or on icy surfaces. 9 OI children should leave 5 min before the end of classes in order to avoid crowds. 9 In physical education, OI children should not participate in any contact sport. However, participation in physical education should be strongly encouraged but with respect to the child's limits. 9 The equipment used should be soft (e.g., balls). 9 The child should rest when he or she is tired. 9 The child should wear ortheses at all times. 9 Other elements facilitating school integration include adjustable desks, wheelchairs with trays, floor mats for rest periods, adapted toilets, and reachers. Given the complexity of the clinical management of OI, a multidisciplinary clinical team approach to treatment is of greatest value for both the patient and the field. Not only are there significant orthopedic and medical issues but also problems of daily living are pervasive. Proper handling during infancy, mainstreaming within schools, driving an automobile, attending college, scoliosis and pulmonary insufficiency, neurological symptoms, pregnancy and genetic risk, and acceleration of bone disease after menopause are complex problems that are difficult for an individual clinician to manage and require an experienced and broad-based treatment team.
Future t h e r a p e u t i c o p t i o n s Because bisphosphonates do not correct the primary cause of OI and the long-term use and effectiveness of antiresorptive agents are uncertain, steps to correct the underlying genetic mutations are being evaluated in both humans and mice. The rationale for gene therapy in OI is derived from the analysis of individuals who are somatic mosaic for an OI mutation but do not have evidence of bone disease. This clinical phenomenon suggests that the deleterious effect of OI cells can be countered by the presence of normal cells. Thus, if it were possible to introduce normal cells into an individual with OI, the severity of bone disease would be reduced. This treatment strategy requires the ability to introduce cells from the osteoblast lineage into OI subjects, with the attendant problems of immune rejection unless a tissuematched donor can be identified. Human transplant studies with bone marrow cells have been performed in a limited number of children with severe OI [283,284]. The success of these initial studies is difficult to assess [285], and a proof of principle experiment in animals is required before human experimentation is undertaken. Perhaps developments using partially differentiated em-
bryonic stem cell will provide another approach for cell engraftment. Assuming that the immune problems related to bone cell transplantation will be a major impediment for longterm engraftment of bone, strategies are being developed to correct in vitro the primary defect in type I collagen production of an individual with OI followed by reintroduction of the corrected cells into the affected host. This requires a two-step process in which the output from the mutant collagen allele is inhibited and a replacement collagen gene for the inactivated mutant gene is inserted. Once corrected, the engineered cells must have the ability to engraft bone, proliferate, and participate in new bone formation. Each facet of these problems is in itself a major research undertaking. Allele-specific suppression of a mutant collagen gene is potentially possible at the genetic or the RNA level. Targeting the endogenous gene with a chimeric R N A DNA oligonucleotide [286] can correct the specified sequence. Although the frequency of this modification is variable, the change is permanent and the output of collagen production is restored to a normal level. The other option is to reduce the output by targeting the RNA from the mutant allele. Although antisense constructs to a RNA transcript is unlikely to have allele-specific discrimination, other strategies, such as hammerhead [287] and hairpin [288] ribozymes, U lsnRNA [289], RNA transplicing [290,291], and RNase P [292], have such a potential. Yet to be evaluated is vector expression RNAi, a strategy that is widely used in lower organisms and has recently been shown to be effective in mammalian cells [293-295]. A detailed description of the background and mechanism of each anti-RNA effector is beyond the scope of this chapter. It is unlikely that any one approach will have sufficient strength and specificity to inhibit the mutationcontaining transcript sufficiently to have a major impact on disease severity [296]. However, combining two or more anti-RNA approaches that act in different compartments within the cell and by different molecular mechanisms may attain this goal. Another challenge is the introduction of a procoUagen cDNA expression construct to replace the lost activity of the suppressed transcript. Collagen gene expression has problems that differ from those of gene replacement for an enzyme or clotting factor. In bone, type I collagen production can account for 20% of total protein synthesis so that an extremely strong promoter is required to drive the replacement cDNA. Another consideration is the vector that delivers the correcting construct. Although a collagen cDNA has been strongly expressed from an adenoviral construct [297], this approach does not achieve permanent expression. Retrovectors have the
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size capacity and permanent integration needed to contain and express a collagen promoter-collagen c D N A construct. However, expression of the transgene can be suppressed after the transduced cells are reintroduced into the host [298]. Modification of the retrovector that removes sequences responsible for suppression appears to overcome this problem, allowing for osteoblastspecific expression of the transgene throughout the life of the mouse [299]. It remains to be demonstrated that this vector approach can achieve the level of collagen c D N A expression equivalent to that of an endogenous collagen allele. The most severe problem for somatic gene therapy for OI is reintroduction into a host of cells that are capable of homing to bone and participating in new bone formation. Although the ability of marrow stromal cells to differentiate into mature osteoblasts in vitro or in subcutaneous implants is well-known [300,301], demonstration that this is possible when administered systemically is still unconvincing. Most studies in man and mouse can demonstrate a low degrees (1-5%) of engraftment of bone or bone marrow stroma as assessed by a transgenic or unique endogenous genetic marker [302-304]. In many cases, the marker gene does not discriminate whether this cell arises from a mesenchymal or macrophage lineage. Even when relatively pure populations of stromal cells are used for transplantation, minor contamination from the myeloid lineage could belie stromal cell engraftment. Only one study has demonstrated expression of a transgene that is a marker of a differentiated osteoblast [305], although the level of engraftment and its contribution to bone formation are difficult to assess. To complicate the analysis, stromal cells appear to have the ability to fuse with resident cells [285]. Fortunately for patients with OI, bisphosphonates have provided a therapeutic choice for improving bone health at a time when the promise of cell or gene therapy is yet to be demonstrated. These drugs have raised the bar for assessing the success of any new therapy because it will be necessary to demonstrate that an experimental approach has either short- or long-term advantages over existing pharmacological regimens. Animal studies are necessary to demonstrate that gene or cell therapy does offer options, and objective measures of success need to be developed that allow comparison of two treatment modalities.
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I19I Sclerosing Bony Dysplasia L. LYNDON KEY,JR., and WILLIAM L. RIES Department of Pediatrics, Medical University of South Carolina Charleston, South Carolina
INTRODUCTION As noted by Michael Whyte [1], osteosclerosis and hyperostosis refer to trabecular and cortical thickening, respectively. This descriptive approach to bony dysplasias has provided a means of classifying these disorders into two major groups: those resulting from defects in endochondral bone formation (primarily affecting all bone, trabecular and cortical) and those resulting from defects in intramembranous bone formation (primarily affecting cortical bone) [2]. Clinically, these disorders follow two distinctive patterns. Those affecting endochondral bone formation result in a marked deformity of the skeleton due to defects in formation, modeling, and remodeling of bone. These defects affect the entire skeleton. Defects in intramembranous bone do not affect growth and modeling. Thus, these patients tend to have more localized skeletal defects. Until recently, it has only been possible to determine that these defects result primarily from an imbalance between formation and resorption (Fig. 1). Although the etiology remains an imbalance between formation and resorption, this chapter introduces the concept, based on known genetic defects [3-2], that the skeletal dysplasias result from specific genetic defects resulting in excessive formation of bone by osteoblasts or from defects in bone resorption by osteoclasts (Table 1). Although there are a variety of conditions for which no specific defect is known (Table 2), it is likely that specific genetic abnormalities in resorption and formation will be found alone or in combination in the various sclerosing bony dysplasias.
PediatricBone
FIGURE 1 Graphic depiction of the bone resorption and formation cycle. If the amount of bone formed exceeds the amount of bone that is being resorbed, bone will accumulate. The imbalance between bone formation and resorption is the basis for the generation of sclerosing bony dysplasias (reproduced with permission from Baron [41]).
DEFECTS IN OSTEOCLASTIC BONE RESORPTION Two sclerosing bony dysplasias have genetic defects in osteoclastic function. Defects that result in failure of osteoclasts to resorb bone normally have been shown to result in osteopetrosis and pycnodysostosis. Both of these diseases result in a global lack of modeling and remodeling. The genetic defects identified in the severe forms of osteopetrosis result in an inability of the osteoclasts to acidify the ruffled border space [10-12]. In milder forms, defects in the processes that allow acid production to continue [13] and defects in stimulating osteoclastic activation [9] have been characterized. In pycnodysostosis, a defect in the production of cathepsin K has been identified [8]. Cathepsin K is a lysosomal
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Copyright2003,ElsevierScience(USA). All rightsreserved.
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L. Lyndon Key,Jr., and William L. Ries TABLE 1
Classification of pediatric sclerosing bony dysplasias
Disorders I.
Endochondral bone dysplasias
Osteopathia striata (Voorhoeve's disease) with cranial stenosis (sclerosis?) Osteopetrosis Infantile malignant Carbonic anhydrase II deficiency Anhydrotic ectodermal dysplasia with immunodeficiencyand lymphoedema Osteopoikilosis Pcynodysostosis II.
Membranous bone dysplasias
Endosteal hyperostosis Hyperostosis corticalis generalisata (van Buchem disease) Sclerosteosis (cortical hyperostosis with syndactyly) Hereditary multiple diaphyseal sclerosis (Ribbing disease) Progressive diaphyseal dysplasia (Camurati-Engelmann disease) Endochondral and intramembranous bone dysplasias (Mixed bone dyplasias)
Craniodiaphyseal dysplasia Craniometaphyseal dysplasia Dysosteosclerosis Melorheostosis Metaphyseal dysplasia (Pyle's disease)
TABLE 2
cystine protease that is localized almost exclusively in the osteoclast and is capable of degrading bone proteins. The defects identified in these two sclerosing dysplasias indicate that specific defects in osteoclastic bone resorption cause an accumulation of excessive bone and a generalized defect in bone modeling and remodeling. In the following sections, the clinical syndromes are discussed, including a clinical description of the different disorders and the specific defects involved.
Osteopetrosis In osteopetrosis [14], failure to resorb bone and calcified cartilage results in a pathognomic histological finding--the presence of remnants of the primary spongiosa. Primary spongiosa is generated from the cartilage that was mineralized as a precursor to endochondral bone formation. This calcified cartilage should be resorbed by chondroclasts and osteoclasts present during fetal life, leading to the eventual formation of lamellar bone by the developing osteoblasts. The presence of these cartilaginous islands suggests that the defect(s) is present from the earliest stage of skeletal development. A variety of clinical types of osteopetrosis have been described. The original description of osteopetrosis was published by Albers-Sch6enberg in 1904 [15]. Two major types have been described: an autosomal dominant adult type and an autosomal recessive infantile form [16]. The
Genetic defects in sclerosing bony dysplasias Gene/protein
Inheritance mode
Congenital
Endochondral bone formation Osteopetrosis Infantile malignant
AR
Yes
CLCN7/C1C-7
chloride channel absence Infantile malignant
AR
Yes
TCIRG1/vacuolar pump subunit defect
AR
Yes
CA2/carbonic
X-R
Yes
IKBKG/impaired NF-•I3 signaling through NEMO
Pcynodysostosis Intramembranous bone formation
AR
Yes
CTSK/absence of cathepsin K cysteine protease
Camurati-Engelmann (Progressive diaphyseal dyplasia)
AD
No
TGFB 1/latency-associated peptide defect
Sporadic
No
TGF-13induced adhesion protein (13ig-h3) deregulation
AR
No
SOST/absence of sclerostin generation
Disorder
Carbonic anhydrase II deficiency anhydrase II absence Including anhydrotic ectodermal dysplasia with immunodeficiency and lymphoedema
Melorheostosis Sclerosteosis
19. Sclerosing Bony Dysplasia
adult type, although usually associated with less severe findings, can present early in life and result in substantial disabilities; however, it may be so mild as to go unrecognized and rarely results in premature death. The infantile malignant or progressive form of osteopetrosis is diagnosed in infancy and is usually fatal during the first decade of life [17]. A third, milder form of osteopetrosis (MIM 259710) was described by Kahler et al. [18]. This form is present at birth but has a slower progression. The life span of patients with this form is unknown, but several patients have lived into their third or fourth decade. A fourth group of osteopetrotic patients has an autosomal recessive syndrome characterized by renal tubular acidosis and cerebral calcifications (MIM 300301). A transient form has been described that presents in infancy and gradually improves during the first year of life. No long-term sequellae seem to remain and the radiographic appearance and shape of the bones return to normal by 1 year of life [19]. The fifth clinical syndrome results in a severe, lethal form that is associated with a neuronal storage disease (MIM 259730) [20]. A sixth syndrome, with X-linked inheritance, has recently been described with anhidrotic ectodermal dysplasia and immunodeficiency (MIM 300301) [9]. The genetic defects associated with osteopetrosis are all related to the dysfunction of the osteoclast. This is not surprising since osteopetrosis was found in 1980 to be due to an osteoclastic defect. A successful bone marrow transplantation with an opposite-sex donor resulted in the reversal of osteopetrosis and the presence of osteoclasts containing a Y chromosome in a female recipient [21]. Since this replication of the original experiments performed by Walker et al. in 1973 in osteopetrotic animals, it has been clear that defective osteoclasts can cause osteopetrosis. In 1996, Lajeunesse et al. [22] reported that replacing the osteoblasts by bone marrow transplantation cured two individuals with osteopetrosis who lacked osteoclasts secondary to a defect in M-CSF production. However, precise defects associated with a variety of clinical syndromes have only recently been described. Since osteopetrosis appears to result from defective osteoclastic function, the increased bone appears to represent more formation of bone than resorption. The resulting accumulation of bone is the cause of abnormalities seen in osteopetrosis, resulting from an inability to resorb cartilage and bone during initial formation, modeling, and remodeling of bone.
Carbonic Anhydrase Deficiency: The First Osteopetrotic Defect Defined in Humans Since 1983 [14], carbonic anhydrase type II deficiency has been the only known genetic cause of defective bone resorption. The syndrome is classified in the group of dysplasias that affect all areas of bone formation, mod-
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eling, and remodeling. This type of "global" defect in bone resorption results in clinical features such as a defect in endochondral bone formation. Associated clinical features include short stature, abnormally shaped bones (especially dental malocclusion), increased bone density and fragility (leading to fracture), and an inability to remodel cranial nerve foramina, resulting in compression of cranial nerves. Carbonic anhydrase II catalyzes the reaction converting CO2 + H20 H2CO3. This is followed by the liberation of a hydrogen ion, H2CO3 ~ H + + HCO3-. The hydrogen ion generated is then excreted into the ruffled border space by the proton pump. This acidifies the ruffled border space, resulting in the dissolution of hydroxyapatite. In the absence of this enzyme, patients have radiographic features of osteopetrosis. Despite these abnormalities, the condition is one of the milder forms of osteopetrosis, perhaps because the defect in carbonic anhydrase type II slows but does not completely preclude the formation of acid within the ruffled border space. An exceptional feature is the high frequency of intracranial calcification, rarely observed in other forms of osteopetrosis. Patients frequently present in childhood with a fracture or with cranial nerve entrapment; however, the life span is not shortened, with patients surviving into adulthood. Patients also have a distal renal tubular acidosis (RTA) due to an inability to acidify urine in the kidney. Aminoaciduria and glycosuria are not present, suggesting a specific rather than global renal tubular defect. Patients may also have mild to moderate deficits in cognitive functioning, but intellect is quite variable. No therapy has been completely successful in reversing this disease; however, the RTA may be treated with oral bicarbonate solutions. McMahon et al. [23] demonstrated that bone marrow transplantation in this disorder may reduce central nervous system calcifications, cures the bone disease, but does not reverse the nephrocalcinosis and RTA.
Adult Forms of Osteopetrosis Adult forms of osteopetrosis are often referred to as benign osteopetrosis. There are two clinically recognized forms. Massive sclerosis of the skull and a diffusely increased thickening of the long bones and spine characterize type 1 adult osteopetrosis (ADO1 MIM 259700). Bone strength, unlike that seen in the dense bones in the more severe forms, is excellent with few fractures. Type 2 adult osteopetrosis (ADO2, MIM 166600) is characterized by massive sclerosis of the base of the skull, hypersclerotic end plates of the vertebral spine, and dense long bones. The increased density in the spine results in the "rugger-jersey" sign, characterized by the alternating dense end plates and less dense bodies of the vertebrae. In ADO2, the bone turnover is decreased, which leads to
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a reduction in tensile strength. Fractures are common. However, in each type, approximately 40% of patients have relatively silent disease, frequently not diagnosed until an incidental radiograph is taken. A major complication of the adult forms of osteopetrosis is frequent bony infections (especially osteomyelitis of the mandible). The frequency of mandibular infection is a result of poor tooth eruption and disordered formation of the root structures and crown of the tooth. These defects often make it necessary to extract the teeth. Poor angiogenesis within the sclerotic bone results in an inability to recruit osteoblasts and inflammatory cells, leading to incomplete repair of the bone and an increase in infection. As a result, attempts to treat mandibular infections with antibiotics have been disappointing. The use of early and frequent debridement may help to cure or stabilize the problem. There is no established treatment for these diseases. Bone marrow transplantation is not recommended due to the relatively high rates of morbidity and mortality (up to 50%). Calcitriol in relatively high doses (up to 32 lag per day) markedly increases bone resorption in 25-35% of patients. In 1991, Gram et al. [24] described an elevation in the creatine kinase BB isozyme in patients with type 2 adult osteopetrosis. The creatine kinase BB isozyme (CK-BB) is normally concentrated in the brain, but it is also the major isozyme of CK that is found in osteoclasts [25,26]. This isozyme is not normally measurable in the serum. When elevated in patients with osteopetrosis, this finding is diagnostic of the ADO2 phenotype. Tartrate-resistant acid phosphatase (TRAP) is also elevated in the serum of patients with ADO2 [27. These elevations in serum levels of TRAP and CK-BB may result from leakage of the enzyme from the ruffled border of defective osteoclasts. Measurement of these two enzymes is recommended to establish the type of adult osteopetrosis. Whether these enzymes are elevated in all types of osteopetrosis is controversial; most clinicians have not found these isozymes in ADO 1 or in the infantile forms. Although the precise defect causing reduced bone resorption is unknown, it has been suggested that the osteoclast may not adhere normally to the bone and that the leakage of enzymes occurs at the level of the ruffled border.
Progressive Form of Osteopetrosis (MIM 259700) The most severe form, malignant infantile osteopetrosis (frequently called the progressive form), has the following features (Fig. 2): a reduction in bone marrow space; bone fragility; pancytopenia; cranial nerve compression; infections, frequently with septicemia; and death in the first decade. In patients with the progressive
FIGURE 2 Although there are a variety of osteopetrotic conditions, the hallmark of these disorders is a generalized defect in bone resorption. This results in total skeletal sclerosis, poor growth, and malformed, poorly modeled and remodeled bones (reproduced with permission from Beighton and Cremin [42], p. 25, Fig. 4).
form, inheritance is autosomal recessive. Interferon-7 (IFN-7) with high-dose calcitriol and bone marrow transplantation are effective therapies. A number of genetic defects have been described that appear to be causative in patients with the progressive form of osteopetrosis. The defects are related to the ability of the osteoclast to acidify the ruffled border space, a necessary chemical step in removing mineral from the matrix. In one group, the defect is a mutation in the gene encoding the OC116 subunit of the proton pump (MIM 604592) [10,11], and in another group there is a defect in the gene encoding the chloride channel (C1C-7, MIM 602727)[12]. The mutation in OC 116 encodes the ct3 subunit of the H+-ATPase proton pump [10]. This defect appears to result in a very severe form of osteopetrosis, with early
19. Sclerosing Bony Dysplasia
bone marrow failure and optic atrophy heralding the most severe phenotype. If not treated with bone marrow transplantation or a combination of calcitriol and IFN-y, these patients will universally die before the end of the first year of life. The second group, with a defect in the C1- channel (C1C-7), causes severe disease. The phenotype features cranial nerve compression; however, bone marrow failure and early death are less likely than in the proton pump abnormality. According to the literature, 50-75% of all cases of severe, progressive osteopetrosis are caused by these two mutations [10-12]. However, this analysis is based on a limited number of patients screened. In the remainder of patients with a similar phenotype, no genetic defect has been found.
Ectodermal Dysplasia with Osteopetrosis and IKBKG gene-NF-KB Signaling Abnormality (MIM 300301) Doffinger et al. [9] described a new form of osteopetrosis characterized by ectodermal anhydrotic dysplasia and osteopetrosis associated with an immune deficiency (EAD/ID) and lymphoedema. The disease has an X-linked recessive inheritance pattern. This form of osteopetrosis results from a defect in the I K B K G gene for the NEMO protein, the regulatory subunit of the IK kinase. This defect impairs but does not completely abolish NF-~cB activity. The disorder is characterized by a reduction in the number of sweat glands, poor tooth eruption, and, if erupted, conical teeth. Patients have sparse hair, lymphoedema, and osteopetrosis. Sometimes, there is a related cleft lip and/or palate. Although most patients have a mild immunodeficiency, some have severe, multiple life-threatening infections, including opportunistic infections such as atypical mycobacteria. Although no patients have been found to have a specific immunological abnormality, a 3-year-old with this syndrome was evaluated after multiple septicemias with gram-negative organisms. The evaluation demonstrated a defect in the generation of IFN-y and interleukin-2 by activated T cells. The patient subsequently contracted mycobacterium avium intracellulare and died of pulmonary proteinosis. Interestingly, the addition of IFN-y to his regimen cleared his mycobacterial infection and no organism was seen or cultured postmortem, suggesting that treatment with IFN-y earlier in the course of the disease may reduce the risk of life-threatening infections. P y c n o d y s o s t o s i s : A Defect in C a t h e p s i n K Function (MIM 2 6 5 8 0 0 ) Pycnodysostosis is a skeletal dysplasia that was reputed to be the cause of the deformities of TulouseLautrec (1864-1901) [28]. The disorder is transmitted in
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an autosomal recessive pattern [29]. Patients have a disproportionate short stature, a deformed rib cage with pectus excavatum, and kyphoscoliosis. Unique features of the disorder include an obtuse angle of the mandible leading to malocclusion, a narrowed upper portion of the rib cage, and failure of the sutures and fontanel to fuse. There are also a variety of facial anomalies, including proptosis, failure to lose the primary teeth, a beaked nose, and frontal bossing. The hands have clubbed fingers due to abnormalities in the distal phalanx (Fig. 3). The radiographic appearance of the bones is dense, with abnormalities of both the medullary canal and the cortex of the long bones. The body, but not the transverse processes, of the vertebrae is dense. There is hypoplasia of the sinuses and the midface, characterized by a depressed nasal bridge. Although this is similar to the generalized involvement seen in osteopetrosis, there are no endobones (primordial bone remnants within the more mature bone) and no cartilaginous islands. Some patients have a reduced level of intelligence, and final adult stature is usually approximately 4 ft, 6 in. (+ 5 in.)[1]. All patients with this syndrome have defined mutations in the production or targeting of the osteoclastic protease cathepsin K, which is a lysosomal protease localized exclusively within the osteoclast [9]. Mutations in both the region encoding cathepsin K and the coding region for the signal peptide that is a component of the prepro-cathepsin K have been identified [9]. An alanineto-valine substitution at residue 277 of cathepsin K was found to create a preprotein that was unable to be activated. The genetic defect was due to a C-to-A substitution at nucleotide 935. Similar mutations have been found in a variety of patients with pycnodysostosis [9]. Other patients have mutations caused by the deletion of a base pair at nucleotide 531, resulting in a truncated protein that lacks the active enzymatic site. A third defect in the prepropetide for cathepsin K was found in the signal peptide region at nucleotide 131. This defect results in an inability to target the mature molecule to the endoplasmic reticulum, making it impossible for it to cross the membrane, a necessary step for secretion [9]. In all three cases, a reduction in cathepsin K activity results in the same phenotype. This defect, unlike the defects in osteopetrosis, involves only one of several proteins that are used to remove matrix proteins during bone resorption. This may be the reason why this defect, although quite severe, does not result in the classic osteopetrotic phenotype or the presence of cartilaginous islands. This lack of cartilaginous islands suggests that the cartilage and matrix can be removed. The presence of the pycnodysostosis phenotype with severe bony sclerosis suggests that the defect retards bone resorption.
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L. Lyndon Key, Jr., and William L. Ries
FIGURE 3 Note the open sutures in an otherwise mature-appearing skull in pycnodysostosis. The radiograph of the hand shows the foreshortened distal phalanges, although all the phalanges are shortened (reproduced with permission from Beighton and Cremin [42], pp. 37 and 38, Figs. 3 and 5).
SKELETAL ABNORMALITIES RELATED TO OVERPRODUCTION OF BONE: INVOLVEMENT OF TRANSFORMING GROWTH FACTORS In three dysplasias of intramembranous bone formation, excessive bone formation appears to be present in the absence of defects in osteoclastic activity. In each, there is an abnormality in protein s of or regulating members of the transforming growth factor-13 (TGF-13) superfamily of proteins. In Camurati-Engelmann disease, there is an abnormal propeptide of TGF-131 [6,7]. In melorheostosis, there is downregulation of a TGF-13-inducible cell adhesion molecule. This molecule appears to inhibit bone formation induced by TGF-13, leading to overly abundant production of bone [5]. In 2001, two groups of researchers independently discovered a novel protein, sclerostosin, which appears to
inhibit bone formation [3,4]. Loss of this protein has been associated with the clinical syndrome of sclerostosis. These three conditions, unlike those affecting osteoclasts, appear to result from the overproduction of bone by osteoblasts (Fig. 1). M e l o r h e o s t o s i s (MIM 155950) The term melorheostosis derives from the Greek description of flowing wax on the sides of burning candles. In this bony dysplasia, in which usually only one limb (or one side of the body) is affected, there is an increase in cortical bone that appears similar to a dripping candle (Fig. 4). This irregular cortical bone is the hallmark of the condition; however, it is usually associated with a cutaneous lesion or rash overlying the abnormal bone. The rash may resemble that seen in scleroderma or may consist of a fibrolipoma, capillary hemangioma, or an atriovestibular malformation. This disease is usually
19. Sclerosing Bony Dysplasia
FIGURE 4 Camurati-Engelmann disease is characterized by the accumulation of bone within the medullary canal. The deposition is primarily in the diaphyseal region, leading to the alternative name of progressive diaphyseal dysplasia (reproduced with permission from Beighton and Cremin [42], p. 128, Fig. 14).
apparent in childhood and sometimes results in pain and stiffness of the affected limbs. The disease may be progressive, resulting in shortened or contracted limbs in children and progressively worsening pain in both children and adults. The characteristic radiographic features result from irregular deposition of hyperostotic cortical bone in long bones adjacent to the medullary canal [1,5,30]. In 2000, Kim et al. [5] described downregulation of a TGF-13-inducible adhesion molecule, 13ig-h3, in melorheostosis. They created recombinant ~ig-h3 and added the purified compound to cultures of a mouse osteoblastic precursor cell line that forms bone nodules in vitro. The purified 13ig-h3 inhibited the formation of bone nodules, suggesting that 13ig-h3 inhibits osteoblastic activity. Thus, downregulation of this protein is predicted to result in excessive or unregulated production of bone. The sporadic nature of the inheritance of this disorder suggests the presence of spontaneous mutations. This would also be consistent with the predominance of localized disease, but with some patients having a more generalized condition (depending on when the mutation appeared in early gestation).
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by LAP-J31. This active TGF-131 stimulates osteoblastic bone-forming activity, with a resultant hyperostosis [6, 7]. Cockayne characterized Camurati-Engelmann disease (CED) in 1920 [1,31,32]. Camuarati described the disease as being a genetic syndrome, and Engelmann detailed the classical syndrome. CED is an autosomal dominant condition characterized by hyperostosis of the diaphyseal region of the long bones and the intramembranous bones of the skull (Fig. 5). Severely affected patients have a characteristic body habitus that includes an enlarged head with a prominent forehead, proptosis, and thin limbs with wasted musculature. Cranial nerve palsies occur in some patients, although much more rarely than in osteopetrosis. Increased intracranial pressure is common. Because of the heavy bones, weak muscles, and some failure to model normally at the hip, there is a waddling gait. The most common symptom is severe pain, which can be excruciating and immobilizing [1]. Also known as progressive diaphyseal dysplasia, the long bones can develop into large dense bones that are tender to palpation. The clinical course is variable and sometimes improves after the onset of puberty. The radiographic appearance of the bone is that of hyperostosis of the cortical bone with an increase in both endosteal and periosteal bone accumulation. Sometimes,
C a m u r a t i - E n g e l m a n n Disease (MIM 131300) In 2001, two groups independently reported a defect in the propeptide of TGF-131 [6,7]. In processing the mature TGF-131, the propeptide is cleaved into the mature TGF-131 and a latency-associated peptide-131 (LAP-131). LAP-131 is mutated in each of the genetic defects in such a way that it no longer binds to and inactivates TGF-[31. The mutations cause the generation of TGF-[31 that is no longer dimerized to and inhibited
FIGURE 5 Melorheostosis is characterized by the presence of periosteal and endosteal bone deposition that resembles dripping wax. This is easily seen in these radiographs of the leg (reproduced with permission from Beighton and Cremin [42], p. 146, Fig. 17).
480
L. Lyndon Key, Jr., and William L. Ries
there is involvement of the metaphysis, but the epiphysis is rarely affected. Accumulation of the bone causes a widening of the diaphyseal region of the bones. No fractures are seen in the painful bony areas; however, bone scans frequently demonstrate areas of enhancement, which resemble that seen in fractures (possibly microfractures). Laboratory parameters are usually normal except for markers of new bone formation. Increased thickness of the skull, pelvis, and ribs is also seen. As noted previously, failure of TGF-131 to be inhibited appears to lead to continued stimulation of osteoblastic bone resorption. The effect on the skeletal muscle may also result from this same effect because it has been suggested that TGF-131 may inhibit the proliferation of skeletal muscle precursors, resulting in an effect that counters that of insulin-like growth factor-1 and insulin [33]. Thus, this is another disorder in which an increased effect of TGF-131 appears to stimulate the formation of abnormal bone. Why there is a differential pattern between these two conditions is unknown.
members such as various bone morphogenic proteins [36]. Mutations in the S O S T gene result in nonsense mutations, whereas the splice site mutations are loss-offunction mutations. Although the precise function of SOST remains unknown, an inhibitory effect on bone formation can be proposed since pathophysiological analyses indicate that sclerosteosis is primarily a disorder of increased formation of normal bone [4,37]. Van Buchem disease also maps to the same area of chromosome 17q12-21 [4]; however, the S O S T gene locus is not mutated. Van Buchem disease is also an autosomal recessive disorder. Symptoms are very similar to those seen in osteosclerosis, including progressive asymmetric enlargement of the jaw with a wide angle but with minimal or no prognathism. Frequently, facial nerve palsies are present. Long bones may become painful with applied pressure but are not excessively fragile. Unlike patients with sclerosteosis, patients with Van Buchem disease have no syndactyly and are not excessively tall [1,38].
S c l e r o s t e o s i s (MIM 2 6 9 5 0 0 ) Perhaps the most intriguing evidence for the hypothesis that TGF-13 is involved in stimulating bone formation resulting in osteosclerosis derives from the loss-offunction mutations in the S O S T gene. Sclerosteosis is a craniotubular disorder inherited as an autosomal recessive disease. The condition was first described by Truswell [34] and is characterized by skeletal overgrowth most prominent in the skull and mandible. The clinical features of sclerosteosis include severe facial distortion; tall stature; and hand malformations with syndactyly of the digits, absent nails, and radial deviation of the terminal digits [35]. Increased intracranial pressure may lead to sudden death [4]. Facial distortion leads to entrapment of cranial nerves seven and eight. The major radiological abnormalities are widening of skull and thickening of the sclerotic jaw (Fig. 6). There is marked prognathism. The long bones have modeling defects, resulting in thickened cortices and dense bones. The vertebral pedicles, ribs, pelvis, and tubular bones are all dense. Histomorphometry of the trabecular bone has shown increased bone formation [1]. Elevated levels of alkaline phosphatase (an osteoblastic enzyme) and increased rates of bone formation suggest that the defect results from excessive bone formation rather than excessive bone resorption [3]. The SOST protein is a novel cystine knot-containing protein that appears to inhibit TGF-13 proteins, which have been shown to stimulate bone formation [3,4]. The protein superstructure is similar to that of proteins such as noggin and chordin and members of the dan family [3], which have been shown to inhibit TGF-13 superfamily
SUMMARY It appears that defects in osteoclastic enzymes that are necessary for the biochemical dissolution of bone, especially those responsible for acid production, result in defects in bone resorption. This increases bone despite a relatively normal or even slowed rate of formation. The resulting disorders tend to have a generalized sclerosis that affects formation and modeling of the bones. Diseases that share these defects are osteopetrosis, osteopetrosis with epidermal hypoplasia and immune deficiency, and pycnodysostosis. A specific enzyme defect has been determined for many patients with these disorders, but defects have not been found in all patients with osteopetrosis. We predict, however, that in these cases defects are in the osteoclast. Conversely, a variety of defects seem to result from excessive bone formation. In these diseases, there are usually normally modeled bones, but there is an increase in the pericortical bone. The disorders in which there are genetic defects are melorheostosis, CamuratiEngelmann disease, and sclerosteosis. These defects all seem to result in higher than normal stimulation of bone by TGF-131, leading to increased amounts of bone formation. Until recently most investigators assumed that the defects in sclerotic bony disorders resulted in alteration of the balance between bone formation and bone resorption, resulting from defective osteoclastic function. Although this is partially correct since bone formation is in excess of resorption in all the disorders, the concept
481
19. Sclerosing Bony Dysplasia
FIGURE 6 Sclerostosis is a disease characterized by a thickened mandible and skull. Patients with this disorder frequently have syndactyly, sclerotic long bones, and gigantism (reproduced with permission from Beighton and Cremin [42], pp. 121-122, Figs. 120 and 121).
that these diseases can be characterized along two separate lines is extremely exciting. To date, little has been done to effectively treat these disorders, except osteopetrosis. We have used calcitriol for a variety of these disorders with success. As gene defects are discovered, it may be possible to tailor treatment strategies to specific genetic defects. Until this occurs, we may have to use therapies that either inhibit bone formation (none in use) or stimulate bone resorption, such as calcitriol, parathyroid hormone, and IFN-ylb (Actimmune) [39]. There is much to learn from osteopetrotic conditions in trying to develop therapies for improving bone density in osteoporosis. The discovery of the role of TGF-[31 in these disorders suggests that one strategy might be to stimulate the activity of this enhancer of bone formation by altering proteins that cause activation of TGF-131 and/ or enhance its action. Conversely, inhibitors of acid secretion (especially those related to a reduction in car-
bonic anhydrase II activity or the proton pump) may reduce osteoclastic activity. In either case, the density of the bone would be enhanced [40]. Although we are still in the early stages of molecular diagnosis of metabolic bone disorders, the fact that the causes of sclerotic bony dysplasias share similarities suggests that manipulation of these factors may allow the development of rational therapy that is more effective in reducing the pain, disfigurement, morbidity, and mortality of patients with these severe conditions.
References 1. Whyte, M. P. (1999). Sclerosing bony disorders. In Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism
(M. J. Favus, Ed.). Lippincott Williams & Wilkins, Philadelphia. 2. Vanhoenacker, F. M., De Beuckeleer, L. H., Van Hul, W., Balemans, W., Tan, G. J., Hill, S. C., and De Schepper, A. M. (2000). Sclerosing bone dysplasias: Genetic and radioclinical features. Eur. Radiol. 10, 1423-1433.
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3. Brunkow, M. E., Gardner, J. C., Van Ness, J., Paeper, B. W., Kovacevich, B. R., Proll, S., Skonier, J. E., Zhao, L., Sabo, P. J., Ying-Hui, F., Alisch, R. S., Gillett, L., Colbert, T., Tacconi, P., Galas, D., Hamersma, H., Beighton, P., and Mulligan, J. T. (2001). Bone dysplasia sclerosteosis results from loss of the sost gene product, a novel cystine knot-containing protein. Am. J. Hum. Genet. 68, 577-589. 4. Balemas, W., Eberling, M., Patel, N., Van Hul, E., Olson, P., Dioszegi, M., Lacza, C., Wuyts, W., Van Den Ende, J.,llems, P., Paes-Alves, F., Hill, S., Bueno, M., Ramos, F. J., Paolo, T., Dikkers, F. G., Stratakis, C., Lindpaintner, K., Vickery, F., Foezrnzler, D., and Van Hul, W. (2001). Increased bone density in sclerosteosis due to the deficiency of a novel secreted protein (SOST). Hum. Mol. Genet. 10, 537-543. 5. Kim, J.-E., Kim, E.-H., Han, E.-H., Park, R.-W., Park, I.-H., Jun, S.-H., Kim, J.-C., Young, M. F., and Kim, I.-S. (2000). A TGF-13inducible cell adhesion molecule, 13ig-h3, is downregulated in melorheostosis and involved in osteogenesis. J. Cell. Biochem. 77, 169-178. 6. Janssens, K., Gershoni-Baruch, R., Guanabens, N., Migone, N., Ralston, S., Bonduelle, M., Lissens, W., van Maldergem, L., Vanhoenacher, F., Verbruggen, L., and van Hul, W. (2000). Mutations in the gene encoding the latency-associated peptide of TGF-13 1 cause Camurati-Engelmann disease. Nature Genet. 26, 273-275. 7. Saito, T., Kinoshita, A., Yoshiura, K., Makita, Y., Wakui, K., Honke, K., Niikawa, N., and Taniguchi, N. (2001). Domainspecific mutations of a transforming growth factor (TGF)-13 1 latency-associated peptide cause Camurati-Engelmann disease because of the formation of a constitutively active form of TGF-131. J. Biol. Chem. 15, 11469-11472. 8. Fujita, Y., Nakata, K., Yasui, N., Matsui, Y., Kataoka, E., Hiroshima, K., Shiba, R.-I., and Ochi, T. (2000). Novel mutations of the cathepsin K gene in patients with pycnodysostosis and their characterization. J. Clin. Endocrinol. Metab. 85, 425-431. 9. Doffinger, R., Smahi, A., Bessia, C., Geissmann, F., Finberg, J., Durandy, A., Bodemer, C., Kenwrick, S., Dupuis-Girod, S., Blanche, S., Wood, P., Rabia, S. H., Headon, D. J., Overbeek, P. A., Le Deist, F., Holland, S. M., Belani, K., Kumararatne, D. S., Fischer, A., Shapiro, R., Conley, M. E., Reimund, E., Kalhoff, H., Abinun, M., Munnich, A., Israel, A., Kourtois, G., and Casanova, J.-L. (2001). X-linked anhydrotic ectodermal dysplasia with immunodeficiency is caused impaired NF-•I3 signaling. Nature Genet. 27, 277-285. 10. Kornak, U., Schulz, A., Friedrich, W., Uhlhaas, S., Kremens, B., Voit, T., Hassan, C., Bode, U., Jentsch, T. J., and Kubisch, C. (2000). Mutations in the a3 subunit of the vacuolar H+-ATPase cause infantile malignant osteopetrosis. Hum. Mol. Genet. 9, 2059-2063. 11. Frattini, A., Orchard, P. J., Sobacchi, C., Giliani, S., Abinun, M., Mattsson, J. P., Keeling, D. J., Andersson, A.-K., Wallbrandt, P., Zecca, L., Notarangelo, L. D., Vezzoni, P., and Villa, A. (2000). Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis [Letter]. Nature Genet. 25, 343-346. 12. Kornak, U., Kasper, D., Bosl, M. R., Kaiser, E., Schweizer, M., Schulz, A., Friedrich, W., Delling, G., and Jentsch, T. J. (2001). The loss of the C1C-7 chloride channel leads to osteopetrosis in mice and man. Cell 104, 205-214. 13. Sly, W. S., Hewett-Emmett, D., Whyte, M. P., et al. (1983). Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proc. Natl. Acad. Sci. USA 80, 2752-2756. 14. Ravel, P. A. (1986). Pathology of Bone. Springler-Verlag, Berlin.
15. Albers-Sch6enberg, H. (1904). Rontgenbilder einer celtenen, knochenerkrankung. Munch. Med. Wochenschr. 51, 365. 16. Johnston, C. C., Lavy, N., Lord, T., et al. (1968). Osteopetrosis: A clinical, genetic, metabolic, and morphologic study of the dominantly inherited, benign form. Medicine 47, 149-167. 17. Gerritsen, E. J., Vossen, J. M., von Loo, I. H., Hermans, J., Helfrich, M. H., and Griscelli, C. (1994). Autosomal recessive osteopetrosis: Variability of findings at diagnosis and during the natural course. Pediatrics 93, 247-253. 18. Kahler, S. G., Burns, J. A., and Aylsworth, A. S. (1984). A mild autosomal recessive form of osteopetrosis. Am. J. Med. Genet. 17, 451-464. 19. Reeves, J. D., Huffer, W. E., August, C. S., Hathaway, W. E., Koerper, M., and Walters, C. E. (1979). Hematopoietic effects of prednisone therapy in four infants with osteopetrosis. J. Pediatr. 94, 210-214. 20. Jagadha, V., Halliday, W. C., Beker, L. E., and Hinton, D. (1988). The association of infantile osteopetrosis and neuronal storage disease in two brothers. Acta Neruolopathol. 75, 233-240. 21. Coccia, P. F., Krivit, W., Cervenka, J., Clawson, C., Kersey, J. H., Kim, T. H., Nesbit, M. E., Ramsay, N. K. C., Warkentin, P. I., Teitelbaum, S. L., Kahn, A. J., and Brown, D. M. (1980). Successful bone-marrow transplantation for infantile malignant osteopetrosis. N. Engl. J. Med. 302, 701-708. 22. Lajeunesse, D., Busque, L., Menard, P., Brunette, M. G., and Bonny, Y. (1996). Demonstration of an osteoblast defect in two cases of human malignant osteopetrosis: Correction of the phenotype after bone marrow transplant. Bone 98, 1835-1842. 23. McMahon, C., Will, A., Hu, P., Shah, G. N., Sly, W. S., and Smith, O. P. (2001). Bone marrow transplantation corrects osteopetrosis in carbonic anhydrase II deficiency syndrome. Blood 97, 1947-1950. 24. Gram, J., Antonsen, S., Horder, M., and Bollerslev, J. (1991). Elevated serum levels of creatine kinase BB in autosomal dominant osteopetrosis type II. Calcif. Tissue Int. 48, 438-439. 25. Funanage, V. L., Carango, P., Shapiro, I. M., Tokuoka, T., and Tuan, R. S. (1992). Creatine kinase activity is required for mineral deposition and matrix synthesis in endochondral growth cartilage. Bone Miner. 17,228-236. 26. Sistermans, E. A., de Kok, Y. J., and Peters, W. (1995). Tissue- and cell-specific distribution of creatinine kinase B: A new and highly specific monoclonal antibody for use in immunohistochemistry. Cell Tissue Res. 280, 435-446. 27. Whyte, M. P., Chines, A., Silva, D. P., Landt, Y., and Landenson, J. H. (1996). Creatine kinase brain isoenzyme (BB-CK) presence in serum distinguishes osteopetrosis among the sclerosing bone disorders. J. Bone Miner. Res. 11, 1438-1443. 28. Maroteaux, P., and Lamy, M. (1965). The malady of ToulouseLautrec. J. Am. Med. Assoc. 191, 715-717. 29. Elmore, S. M. (1967). Pycnodysostosis: A review. J. Bone Joint Surg. 49, 153-162. 30. Campbell, C. J., Papademetriou, T., and Bonfiglio, M. (1968). Melorheostosis: A report of the clinical roentgenographic, and pathological findings in fourteen cases. J. Bone Joint Surg. 50, 1281-1304. 31. Cockayne, E. A. (1920). A case for diagnosis. Proc. R. Soc. Med. 13, 132-136. 32. Englemann, G. (1929). Ein fall vone osteopathia hyperostotica (sclerostisans) multiplex infantalis. Fortschr. Geb. Rontgen. 39, 1101-1106. 33. Xu, P. and Gu., X. (2000). The effects of EGF, TGF-beta and insulin on the growth of rabbit's skeletal muscle satellite cell. Chung-Hua Kou Chiang i Hsueh Tsa Chih Chinese J. Stomatology
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19. Sclerosing Bony Dysplasia 34. Truswell, A. S. (1958). Osteopetrosis with syndactyly, a morphologic variant of Albers-Sch/Snberg disease. J. Bone Surg. 40, 208-218. 35. Beighton, P. (1988). Sclerosteosis. J. Med. Genet. 25, 200-203. 36. Hsu, D., Economides, A., Wang, X., Eimon, P., and Harland, R. (1998). The Xenopus dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities. Mol. Cell. 1, 673-683. 37. Setin, S. A., Witkop, C., Hill, S., Fallow, M. D., Viemstein, L., Gucer, G., McKeever, P., Long, D., Altman, J., and Miller, N. R. (1983). Sclerosteosis: Nogenetic and pathophysiologic analysis of an American kinship. Neurology 33, 267-277. 38. Van Hul, W., Balemans, W., and Van Hul, E. (1998). Van Buchem disease (hyperostosis corticalis gereralisata) maps to chromosome 17q12-21. Am. J. Hum. Genet. 62, 391-399.
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39. Key, L. L., Rodriquiz, R. M., Willi, S. M., Wright, N. M., Griffin, P., Eyre, D., and Ries, W. L. (1995). Recombinant human interferon gamma therapy for osteopetrosis. N. Engl. J. Med. 332, 1594-1599. 40. Lazner, F., Gowen, M., Pavasovic, D., and Kola, I. (2001). Osteopetrosis and osteoporosis: Two sides of the same coin. Hum. Mol. Genet. 8, 1839-1846. 41. Baron, R. (1999). Anatomy and ultrastructure of bone. In Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism (M. J. Favus, Ed.). Lippincott Williams & Wilkins, Phila-
delphia. 42. Beighton, P., and Cremin, P. J. (1980). Sclerosing Bony Dysplasia. Springer-Verlag, New York.
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12ol Parathyroid Disorders MURAT BASTEPE,* HARALD JOPPNER,* and RAJESH V. THAKKERt *Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts *Molecular Endocrinology Group, Nuttfield Department of Medicine, John Radcliffe Hospital Headington, Oxford, United Kingdom
INTRODUCTION
associated with some forms of hypoparathyroidism; mutations in the PTH/PTHrP receptor gene have been identified in patients with two rare genetic disorders, Jansen's and Blomstrand's chondrodysplasia; and mutations in GNAS1, the gene encoding the stimulatory G protein ~ subunit (Gs~) and splice variants thereof, have been found or are suspected in an increasing number of disorders, including, McCune-Albright syndrome, pseudohypoparathyroidism types Ia and Ib, pseudo-pseudohypoparathyroidism, and the plate-like osteoma cutis variant of progressive osseous hyperplasia. Germ-line and somatic mutations of a putative tumor-suppressor gene, designated HRPT2, have been associated with a syndrome characterized by hereditary hyperparathyroidism and jaw tumors. Candidate genes have been identified for DiGeorge syndrome, and the chromosomal locations of the susceptibility genes responsible for other less frequent variants of FBH, DiGeorge, and Williams syndrome have been determined. Molecular genetic studies have thus provided unique opportunities to elucidate the pathogenesis of rare disorders of calcium homeostasis. These advances, together with the exploration of gene structures and functions of PTH, PTHrP, the PTH/PTHrP receptor, the calcium-sensing receptor, and the stimulatory G protein, are reviewed in this chapter.
Extracellular calcium ion concentration is tightly regulated through the actions of parathyroid hormone (PTH) on kidney and bone. The intact peptide is secreted from the parathyroid glands at a rate that is appropriate to and dependent on the prevailing extracellular calcium ion concentration. Hypercalcemic or hypocalacemic disorders can be classified according to whether they occur from an excess or deficiency of PTH, a defect in the PTH receptor [i.e., the PTH/PTH-related peptide (PTHrP) receptor], or insensitivity to PTH caused by defects downstream of the PTH/PTHrP receptor (Table 1). Recent advances in understanding the biological importance of key proteins involved in the regulation of PTH secretion and the responsiveness to PTH in target tissues have led to the identification of molecular defects in a variety of disorders and thus enabled the characterization of some of the mechanisms involved in the regulation of parathyroid gland development, parathyroid cell proliferation, PTH secretion, and PTH-mediated actions in target tissues (Fig. 1). For example, mutations in the calcium-sensing receptor gene have been reported in patients with familial benign (hypocalciuric) hypercalcemia [familial benign hypercalcemia (FBH) or familial hypocalciuric hypercalcemia (FHH)], neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia. Furthermore, the roles of the oncogene PRAD1, which encodes a novel cyclin, and of the multiple endocrine neoplasia type 1 (MEN1) gene in the pathogenesis of some parathyroid tumors have been determined. In addition, mutations in the PTH gene and the mitochondrial genome have been demonstrated to be
PediatricBone
PTH GENE STRUCTURE AND FUNCTION The PTH gene is located on chromosome l lp15 and consists of three exons that are separated by two
485
Copyright 2003, Elsevier Science (USA). All rights reserved.
TABLE 1 Parathyroid Diseases and Their Chromosomal Locations Disease
Metabolic abnormality Hypocalcemia
Isolated hypoparathyroidism
Inheritance
Gene product
Autosomal dominant
PTH
Autosomal recessive
PTH
X-linked recessive
Unknown
Ausomal recessive
GCMB
Hypocalcemic hypercalciuria
Autosomal dominant
CaSR
Hypoparathyroidism associated with polyglandular autoimmune syndrome (APECED)
Autosomal recessive
AIRE
Hypoparathyroidism associated with Kearns-Sayre and M E L A S ~
Maternal
Mitochondria1 genome
DiGeorge
Autosomal dominant
mex4Oc
Blomstrand lethal chondrodysplasia
Autosomal recessive
Kemeyxaffey
Autosomal dominantd
Unknown
AR-Kenney-Caffey
Autosomal recessive
TBCE
Barakat
Autosomal recessived
Unknown
Lymphoedema
Autosomal recessive
Unknown
Nephropathy, nerve deafness
Autosomal dominantd
GATA3
Dysmorphology, growth failure
Autosomal recessive
Unknown
Pseudohypoparathyroidism type l a
Autosomal dominant, paternally imprinted
Gsu
Pseudohypoparathyroidism type Ib
Autosomal dominant, paternally imprinted
Unknown
Hypoparathyroidism associated with complex congenital syndromes
ne~2.2-nex3~
Progressive osseous hyperplasia (POH)
WHIR
Gsu
Plate-like osteoma cutis Hypercalcemia
Multiple endocrine neoplasia type 1
Autosomal dominant
MENIN
Multiple endocrine neoplasia type 2
Autosomal dominant
RET
Hereditary hyperparathyroidism and jaw tumors (HPT-JT)
Autosomal dominant
HRPT2
Sporadic hyperparathyroidism
Sporadic
PRADI/CCNDI Retinoblastoma Unknown
Familial benign hypercalcemia FBH3q
Autosomal dominant
CaSR
FBH19p
Autosomal dominant
Unknown
FBHOk
Autosomal dominant
Unknown
Neonatal severe hyperparathyroidism (NSHPT)
Autosomal recessive
CaSR
Autosomal dominant Jansen's disease
Autosomal dominant
Williams syndrome
Autosomal dominant
Elastin (and other genes)
McCune-Albright syndrome
Mutations during early embryonic development?
Gsu
"Mutations of the PTH gene identified only in some families. b . M~tochondrialencephalopathy, stroke-like episodes, and lactic acidosis. 'Most likely candidate genes. d ~ o s likely t inheritance shown.
PTH l R
Chromosomal location
20. Parathyroid Disorders ldmtg(EIIIIdlm ~
d Function
Exon 2 is 90 bps in length and encodes the initiation (ATG) codon, the prehormone sequence, and part of the p r o h o r m o n e sequence. Exon 3 is 612 bps in length and encodes the remainder of the p r o h o r m o n e sequence, the mature PTH peptide, and the 3' untranslated region [2]. The 5' regulatory sequence of the h u m a n P T H gene contains a vitamin D response element 125 bps upstream of the transcription start site that downregulates P T H m R N A transcription in response to vitamin D receptor binding [3,4]. P T H gene transcription (as well as PTH peptide secretion) is also dependent on the extracellular calcium and phosphate concentration [5-8], although the presence of specific upstream calcium or phosphate response element(s) has not been demonstrated [9,10]. The secretion of mature PTH, an 84 amino-acid peptide, from the parathyroid chief cell is regulated through a G protein-coupled calcium-sensing receptor that is also expressed in renal tubules and in several other tissues, albeit in lower abundance. P T H m R N A is first translated into a pre/pro-PTH peptide. The pre-sequence consists of a 25-amino acid signal peptide (leader sequence) that is responsible for directing the nascent peptide into the endoplasmic reticulum to be packaged for secretion from the cell [11]. The pro-sequence is 6 amino acids in length, and although its function is less well defined than that of the pre-sequence, it is also essential for correct P T H processing and secretion [11]. After the 84-amino acid mature P T H peptide is secreted from the parathyroid cell, it is cleared from the circulation with a short half-life of approximately 2 min via nonsaturable hepatic uptake and renal excretion. The P T H gene shares significant homology with the gene encoding PTH-related peptide (PTHrP also known as PTH-related hormone, P T H r H ) [12,13]. Both peptides mediate their actions through a c o m m o n receptor [14,15]. This P T H / P T H r P receptor, also termed P T H 1 R (Fig. 1), is a member of a subgroup of G protein-coupled receptors, and its gene is located on chromosome 3p21-p24 [16,17]. The P T H 1 R receptor is closely related to P T H 2 R (the gene for P T H 2 R is located on chromosome 2q33), which binds the 39-amino acid tuberoinfundibular peptide (TIP39) with high affinity [18]. The h u m a n but not the rat P T H 2 R also interacts with P T H [19-21].
hylXrCak'iuria
FBH, NSHPT
Hypocalcemia with
MEt.AS, KSS
MENI,HlrrJT
PRADI. Ret
Hypelxumhyroidism
~oeorge=yndmme
Williams
A
PTH
PTH or PTHrP
PTH/PTHrP
L_~r~tor
LOSS of Function
Gain of Function
Blomslrand's
Jansen'=;
~ipo~_~: sla chondrodysplasia momt,y,NI MoCune-Albright
Syndrome
1~'~1~ x
B
FBH,NSHPT
Hypocalceml. with Hypercalcluda
CaSR
FIGURE 1 Schematic representation of some of the components involved in calcium homeostasis. Panel A: Parathyroid cell. Alterations in extracellular calcium are detected by the calcium-sensing receptor (CaSR), a 1078-aminoacid G protein-coupled receptor. Panel B: Target cell. The PTH/PTHrP receptor, which mediates the actions of PTH and PTHrP, is also a G protein-coupled receptor. Thus, Ca2+ and PTH and PTHrP involve G protein-coupled signaling pathways and interaction with their specific receptors can lead to activation of Gs, Gi, and/or Gq. Gs stimulates adenylylcyclase (AC), which catalyzes the formation of cAMP from ATP. Gi inhibits AC activity, cAMP stimulates PKA, which phosphorylates cell-specific substrates. Activation of Gq stimulates PLC, which catalyzes hydrolysis of the phosphoinositide (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG), increasing intracellular calcium, and activating PKC. These proximal signals modulate downstream pathways, resulting in specific physiological effects. Abnormalities in several genes, which lead to mutations in proteins in these pathways, have been identified in specific disorders of calcium homeostasis (Table 1) (adapted with permission from Thakker and Jiippner [210]).
introns [1]. Exon 1 of the P T H gene is 85 base pairs (bps) in length and is untranslated (Fig. 2), whereas exons 2 and 3 encode the 115-amino acid pre/pro-PTH peptide.
Un-
.
Signal
translated
gene
Pro-
peptide /sequence\
P'rH Unpeptide translated
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FIGURE 2 Schematicrepresentation of the PTH gene, PTH mRNA, and PTH peptide. The PTH gene consists of three exons and two introns; the peptide is encoded by exons 2 and 3. The PTH peptide is synthesized as a precursor that contains a "pre-" (signal peptide) and a "pro-" sequence. The mature PTH peptide, which contains 84 amino acids, and larger carboxy-terminalPTH fragments are secreted from the parathyroid cell (adapted with permission from Parkinson and Thakker [33]).
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Interestingly, PTH1R also binds TIP39, but the peptide acts as an antagonist at this receptor [22,23]. The biological roles of PTH2R are poorly understood; it is only expressed in a few tissues, including the hypothalamus, pancreas, placenta, and testis [19,24]. In contrast, PTH1R shows a broader expression profile, with highest expression in kidney and bone, in which it mediates the endocrine actions of PTH. However, the most abundant expression of PTH1R occurs in chondrocytes of the metaphyseal growth plate, in which it mediates predominantly the autocrine/paracrine actions of PTHrP [2528]. Mutations involving the genes that encode PTH, the calcium-sensing receptor, the PTH/PTHrP receptor, and Gs~ all affect the regulation of calcium homeostasis and can thus be associated with genetic disorders characterized by hypercalcemia or hypocalcemia (Table 1).
HYPOCALCEMIC DISORDERS M a n i f e s t a t i o n s of h y p o c a l c e m i a a n d t r e a t m e n t of h y p o c a l c e m i c disorders Typical clinical features of hypocalcemia reflect increased neuromuscular irritability and include perioral paresthesias and cyanosis, which may extend to the entire face, tingling of the fingers and toes, and/or tetany. Tetany may present as rigid spasm of the muscles of the upper and lower extremities; fixation of the wrist in flexion with extension of the thumb is typical of carpal spasm. The spastic phenomenon may progress into tonic-clonic activity of the extremity or even generalized convulsions. In infants signs are less specific and only jitteriness and irritability can be evident; laryngospasm can be the presenting manifestation of hypocalcemia and generalized seizures may develop also in this age group. Latent tetany may be precipitated by maneuvers of pressure or ischemia. For example, percussion of the facial nerve, just anterior to the ear, with a finger or reflex hammer may result in twitching of facial muscles around the eye and mouth (Chvostek's sign). Induction of ischemia for 2-3 minutes by inflation of a blood pressure cuff over the brachial artery at pressures just above systolic blood pressure readings can precipitate carpal spasm (Trousseau's sign). The acute management of symptomatic hypocalcemia consists of slow intravenous infusion (less than 1 ml/min) of 10% calcium gluconate solution, which is equal to 9 mg/ml elemental calcium (1-2 ml per kg body weight; up to a maximum of 20ml per bolus infusion). For longterm infusion, 4-6 ml of 10% calcium gluconate per kg per 24 hours can be used, providing 36-54 mg of elemental calcium per kg body weight. Calcium chloride is less preferential for intravenous use, as it is more irritating
than calcium gluconate. Bicarbonate and phosphate should not be infused with calcium as calcium salts of these anions may precipitate in tubing or the patient's intravascular space. Correction of low serum calcium levels may be refractory to the above therapeutic approaches in the setting of hypomagnesemia. If renal function and urinary output are normal, magnesium is given slowly intravenously or intramuscularly (magnesium sulfate septahydrate is available as a 50% solution, containing 48 mg of elemental magnesium per ml). The dose for infants is 5-10 mg per kg body weight, for older individuals up to 2.4 mg per kg body weight of elemental magnesium (to a maximum of 180rag). Magnesium levels should be monitored to avoid toxicity. As with calcium therapy, cardiac monitoring should be performed. For chronic treatment of hypocalcemia, calcium salts are usually given in combination with vitamin D or the biologically active vitamin D analog 1,25(OH)2D3. However, the goals for chronic therapy vary and depend on the underlying disorder. For example, when the calciumretaining effect of PTH is absent as in patients with hypoparathyroidism, treatment with calcium salts and vitamin D analogs may lead to urinary calcium excretion that is inappropriately high for the serum calcium concentration. For such individuals, fasting serum calcium concentration should be maintained within an asymptomatic range, generally between 1.95 to 2.12mmol/L (7.8 to 8.5mg/dl). In experimental protocols, certain hypocalcemic disorders have been successfully treated with parental PTH(1-34) in order to minimize the urinary calcium losses, and thus the risk of developing or worsening nephrocalcinosis. Other conditions, such as pseudohypoparathyroidism, can be treated more safely with higher doses of 1,25(OH)2D3, as long as urinary calcium excretion usually remains within normal limits. Kidney ultrasounds repeated annually allow to monitor possible nephrocalcinosis. Chronic magnesium supplementation may be required in individuals with ongoing renal or intestinal losses. Four daily doses are recommended (3-6 mg of elemental magnesium per day, but not more than 500 mg/day), if renal function is normal. Hypoparathyroidism Hypoparathyroidism may occur as part of a pluriglandular autoimmune disorder or as a complex congenital defect, as in DiGeorge syndrome. In addition, hypoparathyroidism may develop as a solitary endocrinopathy called isolated or idiopathic hypoparathyroidism. Familial occurrences of isolated hypoparathyroidism with autosomal dominant, autosomal recessive, and X-linked recessive inheritances have been established.
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PTH Gene Abnormalities DNA sequence analysis of the PTH gene (Fig. 2) from one patient with autosomal dominant isolated hypoparathyroidism revealed a single base substitution ( T ~ C ) in exon 2 [29], which resulted in the substitution of arginine (CGT) for cysteine (TGT) in the signal peptide. The presence of this charged amino acid in the midst of the hydrophobic core of the signal peptide impeded the processing of the mutant pre/pro-PTH, as demonstrated by in vitro studies. These studies revealed that the mutation impaired the interaction with the nascent protein and the translocation machinery, and that cleavage of the mutant signal sequence by solubilized signal peptidase was ineffective [29,30]. In kindreds with autosomal recessive isolated hypoparathyroidism, two different PTH gene abnormalities have been identified [31,32]. In one family, an abnormality involving a donor splice site at the exon 2-intron 2 boundary was identified [33]. This mutation involved a single base transition (g~c) at position 1 ofintron 2, and an assessment of the effects of this alteration in the invariant gt dinucleotide of the Y donor splice site consensus on mRNA processing revealed that the mutation resulted in exon skipping, in which exon 2 of the PTH gene was lost and exon 1 was spliced to exon 3. The lack of exon 2 would lead to a loss of the initiation codon (ATG) and the signal peptide sequence (Fig. 2), which are required for the commencement of PTH mRNA translation and for the translocation of the PTH peptide, respectively. In the other family, a single base substitution ( T ~ C ) involving codon 23 of exon 2 was detected. This resulted in the substitution of proline (CCG) for the normal serine (TCG) in the signal peptide [32]. This mutation alters the - 3 position of the pre/pro-PTH protein cleavage site [34]. The presence of the mutant proline at this position likely disrupts cleavage of the pre/pro-PTH that would subsequently be degraded in the rough endoplasmic reticulum (RER).
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ization of this genomic region led to the identification of a molecular deletional insertion involving chromosomes Xq27 and 2p25 as a cause of this disorder [40].
Pluriglandular Autoimmune Hypoparathyroidism Hypoparathyroidism may occur in association with candidiasis and autoimmune Addison's disease, and the disorder has been referred to as either the autoimmune polyendocrinopathy--candidiasis--ectodermal dystrophy (APECED) syndrome or the polyglandular autoimmune type 1 syndrome [41]. This disorder has a high incidence in Finland, and a genetic analysis of Finnish families indicated autosomal recessive inheritance of the disorder [42]. In addition, the disorder has been reported to have a high incidence among Iranian Jews, although the occurrence of candidiasis was less common in this population [43]. Linkage studies of Finnish families mapped the APECED gene to chromosome 21q22.3 [44]. Further positional cloning studies led to the isolation of a novel gene referred to as AIRE (autoimmune regulator). This gene encodes a 545-amino acid protein that contains motifs suggestive of a transcriptional factor and includes two zinc finger motifs, a proline-rich region, and three LXXLL motifs [45,46]. In the APECED families, a number of different mutations throughout the coding exons of AIRE have been reported [47-50], although a codon 257 (Arg~stop) mutation was the predominant abnormality in 82% of the Finnish families [45,46]. Recent work on the cellular functions of the AIRE protein has revealed that it specifically binds certain DNA motifs in dimeric or tetrameric conformations [51]. In addition, AIRE has been demonstrated to exert transcriptional transactivation and to interact with the coactivator cyclic AMP-response element binding protein [52]. The identification of the precise roles of AIRE and additional defects causing APECED will not only facilitate genetic diagnosis but also enhance the elucidation of the mechanisms causing this and possibly other autoimmune diseases.
X-Linked Recessive Hypoparathyroidism X-linked recessive hypoparathyroidism has been reported in two multigenerational kindreds from Missouri [35,36]. In this disorder, only males are affected and they suffer from infantile onset of convulsions and hypocalcemia, which is due to an isolated defect in parathyroid gland development [37]. Relatedness of the two kindreds has been established by the demonstration of an identical mitochondrial DNA sequence, which is inherited via the maternal lineage, in affected males from the two families [38]. Studies utilizing X-linked polymorphic markers in these families localized the mutant gene to chromosome Xq26-q27 [39]. Further character-
Mitochondrial Disorders Associated with Hypoparathyroidism Hypoparathyroidism has been reported to occur in two disorders associated with mitochondrial dysfunction: Kearns-Sayre syndrome (KSS) and the MELAS syndrome. KSS is characterized by progressive external ophthalmoplegia and pigmentary retinopathy before the age of 20 years, and it is often associated with heart block or cardiomyopathy. The MELAS syndrome consists of a childhood onset of mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes. In addition, varying degrees of proximal myopathy can be seen in both
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conditions. Both KSS and MELAS syndromes have been reported to occur with insulin-dependent diabetes mellitus and hypoparathyroidism [53,54]. A point mutation in the mitochondrial gene tRNA leucine (UUR) has been reported in one patient with MELAS syndrome who also suffered from hypoparathyroidism and diabetes mellitus [55]. A large deletion, consisting of 6903 bps and involving 39% of the mitochondrial genome, has been reported in another patient who suffered from KSS, hypoparathyroidism, and sensorineural deafness [54]. The role of these mitochondrial mutations in the etiology of hypoparathyroidism remains to be elucidated.
DiGeorge Syndrome Patients with DiGeorge syndrome (DGS) typically suffer from hypoparathyroidism, immunodeficiency, congenital heart defects, and deformities of the ear, nose, and mouth. The disorder occurs due to a congenital failure in the development of the derivatives of the third and fourth pharyngeal pouches with resulting absence or hypoplasia of the parathyroids and thymus. Most cases of DGS are sporadic, but an autosomal dominant inheritance of DGS has been observed and an association between the syndrome and an unbalanced translocation and deletions involving 22ql 1.2 have also been reported [56]. In some patients, deletions of another locus on chromosome 10p have been observed in association with DGS (DGS2) [57]. Mapping studies of the DGS deleted region on chromosome 22ql 1.2 have defined a 250-kb minimal critical region [58], and cloning of the translocation break point on 22ql 1.21 from a DGS patient [59] has revealed that two genes with opposite transcriptional directions are probably disrupted by this break point [60]. A coding portion of one of these genes, designated rnex40, has homology to the mouse and rat androgen receptors and contains a leucine zipper motif, suggesting that the DGS candidate gene may be a DNA-binding protein. Eleven nucleotides of rnex40 are deleted at the translocation junction, suggesting that loss of function of this gene is responsible for at least part of the DiGeorge phenotype [60]. Another partial transcript, referred to as nex2.2nex3, has also been identified from this break point. Both rnex40 and nex2.2-nex3 are deleted in all DGS patients with 22ql 1 deletions, and studies assessing the presence of hemizygosity and mutations in these genes in DGS patients who do not have detectable 22ql 1 deletions are required to determine the role of these genes in the etiology of DGS. Such studies have been performed for a human homolog of a yeast gene that encodes a protein involved in the degradation of ubiquinated proteins, referred to as UDF1L [61]. UDF1L is located on 22ql 1 and has been found to be deleted in all 182 patients with the 22qll deletion syndrome, which
includes patients with DGS, the velocardiofacial, and conotruncal anomaly face syndromes [56,58]. However, a smaller deletion of approximately 20 kbs that removed exons 1-3 of UDF1L has been detected in 1 patient [61]. This patient, who had a de novo deletion resulting in haploinsufficiency of UDF1L, suffered from neonatalonset cleft palate, a small mouth, low-set ears, a broad nasal bridge, an interrupted aortic arch, a persistent truncus arteriosus, hypocalcemia, T lymphocyte deficiency, and syndactyly of her toes. These results indicate that abnormalities of the UDF1L gene likely contribute to the etiology of DGS. Patients with late-onset DGS have recently been described [62,63]. These patients presented later in childhood or during adolescence with symptomatic hypocalcemia but only subtle phenotypic abnormalities. These late-onset DGS patients were shown to have microdeletions in the 22qll region similar to those observed in other individuals affected by this disorder. The molecular definition of these variants of DGS may provide additional insight into the regulation of PTH secretion and/or parathyroid gland development. Several recently generated mouse models have implicated TBX1 in DGS, which is a member of the transcription factor family containing a conserved DNA binding domain termed T-box [64-66]. Heterozygous deletion of Tbxl in mice, which is located in a chromosomal region syntenic to the human chromosome 22q11, caused a varying degree of aortic arch malformations [65,66]. Furthermore, homozygous deletion of this gene resulted in the presentation of all testable DGS features [65]. These findings suggest that haploinsufficiency of TBX1 likely plays a significant role in the cardiac defects. Deletion of Crkl, a gene that encodes an adaptor protein implicated in growth factor and focal adhesion signaling, has also led to a phenotype in mice quite similar to DGS [67], although mice carrying a single intact allele of this gene appeared to have no DGS phenotype. It thus appears that CRKL can also contribute, as the second distinct loci within 22q 11, to the development of the features of DGS.
Hypoparathyroidism Gcm2 (glial cells missing 2), the mouse homolog of the Drosphilia gene Gcm, is expressed exclusively in the parathyroid glands, suggesting that it may be a specific regulator of parathyroid gland development [68,69]. In order to investigate this, mice that are null for Gcm2 have been generated by homologous recombination. Mice heterozygous ( + / - ) for the deletion were normal, whereas mice lacking both copies of Gcm2 ( - / - ) did not have parathyroid glands and developed hypocalcemia and hyperphosphatemia, as observed in patients affected by hypoparathyroidism [68]. However, despite their lack of
20. Parathyroid Disorders
parathyroid glands, Gcm2 - / - mice did not have undetectable serum PTH levels. In fact, PTH levels were indistinguishable from those of normal (+/+, wild-type) mice. However, the concentration of endogenous PTH in the Gcm2 - / - mice appeared insufficient to fully correct the hypocalcemia. Only the administration of exogenous PTH fully corrected the changes in mineral ion metabolism [68], indicating that Gcm2 - / - mice have a normal response (and are not resistant) to PTH. The auxiliary source of PTH was determined by combined expression and ablation studies, which revealed a cluster of PTHexpressing cells under the thymic capsule in both the Gcm2 - / - and wild-type i+/+) mice. These thymic PTHproducing cells also expressed the calcium-sensing receptor (CaSR), and long-term treatment of Gcm2-deficient mice with 1,25(OH)2 vitamin D3 restored the serum calcium concentrations to normal and reduced the serum PTH levels, indicating that the thymic production of PTH can be downregulated. However, it appears that the thymic production of PTH cannot be sufficiently upregulated to fully correct the hypocalcemia in the Gcm2-deficient mice. This absence of upregulation of PTH expression would be consistent with the very limited number of thymic PTH-producing cell clusters, which is vastly different from the number of hormoneproducing cells in a normal parathyroid gland. The development of the thymic PTH-producing cells also likely involves Gcml, which is the other mouse ortholog of Drosophila Gcm [69]. Gcml expression, which could not be detected in parathyroid glands, colocalized with PTH expression in the thymus [68]. Thus, Gcm2 specifically controls the differentiation of cells of the third pharyngeal pouch into parathyroid glands, and Gcml regulates an auxiliary developmental pathway that involves differentiation of PTH-producing cells in the thymus. These findings may also help explain the high incidence of PTH-producing tumors in the thymus. Gcm genes likely have similar roles in human parathyroid development because a recent report noted that a homozygous intragenic deletion of G C M B , one of the two human orthologs of the Gcm genes, has been identified in a familial case of autosomal recessive hypoparathyroidism [70]. The patient and her affected cousin developed severe hypocalcemia and hypoparathyroidism at a very early age; however, unlike the findings in the Gcm2-deficient mice, the PTH levels in these individuals were markedly reduced or negligible. Although caveats about different PTH assay conditions exist, it appears likely that thymus may not contribute to PTH secretion as much as it does in mice. Further study of GCM proteins and associated regulatory pathways will enhance our knowledge of the processes that underlie the development of the parathyroid glands.
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Hypoparathyroidism, Deafness, and Renal Anomalies Syndrome The combined inheritance of hypoparathyroidism, deafness, and renal dysplasia (HDR) as an autosomal dominant trait was reported in one family in 1992 [71]. Patients had asymptomatic hypocalcemia with undetectable or inappropriately normal serum concentrations of PTH and displayed the normal sharp increases in plasma cAMP in response to the infusion of PTH. The patients also had bilateral, symmetrical, sensorineural deafness involving all frequencies. Renal abnormalities consisted mainly of bilateral cysts that compressed the glomeruli and tubules and led to renal impairment in some patients. Cytogenetic abnormalities were not detected, and abnormalities of the PTH gene were excluded [71]. However, cytogenetic abnormalities involving chromosome 10pl4-10pter have been identified in two unrelated patients with laboratory and clinical features consistent with HDR. These patients suffered from hypoparathyroidism, deafness, and growth and mental retardation. One patient also had a solitary dysplastic kidney with vesicoureteric reflux and a uterus bicornis unicollis [72]. The other patient, who had a complex reciprocal, insertional translocation of chromosomes 10p and 8q, had cartilaginous exostoses [73]. Neither of these patients had immunodeficiency or heart defects, suggesting a genetic locus different from that of DGS2, and further studies defined two nonoverlapping regions; thus, the DGS2 region was located on 10p13-14 and HDR on 10pl4-10pter. Deletion mapping studies in two other HDR patients further defined a critical 200-kb region that contained GATA3 [74], which belongs to a family of zinc finger transcription factors involved in vertebrae embryonic development. DNA sequence analysis in other HDR patients identified mutations that resulted in haploinsufficiency and loss of GATA3 function [74]. The HDR phenotype is consistent with the expression pattern of GATA3 during human and mouse embryogenesis in the developing kidney, otic vesicle, and parathyroids. However, GATA3 is also expressed in the developing central nervous system (CNS) and the hematopoietic organs in man and mice, suggesting that GATA3 may have a more complex role. Indeed, homozygous GATA3 knockout mice have defects of the CNS and a lack of T cell development, whereas heterozygous GATA3 knockout mice appear to have no abnormalities [75]. It is important to note that HDR patients with GATA3 haploinsufficiency do not have immune deficiency, suggesting that the immune abnormalities observed in some patients with 10p deletions are most likely caused by other genes on 10p. Similarly, the facial dysmorphism and growth and development delay commonly seen in patients with larger 10p deletions were absent in HDR
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patients with GATA3 mutations, indicating that these features are likely due to disruption of other genes on 10p [74]. These studies of HDR patients clearly indicate an important role for GATA3 in parathyroid development and in the etiology of hypoparathyroidism.
Additional Familial Syndromes Several familial syndromes have been reported in which hypoparathyroidism forms a component of a complex multisystem developmental disorder that is unique to that kindred (Table 1). For example, hypoparathyroidism can occur in association with sensorineural deafness but in the absence of renal dysplasia [76]. In addition, an autosomal recessive form ofhypoparathyroidism with growth retardation, developmental delay, and dysmorphic features has also been reported [77]. This syndrome, which was identified in families of Bedouin origin, was mapped to chromosome lq42-43 by homozygosity and linkage-disequilibrium mapping studies. In other families, the inheritance of the disorder has been established and abnormalities of the PTH gene excluded [78], but the chromosomal location and the defective gene(s) remain to be determined. C a l c i u m - S e n s i n g R e c e p t o r Abnormalities CaSR mutations that result in a loss of function are associated with familial benign (hypocalciuric) hypercalcemia [79-85], and it has therefore been postulated that CaSR mutations that result in a gain of function may lead to hypocalcemia with hypercalciuria. Investigations of kindreds with autosomal dominant forms of hypocalcemia have led to the identification of such CaSR mutations [85-91]. The hypocalcemic individuals generally had normal serum intact PTH concentrations and hypomagnesemia, and treatment with vitamin D or its active metabolites to correct the hypocalcemia resulted in marked hypercalciuria, nephrocalcinosis, nephrolithiasis, and impaired renal function. The majority (>80%) of CaSR mutations that result in a functional gain are located within the extracellular domain [85-91], which is different from findings in other disorders caused by activating mutations in different G protein-coupled receptors. Pseudohypoparathyroidism The term pseudohypoparathyroidism (PHP) describes patients with hypocalcemia and hyperphosphatemia due to PTH resistance rather than PTH deficiency [92]. Affected individuals show partial or complete resistance to biologically active, exogenous PTH as demonstrated by a failure to increase urinary cyclic AMP and urinary phosphate excretion in response to this hormone; this
condition is referred to as PHP type I [93-95]. If associated with other endocrine deficiencies and characteristic physical stigmata, collectively termed Albright's hereditary osteodystrophy (AHO), the condition is referred to as PHP type Ia. The latter syndrome is associated with heterozygous inactivating mutations in G N A S 1 located on chromosome 20q13.3. This gene gives rise to at least five differently spliced mRNAs, including Gs~. Heterozygous mutations in one of the exons 1-13 of G N A S 1 lead to an approximately 50% reduction in one of the Gs~ activity/protein, partially explaining the resistance toward PTH and other hormones that mediate their actions through G protein-coupled receptors [93-95]. A similar decrease in Gsu activity/protein is also found in patients with pseudo-pseudohypoparathyroidism (PPHP), who have the same physical appearance as individuals with PHP-Ia but lack endocrine abnormalities, including resistance to PTH. Thus, mutations in the Gsuspecific exons of G N A S 1 are thought to be necessary but not sufficient to fully explain either PHP-Ia or PPHP [93-98]. In fact, a retrospective analysis of numerous published cases with either PHP-Ia or PPHP indicated that both disorders are typically found within the same kindred but never within the same sibship [99]. Furthermore, hormonal resistance is paternally imprinted; that is, PHP-Ia occurs only if the defective gene is inherited from a female affected by either PHP-Ia or PPHP, and PPHP occurs only if the defective gene is inherited from a male affected by either of the two disorders [99,100]. Observations consistent with some of these findings in humans have recently been made in mice heterozygous for disruption of exon 2 of the Gnas gene. Animals that inherited the mutant allele from a female showed decreased blood calcium concentration due to resistance toward PTH. In contrast, offspring that obtained the mutant allele lacking exon 2 from a male showed no evidence of endocrine abnormalities [101]. Moreover, mice that carried the ablated allele on their maternal chromosome had undetectable Gs0c protein in the renal cortex but not in many other tissues. Tissue- or cellspecific Gs0~ expression from a single parental allele is thus almost certainly involved in the pathogenesis of PHP-Ia and PPHP, and it provides a reasonable explanation for the finding that heterozygous G N A S 1 mutations result in a dominant phenotype with regard to the hormonal resistance. Mutations in patients with PHP-Ia are spread throughout most of the Gsu coding exons of G N A S 1 . More than 35 different mutations have been identified, and a 4-bp deletion in exon 7 is the most frequently reported mutation [95,102]. No direct phenotypegenotype correlation has been established for patients with PHP-Ia, except for a missense mutation identified in two unrelated boys with PHP-Ia and testo-
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toxicosis [103]. This Ala-to-Ser substitution at codon 366 generates a temperature-sensitive Gs~ mutant that is constitutively active at cooler temperatures due to accelerated GDP release, but it results in a loss of function at ambient body temperature due to thermolability [103]. Heterozygous inactivating mutations of the Gs0ccoding GNAS1 exons or reduced Gs0~ levels have also been identified in some patients with progressive osseous heteroplasia (POH), a congenital disorder ofectopic bone formation that, unlike the ossification in AHO, affects deep connective tissue and skeletal muscle [104]. Initially, a case with severe plate-like osteoma cutis, a variant of POH, has been associated with a mutation found in several patients with PHP-Ia and PPHP, although no other AHO-like features or hormone resistance were documented [104]. Furthermore, a POH patient with a unique exon 1 mutation was reported who also had mild brachydactyly but lacked any additional features of AHO [105]. POH was also present in another case who presented with PHP-Ia and showed reduced Gs0~ levels [105]. Recent study of several familial cases of POH with Gs0~ mutations revealed an interesting paternal-exclusive inheritance pattern [106]. In a large three-generation kindred, affected individuals who inherited from their father the same 4-bp deletion in GNAS1 exon 7, which was previously found in patients with PHP-Ia and PPHP
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[95,102], demonstrated extraskeletal ossification without developmental and hormonal abnormalities. In contrast, the affected children of female disease gene carriers had clinical features of AHO, but the maternal transmission of the mutation did not lead to hormonal resistance. These findings are surprising and unexpected, and it remains to be determined whether undefined genes that modify the actions of Gs0~may explain the variation in the degree of extraskeletal ossification among patients with the same mutations, and why maternal inheritance of GNAS1 mutations affecting the exons that encode Gs0~ do not invariably lead to PHP-Ia (i.e., AHO combined with hormonal resistance). The G N A S I gene was recently shown to be considerably more complex than previously thought because alternative promoter use and splicing result in several different mRNAs (Fig. 3). Some of these transcripts are derived from either the paternal or the maternal allele, whereas others show biallelic expression. It thus appears likely that the complexity of the G N A S I gene contributes to the unique phenotypic abnormalities in patients with PHP (Fig. 3). Gs0~is encoded by exons 1-13 of the G N A S I gene and mediates the biological functions of a large variety of G protein-coupled receptors, including the PTH/PTHrP receptor. The Gs0~ transcript shows a nonimprinted expression profile in most tissues [107-110].
FIGURE 3 Intron/exon organization of the G N A S I gene and depiction of different mRNAs that are derived from alternative splicing. The mRNA encoding Gsu is thought to be expressed in most tissues from both alleles; however, in the renal cortex transcripts appear to be predominantly maternal in origin. The mRNAs encoding the splice variants XL~,s, AS, A/B, and NESP55 are derived either from the paternal allele or from the maternal allele (see text for details).
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Nonetheless, its expression appears to occur only from the maternal allele in several tissues including the renal proximal tubule [101], although a recent study analyzing human fetal tissue demonstrated biallelic Gs~ expression at this site as well [111]. A second transcript, XL~s, comprises a novel first exon (XL) that splices onto exons 2-13. The encoded approximately 92-kDA protein shares amino acid sequence identity with Gsct in its carboxyl-terminal portion [112], and it appears capable of functioning as a stimulatory G protein in vitro [113,114]. The m R N A encoding XL~s is found at numerous sites; particularly high concentrations have been identified in endocrine and neuroendocrine cells [112,115], and in all investigated tissues it appears to be transcribed only from the paternal allele [107,108,110]. From the same XL~s m R N A , an additional protein with an electrophoretic mobility of approximately 48 kDA appears to also be translated using a second open reading frame; this protein, termed ALEX (alternative gene product encoded by XL-exon), does not share homology with Gs~ or XL~s [116]. The third transcript, NESP55 [117], expresses from the maternal allele only [108,110], and is encoded by another exon of the G N A S I gene that is located upstream of exon XL and the Gsu-specific exons 1-13. NESP55, which is a chromogranin-like neuroendocrine secretory protein [117], shares no amino acid sequence homology with either XL~s or Gsct, but its m R N A contains Gscz-specific exons in the 3' noncoding region. Another transcript with broad paternal-specific expression is A/B (also known as 1' or 1A), which uses a unique promoter and first exon located approximately 2.5 kb upstream of Gs~ [118-120]. The A/B transcript also shares exons 2-13 with the Gs0c transcript, but it is uncertain whether the former is translated into a protein. A recently identified transcript reads from the opposite strand of the GNAS1 gene and is hence termed the antisense transcript (AS). As with the A/B transcript, whether AS m R N A leads to a translated protein is unknown [121,122]. Consistent with the parentspecific expression profiles of its individual transcripts, GNAS1 shows allele-specific methylation. Although the promoter of Gs~ lacks methylation, promoters of the transcripts with parent-specific expression are methylated on the inactive, silenced allele. Because of the complexity of the G N A S I gene and the use of different allele- and strand-specific promoters, it appears plausible that mutations in the Gsct-specific exons 1-13 can affect not only the functional properties of Gsct but also those of XL~s, NESP55, and the A/B transcript [93-98,102]. Mutations in the exons encoding Gs~ have not been detected in PHP type Ib (PHP-Ib), a disorder in which affected individuals show PTH-resistant hypocalcemia and hyperphosphatemia but lack developmental defects and additional endocrine abnormalities [93-95].
Furthermore, individuals with PHP-Ib frequently show a normal osseous response to PTH or even biochemical and radiological evidence of increased bone turnover and osteoclastic bone resorption, indicating that the PTHdependent actions on osteoblasts are not impaired [93,94,123,124]. Moreover, PHP-Ib patients show no abnormalities in growth plate development and thus have normal longitudinal growth, indicating that the PTHrPdependent regulation of chondrocyte growth and differentiation is normal. These latter findings indicate that it is unlikely that defects in the PTH/PTHrP receptor can lead to PHP-Ib, and indeed many studies of the PTH/ PTHrP receptor gene and mRNA in PHP-Ib patients have failed to identify mutations [125-128]. In one study, however, a single amino acid deletion in the carboxyl-terminal region of the PTH/PTHrP receptor, de1382Ile, was demonstrated in three siblings with isolated PTH resistance [129]. This mutation appears to uncouple Gs~ from the PTH/PTHrP receptor only, leaving the function of several other Gs-coupled receptors intact. It may thus be the cause of an unusual variant of PHP-Ib in this kindred, although the advanced bone age documented for two of the affected children needs to be investigated further to disprove its association with the isolated PTH resistance. On the other hand, a genomewide scan in four unrelated kindreds mapped the PHP-Ib locus to chromosome 20q13.3, which contains the GNAS1 gene [130]. In this study, it was also shown that the genetic defect is paternally imprinted and is thus inherited in the same mode as the PTH-resistant hypocalcemia in kindreds with PHP-Ia and/or PPHP. The simplest explanation for these observations is that PHP-Ib is caused by a defect in a tissue- or cell-specific enhancer or promoter of the GNAS1 gene, which could directly or indirectly affect the expression levels of the Gs~-specific transcripts and/or the transcripts encoding XL~s and NESP55 (Fig. 3). Alternatively, PHP-Ib could be caused by a defect in a gene close to the GNAS1 locus that is transcribed only from the maternal allele and affects PTH/PTHrP receptor or Gs~ expression and/or function in some renal cells. Recent investigation of the methylation status of GNAS1 in nine sporadic and two familial cases of PHPIb demonstrated a specific loss of methylation at the exon A/B differentially methylated region [131]. This epigenetic defect, which was not present in healthy controls and patients with AHO, has also been found in affected individuals from a number of unrelated PHP-Ib kindreds in whom the genetic defect mapped to the previously defined locus on 20q13.3 [132]. These findings suggest that PHP-Ib is caused by a mutation in a putative cisacting element required for establishment and/or maintenance of the methylation imprint at GNAS1 exon A/B. Through more detailed haplotype analysis of one of the
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previously described kindreds, it was recently shown that this mutation is likely located at least 56 kbs centromeric of the abnormally methylated A/B region [132]. The importance of the epigenetic regulation of G N A S 1 in PHP-Ib has been further emphasized by findings in a child with paternal uniparental isodisomy of chromosome 20q who presented with PTH-resistant hypocalcemia at an early age [133]. This patient, who also had craniosynostosis, mild hypothyroidism, and a moderately elevated serum calcitonin level, but no evidence of A H O or Gs0~-specific mutations, demonstrated a methylation pattern at the G N A S 1 locus that is typically observed for the paternal allele. As a consequence, methylation at the A/B locus was lacking on both alleles, thus making it likely that loss of methylation in this region alone, presumably in the absence of any mutation, can be sufficient to lead to PTH resistance. Blomstrand's Disease Blomstrand's chondrodysplasia is an autosomal recessive disorder characterized by early lethality, dramatically advanced bone maturation, and accelerated chondrocyte differentiation [134]. Affected infants are typically born to consanguineous healthy parents (only in one case have unrelated healthy parents had two affected offspring) [135-139], show pronounced hyperdensity of the entire skeleton (Fig. 4) and markedly advanced ossification, and particularly the long bones are extremely short and poorly modeled. Recently, P T H / P T H r P receptor mutations that impair its functional properties have been identified as the most likely cause of Blomstrand's disease. One of these defects is a nucleotide exchange in exon M5 of the maternal PTH/ P T H r P receptor allele, which introduces a novel splice acceptor site and thus leads to the synthesis of a receptor mutant that does not efficiently mediate the actions of PTH or PTHrP, despite seemingly normal cell surface expression (Fig. 5). For unknown reasons, the patient's paternal P T H / P T H r P receptor allele is only poorly expressed [140]. In a second patient with Blomstrand's disease, the product of a consanguineous marriage, a proline residue located at position 132 was changed to leucine due to a missense mutation [141,142]. Despite reasonable cell surface expression, the resulting PTH/ P T H r P receptor mutant showed severely impaired binding of radiolabeled PTH and P T H r P analogs, greatly reduced agonist-stimulated cAMP accumulation, and no measurable inositol phosphate response. Additional loss-of-function mutations of the P T H / P T H r P receptor have been identified in three unrelated patients with Blomstrand's disease. Two of these mutations led to a frameshift and a truncated protein due to either a homozygous single nucleotide deletion in exon EL2 [143] or a
FIGURE 4 Radiological findings in a patient with Blomstrand's disease. A: Spine, AP view; B: Spine, lateral view; C: upper extremities; D: lower extremities. Note the markedly advanced ossification of all skeletal elements and the extremely short limbs, despite the comparatively normal size and shape of hands and feet. Furthermore, note that the clavicles are relatively long and abnormally shaped (reproduced with permission from Leroy et al. [136]).
FIGURE 5 Schematicrepresentation of the human PTH/PTHrP receptor. The approximate locations of heterozygous missense mutations that lead to constitutive receptor activation in patients with Jansen's disease are indicated by open circles. Mutations identified in patients with Blomstrand's disease are indicated by closed circles or boxes (see text for details). H, histidine; R, arginine; T, threonine; P, proline; I, isoleucine; L, leucine; X, termination codon.
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27-bp insertion between exons M4 and EL2 [144]. The other defect was a nonsense mutation at residue 104 and thus resulted in a truncated receptor protein [144]. As in Jansen's disease, the identification of mutant PTH/ PTHrP receptors has provided a plausible explanation for the severe abnormalities in endochondral bone formation in patients with Blomstrand's chondrodysplasia. The disease is lethal, but it is likely that in addition to striking skeletal defects, affected infants show abnormalities in other organs, including secondary hyperplasia of the parathyroid glands presumably due to hypocalcemia. In addition, analyses of fetuses with Blomstrand's disease have revealed abnormal breast development and tooth impaction, highlighting the involvement of the PTH/PTHrP receptor in the normal development of breasts and teeth [145].
HYPERCALCEMIC DISEASES M a n i f e s t a t i o n s of h y p e r c a l c e m i a a n d t r e a t m e n t of h y p e r c a l c e m i c d i s o r d e r s Mild hypercalcemia (>11 mg/dl) can be associated with little or no symptoms. More severe hypercalcemia (>13 mg/dl) can be accompanied by failure to thrive, anorexia, nausea, abdominal pain (vomiting), somnolence, stupor, constipation, muscle weakness, polydipsia, and polyuria. The complications of long-standing hypercalcemia can include nephrocalcinosis, renal stones, and renal failure.
Acute treatment of severe hypercalcemia (> 15.0mg/ dl) requires forced diuresis with intravenous normal saline (1.5 times maintenance) and, after achieving adequate hydration, furosemide (1 mg/kg every six hours); peritoneal dialysis or hemodialysis with low calcium concentration in dialysate may be necessary. Since hypercalcemia is usually caused by excessive bone resorption, treatment with a bisphosphonate (for example, pamidronate 0.5-2.0 mg/kg body weight i.v. over 4 hours; usually a single treatment is sufficient to normalize serum calcium concentration within 24-48 hours) should be considered to reduce osteoclast activity; calcitonin (salmon calcitonin, 4 IU/kg body weight every 12 hours IM or SC) may also be effective. For treatment of persistent mild hypercalcemia dietary restriction of calcium (to <400 mg/day) and elimination of vitamin D supplementation may be sufficient; a special low-calcium, low vitamin D infant formula is available. Supportive therapy with corticosteroids (Prednisone at 1-2 mg/kg body weight/day) may be effective in reducing intestinal calcium absorption, but long-term treatment is not recommended. Similar to findings in other tumor syndromes, the abnormal expression of an oncogene or the loss of a tumor-suppressor gene can result in an abnormal proliferative activity of parathyroid cells, and the molecular exploration of these genes has provided important novel insights into the pathogenesis of different forms of hyperparathyroidism (Fig. 6). Oncogenes are genes whose abnormal expression can transform a normal cell into a tumor cell. The normal form of the gene is referred to as a protooncogene, and a single mutant allele may
FIGURE 6 Schematicillustration of the molecular defects that can lead to the development of parathyroid tumors. (A) A somatic mutation (point mutation or translocation) affecting a protooncogene (e.g., PRAD1 or RET) results in a growth advantage of a single parathyroid cell and thus its clonal expansion. (B) An inherited single point mutation or deletion affecting a tumor-suppressor gene (first hit) makes the parathyroid cell susceptible to a second, somatic "hit" (point mutation or deletion; i.e., LOH),whichthen leads to the clonal expansion of a single cell.
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affect the phenotype of the cell; these genes may also be referred to as dominant oncogenes (Fig. 6A). The mutant versions (i.e., oncogenes), which are usually excessively or inappropriately active, may occur because of point mutations, gene amplifications, or chromosomal translocations. Tumor-suppressor genes, also referred to as recessive oncogenes or antioncogenes, normally inhibit cell proliferation, whereas their mutant versions in cancer cells have lost their normal function. In order to transform a normal cell to a tumor cell, both alleles of the tumorsuppressor gene must be inactivated. Inactivation occurs due to point mutations or, alternatively, deletions that can involve substantial genomic portions or a whole chromosome. Larger deletions may be detected by cytogenetic methods, Southern blot analysis, or polymerase chain reaction-based analysis of polymorphic markers. Typically, compared to genomic D N A from other cells (e.g., lymphocytes), genomic DNA from the patient's tumor cells lacks certain chromosomal regions, a finding referred to as loss ofheterozygosity (LOH) (Fig. 6B). Because both alleles of the tumor-suppressor gene must be inactivated to transform a normal cell to a tumor cell, the finding of LOH suggests a point mutation in the other allele. For all these somatic mutations, a single point mutation or a deletion provide a growth advantage of a single parathyroid cell and its progeny, leading to their clonal expansion. Parathyroid Tumors Parathyroid tumors can occur as an isolated and sporadic endocrinopathy as part of inherited tumor syndromes such as multiple endocrine neoplasias (MENs) [ 146] or HPT-JT [147], or in response to chronic overstimulation such as in uremic hyperparathyroidism [148]. Genetic analyses of kindreds with MEN1 and MEN2A and of tumor tissue from patients with single parathyroid adenomas have shown that some of the molecular mechanisms known to be involved in tumor genesis can also be responsible for the development of hyperparathyroidism. Based on our current understanding, sporadic parathyroid tumors are caused by single somatic mutations that lead to the activation or overexpression of protooncogenes, such as PRAD1 (parathyroid adenoma 1) or RET (Fig. 6A). Furthermore, in a significant number of patients, LOH has been documented for one of various chromosomal loci that comprise different tumorsuppressor genes predicted to affect the parathyroid glands. In hereditary forms of the disease, two distinct, sequentially occurring molecular defects are observed. The first "hit" (point mutation or deletion) is an inherited genetic defect that affects only one allele of a gene encoding an antioncogene (Fig. 6B). Subsequently, a somatic
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mutation or deletion affecting the second allele occurs in a single parathyroid cell, and because of the resulting growth advantage this mutation leads to its monoclonal expansion and thus the development of a parathyroid tumor. Examples of the latter molecular mechanism in the development of hyperparathyroidism include the inactivation of tumor-suppressor genes, such as the multiple endocrine neoplasia type 1 (MEN1) gene, or the retinoblastoma (Rb) gene. PRAD1 Gene
Investigations of the PTH gene in sporadic parathyroid adenomas have revealed abnormally sized restriction fragment length polymorphisms with a DNA probe for the 5' part of the PTH gene in some adenomas [149], indicating disruption of the gene. Further studies of the tumor DNA demonstrated that the first exon of the PTH gene (Fig. 2) was separated from the fragments containing the second and third exons, and that a rearrangement had occurred juxtaposing the 5' PTH regulatory elements with new non-PTH D N A [150]. This rearrangement was not found in the DNA from the peripheral leukocytes of the patients, indicating that it represented a somatic event and not an inherited germline mutation. This rearranged DNA sequence was localized to chromosome 11 ql 3, and detailed analysis revealed that it was highly conserved in different species and expressed in normal parathyroids and parathyroid adenomas. The protein expressed as a result of this rearrangement, designated PRAD1, was demonstrated to encode a 295-amino acid member of the cyclin D family of cell cycle regulatory proteins. Cyclins were initially characterized in the dividing cells of budding yeast, in which they control the Gl-tO-S transition of the cell cycle, and in marine mollusks, in which they regulate the mitotic phase (M-phase) of the cell cycle [151]. Cyclins are also present in man and play an important role in regulating many stages of cell cycle progression. Thus, PRAD1, which encodes a novel cyclin referred to as cyclin D 1, is an important cell cycle regulator, and overexpression of PRAD 1 may be important in the development of at least 15% of sporadic parathyroid adenomas [152]. Interestingly, >66% of transgenic mice overexpressing PRAD 1 under the control of a mammary tissue-specific promoter have been found to develop breast carcinoma in adult life [153]. Furthermore, expression of this protooncogene under the control of the 5' regulatory region of the PTH gene has provided a good model for primary hyperparathyroidism, because it results in abnormal parathyroid cell proliferation and mild to moderate chronic hyperparathyroidism with altered PTH response to variations in serum calcium concentration [154]. Taken together, these findings in transgenic animals provide further evidence that
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P R A D 1 may be involved in the development of a signifi-
cant number of parathyroid adenomas. MEN1 gene
MEN 1 is characterized by the combined occurrence of tumors of the parathyroids, pancreatic islet cells, and anterior pituitary (Table 2) [146,155]. Parathyroid tumors occur in 95% of MEN1 patients, and the resulting hypercalcemia is the first manifestation of MEN1 in approximately 90% of patients. Pancreatic islet cell tumors occur in 40% of MEN 1 patients, and gastrinomas leading to Zollinger-Ellison syndrome are the most common type and also the most important cause of morbidity and mortality in MEN1 patients. Anterior pituitary tumors occur in 30~ of MEN1 patients, with prolactinomas representing the most common type. Associated tumors that may also occur in MEN1 include adrenal cortical tumors, carcinoid tumors, lipomas, angiofibromas, and coUagenomas [155,156]. The gene causing MEN1 was localized to a <300-kb region on chromosome 1 lql 3 by genetic mapping studies that investigated MEN 1-associated tumors for LOH and by segregation studies in MEN1 families [157]. The results of these studies, which are consistent with Knudson's model for tumor development, indicate that the M E N 1 gene represents a putative tumor-suppressor gene (Fig. 6B). Characterization of genes from this region led to the identification of the M E N 1 gene [158,159], which consists of 10 exons that encode a novel 610amino acid protein referred to as MENIN. The majority (>80%) of the germline M E N 1 mutations in the families are inactivating and are consistent with its role as a tumor-suppressor gene. These mutations are diverse, and approximately 25% are nonsense, approximately 45% are deletions, approximately 15% are insertions, <5% are donor-splice mutations, and approximatelyl 0% are missense mutations [157]. In addition, M E N 1 mutations are scattered throughout the 1830-bp coding region of the gene, with no evidence of clustering. There does not appear to be any correlation between the location of the M E N 1 germline mutations and the clinical manifestations of the disorder [160]. Tumors from MEN1 patients and non-MEN1 patients have been observed to harbor the germline mutation together with a somatic LOH involving chromosome 1 l q13 or small intragenic deletions, as expected from Knudson's model and the proposed role of the M E N 1 gene as a tumor suppressor [161-171]. MENIN is located in the nucleus [172], where it directly interacts with the N-terminus of the AP1 transcription factor JunD to suppress JunDactivated transcription [173]. MENIN has also been shown to interact with NF-~:B proteins and to modulate NF-~:B transactivation [174]. Thus, the tumor-suppresser
TABLE 2 Multiple Endocrine Neoplasia (MEN) S y n d r o m e s and Their Characteristic Tumors and Associated Genetic Abnormalities a
Type (chromosomal location) MEN1 (llq13)
Tumors
Parathyroids Pancreatic islets Gastrinoma Insulinoma Glucagonoma VIPoma PPoma Pituitary (anterior) Prolactinoma Somatotrophinoma Corticotrophinoma Nonfunctioning Associated tumors Adrenal cortical Carcinoid Lipoma Angiofibromas Collagenomas
MEN2 (10 cen-10q.11.2) MEN2a
Medullary thyroid carcinoma Pheochromocytoma Parathyroid
MTC-only
Medullary thyroid carcinoma
MEN2b
Pheochromocytoma Associated abnormalities Mucosal neuromas Marfanoid habitus Medullated corneal nerve fibers Megacolon
aAutosomal dominant inheritance of the MEN syndromes has been established.
activity of MENIN involves control of cell proliferation via the transcriptional regulation pathway. A mouse model of M E N 1 has been generated by the targeted ablation of the mouse homolog Men1 [175]. Homozygous ablation was embryonically lethal, and mice with heterozygous disruption of Men1 developed a wide range of endocrine tumors later in adult life. Moreover, all the tumors tested in these mice demonstrated a loss of the wild-type Menl [175]. Thus, these findings provide further support for the role of MENIN as a tumor suppressor.
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MEN2 Gene (c-ret)
MEN2 describes the association of medullary thyroid carcinoma (MTC), pheochromocytomas, and parathyroid tumors (Table 2) [146,157]. Three clinical variants of MEN2 are recognized: MEN2a, MEN2b, and MTConly. MEN2a is the most common variant, and development of MTC is associated with pheochromocytomas (50% of patients), which may be bilateral, and parathyroid tumors (20% of patients). MEN2b, which represents 5% of all MEN2 cases, is characterized by the occurrence of MTC and pheochromocytoma in association with a Marfanoid habitus, mucosal neuromas, medullated corneal fibers, and intestinal autonomic ganglion dysfunction leading to multiple diverticulae and megacolon. Parathyroid tumors do not usually occur in MEN2b. MTC-only is a variant in which medullary thyroid carcinoma is the sole manifestation of the syndrome. The gene causing all three MEN2 variants was mapped to chromosome 10cen-10ql 1.2, a region containing the c-ret protooncogene, which encodes a tyrosine kinase receptor with cadherin-like and cysteine-rich extracellular domains and a tyrosine kinase intracellular domain [176,177]. Specific mutations of c-ret have been identified for each of the three MEN2 variants. Thus, in 95% of patients, MEN2a is associated with mutations of the cysteine-rich extracellular domain, and mutations in codon 634 (Cys~Arg) account for 85% of these mutations. However, codon 634 mutations do not appear to be present in sporadic non-MEN2a parathyroid adenomas [178,179]. MTC is also associated with missense mutations in the cysteine-rich extracellular domain and most mutations are in codon 618. However, MEN2b is associated with mutations in codon 918 (Met~Thr) of the intracellular tyrosine kinase domain in 95% of patients. Interestingly, the c-ret protooncogene is also involved in the etiology of papillary thyroid carcinomas and in Hirschsprung's disease. Mutational analysis of c-ret to detect mutations in codons 609,611,618,634, 768, and 804 in MEN2a and MTC-only and codon 918 in MEN2b has been used in the diagnosis and management of patients and families with these disorders [177,180].
Rb Gene
The Rb gene, a tumor-suppressor gene located on chromosome 13q 14 [181], is involved in the pathogenesis of retinoblastomas and a variety of common sporadic human malignancies, including ductal breast, small cell lung, and bladder carcinomas. Allelic deletion of the Rb gene has been demonstrated in all parathyroid carcinomas and in 10% of parathyroid adenomas [182], and it was accompanied by abnormal staining patterns for the Rb protein in 50% of parathyroid carcinomas but in
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none of the parathyroid adenomas [182]. These results demonstrate an important role for the Rb gene in the development of parathyroid carcinomas, and it may be of help in the histological distinction of parathyroid adenoma from carcinoma [182]. However, findings of extensive deletions of the long arm of chromosome 13 (including the Rb locus) in some parathyroid adenomas and carcinomas [183], and similar findings in pituitary carcinomas [184], suggest that other tumor-suppressor genes on chromosome 13q may also play a role in the development of such tumors. Gene on Chromosome l p
LOH studies have revealed allelic loss of chromosome l p32-pter in 40% of sporadic parathyroid adenomas [185]. This region is estimated to be approximately 110 cM, equivalent to approximately 110 million base pairs (Mbps) of DNA. However, recent studies have narrowed the interval containing this putative tumorsuppressor gene(s) to approximate 4cM (i.e., approximately 4 Mbps) [186]. Autosomal Dominant Hyperparathyroidism Syndromes
Hereditary HPT-JT syndrome is an autosomal dominant disorder characterized by the occurrence of parathyroid adenomas and carcinomas in association with fibroosseous mandibular or maxillary jaw tumors and occasional Wilms tumors or adult nephroblastomas [147]. Genetic linkage studies of five HPT-JT families mapped the gene causing this disorder, designated H R P T 2 , to chromosome lq21-q31 [147], and a subsequent positional candidate approach identified thirteen different heterozygous, germline, inactivating mutations in a single gene in fourteen kindreds with HPT-JT [212]. In support of the hyporthesis that the protein encoded by H R P T 2 , termed parafibromin, acts as a tumor suppressor, somatic inactivating mutations were identified in parathyroid adenomas with cystic features. Parafibromin shows ubiquitous expression and evolutionary conservation. There appear to be no homologies to known protein domains, but the amino acid sequence of parafibromin displays similarity to a protein of Saccharomyces cerevisiae possibly involved in transcriptional initiation and elongation. Familial isolated primary hyperparathyroidism (FIPH) has been reported in several kindreds. Some families with this disorder were previously shown to harbor mutations involving the M E N 1 gene [161,187], while in other families, which show a high incidence of early onset parathyroid carcinomas, linkage to polymorphic loci from chromosome l q21-q31 was established [188]. In addition, analysis of parathyroid tumors
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from FIPH patients had revealed LOH involving chromosome lq21-q31 loci, i.e. the region comprising H R P T 2 . Furthermore, one of the three somatic inactivating mutations in H R P T 2 was identified in a kindred with FIPH, and a germline mutation affecting an amino acid of parafibromin different from those mutated in HPT-JT has been identified in another FIHP kindred [212]. It thus appears that HTP-JT and some forms of FIPH are allelic variants, and correlating the different mutations of H R P T 2 in these disorders to phenotypic variations will be important. Hyperparathyroidism in Chronic Renal Failure Chronic renal failure is often associated with a form of secondary hyperparathyroidism that may subsequently result in the hypercalcemic state of tertiary hyperparathyroidism. The parathyroid proliferative response in this condition initially suggested that the autonomous parathyroid tissue might have undergone hyperplastic change and therefore be polyclona! in origin. However, studies of X chromosome inactivation in parathyroids from patients on hemodialysis with refractory hyperparathyroidism have revealed at least one monoclonal parathyroid tumor in >60% of patients [149]. In addition, LOH involving several loci on chromosome Xpl 1 was detected in one of these parathyroid tumors, suggesting the involvement of a tumor-suppressor gene from this region in the pathogenesis of such tumors [149]. Interestingly, none of the parathyroid tumors from these patients with chronic renal failure had LOH involving loci from chromosome 11ql 3. This unexpected finding of monoclonal parathyroid tumors in the majority of patients with tertiary hyperparathyroidism suggests that an increased turnover of parathyroid cells in secondary hyperparathyroidism may possibly render the parathyroid glands more susceptible to mitotic nondisjunction or other mechanisms of somatic deletions, which may involve loci other than those located on chromosome 11ql 3 ( M E N 1 and PRAD1). Disorders of the Calcium-Sensing Receptor Two hypercalcemic disorders due to mutations of the CaSR have been reported [79-85]: FBH (also referred to as FHH) and neonatal severe hyperparathyroidism (NSHPT). Mutational analyses of the human CaSR, a G protein-coupled receptor located on chromosome 3ql 3-q21 [189], have revealed different mutations that result in a loss of function of the CaSR in patients with FBH and NSHPT [79-85]. Many of these mutations cluster around the aspartate- and glutamate-rich regions (codons 39-300) within the extracellular domain of the receptor, and this has been proposed to contain low-
affinity calcium binding sites based on similarities with calsequestrin, in which the ligand-binding pockets also contain negatively charged amino acid residues [190]. Approximately two-thirds of the FBH kindreds investigated have unique heterozygous mutations of the CaSR, and expression studies of these mutations have demonstrated a loss of CaSR function whereby there is an increase in the calcium ion-dependent set point for PTH release from the parathyroid cell [79,84,85, 191,192]. NSHPT occurring in the offspring of consanguineous FBH families is due to homozygous CaSR mutations [79,80,82,193]. However, some patients with sporadic neonatal hyperparathyroidism have been reported to have de novo heterozygous CaSR mutations [81,194], suggesting that factors other than mutant gene dosage may also play a role in the phenotypic expression of a CaSR mutation in the neonate [193], such as the degree of set point abnormality, the bony sensitivity to PTH, and the maternal extracellular calcium concentration. The remaining one-third of FBH families in whom a mutation within the coding region of the CaSR has not been demonstrated may have either an abnormality in the promoter of the gene or a mutation at one of the two other FBH loci that have been revealed by family linkage studies. One of these FBH loci, FBH19p, is located on chromosome 19p [195]. Studies of another FBH kindred from Oklahoma who also suffered from progressive elevations in PTH, hypophosphatemia, and osteomalacia [196,197] demonstrated that this variant, designated FBHok, was linked to chromosome 19q13 [198]. These three FBH loci, located on chromosomes 3q, 19p, and 19q, have also been referred to as FBH (or FHH) types 1-3, respectively [198]. Jansen's Disease Jansen's disease (Figs. 5 and 7) is an autosomal dominant disease characterized by short-limbed dwarfism caused by an abnormal regulation of chondrocyte proliferation and differentiation in the metaphyseal growth plate and by an associated severe hypercalcemia and hypophosphatemia, despite normal or undetectable serum levels of PTH or PTHrP [199-201]. These abnormalities are caused by mutations of the PTH/PTHrP receptor that lead to constitutive PTH- and PTHrPindependent receptor activation. Three different mutations of the PTH/PTHrP receptor have been identified, involving codon 223 (His-~Arg), codon 410 (Thr-~Pro), and codon 458 (Ile-§ [199,200,202,203] (Fig. 5). Expression of the mutant receptors in COS-7 cells resulted in constitutive, ligand-independent accumulation of cAMP, whereas the basal accumulation of inositol phosphates was not increased [199-202]. Since the PTH/PTHrP receptor is most abundantly expressed in
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FIGURE 7 Hand radiographs of the patient first described by Jansen at age 10 (left) and age 44 (right) (reproduced with permission from De Haas et al. [211]).
kidney and bone, and in the metaphyseal growth plate, these findings provide a plausible explanation for the abnormalities observed in mineral homeostasis and growth plate development in this disorder. This conclusion is supported by observations in mice that express the human PTH/PTHrP receptor with the H223R mutation under the control of the rat ~l(II) promoter [26]. This promoter targeted expression of the mutant receptor to the layer of proliferative chondrocytes, delayed their differentiation into hypertrophic cells, and led to a mild impairment in growth of long bones, at least in animals with multiple copies of the transgene. These observations are consistent with the conclusion that expression of a constitutively active human PTH/PTHrP receptor in growth plate chondrocytes causes the characteristic metaphyseal changes in patients with Jansen's disease.
in mice results in vascular abnormalities similar to those observed in patients with Williams syndrome [207]. However, the microdeletions that have been reported also involve another gene, designated LIM-kinase, that is expressed in the CNS [208]. The calcitonin receptor gene, located on chromosome 7q21, is not involved in the deletions in Williams syndrome and is therefore unlikely to be implicated in the hypercalcemia of such children [209]. While the involvement of the elastin and LIMkinase genes in the deletions of Williams syndrome patients can explain the respective cardiovascular and neurological features of this disorder, it seems likely that another, as yet uncharacterized gene within this contiguously deleted region is involved in the abnormalities of calcium metabolism. CONCLUSION
Williams S y n d r o m e Williams syndrome is an autosomal dominant disorder characterized by supravalvular aortic stenosis, elfin-like facies, psychomotor retardation, and infantile hypercalcemia. The underlying abnormality of calcium metabolism is unknown, but abnormal 1,25-dihydroxy vitamin D3 metabolism or decreased calcitonin production have been implicated, although none have been consistently demonstrated. Studies have demonstrated hemizygosity at the elastin locus on chromosome 7ql 1.23 in more than 90% of patients with the classical Williams phenotype [204-206], and only one patient had a cytogenetically identifiable deletion, indicating that the syndrome is usually caused by a microdeletion of 7ql 1.23 [206]. Interestingly, ablation of the elastin gene
Considerable advances have occurred during the past few years in identifying key proteins involved directly or indirectly in the regulation of PTH synthesis or secretion and in mediating its hormonal actions in the different target tissues. The subsequent identification of mutations in several of these proteins provided a plausible molecular explanation for a variety of familial and sporadic disorders of mineral ion homeostasis and/or bone development. In addition to these advances, genetic loci and/ or candidate genes have been identified for multiple inherited disorders, and it is likely that the molecular definition of these familial disorders, greatly aided by the rapid progress of the Human Genome Project, will continue to provide important insights into the regulation of blood calcium and phosphate.
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Acknowledgments RVT is grateful to the Medical Research Council and Wellcome Trust (United Kingdom) for support. HJ is supported by grants from the National Institutes of Health, NIDDK (DK-46718-06 and DK50708-01). MB is a recipient of a fellowship from the National Kidney Foundation.
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1211 Fibrous Dysplasia PAOLO BIANCO,* PAMELA GEHRON ROBEy,t and SHLOMO WIENTROUB $ *Department of Experimental Medicine and Pathology, Division of Pathology, Medical School La Sapienza University, Rome, Italy t Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland $Department of Pediatric Orthopedic Surgery, Dana Children's Hospital Tel A viv Medical School Tel Aviv, Israel
INTRODUCTION
the disease, remain the most common, the most severe, the least understood, and the least treatable. Today, the McCune-Albright syndrome (OMIM #174800) eponym is kept alive mainly by virtue of its popular use among endocrinologists to refer to what is in fact only one of several clinical expressions, and indeed of several syndromes, whereby a protean disease presents in different subsets of patients. In pathology textbooks, little reference is made to the systemic nature of the disease, and fibrous dysplasia is commonly described as an overgrowth of fibrous tissue in bone, reflecting a developmental disorder, and associated with an arrested differentiation of bone cells. Deposition of bone is described as occurring in the absence of osteoblasts by some vicarious (and obscure) metaplasia of an immature fibrous tissue. This definition is as wrong as the assumption that bone can be formed in the absence of bone-forming cells (osteoblasts) from a tissue that is nonosteogenic (fibrous) or undifferentiated in nature. Furthermore, fibrous dysplasia may be seen as either a developmental disorder of the whole organism, or an entirely postnatal localized disease. It does not represent per se an impairment in the prenatal organogenesis of bone or in deposition of bone by osteoblasts. Finally, in textbooks on metabolic bone diseases, fibrous dysplasia is most commonly defined as a high turnover disease of bone remodeling. The "bone expert" definition suffers from the adoption of a reading key (rate of turnover) that is commonplace in the field but does not necessarily conform to the specific biology of the disease. Even though turnover of bone is indeed
Recognition of fibrous dysplasia as a distinct skeletal disease is commonly attributed to the description of an osteitisfibrosa disseminata occurring in conjunction with various endocrinopathies and skin pigmentation by Albright et al. [1,2] and by McCune and Bruch [3] in 1937. The term osteitisfibrosa, chosen by Albright, alluded to the perceived fibrous nature of the changes observed in bone but was also meant to convey their resemblance to von Recklinghausen's osteitisfibrosa cystica [4] (hyerparathyroid bone disease) and at the same time their distinction from it. Albright's osteitis would be fibrosa but not cystica, because the cysts seen in hyperparathyroidism would not be a feature of Albright's newly proposed entity. We now know that cysts are very common in the disease for which Albright helped identify, whereas brown tumors, a common but not exclusive cause of radiographically cystic lesions in hyperparathyroidism, are not. Lichtenstein [5] and Lichtenstein and Jaffe [6] described the same disorder and recognized that the same skeletal changes described by Albright could occur as single or multiple lesions, with or without associated extraskeletal disorders; thus, they provided the first unifying concept of the disease. This concept was to prove correct and to withstand a molecular genetic redefinition of the condition approximately 50 years later. For this disorder, Lichtenstein coined the term fibrous dysplasia of bone (FD) [5], and Lichtenstein and Jaffe [6] recommended its use in all cases, which is justified today by the fact that the bone lesions, among all others occurring in
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altered in FD, accelerated turnover is an epiphenomenon and not a cause of the disease or of its individual clinical expressions. The bone that is turned over more rapidly in FD is abnormal, qualitatively and quantitatively, in many other critical features ranging from primary modeling to chemical composition, which more directly translate into clinically adverse effects. During the past 10 years, important advances in the molecular pathogenesis of the disease have established that fibrous dysplasia of bone is a genetic, noninherited disease of bone and bone marrow caused by activating mutations of the GNAS1 gene, which encodes the 0t subunit of the stimulatory G protein, Gs [7,8]. The mutation occurs postzygotically and results in a somatic mosaic state [7]. Mutated cells are exposed to the effects of excess endogenous cAMP production [9], which results from the inappropriate stimulation of adenylyl cyclase by the mutated Gs0t. In bone and marrow, the mutation affects cells of the osteogenic lineage at various stages of maturation [10-13], causing different types of dysfunction. The disease is one of bone growth and modeling more than bone remodeling, even though remodeling of the abnormal FD bone is high and contributes to the natural history of the skeletal lesions. FD is a disease of excess, abnormal, and imperfect bone growth. The disease produces excess bone growth by causing a localized increase in bone tissue within bone (or local bone mass). The disease causes abnormal bone growth because bone formation does not adhere to the architectural design of the affected, growing bone segments. The territorial definition of cortical bone, cancellous bone, and marrow space is lost, and bone is formed with haphazard trabecular architecture and an irregular internal structure and is mechanically unsound. The disease causes imperfect bone growth because the matrix deposited has an abnormal chemical composition, an abnormal "tricotage," and an abnormal mineral content. As a result, the abnormal bone is not only fragile but also excessively compliant. Fracture and deformity ensue. Because of abnormal bone formation, dysmorphisms at specific anatomic sites may jeopardize the integrity of critical structures such as cranial nerves. Secondary changes, such as cysts and hemorrhage, are further consequences of specific changes in the structure of the fibrous dysplastic bone and the fibrous dysplastic marrow, and they may significantly, sometimes dramatically, affect the clinical course of the disease.
CLINICAL FEATURES Skeletal Lesions As classically recognized by Lichtenstein and Jaffe [6], the skeletal disease may be monostotic or polyostotic and
affect the craniofacial, axial, or appendicular skeleton in variable combinations. The ratio of monostotic to polyostotic forms is approximately 10:1. Polyostotic disease of limb bones may be unimelic or polymelic and ispilateral or amphilateral. In the most severe cases, the entire skeleton may be affected (panostotic disease) [14]. As a result of the highly variable number of lesions, disease severity ranges from subclinical, incidentally discovered forms to rare lethal forms. In the latter, different kinds of extraskeletal complications occur either from the associated endocrine disease(s) (e.g., opportunistic infections in neonates with Cushing's syndrome) or from the skeletal disease (e.g., restrictive respiratory failure and bronchopneumonia from severe thoracic disease). Most lesions are not congenital. In the rare instances of perinatal disease, unusual bone changes not immediately related to the common histological appearance of postnatal FD may be observed, including growth plate abnormalities (unpublished observations). FD lesions develop mostly during the period of bone growth. Monostotic disease tends to appear in adolescence, and polyostotic disease occurs in infancy. In general, the more widespread the disease, the earlier the time of clinical presentation. Major skeletal lesions do not usually develop de novo after puberty but do remain capable of growth, and additional small sites of separate involvement of the same bone can develop as well. Monostotic lesions may be asymptomatic and accidentally discovered or present with a pathologic fracture, bone deformity, or bone pain of long duration. Severe polyostotic forms usually present with a varied combination of pain, pathologic fracture, deformity, and associated extraskeletal disorders and complications. Presentation of associated extraskeletal disease, mostly precocious puberty in females, may precede the appearance or the discovery of the skeletal disease. In patients with polyostotic disease, craniofacial deformity may be compounded by a characteristic facies fibrodysplastica (Fig. 1) (macrocephaly, frontal bossing, malar prominence, elongation and widening of the midface, and hypertelorism), which represents a remarkable example of leontiasis ossea in Virchow's original semeiological sense. Less generalized or less uniform involvement of the craniofacial skeleton is common, sometimes leading to a pseudocherubic facies, facial and orbital asymmetry, proptosis, or localized tumor-like growths in gnathic bones. Involvement of the sphenoid, orbital processes of frontal bones, and the temporal bone may impinge on cranial nerves and cause visual or auditory loss or impairment. Whereas craniofacial deformity solely represents the effects of the overgrowth of fibrous dysplastic bone, limb deformity may result from a combination of excess compliance of the abnormal bone and a complex sequence of
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upper third of the femur (the shepherd's crook deformity) (Figs. 2 and 3), a time-honored radiographic sign of FD. Limb length discrepancy and long bone bowing are the most common expressions of limb bone deformity. It should be noted that bowing is not restricted to weightbearing bones, even in the absence of fractures, and may in these cases solely reflect the malacic compliance of the abnormal bone (Fig. 4). R a d i o g r a p h i c Features
FIGURE 1 The facies fibrodysplastica. Note the prominent frontal bossing and malar prominence, associated with widening and elongation of the midface, and depression of the nasal bridge. (see color plate.)
pathologic or fatigue fractures. C o x a vara may result from fatigue fracture in the femoral neck or non-ad u n g u e m reduction of a fracture. Combined with overgrowth and excess pliability of FD bone, these events may evolve into a more complex varus deformity of the
Craniofacial lesions are usually sclerotic in a radiological sense. This reflects the florid bone formation that occurs within the lesional tissue of craniofacial bones as a site-specific characteristic and a greater tendency of the lesional craniofacial bone matrix to mineralize compared to the lesional bone matrix at other sites. In this respect, it should be noted that normal craniofacial bones, especially gnathic bones, are more densely mineralized (per equal mass of bone matrix) than the rest of the skeleton in normal subjects. In severe cases of generalized disease of the craniofacial skeleton, a unique radiographic picture can be produced. In an anterior projection, the marked sclerotic changes blur and efface all anatomical features and radiographic detail of skull bones (which we named the "iron mask inside" sign) (Fig. 5). In lateral projections, the contour of calvarial bones appears fuzzy and hairy due to excess bone formation resulting in a thicker than normal but noncompact bone (conceptually akin to, but
FIGURE 2 Development of the shepherd's crook deformity as a result of FD of the femur. A ground glass lesion with significant superimposed lytic changes (left) has evolved into a marked varus deformity (right) over 3 years.
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FIGURE 3 Severeshepherd's crook deformity of the femur, with superimposed cystic changes. FIGURE 4 Bowing of non-weight-bearing bones: humerus of a 24-year-oldpatient with severepolyostoticFD and hypophosphatemia. clearly distinct from, the "crew haircut" picture of congenital hemolytic anemias and the underlying hyperostosis porotica [15]). In less severe cases, a marked thickening of the calvarium similar to what is seen in Paget's disease can be observed. Lesions of the long bones may be diaphyseal or metaphyseal and usually spare the epiphyses. However, epimetaphyseal involvement with crossing of the physis was demonstrated in one of the original cases described by Albright in 1937 [1,16], which highlights the nonabsolute value of the epiphysis-sparing criterion in clinical practice. What seems to be spared in all cases is the articular cartilage, which may, however, be involved in severe arthritic changes secondary to bone deformity. Likewise, whereas fibrous dysplasia generally does not interrupt the continuity of the cortex and remains confined by a thinned cortex, exophytic and paraosteal forms do occur,
particularly in short tubular bones of hands and feet (fibrous dysplasia protuberans [17]). Uncomplicated long bone lesions appear as medullary, expansile lesions with a characteristic ground glass appearance, variable degrees of expansion of the bone contours, cortical thinning, and scalloping (Fig. 6). This appearance may be altered by superimposed secondary changes, thus generating complex pictures significantly different from baseline (Fig. 7). Focal rarefaction of the ground glass density may result from small areas of cartilage, from small hemorrhagic and cystic changes, or simply from the local predominance of resorptive events. Focally increased density, in contrast, may reflect local predominance of bone formation within a lesion or different histological events. Fine or coarse stippling,
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FIGURE 5 Severe craniofacial fibrous dysplasia. (a) The "iron mask inside" sign in a patient with facies fibrodysplastica, polyostotic FD, and hypophosphatemia. Note the blurring and effacement of all radiographic detail expressing normal anatomical features of craniofacial bone. (b) A lateral view of the skull of the same patient demonstrating the hairy and fuzzy contour of the calvarium. (c) Computed tomography scan demonstrates the massive involvement of craniofacial bones by FD. (d) The marked thickening of the calvarium in another patient.
or conspicuous patches of densely mineralized material appearing in the context of the ground glass background, may represent a nonosseous mineralized phase [18]. In lesions of long duration (i.e., especially in adults) that do not sustain either fracture or surgery, ring-like sclerotic rims (sometimes polycyclic) may encircle the original lesional areas (rinds) (Fig. 8). Direct histological observation of these structures indicates that they likely represent bone scars signaling the arrested growth of the lesion, akin to the prominent growth scars observed in the physis of certain mammals. Hemorrhage and cyst formation generate a significant enlargement and rarefaction of the lesion and promote additional expansion and thinning of the bone contour. A s s o c i a t e d Extraskeletal Lesions Endocrine and nonendocrine extraskeletal changes may be associated with bone lesions. Hyperfunctional
endocrinopathies and skin pigmentation are associated with polyostotic FD in McCune-Albright syndrome (MAS), but each may also occur with monostotic or polyostotic FD in the absence of the other. Overt endocrinopathies are present in approximately 70% of patients with polyostotic FD but are much less frequent in patients with monostotic disease. However, subtle and subclinical hormonal imbalances may remain undetected unless careful and complete testing for endocrine function is performed. All endocrinopathies reflect the direct effect of the sustained, ligand-independent generation of cAMP brought about by the constitutive activity of the mutated Gs~, which mediates the transduction of signals generated by heptatransmembrane domain receptors (TSH, ACTH, gonadotropin, and GH-RH) in the normal cell types of endocrine systems. Precocious puberty is the most common accompanying endocrinopathy, is gonadotropin-independent in most cases, and is much more commonly seen in females
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FIGLIRE 7 Complexand diffuse FD lesions of tibia and fibula. Note the marked bowing, multiple lytic lesions, and focal cystic and sclerotic changes.
FIGURE 6 FD lesion of the femur showing the typical ground glass appearance in the upper third and rarefaction of the ground glass appearance in the middiaphysis. The inset shows a detail of the diaphyseal cortical bone, demonstrating scalloping and internal erosion (arrows).
than in males [19-23]. It is caused by ovarian follicular cysts, in which the mutation can be directly demonstrated [24]. There may be multiple cysts, and each may be present and active only transiently, explaining the intermittency or remittance of precocious puberty symptoms in some cases. Central puberty may follow in some cases. Regardless of its obvious effects on bone age, whether and how precocious puberty affects the development and growth of bone lesions is unclear. Growth hormone (GH) excess is also c o m m o n in patients with FD. However, somatotroph adenomas are detectable in only a minority of patients with F D and G H excess [25], indicating that either non-tumor-forming hyperplasia or cell hypersecretion without hyperplasia may be the most c o m m o n effect of G N A S 1 mutations in the pituitary. This is in agreement with the observation that gsp + somatotroph adenomas not associated with F D are usually smaller than gsp- adenomas [26]. Cases of gigantisrn are rare [27-30], whereas acromegalic traits (which are untimely in growing individuals and young adults) [31,32] may occur simultaneously with the
FIGLIRE 8 Nondeformed femur of a 50-year-old patient with FD. The lesional areas are outlined by a typical rind (arrows).
21. FibrousDysplasia native faciesfibrodysplastica and contribute to craniofacial deformity. GH excess allows the effects of precocious puberty on stature to be compensated (M. T. Collins, personal communication). Cushing's syndrome is typically seen in infants and young children, and it reflects the occurrence of GNAS1 mutation in the adrenal and the development of multinodular adrenocortical hyperplasia. It appears to subside spontaneously with time [33-35]. Neonatal/infantile Cushing's syndrome caused by GNAS1 mutation in the adrenal may cause fracture as a result of glucocorticoid-induced osteopenia, totally independent of the development of FD. There is only one recorded case of true infantile Cushing's disease reflecting a basophilic corticotroph pituitary adenoma in which an R201 GNAS1 mutation (i.e., the kind of GNAS1 mutation occurring in FD) was demonstrated [36], whereas Q227 mutations are more common [37]. Thyroid hyperfunction is more common than GH excess and may be caused either by goiter (more commonly) or by welldefined adenoma [22,38]. All endocrinopathies associating with FD, and even major physiological changes in the hormonal climate such as pregnancy [32,39], diversify the clinical picture in different patients and may affect the course of the bone disease in many ways. The specific manner in which these effects may occur needs to be precisely determined. Cutaneous pigmented macules (Fig. 9) are commonly referred to as caf6 au lait spots with a "coast of Maine" profile typical of neurofibromatosis (as opposed to the "coast of California"), and they are of similar hue to those occurring in neurofibromatosis. They represent the autonomously enhanced, MSH-independent synthesis and transfer of melanin by GNASl-mutated melanocytes that distribute according to a pattern reflecting developmental migratory events, cAMP levels are elevated in mutated melanocytes and expression of the tyrosinase gene is upregulated [40].
FIGURE9 Caf6au lait maculesin the skin of a child with MAS.Note that the macules arrest at the midline, which is a common but not an obligatory feature. Also note the breast bud development induced by precocious puberty. (see color plate.)
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The association of skeletal myxomas with FD was first recognized by Henschen and Fallvon [41]. Myxomas may appear as single or, more commonly, multiple lesions, which in turn are associated with single or multiple FD lesions, with or without endocrinopathies, in what is now known as Mazabraud's syndrome [42,43]. They may occur in the same limb segment as FD lesions but tend to appear after the establishment of an FD lesion [44-48]. GNAS1 mutations have been demonstrated in skeletal myxomas occurring in conjunction with FD, but they have also been demonstrated in its absence [49]. Interestingly, Gs~ is a negative regulator of myogenic differentiation [50], and histological pictures indistinguishable from skeletal myxomas have also been observed within FD bone as local changes in the fibrous tissue. Cholestatic liver disease has been seen in neonates and infants [33,52] and is associated with marked biliary ductular proliferation (unpublished observation). It appears to subside spontaneously over time. Other less frequent patterns of organ involvement may occur [22,51], and the spectrum of changes directly dependent on the local expression of mutated Gsct may be broader than currently appreciated. Renal phosphate wasting and hypophosphatemia with low levels of 1,25(OH)2D3 occurs in variable degrees in approximately half of patients with FD/MAS [53], making it one of the most common metabolic derangements associated with skeletal lesions. In the most severe instances, hypophosphatemic rickets may occur simultaneously with the FD-related changes in bone [54]. Direct involvement of the mutated Gs~ in the proximal tubule of the kidney as the cause of phosphate wasting has not been conclusively ruled out [55]. However, indirect evidence supports the view that a putative and unidentified humoral factor, produced in the FD tissue, may be the cause of FD-associated renal phosphate wasting [53,56], similar to oncogenic osteomalacia. Preliminary data indicate that FGF-23, one of the putative factors involved in osteogenic osteomalacia [57], is in fact expressed in FD tissue (M. Riminucci, personal communication). Whereas skeletal lesions are believed to be mostly synchronous, the combination of skeletal and extraskeletal diseases may develop synchronously or metachronously. In a recent series of 32 patients with MAS [58], skin lesions were regularly present at birth and precocious puberty appeared by age 4 in 50% of patients. Bone lesions first appeared by age 8 in 50% of patients and increased in number over time. Although rare, skin lesions can develop in late adolescence (P. Bianco, personal observation), and an isolated FD lesion may develop in adulthood, decades after precocious puberty, presenting as an isolated endocrine dysfunction [59].
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These rare cases are the only ones in which adult metabolic remodeling, rather than growth and modeling of bone, may be the trigger for development of a skeletal lesion. Conversely, endocrine dysfunction or lesions, such as a GH-secreting adenoma, may develop decades after the presentation of skeletal disease [60]. The apparent occurrence of certain patterns of multiorgan involvement at specific ages (e.g., liver and adrenal disease in early infancy, prior to the development of apparent bone lesions) may signify the existence of age-specific syndromic associations.
MOLECULAR GENETICS The G N A S 1 G e n e a n d Its P r o d u c t s The gene encoding Gs~, G N A S 1 (OMIM 139320), is located at chromosome 20q13 and was originally described as a 20-kilobase gene containing 13 exons and 12 introns [61]. Multiple promoters and at least 5 alternative first exons give rise to a complex family of transcripts [62]. Transcription of exons 1-13 from the most downstream promoter generates a Gs~ species with an apparent molecular weight of ~52 kDa. Splicing out exon 3 gives rise to a short form of Gs~ (~45 kDa), whereas insertion of CAG (coding for serine) by the use of two alternative splice acceptors (TG instead of the consensus AG) 3 base pairs upstream of exon 4 generates additional splice variants [63-65]. Thus, the following four major Gs~ isoforms can be formed: Gs~ 1 (long), Gs0t 2 (short), Gs~ 3 (long + Ser), and Gscz 4 (short + Ser)]. These are all functionally active and generated by transcription from a single promoter and alternative splicing. Gs~ 1 and 3 (long forms) predominate in kidney, placenta, adrenal medulla, cortex, and cerebellum, whereas isoforms 2 and 4 (short forms) predominate in heart, liver, neostriatum, and platelets [66]. Both the long and short forms of G ~ are expressed in osteogenic cells (M. Riminucci and L. W. Fisher, unpublished results). The alternative promoters and first exons in the G N A S 1 gene, spliced onto the common set of exons 2-13, generate transcripts that have not been identified in bone [61,67-71]. However, at least two of them (XL~s and NESP55) are expressed in endocrine and neuroendocrine tissues that may be related to extraskeletal diseases associated with FD [72-75]. The role of different Gs~ isoforms in disease in unclear and may require attention. It has been postulated that different isoforms may be functionally different with respect to their ability to interact with different downstream effectors. In principle, they may also differ in relative abundance in involved and uninvolved tissues, posttranslational modifications, membrane trafficking, association with receptors and the [37
subunit, and, finally, in their interactions with adenylyl cyclase [66].
Causative Mutations Activating missense mutations of the G N A S 1 gene associated with fibrous dysplasia consist of single base substitutions at codon R201 in exon 8 (Fig. 10). R201C and R201H mutations of the G N A S 1 gene were first identified, along with mutations at codon Q227, in isolated endocrine tumors [76-80]. Hence, the mutated gene was designated the gsp oncogene [77]. However, this label should be used with caution since there is no evidence that G N A S 1 mutations are indeed transforming. Most of the endocrine tumors associated with G N A S 1 activating mutations are benign, and there is no evidence of a causative role of G N A S 1 mutation in their development. In vitro studies suggest that the gain in proliferative activity induced by transfection of the putative gsp oncogene is too modest to account for the development of a tumor or even to predispose proliferating cells to an increased likelihood of a second transforming event [81]. Excessive cAMP-induced protein kinase A (PKA) activation, the putative mediator of the transforming
FIGURE 10 (A) Mutations at codon 201 demonstrated by sequencing of the relevant PCR-amplified region of exon 8. CGT~CAT transition (left, asterisk) results in the R201H mutation; CGT~TGT transition (right, asterisk) results in the R201C mutation. (B) Selective amplification of the mutated allele by PCR in the presence of PNA oligos blockingthe amplificationof the normal allele [85]. This method allows the demonstration of low amounts of the mutated genotype(low numbers of mutated cells). (C) Reverse transcriptase-PCR analysis of normal and mutated stromal cell strains. Only the normal genotype is demonstrated in normal cells; both the normal and mutated alleles are expressed in FD samples [13]. (see color plate.)
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effect, may even counter, rather than promote, cell transformation in certain systems [82]. R201 mutations (R201H and R201C) identical to those originally detected in isolated endocrine tumors were later demonstrated in patients with MAS [7,8], in which FD is associated with various hyperfunctional disorders. Soon thereafter, it was shown that the mutations could be demonstrated directly in fibrous dysplastic bone [83] and in cells grown in culture from fibrous dysplastic bone [11]ma finding suggesting a direct involvement, in the context of a complex endocrine disorder, of the disease genotype in the pathogenesis of the skeletal disease. It is now established that R201H and R201C mutations are consistently detected not only in the bone lesions of MAS but also in monostotic and polyostotic forms of fibrous dysplasia that occur in the absence of any apparent endocrine abnormality [12,84,85]. Thus, all forms of fibrous dysplasia, all forms of FD-associated hyperfunctional endocrinopathy, and some sporadic, isolated endocrine tumors appear to share the same disease genotype. Rarely, different amino acid substitutions for R201 occur. R201S and R201G mutations have been reported in patients with polyostotic FD and MAS, respectively [86,87], and R01S and R201L mutations have been reported in patients with isolated endocrine tumors in the absence of skeletal disease [88,89]. Thus, of the predicted missense mutations at codon 201, only R201P remains undetected. It has been noted that the diversity of amino acid substitutions encoded by the missense mutations detected to date (basic, uncharged polar, and nonpolar), in association with consistent gain-of-function effects in different clinical disorders, highlights the critical significance of replacement of R201 per se in determining a functional pathogenic effect [87]. Mutability of t h e R201 C o d o n The disease genotype is not inherited; therefore, each patient represents a de novo mutational event. This observation, together with the consistency of the mutation observed, suggests that the R201 codon (5'-CGT-35 is a mutational hot spot, and that extremely frequent mutations cause a remarkably rare disease. The R201C and R201H mutations, which account for almost all cases of FD, involve C G ~ T G and CG---,CA transitions, respectively. These two transitions of CG dinucleotides represent 32% of all point mutations known to cause human disease, a 12% higher frequency than that predicted from random expectations [90]. All CG dinucleotides are thus indeed mutational hot spots, consistent with a chemical model of mutation in which methylation and deamination generate the relevant base transitions. However, the overall frequency of transitions at CG dinucleotides in
human disease also exceeds that predicted by the methylation-deamination model and may postulate the concurrence of additional mechanisms, such as nucleotide misincorporation as a result of transient misalignment of bases at the replication fork [90]. The nonrandom nature of CG mutations might be used to predict the rate of mutation and even the prevalence of human disease [90], which would be of direct clinical relevance for FD, for which no epidemiological assessments have been performed. Functional C o n s e q u e n c e s of t h e GALAS 1 M u t a t i o n s Heterotrimeric G proteins couple receptors for a variety of extracellular signals to intracellular effectors and consist of three different polypeptides [91-93]. The subunits bind guanine nucleotides with high affinity and specificity, whereas the 13and y peptides are noncovalently associated with one another in a functional dimer subunit. The ~13y heterotrimer associates with the inner aspect of the plasma membrane and is coupled with high affinity to the relevant receptor. Upon ligand binding to the receptor, GTP is exchanged for GDP due to a conformational change in Gs~, the heterotrimer dissociates from the plasma membrane, and the GTP-binding subunit dissociates from the 13y subunit. GTP-binding Gs~ binds and activates adenylyl cyclase (AC), thus generating cAMP. AC remains active only as long as it is bound to activated Gs~. The amount of time that this association exists is dependent on the rate at which Gs~bound GTP is hydrolyzed to GDP by the intrinsic GTPase activity of Gs~. Hydrolysis of GTP has been found to rely primarily on Q227 and R201 [93], which are thought to be involved in the maintenance of the structural requirements for GTP binding and in the regulation of the timing of its hydrolysis, respectively [94]. The constitutive activation of AC induced by cholera toxin is dependent on ADP ribosylation of R201 and is also coupled to suppression of GTP hydrolysis [95]. R201 (or a homologous residue) is conserved in homologous regions in all proteins with GTPase activity, further highlighting its importance [94]. Mutations of Q227 (in endocrine tumors) and R201 (in endocrine tumors and fibrous dysplasia) reduce the kcat- of the GTPase activity inherent to Gs0~ [94]. Prolonged binding of GTP to mutated Gs~ induces a constitutive activation of adenylate cyclase resulting in constitutive elevation of cAMP levels. In cells transfected with wild-type or R201C mutated Gs~, immunofluorescence studies document that although Gs~ shuttles between the plasma membrane and the cytoplasm depending on activation/inactivation cycles induced by receptor stimulation, mutated Gs~ is stably localized in
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the cytoplasm with a diffuse distribution [96]. Sustained basal levels of cAMP measured in cultures of cells grown from FD tissue [9] are apparently consistent with the induction of a constitutive activation of AC by the mutated Gs~, and also with the inference that a cAMP-ependent pathway(s) may mediate many of the phenotypic effects of the mutation in bone. However, the abnormal cAMP response induced by the mutated Gsct may be subject to significant modulation. Furthermore, in many systems cAMP may act as a gating mechanism, of which the net effect on cellular physiology may be highly diversified [97]. Concurrent upregulation of phosphodiesterases induced by cAMP may buffer the effects of the constitutive activity of AC in some systems, resulting in normal or near-normal levels of cAMP. Basal levels of cAMP may be similar in gsp + or gsppituitary tumors [98]. However, the increase in cAMP concentration induced by specific phosphodiesterase blockade is markedly higher in gsp + tumors compared to gsp- lesions, and a sevenfold higher level of phosphodiesterase activity has been detected in human pituitary GH-secreting adenomas [98]. Likewise, either the basal cAMP levels or the mitogenic response induced by mutated Gs~ require blockade of phosphodiesterases in order to be demonstrated [99,100]. Thus, the presence of an activating mutation of Gs~ does not necessarily translate into constitutively high levels of cAMP under all conditions, and a suitable context of extracellular stimuli and intracellular permissive conditions (many of which remain to be dissected and may in theory include posttranslational modification, interaction with associated proteins, phosphorylation status, and changes in intracellular trafficking) modulate the elementary and fundamental phenotypic effect of the mutation. Furthermore, expression of Gs~ may be up- or downregulated in mutated cells [89,101].
of the lines of Blaschko, which represent directions of dorsoventral migration of embryonic cells [102]. The cutaneous lesions reflect the discontinuous distribution of dysfunctional melanocytes, each of which produces and transfers to neighboring keratinocytes an excessive amount of melanin. Melanocytes are neural crest derivatives, and they follow a dorsoventral migration pattern. Thus, the nonuniform distribution of dysfunctional and normally functional melanocytes indicates in FD, as in other unrelated diseases, the existence of a dual population of the same cell type that segregates during embryonic development prior to the cell migration events reflected into Blaschko lines. This observation led Happle [102] to postulate, long before the recognition of the causative GNAS1 mutations, that the functional duality of cells established in embryonic development in patients with MAS would express a genetic mutation translating into a somatic mosaic state. Since the disease is never inherited, Happle also postulated that the disease genotype would be embryonic lethal if transmitted via the germline (i.e., if all cells in the zygote were mutated) but compatible with cell survival if mutated cells were intermingled with normal cells (i.e., if a postzygotic mutation arose in a somatic cell). Happle's predictions on a somatic mosaic state were confirmed by the demonstration of two genotypes (the disease genotype and the normal genotype) in tissues from patients with MAS [7]. Happle also predicted that the somatic mosaic state would allow the intermingling of normal and mutated cells, and this would provide an essential survival factor for embryonic cells exposed to the effect of an inherently lethal mutation. This prediction has also found some experimental support [13], which interestingly may extend the principle of survival through mosaicism to mutated cells in the postnatal organism. Site a n d Time of Origin of t h e M u t a t e d Clone
DETERMINANTS OF PHENOTYPIC VARIABILITY Phenotypic variability at the organism level, rather than at the single cell level, expresses the effect of multiple additional determinants resulting in the varied spectrum of clinical expression of the same disease genotype. Although incompletely identified or understood, these determinants likely include developmental and epigenetic phenomena. Somatic Mosaicism The cutaneous pigmented lesions observed in MAS, or in association with FD without MAS, sometimes distribute in a systematized fashion, closely reminiscent
Happle also suggested that widespread vs locally restricted distribution of the progeny of the original mutated somatic cell underlies the clinical variability of the disease (e.g., monostotic or polyostotic forms of skeletal disease, single or multiorgan endocrine dysfunction, and single or multiple skin pigmented lesions) and in turn reflects the time of occurrence of the mutational event. Early mutation in this view results in disseminated disease, and late mutation results in unifocal disease. However, the clinical diversity of the disease phenotype suggests a more complex picture and a less stringent correlation of genotype and phenotype, as well as phenotype with embryonic time of mutation, than is assumed in Happle's prediction. The spectrum of clinical phenotypes generated by GNAS1 mutations can be classified based on the embryonic origin of the tissue involved.
21. FibrousDysplasia Severe panostotic and polyostotic forms associated with endocrine and cutaneous abnormalities express a disease phenotype in tissues derived from more than one germ layer. Assuming by default the occurrence of a single mutation event resulting in the development of a single clone of mutated cells, these forms imply that a mutation occurs prior to germ layer separation (pangermal disease). The concurrence of lesions in multiple tissues that are ectodermal and mesodermal, but not endodermal, in origin would be consistent with mutation arising in a mesectodermal cell (digermal disease). An isolated monostotic form of FD and an isolated lesion in one endocrine organ would conversely be consistent with mutation occurring at later times, inasmuch as they might represent the effects of a mutated clone arising in one germ layer or in later derivatives thereof (monogermal disease). Size a n d Viability of t h e M u t a t e d Clone The isolated involvement of only two sites in the skeleton may signify markedly different embryological situations. Since all different bones of a limb and the cognate limb girdle are derived from the same spatial specification of the lateral mesoderm, two lesions in two bones of the same limb (a polyostotic monomelic form of fibrous dysplasia) might be consistent with monogermal disease and with mutation arising in the limb bud, a relatively late mutational event. Two lesions involving the craniofacial skeleton and one femur, respectively, would instead imply pangermal or digermal disease, with mutation arising in the mesectoderm or earlier (in the inner cell mass) since the craniofacial bones are neuroectodermal (neural crest) in origin and the limb bones are mesodermal. Therefore, mutations occurring at comparable embryonic stages may result in vastly different dissemination of the disease phenotype (restricted to two skeletal sites or involving the whole skeleton plus extraskeletal sites). Conversely, mutations occurring at different embryonic times and sites may result in phenotypes of comparable clinical severity. The existence of disease limited to two or three embryologically unrelated sites may indicate that many other tissues either clear themselves of mutated cells during development and growth or carry a silent disease genotype in phenotypically normal tissues. Thus, the demise of the mutated genotype or its occurrence in a nonpathogenic form in different tissues is implied. Individual cell clones arising from equipotent and synchronous normal embryonic cells may vary significantly in size; thus, variability of disease extent may also directly express the variable size of the mutated clone. Further complicating the issue, inferences based on basic tenets
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of classical embryology (such as the dogma of germ layer differentiation fidelity) are indeed challenged by current developments in the field of stem cell biology [103,104]). We now know, for example, that a normal mesodermal cell can give rise to tissues normally derived from the endoderm under certain conditions [105], and we cannot rule out that an activating G N A S 1 mutation might alter the differentiation capability and fate of an embryonic cell. For example, cAMP enhances the differentiation of F9 teratocarcinoma cells to parietal endoderm [106]. In principle at least, one cannot exclude that even a widely disseminated disease might reflect a mutation in a postembryonic, migratory stem cell rather than a specific time of mutation. The current awareness of the identity of the disease genotype and the availability of simple methods for mutation analysis provide the means to prove or refute some of these conjectures. The simple detection of the mutation at embryologically distant and phenotypically normal sites in patients with monostotic disease would, for example, disprove the tight genotypephenotype-time of mutation link that is assumed by many.
Imprinting Differential methylation between maternal and paternal alleles occurs within CpG islands of some autosomal genes [107]. The resulting repression of transcription from the methylated allele determines the maternal or paternal epigenotype, and imprinted genes are involved in certain human diseases [71,107]. The multiple transcripts derived from the G N A S 1 gene are separately imprinted. XL~s is only transcribed from the paternal allele, as a result of methylation of the maternal allele. NESP55 is oppositely imprinted: the maternal allele is transcribed and the paternal allele is methylated [68,69]. A tissue-specific pattern of imprinting of Gs~ was originally surmised based on the evidence for a parental pattern of inheritance of renal resistance to parathyroid hormone (PTH) (and to a lesser extent peripheral resistance to TSH and gonadotropins) associated with loss-offunction mutations of Gs~ [62,70]. Tissue-specific imprinting with monoallelic expression of Gs~ was then demonstrated for certain tissues (kidney and brown and white adipose tissue) in the mouse [108,109]. Recently, the first direct evidence for tissue-specific imprinting of G ~ in humans was obtained. Only the maternal allele appears to be expressed in the normal pituitary, and in 21 of 22 gsp + GH-secreting adenomas the mutation was demonstrated in the maternal allele [88]. Although predicted by clinical observation in patients with pseudohypoparathyroidism type Ia and inferred from direct evidence in the mouse, G ~ imprinting in the human kidney has not been demonstrated to date, and in fact
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biallelic expression was observed in immature human kidneys in one study [110]. It is conceivable that Gs0~ imprinting in the human kidney is developmentally (temporally) regulated [62] and continues or even occurs postnatally (imprinting of many genes is developmentally regulated). Interestingly, although only the maternal allele is transcribed in normal somatotrophs, relaxation of (release from) imprinting occurs in GH-secreting pituitary adenomas, and both Gs~ alleles are transcribed as a result [88]. Taken together, these data imply that imprinting of Gsc~ may be acquired or lost during normal development or abnormal growth of tissues. In principle, tissue-specific imprinting of G~0~, or its developmental or timed occurrence and loss, may greatly affect the phenotypic expression of the heterozygous gain-of-function mutations underlying FD. Mutation of the allele that is specifically expressed or silenced in specific extraskeletal tissues may include or exclude those tissues from the range of anatomical sites where a mutation-dependent lesion or dysfunction would develop. Conversely, evidence of a mutation-dependent lesion in a tissue with a known pattern of tissue-specific imprinting would allow prediction of the parental allele that underwent mutation. Based on the recent demonstration of maternal expression of Gsc~ in the pituitary, for example, one would predict that in FD patients suffering from associated GH excess (~20% [25]), there is not only the inclusion of mutated cells in the anterior pituitary anlage during development but also the occurrence of mutation in the maternal allele. Relaxation of imprinting, on the other hand, might induce expression of a mutated but silenced GNAS1 allele and the resulting downstream effects. Thus, it would be interesting to determine whether relaxation of imprinting can occur in any physiological circumstances besides tumor growth and contribute to the development of a GNAS1dependent organ lesion. Although Gsc~ is thought to be biallelically expressed in most tissues including bone [111], a direct and thorough analysis of its potential imprinting in different tissues has not been conducted. At least some clones of osteoprogenitor cells derived from FD express mRNAs transcribed from both the normal and the mutated allele [13], consistent with biallelic expression of Gs0~ in FD osteogenic cells. It must also be kept in mind that imprinting may not only be tissue specific but also cell type specific, as in the case of the mouse kidney cortex, in which it is restricted to the proximal tubule [109,112]. Thus, in bone, as in any organ, Gs0t might be imprinted in specific histological structures and still be seen as biallelically expressed if assayed in whole tissues or organs.
PATHOLOGY M o d e l i n g of FD B o n e Fibrous dysplasia affects both cortical and cancellous bone and the bone marrow in a focal or diffuse manner. In FD, the spatial definition and structural distinction of cortical bone, cancellous bone, and bone marrow that is achieved through normal modeling is blurred and the distinct territories tend to become structurally continuous and homogeneous. An excess of perivascular marrow space develops in the cortex and an excess of abnormal and undermineralized cancellous bone develops in the marrow cavity, giving rise to a continuous bony structure of plexiform architecture. This structural pattern, readily apparent in macroscopic and submacroscopic samples of FD bone [16,113] (Fig. 11), is established through abnormal modeling of a growing skeletal
FIGURE 11 (Top) Normal anatomical specimen of the frontal bone for comparison to the fibrous dysplastic frontal bone shown in the back-scattered electron image at the bottom. The specimens are seen from above, and the frontal tuberosity has been removed to expose the cortical and diploic structures. The widening of intracortical vascular space (arrows) results in a cancellous rather than compact architecture of cortical bone, whereas the expanded diploe (d) is occupied by an excess of fibrous dysplastic trabecular bone. Note the direct continuity of the intracortical and intradiploic vascular/medullary space. Arrowheads indicate the discontinuities in the bony wall of the frontal sinus that occur as a result of the fibrous dsyplastic process. Through these discontinuities, highly vascular FD tissue herniates and bleeds, leading to pseudocyst formation (see Fig. 16) (photographs courtesy of Professor Alan Boyde, University College London).
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site but may involve remodeling of an established bony structure such as a femoral cortex. Depending on the specific architectural plan of the individual bone affected and the phase of skeletal growth affected by the development of the FD lesion, varied gross and radiographic effects ensue. In the calvarium of a growing child, in whom cortical bone is thin under normal conditions, FD modeling results in the complete replacement of both cortical plates and the internal diploe by a thick and continuous, vaguely cancellous bony structure. In a long bone of an adolescent, in whom a more conspicuous cortex has been established, the inner portion of the cortex and the marrow cavity become occupied by FD bone. The marrow spaces located between endomedullary FD trabeculae, or within the "spongiosized" cortical bone, are occupied by an abnormal marrow stroma commonly defined as fibrous but in fact consisting of cells of osteogenic nature [10]. This tissue excludes hematopoiesis and does not contain marrow adipocytes. Thus, among the elementary events of bone modeling, it is the development of red and yellow marrow and the differentiation of the major stromal cell types therein (hematopoiesis supporting reticular cells and adipocytes), and not the development of bone tissue or the differentiation of osteoblasts, that is arrested in FD. FD bone is an abnormal trabecular bone in which the marrow does not differentiate correctly. D e p o s i t i o n a n d Internal S t r u c t u r e of FD B o n e Like all kinds of bone, FD bone is deposited by boneforming cells (i.e., osteoblasts). These cells are not easily recognized in tissue sections simply because of their unusual retracted cell shape, a direct in vivo correlate of the effects of cAMP on osteoblast-like cells in culture [10,114,115]. The bone trabeculae resulting from FD bone formation are woven in structure. Detection of lamellar trabeculae within FD indicates their origin from resorption of preexisting normal bone [10]. The edge of the FD trabeculae is noted for arrays of collagen bundles running perpendicular to the trabecular surface instead of parallel to it [10,114]. These bundles are identical to Sharpey fibers, a normal feature of sutural bone growth in cranial bones and of sites of tendon and ligament insertion into bone. Stellate, retracted osteoblasts and their cytoplasmic processes are closely associated with Sharpey fibers (Fig. 12). This peculiar osteoblastic morphology and the Sharpey fiber pattern combine to form a unique morphology of the bone-formation sites in FD, represent the most consistent histological findings in FD bone, and can be detected regardless of the site of skeletal involvement and the overall histological pattern [114].
FIGURE 12 Sharpey fibers and osteoblast cell shape in fibrous dysplasia. (Top) H&E section demonstrating multiple bundles of collagen (Sharpey fibers) running perpendicular to the trabecular surface into the adjacent fibrous tissue. (Bottom) Undecalcified plastic section of FD bone. The better resolution of plastic sections allows one to discern retracted osteoblasts along the osteoid surface. The processes of these cells outline round features that represent cross sections of Sharpey fibers (arrows). (see color plate.)
Mineralization of FD Bone The FD calvarium of a child may be 10-fold thicker than a normal one, but it can be cut with a scalpel. The FD vertebra of a child can be shaved and cut with a nail. These known characteristics of FD bone [16] can only be explained as an effect of its undermineralization. Studies on bone biopsies taken from the affected iliac crest of patients with FD have shown that a severe mineralization defect of lesional bone occurs in most cases [85,116]. This feature of FD bone has remained largely unrecognized primarily because samples of FD taken at surgery are usually decalcified and embedded in paraffin for routine histology. The use of plastic embedding of undemineralized samples, as per routine procedures commonly in use for processing iliac crest bone biopsies,
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readily reveals that FD bone is undermineralized more than it is reduced in mass (Fig. 13). This finding has immediate clinical relevance because it explains the abnormal compliance of FD bone, which leads or contributes to bone deformity and fatigue fractures. Curiously, even one of the most classical clinical features of severe FD, the shepherd's crook deformity of the femur, directly indicates the abnormal compliance of FD bone, and yet the inherent undermineralization of FD bone had not been clearly recognized. Analysis of the severely undermineralized FD bone with polarized light microscopy also demonstrates that the excess osteoid is composed of woven bone matrix and Sharpey fibers. Normal woven bone (fetal woven bone) mineralizes more rapidly than lamellar bone due to its higher content in water and hydrophilic noncollagenous proteins [including the putative mineral nucleator bone sialoprotein (BSP)] and relatively lower content in collagen [117,118]. Excess woven osteoid in FD thus expresses an impairment in mineralization even more severe than the same amount of lamellar osteoid. Values of osteoid thickness and osteoid surfaces in FD bone are well within the range conventionally considered diagnostic for a true osteomalacic change in lamellar bone [116], and preliminary data
with dual tetracycline labeling in FD bone reveal the absence of dual labels and a smeared pattern of single labeling, again consistent with a genuine osteomalacic state. Studies with quantitative back-scattered electron imaging, a technique that reliably measures mineral density in bone [119], also show that the degree of mineralization attained even in mineralized portions of the FD bone is lower than normal [116], at variance with other genetic diseases of the skeleton that, like FD, are noted for high levels of bone turnover and deposition of immature and fragile bone, such as osteogenesis imperfecta [120]. Renal phosphate wasting, hyphosphatemia, and low levels of 1,25(OH)2D3 [53] are major determinants of the mineralization defect observed within FD lesions, even though in most cases they are not severe enough to generate a systemic osteomalacic change. The potential additional intrinsic local mechanisms of impaired mineralization of FD bone remain to be clarified. R e m o d e l i n g of FD B o n e Internal remodeling and resorption of FD bone gives rise to bizarre trabecular shapes (C-shaped and S-shaped
FIGURE 13 Osteomalacic change in FD bone. (a, b) Samples of the same biopsy of an FD affected iliac crest processed separately for paraffin embedding after decalcification (a) and for undecalcified methyl metacrylate embedding (b). (a) A paraffin section stained with H&E in which the total amount of bone plus osteoid was imaged in fluorescence. (b) A plastic section stained with von Kossa. The paucity of mineralized bone, but not that of total bone matrix, is readily apparent. (c, d) Transmitted and polarized light views of a plastic section of FD demonstrating the huge excess of osteoid and the woven texture of the osteoid. (see color plate.)
21. FibrousDysplasia trabeculae--so-called "Chinese writing" or "alphabet soup" patterns (Fig. 14) [16,18]. These patterns are more commonly seen in long-standing F D lesions, in which multiple cycles of remodeling have occurred. Hence, they are more common in the axial and limb bones than in craniofacial bones [113], in which histological patterns produced by modeling prevail. Lesions of gnathic bones, for example, often exhibit a unique pattern, termed hyperosteocytic [114], in which parallel rods of FD bones are laid down. The location of osteoblasts at single and homologous sides of the individual rods indicates a modeling drift as a contributor to the origin of the peculiar histological pattern. F D bone undergoes extensive tunneling resorption (resorption
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from within), which is the standard pattern of resorption leading to Haversian remodeling in normal cortical bone. In normal cancellous bone, resorption predominantly occurs over trabecular surfaces, but tunneling resorption of trabeculae is a common finding in hyperparathyroidism [121]. Tunneling resorption may reflect the recruitment and activation of osteoclasts not on the trabecular surface but along the bone surface bordering intratrabecular vascular spaces, and it is a major determinant of the bizarre trabecular patterns observed in FD. Histomorphometric indices of bone resorption (osteoclast numbers and surfaces) are higher in FD bone than agematched reference values and correlate strictly with urinary pyridinium cross-links [116].
FIGURE 14 Histologicalpatterns of FD. (a) The parallel arrangement of FD trabeculae commonly seen in jawbones, expressing a local modeling drift. Osteoblasts in these structures are always located on homologous sides, and large numbers of conjoined osteocytelacunae are seen (hyperosteocyticbone). (b) Note the excavation of FD trabeculae from within (tunneling resorption; arrows). (C) The conventional "Chinese writing," which is mainly the result of extensive tunneling resorption of primary FD bone. (d) Intracortical erosion by FD tissue of the prominent vascularity (arrows), also seen in e. (f) Detail of an irregular FD trabecula extensively excavated from within. (see color plate.)
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FIGURE 15 Imagesfrom a 24-year-oldpatient with polyostoticFD and hypophsphatemia,low 1,25(OH)2D3, and secondary hyperparathyroidism. Hyperparathyroidism-induced changes include a prominent pattern of tunneling resorption, unusually high numbers of osteoclasts (arrows), and the formation of solid clusters of osteoclasts (bottom). bv, blood vessel. (see color plate.)
Furthermore, their tight correlation with levels of circulating PTH is indicated in studies [116]. Indeed, hyperparathyroidism secondary to low 1,25(OH)2D3 can be predicted from analysis of the FD biopsy based on the frequency of hyperparathyroid-like histological features (Fig. 15). In these cases, tunneling resorption is more obvious and extensive than usual, and large clusters of osteoclasts and mononuclear TRAPpositive cells (mini-brown tumors) are observed. These findings indicate that the FD lesion remains hormonally responsive, and that the tissue changes observed in histological material reflect the systemic hormonal status in addition to the inherent dysfunction of local mutated skeletal cells. Further analysis is necessary to determine whether other common hormonal imbalances or changes in MAS/FD patients (hyperthyroid states, GH excess, puberty or precocious puberty, and pregnancy) are in turn reflected in recognizable changes in the lesion histology that may be of clinical relevance for the course of the bone disease.
Vascularity A marked increase in vascularity characterizes FD lesions (Fig. 14). Both arterial capillaries and venous sinusoids are increased in number per unit volume of tissue, and ectatic capillaries engorged with blood are commonly seen along and even inside the trabecular surfaces. These vessels are extremely prone to bleeding, and microhemorrhages are common. This is reflected in the common experience of unusually high bleeding of FD bone at surgery and explains the frequency with which posthemorrhagic aneurysmal bone cysts secondarily engraft on FD lesions [122-124]. Occasionally, even microscopic but otherwise histologically typical aneurysmal bone cysts can be detected at biopsy. Engrafted aneurysmal bone cysts generate a marked expansion of the lesion and the bone contour. Bleeding within a lesion per se always generates a sudden change in local tissue volume. In the craniofacial skeleton, this can in turn cause herniation of the suddenly swollen tissue through anatomic
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foramina or through vascular passages traversing the normally thin cortex of cranial bones or the highly porous FD bone. Constriction of venous channels through the pores of the FD craniofacial bones may cause bleeding. Acute compressive events of the optic nerve or chiasma leading to sudden blindness are the most severe complications. Pseudocysts with air-fluid level forming in sinusal airspace (Fig. 16) (inappropriately referred to as either aneurysmal bone cysts or mucoceles) [125-127] may be much less ominous but do indicate the same kind of event. Rare instances of high-output cardiac failure in FD may be attributed to extensive arterovenous shunts generated by vascular remodeling of the unusually rich local vascularity [128]. G r o w t h of Lesions Examination of the edges of expanding lesions demonstrates a marked increase in vascularity, loose perivascular edema of tissues, and two distinct changes along the bone surfaces. Osteoclasts are focally increased in number and clustered, whereas large portions of the trabecular profile exhibit a distinct layer of so-called endosteal fibrosis, separating bone from the adjacent marrow. The cells in the endosteal fibrosis exhibit strong alkaline phosphatase activity and, interestingly, the pericyte marker ~-smooth muscle actin [129]. In both respects, the endosteal fibrosis of FD closely resembles that observed in hyperparathyrodism [130,131]. Interestingly, microvascular walls and the cellular lining of bone trabeculae, the two microanatomical sites at which one observes a change in early developing FD lesions, coincide with the sites at which Gs~ is expressed at high levels [10].
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Cartilage Occasionally, islands of cartilage appear in an otherwise typical fibroosseous FD background. This is especially common in bones of endochondral origin in young patients. In long bones, these islands may be continuous with the growth plate (and thus be cartilage peninsulae rather than islands). These blocks of cartilage remain unmineralized and populated with nonhypertrophic chondrocytes, indicating that they escaped the maturational sequence that chondrocytes undergo in a normal growth plate. Keeping in mind that Gs~ mediates the effects of PTH/PTH-related protein (PTHrP) in bone and cartilage, this finding is of interest in view of the effects of PTH/PTHrP signaling in the normal growth plate. Expression of a constitutively active PTH/PTHrP receptor in cartilage results in a retarded maturation and elongation of the growth plate [132]. It is common to ascribe to cartilage islands the spotty calcifications frequently seen in plain radiographs of FD lesions. Cartilage islands in FD are rare, and they are much less frequent than the spotty mineralized phase observed in radiographs. As a rule, cartilage in FD is unmineralized and therefore undetectable radiographically. The term fibrocartilaginous dysplasia [133,134] is sometimes used to note instances in which hyaline cartilage is more than a sporadic finding and forms significant portions of the lesional tissue. It is unclear how many of these cases, if any, represent a histological variation in true FD vs a distinct, benign but occasionally locally aggressive bone lesion. Availability of a known mutation as a genetic marker of FD provides a tool for distinguishing between the two possibilities.
FD vs O t h e r F i b r o o s s e o u s Lesions
FIGURE 16 Hemorrhagic pseudocyst (pc) with air-fluid level in the maxillary sinus secondary to FD of the maxillary bone. Compare with Fig. 11.
Assessment of GNAS1 mutations should be useful to unequivocally define other bone lesions whose distinction from FD is vague or uncertain. It is now clear that the lesion called osteofibrous dysplasia (ossifying fibroma of long bones or Campanacci's lesion) [18,135]), which affects the tibia and fibula of young children in a highly restricted fashion and is clearly histologically different from FD, is not associated with GNAS1 mutations [136]. Likewise, ossifying and cementoossifying fibroma of jawbone, often referred to as part of a common spectrum of benign fibroosseous lesions that include FD [137], can be distinguished from FD based on the absence of GNAS1 mutations [138]. Nonetheless, true mutation-positive FD of jawbone may share significant histological features with cementoossifying fibroma, including the formation of cementum and
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psammoma bodies, which is not rare in FD lesions outside of the gnathic bones, indicating that they have nothing to do with true dental cementum. These structures represent the onset and subsequent spread of single cell mineralization occurring separately and independently of any bone-formation event. They occur in areas of nontypical cellularity and may coalesce to form large masses of mineralized nonosseous material, which can be prominent on plain radiographs [18]. Nonossifying fibromas of long bones may both mimic and be mimicked by FD. A storiform (Latin storea, mat) arrangement of fibrohistiocytic cells with interspersed giant cells, foam cells, and hemosiderin deposits may be seen in "aged" FD lesions and are occasionally indistinguishable from a nonossifying fibroma (NOF) when there is radiographic evidence of cortical involvement. NOF-like changes reflect posthemorrhagic reactions in FD. It is important to remember that multiple NOFs occur along with skin pigmentation and various extraskeletal disorders in Jaffe-Campanacci syndrome (JCS) [139], and that the multiple bone lesions may be unilateral both in JCS and in polyostotic FD. Thus, a differential diagnosis between FD and NOF is in order when dealing either with individual lesions or with multifocal, polyostotic disease. There have been no attempts to demonstrate G N A S 1 mutations in JCS as a potential cause. Interestingly, many, if not all, fibrous or fibroosseous lesions for which a differential diagnosis with FD is entertained seem to represent the expression of an unknown genetic defect. Tumors in FD Occasionally, malignant bone tumors develop from preexisting FD lesions of bone. Given the sporadic nature of the event and of its records in the literature, it is difficult to estimate the frequency of malignant change in FD. In a study from the Mayo Clinic, a total of 28 cases of malignant bone tumors complicating FD were found in a series of 1122 cases [140]. Interestingly, the vast majority of these tumors complicated monostotic FD. Neither the extent of the disease nor the concurrence of endocrine dysfunction predicts the risk for malignant change in FD. A history of radiation therapy was available for approximately half of the tumors, indicating that transformation may occur independent of irradiation. Different types of clonal chromosomal aberrations [t (6;11), +2, rearrangements involving chromosome band 12p13, and others) [141-144] have been observed in FD and used to argue for a true neoplastic nature of FD. Although the contention may not be tenable, these observations may represent the accidental detection of a phase of clonal evolution of FD that occasionally, and depending on the nature of the associated chromosomal
changes, may ultimately, but not necessarily, result in the development of a malignant tumor upon a second hit as per the classical Knudson's two-hit hypothesis. Tumors complicating FD most commonly arise in the craniofacial skeleton, usually behave aggressively, and have a poor prognosis [145]. Osteogenic sarcoma is the most common type of tumor, followed by chondrosarcoma, fibrosarcoma, and malignant fibrous histiocytoma [146-153]. Rarely, angiosarcoma has been reported as well [154]. Interestingly, this spectrum of tumor phenotypes mirrors the involvement of skeletal progenitor cells (osteo-, chondro-, and fibrogenic progenitors) as the target of the transforming events, whereas the occurrence of tumors of angiogenic lineage highlights the inclusion of the bone/bone marrow vascularity among the targets of G N A S ! mutation with physiologically significant impact. FD and certain malignant bone tumors may mimic each other clinically and histologically. Low-grade central osteogenic sarcoma (LGCOS) may occasionally be disguised as FD clinically and radiographically and be misdiagnosed as such [155]. A subset of these tumors is even alluded to in the pathology literature as FD-like low-grade osteogenic sarcoma to emphasize the similarity of the histological patterns. Recently, a G N A S 1 mutation was demonstrated in one case of FD-like osteogenic sarcoma [155], which in fact complicates the use of mutation analysis for differential diagnosis between FD and LGCOS but suggests that FD may evolve into a tumor retaining resemblance to the original FD lesion.
PATHOGENESIS Expression of Gso~in t h e B o n e / B o n e M a r r o w Environment In some tissues and organs of patients with PFD or MAS, the presence of mutated cells may remain pathologically and clinically silent. Although Gs~ is thought to be ubiquitously expressed, marked differences in the levels of expression exist across tissues and within individual tissues that may help explain why some organs bear or do not bear adverse consequences of carrying G N A S 1 mutated cells. Immunolocalization and in situ hybridization studies have shown that in normal bone, Gs~ is expressed at comparatively low levels in preosteoblasts (such as cells in the cambial layer of the prenatal periosteum) and at much higher levels in mature osteoblasts, osteoclasts, and cells of the marrow microvasculature [10]. A similar pattern is observed in FD [10]. Osteogenic cells bordering the abnormal bone trabeculae and pericytes of arterial capillaries exhibit the highest levels of signal for Gs~, whereas the "fibroblastic cells"
2 I. Fibrous Dysplasia
filling the marrow spaces (which are preosteoblastic in phenotype) exhibit comparatively lower levels of Gs~ mRNA and protein. The increased levels of Gs~ signal in mature osteoblasts compared to less mature osteogenic cells may reflect either the increase in total RNA associated with osteoblast maturation [156] or a specific upregulation of the Gs~ mRNA transcription. In either case, the net effect is a higher expression of Gs~ protein in mature osteoblasts involved in the deposition of new bone matrix. Hence, it is logical to assume that mutated Gs~ is also expressed at a comparatively high level in mature osteoblasts. Given the increased AC-stimulating activity of mutated Gscz, the maturation process of osteoblasts amplifies the effect of carrying a mutated Gs~, thus exposing the individual cell to a much enhanced stimulation of AC and production of cAMP. Therefore, the differentiation of osteoblasts from their progenitors by default exposes bone tissue to the effects of the disease genotype. Although the effects of mutated Gs~ on osteoclasts have not been investigated and may require attention, cAMP-mediated effects on normal osteoclasts are largely inhibitory in nature, as best exemplified by the cAMPmediated effects of calcitonin, which induces cell retraction of osteoclasts, thus arresting their resorptive activity. At least in principle, one can expect that mutated osteoclasts are less active in resorption than nonmutated ones on a per cell basis. The higher resorptive activity observed in FD bone in most cases cannot easily be envisioned as a direct, cell-autonomous effect of Gs~ mutation in osteoclasts and rather postulates an effect of enhanced osteoclast differentiation mediated by osteogenic cells. The sustained expression of Gs~ in the marrow microvasculature is not surprising since adrenergic receptors are G protein coupled. Recent observations indicate that pericytes in the marrow microvasculature are a prime source of osteoprogenitor cells in the marrow [104]; hence, the expression of mutated Gs~ in marrow arterial capillaries may be significant for the development of FD lesions. The increased vascularity of FD marrow compared to normal marrow and the observation of a pericyte-like phenotype in FD stromal cells in vivo and in vitro [129] may be consistent with this view.
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human bone marrow, they are noted for an incomplete osteogenic phenotype and express alkaline phosphatase and certain noncollagenous proteins of bone. Some of the abnormal stromal cells also express the pericyte marker ~-smooth muscle actin, which is commonly expressed by marrow stromal fibroblasts in culture but highly restricted to pericytes of arterial capillaries in the normal human bone marrow in situ. s-Smooth muscle actin [130] and ALP activity [131] are highly expressed in the endosteal fibrosis of hyperparathyroidism, in which the fibrosis of FD has a direct phenotypic and functional match. Like the normal bone marrow stroma, the fibrous tissue seen in FD is composed of clonogenic cells [13], among which skeletal progenitor cells are found under normal conditions [103]. Assessment of the frequency of clonogenic cells in the FD marrow stroma compared to normal stroma shows a several-fold enrichment in clonogenic cells in FD. Cbfal, a pivotal transcription factor regulating osteogenic differentiation [158], is expressed in mutated stromal colonies like in normal clones of marrow-derived fibroblasts [159]. Taken together, the phenotype exhibited by FD fibroblastic cells in situ and the clonogenic efficiency of the FD fibrotic marrow stroma make the fibrosis of FD a modified, expanded, osteogenic bone marrow stroma [160]. The absence of hematopoiesis in the FD marrow indicates that the FD stroma is not efficient in supporting the homing and differentiation of hematopoietic progenitors and in establishing a hematopoietic microenvironment. The absence of adipocytes in the FD tissue in vivo in turn indicates that the conversion of marrow reticular/fibroblastic cells to adipocytes is also impaired [161], consistent with the known effects of Gs~ activity, cholera toxin, and cAMP on the adipose conversion in a variety of fibroblastic/preadipocytic cell systems [162,163]. When normal marrow stromal cells are transplanted ectopically in immunocompromised mice, they establish a complete "ossicle" in which normal bone tissue, a normal hematopoietic microenvironment, and marrow adipocytes develop from the transplanted cells [103]. Under the same experimental conditions, FD cells generate abnormal woven bone but fail to establish a hematopoietic microenvironment and to give rise to adipocytes [13] (Fig. 17).
N a t u r e of t h e FD Fibrous Tissue
D o w n s t r e a m Effects of Excess cAMP in FD O s t e o g e n i c Cells
The spaces between abnormal FD trabeculae can be equated to hematopoietically inactive marrow spaces. The fibrous tissue, which fills the space, is in turn composed of cells resembling marrow stromal cells in various respects. Like reticular-fibroblastic cells (sinusoidal pericytes and adventitial reticular cells) [157] in the normal
In cells grown in culture from FD lesions, higher levels of cAMP are observed compared to those of controls [9]. Downstream effects of excess cAMP production in bone cells have only partially been characterized. It was initially shown that/n situ FD cells express high levels of c-los [164]. Fibrous changes and abnormal osteogenesis
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FIGLIRE 17 Strains of stromal cells derived from normal bone marrow or from FD were transplanted ectopically into the subcutaneous tissue of im_munocompromised mice using hydroxyapatite/tricalcium phosphate particles as a carrier. Eight weeks later, normal bone and hematopoietic marrow with adipocytes formed in transplants of normal cells (a, b), and abnormal bone and fibrous marrow depleted of hematopoiesis and adipocytes formed in transplants of FD cells (c, d). Undecalcified methyl-metacrylate embedding; Goldner's stain, hac, hydroxyapatite carrier. (see color plate.)
are observed in mice overexpressing c-fos [165], which further supports a role for it as one of the mediators of the FD phenotype. Furthermore, c-fos is regulated by cAMP and in turn may regulate the expression of cytokines relevant to bone physiology, such as interleukin-6 (IL-6), that are directly involved in the promotion of osteoclast differentiation. Murine bone cells transfected with mutated Gs0~produce sustained levels of IL-6 [166], and enhanced production of IL-6 in human FD cells has been shown in two studies [9,167]. From a total of four patients, levels of IL-6 production as assessed by enzyme-linked immunosorbent assay was increased in nonclonal populations of lesion-derived cells in three patients. In situ, high levels of IL-6 production are observed in FD tissue in a highly localized fashion, which coincides with the sites of excess osteoclast differentiation, both along
trabecular bone surfaces and ectopically. However, both stromal/osteoblastic cells and nonosteoblastic cells appear to express IL-6 in FD tissue in situ [168]. Analysis of production of IL-6 by clonal strains of PTHstimulated or-unstimulated human mutated stromal/ osteogenic cells indicates a marked variability across different clones, which may reflect the natural heterogeneity of stromal cells that has been noted in normal donors. Although consistent with a role of IL-6 in supporting osteoclastogenesis in FD tissue, these data suggest that IL-6 production may be highly modulated in Gsct mutated cells rather than an obligate, autonomous, and consistent downstream effect of the mutation. Constitutively high levels of expression of PTHrP by FD cells may also concur in promoting osteoclastogenesis and may, on the other hand, amplify the effects of the mutation on the osteogenic cells within the FD tissue [169].
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Excess cAMP has dramatic effects on osteoblast cell shape and interaction with the extracellular matrix. When exposed to exogenous dibutyryl-cAMP, a cellpermeant form of cAMP, or to PTH under serum-free conditions, normal bone cells undergo a rapid and reversible change in cell shape and assume a retracted, osteocyte-like morphology [115]. This effect is mediated by the disruption of actin filaments, the disappearance of stress fibers, and the reorganization of F-actin into unusual patterns (unpublished observations). The same effect is brought about in clonal populations of human mutated osteogenic cells upon simple serum withdrawal, independent of PTH stimulation or addition of dbcAMP, suggesting that endogenous excess cAMP is sufficient to mediate this effect in mutated cells. These observations represent a direct in vitro correlate to the abnormal osteoblast shape observed in FD tissue in vivo and provide a simple explanation for this aspect of FD histology. Furthermore, cAMP-induced cell retraction may in turn correlate with a number of other effects on cell function that have not been elucidated. Interestingly, high levels of IL-6 mRNA are observed specifically in retracted cells in situ, and osteogenic cells topographically associated with sites of enhanced osteoclast differentiation, both in FD and in hyperparathyroidism [131], exhibit a retracted morphology. In a variety of cell systems, disruption of actin cytoskeletal filaments and retraction of the cell body are linked to the release of adhesion to the extracellular matrix and marked changes in expression and activation of matrix-degrading enzymes, such as matrix metalloproteinases [170-173]. This suggests that inordinate levels of endogenous cAMP in mutated osteogenic cells may cause enhanced matrix degradation. Preliminary data obtained using clonal strains of human mutated cells in an in vitro collagen degradation assay support this suggestion [158]. This finding is relevant to the observation that in FD bone, cell retraction is physically associated with the formation of Sharpey fibers, and it provides an interesting key to the mechanism of their formation as an effect of locally increased degradation of unmineralized collagen. This implies that an enhanced turnover (deposition and degradation) of unmineralized collagen may occur in FD as a unique form of accelerated bone turnover centered on unmineralized osteoid matrix rather than on mineralized bone. The bone matrix that is newly formed in FD bone differs in its immunohistochemical profile from both adult trabecular bone matrix and normal fetal bone matrix. Fetal bone formed de novo is characterized by very high levels of production and deposition of certain noncollagenous proteins, such as BSP and osteopontin [117]. In situ hybridization and immunolocalization data show that in FD bone formed de novo, these proteins and
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their mRNA are not highly expressed, whereas other proteins, such as osteonectin and versican, are abundantly produced [10]. These observations may be relevant to other characteristics of the FD bone, such as its apparently lower propensity to rapid mineralization (which is conversely characteristic of fetal woven formed de novo) or its unique pattern of collagen texture ("combed" bone and Sharpey fibers). One study suggested that FD cells grown in culture may proliferate more rapidly compared to normal bone cells [12]. Data obtained with clonal strains of mutated cells suggest that a markedly enhanced proliferation may not be a direct effect of the mutation, may not be uniform in mutated cells, or may be induced in nonmutated cells coexisting in the tissue and in the nonclonal cultures rather than being inherent to the mutated cell population per se. In situ, markers of S-phase that are commonly used to assess cell proliferation in human tumors are expressed in FD tissue at levels higher than those in normal nongrowing bone but orders of magnitude lower than those at sites of physiological skeletal growth. Interestingly, the highest proportion of DNA-synthesizing cells within FD tissue is observed within or in the immediate vicinity of vascular walls, suggesting that the bone and bone marrow microvasculature could represent a "hot spot" of cell proliferation in FD, consistent with the locally high levels of expression of Gsct [129].
Mosaicism and Viability of FD Cells and Tissue Mutation analysis conducted on multiple individual clones of stromal cells derived from FD tissue demonstrates that a combination of mutated and nonmutated cells, including clonogenic cells and their progeny, exists in individual lesions [13]. Thus, each lesion is a mosaic rather than simply the "bad" (and clonal) piece of an organismic mosaic. In another study, the disease genotype could be demonstrated in cells derived in culture from the endosteum of an FD lesion, whereas cells derived from the periosteum only demonstrated the normal genotype [174]. Ectopic transplantation of mosaic strains of mutated and nonmutated FD-derived stromal cells in immunocompromised mice results in the generation of an ectopic FD-like ossicle, whereas transplantation of pure strains of mutated cells results in the loss of transplanted cells from the transplantation site. These data imply that even in the postnatal organism, mosaicism is a survival factor for the mutated cells, whereas a homogeneous population of mutated cells would be lethal. During development, abnormal cells are eliminated by apoptosis. Studies using the TUNEL technique for detection of apoptotic cells in FD tissue have yielded
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surprisingly high frequencies of apoptotic osteoblasts in FD bone [175], a finding corroborated by the frequency of clear-cut apoptotic bodies along FD trabecular surfaces and in osteocyte lacunae. Conversely, the FD fibrous tissue remains unlabeled. Taken together, the unusually high rate of osteoblastic apoptosis, the high rate of osteoclastic resorption of FD bone that reaches a sufficient mineralization state, and the nonosteoclastic degradation of FD osteoid indicate that FD bone is a nonviable tissue deposited by nonviable cells, which is not surprising in view of the contended cell-lethal nature of the disease genotype. In a variety of organ systems, hyperplasia of progenitor cells associates with the production of imperfect end products, which undergo untimely removal by scavenging mechanisms and/or apoptotic demise. This applies to genetic and acquired diseases of nonskeletal lineages dependent on system-specific stem cells, such as the hematopoietic system or gastrointestinal mucosa. In FD, an expansion in the compartment of clonogenic osteogenic cells (reflected by an excess of marrow stromal cells) is associated with excessive rates of demise and removal of the mature osteogenic cells and the abnormal bone tissue they deposit. The most convenient analogy is drawn between FD and the so-called ineffective erythropoiesis of thalassemic syndromes or pernicious anemia. In both cases, a marked expansion of the immature progenitors of red blood cells is complemented by accelerated demise of the mature red cell or its immediate precursors. The paradigm best suited to convey the nature of the skeletal disease in FD is thus that of an ineffective osteogenesis rather than the cbnventional "high turnover bone disease" or "arrested maturation of primitive mesenchymal tissue." Mutation analysis at the clonal level on a series of patients demonstrated that the frequency of mutated clonogenic cells is highly variable in different lesions (manuscript in preparation). Interestingly, a clear-cut inverse correlation of the frequency of mutated clones with patient age can be demonstrated. No mutated clonogenic cells seem to be detectable in many clinically (i. e., radiographically) overt FD lesions of patients older than 35 years. Even more interesting, a "normalized" histology is observed in some of these patients, as if the lesion had burned out while leaving behind gross deformity and abnormal radiographic appearance, in agreement with recent observations [176]. These data provide a biological explanation for the anecdotal experience that the disease improves, mitigates, or subsides with age. More important, these data imply phases of expansion and consumption of mutated skeletal progenitor cells in the postnatal life as the mechanisms underlying the development, maintenance, and burning out of individual lesions in different age ranges. Frequencies of
mutated cells in this model would vary as a function of age and phases of skeletal physiology, as indeed seems to be the case. Reaching a pathogenic threshold of mutated cell frequency would be required for the development of a lesion during skeletal growth, and a lethal threshold would be attained in the phase of maximal lesion growth. Depletion of mutated cells would then ensue and leave room for remodeling of FD bone into histologically near-normal but macroscopically abnormal bone. It should be noted that some extraskeletal effects of the mutation also appear to follow a temporal sequence of flare and remission. This applies to liver changes, adrenal hyperplasia, and ovarian cysts. Thus, similarity in the natural history of the mutated cells, even though unfolding over significantly different time intervals and at different organism ages, may underlie different organ lesions. MANAGEMENT AND TREATMENT One of the main problems in the management of FD patients derives from the fact that disorders as diverse as precocious puberty and bone deformity may be brought to the attention of different specialists at disease presentation but also during the life-long course of the disease. Because it is not a solely endocrine, solely orthopedic, or solely pediatric disease, FD requires expert monitoring by a multidisciplinary team, each member of which should ideally have a specific awareness of the natural history of the disease, which remains to be written in large part. Initial evaluation should include obtaining conclusive evidence supporting the diagnosis of FD, accurate determination of the extent of the skeletal disease, and assessment of associated endocrine and metabolic imbalances. In most cases, reliable diagnostic accuracy is attained by expert evaluation of clinical features, plain X-rays, or other available imaging material, and it is always straightforward in patients with MAS. Nonetheless, it should be kept in mind that radiological and even histological misdiagnosis of FD is much more common than one would surmise, and the very boundaries of FD vs other fibroosseous lesions of the skeleton are ill defined and poorly understood. Expert review of imaging and histological material is often an important step. Mutation analysis may contribute to diagnostic evaluation. The extent of the disease is conveniently determined using technetium-99 bone scans. Once the diagnosis of FD is conclusively established, the function of the pituitary, adrenal, thyroid, parathyroid, and gonads must be carefully evaluated, monitored over time in all patients, and treated as appropriate. Aromatase inhibitors are used to treat precocious puberty in females [177,178], antithyroidal agents are
21. FibrousDysplasia used to for hyperthyroidism, and surgery is performed for isolated "hot" or "dominant" thyroid nodules. Surgery may be considered for pituitary adenomas, but it is often difficult due to the extent of skeletal disease in the skull base, and medical correction of GH excess in the absence of detectable adenomas may also be necessary. Computed tomography and magnetic resonance imaging of the cranium and ophtalmological and ENT evaluation must be performed for patients with craniofacial disease. Visual and auditory functions must be monitored over time as appropriate. In addition to markers of bone metabolism (alkaline phosphatase, bone-specific alkaline phosphatase, and urinary pyridinium cross-links), which must be assessed and are always elevated in FD [179], evidence of renal phosphate wasting, hypophosphatemia, and low levels of 1,25 (OH)zD3 must be specifically sought at initial evaluation, corrected even if overt radiographic evidence of rickets or osteomalacia are missing, and monitored over time. M u t a t i o n Analysis in Clinical Practice A number of methods have been developed to determine the presence of mutations at codon R201 in clinical material [7,13,85,86]. Direct sequencing of the relevant PCR amplification product of genomic DNA extracted from tissue is the simplest method. Because of the variable proportion of mutated to nonmutated cells in affected tissues, the sensitivity of this method may be too low to detect the mutation in some instances. More sensitive methods may be applied, and many such methods are being developed. However, the diagnostic value of these procedures must not be overestimated. The diagnosis of FD should not rely primarily on mutation analysis in most cases, and the clinical significance of detecting even low numbers of mutated cells may be limited until a more direct understanding of the biological significance of different mutational loads in different tissues is gained. Local variability and random sampling detract applicative clinical value from attempts to quantify mutation. Mutation analysis, especially analysis of the variable frequency of mutation, remains largely an investigative procedure aimed at understanding the biology of a lesion's development. However, mutation analysis may be diagnostically critical in specific circumstances. Failure to detect mutation in instances in which clinical, radiological, and histological findings leave doubts about the diagnosis may help to rule out FD. Conversely, when radiographic and histological findings are strongly indicative of FD and no R201 mutation is detected, the diagnosis of FD is not disproven. In these cases, potential mutations at codon Q227 should be investigated. Such mutations have never been detected in FD but are known to occur in isolated
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endocrine tumors, and it is not inconceivable that they may be found in FD. Surgery Monostotic focal lesions of orthopedic concern may be treated by curettage and bone grafting, which are more effective in older patients. However, grafts of autologous bone are readily resorbed [180], resulting in complete or partial removal of the grafted bone depending on the predominantly cancellous or cortical architecture of the graft. Alternative grafting procedures using, for example, osteoconductive bioceramic scaffolds that are less resorbable than bone, with or without marrow tissue or cells, have not been carefully evaluated. Extensive lesions in a limb bone may require surgery for correction of deformity or fracture nonunion [181-183], although the frequency of the latter is probably grossly overestimated in the literature. Internal fixation using an intramedullary nail or an external plate is used in these cases [184], but the nature of the FD bone often makes anchorage of fixation devices poor. Resection in conjunction with a vascularized bone graft followed by fixation may occasionally be required in aneurysmal bone cysts engrafted onto FD in cases in which a suspicion of malignancy is justified or in cases in which multiple fractures recur at the same site. In general, the outcome of any surgical procedure can only be predicted based on a thorough understanding of the actual pathologic status of the surgical bed. This requires a better correlation of radiographic and pathologic findings so that specific features observed in imaging material can be interpreted correctly in individual case. Thus, an adequate understanding of the tissue and cell biology of FD lesions is important for improving the effectiveness of surgery. Neurosurgeons and maxillofacial surgeons are confronted with additional problems when dealing with FD. Resection of affected bone should be restricted to cases with reliable prediction of impending loss of major functions (vision, hearing, and airway patency). Most experts advocate a more conservative attitude or the use of conservative procedures [185]. Bisphosphonates Intravenous pamidronate has been used in open studies for the medical treatment of FD both in adults and in children and adolescents [186-192]. Several reports are in agreement with regard to its ability to induce a reduction of metabolic parameters of bone formation and resorption and a reduction of bone pain. In some studies, changes in radiographic density of individual lesions have been reported and considered as possible evidence of improved bone quality. Double-blind,
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placebo-controlled, randomized studies on the effects of third-generation bisphosphonates are under way. While awaiting the results of these studies, the ethical need for medical intervention in a disease that is occasionally devastating justifies, in the face of proven safety, an attempt to treat. A caveat in the use of bisphosphonates in FD concerns the high frequency of hypophosphatemia in FD patients. Careful monitoring of relevant metabolic parameters is in order, as is the concurrent administration of phosphorus and vitamin D supplements. A second caveat concerns the undocumented effect of bisphosphonates in FD lesions with a predominant sclerotic nature, such as those in the craniofacial skeleton. A more general question that remains open is whether reducing remodeling of FD bone per se should be necessarily seen as a long-term beneficial effect, regardless of patient age. The observation of disease burnout via remodeling, if confirmed and further substantiated, may suggest that remodeling may be beneficial in FD, at least at certain ages and stages of disease evolution. At a minimum, an understanding of the agerelated differences in the disease biology would allow identification of the correct time flames in which to consider treatment. The same applies to surgery. However, one must keep an open mind as to the actual benefit of reducing remodeling even when excess bone resorption is but a secondary effect and not the prime key to a disease mechanism. The beneficial effects of bisphosphonate treatment in patients with osteogenesis imperfecta [193-195] would support this attitude. Some of these effects, such as the apparent increase in bone formation, may represent more than an epiphenomenon of reduced resorption. Potential effects of bisphosphonates on cells of osteogenic lineage are poorly understood and may be compounded in a reduction of cell growth and enhanced cell differentiation, as suggested by in vitro studies. Nitrogen-containing bisphosphonates (such as pamidronate and alendronate, the two main bisphosphonates used for treating FD in open studies and controlled trials, respectively) inhibit protein prenylation [196], a type of posttranslational modification critical to a variety of GTP binding proteins. Gs~ is pamitoylated [197], whereas the y subunit is prenylated [198-200]. Palmitoylation and depalmitoylation regulate membrane association and redistribution to the cytosol of Gs~ [197], and turnover of palmitate is greatly enhanced in mutated Gs~ [201,202]. Prenylation of the [3y subunit affects its affinity for Gs~ and stabilizes and enhances the effects of palmitoylation with respect to membrane anchorage of Gs~ [203]. Additional GTP binding proteins that are prenylated interfere with downstream effects of cAMP, such as cytoskeletal remodeling. Analysis of the effects ofbisphosphonates on mutated osteogenic cells from FD thus appears warranted.
Strategies for Innovative T r e a t m e n t
Medical Pending further evidence, all existing options for treating FD are palliative, and the nature of the disease certainly calls for the development of treatments more adherent to its pathogenetic mechanism. Ideally, a suitable medical treatment would obviously solve the general problem of systemic FD disease and deal with specific inadequacies of surgery, limiting the need for surgery. The simplest approach in principle involves the use of inhibitors of either cAMP or Gs~ activity. Animal models are necessary to test any candidate compounds. A model based on ectopic transplantation of osteoprogenitor cells from FD into immunocompromised mice has been developed [204] and lends itself to investigate the effects of treatment on mutated osteogenic cells in vivo. Additional models based on murine transgenesis will enable long-term experimentation over the entire life span of the organism, the same temporal background against which the natural history of the disease genotype unfolds. These models will also allow investigation of the effects of the mutation on organogenesis of bone as well as the interplay of skeletal and nonskeletal effects of the mutation. A separate avenue may relate to the viability of the mutated clone and a way to selectively promote the demise of mutated cells. To this end, available pure strains of mutated cells might be used to investigate potential mechanisms for promoting their selective apoptosis.
Cell Therapy and Gene Therapy The notion that the skeleton depends on a compartment of progenitor/stem cells (marrow stromal stem cells [103]) for growth and turnover makes any genetic disease of osteogenic cells a theoretical candidate for cell therapy approaches. However, systemic administration of skeletal progenitor cells is not a reasonable approach. There is no evidence from animal studies that skeletal progenitor cells efficiently (i.e., to a biological effect) engraft when infused via a systemic route, despite some instances in which this kind of procedure has been used on patients with other genetic diseases of the skeleton. The notion of a somatic mosaic state in FD would allow for the use of autologous cells if efficient routes of engraftment were available, but significant basic and preclinical work is required. Autologous marrow stromal cells could instead be used for local transplantation at sites requiring surgical intervention. The relative ease with which clonal strains of normal and mutated cells can be separately grown implies the possibility of expanding normal cells selectively e x vivo for use in local transplantation in conjunc-
21. Fibrous Dysplasia tion with a suitable b i o m a t e r i a l . Based on extensive preclinical ( a n d limited clinical) experience with e n g i n e e r i n g of b o n e tissue using m a r r o w - d e r i v e d p r o g e n i t o r cells [205], satisfactory r e g e n e r a t i o n o f b o n e m a y be expected f r o m these p r o c e d u r e . H o w e v e r , r e g r o w t h o f lesional tissue (as h a p p e n s with b o n e grafts) at the site o f transp l a n t a t i o n c a n n o t be ruled out. D e v e l o p m e n t s in the use o f vectors suitable for efficient gene transfer in h u m a n cells a n d in the area o f antisense a n d r y b o z y m e R N A s [206,207] m a y be relevant to future t r e a t m e n t s o f F D b u t do n o t c u r r e n t l y r e p r e s e n t a viable o p t i o n o f t h e r a p e u t i c interest. Also, a d o m i n a n t g a i n - o f - f u n c t i o n m u t a t i o n in an essential a n d ubiquitously expressed gene represents p e r h a p s the worst-case scenario (the m o s t difficult challenge) for gene t h e r a p y . M a j o r efforts are r e q u i r e d in b o t h clinical a n d basic areas o f research before o n e or m o r e r a t i o n a l a n d effective t h e r a p e u t i c o p t i o n s b e c o m e available. T h e rarity of the disease is a m a j o r limiting step to the acquisition o f the necessary k n o w l e d g e a n d requires c o o r d i n a t i o n o f efforts worldwide. As Di G e o r g e [208] n o t e d , F D is "a rare disorder, yes, an u n i m p o r t a n t one, n e v e r , " given its d e v a s t a t i n g consequences.
Acknowledgments We are indebted to Dr. Mara Riminucci (University of L'Aquila, Italy) for extensive discussions, critical reading of the manuscript, and liberal sharing of unpublished data and observations. We acknowledge insightful discussions with Professor Alan Boyde (University College London), who also generously provided photographs in Fig. 11. His contributions and those of Drs. Sergei Kuznetsov, Larry Fisher, Michael T. Collins, and Kenn Holmbeck (NIDCR, NIH NIDCR, NIH) and of Dr. Alessandro Corsi (University of L'Aquila) to unpublished work mentioned in this chapter are also acknowledged. Published and unpublished personal investigations mentioned in this chapter were supported in part by Grants E.519 and E.1029 to PB. We are most grateful to the patients who enrolled in the current NIDCR, NIH, studies on fibrous dysplasia for their willingness to help future patients and for their trust in basic and clinical investigations, which to all patients with fibrous dysplasia are committed.
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122[ Nutritional Rickets JOHN M. PETTIFOR MRC Mineral Metabolism Research Unit, Department of Pediatrics, University of the Witwatersrand and Chris Hani Baragwanath Hospital Johannesburg, South Africa
INTRODUCTION
grouped together under the term vitamin D resistant rickets, have now been classified into a number of different etiologies. In this chapter, the general features of rickets are reviewed and a classification of the various causes of the disease is presented. Finally, nutritional rickets is discussed in detail.
Rickets has been a public health problem for children living in temperate climates for many centuries. Although it is unclear when rickets was first differentiated from other forms of deforming bone diseases, Daniel Whistler and Francis Glisson from England provided detailed and accurate descriptions of the clinical features of the disease in excellent treatises in the mid-17th century. At the turn of the 20th century, rickets was an almost universal finding at autopsies conducted on children who died during the winter months in northern Europe. Even during summer, the prevalence of rickets remained high. Although the scourge of nutritional rickets has been almost eliminated in many developed countries, it remains a major public health problem in a number of developing countries, and there has been an increase in the incidence of the disease in a number of countries from which it had almost been totally eradicated. In the late 1920s and early 1930s, it was established that nutritional rickets could be prevented by ultraviolet light irradiation, sunlight, or the provision of cod liver oil. In the 1920s, vitamin D was isolated and its role in the pathogenesis of rickets was established [1,2]. These discoveries provided cheap and effective means for prevention and treatment. During the past 30 years, considerable progress has been made in our understanding of the metabolism and functions of vitamin D [3-5] and the factors influencing mineral and bone homeostasis. These advances have resulted in a much clearer understanding of the pathogenesis of rickets and a more rational approach to the management of the various forms of the disease. The nonnutritional causes of rickets, which were originally
PediatricBone
DEFINITION OF RICKETS Rickets is a clinical syndrome characterized by a failure of or delay in endochondral calcification at the growth plates of long bones, resulting in deformation of the growth plate, a reduction in longitudinal growth, and the development of bone deformities. The disease is also associated with osteomalacia, which is a failure of mineralization of preformed osteoid on the trabecular and cortical bone surfaces of all bones. Thus, children who present with rickets have histological features of both rickets and osteomalacia, whereas once the growth plates have fused and growth has ceased, only features of osteomalacia are found [6]. Although it might be considered pedantic to distinguish between rickets and osteomalacia, the structures and cells involved are different. In rickets, the primary organ involved is the growth plate, with its chondrocytes and chondrocyte-derived extracellular matrix rich in proteoglycans and collagens type II and X. Mineralization of the cartilage matrix initially occurs in the matrix vesicles, which act as a naidus for further mineralization. In osteomalacia, the osteoblast and its extracellular product, osteoid rich in collagen type I and other noncollagenous proteins such as osteocalcin, are affected. These
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Copyright 2003, Elsevier Science (USA). All rights reserved.
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divisions are based on whether or not the primary defect results in hypocalcemia (calciopenic rickets), hypophosphatemia (phosphopenic rickets), or direct inhibition of mineralization (Table 1). Table 1 is not complete because isolated and unusual descriptions of rickets have not been included. Chapters 22-28 discuss the various types of rickets in more detail. The classification helps to categorize the causes into broad groups, each of which has characteristic biochemical changes that help in establishing the pathogenesis of the disease in an individual child. In the calciopenic forms of rickets, the typical biochemical changes include hypocalcamia and hyperparathyroidism, whereas in the phosphopenic form hypophosphatemia with normal parathyroid hormone concentrations is characteristic (Table 2).
two target organs may respond differently to rachitic factors and to various therapeutic regimens. For example, hypophosphatemic bone disease is characterized by osteomalacia and bone deformities but with no radiological evidence of rickets at the growth plate [7]. In X-linked hypophosphatemic rickets, it appears that the rachitic features at the growth plate respond more rapidly and completely to therapy with vitamin D analogs and phosphate supplements than does the osteomalacic component on the trabecular bone surfaces because bone biopsies may show continued osteomalacia when the growth plate shows no radiological features of rickets [8].
CLASSIFICATION OF RICKETS The causes of rickets may be classified into three major categories based on their pathogenetic mechanisms. Normal mineralization of the growth plate and of osteoid at the trabecular and cortical bone surfaces is dependent on a number of factors, including the presence of normal concentrations of both calcium and phosphorus and of alkaline phosphatase. Thus, the broad
NUTRITIONAL RICKETS Historical Perspective The first accurate descriptions of nutritional rickets were published by Whistler in Leyden in 1645 and by
TABLE 1 A classification of t h e c a u s e s of rickets b a s e d o n t h e p r i m a r y p a t h o g e n e t i c m e c h a n i s m s . N o t all t h e c a u s e s of rickets h a v e b e e n i n c l u d e d in t h e table.
Calciopenic
Inhibition of mineralization
Phosphopenic Dietary phosphorus deficiency
Hereditary hypophosphatasia
9 Vitamin D deficiency
Impaired intestinal phosphate absorption
Aluminium toxicity
9 Increased catabolism o f 25-OHD
Increased renal phosphate loss:
Fluoride toxicity
Alterations of vitamin D metabolism:
First generation bisphosphonates
9 Decreased production of 1,25-(OH)aD
9 PHEX gene abnormalities (X-linked hypophosphataemia)[209]
9 End-organ resistance to vitamin D
9 F G F 23 gene abnormalities (Autosomal dominant hypophosphataemia)[210] 9 Fanconi syndrome
Dietary calcium deficiency
9 Distal renal tubular acidosis 9 Tumour associated rickets 9 Neurocutaneous syndromes 9 Polyostotic fibrous dysplasia
TABLE 2
Typical b i o c h e m i c a l f e a t u r e s of t h e v a r i o u s c a t e g o r i e s o f rickets
Phosphopenic
Calciopenic 9 Hypocalaemia
9 Hypophosphataemia
9 Hyperparathyroidism
9 Normal PTH
9 hypocalciuria
9 increased TmP/GFR in the dietary and intestinal causes 9 decreased TmP/GFR in the renal causes
Impaired mineralization 9 May have normal serum calcium and phosphorus conc.
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Glisson in London in 1650. They highlighted the fact that the disease was most prevalent in infants and young children between the ages of 6 months and 21/2 years. Glisson considered rickets to be a new disease because it had not been previously described in books on diseases of children. Interestingly, at that time rickets was more common in infants of wealthy families than those of the poor [9,10]. The reason for this was probably that children of wealthy parents were not encouraged to play outdoors, whereas children of poor families spent time outside with their mothers, who worked on farms, and thus received adequate sunlight exposure. With the onset of the industrial revolution, and the movement of large numbers of people from rural farming areas to rapidly developing, overcrowded, and squalid towns and cities, the disease became more prevalent in the urban poor [9,11]. During the 19th century, several studies in England [9], Scotland [12], and Europe confirmed the widespread to almost universal prevalence of the disease among children. Although the disease was known as the English disease on the continent because of its high prevalence in England, continental children were not immune; for instance, in Vienna after World War I, rickets was a major public health problem among children [13]. Prior to the 20th century, the pathogenesis of nutritional rickets was generally thought to be related to poor hygiene, poor diet, and bad air, even though Trousseau in Paris in the mid-19th century suggested that osteomalacia was adult rickets, and that rickets was caused by nutritional problems and a lack of sunlight. He also proposed the use of cod-liver oil and prolonged breastfeeding for the treatment of the disease [14]. Interestingly, fish oils, especially cod-liver oil, had been used for the treatment of rickets as early as the 18th century. However, it was not until the 1920s that sunlight and cod-liver oil were accepted as effective forms of treatment. At that time, work by McCollum and Park in the United States and Mellanby in England, among others, highlighted the importance of ultraviolet irradiation and the role of vitamin D in the pathogenesis of rickets [15]. Chick, who worked in Austria immediately after World War I, provided a vivid description of the effect of either cod-liver oil or ultraviolet light on the healing of rickets in young infants [13]. With the general acceptance of the role of vitamin D in the pathogenesis of nutritional rickets, commercial breast milk substitutes were vitamin D fortified at levels of 400 IU per liter or quart in most developed countries. Furthermore, several countries enriched other foods, such as cow's milk, cereals, and bread. As a result of these policies, nutritional rickets was almost eradicated from countries such as the United States and Canada. In the United Kingdom, the excessive fortification of a
number of foods (dried milk powders and cereals) in the early 1950s was considered to be a possible cause of the unexplained increase in idiopathic hypercalcemia diagnosed in infants at that time. Daily intakes of vitamin D were estimated to be between 3000 and 4000 IU. As a result, vitamin D fortification was reduced in 1957 [16]. Nevertheless, the evidence to suggest a causal link between vitamin D toxicity and infantile hypercalcemia is tenuous. There is now evidence that infants who have idiopathic infantile hypercalcemia in association with the characteristic dysmorphic features of Williams syndrome have mutations in the elastin gene [17]. During the past 20 years, concern has been expressed by many researchers about an apparent resurgence of rickets in certain communities. Furthermore, the problem of rickets in a number of developing countries has also been highlighted. These problems are discussed later.
Epidemiology
Vitamin D Deficiency Vitamin D deficiency is generally considered the primary cause of nutritional rickets in most communities. Thus, nutritional rickets and/or osteomalacia are diseases with peak prevalences at the two extremes of life, the young and the elderly, to a large extent as a result of these two age groups' lack of independent mobility and thus their inability to spend sufficient time outdoors to obtain adequate skin exposure to sunlight. Although traditionally grouped with the vitamins, vitamin D is more appropriately considered a prohormone because it needs to be metabolized to 1,25dihydroxyvitamin D (the hormone) mainly in the kidney before inducing its physiological effects. Furthermore, the production of 1,25-dihydroxyvitamin D [1,25-(OH)2D] by the kidney is tightly regulated by a number of factors, including serum parathyroid hormone and phosphorus concentrations [18]. Finally, the normal diet of most communities does not contain adequate quantities of vitamin D to maintain vitamin D sufficiency in humans, who are dependent on the epidermal formation of vitamin D from 7-dehydrocholsterol under the influence of ultraviolet (UV) light [19]. Vitamin D biology is discussed in detail in Chapter 7; this section focuses on factors influencing the formation of vitamin D and the maintenance of vitamin D sufficiency. Vitamin D production in the skin is dependent on the conversion of provitamin D (7-dehydrocholesterol) to previtamin D under the influence of UV-B radiation (290-315 nm). Previtamin D then undergoes thermal isomerization to vitamin D3 (cholecalciferol) within the skin. This process is relatively slow, taking approximately
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36-48 hr to equilibrate at body temperature [20]. Once formed, vitamin D is removed from the skin through the dermal capillaries and transported attached to the vitamin D binding protein to the liver for 25-hydroxylation. Continued or excessive UV-B radiation converts previtamin D to other isomers such as lumisterol and tachysterol, which are.physiologically inert. Thus, the skin production of vitamin D under the influence of UV-B radiation is well adapted to human needs. The slow isomerizafion of previtamin D to vitamin D prevents rapid surges of vitamin D being released into the circulation when an individual is exposed to sunlight. Furthermore, not only does the conversion of previtamin D to inert isomers prevent the formation of toxic amounts of vitamin D when UV-B exposure is excessive but also a similar degradation of vitamin D to suprasterols and 5,6-trans-vitamin D occurs [21]. There are no reports of vitamin D toxicity resulting from excessive sunlight exposure, except in disease states such as sarcoidosis. Factors that play an important role in influencing the amount of vitamin D formed in the skin are listed in Table 3. The most important factors influencing the vitamin D status of individuals are the duration of time spent outdoors, the amount of skin exposed to UV-B radiation, and the solar zenith angle, which inversely influences the amount of UV-B reaching the earth [19]. Studies conducted in North America have shown that sunlight during the winter months in Boston (42~ and Edmonton (52-~ is unable to synthesize vitamin D from 7-dehydrocholesterol. In the Southern Hemisphere, similar studies conducted in Johannesburg (26-~ and Cape Town (32-~ showed that vitamin D synthesis was significantly reduced during the winter months in Cape Town when compared to Johannesburg [22], and in two cities in Argentina at 34 and 55-~ respectively, similar
TABLE 3
Factors influencing the amount of vitamin D produced in the skin
The amount of UV-B reaching the earth:
9 9 9 9
The latitude of the country The season of the year Atmosphericpollution The average sunshine hours/day
The amount of vitamin D formed in the skin:
9 9 9 9 9
The amount of skin exposed to sunlight The duration spent in sunlight The use of sunscreens The degree of melanin pigmentation The age of the individual
seasonal trends were seen with a prolonged "vitamin D winter" in the southernmost city of Ushuaia [23]. Melanin pigmentation may also be a limiting factor in dark-skinned populations living in countries in northern latitudes, where the amount of UV-B radiation reaching the earth is not optimal. Edmonton (52-~ is unable to synthesize vitamin D from 7-dehydrocholesterol. In the Southern Hemisphere, similar studies conducted in Johannesburg (26-~ and Cape Town (32-~ showed that vitamin D synthesis was significantly reduced during the winter months in Cape Town when compared to Johannesburg [22], and in two cities in Argentina at 34 and 55-~ respectively, similar seasonal trends were seen with a prolonged "vitamin D winter" in the southernmost city of Ushuaia [23]. Melanin pigmentation may also be a limiting factor in dark-skinned populations living in countries in northern latitudes, where the amount of UVB radiation reaching the earth is not optimal. The peak incidence of vitamin D deficiency rickets in children is between 3 and 18 months of age [24-26]. Infants younger than 3 months of age are partially protected from developing symptomatic vitamin D deficiency by the transfer of vitamin D metabolites across the placenta in utero. 25-Hydroxyvitamin D readily crosses the placenta so that fetal and newborn levels are approximately two-thirds of the maternal concentrations [27,28] (see Chapter 23). It is unclear whether vitamin D crosses the placenta to any extent; however, because circulating maternal levels are usually low, they are unlikely to contribute significantly to the vitamin D status of the newborn. 25-Hydroxyvitamin D has a circulating half-life of between 3 and 4 weeks; thus, concentrations decrease rapidly in the neonatal period unless an exogenous source of vitamin D is provided. In one study of breast-fed infants conducted during the winter months, serum concentrations of 25-OHD declined to very low levels by 6 weeks of age in infants whose mothers or they themselves were not supplemented with vitamin D [29], but this is not a universal finding [30,31]. Evidence that the maternal transfer of vitamin D metabolites to the fetus is protective in the first few months of life is provided by the fact that congenital rickets has been described in neonates born to vitamin D-deficient mothers [32-341. Prior to the era of vitamin D-fortified infant milk formulas, breast-fed infants were less likely to develop nutritional rickets than those who were fed breast milk substitutes, despite the low vitamin D activity of breast milk [35]. It has been suggested that the lower prevalence of rickets in breast-fed infants was due to the fact that breast milk contained considerable quantities of watersoluble vitamin D sulfate (9-22lag/liter), which was thought to be biologically active [36,37]; however, these findings have not been confirmed [38]. Recently, low
22. Nutritional Rickets vitamin D activity in breast milk has been confirmed by newer methods and has been estimated by several researchers to be the equivalent of approximately 2060 IU of vitamin D per liter of breast milk in normal circumstances [39-42]. Thus, if an infant consumes approximately 600-700 ml of breast milk daily, the vitamin D intake from this source will generally be less than 40IU, which is insufficient to maintain the normal vitamin D status of the infant. The vitamin D content of breast milk is dependent on the maternal vitamin D status [43] and can be increased to maintain vitamin D sufficiency in the suckling infant if the mother's daily intake of vitamin D reaches approximately 10002000 IU [29,44,45]. However, in normal circumstances, serum 25-OHD levels in the breast-fed infant mirror skin exposure to sunlight rather than breast milk vitamin D concentrations [46,47], emphasizing the importance of sunlight exposure in the maintenance of vitamin D sufficiency in the breast-fed infant. Specker and coworkers [42] showed that vitamin D activity in the breast milk of African American mothers is lower than that of white mothers and suggested that the lower vitamin D activity is a result of decreased vitamin D synthesis in the skin due to either decreased sun exposure or increased melanin pigmentation. The reduced vitamin D activity might contribute to the higher prevalence of nutritional rickets in breast-fed African American infants than in their white peers. The same group of researchers in Cincinnati (latitude 3 9 ~ that in order to maintain vitamin D sufficiency in breast-fed infants during the summer months, infants need to be exposed to sunlight for 30 min/week if wearing only a diaper or for 2 hr/week if fully clothed but not wearing a hat [47]. The possible explanation for the reported lower incidence of rickets in breast-fed infants compared to those fed unfortified cow's milk preparations may relate to the
TABLE 4 Country
United Kingdom South Africa Tibet China Greece Nigeria Turkey Iran Yemen Ethiopia
Year
545
more physiological calcium:phosphorus ratio in breast milk [48]. The calcium:phosphorus ratio in breast milk is approximately 2:1, whereas in unmodified cow's milk preparations it is approximately l:l. The lower phosphate load in breast milk assists in maintaining normal serum concentrations of calcium and phosphorus for longer, even in situations of vitamin D deficiency. Since the introduction of the universal policy of fortifying all infant milk formulas with at least 400 IU vitamin D/liter or quart, a reversal of patterns has occurred, with prolonged breast-feeding becoming a risk factor in the pathogenesis of nutritional rickets [49-51]. Thus, vitamin D deficiency is more common in infants who are breastfed, do not receive vitamin D supplements, and not exposed to adequate amounts of sunlight. With the discovery of vitamin D and the fortification of infant milk formulas with vitamin D, the prevalence of rickets declined dramatically in developed countries. This occurred at a time when exclusive breast-feeding of infants was an uncommon practice in most industrialized communities. However, in a number of countries nutritional rickets has been reported to be a continuing public health problem (Table 4). For example, in 1968 in Glasgow in the United Kingdom, it was estimated that approximately 9% of young children had radiological features of rickets [52], whereas in 1996 in England between 20 and 34% of 2-year-old Asian children had 25O H D concentrations lower than 25 nmol/liter [53]. In 2001 in Tibet, 66% of children older than 2 years of age had clinical features of rickets [54]. It should be noted that rickets is a problem not only in temperate climates or northern latitudes, such as in Tibet or the United Kingdom, but also in tropical and subtropical countries such as Nigeria. An extensive review of nutritional rickets in the tropics has been provided by Bhattacharyya [55]. In many of these tropical and subtropical countries,
The prevalence of rickets in a number of countries Prevalence
1968 9% radiological changes 1976 45% biochemical changes 1969 17% clinical features 1994 66% clinical features 1977-83 41% clinical features 1968 15% biochemical changes 1998 9% clinical features 1994 10% 1975 15% 1997 50% 1997 42% radiological changes
Sample
Reference
12-24 months random sample 9-16 yrs Asian schoolchildren 3-8 months immunization attendance during winter >24 months representative sample <3 yrs of age community sample <12 months 6-36 months community sample <36 months community sample <5 yrs consecutive X-rays <5 yrs admitted with pneumonia <Syrs admitted with pneumonia
[52] [75] [211] [54] [212] [213] [214] [60] [24] [137] [136]
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the pathogenesis of rickets appears to be classic vitamin D deficiency related to overcrowding in urban communities or social, religious, or clothing customs, such as purdah, which preclude adequate sunlight exposure. In Kuwait, poor maternal education, lack of exposure to sunlight, and a poor weaning diet with prolonged breastfeeding are factors that have been incriminated in the pathogenesis of the disease [26]. Similar contributing factors appear to be responsible in Iran and Saudi Arabia [24,56]. In Ethiopia, in addition to the contributing factors mentioned previously, malnutrition is strongly associated with rickets [57]. Low serum levels of 25-hydroxyvitamin D were documented in mothers of rachitic infants in Saudi Arabia, and these could have contributed to the low vitamin D status of the breast-fed infants [56]. In several studies, a familial occurrence of rickets has been noted. Although it is likely that children within the same family are exposed to similar environmental and social factors, and thus to the same rachitogenic factors, Doxiadis and coworkers [58] suggested that there might be a genetic element in some families. Onethird of the infants they studied who had rickets had persistent amino aciduria after the rickets healed, and many of their parents had renal tubular abnormalities manifesting as phosphaturia and increased a-amino nitrogen excretion. Furthermore, a possible sex-linked genetic factor is suggested by the fact that nutritional rickets has been reported to be more common in male than female infants [24,59], although this finding is not supported by all studies [60]. A number of reports have appeared suggesting that there has been an upsurge of rickets among minority groups and communities in several developed countries. In the United States, approximately 20 years ago attention was drawn to the association of rickets with prolonged unsupplemented breast-feeding, strict vegetarianism in mothers, and fad diets [49-51,61-64]. The vast majority of infants were African American [65-67]. Several factors appear to be responsible for the increased prevalence of rickets in this community: The public is being made aware of the benefits of exclusive breastfeeding and is being encouraged to breast-feed, there is a belief that breast milk contains all the nutrients a young infant requires for normal growth and development, the increased skin pigmentation probably reduces cutaneous vitamin D production in situations of marginal UV radiation, and there is increasing concern about the risk of skin cancer as a result of sunlight exposure. Furthermore, the American Academy of Pediatrics does not recommend universal vitamin D supplementation for breast-fed infants; rather, it suggests that "vitamin D may need to be given before 6 months of age in selected groups of infants (for infants whose mothers are vitamin
D deficient or those infants not exposed to adequate sunlight)" [68]. However, this policy is not supported by all [69]. Not just in the United States does increased melanin pigmentation place infants and toddlers at increased risk of rickets. The disease has been reported in young children of dark-skinned immigrants in New Zealand [70], Canada [71], and a number of European countries [72,73]. Furthermore, in the United Kingdom rickets remains a major problem in the Indian/Pakistani/Bangladeshi (Asian)community [53,74,75]. In The Netherlands, children from Morocco and Turkey have lower 25-OHD levels and higher parathyroid hormone (PTH) values than Caucasian children [76]. These biochemical perturbations are considered to be due to differences in skin pigmentation and calcium intakes. A number of researchers have noted the role of macrobiotic or vegetarian diets in exacerbating vitamin D deficiency and the development of rickets [77-79]. It is suggested that the pathogenesis of the bone disease relates to the low vitamin D content of most vegetarian diets and the low dietary calcium bioavailability, which aggravates the vitamin D deficiency.
Induction of Vitamin D Deficiency by Low Dietary Calcium Intake In the United Kingdom, the Asian communities (Pakistani, Indian, and Bangladeshi) are particularly at risk for developing rickets, not only in the infant and toddler age groups but also throughout childhood, adolescence [80], and into adulthood [81]. Of interest is the finding that other dark-skin immigrants such as West Indians do not have the same risk of developing rickets [82]. Attention was drawn to the problem among Asian immigrants in the 1960s [52,83], and since that time a large number of studies have been conducted to determine the pathogenesis and the best methods of management. It is clear that vitamin D deficiency is an essential component of the pathogenesis [75,84-87]; however, other factors that exacerbate the development of vitamin D deficiency appear to be involved [12,88], including vegetarianism [89,90], poor calcium and high phytate contents of the diet [81,91,92], and extensive skin coverage by clothing [93]. In 1989, Clements [88] proposed that the pathogenesis of Asian rickets was related to the induction of vitamin D deficiency as a result of the effects of the low dietary calcium content and its poor bioavailability on the catabolism of vitamin D and its metabolites (Fig. 1). Studies in rats have shown that dietary calcium deficiency or the addition of phytate to the diet reduce the half-life of circulating 25-OHD without altering the metabolic clearance of 1,25-(OH)zD [94]. This is achieved by an increase in the inactivation of 25-OHD by the liver through the
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22. Nutritional Rickets
y Poor calcium intake
Vegetarian diet
I
High phytate content
Inadequate calcium absorption
1
Increased PTH concentrations
Increased 1,25-(OH)2D production
l
Increased 25-OHD catabolism
Low dietary vitamin D
/
Marginal vitamin D status
iv
Vitamin D deficiency
.~ Rickets
Poor skin exposure FIGURE 1
The pathogenesis of rickets in Asian children in the United Kingdom.
promotion of the hepatic conversion of vitamin D to polar inactivation products, which are excreted in the bile. The effect is mediated by elevated levels of 1,25(OH)2D in response to secondary hyperparathyroidism. Halloran and coworkers [95,96] showed that the reduction in serum 25-OHD levels associated with the chronic infusion of 1,25-(OH)2D could be explained by the increase in metabolic clearance rate of 25-OHD, and they suggested, unlike Clements and coworkers, that this probably occurs through the 24-hydroxylation pathway at sites other than the liver. The effects of elevated levels of 1,25-(OH)2D on serum concentrations of 25OHD are similar in humans to those demonstrated in rats and can occur within 24 hr of administration of 1,25(OH)2D [97]. Furthermore, intestinal malabsorption syndromes and high-fiber diets have been shown to reduce the plasma half-life of 25-OHD [98,99]. Thus,
the high prevalence of rickets in the Asian population in the United Kingdom is related to the marginal vitamin D status of most inhabitants due to the high latitude of the country, which is aggravated in the Asian community by vegetarian diets that are low in calcium and high in phytates. The low calcium bioavailability results in secondary hyperparathyroidism and elevated 1,25-(OH)2D levels, with the latter causing increased catabolism of 25OHD, resulting in a shortened half-life and vitamin D deficiency [88] (Fig. 1). The interaction of poor vitamin D status with low calcium bioavailability in inducing vitamin D deficiency and rickets probably does not just occur in the Asian community in the United Kingdom but may also play a role in many situations in which calcium intakes are poor and vitamin D status marginal, as may be the situation in African Americans. In fact, this combination of factors
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John M. Pettifor
may be a more common cause of rickets than isolated vitamin D deficiency. This mechanism may also explain the relatively high prevalence of rickets during adolescence; it is during this period that calcium requirements increase to meet the demands of the rapidly growing skeleton [100-103].
Dietary Calcium Deficiency Although nutritional rickets has generally been considered to be associated with vitamin D deficiency, during the past 25 years a number of studies from developing countries have highlighted the role of dietary calcium deficiency as a possible cause of rickets in the face of an apparently normal vitamin D status. Isolated cases of rickets, caused by very low calcium diets used in the management of allergies or gastrointestinal malabsorption, have been reported from the developed world [104,105]. However, until recently, the typically low calcium content of staple diets in many developing countries was considered to cause few problems [106-108]. Recent studies suggest that this is not the case. Children described with dietary calcium-deficiency rickets in studies conducted in South Africa [109,110], Nigeria [111-113], and Bangladesh [114] are typically older than the infants and toddlers who are at risk of developing vitamin Ddeficiency rickets. In South Africa, the children were 416 years, whereas the mean age of those in Nigeria was approximately 4 years and in Bangladesh it was 51/2 years. The diets are exceptionally low in calcium, with an estimated calcium intake of approximately 150mg/ day. Further compromising calcium absorption is the high phytate content of the typically cereal-based diet (corn or rice). Characteristic of the diet is the absence of dairy products [115,116]. It is of interest that reports from Bangladesh suggest that the disease is of recent origin (during the past 20 years) in that country [114]. It is possible that with population increases and more intensive farming methods, the staple diet of children in the area has become less varied and calcium intakes may have declined. The biochemical features of the disease are discussed later; however, a striking hallmark of the rickets described in these three developing countries is the normal or near-normal circulating levels of 25-OHD, thus excluding vitamin D deficiency as a factor in the pathogenesis of the disease. Although there is general consensus among researchers in these areas that low dietary calcium intakes are a major pathogenetic factor because the disease responds to calcium supplements, it is possible that there might be other contributing factors in some areas. Studies conducted by Thacher et al. [112,116] have consistently not been able to show a difference in calcium intakes between rachitic subjects and age-matched controls. It is possible that rachitic children
are less able to adapt to the stress of low calcium intakes than their normal age-matched controls. In this regard, a possible difference in the frequency of vitamin D receptor alleles has been found in subjects compared to community controls [117]. Further work is needed to establish the factors responsible for making some children on low-calcium diets susceptible to rickets. During the past 20 years, it has become apparent that the pathogenesis of nutritional rickets may be viewed as a spectrum of causes, with pure vitamin D deficiency at one end of the spectrum and dietary calcium deficiency at the other. In between, combinations of relative vitamin D insufficiency and decreased calcium bioavailability result in the development of vitamin D deficiency and the exacerbation of the development of rickets. Clinical P r e s e n t a t i o n Rickets presents clinically as a consequence of hypocalemia, bone abnormalities associated with rickets and the accompanying osteomalacia, or the effects of vitamin D deficiency on other systems, such as the muscular or immune systems. Furthermore, the presentation differs depending on the age of the child and on the bones that are subjected to weight bearing or under bending stress. The progression of vitamin D deficiency has been categorized into three stages based on the biochemical changes that occur as the disease becomes more severe [118]. Although there is considerable overlap between the three stages and stage 1 may not be seen in all patients with rickets, the concept is clinically useful. Stage 1 is characterized by hypocalcemia and usually occurs very transiently in the early phase of vitamin D deficiency prior to the onset of hyperparathyroidism. Clinically, the features are those of hypocalcemia and are usually seen in infants younger than 6 months of age. They may present with apnoeic episodes [119], convulsions [120], tetany [121], or stridor. Bony features of rickets are characteristically absent at this stage. It appears that symptomatic hypocalcemia in vitamin D deficiency may be precipitated by an acute illness [122,123]. Late neonatal hypocalcemia occurs more commonly in neonates who are vitamin D deficient as a result of maternal deficiency [124,125]. Maternal subclinical vitamin D deficiency may also result in impaired infant growth [126], impaired neonatal cardiac function [127], and enamel hypoplasia [121]. During stage 2, hypocalcemia improves to nearnormal or normal levels and the bony changes of rickets become apparent. In stage 3, the clinical features of rickets become progressively more severe and hypocalemia may once again become symptomatic as the homeostatic mechanisms attempting to maintain normocalcemia fail. The initial bony changes of rickets are
22. Nutritional Rickets
subtle, occurring most prominently at the growth plates of the rapidly growing long bones. Thus, the changes are typically noted first at the wrist, with visible and palpable enlargement of the distal ends of the radius and ulna, and around the knee, with enlargement of the distal femur and proximal tibia, although changes at the latter two sites are often difficult to assess unless severe. As the disease becomes more prolonged and severe, deformities of the appendicular skeleton occur at sites affected by weight bearing or stress. The types of deformities depend on the age of the child and whether or not the child is walking. Thus, in the very young infant, deformities and fractures of the distal third of the radius and ulna may be associated with swaddling, and anterior bowing of the distal tibia is associated with the infant lying with one leg crossed over the other. As the child starts to stand and walk, the physiological bowing around the knees becomes accentuated and bowlegs (genu varum) become prominent. Bowlegs are associated with the development of bilateral tibial torsion and intoeing. In the older child, knock-knees (genu valgum) may be the more common feature as physiological bowlegs spontaneously disappear with age. Rarely, a combination of valgus and varus deformities may be found the so-called "windswept" deformity. In long-standing and severe rickets, coxa vara of the femoral neck may occur, leading to gait abnormalities, and the development of pelvic deformities may result in narrowing of the pelvic outlet and obstructed labor once adulthood is reached [32]. Although the typical long bone features of rickets are most marked in the legs, upper limb deformities do occur as a result of weight bearing or pressure in children with long-standing or severe rickets. Costochondral enlargement becomes clinically apparent as beading along the anterolateral aspects of ribs to form the rachitic rosary. Harrison's sulcus develops as a result of the muscular pull of the costal attachments of the diaphragm on the lower ribs (Fig. 2). As the ribs progressively soften, the intermittent intrathoracic negative pressure associated with breathing narrows the chest in the lateral diameter, giving the chest an appearance of a violin case (Fig. 2). The resultant chest deformities in association with muscle hypotonia may result in severe respiratory distress. In very severe cases of rickets, vertebral abnormalities with the development of kyphoscoliosis become apparent. Much of the curvature may be a result of severe muscular hypotonia, but vertebral body collapse may also occur. Rickets is associated with delayed closure of the cranial fontanelles, with the anterior fontanelle being excessively large for age. The skull develops the hot crossed bun appearance due to frontal and parietal bossing. In the infant, the presence of craniotabes (softening of the
549
FIGURE 2 Clinical features of rickets in an infant with vitamin D deficiency rickets. Note the Harrison's sulcus and violin case deformity of the thoracic cage, the frontal bossing, and the protuberance of the abdomen (reproduced with permission from Pettifor and Daniels [215]).
skull bones behind the ear over the occipital region) is considered to be very suggestive of rickets [128]; however, it may be a normal feature in infants 3 months of age or younger [129,130]. Eruption of primary dentition is delayed, and if the mother was vitamin D deficient during pregnancy, the primary dentition may show signs of enamel hypoplasia [121]. Asymmetry of the skull (plageocephaly) develops as a result of the softness of the cranial vault and the hypotonia, which is characteristic of vitamin D-deficiency rickets. Craniosynostosis of the coronal or multiple sutures has been reported to occur in approximately 25% of patients with severe vitamin D-deficiency rickets who have been followed up after treatment [131]. Pseudotumor cerebri and cataracts have been described in a young infant with severe rickets and hypocalcemia [132]. In addition to the bony deformities, vitamin D-deficiency rickets is characterized by a delay in gross motor milestones as a result of hypotonia and muscle weakness; thus, children often present with a history of delayed sitting, crawling, or walking. Bone pain may also limit ambulation. In young infants, the abdomen may appear
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John M. Pettifor
distended as a result of hypotonia of the abdominal musculature. In older children and adolescents, the myopathy is described as mainly a proximal myopathy, which may result in difficulty climbing stairs or getting out of chairs. Despite the hypotonia, deep tendon reflexes may be brisk. The pathogenesis of hypotonia is thought to be the result of vitamin D deficiency on muscle function [133] rather than a consequence of hypophosphatemia. Cardiac abnormalities, including dilated cardiomyopathy [134], electrocardiographic changes, and left ventricular dysfunction [135], have also been described in children with vitamin D deficiency. These abnormalities improve on treatment and are thought to be a consequence of hypocalcemia. Profuse sweating is also described as a feature of severe rickets in young children, and it probably reflects the increased work of breathing associated with the chest deformities and the increased flexibility of the ribs. Vitamin D-deficiency rickets in young children appears to be associated with an increased risk of respiratory and gastrointestinal infections [60]. Many reported series of children with rickets have been derived from hospital admission data, and the majority of children were not admitted because of rickets but rather because of respiratory or gastrointestinal infections [24,59]. The results of a case-control study of children with pneumonia admitted to a hospital in Ethiopia emphasized the importance of rickets as a predisposing factor (rickets was 13 times more common in children with pneumonia than in controls) [136]. Although no increase in mortality was found to be associated with the presence of rickets, another study, which assessed factors responsible for the high mortality from pneumonia in Yemen, found that rickets together with anemia and malnutrition were associated with an increased risk of dying [137]. The mechanisms by which vitamin D deficiency causes an increase in infection risk are probably a combination of structural and immunological factors. Physical factors, such as rib softening, enlargement of the costochondral junctions with pressure on the underlying lung, and muscle weakness, may all contribute to the increased risk of respiratory infections due to an inability to adequately clear the lung of pathogens and mucus. The role of vitamin D, or more specifically 1,25-(OH)zD, in immune modulation is well described [138]. A number of abnormalities of the immune system have been described in vitamin D deficiency, including impaired phagocytosis [139] and neutrophil mobility [140], which could predispose vitamin D-deficient children to increased risk of infection. Other hematological abnormalities reported in children with rickets include von Jacksch-Luzet syndrome, which is associated with anemia, thrombocytopenia, leukocytosis, myeloid metaplasia, and hepatosplenomegaly [141,142]. Myelofibrosis has also been described in
long-standing vitamin D deficiency [143]. In several studies, an association has been found between the presence of iron-deficiency anemia and vitamin D deficiency [53,144]. The mechanisms are not clearly understood, but it is possible that iron deficiency impairs intestinal mucosal function, resulting in vitamin D and/or calcium malabsorption. Maternal vitamin D deficiency has been shown to impair neonatal calcium homeostasis, resulting in an increase in symptomatic neonatal hypocalcemia, and to delay fetal bone ossification and tooth enamel formation [145]. Furthermore, there is evidence that both intrauterine and postnatal growth might be affected [126,146]. The clinical features of rickets due to dietary calcium deficiency are similar to those described for vitamin D deficiency; however, the peak age of dietary calcium deficiency is older than that of vitamin D deficiency, and the degree of rickets tends to be less severe. Thus, as noted in Table 5, the features are more often related to limb deformities and growth plate enlargement rather than craniotabes, delayed motor milestones, and open fontanelles. Furthermore, hypotonia and muscle weakness appear to be less prominent in dietary calcium deficiency. In studies in South Africa, lack of the two previously mentioned features is used to help differentiate dietary calcium deficiency from vitamin D deficiency. A possible reason for the absence of muscle symptoms is the presence of elevated 1,25-(OH)2D concentrations in dietary calcium deficiency.
TABLE 5 The prevalence of various presenting s y m p t o m s and signs in children a g e d 18 m o n t h s and older with radiological rickets in Nigeria Prevalence (%)
Characteristic Symptoms
N=278 Weakness
65%
Leg pain when walking
60%
Excessive falling
58%
Unable to walk
11%
Previous fracture Signs
Enlarged costochondral junctions
9% 77%
Enlarged wrists
75%
Genu varum
48%
Enlarged ankles
38%
Genu valgum
32%
Rib cage deformities
15%
Windswept deformities
14%
Open anterior fontanelle
12%
Dental enamel defects
11%
22. Nutritional Rickets
A child with severe active rickets is not difficult to diagnose; however, assessment of the prevalence of rickets in a community is much more difficult without the use of radiology and/or biochemical markers. A number of the studies listed in Table 4 that assessed the prevalence of rickets in a community were based only on clinical features. Depending on the clinical features used for diagnosis, it is highly possible that the prevalence of active rickets is overestimated because the features are likely not specific for rickets and even less so for active disease. However, the need to be able to diagnose active rickets accurately without the use of radiology is of importance to health planners and to health care professionals in a primary health care setting [147]. Even the use of biochemical tests, such as an alkaline phosphatase level, to help confirm the diagnosis is problematic [147]. In a recent study conducted in Nigeria of more than 700 children older than 18 months of age, who were referred because of leg deformities and because they were not able to walk and who were suspected of having rickets, various clinical features were assessed for sensitivity and specificity to diagnose active rickets against radiological confirmation (Table 5; T. D. Thacher, P. R. Fischer, and J. M. Pettifor, personal communication). The following features were found to be independently predictive of active rickets: age younger than 5 years, height for age less than 2 SD below the mean, leg pain during walking, wrist enlargement, and costochondral enlargement. When three or more of these clinical features occurred simultaneously, 87% of the children who had active rickets were identified. When less than three features occurred simultaneously, 76% of the children who did not have active rickets were accurately identified. Although these findings suggest reasonable sensitivity, 24% of children with less than three signs were incorrectly classified as not having active rickets. A similar study was conducted in a group of younger subjects (3-30 months) attending an outpatient clinic in South Africa. The study used a clinical scoring system that included the presence or absence of craniotabes, frontal and parietal bossing, thickened costochondral junctions, Harrison's sulcus, and thickened wrists [128]. At that time, vitamin D-deficiency rickets was common in the community and more than 30% of children were diagnosed radiologically and biochemically as having rickets. The researchers found that enlargement of the costochondral junctions was the most reliable clinical sign, followed by enlargement of the wrists. In children older than 12 months of age, delayed closure of the fontanelle was a useful sign. However, the study concluded was rickets could not be diagnosed or excluded with certainty using clinical signs alone because 31% of the infants diagnosed clinically as having rickets did not
551
have biochemical or radiological evidence of the disease, and 13% of infants with no clinical signs has radiological and biochemical evidence of the disease. Another study conducted in South Africa assessed the usefulness of craniotabes in the diagnosis of rickets in infants 3-12 months of age [130]. In infants 3-6 months of age, 40% of those with craniotabes had radiological evidence of rickets, whereas in the older group aged 7-12 months, 60% with craniotabes had rickets. In a control group, none of the infants aged 3-6 months who did not have craniotabes had rickets, whereas 35% of those 7-12 months old without craniotabes had radiological evidence of rickets. Thus, the finding of craniotabes in older infants is more suggestive of rickets than that in younger infants. These various studies indicate that it is unlikely that a single set of clinical signs or symptoms can be used to diagnose rickets at all ages because some of the signs are age dependent, mild/early rickets is difficult to diagnose clinically, and the usefulness of the clinical signs depends on the prevalence of rickets in the community.
Biochemical Changes Associated with Nutritional Rickets The classic biochemical abnormalities of nutritional rickets, whether due to vitamin D deficiency or lack of dietary calcium, relate to the fact that the basic defect is an inability to maintain normal calcium homeostasis. Thus, as described by Fraser and coworkers [118], the initial biochemical abnormality is hypocalcemia. As discussed earlier, this early phase in vitamin D deficiency may not be clinically apparent because it may be transient and relatively mild; however, it is sufficient to induce secondary hyperparathyroidism. With the development of elevated PTH levels, the biochemical abnormalities, which are typically associated with calciopenic rickets, become apparent. Thus, increased renal tubular loss of phosphate results in hypophosphatemia. Other effects of increased parathyroid activity on the renal tubules include decreased urinary calcium loss, increased cyclic AMP excretion, generalized amino aciduria [148], and increased bicarbonate loss. Although stage l vitamin D deficiency is considered to be associated with normal PTH levels [118,120], other workers [149] have found elevated PTH concentrations and increased urinary cyclic AMP excretion. This should not be surprising because the three stages of vitamin D deficiency are a continuum; thus, as stage 1 merges with stage 2, elevated PTH levels would be expected [150] (Fig. 3). Hypocalcemia (low ionized calcium concentration) is the hallmark of calciopenic tickets; nevertheless, many children with rickets due to either vitamin D or dietary calcium deficiency have total calcium values within the
5 5 2.
John M. Pettifor
PROGRESSIVE DEPLETION OF 25OHDa
FIGURE 3 The progression of biochemical changes that occur in vitamin D deficiency (reproduced with permission from Arnaud et al. [216]).
normal range. In the majority of cases, these are at the lower end of normal. On treatment, the values increase and then stabilize at a new steady state, suggesting that for the individual child the low normal values are not normal. As discussed earlier, symptomatic hypocalemia may occur in both the early and late stages of the disease. In the early stage, a lack of PTH response to the hypocalcemia has been suggested as the mechanism [118], although Kruse [149] provides evidence for PTH resistance. In the more severe forms of the disease, an inability to maintain calcium homeostasis probably results from a failure to adequately mobilize mineral from bone [151]. Although hypophosphatemia is a characteristic biochemical feature in children with vitamin D-deficiency rickets, elevated or normal serum phosphate levels have been described not only in stage 1 of the disease (as might be expected because of normal PTH values) but also in some patients with more severe forms of the disease in which PTH levels are elevated [149,152-154]. Normal or elevated levels of serum phosphate have also been described in children suffering form rickets due to dietary calcium deficiency [109,155,156]. In these children, the tubular reabsorption of phosphate is high and is unresponsive to infusions of PTH (J. M. Pettifor, unpublished results). A similar finding has been noted in adolescent vitamin D-deficient children with hyperphosphatemia [154]. Correction of the associated
hypocalcemia restores the renal responsiveness to PTH and serum phosphate concentrations return to normal (J. M. Pettifor, unpublished results). Traditionally, bone turnover in children has been assessed by measuring serum total alkaline phosphatase as a marker of bone formation and the urinary excretion of hydroxyproline as a reflection of bone resorption. Total alkaline phosphatase has been used extensively as a biochemical marker of rickets in children because in the vast majority of cases levels are generally elevated [128], but it does lack specificity [147]. Because the liver isoenzyme contributes a substantial amount to the total value, hepatic diseases, especially those associated with cholestasis or biliary obstruction, may make the interpretation of results difficult. Furthermore, other factors, such as growth rates, the stage of puberty, and the nutritional status of the child [55], may influence the results. It is unclear why alkaline phosphatase levels are elevated in rickets because osteoblastic activity is decreased on bone histology [6]. The measurement of urinary hydroxyproline excretion is also problematic because the collection of complete 24-hr urine samples is difficult in children, there is considerable fluctuation in daily urinary excretion, and the amount ofhydroxyproline excreted in the urine is a reflection of not only bone collagen breakdown but also collagen breakdown elsewhere in the body and from that ingested in food. Nevertheless, hydroxyproline excretion is consistently increased in children with vitamin Ddeficiency rickets, reflecting the increased bone resorption associated with secondary hyperparathyroidism [149]. During the past 20 years, a number of new markers of bone formation and resorption have been described and are readily measured in either serum or urine [157,158]. Recently, they have been measured in normal children and in those with metabolic bone diseases [159-162]. The newer markers of bone formation include bone-specific alkaline phosphatase (ALP), osteocalcin (OC), and carboxy-terminal propeptide of type I procollagen (PICP), whereas the markers of bone resorption include pyridinoline (PYD) and deoxypyridinoline (DPD) cross-links of collagen, the cross-linked carboxy-terminal telopeptide of type I collagen (CTX-I), and the cross-linked N-telopeptides of type 1 collagen (NTX-I). Many of the markers show considerable variation dependent on age of the child and pubertal development, reflecting changes in bone turnover associated with varying rates of bone growth [160]. Furthermore, a number of markers, such as serum OC and urinary DPD and PYD, also show a circadian rhythm. Only a few studies have reported on the changes of these newer markers of bone turnover in children with nutritional rickets [161-163]. As is to be expected with secondary hyperparathyroidism and increased bone
22. Nutritional Rickets
resorption, serum CTX and urine NTX levels are elevated prior to treatment in children with vitamin Ddeficiency rickets [162]. With treatment, levels increase initially and then decline to the normal range within 4-6 weeks. Markers of bone formation show divergent patterns: Alkaline phosphatase levels decline from elevated to normal values within 8 weeks; serum PICP levels are only slightly elevated on admission, increase significantly during the first 2 weeks of treatment, and then return slowly to normal values by 8 weeks; and osteocalcin levels are significantly lower than normal on admission, increase sharply in the first 2 weeks, and then decline to normal values by 6 weeks. The discordance between alkaline phosphatase and osteocalcin levels in rickets has been noted by a number of researchers [163,164], suggesting that in vitamin D deficiency there might be an arrest in the maturation of osteoblasts prior to the mineralization phase during which osteocalcin is secreted [162]. Osteocalcin levels in rickets are in keeping with the histological evidence of decreased osteoblastic activity [6]. A number of bone turnover markers have been measured in children with dietary calcium-deficiency rickets [161,163,165]. Scariano and coworkers [161] reported that CTX and OC values are elevated in Nigerian children with presumed dietary calcium deficiency; however, elevated OC levels prior to treatment have not been confirmed in other studies [111,163]. The Nigerian workers also reported an increase in NTX and PTH levels with calcium treatment [165]. Despite the fact that there are a number of markers available to measure bone turnover in children with rickets, serum alkaline phosphatase probably represents the cheapest and most appropriate way to follow the response to therapy in a child with nutritional rickets [162]. Considerable attention has been paid to the concentrations of various vitamin D metabolites in rickets. Vitamin D deficiency is characterized by low circulating levels of 25-OHD, but what levels are normal is currently being debated [166-168]. In children, the range in normal pediatric populations is 12-50ng/ml (30-125 nmol/liter) [46,47,169,170]. At question is whether or not this normal range is appropriate for a population of children to optimize mineral homeostasis and bone growth. In children with untreated florid rickets, serum 25-OHD levels are typically <12ng/ml [82,171-173] and in many cases <5ng/ml [84,149]. In subclinical rickets, similar values have been found [174], although in one study higher values were documented [82]; however, the very mild radiological rickets in this group of children healed spontaneously within 6 weeks, suggesting that these higher values might well have reflected those of healing rickets. It must be emphasized that vitamin D-deficiency
553
rickets may be associated with a range of 25-OHD levels because rickets is an end result of a process of diminished calcium absorption that occurs over a period of time. The rate at which clinical rickets develops is dependent on the severity of the vitamin D deficiency, the calcium demands of the growing skeleton, and the dietary calcium content and its bioavailability. Several authors have used the term vitamin D deficiency to refer to serum 25-OHD concentrations associated with rickets or osteomalacia, whereas vitamin D insufficiency has been reserved for serum 25-OHD concentrations that are associated with perturbations of calcium homeostasis but without osteomalacia or rickets [168]. A number of studies have documented changes in serum PTH and/or calcium concentrations in association with seasonal fluctuations in serum 25-OHD levels [100,175-178]. In two of these studies, serum 25-OHD concentrations of ~ 12 [177] and ~ 16 ng/ml [178] were calculated as the levels below which serum PTH started to increase. A similar value of 12 ng/lm was found to be the point below which neonates developed hypocalcemia and secondary hyperparathyroidism [145]. Using a different technique to assess the desirable 25-OHD concentration in prepubertal children, Docio and coworkers [179] estimated the level to be between 12 and 20 ng/ml. Thus, in the majority of studies conducted in children, vitamin D sufficiency appears to equate to a serum 25OHD concentration higher than approximately 12 ng/ ml. However, little is known about the effect that differing calcium intakes might have on the serum 25-OHD concentration required to maintain normal calcium homeostasis. Studies by Clements have highlighted the role that low dietary calcium content and high dietary phytate play in inducing vitamin D deficiency and rickets [88,94]. Although serum 25-OHD levels are consistently low in untreated vitamin D-deficiency rickets, no such pattern appears to exist for serum 1,25-(OH)2D concentrations. Values of the latter metabolite have been reported to be low, normal, or even elevated [149,171,172,180-182]. The finding of normal or elevated levels of 1,25-(OH)zD in children with florid rickets has led some investigators to postulate that other metabolites besides 1,25-(OH)zD might be necessary to prevent the development of rickets and osteomalacia [181,183,184]. Certainly there is evidence that 24,25-dihydroxyvitamin D [24,25-(OH)zD] may be necessary for normal fetal bone development [185], but there are little data to suggest that vitamin D metabolites other than 1,25-(OH)zD are necessary for normal calcium homeostasis. Another interpretation of the normal or elevated levels of 1,25-(OH)zD seen in some children with florid rickets is that these values are actually inappropriately low for the degree of
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secondary hyperparathyroidism present [149,182]. A plausible explanation for the differing 1,25-(OH)2D concentrations in various studies is provided by Arnaud [150] and Kruse [149] (Fig. 3). In the early stages of vitamin D deficiency (stage 1), serum 1,25-(OH)2D concentrations decrease to levels insufficient to maintain intestinal calcium absorption at the required level for normocalcemia and growth; thus, ionized calcium values decrease and secondary hyperparathyroidism develops, increasing l a-hydroxylase activity in the kidney (transition between stages 1 and 2). Increased activity leads to a return of 1,25-(OH)2D levels to normal [149], which increases intestinal calcium absorption and bone resorption, partially correcting the hypocalcemia (stage 2). As the substrate (25-OHD) for 1,25-(OH)zD production continues to decline, a stage is reached in which 25OHD concentrations decline to such a level that 1,25( O H ) z D concentrations are unable to be maintained and metabolite levels decline. That the 1,25-(OH)2D levels are inappropriately low for the degree of secondary hyperparathyroidism is evidenced by the rapid increase in levels induced by small doses of vitamin D [180]. Thus, the diagnosis of vitamin D deficiency is based on the measurement of circulating levels of 25-OHD and not 1,25-(OH)2D. Serum 24,24-(OH)zD concentrations are normally closely related to the serum levels of 25-OHD, being less than 10% of the latter in vitamin D-replete individuals. In vitamin D deficiency, 24,25-(OH)zD levels are low or undetectable [171,172,180,182,186], as expected with low substrate levels. In dietary calcium-deficiency rickets, the characteristic pattern of serum vitamin D metabolites is normal or low normal levels of 25-OHD and elevated levels of 1,25(OH)zD. Normal levels of 25-OHD and the biochemical and radiological response to an improved calcium intake distinguish dietary calcium-deficiency from vitamin Ddeficiency rickets [187]. Mean serum concentrations of 25-OHD are reported to be lower than those of controls but within the normal range in children suffering from dietary calcium-deficiency rickets in a number of studies conducted in Nigeria, Bangladesh, and South Africa [109,111,113,114,116] (Table 6). Mean values are typically higher than 30 pmol/liter, which has been suggested as the lower limit of vitamin D sufficiency in children. In only one small study of 10 rachitic children in Nigeria was the mean 25-OHD level suggestive of vitamin D insufficiency [188]. The reason for the poorer vitamin D status in rachitic children than controls has not been well studied. In Nigeria, neither sociocultural factors nor lack of exposure to sunlight appear to be responsible [116,189]. A possible explanation may be the fact that 1,25-(OH)zD concentrations are higher in rachitic subjects than in controls (Table 6) and thus could be responsible for in-
creased catabolism of circulating 25-OHD with a resultant decrease in serum levels [88]. Support for this hypothesis derives from the results of a randomized controlled trial in which children with active rickets were treated for 6 months with vitamin D, calcium suppleanents, or both. In the group receiving calcium supplements alone (1000mg Ca daily), mean serum 25-OHD concentrations increased from 16 to 21 ng/ml, whereas 1,25-(OH)2D levels declined from 130 to 109 pg/ml [112], possibly indicating a decrease in 25-OHD catabolism associated with the decrease in 1,25-(OH)2D. As mentioned previously, serum 1,25-(OH)2D concentrations are typically elevated in children suffering from dietary calcium-deficiency rickets [111,113,114, 116,190]. Not only are the values two- or threefold higher than reference values derived from children in developed countries but also in the majority of studies they are significantly higher than those of age-matched controls from the same communities (Table 6). It is presumed that 1,25-(OH)2D levels increase in children with dietary calcium deficiency in response to the habitually low dietary calcium content (approximately 150-200 mg/day) and the consequent hypocalcemia, which would be expected to induce secondary hyperparathyroidism. PTH concentrations have been measured in a number of studies, and although mean levels are higher than control values and the manufacturers' reference ranges in the majority of studies, many rachitic children have PTH values within the normal range [111,113,116,161,188,190]. In these children, the stimulus for increasing 1,25-(OH)2D production is
TABLE 6 C o m p a r i s o n of rachitic a n d control s u b j e c t s in a Nigerian s t u d y of children with dietary calcium deficiency
(adapted from [1 16]). Rachitic subjects N=123
Variable
Control subjects N= 123
P value
Age (months)
46 (34,63) a
42 (25,70)
0.14
Age at stopping breast feeding (mo)
16.0 + 5.3 b
17.3 + 4.5
0.04
Daily calcium intake (mg)
217 + 88
214 + 77
0.6
Serum Ca (mmol/1)
1.93 + 0.22
2.24 + 0.15
<0.0001
Serum Pi (mmol/1)
1.67 + 0.61
1.92 + 0.59
Alkaline phosphatase (U/l)
707 (545,1021)
235 (196,278)
<0.0001
25-OHD (nmol/1)
32 (22,40)
50 (42,62)
<0.0001
1,25-(OH)eD (pmol/1)
322 + 96
278 + 91
0.0007
PTH (pmol/1)
20 (13,31)
12 (11,16)
0.006
a
median and
25 th
and 75th percentiles;
b
0.0017
mean + standard deviation
22. Nutritional Rickets unclear, as is why PTH values remain within the normal range despite persistent hypocalcemia. It is possible that PTH secretion is suppressed to a certain extent by the elevated 1,25-(OH)zD concentrations [191,192]; thus, PTH values would be expected to be lower in dietary calcium-deficiency rickets than those in vitamin D-deficiency rickets, but values well within the normal range are difficult to explain. Further studies are required to elucidate possible mechanisms.
Radiological Diagnosis of Rickets As discussed earlier, several different aspects make up the disease and manifest radiologically: (i) most prominent in children is the failure of endochondral calcification manifesting at the growth plates; (ii) the failure of or delay in mineralization of preformed osteoid at the sites of bone turnover and of intramembranous mineralization (osteomalacia); and (iii) the effects of secondary hyperparathyroidism. The most prominent feature of rickets is the defect in mineralization that occurs at the cartilaginous growth plate of growing bones. The earliest radiographic feature described by Goel and coworkers [82] is a loss of the welldemarcated zone of provisional calcification at the distal end of the metaphysis. Thus, the distinction between the unmineralized growth plate and the distal end of the calcified metaphysis becomes blurred. As the disease progresses, there is widening of the growth plate, as evidenced by an increase in distance between the distal end of the metaphysis and the proximal end of the epiphysis [193] (Fig. 4). At the wrist, the distance between the distal radial metaphysis and the radial epiphysis is never more than l mm in normal children [t94].
555
Widening of the growth plate is associated with lateral expansion of the plate as a result of weight bearing and stresses, and this becomes clinically visible as widening of the distal ends of the long bones and the costochondral junctions. Radiographically, these features manifest as soft tissue swellings at the distal end of the long bones and bulbous expansion of the anterior ends of the ribs. The distal metaphyses become cupped and splayed, and the junction between the growth plate and the metaphyses appears frayed with spur formation (Fig. 4). The epiphyses, if visible, are poorly developed, small, and osteopenic; thus, the bone age in children with calciopenic rickets is typically delayed. The radiographic features of rickets are best seen in rapidly growing long bones; thus, in the young child the distal radius and ulna are probably the best bones for diagnosing early rickets. In the older child, the bones around the knee are better suited for diagnosis because they grow rapidly. Although the appearance of the costochondral junctions has been used to diagnose rickets, early changes are often difficult to interpret and may lead to overdiagnosis of the disease unless suspicious features are confirmed by biochemical or other radiographic evidence [174]. Once long bone epiphyses fuse during adolescence, the diagnosis of rickets is more difficult. In children with rickets after puberty, the secondary ossification centers of the iliac crest and ischium, which appear at puberty and normally unite with the rest of bone between 15 and 25 years of age, may provide radiological evidence of rickets [195]. The appearance of the apophyses at the iliac crest has been likened to a pair of eyebrows. The radiographic features of osteomalacia develop more slowly than those of rickets and are less distinctive. The pathognomonic feature of osteomalacia is
FIGURE 4 Radiographicfeatures of vitamin D deficiency rickets in an 18-month-oldchild. (Top) The wrist shows marked widening of the growth plates at the distal radius and ulna with apparent soft tissue swelling.The distal metaphysesare splayed, cupped, and frayed. The developmentof the distal radial epiphysisis delayed. The trabecular structure of the metaphysesis coarsened and the cortices are ill defined. (Bottom)Radiographs of both knees in the same child showingsimilar features as those described for the upper limbs.
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the Looser's zone (pseudofracture), which is a translucent strip of unmineralized osteoid running perpendicularly through the bone cortex (Fig. 5). The classical sites at which Looser's zones may be found are the medial portion of the femoral neck, the pubic rami, the lateral border of the scapula, and the ribs [194]. The pathogenesis of Looser's zones is unclear, although they are thought to occur at sites of skeletal stress. Because a considerable amount of the surface of trabecular and cortical bone is covered in unmineralized osteoid, the bone will have an osteopenic appearance on radiographs. Deformities of long bones are typical of long-standing rickets and result from bending of the shafts of the softened osteomalacic long bones or deformation occurring at the widened and unmineralized growth plates. Deformities result from the stresses placed on bone by muscle attachments or by weight bearing; thus, the lower limbs are generally more severely affected than the upper limbs. Genu varum (bowlegs) tends to occur in younger children than does genu valgum (knock knees). Long-standing and severe rickets may also be associated with pelvic deformities (triradiate deformity) and protrusio acetabulae. The femoral neck may also be
FIGURE 5 A Looser's zone on the medial cortex of the femur in an adult with osteomalacia. Note the radiolucent band surrounded by sclerosis and thickening of the cortex. It is differentiated from a fracture by the fact that only one cortex is involved and there is lack of callus.
affected, with coxa vara being more common than coxa valga [196]. Greenstick and pathological fractures may occur as a result of osteopenia and softening of the bones. Vertebral compression fractures may also be noted, but the kyphoscoliosis seen in some children with severe rickets is more often due to muscle weakness rather than structural deformities. Features of secondary hyperparathyroidism are often subtle in children with nutritional rickets. The increase in bone turnover associated with elevated PTH levels aggravates the generalized osteopenia, which is apparent on radiographs. Specific features of secondary hyperparathyroidism, such as subperiosteal erosions on the radial border of the middle phalanges of the fingers, cortical erosions at the outer ends of the clavicles, at the symphysis pubis, and at the sacroiliac joints, and the development of the pepper pot appearance to the cranial vault, are much less commonly seen. Coarsening of the trabecular pattern at the ends of the long bones and cortical tunneling may be noted. Loss of the lamina dura around the teeth is frequently seen in older children with calciopenic rickets; however, the loss is not specific for hyperparathyroidism. There are no documented distinguishing radiographic features between rickets due to vitamin D deficiency and that due to dietary calcium deficiency because the pathogenesis of the bone disease is similar in both cases. Nevertheless, many of the radiographs of the hands of children diagnosed with dietary calcium deficiency have a loss of wasting of the metacarpals so that the metacarpals appear sausage shaped. It is postulated that the endosteal expansion is a manifestation of chronic secondary hyperparathyroidism. It may be useful in clinical trials to assess the severity of the radiographic changes of rickets objectively. A recently developed scoring system [197] for the changes at the wrist and knee is useful for assessing the response to therapy [112]. The degree of severity at the wrist is graded 1-4 and that at the knee is graded 1-6, giving a maximum score of 10 for a child with severe rickets (Fig. 6 and Table 7). Another scoring system has also been reported, which was used to assess response to therapy in children with calcium-deficiency rickets, but no details of the method were provided [198]. During the process of healing, the earliest sign of response to therapy is the development of a broad band of irregular, dense mineralization at the end of the metaphysis (Fig. 7). This is followed by a gradual filling in of the undermineralized metaphysis. Gradually, the coarse trabecular pattern at the metaphyses becomes finer and more numerous. Periosteal new bone formation may be seen along the shafts of the long bones as the unmineralized osteoid that had been laid down during growth gradually mineralizes. With appropriate treatment,
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22. Nutritional Rickets A
B
C
TABLE 7 Ten-point radiographic scoring m e t h o d for the assessment of the severity of rickets. (Reproduced with permission from [ 197]) Ten-point radiographic scoring method for rickets
Grade
1
0
2
WRIST"- score both radius and ulna separately
Grade 1
Widened growth plate, irregularity of the metaphyseal margin, but without concave cupping. Grade 2 Metaphyseal concavity with fraying of margins 2 bones x 2 points = 4 points possible Knee
Grade
0
1
2
3
FIGURE 6 Schematic drawing of the scoring system used to grade the severity of rickets at the wrist and knee. The stippled areas indicate poorly mineralized or completely unmineralized metaphyses. Arrows indicate areas of lucency at the medial or lateral aspects of the knee (reproduced with permission from Thacher et al. [197]).
KNEE"- score both femur and tibia separately Multiply the grade in A by the multiplier in B for each bone, then add the femur and tibia scores together. A: Grade Degree of lucency and widening of zone of provisional calcification 1 Partial lucency, smooth margin to metaphysis visible 2 Partial lucency, smooth margin to metaphysis not visible 3 Complete lucency, epiphysis appears widely separated from distal metaphysis B: Multiplier Portion of growth plate affected 0.5 < 1 condyle or plateau 1 2 condyles or plateaus 2 bones x 1 point x 3 points = 6 points possible Total: 10 points possible
ascore the worst knee and the worst wrist.
Treatment
and Prevention
As discussed earlier, the disease is caused by a spect r u m o f conditions, with vitamin D deficiency at one end o f the s p e c t r u m a n d dietary calcium deficiency at the other. In between the two extremes are varying c o m b i n ations o f relative vitamin D insufficiency a n d low dietary calcium intake.
Treatment
FIGURE 7 Radiograph of s wrist showing features of early healing. Note the irregular sclerotic band at the distal end of the metaphysis and periosteal new bone formation on the ulna.
r a d i o g r a p h i c evidence o f healing m a y be seen within 1 m o n t h , but complete healing o f the m e t a p h y s e a l and g r o w t h plate changes m a y take several m o r e m o n t h s . The long b o n e deformities m a y i m p r o v e gradually over several years as m o d e l i n g reshapes the bones to the m o r e n o r m a l stresses.
F o r m o r e t h a n a century, the beneficial effects o f sunlight for healing o f rickets have been k n o w n alt h o u g h n o t generally accepted. D e n t and c o w o r k e r s [199] showed that vitamin D-deficiency rickets in an Asian child could be very effectively cured by U V irradiation to the whole b o d y for 2 weeks. Similarly, significant i m p r o v e m e n t s in vitamin D status a n d biochemical abnormalities are seen during the s u m m e r m o n t h s in communities in areas of high latitude [86]. Holick [19] estimates that a y o u n g adult exposed to a whole b o d y dose o f sunlight, which causes minimal e r y t h e m a , receives an equivalent to an oral dose of 10,000 I U o f vitamin D3. Thus, children with vitamin D-deficiency
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rickets can be treated effectively by exposing them to adequate amounts of UV irradiation. However, vitamin D-deficiency rickets is probably more effectively managed by the oral administration of small doses of vitamin D2 or D3. There is little evidence to suggest that the response to either form of vitamin D differs; however, a study has shown that vitamin D3 is more effective than vitamin D2 in increasing 25-OHD concentrations [200]. Using small daily doses of vitamin D (200-450 IU), Stanbury et al. [180] showed in subjects with vitamin D deficiency that 1,25-(OH)2D levels increased to values approximately fivefold higher than normal within 7-10 days and that 25-OHD concentrations also increased during the same period. Furthermore, during the same period, serum calcium and PTH levels started to return to normal. Similar findings relating to 1,25-(OH)2D levels were reported in infants receiving 400 IU of vitamin D [201]. Recovery occurs more quickly with the use of larger doses, and it is recommended that doses of 5000-15,000 IU/day be used for a period of 4-8 weeks. Using these doses, serum calcium, phosphorus, and PTH values rapidly return to normal within 2 or 3 weeks [149], although 1,25-(OH)2D and alkaline phosphatase levels remain elevated for several months [149,171,180]. Many clinicians provide a calcium supplement of 500-1500mg/day (depending on the size of the child) during the initial stages of healing to ensure that the calcium intake is adequate to meet the demands of the skeleton [149,202]. Although this is probably unnecessary in children who have calcium intakes similar to the daily recommended intake or recommended daily allowance for age (600-1000mg/day), in children who are vegetarian or have low calcium intakes for other reasons, calcium supplementation is prudent. All children who have symptomatic hypocalemia should receive oral calcium supplements and, if necessary, be given calcium as calcium gluconate (1 or 2 ml/kg of a 10% solution) in a slow intravenous infusion. A single large dose of vitamin D (200,000-600,000 IU) given either orally or intramuscularly has been used to treat patients with active rickets in central Europe for many years. It is suggested that the intramuscular route is less effective than the oral route because the response is delayed when vitamin D is given intramuscularly [142]. An oral dose of 600,000 IU of vitamin D results in rapid improvement in the biochemical abnormalities within 4-7 days and radiographic evidence of healing within 2 weeks in the majority of patients [123,202]. The dose can be repeated after several months if an incomplete response to therapy is obtained. There is no evidence that vitamin D toxicity can occur when large doses such as those mentioned previously are used for the treatment of vitamin D deficiency. The previously
discussed method of treatment is considered by some authors [142,202] to have an advantage over treatment with smaller daily doses because compliance is ensured in the single-dose method, which is not the case in the daily dose regimen [59]. It has been customary to treat children with rickets in developing countries with vitamin D combined occasionally with calcium supplements. This is an acceptable policy in countries such as Tibet, China, and those of the Middle East and Persian Gulf, in which it is known that the pathogenesis of rickets is related to vitamin D deficiency rather than to lack of dietary calcium. However, this form of therapy is probably not ideal for patients older than infant and toddler ages suffering from rickets in countries such as Nigeria, Bangladesh, and South Africa, where dietary calcium deficiency appears to be common. In such situations, calcium supplements (1000mg/day) for 6 months are recommended [109,111,112,188,198]. In a randomized, controlled trial, the addition of vitamin D (600,000 IU given three times a month) to calcium supplements did not produce significantly better results with regard to the completeness or speed of response [112]. Vitamin D therapy alone, on the other hand, was significantly less effective, although an improvement in biochemical and radiological evidence of rickets did occur. Healing of active rickets due to presumed dietary calcium deficiency has also been achieved by the addition of readily available calcium-rich foods such as ground dried fish to the diet or by using lower doses of calcium supplements (500 mg/day) (T. D. Thacher, personal communication). However, no data are available to compare the efficacy of these treatments with those used in the randomized, controlled trail mentioned previously.
Prevention Effective and cheap means of preventing vitamin D deficiency exist, but nutritional rickets remains a public health problem in a number of countries for a number of reasons. One major reason in developed countries is that the individuals at risk are often members of minority groups that may not have integrated well into the larger community or that have dietary patterns that might be considered alternative by the majority of the population. In developing countries, rickets is often part of much broader problems of overcrowding, poverty, undernutrition, and poor primary health care facilities, thus making its eradication difficult because it may not be seen as a public health priority when there are so many other needs. Both the United Kingdom and the United States recently reassessed the dietary requirements of vitamin D. The United Kingdom continues to recommend a refer-
22. Nutritional Rickets
ence nutrient intake (RNI) of 8.5 lag/day (340 IU/day) for infants younger than 6 months of age and 7 lag/day (280 IU/day) for children between 6 months and 3 years of age [203]. The report notes that unless adequate sun exposure is ensured, many infants and young children will not maintain an adequate vitamin D status, and it supports the recommendation of an earlier Department of Health Report on Weaning and Weaning Diet that routine vitamin D supplementation provides an effective safety net for groups at risk. An RNI is not set for older children because adequate sunlight exposure rather than diet is the major source of vitamin D. In 1997, the Institute of Medicine in Washington, DC [204], recommended an adequate intake of 200 IU (5 lag)/day for infants and children 0-18 years of age. Few dietary sources contain adequate amounts of vitamin D for humans to maintain vitamin D sufficiency without a supplement, ingesting vitamin D-fortified foods such as infant milk formulas, or being exposed to UV irradiation. Thus, in countries or communities in which adequate UV exposure is difficult to achieve because of the latitude or because of social or religious customs or personal attributes, such as infirmity, increased melanin pigmentation, or overcrowding, vitamin D supplementation should be considered. The exclusively breast-fed infant is particularly at risk because of the low vitamin D content of breast milk and the child's dependence on being exposed to sunlight by the caregiver. In the United States, some clinicians recommend that all breast-fed infants be supplemented with 400 IU/day of vitamin D [69]; however, the American Academy of Pediatrics suggests that only at-risk groups (those infants whose mothers are vitamin D deficient or those infants not exposed to adequate sunlight) should be considered for supplementation [68]. Certainly, in countries in which vitamin D-deficiency rickets is common among breast-fed infants, such as China and Tibet, routine supplementation should be considered. Results of a study conducted in China showed that a vitamin D supplement of 400 IU/day in infants for 6 months after birth maintained serum 25-OHD levels in a more normal range than supplements of 100 or 200 IU/ day [173]. In most countries, infant milk formulas are fortified with 400 IU/liter of vitamin D when reconstituted. Therefore, if an infant drinks ~600 ml/day, he or she will receive more than 200 IU from this source alone. Thus, there is no reason for the artificially fed infant to be vitamin D supplemented; however, if given, a supplement of 400 IU would not cause vitamin D toxicity [166]. The best method of providing vitamin D supplement to those children who require it is controversial. As with any medication that requires a daily dose, long-term compliance may be a problem [59]. In a number of
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central European countries, stosstherapie has been used to prevent vitamin D deficiency in young children. The dose, usually 600,000IU, is administered every 3-5 months for the first 18 months of life. There is little information on the efficacy of this prevention strategy; however, one study found that 34% of infants developed hypercalcemia at some stage, leading the authors to suggest that this dosage was excessive and unsafe [205]. Since that time, further studies using intermittent large doses to prevent vitamin D deficiency have been conducted [206]. Researchers recommended that administration of 2.5 mg (100,000 IU) of vitamin D every 3 months from birth was the most efficacious way of preventing vitamin D deficiency. This form of therapy might be very appropriate if it is linked with an immunization program in countries in which vitamin D deficiency in infants remains a major public health problem (e.g., Tibet and Mongolia). In many developed countries, one or more foods are vitamin D fortified. For example, in the United States dairy milk and all infant formulas are fortified, but this does not address the prevalence of vitamin D-deficiency rickets in certain groups, such as vegetarians and African Americans. In the United Kingdom, margarine and other fat spreads and some cereals are fortified; however, this fortification does not prevent the high prevalence of vitamin D deficiency in the Asian community because the foods that are fortified do not make up a major component of the diet of Asians. In the 1970s, a study showed the effectiveness of fortifying chapatti flour at 6000 IU/kg [207] as a cheap means of preventing vitamin D deficiency in the Asian community. This fortification increased 25-OHD levels into the normal range and corrected biochemical abnormalities indicative of vitamin D deficiency; however, the concept never became policy. Although fortification may be an effective means of addressing widespread specific nutrient deficiencies in a community, the process needs to be carefully regulated to ensure that food manufacturers comply with the regulations, that the targeted communities actually utilize the fortified food, and that toxicity is not induced in some members of the community. The prevention of dietary calcium-deficiency rickets is a major problem because the disease occurs in developing countries and is associated with lack of food variety in the diet of families and particularly children suffering from the disease. The diet of these families is based on calcium-poor staples (maize, rice, cassava, yams, and plantain) and contains little or no dairy products. In many of the areas in which the disease occurs, dairy herds are not farmed and milk and other dairy products are not affordable for most families. Trials are currently under way in Nigeria to determine whether local calcium-rich foods (powdered whole fish or limestone)
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added to the diet will be culturally acceptable and reduce the incidence of rickets in young children (T. D. Thacher, personal communication). In South Africa, a daily calcium supplement of 500mg increased serum calcium values and lowered alkaline phosphatase values during a period of 3 months in children attending school in a community in which biochemical evidence of dietary calcium deficiency was prevalent [208].
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18. Reichel, H., Koeffler, H. P., and Norman, A. W. (1989). The role of the vitamin D endocrine system in health and disease. N. Engl. J. Med. 320, 980-991. 19. Holick, M. F. (1991). Photosynthesis, metabolism, and biologic actions of vitamin D. In Rickets (F. H. Glorieux, Ed.), pp. 1-20. Raven Press, New York. 20. Holick, M. F., MacLaughlin, J. A., Clark, M. B., Holick, S. A., Potts, J. T., Jr., Anderson, R. R., Blank, J. H., Parrish, J. A., and Elias, P. (1980). Photosynthesis of previtamin D3 in human skin and the physiologic consequences. Science 210, 203-205. 21. Holick, M. F., MacLaughlin, J. A., and Doppelt, S. H. (1981). Factors that influence the cutaneous photosynthesis of previtamin D3. Science 211, 590-593. 22. Pettifor, J. M., Moodley, G. P., Hough, F. S., Koch, H., Chen, T., Lu, Z., and Holick, M. F. (1996). The effect of season and latitude on in vitro vitamin D formation by sunlight in South Africa. S. Afr. Med. J. 86, 1270-1272. 23. Ladizesky, M., Lu, Z., Oliveri, B., Roman, N. S., Diaz, S., Holick, M. F., and Mautalen, C. (1995). Solar ultraviolet B radiation and photoproduction of vitamin D3 in central and southern areas of Argentina. J. Bone Miner. Res. 10, 545-549. 24. Salimpour, R. (1975). Rickets in Tehran. Arch. Dis. Child. 50, 63-66. 25. el Hag, A. I., and Karrar, Z. A. (1995). Nutritional vitamin D deficiency rickets in Sudanese children. Ann. Trop. Paediatr. 15, 69-76. 26. Molla, A. M., Badawi, M. H., al-Yaish, S., Sharma, P., and elSalam, R. S. (2000). Risk factors for nutritional rickets among children in Kuwait. Pediatr. Int. 42, 280-284. 27. Hillman, L. S., and Haddad, J. G. (1974). Human perinatal vitamin D metabolism 1: 25-Hydroxyvitamin D in maternal and cord blood. J. Pediatr. 84, 742-749. 28. Gertner, J. M., Glassman, M. S., Coutsan, D. R., and Goodman, D. B. P. (1980). Fetomaternal vitamin D relationships at term. J. Pediatr. 97, 637-640. 29. Rothberg, A. D., Pettifor, J. M., Cohen, D. F., Sonnendecker, E. W. W., and Ross, F. P. (1982). Maternal-infant vitamin D relationships during breast-feeding. J. Pediatr. 101, 500-503. 30. Roberts, C. C., Chan, G. M., Folland, D., Rayburn, C., and Jackson, R. (1981). Adequate bone mineralization in breast-fed infants. J. Pediatr. 99, 192-196. 31. Birkbeck, J. A., and Scott, H. F. (1980). 25-Hydroxycholecalciferol serum levels in breast-fed infants. Arch. Dis. Child. 50, 691-695. 32. Maxwell, J. P. (1934). Further studies in adult rickets (osteomalacia) and foetal rickets. Proc. R. Soc. Med. 28, 265-300. 33. Ford, J. A., Davidson, D. C., McIntosh, W. B., Fyfe, W. M., and Dunnigan, M. G. (1973). Neonatal rickets in Asian immigrant population. Br. Med. J. 2, 211-212. 34. Moncrieff, M., and Fadahunsi, T. O. (1974). Congenital rickets due to maternal vitamin D deficiency. Arch. Dis. Child. 49, 810-811. 35. Harris, R. S., and Bunker, J. W. M. (1939). Vitamin D potency of human breast milk. Am. J. Public Health 29, 744-747. 36. Lakdawala, D. R., and Widdowson, E. M. (1977). Vitamin D in human milk. Lancet 1, 167-168. 37. Asano, T., Hasegawa, T., Sukuzi, K., Masushige, S., Nose, T., and Sukuzi, T. (1981). Determination of vitamin D-sulfate in milk by high performance liquid chromatography. Nutr. Rep. Int. 24, 451-457. 38. Hollis, B. W., Roos, B. A., Draper, H. H., and Lambert, P. W. (1981). Occurrence of vitamin D sulfate in human milk whey. J. Nutr. 111, 384-390. 39. Hollis, B. W., Roos, B. A., Draper, H. H., and Lambert, P. W. (1981). Vitamin D and its metabolites in human and bovine milk. J. Nutr. 111, 1240-1248.
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162. Baroncelli, G. I., Bertelloni, S., Ceccarelli, C., Amato, V., and Saggese, G. (2000). Bone turnover in children with vitamin D deficiency rickets before and during treatment. Acta Paediatr. 89, 513-518. 163. Daniels, E. D., Pettifor, J. M., and Moodley, G. P. (2000). Serum osteocalcin has limited usefulness as a diagnostic marker for rickets. Eur. J. Pediatr. 159, 730-733. 164. Greig, F., Casas, J., and Castells, S. (1989). Changes in plasma osteocalcin concentrations during treatment of rickets. J. Pediatr. 114, 820-823. 165. Scariano, J. K., Vanderjagt, D. J., Thacher, T., Isichei, C. O., Hollis, B. W., and Glew, R. H. (1998). Calcium supplements increase the serum levels of crosslinked N-telopeptides of bone collagen and parathyroid hormone in rachitic Nigerian children. Clin. Biochem. 31, 421-427. 166. Vieth, R. (1999). Vitamin D supplementation, 25-hydroxyvitamin D concentrations, and safety. Am. J. Clin. Nutr. 69, 842-856. 167. Pettifor, J. M. (2000). What is the optimal 25 (OH)D level for bone in children? Vitamin D endocrine system: Structural, biological, genetic and clinical aspects. In 11th Workshop on Vitamin (D. A. W. Norman, R. Bouillon, and M. Thomasset, Eds.), pp. 903-907. Univ. of California Press, Riverside. 168. Heaney, R. P. (2000). Vitamin D: How much do we need, and how much is too much? Osteoporosis Int. 11, 553-555. 169. Haddad, J. G., and Chyu, K. J. (1971). Competitive protein binding radioassay for 25-hydroxycholecalciferol. J. Clin. EndocrinoL 33, 992-995. 170. Pettifor, J. M., Ross, F. P., Moodley, G. P., and Margo, G. (1978). Serum calcium, magnesium, phosphorus, alkaline phosphatase and 25-hydroxyvitamin D concentrations in a paediatric population. S. Afr. Med. J. 53, 751-754. 171. Markestad, T., Halvorsen, S., Seeger Halvorsen, K., Aksnes, L., and Aarskog, D. (1984). Plasma concentrations of vitamin D metabolites before and during treatment of vitamin D deficiency rickets in children. Acta Paediatr. Scand. 73, 225-231. 172. Garabedian, M., Vainsel, M., Mallet, E., Guillozo, H., Toppet, M., Grimberg, R., Nguyen, T. M., and Balsan, S. (1983). Circulating vitamin D metabolite concentrations in children with nutritional rickets. J. Pediatr. 103, 381-386. 173. Specker, B. L., Ho, M. L., Oestreich, A., Yin, T., Shui, Q., Chen, X., and Tsang, R. C. (1992). Prospective study of vitamin D supplementation and rickets in China. J. Pediatr. 120, 733-739. 174. Pettifor, J. M., Isdale, J. M., Sahakian, J., and Hansen, J. D. L. (1980). Diagnosis of subclinical rickets. Arch. Dis. Child. 55, 155-157. 175. Olivieri, M. B., Ladizesky, M., Mautalen, C. A., Alonso, A., and Martinez, L. (1993). Seasonal variations of 25 hydroxyvitamin D and parathyroid hormone in Ushuaia (Argentina), the southernmost city of the world. Bone Miner. 20, 99-108. 176. Guillemant, J., Cabrol, S., AUemandou, A., Peres, G., and Guillemant, S. (1995). Vitamin D-dependent seasonal variation of PTH in growing male adolescents. Bone 17, 513-516. 177. Guillemant, J., Le, H.-T., Maria, A., Allemandou, A., Peres, G., and Guillemant, S. (2001). Wintertime vitamin D deficiency in male adolescents: Effect on parathyroid function and response to vitamin D3 supplements. Osteoporosis Int. 12, 875-879. 178. Outila, T. A., Karkkainen, M. U., and Lamberg-Allardt, C. J. (2001). Vitamin D status affects serum parathyroid hormone concentrations during winter in female adolescents: Associations with forearm bone mineral density. Am. J. Clin. Nutr. 74, 206-210. 179. Docio, S., Riancho, J. A., Perez, A., Olmos, J. M., Amado, J. A., and Gonzales-Macias, J. (1998). Seasonal deficiency of vitamin D in children: A potential target for osteoporosis-preventing strategies? J. Bone Miner. Res. 13, 544-548.
180. Stanbury, S. W., Taylor, C. M., Lumb, G. A., Mawer, E. B., Berry, J., Hann, J., and Wallace, J. (1981). Formation of vitamin D metabolites following correction of human vitamin D deficiency: Observations in patients with nutritional osteomalacia. Miner. Electrolyte Metab. 5, 212-227. 181. Eastwood, J. B., de Wardener, H. E., Gray, R. W., and Lemann, J. R., Jr. (1979). Normal plasma-l,25-(OH)2_vitamin-D concentrations in nutritional osteomalacia. Lancet 1, 1377-1378. 182. Chesney, R. W., Zimmerman, J., Hamstra, A., DeLuca, H. F., and Mazess, R. B. (1981). Vitamin D metabolite concentrations in vitamin D deficiency. Am. J. Dis. Child. 135, 1025-1028. 183. Rosen, J. F., and Chesney, R. W. (1983). Circulating calcitriol concentrations in health and disease. J. Pediatr. 103, 1-17. 184. Rasmussen, H., Baron, R., Broadus, A., DeFronzo, R., Lang, R., and Horst, R. (1980). 1,25(OH)2D3 is not the only D metabolite involved in the pathogenesis of osteomalacia. Am. J. Med. 69, 360-368. 185. St.-Arnaud, R., and Glorieux, F. H. (1997). Vitamin D and bone development. In Vitamin D (D. Feldman, F. H. Glorieux, and J. W. Pike, Eds.), pp. 293-303. Academic Press, San Diego. 186. Nguyen, T. M., Guillozo, H., Garabedian, M., Mallet, E., and Balsan, S. (1979). Serum concentrations of 24,25-dihydroxyvitamin D in normal children and in children with rickets. Pediatr. Res. 13, 973-976. 187. Pettifor, J. M. (1991). Dietary calcium deficiency. In Rickets (F. H. Glorieux, Ed.), pp. 123-143. Raven Press, New York. 188. Thacher, T., Glew, R. H., Isichei, C., Lawson, J. O., Scariano, J. K., Hollis, B. W., and Vanderjagt, D. J. (1999). Rickets in Nigerian children: Response to calcium supplementation. J. Trop. Pediatr. 45, 202-207. 189. Akpede, G. O., Omotara, B. A., and Ambe, J. P. (1999). Rickets and deprivation: A Nigerian study. J. R. Soc. Health 119, 216-222. 190. Pettifor, J. M., Ross, F. P., Travers, R., Glorieux, F. H., and DeLuca, H. F. (1981). Dietary calcium deficiency: A syndrome associated with bone deformities and elevated serum 1,25-dihydroxyvitamin D concentrations. Metab. Bone Rel. Res. 2, 301-305. 191. Pocotte, S. L., Ehrenstein, G., and Fitzpatrick, L. A. (1991). Regulation of parathyroid hormone secretion. Endocr. Rev. 12, 291-301. 192. Marks, K. H., Kilav, R., Naveh-Many, T., and Silver, J. (1996). Calcium, phosphate, vitamin D, and the parathyroid. Pediatr. Nephrol. 10, 364-367. 193. Oestreich, A. E., and Ahmad, B. S. (1993). The periphysis and its effect on the metaphysis. II. Application to rickets and other abnormalities. Skeletal Radiol. 22, 115-119. 194. Steinbach, H. L., and Noetzli, M. (1964). Roentgen appearance of the skeleton in osteomalacia and rickets. Am. J. Roentgenol. 91, 955-972. 195. Hunter, G. J., Schneidau, A., Hunter, J. V., and Chapman, M. (1984). Rickets in adolescence. Clin. Radiol. 35, 419-421. 196. Adams, J. E. (1997). Radiology of rickets and osteomalacia. In Vitamin D (D. Feldman, F. H. Glorieux, and J. W. Pike, Eds.), pp. 619-642. Academic Press, San Diego. 197. Thacher, T. D., Fischer, P. R., Pettifor, J. M., Lawson, J. O., Manaster, B. J., and Reading, J. C. (2000). Radiographic scoring method for the assessment of the severity of nutritional rickets. J. Trop. Pediatr. 46, 132-139. 198. Oginni, L. M., Sharp, C. A., Worsfold, M., Badru, O. S., and Davie, M. W. (1999). Healing of rickets after calcium supplementation. Lancet 353, 296-297. 199. Dent, C. E., Round, J. M., Rowe, D. J. F., and Stamp, T. C. B. (1973). Effect of chapattis and ultraviolet irradiation on nutritional rickets in an Indian immigrant. Lancet 1, 1282-1284.
22. Nutritional Rickets 200. Trang, H. M., Cole, D. E. C., Rubin, L. A., Pierratos, A., Siu, S., and Vieth, R. (1998). Evidence that vitamin D3 increases serum 25-hydroxyvitamin D more efficiently than does vitamin D2. Am. J. Clin. Nutr. 68, 854-858. 201. Venkataraman, P., Tsang, R. C., Buckley, D. D., Ho, M., and Steichen, J. J. (1983). Elevation of serum 1,25 dihydroxyvitamin D in response to physiologic doses of vitamin D in vitamin D deficient infants. J. Pediatr. 103, 416-419. 202. Shah, B. R., and Finberg, L. (1994). Single-day therapy for nutritional vitamin D-deficiency rickets: A preferred method. J. Pediatr. 125, 487-490. 203. Department of Health (1998). Nutrition and Bone Health. Stationary Office, London. 204. Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine (1997). Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. National Academy Press, Washington, DC. 205. Markestad, T., Hesse, V., Siebenhuner, M., Jahreis, G., Aksnes, L., Plenert, W., and Aarskog, D. (1987). Intermittent high-dose vitamin D prophylaxis during infancy: Effect on vitamin D metabolites, calcium, and phosphorus. Am. J. Clin. Nutr. 46, 652-658. 206. Zeghoud, F., Ben-Mekhbi, H., Djeghri, N., and Garabedian, M. (1994). Vitamin D prophylaxis during infancy: Comparison of the long-term effects of three intermittent doses (15,5, or 2.5 mg) on 25-hydroxyvitamin D concentrations. Am. J. Clin. Nutr. 60, 393-396. 207. Pietrek, J., Preece, M. A., Windo, J., O'Riordan, J. L. H., Dunnigan, M. G., Mclntosh, W. B., and Ford, J. A. (1976). Prevention of vitamin-D deficiency in Asians. Lancet 1, 1145-1148.
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208. Pettifor, J. M., Ross, F. P., Moodley, G. P., and Shuenyane, E. (1981). The effect of dietary calcium supplementation on serum calcium, phosphorus and alkaline phosphatase concentrations in a rural black population. Am. J. Clin. Nutr. 34, 2187-2191. 209. Dixon, P. H., Christie, P. T., Wooding, C., Trump, D., Grieff, M., Holm, I., Gertner, J. M., Schmidtke, J., Shah, B., Shaw, N., et al. (1998). Mutational analysis of PHEX gene in X-linked hypophosphatemia. J. Clin. Endocrinol. Metab. 83, 3615-3623. 210. White, K. E., Evans, W. E., O'Riordan, J. L., Speer, M. C., Econs, M. J., Lorenz-Depiereux, B., Grabowski, M., Meitinger, T., and Strom, T. M. (2000). Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nature Genet. 26, 345-348. 211. Robertson, I. (1969). Survey of clinical rickets in the infant population in Cape Town 1967-1968. S. Afr. Med. J. 43, 1072-1076. 212. Zhou, H. (1991). Rickets in China. In Rickets (F. H. Glorieux, Ed.), pp. 253-261. Raven Press, New York. 213. Lapatsanis, P., Deliyanni, V., and Doxiadis, S. (1968). Vitamin D deficiency rickets in Greece. J. Pediatr. 73, 195-202. 214. Pfitzner, M. A., Thacher, T. D., Pettifor, J. M., Zoakah, A. I., Lawson, J. O., Isichei, C. O., and Fischer, P. R. (1998). Absence of vitamin D deficiency in young Nigerian children. J. Pediatr. 133, 740-744. 215. Pettifor, J. M., and Daniels, E. D. (1997). Vitamin D deficiency and nutritional rickets in children. In Vitamin D (D. Feldman, F. H. Glorieux, and J. W. Pike, Eds.), pp. 663-678. Academic Press, San Diego. 216. Arnaud, S. B., Arnaud, C. D., Bordier, P. J., Goldsmith, R. S., and Flueck, J. A. (1975). The interrelationships between vitamin D and parathyroid hormone in disorders of mineral metabolism in man. In Vitamin D and Problems of Uremic Bone Disease, pp. 397-416. de Gruyter, New York.
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1231 Metabolic Bone Disease of Prematurity NICK BISHOP* and MARY FEWTRELLt *Academic Unit of Child Health, University of Sheffield, Sheffield Childrens Hospital Western Bank, Sheffield, United Kingdom t M R C Childhood Nutrition Research Centre, Institute of Child Health, London, United Kingdom
INTRODUCTION
to provide adequate mineral substrate intake remain important. An infant born before 37 weeks of completed pregnancy is by definition preterm. With modern neonatal intensive care, survival is possible after as few as 23 weeks of gestation. These tiny infants present a major challenge in nutritional management since they are born during a period of extremely rapid fetal growth; the fetus normally trebles in weight between 24 and 36 weeks of gestation, gaining 15-20 g/kg/day. Many nutrients are laid down late in gestation so that preterm infants are born with low body stores. For example, body fat content increases from 1% of body weight at 20 weeks of gestation to 15% at term. Carbohydrate stores are also laid down relatively late, with an estimated 9 g at 33 weeks and 34 g at term [1]. These low reserves, combined with immature metabolic responses, have important consequences for the ability of preterm infants to adapt to postnatal life and withstand starvation. It has been calculated that a 1-kg infant can survive only 4 or 5 days of starvation compared to 1 month for a term infant and 3 months for a healthy adult.
Preterm infants are at risk of developing metabolic bone disease during the first weeks of life due to an inadequate supply of phosphorus and calcium. The disease is characterized by a sequence of events beginning with biochemical evidence of disturbed mineral metabolism and followed by reduced bone mineralization leading to abnormal bone remodeling and reduced linear growth. It is often asymptomatic, being detected biochemically or incidentally on X-ray films taken for other purposes. Many studies have demonstrated low bone mass in preterm infants during the neonatal period, with the lowest values in those receiving diets lowest in minerals, such as unsupplemented human milk. Some studies suggest that preterm infants show catch-up in bone mass during infancy and childhood. However, a randomized trial found higher bone mass at 5 years and higher bone formation at 9-12 years in children who received the lowest mineral and nutrient intakes during the neonatal period. Moreover, height at age 9-12 years was significantly lower in children with evidence of neonatal metabolic bone disease. Longer-term follow-up is required to examine the effect of metabolic bone disease on peak bone mass and bone turnover, which could have implications for the risk of osteoporosis later in life. Although modern neonates generally receive better nutrition and mineral intakes than those included in many of the published studies, they may be smaller, more preterm, and, importantly, receive corticosteroids that adversely affect short-term growth and mineralized bone mass accretion. Thus, despite many advances in neonatal care, measures
PediatricBone
IN UTERO MINERAL ACCRETION AND BONE GROWTH During fetal life, plasma levels of calcium and phosphorus are higher than those after delivery. Compared to the serum concentrations seen in adult life, the fetus also has increased levels of circulating calcitonin, with low levels of parathyroid hormone (PTH) and the active
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Copyright 2003, Elsevier Science (USA). All rights reserved.
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Nick Bishop and Mary Fewtrell
metabolites of vitamin D, 25-OHD and 1,25-D [2,3]. Of the active metabolites, 25-OHD crosses the placenta, whereas 1,25-D does not but is produced in the placenta. Umbilical cord 25-OHD concentration is thought to reflect the degree of maternal vitamin D sufficiency, with concentrations greater than 20ng/ml regarded as adequate [4,5]. At birth, :99% of body calcium and approximately 80% of phosphorus is in the skeleton. It is generally agreed that at least 80% of this skeletal mineral is deposited between 25 weeks of gestation and term, with estimated daily fetal accretion rates of 2.6-3.2 mmol/kg/ day for calcium and 2.1-2.5mmol/kg/day for phosphorus [6]. There is an early peak of accretion at 27 or 28 weeks of gestation, followed by a slight decline and then an exponential increase, with a second peak at 37 or 38 weeks of gestation (calculated from the data of Ziegler [7,8]; Fig. 1). Calcium and phosphorus are accreted in an almost constant molar ratio of 1.2-1.3:1 during the entire period. Preterm infants are thus born during an extremely rapid phase of mineral accretion and have low skeletal mineral stores at birth compared to term infants.
PHYSIOLOGICAL CHANGES IN MINERAL HOMEOSTASIS AT BIRTH Term Infant The supply of calcium and phosphorus halts abruptly at birth when the umbilical cord is cut. Whole blood ionized calcium declines rapidly, reaching a nadir of
Mineral accretion mmol/kg/day calcium
_
"---"0~
phosphate
I
20 FIGURE 1 life.
30 Gestation (weeks)
I
40
Accretion rates for calcium and phosphorus during fetal
1.0-1.2 mmol/liter by approximately 16 hr of age. The rapid decline in circulating calcium is thought to reflect continued incorporation of mineral substrate into bone in the face of reduced intake, with initially low levels of PTH and 1,25-D. Paradoxically, in the face of a declining calcium supply, plasma calcitonin increases rapidly after birth in infants. The mechanism responsible for this surge is unclear. In the adult, calcitonin is secreted in response to hypercalcemia and in response to elevated plasma gastrin concentrations. The infant surge in plasma calcitonin peaks at approximately 12 hr after birth and is greater in preterm infants than in term small for gestational age (SGA) infants [9-11]. The surge can be ameliorated in preterm infants by the provision of large supplements of calcium (2 mmol/kg/day) [12]. PTH concentrations increase immediately after birth as plasma calcium declines and increase severalfold during the first 24-48hr [13]. In experimental models, the initial response to infused PTH is rapid and does not require protein synthesis. There is an increase in osteoclastic metabolic activity within 30-90min of the commencement of infusion [14]. The subsequent response of bone to infused PTH is an increase in osteoclast numbers and activity. This secondary response is mediated through the increased expression of RANKligand by osteoblast lineage cells in response to both PTH and 1,25-D [15]. It is likely that in newborn infants similar mechanisms are employed to maintain calcium homeostasis. There is no indication that tumor necrosis factor-~ or interleukin-6 play any role in osteoclast activation in the immediate postnatal period. The recruitment, activation, and fusion of osteoclastic precursors, and their subsequent activity in response to osteoblastderived humoral factors, provide a mechanism to ensure that longer term calcium requirements are met. The prolonged neonatal hypocalcemia experienced by some infants appears to be in large part related to maternal vitamin D sufficiency [16], indicating the importance of having both PTH and 1,25-D available for calcium homeostasis in the immediate postnatal period. The result of the resorptive process is to produce calcium, phosphorus, and the breakdown products of bone matrix. Although the mechanism coupling bone resorption to new bone formation is unclear, the increase in osteoclastic activity stimulated by PTH following delivery should be matched by appropriate new bone formation. However, if mineral substrate supply is inadequate at this stage, net loss of bone mineral could begin to occur. In addition to the supply of adequate mineral substrate to normally functioning osteoblasts, a favorable local environment for bone mineralization is also crucial to the growth and remodeling of bone. Many factors
23. Metabolic Bone Disease of Prematurity
have been identified as influencing this process, but there is strong evidence for a principal role for matrix vesicles in determining the initiation and subsequent propagation of mineral crystallization [17]. Matrix vesicles are discrete sacs thought to be derived from the osteoblast cell membrane either by budding or during the process of osteoblast cell death. They are formed from a bilayered lipid membrane rich in phosphatase enzymes including alkaline phosphatase. They accumulate specifically at the growing front of bone and are seen in larger numbers in phosphorus-deficient states. Alkaline phosphatase, as its name implies, is a phosphatase enzyme only in an alkaline environment (pH 9 or 10). Anderson [17] hypothesized that at the physiological pH of the bone mineralization front, alkaline phosphatase functions as a transmembranous phosphotransferase, acting as a channel through which phosphate residues cleaved by other phosphatase enzymes in the matrix vesicle membrane can be taken up into the vesicle sap. The vesicles have a high content of phosphatidyl serine, which may trap calcium ions on the inner membrane of the vesicle. The influx of phosphate residues promoted by alkaline phosphatase, together with the high local concentration of trapped calcium ions, creates a supersaturated solution in which the formation of amorphous calcium-phosphate crystals can occur. Electron microscopy has shown the growth of such crystals on the inner surface of vesicles, with subsequent vesicular disruption as the ends of the crystal pierce the bilayered membrane. The crystals then seed into the fluid at the bone mineralization front and, given a sufficient quantity of mineral substrate, act as foci for further crystallization [18]. The rate of turnover of the vesicles with release of their membrane constituents therefore reflects the rate of initiation of crystallization. Laboratory studies using animal models have shown greater numbers of matrix vesicles in rachitic growth plates, supporting the concept that increased plasma alkaline phosphatase activity reflects increased matrix vesicle turnover in states of substrate or vitamin D deficiency [19]. Figure 2 shows how the processes described previously might converge at approximately the time of birth, and it provides a framework for what is known about metabolic bone disease in premature infants. P r e t e r m Infant The term infant has built up large stores of minerals during the last trimester and receives sufficient mineral substrate after delivery for the needs of normal growth and development to be satisfied. The preterm infant differs in the following ways, which render him or her susceptible to mineral deficiency and metabolic bone disease:
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9 Born during a phase of rapid growth and mineral accretion. 9 Poor early nutrition/mineral intake. 9 Frequently ill during the neonatal period (respiratory distress syndrome, sepsis, and necrotizing enterocolitis). Lack of movement due to sedation or paralysis associated with ventilatory support: Moyer-Mileur et al. [20] found that a daily 5-to 10-min program of passive limb exercise in very low-birth-weight preterm infants produced significant improvements in weight gain, forearm length, bone area, bone mineral content, and fat-free mass. 9 Frequent use of drugs that may alter bone mineralization (e.g., steroids and diuretics): Dexamethasone treatment (commonly used in preterm infants with chronic lung disease) is associated with a suppression of bone turnover, calcium absorption and retention, bone mineralization, and linear growth [21-25]. Although markers of bone turnover appear to return to normal values when treatment is stopped [24,25], the long-term effects of this early suppression are unknown.
METABOLIC BONE DISEASE Historical P e r s p e c t i v e The problems associated with mineral metabolism in preterm infants were in fact identified in the first half of the 20th century. Benjamin et al. [26] performed mineral balance studies in preterm infants and concluded that human milk was an insufficient source of minerals for this population; that phosphorus was the limiting factor for bone mineralization; and that its deficiency resulted in calcium wasting in the urine, with secondary calcium deficiency. Von Sydow predicted that preterm infants fed human milk without added phosphorus would develop rickets. He showed hypophosphatemia, hyperphosphatasia, and an increased incidence of radiological changes consistent with the diagnosis in infants receiving human as opposed to cow's milk. He also found that vitamin D supplementation did not influence these findings for either group and suggested that an increased supply of calcium and phosphorus might reduce the incidence of the disease. In 1957, Eek et al. [27] in Oslo, Norway, reported a detailed prospective study of 69 preterm infants weighing less than 2000 g randomized to one of three diets: human milk from the Oslo breast milk bank, cow's milk diluted with water with extra glucose, or human milk with a supplement of dried skimmed milk. Infants in the first two groups also received a protein supplement, and all infants received 800 IU/day of vitamin D and 25 mg/day of vitamin C.
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Nick Bishop and Mary Fewtrell l In utero:-
I
High serum calcium, phosphate, calcitonin Low serum PTH, 1,25 (OH)2 D High mineral accretion rate High, but diminishing, linear growth rate At birth:-
I
Continued linear growth and mineral accretion into bone
Loss of transplacental mineral supply
I Calcitonin surge I
I Hypoccaena] PTH production increased /
Bone effects of PTH
I
Renal effects ~ of PTH ~ ' ~ ~ ~ Urinary reabsorption ~ \ 41 of calcium Increased synthesis I " ) ( [ - . - - - - ~ of 1,25 vitamin D I / \1 Urinary excretion of phosphate
I Bone resorption ~
~~ ~
i
~
i
~.~
I Nutrient supply ....... I [ Mechanical stimuli
/
/
Increased intestinal calcium absorption
/"
Restoration of serum calcium levels
t
Humoral factors
Calcmm ' phosphate released from bone
i
88
New bone formation; remodelling, growth and mineralisation
[I S Mineralised bone mass accretion
FIGURE 2
Events influencing skeletal homeostasis at approximately the time of birth in term infants.
Mean plasma phosphorus was lowest in the infants fed human milk, highest in the group receiving cow's milk, and intermediate in the group receiving the modified human milk diet; the differences between the groups were statistically significant. Plasma alkaline phosphatase activity was also increased in the group fed human milk compared with the other two groups. The clinical observation of "considerable craniotabes" was made in 12 of the 21 infants receiving human milk and in none of the infants receiving cow's milk. No other clinical evidence of abnormal bone remodeling (significant wrist
swelling and rickety rosary) was reported. The mean length gain was 0.116cm/day for the group fed cow's milk versus 0.104cm/day for the group fed human milk, but the difference did not reach statistical significance. Radiological findings in each group (forearm and hand radiographs within 1 week of birth and then every 4 weeks) were also reported. Initially, there was metaphyseal rarefaction, with progressive radiological osteoporosis spreading from the metaphyseal zone toward the midpoint of the diaphysis. Then there was generalized
23. Metabolic Bone Disease of Prematurity
osteoporosis from the age of 10-13 weeks. Periosteal double contours were seen earlier in the infants fed either cow's milk or supplemented human milk than in those fed human milk alone (113 or 105 vs 210 days), and this was interpreted as the first sign of improved mineralization since such findings are also observed in cases of healing rickets. In 1971, Lewin [28] reported four cases of infants born at 27-32 weeks of gestation and weighing 680-1300 g who were fed formula thought to contain sufficient vitamin D for normal bone growth and mineralization. However, all four infants showed radiological evidence of rachitic changes 2-4 months postnatally. The mineral content of the milk (Enfalac liquid, Mead Johnson Laboratories) was not stated; the calculated vitamin D intakes for the infants averaged 100-150IU/day. The authors stated that supplemental vitamin D had not been given to these four infants because it was believed that the amount contained in the formula was sufficient to prevent rickets. They suggested that their normal policy of supplementing all infants with 400 IU would have prevented rickets in these infants. The authors did not, however, consider the role of the formula's mineral content in these cases. In 1974, Tulloch [29] reported two cases of"rickets" in preterm infants receiving vitamin-supplemented adapted cow's milk-based formula. Clinical signs--craniotabes and swollen costochondral junctions--were noted in both infants at 12-16 weeks of age, with osteoporosis of the skull vault and metaphyseal lucency and cupping, splaying, and fraying of the ends of the long bones. Both also had significantly increased plasma alkaline phosphatase activity. They were treated with large doses of vitamin D (5000 IU/day), and changes consistent with healing rickets were found on radiological investigation within 3 weeks, with a concomitant increase in plasma phosphate in one (but not the other) infant. The radiological changes observed in these early studies were often not clearly defined until the postnatal age of 12 or more weeks. The advent of single photon absorptiometry (SPA) adapted for use in this population was a major advancement allowing precise in vivo quantification of the loss of bone mineral during the first few weeks of postnatal life [30-32]. Photon absorptiometry became a widely used tool in this context and facilitated work directed not only at understanding the predisposing factors but also determining the most effective mode of treatment. Despite the early publications pointing to phosphorus deficiency as the most likely primary cause for bone disease in preterm infants, the pathogenesis of the condition was disputed during the 1980s, with calcium deficiency and vitamin D deficiency being favored by some as the primary etiological factors. Rowe et al. [33]
5 71
reported calcium and phosphorus balances in preterm infants receiving either human milk or standard infant formula, and they showed that the premature gut had adequate capacity to absorb ingested minerals, suggesting that dietary intake was the limiting factor rather than gut absorption. Subsequently, many investigators reported metabolic balance studies confirming that phosphorus and calcium are present in inadequate quantities in human milk to support optimal bone mineralization in growing preterm infants. At intakes of 180-200ml/kg/day, human milk provides only 21-28 mg/kg/day (0.7-0.9 mmol/kg/day) of phosphorus and 51-68 mg/kg/day (1.25-1.7 mmol/kg/day) of calcium compared to the daily requirement of up to 74 mg/kg/ day (2.4 mmol/kg/day) of phosphorus and up to 119 mg/ kg/day (3mmol/kg/day) of calcium to meet in utero accretion rates. It is therefore not surprising that preterm infants fed solely on human milk develop phosphorus and calcium deficits.
Clinical, Biochemical, and Radiological Features Metabolic bone disease in preterm infants is characterized by a sequence of events that begins with biochemical evidence of disturbed mineral metabolism, continues with reduced bone mineralization, and results in abnormal bone remodeling and reduced linear growth. Clinical signs of mineral deficiency, with rickets and/or fractures, are unusual and occur relatively late in the neonatal period, often being detected incidentally on X-ray films taken for other purposes. The initial manifestation of disturbed mineral metabolism is hypophosphatemia accompanied by hypophosphaturia with high tubular phosphate reabsorption. Hypercalciuria and hypercalcemia are frequently observed. Phosphate depletion can be monitored using serial urinary calcium:phosphate ratios. This test can be performed on single, untimed samples and the ratio should be less than 1 by 3 weeks of age if the infant is phosphate replete. A later (4-6 weeks of age) feature of disturbed mineral metabolism is increased plasma alkaline phosphatase; plasma concentrations more than five times the upper limit for adults are associated with reduced linear growth persisting up to 12 years [34]. Although all preterm infants are potentially at risk for mineral deficiency, the problem is greatest in the smallest, most immature infants, who are born with the largest mineral deficit. Frequently, signs of mineral deficiency do not develop early in life when the infant is sick and fails to grow. However, once the period of acute illness ends, growth accelerates and with it the likelihood of mineral deficiency unless an adequate mineral supply is ensured.
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Although historically plain radiographs have been used to assess the degree of osteopenia as well as search for evidence of fractures, this is an insensitive method for detecting signs of bone disease since up to 30% of skeletal mass may be lost before changes are apparent. The use of photon absorptiometry has permitted bone mineral content to be assessed in preterm infants receiving various dietary regimens and supplements and in fetuses of comparable gestational age. A large number of studies have demonstrated that preterm infants fed human milk or standard term infant formulas have lower bone mineralization rates than those fed formulas with higher mineral content or reference fetuses. Some of these studies are summarized in the following sections.
SKELETAL HEALTH IN PRETERM INFANTS Birth to Discharge from Hospital Linear Growth
Two studies have reported that preterm infants continue to grow in length during the first few weeks of life despite inadequate mineral intakes and poor bone mineralization [35,36]. Bone Mass
Most studies in which bone mass has been measured have used SPA. The majority of studies in the 1980s and early 1990s thus focused on the accretion of cortical bone. With the advent of dual-energy X-ray absorptiometry (DXA), there have been a number of reports of whole body bone mass accretion and changes in bone mass in the lumbar spine. It is important to remember, however, that the vertebrae are largely cartilaginous at birth, and that the changes occurring in the early neonatal period may reflect changes in the tissue composition of the structures being measured as well as accretion of mineralized bone. Many studies have documented low bone mass in preterm infants during the early neonatal period. Minton et al. [37] measured radial bone mineral content (BMC) using SPA in 42 term and 30 preterm appropriate for gestational age (AGA) infants. The preterm infants were fed standard term infant formula. Sequential measurements in the preterm infants showed that the postnatal increase in BMC was significantly lower than that expected in utero. Greer and McCormick [38] reported similar findings; BMC in preterm infants remained fairly static despite overall body and bone growth. James et al. [39] found low radial BMC in 17 preterm infants at the
equivalent of term, and the difference remained significant after adjusting for weight and length, suggesting that it was not simply a reflection of smaller body size. Lyon et al. [35] measured the BMC of the distal radius (mixed cortical and trabecular bone) weekly in 15 preterm infants of less than 30 weeks of gestation using an "in-house" DXA instrument with contemporaneous measurement of an aluminum step wedge. Mineral content increased during the first week of life but then decreased before increasing slowly from 6 weeks of age, and it was poor compared to that of a fetus at the equivalent postconceptual age. Measured mineral intakes in these infants were considerably lower than calculated intrauterine requirements. Pohlandt [40] measured the BMC of the humerus in 269 preterm infants at a median of 35 weeks postconceptual age and found a significantly lower ratio of BMC: body weight than in the reference fetus of the same body weight. Minton et al. [41] examined the effect of intrauterine growth retardation on BMC in both term and preterm infants and found that although term SGA infants had lower BMC at birth than term AGA infants, there was no difference between preterm SGA and AGA infants at birth. They suggested that retarded skeletal growth might only occur as a result of protracted intrauterine malnutrition. In contrast, Pohlandt and Mathers [42] found lower humeral BMC in both preterm and term SGA infants. However, these differences disappeared when related to birth weight rather than gestation, emphasizing the importance of relating bone mass to body size. Petersen et al. [43] also found lower whole body BMC in SGA term and preterm infants using dual photon absorptiometry. However, when expressed relative to body weight or length, the difference in BMC between preterm SGA and AGA infants was no longer apparent. Greer et al. [44] found that bone mineralization in low-birth-weight infants fed a term infant formula lagged significantly behind intrauterine mineralization rates [37], and that this was avoided by using a nutrient-enriched preterm infant [44] or by supplementing the formula with calcium, phosphorus, and vitamin D [45]. Pittard et al. [46] also found that preterm infants fed a preterm formula had a radial BMC at 8 and 16 weeks of age, which was within the limits of term infants of the same age. Chan et al. [47] studied radial BMC in 36 preterm infants (
23. Metabolic Bone Disease of Prematurity the in utero mineral accretion rate. BMC in infants fed human milk was significantly lower than that in the formula groups. Venkataramen and Blick [48] reported similar findings in a study comparing radial BMC in preterm infants fed unfortified human milk, fortified human milk, or preterm formula. Infants fed unfortified human milk had significantly lower BMC than those fed formula. The compositions of some of the milks available for use in preterm infants are shown in Table 1. Greer and McCormick [49] measured radial BMC during the first 6 weeks of life in preterm infants fed fortified human milk, unfortified human milk, preterm formula, or term formula. BMC was highest in the fortified human milk and preterm formula groups but remained below expected in utero values. Gross [50] compared the BMC of the humerus in 50 preterm infants with birth weight
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the three randomized groups, nor was there any effect of postdischarge diet. Pettifor et al. [51] compared radial BMC in very lowbirth-weight infants (1-1.5 kg) randomly assigned to receive unsupplemented human milk or human milk with a breast milk fortifier until they weighed 1.8 kg. At this point, infants in the fortifier group had significantly higher BMC. After this time, both groups of infants were breast-fed, and at 3 months of age (term equivalent) there was no significant difference in BMC between groups, suggesting that the unsupplemented human milk group had corrected its abnormalities. Lapillone et al. [52] measured whole body BMC in 25 very low-birth-weight infants ( < l . 5 k g at birth) using DXA. At the equivalent of term, whole body BMC was significantly lower than that of term infants at birth, although no adjustment was made in this study for body size. These infants received either a preterm infant formula or banked human milk supplemented with calcium, phosphorus, and magnesium; mean daily intakes of mineral in both groups were 100mg/kg/day calcium and 72 mg/kg/day phosphorus--higher than in the earlier studies. Wauben et al. [53] compared whole body BMC at the equivalent of term using DXA in three groups of preterm infants who received human milk supplemented with calcium and phosphorus (130mg/kg/day calcium and 104 mg/kg/day phosphorus), human milk with a multinutrient fortifier (108 mg/kg/day calcium and 101 mg/kg/ day phosphorus), or a preterm infant formula (102 mg/ kg/day calcium and 68mg/kg/day phosphorus). BMC was not significantly different between the groups and was within the normal range of term infants at birth
TABLE 1 Recommended nutrient intakes a and composition of milks used for preterm infants Recommended intake per kg per day
Energy kcal KJ Protein (g) Carbohydrate (g) Calcium (mg) Phosphorus (mg) Sodium (mg)
110-120 460-502 3.6-3.8 3.8-11.4 120-230 60-140 46-69
Nutrient content per 100 ml Mature human milk I
Preterm infant Formula 2
Term formula 3
Post-discharge formula4
70 293 1.3 7.4 35 15 15
80 336 2.0 7.7 110 63 42
68 284 1.45 6.96 39 27 17
72 301 1.85 7.24 70 35 22
aTsang RC et al. Nutritional needs of the preterm infant. Scientificbasis and practical guidelines. Caduceus Medical Publishers, New York, 1993, pp288-9 1DHSS Reports on Health and Social Subjects No.18. 1980:2 Based on Farley'sOsterPrem: 3 Based on Farley'sFirst Milk: 4 Based on Farley's Prem Care
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when expressed as absolute values and as a function of length or lean body mass. Faerk et al. [54] measured whole body mineral content in preterm infants at 36 weeks postconception and found no significant difference between those randomized to receive 200ml/kg/day of human milk with phosphate, fortified human milk, or a preterm formula. However, all infants had lower BMC (both absolute and per kilogram) than expected for a term infant at birth. Thus, there is general agreement that preterm infants fed diets low in minerals, such as unsupplemented human milk or term infant formula, have lower BMC than those who receive higher mineral intakes (either from fortified human milk or from preterm formula) or compared to fetuses of the same postconceptual age. However, many studies do not relate BMC to body size, so it is not clear whether low BMC is simply commensurate with the infants' smaller size or reflects genuine undermineralization. Preterm infants who receive adequate mineral intakes may be able to achieve the in utero rate of mineral accretion, although variable absorption of minerals, feeding difficulties, and intercurrent illness compromise the provision in many of the smallest infants. Growthretarded infants may have low BMC compared to AGA infants, although the differences frequently disappear when BMC is related to body size rather than gestation. Bone Turnover
A number of investigators have measured markers of bone turnover in preterm infants during the first weeks of life. The range of markers used parallels that in studies of older children and adults, with all the attendant problems. Of particular interest, colorimetric assays used for urinary creatinine estimations may be affected by urinary excretion of conjugated bilirubin and its photoisomers, as occurs in jaundiced infants receiving phototherapy. The picric acid method of creatinine estimation is not affected by bilirubin compounds. Tissue-nonspecific alkaline phosphatase (ALP), usually referred to simply as alkaline phosphatase, is the most widely reported marker. However, the interpretation of values varies as widely as that of the normal ranges quoted for individual assays. Due to the variety of buffer systems used in the kits and systems available, the upper limit of the normal range quoted for adults can vary from 130 to 950 IU/liter. Values are probably easiest to compare "across platforms" when reported as multiples of the upper limit of an assay's normal range. Detailed contemporaneous interpretation is more problematic because the value obtained at any one time is a composite of the modeling activity in the metaphyseal region close to the growth plate and the remodeling activity occurring in the diaphysis of individual bones.
At approximately the time of delivery, ALP activity is relatively low, implying that bone formation activity is also low; this is a surprising result given the rapidity of growth during pregnancy. It is tempting to speculate that lower ALP concentrations reflect normal modeling and remodeling activity, whereas higher concentrations indicate increased turnover of matrix vesicles. The increased turnover would imply that although mineral crystallization could still occur, crystal propagation in the face of diminished substrate supply could not. In studies undertaken in the early 1980s by Lucas and colleagues [55], ALP activity increased during the first 4-6 weeks of life and then plateaued. In infants receiving diets low in minerals (mothers' own expressed breast milk or donor breast milk, neither supplemented in any way), ALP then increased further. In a study by Beyers et al. [56], increased ALP and urinary hydroxyproline excretion were associated with endosteal resorption and a thinning of the humeral cortex as assessed by magnification radiogrammetry. Bhandari et al. [57] evaluated weekly changes from birth in bone-specific alkaline phosphatase activity (BSALP) and C-terminal propeptide of type I collagen (P1CP) as markers of bone formation and growth in 77 infants, 15 of whom also had measurements of osteocalcin (OC). P1CP decreased after birth in association with changes in weight, whereas BSALP increased. These changes did not correlate with changes in serum OC. In contrast, Crofton and colleagues [58] examined the relationships of BSALP, P1CP, N-terminal propeptide of type III procollagen (P3NP), C-terminal telopeptide of type I collagen (ICTP), urinary pyridinoline (Pyd), and deoxypyridinoline (Dpd) with rates of weight gain, length, lower leg length, and BMC measured weekly during the first 10 week of life in a detailed longitudinal study of 25 preterm infants. Each marker showed a distinctive pattern of postnatal change, with P1CP and P3NP increasing soon after birth, whereas ICTP decreased. Markers reached a plateau during weeks 4-10. After this plateau was reached, P3NP was positively correlated and Pyd and Dpd were negatively correlated with rate of weight gain; P3NP was also positively correlated with linear growth. P1CP was strongly correlated with total BMC attained by the end of the study period, and BSALP was positively correlated with the rate of bone mineral accretion. The authors concluded that P3NP was a good marker for overall ponderal and linear growth in preterm infants and that P1CP and BSALP could be regarded as surrogate markers for bone mineralization. This interpretation should be viewed in light of the mineral supply to the infants, which in this study was good. The authors used the data from the study to create standard deviation scores for each marker against which
23. Metabolic Bone Disease of Prematurity
to assess bone turnover in infants receiving the steroid dexamethasone for the treatment of chronic lung disease. The markers of collagen formation and degradation were all depressed by dexamethasone treatment in a dosedependent fashion, but the degree of depression varied widely between infants. Seibold-Weiger et al. [59] noted a significant gender difference in cord blood P1CP, with higher values in male infants. Frequent measurements generated a clear pattern of P1CP values with an initial decrease during the first 3 postnatal days followed by a rapid increase from day 7 to day 28. In addition to associations confirmed in other studies with birth weight and gestation, P1CP in this cohort was positively associated with cord blood insulin-like growth factor-1 concentrations. Shift et al. [60] studied OC, BSALP, P1CP, and ICTP in 20 preterm infants with a mean gestational age of 27 weeks for an average of 11 weeks postnatally. All three markers of osteoblastic activity increased significantly during the first 3 weeks of life and then had a more gradual increase until week 10. ICTP levels increased during the first week and then gradually decreased during follow-up. These results are consistent with increased bone formation in the first 3 months of postnatal life. Ng et al. [25] also reported a gradual increase in plasma BSALP and OC and in urinary DPD during the first weeks of life in preterm infants. Gfatter et al. [61] measured urinary excretion of Pyd, Dpd, and N-terminal cross-linked peptide in preterm and term infants during the first 2 months of life and found significantly higher concentrations of all three markers in preterm compared to term infants. Thus, there is general agreement that markers of both formation and resorption are significantly higher in preterm infants than in those born at term, that concentrations show an inverse relationship with gestational age and with birth weight, and that levels increase during the first few weeks of life. These findings suggest that preterm infants have high bone turnover early in postnatal life. The decreases in markers of bone formation and resorption reported in infants treated with dexamethasone for chronic lung disease appear to be transient, but longer term follow-up is required to substantiate this and to examine whether there are any long-term consequences for bone growth or turnover. Fractures
There are case reports of fractures occurring in preterm infants prior to discharge from hospital. These fractures may be of the ribs, possibly in association with physiotherapy, and at the ends of the long bones at the metaphyseal-diaphyseal junction. In this latter situation, the fractures have been associated with three
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point bending forces occurring during the insertion of peripheral or central venous access lines. There are no reports of metaphyseal corner or bucket-handle fractures, suggestive of twisting and pulling or shearing lateral forces occurring during this initial period of hospitalization. These observations fit with the model proposed by Beyers et al. [56], in whose study progressive cortical thinning rather than loss of trabecular bone was observed during longitudinal measurements of the cortical index of the humerus by magnification radiogrammetry. Endosteal resorption would not unduly weaken the bone until the "buckle ratio" of the cortical thickness to the radius of the bone approached 10:1. Further structural weakening, however, might occur in infants with persistently elevated serum PTH as a result of cortical erosions. Discharge to 2 Years Linear Growth
In a large randomized trial of diet during the early neonatal period in preterm infants, those who had biochemical evidence of metabolic bone disease during the neonatal period (more common in those randomized to receive unsupplemented human milk than a nutrientenriched preterm formula) were significantly shorter at 18 months corrected age after adjusting for other factors known to affect body length [55]. These findings suggest that metabolic bone disease might be associated with later linear stunting or that "programmed" changes in skeletal function may have taken place in association with early nutritional exposure to diets low in minerals, protein, and energy. Bone Mass
In contrast to the large number of studies on bone mineralization during the first few weeks of life prior to full-term, there are fewer reports on outcome during infancy. Salle et al. [62] measured lumber spine BMC in 49 preterm infants at 41,89,184, and 365 days and compared the results to those of 22 full-term infants of the same postnatal age. The authors reported a deficit in BMC in the preterm infants at 1 month of age that was partially corrected at 1 year (21% lower BMC at 1 year as opposed to 46% lower at 1 month). However, no account was taken of the smaller body size of the preterm children or the fact that ages were not corrected for the degree of prematurity. If the BMC results are expressed per kilogram body weight using the data provided by the authors, there is little difference between term and preterm children, particularly after 6 months of age. Koo et al. [63] studied 74 preterm infants weighing approximately 1 kg at birth. Radial BMC was measured
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at 5,14,26, and 40 weeks and 1 year of age. There was no significant difference in BMC between infants who had radiographic evidence of rickets and/or fractures and those who did not, although all infants had BMC values lower than the expected levels. No information was given on diet. Congdon et al. [64] compared radial BMC measurements in 15 preterm and 17 term infants. At the equivalent of term, preterm infants had significantly lower BMC than term infants. However, by 46-71 weeks postconceptual age (6-31 weeks postterm), there was no significant difference between term and preterm infants, suggesting that catch-up in bone mineralization had occurred over a relatively short period. All infants received term formula during this period. Other investigators have examined the influence of diet during the postdischarge period on bone mineralization. Chan and Mileur [65] studied 10 preterm infants fed human milk and 14 fed standard term formula after hospital discharge at 42,48, and 56 weeks postconceptual age (i.e., up to 16 weeks postterm). Breast-fed infants received their mother's milk with supplemental calcium while in the hospital, and formula-fed infants received preterm formula. Radial BMC was identical in the two groups at 42 weeks, but by 56 weeks postconceptual age it was significantly higher in the formula-fed group than in the breast-fed group, despite comparable weight and length gains. Serum calcium, phosphorus, and alkaline phosphatase concentrations were similar in both groups, and no infant had biochemical or clinical evidence of rickets. Abrams et al. [66] studied 17 very low-birth-weight infants during the first year of life and found significantly lower radial BMC at 10,16, and 25 weeks of age in those fed unfortified human milk as opposed to term formula during the initial posthospitalization period (weeks 10-25). After 25 weeks, infants were weaned onto solids and some of the breast-fed infants received formula milk. However, differences in BMC remained at 1 year of age, independent of current weight. The BMC for formulafed preterm infants was within the reference range for term infants, whereas that for the breast-fed infants was low. Breast-fed infants also had significantly lower serum phosphate and higher alkaline phosphatase than formula-fed infants, suggesting they were phosphorus deficient. At 2 years of age, however, BMC was similar in breast-fed and formula-fed groups and within the range reported for term infants in both groups. Thus, infants fed human milk during the neonatal period demonstrated catch-up in bone mineralization by the age of 2 years. Bishop et al. [67] also studied the effect of postdischarge nutrition. Thirty-one formula-fed preterm
infants were randomized to receive a standard term formula versus a nutrient-enriched postdischarge formula (PDF; intermediate in nutrient composition between term and preterm formula) from the time of hospital discharge until 9 months corrected age. Radial BMC was measured by SPA at 3 and 9 months corrected age. Infants fed PDF had significantly higher BMC at both 3 and 9 months, independent of body size (which was also higher in the formula group). Wauben et al. [68] reported that preterm infants who were breast-fed after discharge had significantly lower bone mass at 6 months postterm than infants fed a standard term formula, even after adjustment for body size. Brunton and colleagues [69] studied 60 preterm infants with bronchopulmonary dysplasia who were randomized to either nutrient-enriched formula or standard formula during the postdischarge period, measuring both radial and total body bone mass. At 3 months corrected age, infants fed enriched formula attained greater length, radial BMC, and lean mass. The male infants in the enriched-formula group had greater whole body BMC than did male infants in the standard-formula group. Collectively, these studies suggest that postdischarge nutrition in vulnerable infants might affect bone mass. However, with the exception of the study by Abrams et al. [66], they did not examine whether the effects persisted beyond the first year of life. Bone Turnover
In contrast to the many studies measuring markers of bone turnover during the period of hospitalization in preterm infants, there are no reports of bone turnover during the postdischarge period and infancy. This presumably relates to the relative difficulties of obtaining blood samples from infants in the community compared to those in hospital who undergo regular blood sampling for clinical purposes. Fractures
In Koo et al.'s prospective study [63], fractures did occur after discharge from hospital, but no further fractures appeared after 6 months corrected postnatal age. Amir et al. [70] reported clinically apparent fractures in 1.2% of preterm infants between days 24 and 160 of postnatal life. The true incidence of fractures in this population following hospital discharge will likely remain unknown. Rib fractures are difficult to detect clinically because single fractures may be splinted by adjoining ribs. The finding of apparently unexplained fractures, typically
2.3. Metabolic Bone Disease of Prematurity
seen on a chest X-ray taken during an episode of respiratory illness, will usually initiate child protection proceedings. The difficulty in prospectively identifying those infants at significant risk of fracture for pathological reasons is compounded by the knowledge that infants born prematurely are also at increased risk of abuse. Such cases require a careful dissection of the neonatal notes for clues suggesting the presence or absence of metabolic bone disease.
Two Years a n d O n w a r d s Linear Growth
Several studies have shown that preterm infants remain shorter than their term peers during childhood. In an analysis of factors predicting height at 9-12 years in children born preterm, Fewtrell et al. [34] found that the only factor from the neonatal period associated with childhood height was biochemical evidence of metabolic bone disease during the neonatal period. On average, children with evidence of this condition were 0.12 standard deviations shorter than those without it, suggesting that asymptomatic bone disease may be associated with stunting of linear growth for at least a decade. In a related analysis, children born preterm who had shown the greatest catch-up in height during childhood had the highest bone mass at 9-12 years of age [71], raising the possibility that one way of maximizing later bone mass in individuals born preterm is to maximize their linear growth during childhood. Bone Mass
Rubinacci et al. [72] measured radial BMC in 82 preterm children aged 2 months to 12 years and compared the results to previously published results from term children. BMC values for preterm children aged 2-5 months were lower than the reference range for term infants (although it appears that the preterm infants were measured at actual rather than corrected age, which would put them at a disadvantage), but values for the older preterm children were within the normal range. Nutrition during the neonatal period---either fortified human milk or preterm formula during the neonatal period followed by term formula or breast milk after discharge (details obtained from clinical notes and by maternal recall)mhad no effect on BMC. Hori et al. [73] measured lumbar spine BMC in 21 preterm children at 3 and 4 years of age using DXA and in 16 term children aged 4 years. Preterm infants were fed a mixture of human milk plus term formula (gestation >30 weeks) or preterm formula (gestation <30 weeks). Eleven
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of the preterm infants had previous BMC measurements at the equivalent of term that showed they were osteopenic. There were no significant differences in BMC or BMD between preterm and term children, either in absolute values or after expressing values per kilogram body weight. Helin et al. [74] measured radial BMC using SPA in 75 children aged 4-16 years who were born prematurely and in term children of the same age. BMC was significantly lower in preterm boys compared to term boys, but this difference disappeared after adjusting for weight and height, suggesting the low BMC was a reflection of the smaller body size in preterm boys. Kurl et al. [75] reported lumbar spine BMC and BMD measurements in 38 children at ages 6 and 7 years who were born preterm. All had received banked human milk supplemented with 100-105 mg/100 kcal calcium and 60-70mg/100kcal phosphorus during the neonatal period. After discharge, 27 infants were partially breastfed for a median of 5 months. Yearly data on growth were obtained from each child's health records. At 6 and 7 years, all children were within normal limits for height and weight, having demonstrated catch-up growth from the age of 2 years. Lumbar spine BMC was also within normal limits. Factors that independently predicted bone mass in this study were weight, bone area, gestational age, and weight at 1 year. Children who were lightest at 1 year of age had higher BMC at age 6 and 7 years, after adjusting for current weight. This suggests that preterm children who had the greatest increase in weight between 1 year and 6 or 7 years had the highest BMC and that childhood growth may affect later bone mineralization. Bowden et al. [76] measured bone mass using DXA in 46 ex-preterm infants at 8 years of age. BMC was significantly lower at all skeletal sites than that for age-matched term children; however, differences disappeared when adjusted for the smaller body size of the preterm group. Backstrom et al. [77] randomized 70 preterm infants to receive either 500 or 100 IU of vitamin D per day and calcium- and phosphorus-supplemented or unsupplemented breast milk. Although at 3 months of age mineral-supplemented infants had higher BMC, by ages 9-11 years no differences were seen between the groups. Bishop et al. [78], using SPA, studied 54 children at 5 years of age who were born preterm and randomly assigned to diet during the neonatal period. They had been randomized to either unsupplemented banked human milk (BBM) or a preterm infant formula (PTF) as a supplement to their mother's own expressed breast milk (EBM) during their time in the neonatal unit. Children previously fed BBM had significantly higher radial BMC than those from the PTF group at 5 years of age, both with and without adjustment for current body size.
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BMC also increased as the proportion of EBM in the infant's diet increased, with a significant interaction between randomized diet and the proportion of EBM, such that the effect of increasing amounts of EBM was greatest in infants who received PTF. Children from the BBM group also had significantly higher radial BMC for their body size than term children of the same age. Thus, children who received the lowest mineral intakes during the neonatal period paradoxically had higher BMC 4 years later. Two possible explanations were advanced for these findings. First, the low early mineral intakes might program bone cells to be conservative with minerals, resulting in overmineralization at a later stage when bone mineral intake is normal. Alternatively, one of the many growth factors or hormones in human milk might have a specific enhancing effect on later mineralization. Using DXA, Fewtrell et al. [79] studied 243 children 8-11 years of age who were from the same original preterm cohort. Compared to their term peers, these children had lower lumbar spine, hip, and whole body bone mass, although this was commensurate with their smaller body size. No effect of early randomized diet on bone mass was found at this age. Bone Turnover
Hori et al. [73] reported higher plasma osteocalcin concentrations in 3- and 4-year-old children born preterm compared to those born at term. In our large randomized trial [79], children randomized to the least adequate diets during the neonatal period (both in overall nutrients and in minerals) had significantly higher plasma osteocalcin at 9-11 years than those fed the more optimal preterm formula, suggesting that early diet may influence later bone formation. Urinary deoxypyridinoline concentrations were not significantly different between groups; nevertheless, it seems likely that the increased serum osteocalcin may represent increased bone turnover since bone mass was not increased. Fractures
Dahlenberg et al. [80] found no increase in the prevalence of prematurity in children presenting to a casualty department with fractures compared to children presenting without fractures, although there was a significant trend toward an earlier presentation of fractures (age younger than 2 years) in infants born at less than 32 weeks of gestation. Bowden et al. [76] found that fractures were less common by 8 years of age in a group of 46 ex-preterm infants than in age-matched peers. Fewtrell et al. [79] also found that similar proportions of children born term and preterm had sustained fractures by 12 years of age.
SUGGESTED GUIDELINES FOR THE PREVENTION OF METABOLIC BONE DISEASE IN PRETERM INFANTS The main factor to be addressed in the prevention of metabolic bone disease is the provision of an adequate supply of minerals, particularly phosphate. As with all facets of neonatology, the smallest, most immature, and sickest infants are at greatest risk of deficits. Minerals can be provided parenterally until enteral feeding is established. Organic phosphate solutions (sodium glycerophosphate) make the provision of adequate amounts of phosphate easier while avoiding solubility problems encountered with older solutions. Suitable parenteral intakes are 1.5-2.25 mmol/kg/day for both phosphorus and calcium. All enterally fed preterm infants should receive 2 mmol/kg/day of phosphorus. Human milk has a typical phosphorus content of 0.5mmol/100ml, so infants fed exclusively on human milk require a phosphorus supplement of approximately 1 mmol/kg/day when on full feeds. Phosphate depletion can be monitored by serial calcium:phosphate ratios. Once phosphate supplementation is adequate, a relative deficiency of calcium may become apparent. If both calcium and phosphate are added to human milk, the phosphate should be added first and left to stand for a few minutes before addition of calcium to avoid precipitation. Another alternative is the use of a multinutrient breast milk fortifier. Preterm infant formulas contain adequate calcium and phosphorus, and further supplements should not be necessary. All infants should receive 400 IU of vitamin D per day; there is no evidence that higher doses or the use of active metabolites of vitamin D confer any additional benefit. The optimal mineral intake of preterm infants after they are discharged from the hospital is less clear. Studies have shown improved linear growth in infants fed either preterm formulas or nutrient-enriched postdischarge formulas during this period, and this may partly reflect the higher mineral content of these diets compared to standard term formulas. Preterm infants who are breast-fed after discharge are likely to have the lowest mineral intakes and have been shown to have slower growth and lower rates of bone mineralization at least in the short term. The value of mineral or nutrient supplements in these infants after discharge has not been investigated and would pose practical problems. However, because most breast-fed preterm infants also receive some formula during the first few months, mineral intake could be increased by the use of a nutrientenriched postdischarge formula rather than a standard term formula.
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CONCLUSIONS Metabolic bone disease in infants born prematurely is characterized by a sequence of events that begins with biochemical evidence of disturbed mineral metabolism, continues with reduced bone mineralization, and results in abnormal bone remodeling and reduced linear growth. As with most disorders occurring during this time of life, those most at risk are the smallest, most immature infants. Clinical signs of mineral deficiency, with rickets and/or fractures, are unusual. Such features occur relatively late during the period of hospitalization, often detected incidentally on X-ray films taken for other purposes. Fractures may occur after hospital discharge; the circumstances surrounding the clinical presentation of such fractures require careful consideration and care proceedings may be necessary. Many studies have demonstrated low bone mass in preterm infants early in the neonatal period, with the worst results in those who receive the diets lowest in minerals (e.g., unsupplemented human milk). There is also evidence that diet during the postdischarge period influences bone mass at this time, with higher bone mass in infants receiving formula (particularly nutrient-enriched postdischarge formula)compared to human milk. In the longer term, significant effects of early diet on later bone mass and turnover have been shown in a randomized trial, namely, higher BMC at 5 years and higher bone formation at 9-12 years in children who received the lowest mineral intakes during the neonatal period. Linear growth in children assessed during this latter study was also influenced by the presence of metabolic bone disease (as indicated by serum alkaline phosphatase concentrations) during the period of initial hospitalization. Importantly, growth at age 9-12 years was still independently associated with evidence of early bone disease after adjusting for height at the age of 7 years. This suggests that it may not simply reflect a stunting effect of metabolic bone disease on early growth which then persists, but that there is a continuing or emerging influence resulting in reduced linear growth after 7 years. Although generally asymptomatic during early life, metabolic bone disease in preterm infants appears to have important sequelae persisting into midchildhood. Longer term follow-up is required to determine if there are effects on peak bone mass or bone turnover that might in turn have implications for the risk of osteoporosis later in life. Although modern neonates generally receive better nutrition and mineral intakes than those included in many of the published studies, they may be smaller, more preterm, and, importantly, receive corticosteroids that adversely affect short-term growth and bone mineralization. Thus, despite many advances in
neonatal care, measures to provide adequate mineral substrate intake remain important.
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23. Metabolic Bone Disease of Prematurity 54. Faerk, J., Petersen, S., Peitersen, B., and Michaelsen, K. F. (2000). Diet and bone mineral content at term in premature infants. Pediatr. Res. 47, 148-156. 55. Lucas, A., Brooke, O. G., Baker, B. A., Bishop, N., and Morley, R. (1989). High alkaline phosphatase activity and growth in preterm neonates. Arch. Dis. Child. 64, 902-909. 56. Beyers, N., Alheit, B., Taljaard, J. F., Hall, J. M., and Hough, S. F. (1994). High turnover osteopenia in preterm babies. Bone 15, 5-13. 57. Bhandari, V., Fall, P., Raisz, L., and Rowe, J. (1999). Potential biochemical growth markers in premature infants. Am. J. Perinatol. 16, 339-349. 58. Crofton, P. M., Shrivastata, A., Wade, J. C., Stephen, R., Kelnar, C. J., et al. (1999). Bone and collagen markers in preterm infants: Relationship with growth and bone mineral content over the first 10 weeks of life. Pediatr. Res. 46, 581-587. 59. Seibold-Weiger, K., Wollmann, H. A., Ranke, M. B., and Speer, C. P. (2000). Plasma concentrations of the carboxyterminal propeptide of type 1 procollagen (P1CP) in preterm neonates from birth to term. Pediatr. Res. 48, 104-108. 60. Shiff, Y., Eliakim, A., Shainkin-Kestenbaum, R., Arnon, S., et al. (2001). Measurements of bone turnover markers in premature infants. J. Paediatr. Endocrinol. Metab. 14, 389-395. 61. Gfatter, R., Braun, F., Herkner, K., Kohlross, C., and Hackl, P. (1997). Urinary excretion of pyridinium crosslinks and N-terminal crosslinked peptide in preterm and term infants. Int. J. Clin. Lab. Res. 27, 238-243. 62. Salle, B. L., Braillon, P., Glorieux, F. H., Poloniato, A., Cavero, E., and Meunier, P. J. (1996). Bone mineral content of the lumber spine in preterm infants: A longitudinal study during the first year of life. Pediatr. Res. 294A, 1750A. 63. Koo, W. W. K., Sherman, R., Succop, P., Oestreich, A. E., Tsang, R. C., Krug-Wispe, S. K., and Steichen, J. J. (1988). Sequential bone mineral content in small preterm infants with and without fractures and rickets. J. Bone Miner. Res. 3, 193-197. 64. Congdon, P. J., Horsman, A., Ryan, S. W., Truscott, J. G., and Durward, H. (1990). Spontaneous resolution of bone mineral depletion in preterm infants. Arch. Dis. Child. 65, 1038-1042. 65. Chan, G. M., and Mileur, L. J. (1985). Posthospitalization growth and bone mineral status of normal preterm infants. Feeding with mother's milk or standard formula. Am. J. Dis. Child. 139, 896-898. 66. Schanter, R. J., Burns, P. A., Abrams, S. A., Garza, C. (1992). Bone mineralization outcomes in human milk-fed preterm infants. Pediatr. Res. 31(6), 583-586. 67. Bishop, N. J., King, F. J., and Lucas, A. (1993). Increased bone mineral content of preterm infants fed with a nutrient enriched formula after discharge from hospital. Arch. Dis. Child. 68, 573-578.
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[24J Rickets Due to Hereditary Abnormalities of Vitamin D Synthesis or Action ANTHONY A. PORTALE and WALTER L. MILLER Department of Pediatrics, University of California San Francisco, San Francisco, California
intake of vitamin D and exposure to sunlight. Although 25-OHD is the most abundant form of vitamin D in the blood, it has minimal capacity to bind to the vitamin D receptor and elicit a biologic response. The active form of vitamin D, 1,25(OH)ED, is produced by the l~-hydroxylation of 25-OHD by the mitochondrial enzyme 25-hydroxyvitamin D lct-hydroxylase (lct-hydroxylase or P450clat) (Fig. 1). The circulating concentration of 1,25(OH)2D primarily reflects its synthesis in the kidney; however, l~-hydroxylase activity is also found in keratinocytes, macrophages, and osteoblasts [2-4]. The l ct-hydroxylation is the rate-limiting step in the bioactivation of vitamin D, and enzyme activity in the kidney is tightly regulated by parathyroid hormone (PTH), calcium, phosphorus, and 1,25(OH)ED [1]. Because of the importance of this enzyme in normal physiology and because synthesis of 1,25(OH)ED is impaired in chronic renal insufficiency, Fanconi syndrome, X-linked hypophosphatemic rickets, autosomal recessive vitamin D-dependent rickets type 1, and other disorders, the l~-hydroxylase has been the subject of intense study for approximately 30 years. The other important vitamin D-metabolizing enzyme, the 25-hydroxyvitamin D 24-hydroxylase (24hydroxylase or P450c24), is found in kidney, intestine, lymphocytes, fibroblasts, bone, skin, macrophages, and possibly other tissues [5]. The enzyme can catalyze the 24-hydroxylation of 25-OHD to 24,25(OH)ED and that of 1,25(OH)ED to 1,24,25(OH)3D; both reactions are thought to initiate the metabolic inactivation of vitamin D via the C 24 oxidation pathway. The kidney and intestine are major sites of hormonal inactivation of vitamin D by virtue of their abundant 24-hydroxylase activity.
INTRODUCTION As one of the principal hormonal regulators of calcium and phosphorus metabolism, 1,25-dihydroxyvitamin D [1,25(OH)ED] is critically important for normal growth and mineralization of bone. The classical actions of 1,25 (OH)ED are to stimulate calcium and phosphorus absorption from the intestine, thereby maintaining plasma concentrations of these ions at levels sufficient for normal growth and mineralization of bone. 1,25(OH)2D also has direct actions on bone, kidney, parathyroid gland, and many other tissues unrelated to mineral metabolism [1].
BIOSYNTHESIS OF VITAMIN D A detailed discussion of the metabolism and action of vitamin D is provided in Chapter 7. Briefly, vitamin D exists as either ergocalciferol (vitamin DE) produced by plants or cholecalciferol (vitamin D3) produced by animal tissues and by the action of near ultraviolet radiation (290-320nm) on 7-dehydrocholesterol in human skin. Both forms of vitamin D are biologically inactive prohormones that must undergo successive hydroxylations at carbons 25 and 1 before they can bind to and activate the vitamin D receptor (Fig. 1). The 25-hydroxylation of vitamin D occurs in the liver, catalyzed by one or more enzymes including the mitochondrial enzyme vitamin D 25-hydroxylase (P450c25). The activity of hepatic 25-hydroxylation is not under tight physiologic regulation, and thus circulating concentrations of 25-OHD are determined primarily by dietary
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Copyright 2003, Elsevier Science (USA). All rights reserved.
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Anthony A. Portale and Walter L. Miller
Cholecalciferol
~ c
25 .~ liver +
O ~ ~ ~ p 0(H
c1(~.~ kidney
r
HO
1,25(OH)=D
25OHD
HO
HO
P450c24X~ kidney
HO~
OH
OH
~P450c24 kidney H
OH
( 1),24,25(O H)2(3)D FIGURE 1 Biosynthesis of vitamin D3. Near ultraviolet light (290-320nm) cleaves the B ring of 7-dehydrocholesterol in the skin to yield cholecalciferol (vitamin D3). Vitamin D, which circulates in blood bound to 56-kDa vitamin D binding protein, undergoes 25-hydroxylation in the liver. The resulting 25-OHD, the most abundant form of vitamin D in the human circulation, can undergo l~-hydroxylation in the kidney by P450c10t to yield the active hormonal compound 1,25(OH)2D. Both 25-OHD and 1,25(OH)2D can also undergo 24-hydroxylation in the kidney by P450c24 to yield either 24,25(OH)2D or 1,24,25(OH)3D, respectively.
VITAMIN D BIOSYNTHETIC ENZYMES The vitamin D 25-hydroxylase, the l~-hydroxylase, and the 24-hydroxylase are mitochondrial, or type I, cytochrome P450 enzymes. Such enzymes are hemecontaining, mixed function oxidases that receive electrons from N A D P H via an electron-transfer chain consisting of two proteinsma flavoprotein termed ferredoxin reductase and an iron-sulfur protein termed ferredoxin. Molecular oxygen is the terminal electron acceptor. In contrast, type II P450 enzymes, such as the drug-metabolizing hepatic enzymes or the steroidogenic 21-hydroxylase and 17-hydroxylase enzymes, are found in the endoplasmic reticulum, and they receive electrons from a flavoprotein termed P450 oxidoreductase, sometimes with the allosteric assistance of cytochrome b5 [6].
sera raised against the purified protein to screen a rat liver cDNA expression library, yielding a P450c25 cDNA [7,8]. This enzyme can also hydroxylate carbons 26 and 27 of sterols to initiate bile acid synthesis, and it is often referred to as P450c27 or CYP27 [9]. There is evidence that a second, perhaps more physiologically relevant, vitamin D 25-hydroxylase exists in the endoplasmic reticulum, although the gene encoding such an enzyme has not been cloned [10]. The 24-hydroxylase (P450c24 or CYP24) was the second vitamin Dmetabolizing enzyme to be cloned, again by purifying the protein from rat renal mitochondria, raising a polyclonal antiserum, and screening a rat kidney cDNA expression library [11]. Subsequently, the human cDNA [12] and gene [13] were cloned. Studies with the purified rat kidney enzyme [11] and with cells expressing the human P450c24 cDNA [12] confirmed that this enzyme can catalyze the 24-hydroxylation of both 25-OHD and 1,25(OH)2D.
VITAMIN D 25-HYDROXYLASE AND 24-HYDROXYLASE VITAMIN D I e~-HYDROXYLASE The hepatic 25-hydroxylase was the first of the three vitamin D-metabolizing enzymes to be cloned. This was accomplished by purifying vitamin D 25-hydroxylase from rat liver mitochondria and using polyclonal anti-
Despite the cloning of the 25- and 24-hydroxylases in 1990 and 1991, it was not until 1997 that four groups of investigators working independently and using different
24. Rickets Due to Hereditary Abnormalities
approaches reported the cloning of the human, rat, and mouse vitamin D l~-hydroxylase cDNAs [14-18] and subsequently the human gene P450c1~ (CYP27B1) [15,19]. Efforts to purify the 10~-hydroxylase enzyme had been unsuccessful primarily because there is very little of this protein in renal mitochondria. Hence, the immunologic approaches used to clone the 24- and 25-hydroxylase enzymes could not be used. The group of Portale and Miller [14] approached the problem of low renal abundance of P450c1~ by using a different tissue system--primary cultures of human keratinocytes. These cells, when grown in serum-free medium in the presence of low concentrations of calcium, exhibit substantial l~-hydroxylase activity [2]. Using keratinocytes as a source of RNA enriched for P450c1~ mRNA, degenerate-sequence oligonucleotides were prepared based on the relatively well-conserved sequences of the ferredoxin binding sites and heme binding sites of P450c24 and P450c25 and were used for polymerase chain reaction (PCR) amplification of the keratinocyte cDNA. The resulting 300-bp PCR product was then cloned and sequenced, yielding a partial-length candidate clone for P450c1~. This was used to screen a keratinocyte cDNA library yielding a partial-length, 1.9kb cDNA, whose complete sequence was obtained by rapid amplification of cDNA ends (Y-RACE) [14]. The human P450c1~ cDNA is 2.4 kb in length, encodes a protein of 508 amino acids, and has a predicted molecular mass of 56 kDa [14]. Only six mitochondrial P450 enzymes have been identified to date, three of which are involved in the metabolism of vitamin D; the other three are P450scc, the cholesterol side chain cleavage enzyme, and P450c11, the steroid 11 [3-hydroxylase, and its isozyme P450cl 1AS, the aldosterone synthase. Within the hemebinding region of the P450c1~ enzyme, the predicted amino acid sequence identity is 65-73% of that of human P450c25 and P450c24; however, its overall identity to that of the other mitochondrial P450 enzymes is limited (30-39%) [14]. Although the P450cl ~ cDNA was cloned from human keratinocytes, four lines of evidence demonstrated that the keratinocyte and renal P450c 1~ enzymes are encoded by the same gene [14]. First, when the keratinocyte P450c10~ cDNA was transfected into mouse Leydig MA-10 cells, the transfected cells catalyzed the conversion of 25-OHD3 to authentic 1,25(OH)2D3, as determined by high-pressure liquid chromatography and confirmed by gas chromatography/mass spectrometry of the l~-hydroxylated product. Second, the cloned P450cl ~ had a Km for 25-OHD of 2.7 x 10 .7 M, which closely approximates the concentration of 25-OHD found in vivo. Third, reverse-transcription polymerase chain reaction (RT/PCR) was used to show that those
585
sequences that were cloned from keratinocytes were also expressed in human kidney. Finally, keratinocytes were obtained from a patient with vitamin D-dependent rickets type 1 (VDDR-1) and found to be devoid of 1~hydroxylase activity. Analysis of cloned cDNA and genomic DNA revealed that the patient was a compound heterozygote for two deletion/frameshift mutations that resulted in premature truncation of the protein. Thus, these findings provided genetic proof of the identify of the P450cl ~ and the first proof that VDDR-1 is caused by mutations in the vitamin D 1~-hydroxylase gene [14]. Soon after the P450c1~ cDNAs were cloned, the human gene was cloned [15,19], localized to chromosome 12 by somatic cell hybrid analysis [19], and mapped to 12q13.1-13.3 by fluorescence /n situ hybridization (FISH) [16,20,21]. The human gene for l~-hydroxylase is only 5 kbs in length, is single copy, and comprises nine exons and eight introns (Fig. 2) [19]. Although it is substantially smaller than the genes for other mitochondrial P450 enzymes, its intron/exon organization is very similar, especially to that of P450scc [19]. This strongly suggests that although the mitochondrial P450 enzymes retain only 30-40% amino acid sequence identity with each other, they all belong to a single evolutionary lineage. The mouse P450c1~ gene has also been cloned [22,23].
RICKETS DUE TO ABNORMALITIES OF VITAMIN D METABOLISM I a - H y d r o x y l a s e Deficiency In 1961, Prader et al. [24] described a new form of rickets that differed from the hypophosphatemic rickets first reported by Albright et al. in 1937 [25] by its onset within the first year of life, the presence of severe hypocalcemia but only moderate hypophosphatemia, and reversal of the clinical and laboratory findings of rickets by daily administration of high doses of vitamin D. Initially termed hereditary pseudo-vitamin D-deficiency rickets (PDDR) [24], vitamin D dependency because of its responsiveness to vitamin D [26], or vitamin D-dependent rickets type I, this disease is now known to be caused by defective renal conversion of 25-OHD to 1,25-(OH2)D as a consequence of loss-of-function mutations in the renal 10~-hydroxylase gene P450c1~ [14]. Here, we refer to this form of rickets as vitamin D 1~-hydroxylase deficiency. Clinical Features Patients with l~-hydroxylase deficiency usually are normal at birth but come to medical attention within the first 24 months of life, most commonly because of
586
AnthonyA. Portale and Walter L. Miller 3391 7bp dup I 3396 2bp dup
IVS3+lg--+a gggcg ~cttcgg
I
I s323Y
1921AG 1984z~C I R335P
R107H I G125E
IIT409' IIIi R429P W433X R453C mmmm
mmmm
,oo .
212AG
IVS2+lg~a
i
1 kb
i
i}1,E189o NE189G I,,T32,. I! P382S
I
I E189K D164N P143L
R389C R389G R389H
],v4780 P497R
i
FIGURE 2 Scalediagram of the intron/exonorganization of the human P450c10~gene as reported by Fu et al. [19]. All mutationscausing l~-hydroxylasedeficiencyreported through mid-2002are shown.
growth retardation, poor gross motor development, or generalized muscle weakness. Some infants are irritable when held, presumably due to bone pain, or develop pneumonia or seizures. Physical findings are similar to those observed in rickets due to simple vitamin D deficiency and include enlargement of the costochondral junction of the ribs (rachitic rosary), enlargement of the wrists or ankles, genu varus, and, in some cases, hypotonia, frontal bossing, enlarged sutures and fontanelles, or craniotabes (softening of the parietooccipital area). Muscle traction on the softened rib cage can give rise to thoracic deformity, including pectus carinatum. Dental development is often affected, with delayed eruption, enamel hypoplasia, and early caries. Radiographic examination of the long bones reveals the typical abnormalities of rickets, with widening of the metaphysis; fraying, cupping, and widening of the zone of provisional calcification; and diffuse demineralization. Radiographs of the chest may reveal enlargement of the costochondral junctions. Older children may exhibit bowing of the tibia and femur. Laboratory Features In most patients, hypocalcemia, hypophosphatemia, and increased serum alkaline phosphatase activity and PTH concentrations are observed [24,27-33], as is typical of patients with vitamin D-deficiency rickets. When severe, hypocalcemia can cause tetany and seizures. Metabolic balance studies in patients with l~-hydroxylase deficiency reveal malabsorption of calcium and phosphorus and reduced urinary calcium excretion
[28,29,32,34]. The renal tubular abnormalities of hyperchloremic metabolic acidosis and generalized hyperaminoaciduria are observed in some patients [24,27,29-32], findings also seen in patients with vitamin D deficiency [35]. The hallmarks of l ct-hydroxylase deficiency are greatly reduced serum concentrations of 1,25(OH)2D despite normal concentrations of 25-OHD and the reversal of clinical and laboratory abnormalities by administration of physiologic amounts of 1,25(OH)2D3 [33, 36,37]. These findings support the hypothesis of Fraser e t al. [36] that the genetic defect results from defective renal conversion of 25-OHD to 1,25(OH)2D, and they serve to distinguish patients with 10t-hydroxylase deficiency from those with nutritional vitamin D deficiency, in whom serum levels of 25-OHD are reduced, or hereditary 1,25(OH)2D-resistant rickets (vitamin D-dependent rickets type II), in whom serum levels of 1,25(OH)2D are greatly increased. In some patients with 10t-hydroxylase deficiency, serum concentrations of 1,25(OH)2D are nominally within the normal range [34,38], although such values are inappropriately low given the reduced serum concentrations of calcium and phosphorus and increased concentrations of PTH, all of which should increase renal production of 1,25(OH)2D. Indeed, administration of parathyroid extract failed to increase the serum concentration of 1,25(OH)2D in a child with l~-hydroxylase deficiency, in contrast to the increase induced in control subjects [39]. Serum concentrations of 24,25(OH)2D are normal in patients with l~-hydroxylase deficiency [34,39,40], indicating that the 24-hydroxylase enzyme is intact.
24. Rickets Due to Hereditary Abnormalities
587
Molecular Genetics of 1oL-Hydroxylase Deficiency The hereditary nature of l~-hydroxylase deficiency was observed by Prader et al. in their initial description of the disease [24], with autosomal recessive inheritance reported soon thereafter [27,30,31]. 1~-hydroxylase deficiency is rare in most populations, but it is particularly common among French Canadians, with an apparent carrier rate of 1/26 in the Charlevoix-Saguenay-Lac Saint Jean area of Quebec [41]. Using linkage analysis in French Canadian families with l~-hydroxylase deficiency, the disease was mapped to a region in band 14 of the long arm of chromosome 12 (12q14) [42]. Microsatellite haplotype analysis of 32 affected families mapped the gene proximal to D12S312 and distal to D12S305, D12S104 [43]. It was suggested that analysis of haplotypes (groups of tightly linked markers that segregate together over generations) using the markers D12S90, D12S305, and D12S104 could distinguish the allelic contribution of various founder populations, and that patients from the Charlevoix-Saguenay-Lac Saint Jean region of Quebec and those from eastern Canada (Acadia) derived from independent founder effects [43]. With the cloning of the l~-hydroxylase gene, the molecular genetics of l~-hydroxylase deficiency have now been studied thoroughly by several groups [14,20,21,38,44M6]. Gene localization via human/rodent somatic cell hybrids [19] and FISH [16,20] revealed that the gene maps to 12ql 3.3, consistent with the linkage analysis of Labuda et al. [43] and with the autosomal recessive inheritance of the disease. The first mutation in the l~-hydroxylase gene was identified by Fu and colleagues [14] in a Caucasian American girl with 1~-hydroxylase deficiency who was a compound heterozygote for two deletion/frameshift mutations in exon 2 of the gene that predicted premature truncation of the protein (Fig. 3A). Cultured skin keratinocytes from the patient were devoid of l~-hydroxylase activity (Fig. 3B). Subsequently, four unrelated Japanese patients studied by Kitanaka and colleagues [20] confirmed that mutations in the l~-hydroxylase gene could cause the clinical syndrome of l~-hydroxylase deficiency. However, several questions remained. It was not yet known whether all patients with the typical clinical syndrome of 1~-hydroxylase deficiency had the same disease. Furthermore, although it was established that l~-hydroxylase deficiency was common in French Canada, it was not known whether such patients had two distinct mutations, as had been suggested by linkage analysis [43]. Finally, there was no information relating enzyme structure to its function. Wang et al. [38] studied the P450c1~ genes of 19 patients with the clinical syndrome of l~-hydroxylase
FIGURE 3 Mutation of P450c10t causes 10t-hydroxylase deficiency. (A) Keratinocytes from a healthy person and from a patient with l u-hydroxylase deficiency were used to prepare mRNA, which was reverse transcribed, and the P450c1~ cDNA was PCR amplified using specific primers. (Top) Normal (left) and patient (right) cDNA sequences in the region of codon 211 showing that the G (arrow) in the normal sequence is deleted in the patient. (Bottom) cDNA sequence in the region of codon 211 showing that the normal C (arrow) is deleted in the patient. Thus, the patient was a compound heterozygote for two deletion/frameshift mutations. (B) 10c-Hydroxylase activity in keratinocytes from human neonatal foreskin (N), adult skin (Ad), and skin from a patient with 1~-hydroxylase deficiency (Pt). No activity was detected in the patient. The scale is logarithmic and begins at the level of detection of the assay.
deficiency from 17 families representing multiple ethnic groups, including 5 French Canadian families, 3 Polish families, 4 Caucasian American families, 1 Filipino family, 1 Chinese family, 1 Hatian family, 1 African American family, and 1 Hispanic family [38]. All of the patients were healthy at birth but came to medical attention within the first 24 months of life, most commonly because of growth retardation or poor gross motor development. All patients had typical laboratory findings of l~-hydroxylase deficiency: hypocalcemia, hypophosphatemia, increased serum concentrations of alkaline phosphatase and PTH, normal serum concentrations of 25-OHD, and low or undetectable concentrations of 1,25 (OH)2D. All patients had radiographic evidence of rickets and all responded to physiologic replacement doses of 1,25(OH)2D3. For each family, the parental origin of all P450c1~ mutations was identified and the mutations were correlated with the microsatellite haplotyping of chromosome 12q 13 using the markers D 12S90, D12S305, and D12S104.
588
Anthony A. Portale and Walter L. Miller
As noted previously, Labuda et al. [43] examined the microsatellite genetic markers on chromosome 12 and observed that the French Canadian patients with 1~-hydroxylase deficiency carried one of two haplotypes. Patients from the Charlevoix region of Quebec carried haplotype 4-7-1, whereas patients from eastern Canada (the Acadian population) carried haplotype 6-7-2. Among the five French Canadian families studied by Wang et al. [38], 9 of 10 unique alleles carried the 4-7-1 haplotype, and all 9 of these carried the identical mutation in codon 88, the deletion of guanosine at position 958 (AG958), which changes the reading frame and leads to premature termination of translation. The resultant protein would be predicted to have no enzyme activity. Thus, the finding that haplotype 4-7-1 is strongly associated with the AG958 mutation identifies it as the Charlevoix mutation. This mutation deletes the G in the sequence Y-ACGT-3 I, which is normally recognized by the endonucleases Tai I and M a e II. This feature was used to design a rapid, accurate PCR-based diagnostic test that can detect this mutation in genomic D N A from any source (Fig. 4) [38].
Yoshida et al. [21] characterized the P450c1~ genes of four French Canadian patients and found three to be homozygous for the AG958 mutation and one homozygous for the duplication of a 7-bp sequence in exon 8. Based on the geographic origins of each patient, Yoshida et al. suggested that mutation AG958 is the Charlevoix mutation and that the 7-bp duplication is the Acadian mutation, but they did not perform microsatellite haplotyping to confirm this. Wang et al. [38] found this 7-bp duplication, upstream from codon 441, on seven separate alleles in six families (Fig. 5). Four of these alleles carried the haplotype 9-7-2 but were found in different ethnic groups: Polish, Chinese, and Hispanic. The other three alleles bearing the 7-bp duplication carried the haplotypes 9-6-2, 9-3-3, and 6-6-1 and were found among Filipino, Caucasian American, and African American patients. Only one patient (from Poland) carried the Acadian 6-7-2 haplotype, but this allele carried the missense mutation P497R rather than the 7-bp duplication. Smith et al. [44] identified two unrelated patients from the United Kingdom who were homozygous for this same 7-bp duplication. Thus, the 7-bp duplication arose de novo among many different ethnic groups, and the identity of the Acadian mutation remains to be established. Wang et al. [38] identified a total of 14 different mutations in 19 patients, including 7 missense mutations. To determine the effect of these mutations on enzyme activity, each mutant was recreated using site-directed mutagenesis and expressed in MA-10 cells. None of the missense mutations encoded a protein with 1~-hydroxylase activity significantly above the low endogenous activity of MA-10 cells. Eight additional missense mutations were identified by Kitanaka et al. [20,45] in eight Japanese families, including one in a patient with mild clinical abnormalities, and the activities of these mutants were tested in a promoter/reporter transactivation assay based on activation of the vitamin D receptor by 1,25 (OH)2D. None of the eight mutants showed any
A
B
P438 T439 P440 H441 P442 F443 A444 5' CCC A C C / ~ CAC CCA TTT GCA 3'
/
\
5' CCC ACC CCC CAC CCC CCA CCC ATT TGC 3' P438 T439 P440 H441 P442 P443 P444 1445 C446
FIGURE 4 Genetic diagnosis of the AG958 mutation commonly found among the Charlevoix French Canadian population. Mutation AG958 deletes a Tai I site. A 1458-bp fragment was amplified from genomic DNA from a homozygously affected patient, an obligately heterozygous parent, and a homozygously unaffected normal control and digested with Tai L The patient's DNA, carrying AG958 on both alleles, is not cut; half of the parent's DNA is cut, and the normal DNA is cut to completion.
FIGURE 5 The 7-bp duplication. (A) The sequence CCCACCC is normally duplicated in exon 8, encoding residues 438-442 (Pro-ThrPro-His-Pro). (B) The mutation involves the insertion of a third copy of the CCCACCC sequence, which changes the reading frame, beginning with residue 443. The triplication is arbitrarily shown as an insert at codon 440 between the two normal copies of the CCCACCC sequence. It is not possible to specify which of the three copies in the mutant sequence is new.
24. Rickets Due to Hereditary Abnormalities
activity, consistent with the phenotype of the patients. Two other missense mutations were identified by Smith et al. [44] in a compound heterozygous patient from the United Kingdom; 10~-hydroxylase activity in peripheral blood macrophages from the patient was undetectable, although activity was present in cells from normal individuals and from the obligately heterozygous parents. Considerable phenotypic variation has been observed among patients with l~-hydroxylase deficiency, but the molecular basis of such variation is unknown. To address this question, Wang et al. [46] analyzed six patients with clinical and radiographic features of rickets; in four patients the laboratory abnormalities were typical of 1~hydroxylase deficiency, but in two they were unusually mild [i.e., mild hypocalcemia and normal serum 1,25 (OH)2D concentrations]. Mutations were identified in all patients. One patient was homozygous for a splice site mutation, substitution of an adenine (a) for a guanine (g) in the first nucleotide of intron 2 (IVS2+ l g--,a), which disrupts the splice donor site resulting in retention of intron 2, shift of the reading frame, and premature termination of translation, yielding a truncated peptide devoid of enzymatic activity. Of the three new missense mutations found, mutant R389G ( C G T ~ G G T ) in exon 7 was totally inactive, but mutant L343F (CTC~TTC) in exon 6 retained 2.3% of wild-type activity and mutant E189G ( G A A ~ G G A ) in exon 3 retained 22% of wildtype activity (Fig. 6). The two mutations that confer partial enzyme activity in vitro were found in the two patents with mild laboratory abnormalities, suggesting that such mutations contribute to the variable phenotype observed in patients with 1~-hydroxylase deficiency [46]. 100
100-
589
Structure and Function of I a-Hydroxylase Although vitamin D 1~-hydroxylase deficiency is rare, to date a total of 31 different mutations have been found on 88 distinct chromosomes since the first description of gene mutations in 1997 (Table 1; Fig. 2) [14,46]. The mutations observed most frequently are AG958, commonly found in French Canadian patients [38] due
TABLE 1
Known mutations
o f P 4 5 0 c I oL in p a t i e n t s w i t h
v i t a m i n D- I a - h y d r o x y l a s e
deficiency
Nucleotide Change*
Exon
Mutation
Reference
MissenseMutafions
Q65H
G246T
1
38
R107H
G1016A
2
20,45
G125E
G1070A
2
20
P143L
C1634T
3
45
D164N
G1696A
3
45
E189G
A1772G
3
46
E189K
G1771A
3
38
T321R
C2337G
5
45
$323Y
C2546A
6
44
R335P
G2582C
6
20
L343F
C2605T
6
46
P382S
C2925T
7
20
R389C
C2946T
7
45
R389G
C2946G
7
46
R389H
G2947A
7
38,46
T409I
C3299T
8
38,46
R429P
G3359C
8
38
R453C
C3430T
8
38
V478G
T3680G
9
44
P497R
C3917G
9
38
W241X
G2014A
4
38
W433X
G3372A
8
45
Nonsense Mutations
80v
Deletions
._~ 60-
> .m O
40._> a..., t~
m ID
22
flame shift
212AG
1
38
flame shift
958AG
2
21, 38
flame shift
192lAG
4
14, 38
flame shift
1984AC
4
14, 38
20Insertions
0 Vector
0
flame shift
3399 7bp dup
8
21, 38,44
R389G
frame shift
3399 7bp-2bp dup
8
38
GGGCG897-901CTTCGG
2
46
343F
E 189G
WT
FIGURE 6 10t-Hydroxylase activity of the P450c1~ mutants. The three novel missense mutations, R389G, L343F, and E189G, were recreated in a P450c10t c D N A expression vector and transfected into mouse Leydig MA-10 cells. The mutation R389G had no l s - h y d r o xylase activity, as did the vector control (vector); mutation L343F (patient 4) retained 2.3% of wild-type activity (WT); and mutation E189G (patient 6) retained 22% of wild-type activity [46]. D a t a are expressed as a percentage of the activity of the wild-type cDNA.
Deletion-Insertion
flame shift S~ce-site Mutations
flame shift
IVS2+I g to a (G1083A)
Intron 2
46
frame shift
IVS3+I g to a (G1796A)
Intron 3
45
*Nucleotides are numbered from the transcription start site (19).
590
Anthony A. Portale and Walter L. Miller
to a founder effect [43], and a 7-bp duplication that arose independently in several populations [38]. All of the frameshift and nonsense (premature translation arrest) mutants eliminate the heme binding site of the 1~-hydroxylase, resulting in a protein devoid of enzymatic activity. Of the 20 missense mutations reported to date, all except 2 [46] were totally inactive when assayed for enzymatic activity in vitro. Although the tertiary structures of mitochondrial cytochromes P450 have not been determined, most bacterial P450s are also class I, and the structures of several bacterial P450 enzymes have been determined by X-ray crystallography [47-49]. Comparisons of the structures of these enzymes reveals remarkable conservation of their topology and tertiary structure, despite low amino acid sequence identity [50]. Wang et al. [38] aligned the sequence of the human l~-hydroxYlase with that of the class I bacterial P450s on the basis'of regions of predicted secondary structure rather than amino acid sequence identity (Fig. 7). This permits a preliminary assignment of the locations of the amino acid replacement mutations and an analysis of the mechanism by which such mutations disrupt enzyme activity. Mutation Q65H is in ~ helix A t, T409I is in strand 3 of 13sheet 1, and R389H is in strand 4 of [3sheet 1. Although distinct from one another in terms of their amino acid numbers, these mutations lie in the clustered [3 sheet domain that interacts with the inner mitochondrial membrane and defines the substrate entry channel. Their locations suggest that they probably are conformational mutants that disrupt the ability of the enzyme to bind substrate rather than mutants that disrupt the catalytic site or the redox partner binding site. Mutation E189L lies in the E helix and disrupts the four-helix bundle consisting of the D, E, I, and L helices and thus significantly disrupts the structure of 1~-hydroxylase. Mutant R429P inserts a proline at the junction of the K' helix and the meander, changing the direction of the carbon backbone and grossly disrupting the meander. Mutant R453C, which is two residues away from the thiolate cysteine 455, disrupts a salt bridge that interacts with the heme propionate, much like the corresponding P440C mutation in P450c17, which causes complete 17ct-hydroxylase deficiency [51], and like the R435C mutation in P450arom, which causes complete aromatase deficiency [52]. Mutant P497R lies near strand 3 of sheet 3, which participates in defining the top of the substrate-binding pocket; the directional change in the carbon backbone that results from insertion of a proline could disrupt substrate binding. Three different missense mutations~R389G [46], R389H [38], and R389C [45]~have been described in codon 389, all of which are totally inactive. This would be predicted because R389 aligns with the highly con-
served arginine in the ~1-4 helix of type 1 P450 proteins [38]; this arginine coordinates one of the heme propionate side chains [50,53] and hence is presumed to be essential for catalytic activity. It is not known whether such mutants even retain the capacity to bind heme. The mutation L343F [46] changes leucine, a small uncharged residue, to phenylalanine, a bulky uncharged residue. L343 lies in the J helix, which is a structurally conserved region that is important structurally but not catalytically. The mutation L343F could disrupt activity by creating a conformational mutant. Sawada et al. performed a structure-function analysis of missense mutations identified in Japanese patients; their sequence alignments were virtually identical to those of Wang et al. [38]. All mutations were functionally inactive when expressed in vitro [54]. The authors suggested that mutations of residues R107, G125, and P497, which are located in the substrate-recognition region, would abolish enzyme activity by disrupting the tertiary structure of the substrate-heme pocket. They further suggested that residues R389 and R453 are involved in heme-propionate binding and that residue D 164, which is negatively charged and located in the D helix, would stabilize the four-helix bundle, possibly by forming a salt bridge. Residue T321 was thought to be required for the activation of molecular oxygen [54]. The first missense mutation described that retains partial activity in vitro is mutation E 189G, which retains 22% of wild-type activity (Fig. 6) [46]. Residue E189 lies in the E helix. A change from glutamic acid (E) to glycine (G) removes a three-carbon side chain and replaces an acidic residue with a neutral one; such a change could cause a conformational disturbance that still permits substrate binding and interaction with ferredoxin, albeit at decreased efficiency. The patient who was homozygous for the E 189G mutation [46] came to medical attention due to hypotonia and leg deformity and was found to have secondary hyperparathyroidism, but serum concentrations of calcium, phosphorus, and 1,25(OH)zD were not reduced. The diagnosis of 10~-hydroxylase deficiency was considered when the patient failed to respond to large doses of vitamin D3 but showed rapid improvement with administration of 0.25 lxg per day of 1,25 (OH)ED3. In another patient whose mutation retained 2.3% of wild-type activity, serum concentrations of 25-OHD and 1,25(OH)ED were not reduced, but the diagnosis was more readily considered because of hypophosphatemia and increased serum concentrations of alkaline phosphatase and PTH. Such cases demonstrate that the classical laboratory criteria for the diagnosis of l~-hydroxylase deficiency may fail to identify patients with partial but significant defects in this enzyme; hence, l ct-hydroxylase deficiency syndromes may be more common than previously appreciated.
cia cam terp eryF
MTQTLKYASRVFHRVRWAPELGASLGYREYHSA----------RRSLADIPGPSTPSFLAELFCKG ............................... MTTETIQSNANLAPLPPHVPEHLVFDFDMYNPSNLSA........................................... MDARATIPEHIARTVILPQGYA---................................................... MTTVPDLESDSFHV---a A'
cla cam terp eryF
RLHELQVQGAAHFG---PVWLASFG-TVRTVYVAAPALVEEURQEGPRPER--CS-----------FSPWTEIIRRCRQRAC GVQEAWAVLQESNV--PDLVWTRCNG--GHWIATRGQLIREAYEDYRHFSSE--CP-----------FIPREAGEAY-----DDEVIYPAFKWLRDEQPLAMAHIEGYDPMWIATKHADVMQIGKQPGNAEGSE-----------ILYDQNNEAFMRSIS
59 37 22 14
vR107H
vQ6SH
----DWYRTYAELRETAPVTPVRFLGQD-AWLVTGYDE~SDLRLSSDPKKKYPGVEVEFPAYLGFPEDVRNYFA---aA 81-1 Dl-2 aB Pl-5 aB' VG12SE
vP125L
vDl64N
124 97 92 87
VE 189G/K
cla cam t erp eryF
cla cam terp eryF cla cam terp eryF
cla cam terp eryF
AYPSATVLSQLPLLKAVVKEVLRLYPVVPGNSR-VPDKDIHVGDYIIPKNTLVTLCHYATSRDPAQFPEPNSFRPARWLGEG-P----T 439 ----------- ERIPAACEELLRRFSLV-ADGRILTSDYEFHG-VQLKKGDQILLPSGLDERENACPMHVDFSRQS-------- 347 ----------- ALIPRLWEAVRWTAPVKSFMRTALADTEVRG-QNIKRGDRIMLSYPSDEEVFSNPDEFDITRFPNR-------366 ----------- SALPNAVEEILRYIAPPETTTRFAAEEVEIRG-VAIPQYST~LVANGNRDPKQFPDPHRFDVTRDTRG-------340 -aK PI-4 P2-1 P2-2 pl-3 aK' Meander
cla cam terp eryF FIGURE 7 Alignment of the sequence of human P450cln (top) with the sequences of the crystallographically solved bacterial class I P450 proteins P450cam, P450trp, and P450eryF. The names of the various ct helices and P sheets are given below, with the ct helices highlighted in boldface and the P sheets underlined. The locations of the "meander" and Cys pocket are also shown. 'I'he locations of the known amino acid replacement mutations in P450cla are shown above its sequence with downward arrows.
59:2
Anthony A. Portale and Walter L. Miller
Treatment In the earliest reports of treatment of 1s-hydroxylase deficiency, administration of large doses of vitamin D2 (50,000-200,000 units per day) was associated with reversal of clinical, chemical, and radiographic abnormalities and improvement in growth rate [24,27-29]. However, the availability of activated forms of vitamin D has now rendered such therapy obsolete. Hypocalcemia and hyperparathyroidism were reversed and rickets was healed by administration of 1-3 gg/day (80-100ng/kg) of l s-OHD3 [55], a l s-hydroxylated analog of vitamin D that requires 25-hydroxylation in the liver. The synthetic analog dihydrotachysterol (DHT) is not l shydroxylated but carries a hydroxyl group in the 3s position; rotation of the A ring about the 6-7 carbon bond brings this group into a pseudo-Is-hydroxyl configuration so that DHT is active in the absence of 1s-hydroxylation. Typical doses are 50 mg/kg/day in infancy and 0.5-1.0 mg/day in adults. Currently, most authorities favor the use of physiologic replacement doses of 1,25(OH)2D3 (calcitriol), the most potent and most rapidly acting form of vitamin D. Oral administration of 0.25-2.0 gg/day (10-400 ng/ kg/day) of 1,25(OH)2D3 induced rapid correction of hypocalcemia, secondary hyperparathyroidism, and rickets; restoration of bone mineral content; and repair of bone architecture [33]. The maintenance dosage of 1,25(OH)2D3 is typically lower than that needed to initiate healing of rickets; therapy must be lifelong and is predictably successful [38]. Regardless of the form of vitamin D therapy, it is essential to monitor serum calcium, phosphorus, and PTH concentrations. A substantial calcium intake must be ensured, especially during bone healing that accompanies the initial phase of therapy. One generally aims to increase the total serum calcium concentration into the low-normal range (8.5-9 mg/ dl), which is sufficient to suppress the PTH concentration to values slightly lower than the upper limit of normal; higher calcium values increase the risk of hypercalciuria and nephrocalcinosis. It is important to monitor the urinary excretion of calcium. The ratio of urinary calcium to urinary creatinine in a single urine specimen should remain less than 0.25; the 24-hr urinary excretion of calcium should remain less than 4 mg/kg.
osteomalacia, and osteitis fibrosa in whom serum concentrations of 25-OHD were normal and those of 1,25 (OH)2D were greatly increased. The authors suggested that the disorder resulted from impaired end-organ response to 1,25(OH)2D and proposed that it be called vitamin D-dependent rickets type II (VDDR-II) in order to distinguish it from VDDR-I. In the same year, Marx et al. [57] described a condition with similar clinical and laboratory findings in two sisters in whom rickets first became evident between the ages of 5 and 20 months (Fig. 8). The hypocalcemia in these patients responded to oral administration of 1,25(OH)2D in doses of 1520 ~tg/day, approximately 20 times that needed to reverse hypocalcemia in patients with 1s-hydroxylase deficiency. These authors suggested that the condition could result from a hereditary abnormality in the receptor for 1,25 (OH)2D. Indeed, it is now known that in such patients, resistance of target tissues to vitamin D is caused by mutations in the vitamin D receptor. This disease has been variously referred to as VDDR-II, pseudo-vitamin D
RICKETS DUE TO ABNORMALITIES OF VITAMIN D ACTION
Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets In 1978, Brooks et al. [56] described a young woman with hypocalcemia, secondary hyperparathyroidism,
FIGURE 8 Two sisters, 7 (left) and 3 years of age, with HVDRR and alopecia (reprinted with permission from Rosen et al. [60]).
24. Rickets Due to HereditaryAbnormalities deficiency type II (PDDR II), calcitriol-resistant rickets, hypocalcemic vitamin D-resistant rickets, and hereditary 1,25(OH)zD-resistant rickets (HVDRR). Here, we refer to the disease as HVDRR, as suggested in a recent comprehensive review [58]. Clinical a n d Laboratory Features Most of the clinical, laboratory, and radiographic findings in patients with HVDRR are similar to those in patients with rickets due to 1~-hydroxylase deficiency or nutritional vitamin D deficiency [56,57,59,60]. However, a striking difference is the finding of either sparse body hair or total alopecia in the majority of affected patients [59-63]. The absence of body hair can be present at birth or develop within the first year of life. Patients with alopecia appear to have an earlier age of onset of rickets and greater resistance to 1,25(OH)2D3 treatment [63]. Serum concentrations of 1,25(OH)2D are greatly increased in patients with HVDRR, with values ranging from approximately 3-fold to 30-fold higher than the normal mean value [56,57,59-61,64]. This characteristic feature readily distinguishes such patients from those with l~-hydroxylase deficiency, in whom serum 1,25 (OH)2D is usually undetectable or greatly decreased. In untreated patients with HVDRR, serum concentrations of 24,25(OH)2D are low [59,61,65-67] or undetectable [64]; the basis for this apparent difference is not known. Patients are resistant to treatment with physiological and even supraphysiological doses of vitamin D. Cellular a n d M o l e c u l a r Defect: Vitamin D R e c e p t o r As with many other steroid hormones, the action of 1,25(OH)2D in target tissues depends on its binding to the vitamin D receptor (VDR), a member of the thyroidretinoid group of receptors, which is a subgroup of
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the steroid-thyroid-retinoid gene superfamily of nuclear transcription factors [68]. Upon binding of 1,25(OH)2D, the VDR forms a heterodimer with the retinoid-X receptor (RXR), and the complex then binds to specific short DNA sequences, termed vitamin D-responsive element (VDRE), on target genes, thereby regulating gene transcription. The transcriptional activity of the liganded V D R - R X R heterodimer is influenced by posttranslational modifications and by association with nuclear receptor coactivators. Like other members of its superfamily, the structure of the VDR can be separated into five regions or domains, designated A/B, C, D, and E/F (Fig. 9). Region A/B includes those residues amino terminal to the C region, which is the highly conserved DNA-binding domain that contains two zinc-coordinated finger structures that interact with DNA. The D region, or hinge region, serves as a highly flexible link between the DNAbinding domain and region E/F, the ligand-binding domain. Region D is the least conserved among the nuclear receptors. In addition to its ligand-binding function, domain E/F contains a ligand-dependent transcriptional activation function termed AF-2. This highly conserved region lies at the distal carboxy terminus of the receptor and contains residues critical for binding transcriptional coactivators and activating transcription [69-71]. Ligand binding appears to induce a conformational change of the AF-2 helix that allows recruitment of coactivators of the p160 family of coactivator proteins, including steroid receptor coactivator 1 (SRC-1) [69,72]. Another class of coactivators, vitamin D receptor-interacting protein (DRIP), binds to the liganded VDR, strongly potentiates transcription, and is essential for ligand-dependent transactivation [73]. The VDR is found in numerous tissues, including skin fibroblasts; therefore, skin obtained by biopsy from patients with HVDRR was used in early studies of the nature of their vitamin D "resistance." Two abnormalities were found in cultured skin fibroblasts from affected
DNA 1,25(OH)2D binding binding domain domain FIGURE9 Schematicillustration of the VDR. The protein is composed of 427 amino acids and can be separated into five domains, designated A/B, C, D, and E/F, whose approximate boundaries are indicated. The gray and black shaded regions exhibit strong homology with other members of the nuclear receptor gene family. E1 and AF-2 represent helices within the E/F domain that participate in transactivation.
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patients: High-affinity nuclear uptake of [3H]1,25 (OH)2D3 was not detectable [74,75], and 1,25(OH)2D failed to induce an increase in fibroblast 24-hydroxylase activity [75], a well-characterized biomarker for cellular responsiveness to 1,25(OH)2D. Subsequent studies showed that the cellular defect was heterogeneous. Patients in w h o m [3H] 1,25(OH)2D3 binding was very low or undetectable were designated as receptor negative or "hormone-binding negative [74,76-80], and patients whose cells had detectable [3H]l,25(OH)2D3 binding were designated receptor positive or "hormone-binding positive [76,78,81,82]. In either case, cellular responsiveness to 1,25(OH)2D was greatly impaired [75,77,81]. However, fibroblasts from patients designated receptor negative contained immunoreactive VDR protein, suggesting that the abnormal binding was caused by a defect in the ligand-binding domain of the VDR rather than defective synthesis of the protein [83,83-86]. In patients designated receptor positive, the DNA-binding affinity of the VDR was shown to be defective [66,82,87,88]. In some receptor-positive patients, DNA-binding affinity was normal but the ability of the VDR to localize to the nucleus was impaired [82]. Molecular Genetics of HVDDR Cloning of the avian VDR [89] and human VDR [90] permitted analysis of the molecular genetic basis of HVDRR. The human VDR gene is localized to chromosome 12q13-14 [91], the same region in which the gene encoding the vitamin D l~-hydroxylase is found. The VDR gene spans 75 kbs of DNA and comprises eight coding exons (exons 2-9) and at least three short 5' noncoding exons (exons l a-lc). Exons 2 and 3 encode the highly conserved DNA-binding domain (DBD) of the receptor. The ligand-binding domain (LBD) is encoded by exons 6-9, and the region between the DBD and LBD (the hinge region) is encoded by exons 4-6. Ligand-Binding Positive P h e n o t y p e Hughes et al. [92] identified the first mutations in the VDR gene in two families in which the affected children were homozygous for HVDRR; in these patients, receptor binding of hormone was normal but its affinity for DNA was decreased. One family carried a missense mutation, R73Q, in exon 3 that encodes the second zinc finger region (Fig. 10). (In this chapter, the numbering system of Baker et al. [90] is used to designate the mutations.) The second family carried a missense mutation, G33D, in exon 2, which encodes the first zinc finger region. The mutant residues were created in vitro by site-directed mutagenesis and expressed in COS-1 cells; 1,25(OH)2D-binding activity of the mutant proteins was
normal but DNA-binding affinity was defective, as was observed in receptor isolated from the patients [92]. The mutant receptor was transcriptionally inactive in vitro as well [93]. At least 10 different mutations in the DBD of the VDR have been identified (Table 2; Fig. 10) [94-104]. In most cases, fibroblasts from affected patients had normal [3H]l,25(OH)zD3-binding capacity but reduced DNA-binding affinity, consistent with mutations that affect those regions of the receptor necessary for binding to DNA. Based on structural similarities with the crystal structures of the related glucocorticoid receptor [105], RXR, and thyroid receptor [106], one can predict the likely consequences of the mutations in the VDR. Mutations G33D, H35Q, K45E, G46D, and F47I affect regions of the receptor that contact DNA. Mutation K45E should disrupt the hydrogen bonding between K45 and a guanine residue in the corresponding VDRE. Mutations G46D and G33D introduce a bulky charged amino acid, resulting in unfavorable electrostatic interactions with the negatively charged phosphate backbone of the DNA [107]. Mutations R30X in exon 2 and R73X in exon 3 result in receptors that are truncated in the middle of the first and second zinc fingers, respectively, and thus devoid of both DNA- and ligand-binding functions. Ligand-Binding Negative P h e n o t y p e Ritchie et al. [94] identified the first VDR mutation associated with a ligand-binding negative phenotype in three unrelated patients with HVDRR. The mutation, T295X, resulted in truncation of 132 amino acids of the carboxy terminus of the VDR, deletion of a major portion of the LBD, and creation of the ligand-binding negative phenotype. This mutation has been identified in several additional families [95,96,99]. Twelve additional mutations in the LBD of the VDR have been identified (Table 2; Fig. 11) [65,92,103,104,107-116]. The crystal structure of the LBD of the human VDR was recently described, confirming that its overall topology is similar to that of the other nuclear receptors and is composed of 13 0~helices, H1-H12 and H3n, organized in three layers, and a three-stranded 13 sheet (Fig. 12) [117]. Helices H1 and H3 are connected by two small helices, H2 and H3n. The ligand-binding pocket is bordered by helices H3, H5, H7, H11, and loop-encompassing residues that include Ser a75 (loop H5-13), Trp 286 (if-l), and L e u 233 (H3), with a "lid" formed by H12. The ligand-binding pocket is lined predominately by hydrophobic residues; two of these residues, H305 (loop H6H7) and H397 (loop H11), are hydrogen bonded to the 25-hydroxyl group of the 1,25(OH)2D ligand [117]. This structure predicts that the naturally occurring mutation
24. Rickets Due to Hereditary Abnormalities
FIGURE 10 Model of the DNA-binding domain (DBD) of the VDR and locations of mutations in patients with HVDRR. Shown are the two zinc finger structures and the amino acid composition of the DBD. Conserved amino acids are depicted as shaded circles. The large arrows depict the locations of the mutations. Mutations are shown as large shaded circles. FS, frame shift. Intron D (arrow) separates exons 2 and 3, which encode the two zinc fingers. The numbers indicate the amino acid numbers (modified from Malloy et al. [58]).
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TABLE 2
K n o w n M u t a t i o n s in t h e VDR in p a t i e n t s w i t h HVDRR
Mutation*
Nucleotide Change
R30X
CGA-TGA
2
DBD
G33D
GCC-GAC
2
DBD
H35Q
CAC-CAG
2
DBD
+
+
65
K45E
AAA-GAA
2
DBD
+
+
107
G46D
GGC-GAC
2
DBD
+
+
112
F471
TTC-ATC
2
DBD
+
+
107
R50Q
CGA-CAA
3
DBD
+
+
109
R73Q
CGA-CAA
3
DBD
+
+
92
R73X
CGA-TGA
3
DBD
-
+
99, 103
R80Q
CGG-CAG
3
DBD
+
+
108, 110
-
+
111
E92fs
Exon
Domain
Ligand Binding
intron E
Alopecia
Reference
-
+
113, 114
+
+
92
Q 152X
CAG-TAG
4
hinge
-
+
100
C190W
TGT-TGG
5
LBD
?
+
97
L233fs
GTC-GTG
6
LBD
-
+
103
F251C
TTC-TGC
6
LBD
-
+
104
Q259P
CAG-CCG
7
LBD
+
+
103
R274L
CGC-CTC
7
LBD
+
-
100
Y295X
TAC-TAA
7
LBD
-
+
94, 96
H305Q
CAC-CAG
8
LBD
+
-
101
I314S
ATC-AGC
8
LBD
+
-
102
Q317X
CAG-TAG
8
LBD
-
+
116
R391C
CGC-TGC
9
LBD
+
+
102
E420K
CAA-AAA
9
LBD
+
-
115
LBD
-
+
97
exon 7-9 deletion
*Mutations are numbered according to Baker et al. (90)
FIGURE 1 1 Model of the ligand-binding domain of the V D R and locations of mutations in patients with H V D R R . The helices are depicted as shaded rectangles and the single 13turn (S 1) as an open rectangle. The E 1 and AF-2 regions are shown above the ct helices, fs, frame shift (modified from Malloy et al. [58]).
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24. Rickets Due to Hereditary Abnormalities
located in helix H12 of the LBD, alters the coactivator binding site, preventing proper interaction between the VDR and coactivators SRC-1 and DRIP205 and thus resulting in loss of transactivation [115]. Additional M u t a t i o n s
FIGURE 12 Model of the ligand-binding domain of the human VDR based on a high-resolution crystal structure of the protein complexed with 1,25(OH)2D3. The helices are represented as cylinders and [3 sheets as arrows. The chemical structure of the 1,25(OH)2D3 ligand is depicted (reprinted with permission from Rochel et al. [117]).
A mutation in the hinge region, Q152X, deletes 306 amino acids of the VDR, resulting in a ligand-binding negative phenotype and defective transactivation in vitro [99,100]. The donor splice site mutation eliminates the 5' donor splice site of the fifth intron (E), leading to skipping of exon 4, a shift in the reading frame (E92fs), and premature termination of translation within exon 5 [111]. A second splice site mutation introduces a cryptic 5I donor splice site in exon 6, causing a 56-bp deletion in exon 6, a shift in the reading frame (L233fs), and premature truncation of 194 amino acids of the VDR, leading to a hormone-binding negative, defective transactivation phenotype [103]. A patient with HVDRR in whom exons 7-9 of the VDR gene were deleted has been reported [97]. Hewison et al. [118] described a patient with the usual phenotypic features of HVDRR, including alopecia, but without detectable mutations in the coding region of the VDR gene. In fibroblasts from the patient, [3H]1,25 (OH)2D3 binding was normal, but 1,25(OH)zD3 failed to induce 24-hydroxylase activity in these cells. When the patient's cDNA was expressed in vitro, 1,25(OH)2D3induced transactivation was normal. Given the apparently normal VDR in this patient, the authors suggested that the disease was due to mutation of another protein essential for 1,25(OH)2D-mediated transcriptional activation. Treatment
H305Q, described by Malloy et al. [101], results in defective ligand binding, as observed in fibroblasts from the affected patient. Mutation F251C is located in the E1 region (amino acids 244-263), which overlaps the C-terminal portion of helix H3, loop 3-4, and the N-terminal portion of helix H4 [104]. Found within the E1 region is the strictly conserved hydrophobic residue phenylalanine 251. Mutation of F251 likely disrupts the ligand-binding pocket of the VDR and interferes with conformation required for optimal function, consistent with the reduced [3H]l,25(OH)zD3-binding affinity of the patient's fibroblasts and the defective heterodimerization and reduction in transactivation activity of the mutant receptor recreated and expressed in vitro [104]. Mutations G259P (which occurs in H4), R274L (H5), I314S (H7), and R391C (H 10) should interfere with either ligand binding or dimerization. Mutation E420K,
Patients with HVDRR have been treated with large doses of vitamin D2, 25-OHD, 1~-OHD, or 1,25(OH)zD, with highly variable results. A few patients responded to 4000-40,000 units/day of vitamin D2 [56,57,119]; however, oral administration of vitamin D2 in doses as high as 200,000 units per day was not effective in improving the clinical, chemical, or radiographic features of the disease in other patients [59-61,120]. Patients with HVDRR without alopecia are generally more responsive to treatment with metabolites of vitamin D than are patients with alopecia [63]. Some patients without alopecia responded to treatment with 25(OH)D3 in doses ranging from 20 to 200lag/day and 1,25(OH)2D3 in doses of 17-20 lag/day [57]. In a patient with HVDRR without alopecia, a missense mutation in the LBD of the VDR, H305Q, resulted in a modest decrease in the receptor's affinity for 1,25(OH)2D [101]. Treatment with 12.5lag/day of 1,25(OH)zD3 induced reversal of
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hypocalcemia and secondary hyperparathyroidism and healing of rickets, suggesting that high doses of the hormone overcame the affinity defect and rendered the patient's cells responsive to the hormone [101]. Thus, patients with mutations in LBD are more likely to respond to high-dose vitamin D therapy than those with mutations in the DBD of the receptor [ 101,102]. Although many patients are refractory to treatment [65,76,121,122], some have responded to administration of I~-OHD3 in doses as high as 901~g/day [67,123] or to 1,25(OH)2D3 in doses as high as 20 l~g/day [66,67,76,123-125]. For patients who are refractory to treatment, intravenous administration of large amounts of elemental calcium (400-1400mg/m2/day) for periods up to 3.8 years was associated with resolution of bone pain, normalization of the serum calcium and phosphorus concentrations, reversal of hyperparathyroidism [65,121,122], radiographic and histologic healing of skeletal lesions [121], and increased growth velocity [121,122]. After clinical improvement was induced with intravenous calcium infusions, high-dose oral calcium (3.5-9g/m2/day of elemental calcium) was required to maintain normocalcemia [122]. However, such intensive therapy can be complicated by cardiac arrhythmias, hypercalciuria, nephrolithiasis, and sepsis [122]. The effectiveness of high-dose calcium infusions in such patients supports the formulation that the defect in the intestinal VDR causes failure of intestinal calcium absorption, resulting in hypocalcemia and consequent metabolic bone disease. Thus, although treatment of patients with l cthydroxylase deficiency with physiologic replacement doses of calcitriol is straightforward and uniformly successful [38], treatment of patients with mutations of the VDR remains daunting. References 1. Feldman, D., Malloy, P. J., and Gross, C. (1996). Vitamin D: Metabolism and action. In Osteoporosis (R. Marcus et al., Eds.), pp. 205-235. Academic Press, San Diego. 2. Bikle, D. D., Nemanic, M. K., Gee, E., and Elias, P. (1986). 1,25Dihydroxyvitamin D3 production by human keratinocytes. J. Clin. Invest. 78, 557-566. 3. Adams, J. S., Sharma, O. P., Gacad, M. A., and Singer, F. R. (1983). Metabolism of 25-hydroxyvitamin D3 by cultured pulmonary alveolar macrophages in sarcoidosis. J. Clin. Invest. 72, 1856-1860. 4. Howard, G. A., Turner, R. T., Sherrard, D. J., and Baylink, D. J. (1981). Human bone cells in culture metabolize 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. J. Biol. Chem. 256, 7738-7740. 5. Armbrecht, H. J., Okuda, K., Wongsurawat, N., Nemani, R. K., Chen, M. L., and Boltz, M. A. (1992). Characterization and regulation of the vitamin D hydroxylases. J. Steroid Biochem. Mol. Biol. 43, 1073-1081. 6. Auchus, R. J., Lee, T. C., and Miller, W. L. (1998). Cytochrome b5 augments the 17,20-1yase activity of human P450cl 7 without direct electron transfer. J. Biol. Chem. 273, 3158-3165.
7. Usui, E., Noshiro, M., and Okuda, K. (1990). Molecular cloning of cDNA for vitamin D3 25-hydroxylase from rat liver mitochondria. FEBS Lett. 262, 135-138. 8. Su, P., Rennert, H., Shayiq, R. M., Yamamoto, R., Zheng, Y. M., Addya, S., Strauss, J. F. I., and Avadhani, N. G. (1990). A cDNA encoding a rat mitochondrial cytochrome P450 catalyzing both the 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3: Gonadotropic regulation of the cognate mRNA in ovaries. DNA Cell Biol. 9, 657-665. 9. Cali, J. J., and Russell, D. W. (1991). Characterization of human stero127-hydroxylase. J. Biol. Chem. 266, 7774-7778. 10. Jones, G., Strugnell, S. A., and DeLuca, H. F. (1998). Current understanding of the molecular actions of vitamin D. Physiol. Rev. 78, 1193-1231. 11. Ohyama, Y., Noshiro, M., and Okuda, K. (1991). Cloning and expression of cDNA encoding 25-hydroxyvitamin D3 24hydroxylase. FEBS Lett. 278, 195-198. 12. Chen, K. S., Prahl, J. M., and DeLuca, H. F. (1993). Isolation and expression of human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA. Proc. Natl. Acad. Sci. USA 90, 4543-4547. 13. Chen, K. S., and DeLuca, H. F. (1995). Cloning of the human 1 alpha, 25-dihydroxyvitamin D-3 24-hydroxylase gene promoter and identification of two vitamin D-responsive elements. Biochim. Biophys. Acta 1263, 1-9. 14. Fu, G. K., Lin, D., Zhang, M. Y. H., Bikle, D. D., Miller, W. L., and Portale, A. A. (1997). Cloning of human 25-hydroxyvitamin D-l~-hydroxylase and mutations causing vitamin D-dependent rickets type 1. Mol. Endocrinol. 11, 1961-1970. 15. Monkawa, T., Yoshida, T., Wakino, S., Shinki, T., Anazawa, H., DeLuca, H. F., Suda, T., Hayashi, M., and Saruta, T. (1997). Molecular cloning of cDNA and genomic DNA for human 25-hydroxyvitamin D3 l~-hydroxylase. Biochem. Biophys. Res. Commun. 239, 527-533. 16. St.-Arnaud, R., Messerlian, S., Moir, J. M., Omdahl, J. L., and Glorieux, F. H. (1997). The 25-hydroxyvitamin D 1-alphahydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus. J. Bone Miner. Res. 12, 1552-1559. 17. Shinki, T., Shimada, H., Wakino, S., Anazawa, H., Hayashi, M., Saruta, T., DeLuca, H. F., and Suda, T. (1997). Cloning and expression of rat 25-hydroxyvitamin D3-1at-hydroxylase cDNA. Proc. Natl. Acad. Sci. USA. 94, 12920-12925. 18. Takeyama, K., Kitanaka, S., Sato, T., Kobori, M., Yanagisawa, J., and Kato, S. (1997). 25-Hydroxyvitamin D3 10~-hydroxylase and vitamin D synthesis. Science 277, 1827-1830. 19. Fu, G. K., Portale, A. A., and Miller, W. L. (1997). Complete structure of the human gene for the vitamin D l u-hydroxylase, P450clQt. DNA Cell Biol. 16, 1499-1507. 20. Kitanaka,' S., Takeyama, K., Murayama, A., Sato, T., Okumura, K., Nogami, M., Hasegawa, Y., Nimi, H., Yanagisawa, J., Tanaka, T., and Kato, S. (1998). Inactivating mutations in the 25-hydroxyvitamin D 3 1Qt-hydroxylasegene in patients with pseudovitamin D-deficiency rickets. N. Engl. J. Med. 338, 653-661. 21. Yoshida, T., Monkawa, T., Tenenhouse, H. S., Goodyer, P., Shinki, T., Suda, T., Wakino, S., Hayashi, M., and Saruta, T. (1998). Two novel 1~-hydroxylase mutations in French-Canadians with vitamin D dependency tickets type I. Kidney Int. 54, 1437-1443. 22. Kimmel-Jehan, C., and DeLuca, H. F. (2000). Cloning of the mouse 25-hydroxyvitamin D3-1 ct-hydroxylase (CYP1 at) gene. Biochim. Biophys. Acta 1475, 109-113. 23. Panda, D. K., AI Kawas, S., Seldin, M. F., Hendy, G. N., and Goltzman, D. (2001). 25-Hydroxyvitamin D lalpha-hydroxylase: Structure of the mouse gene, chromosomal assignment, and developmental expression. J. Bone Miner. Res. 16, 46-56.
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Thomasset, Eds.), p. 6. de Gruyter, New York. 98. Rut, A. R., Hewison, M., Rowe, P., Hughes, M., Grant, D., and O'Riordan, J. L. H. (1991). A novel mutation in the steroid binding region of the vitamin D receptor (VDR) gene in hereditary vitamin D resistant rickets (HVDRR). In Vitamin D." Gene Regulation, Structure-Function Analysis, and Clinical Application. Eighth Workshop on Vitamin D (A. W. Norman, R. Bouillon, and M.
Thomasset, Eds.), pp. 94-95. de Gruyter, New York. 99. Wiese, R. J., Goto, H., Prahl, J. M., Marx, S. J., Thomas, M., al-Aqeel, A., and DeLuca, H. F. (1993). Vitamin D-dependency rickets type II: Truncated vitamin D receptor in three kindreds. Mol. Cell. Endocrinol. 90, 197-201.
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100. Kristjansson, K., Rut, A. R., Hewison, M., O'Riordan, J. L., and Hughes, M. R. (1993). Two mutations in the hormone binding domain of the vitamin D receptor cause tissue resistance to 1,25 dihydroxyvitamin D3. J. Clin. Invest. 92, 12-16. 101. Malloy, P. J., Eccleshall, T. R., Gross, C., Van Maldergem, L., Bouillon, R., and Feldman, D. (1997). Hereditary vitamin D resistant rickets caused by a novel mutation in the vitamin D receptor that results in decreased affinity for hormone and cellular hyporesponsiveness. J. Clin. Invest. 99, 297-304. 102. Whitfield, G. K., Selznick, S. H., Haussler, C. A., Hsieh, J. C., Galligan, M. A., Jurutka, P. W., Thompson, P. D., Lee, S. M., Zerwekh, J. E., and Haussler, M. R. (1996). Vitamin D receptors from patients with resistance to 1,25-dihydroxyvitamin D3: Point mutations confer reduced transactivation in response to ligand and impaired interaction with the retinoid X receptor heterodimeric partner. Mol. Endocrinol. 10, 1617-1631. 103. Cockerill, F. J., Hawa, N. S., Yousaf, N., Hewison, M., O'Riordan, J. L., and Farrow, S. M. (1997). Mutations in the vitamin D receptor gene in three kindreds associated with hereditary vitamin D resistant rickets. J. Clin. Endocrinol. Metab. 82, 3156-3160. 104. Malloy, P. J., Zhu, W., Zhao, X. Y., Pehling, G. B., and Feldman, D. (2001). A novel inborn error in the ligand-binding domain of the vitamin D receptor causes hereditary vitamin D-resistant rickets. Mol. Genet. Metab. 73, 138-148. 105. Luisi, B. F., Xu, W. X., Otwinowski, Z., Freedman, L. P., Yamamoto, K. R., and Sigler, P. B. (1991). Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature 352, 497-505. 106. Rastinejad, F., Perlmann, T., Evans, R. M., and Sigler, P. B. (1995). Structural determinants of nuclear receptor assembly on DNA direct repeats. Nature 375, 203-211. 107. Rut, A. R., Hewison, M., Kristjansson, K., Luisi, B., Hughes, M. R., and O'Riordan, J. L. (1994). Two mutations causing vitamin D resistant rickets: Modelling on the basis of steroid hormone receptor DNA-binding domain crystal structures. Clin. Endocrinol. (Oxford) 41, 581-590. 108. Sone, T., Marx, S. J., Liberman, U. A., and Pike, J. W. (1990). A unique point mutation in the human vitamin D receptor chromosomal gene confers hereditary resistance to 1,25dihydroxyvitamin D3. Mol. Endocrinol. 4, 623-631. 109. Saijo, T., Ito, M., Takeda, E., Huq, A. H., Naito, E., Yokota, I., Sone, T., Pike, J. W., and Kuroda, Y. (1991). A unique mutation in the vitamin D receptor gene in three Japanese patients with vitamin D-dependent rickets type II: Utility of single-strand conformation polymorphism analysis for heterozygous carrier detection. Am. J. Hum. Genet. 49, 668-673. 110. Malloy, P. J., Weisman, Y., and Feldman, D. (1994). Hereditary 1~,25-dihydroxyvitamin D-resistant tickets resulting from a mutation in the vitamin D receptor deoxyribonucleic acid-binding domain. J. Clin. Endocrinol. Metab. 78, 313-316. 111. Hawa, N. S., Cockerill, F. J., Vadher, S., Hewison, M., Rut, A. R., Pike, J. W., O'Riordan, J. L., and Farrow, S. M. (1996). Identification of a novel mutation in hereditary vitamin D resistant rickets causing exon skipping. Clin. Endocrinol. (Oxford) 45, 85-92. 112. Lin, N. U., Malloy, P. J., Sakati, N., al-Ashwal, A., and Feldman, D. (1996). A novel mutation in the deoxyribonucleic acidbinding domain of the vitamin D receptor causes hereditary 1,25-dihydroxyvitamin D-resistant rickets. J. Clin. Endocrinol. Metab. 81, 2564-2569. 113. Mechica, J. B., Leite, M. O., Mendonca, B. B., Frazzatto, E. S., Borelli, A., and Latronico, A. C. (1997). A novel nonsense mutation in the first zinc finger of the vitamin D receptor causing
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hereditary 1,25-dihydroxyvitamin D3-resistant rickets. J. Clin. Endocrinol. Metab. 82, 3892-3894. Zhu, W., Malloy, P. J., Delvin, E., Chabot, G., and Feldman, D. (1998). Hereditary 1,25-dihydroxyvitamin D-resistant rickets due to an opal mutation causing premature termination of the vitamin D receptor. J. Bone Miner. Res. 13, 259-264. Malloy, P. J., Xu, R., Peng, L., Clark, P. A., and Feldman, D. (2002). A novel mutation in helix 12 of the VDR impairs coactivator interaction and causes 1,25-dihydroxyvitamin D-resistant rickets without alopecia. Mol. Endocrinol., 16, 2538-2546. Malloy, P. J., Zhu, W., Bouillon, R., and Feldman, D. (2002). A novel nonsense mutation in the vitamin D receptor causes hereditary 1,25-dihydroxyvitamin D-resistant rickets. Mol. Genet. Metab., 77, 314-318. Rochel, N., Wurtz, J. M., Mitschler, A., Klaholz, B., and Moras, D. (2000). The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol. Cell 5, 173-179. Hewison, M., Rut, A. R., Kristjansson, K., Walker, R. E., Dillon, M. J., Hughes, M. R., and O'Riordan, J. L. (1993). Tissue resistance to 1,25-dihydroxyvitamin D without a mutation of the vitamin D receptor gene. Clin. Endocrinol. (Oxford) 39, 663-670. Zerwekh, J. E., Glass, K., Jowsey, J., and Pak, C. Y. C. (1979). A unique form of osteomalacia associated with end organ refractoriness to 1,25-dihydroxyvitamin D and apparent defective synthesis of 25-hydroxyvitamin D. J. Clin. Endocrinol. Metab. 49, 171-175.
120. Feldman, D., Chen, T., Cone, C., Hirst, M., Shani, S., Benderli, A., and Hochberg, Z. (1982). Vitamin D resistant rickets with alopecia: Cultured skin fibroblasts exhibit defective cytoplasmic receptors and unresponsiveness to 1,25(OH)2D3. J. Clin. Endocrinol. Metab. 55, 1020-1022. 121. Balsan, S., Garabedian, M., Larchet, M., Groski, A. M., Coumot, G., Tau, C., Bourdeau, A., Silve, C., and Ricour, C. (1986). Long-term nocturnal calcium infusions can cure rickets and promote normal mineralization in hereditary resistance to 1,25-dihydroxyvitamin D. J. Clin. Invest. 77, 1661-1667. 122. Hochberg, Z., Tiosano, D., and Even, L. (1992). Calcium therapy for calcitriol-resistant rickets. J. Pediatr. 121, 803-808. 123. Kudoh, T., Kumagai, T., Uetsuji, N., Tsugawa, S., Oyanagi, K., Chiba, Y., Minami, R., and Nakao, T. (1981). Vitamin D dependent rickets: Decreased sensitivity to 1,25-dihydroxyvitamin D. Eur. J. Pediatr. 137, 307-311. 124. CasteUs, S., Greig, F., Fusi, M. A., Finberg, L., Yasumura, S., Liberman, U. A., Eil, C., and Marx, S. J. (1986). Severely deficient binding of 1,25-dihydroxyvitamin D to its receptors in a patient responsive to high doses of this hormone. J. Clin. Endocrinol. Metab. 63, 252-256. 125. Kruse, K., and Feldmann, E. (1995). Healing of rickets during vitamin D therapy despite defective vitamin D receptors in two siblings with vitamin D-dependent rickets type II. J. Pediatr. 126, 145-148.
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1251 Familial Hypophosphatemia and Related Disorders INGRID A. HOLM,* MICHAEL J. ECONS, t and THOMAS O. CARPENTER* *Harvard Medical School, Boston, Massachusetts tindiana University School of Medicine, Indianapolis, Indiana Yale University School of Medicine, New Haven, Connecticut
INTRODUCTION
of management and therapeutics are reviewed as well as their long-term complications.
The precise physiologic basis of the hypophosphatemic disorders has eluded clinicians and scientists for years. Key discoveries in recent years have brought us closer to a mechanistic understanding of this group of disorders, but the complete story continues to rely on a degree of speculation. The scientific puzzle presented by the peculiar set of clinical observations in X-linked hypophosphatemia (XLH) is intriguing and challenging; the significance of solving this puzzle is evidenced by the fact that XLH is one of the most common bone diseases seen in children. Furthermore, therapy remains suboptimal, requiring frequent monitoring, and a panoply of chronic features complicate the affected population throughout life. This chapter provides an overview of the prototype hypophosphatemic disease, XLH. Novel and established clinical features of XLH are reviewed. Key elements central to the pathophysiology of XLH and its related disorders are discussed, including the putative function of the mutated endopeptidase PHEX and substances that appear to be important candidates for mediation of the disease. The biochemistry of FGF23 is reviewed with respect to its role in the related disorder, autosomal dominant hypophosphatemic rickets (ADHR). Clinical features of related disorders including ADHR, hereditary hypophosphatemic rickets with hypercalciuria (HHRH), and tumor-induced osteomalacia (TIO) are compared to those observed in XLH. Current strategies
PediatricBone
CLINICAL DESCRIPTION OF DISEASE ENTITIES XLH XLH is characterized by renal phosphate wasting leading to hypophosphatemia and low or normal concentrations of 1,25-dihydroxyvitamin D [1,25(OH)ED], an inappropriate response to hypophosphatemia. In children, the disorder first becomes apparent with the development of rickets, skeletal deformities, short stature, and dental abscesses. In adults, manifestations of XLH include osteomalacia, degenerative joint disease, enthesopathy, bone and joint pain, and continued dental disease [1]. XLH is the most common inherited form of rickets in the United States and the most common inherited defect in renal tubular phosphate transport. The incidence has been estimated at 1 in 20,000 in England [2]. There is a great deal of variability in the manifestations of XLH. In the mildest forms, only hypophosphatemia is evident. In the more severe forms, hypophosphatemia leads to decreased mineralization of newly formed bone and the clinical findings of rickets. Surgical correction of limb deformities is often required. Patients frequently present with leg deformities, especially patients with no family history of XLH.
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Biochemical findings in the serum of XLH patients that differentiate XLH from other causes of rickets or hypophosphatemia include low phosphate levels, normal or low 1,25(OH)2D levels, normal calcium levels, elevated alkaline phosphatase activity, and normal or mildly increased parathyroid hormone (PTH) levels.
or indirectly affect the availability of the high-affinity, low-capacity cotransporter NPT2 [3]. Among these factors, PTH is the most physiologically important regulator, and it acts by inhibiting the uptake of phosphate in the renal proximal convoluted and straight tubules.
Biochemical Findings in XLH Regulation of Phosphate Homeostasis In healthy adults, 65-75% of ingested phosphate is absorbed in the intestine, independent of the amount of phosphate ingested [3]. Thus, the greater the dietary phosphate content, the greater the amount of absorbed phosphate. 1,25(OH)2D promotes intestinal phosphate absorption, although this effect is limited for this primarily passive function. The eventual fate of absorbed phosphate includes incorporation into the mineral phase of bone, organification (into RNA, DNA, phospholipids, etc.), and urinary excretion [3]. Phosphate concentration in the blood is very stable, phosphate absorption in the intestine is not tightly regulated, and most of the absorbed phosphate is excreted in the urine, thus highlighting the importance of the kidney in the regulation of blood phosphate concentrations. Approximately 60% of phosphate reabsorption occurs in the proximal convoluted tubule and approximately 15-20% in the proximal straight tubule [3]. Phosphate transport in the renal tubules occurs via several sodium-phosphate (Na-Pi)-dependent cotransporters in the brush-border membrane of the luminal surface. There is a low-affinity, high-capacity transporter and a high-affinity, low-capacity transporter (NPT2); in the proximal straight tubule there is only a high-affinity, low-capacity system [3]. Active transport of sodium and phosphate into the cell from the lumen of the tubule via these cotransporters is driven by the downhill sodium gradient into the cell. This gradient is maintained by a Na/K-ATPase pump at the basolateral surface of the cell, which pumps Na out of the cell. Phosphate efflux from the cell is poorly understood. Phosphate in the cell is thought to exit passively at the basolateral surface, driven by an anion-exchange pump [3]. There is also a Na-Pi cotransporter at the basolateral surface that transports phosphate into the cell, although this cotransport appears to account for only a small proportion of phosphate transport into the cell [3]. A number of factors regulate phosphate transport. Insulin-like growth factor-1 (IGF-1), growth hormone (through IGF-1), insulin, epidermal growth factor, thyroid hormone, and 1,25(OH)zD stimulate tubular reabsorption of phosphate. PTH, PTHrP, calcitonin, ANF, transforming growth factor-~ (TGF-~), TGF-[3, and glucocorticoids inhibit these renal transport mechanisms [4,5]. All these hormonal factors directly
The biochemical findings in serum that characterize XLH include low phosphorus, normal calcium, inappropriately normal 1,25(OH)zD, normal 25-hydroxyvitamin D3 [25(OH)D], elevated alkaline phosphatase, and a normal or slightly elevated PTH [6].
Hypophosphatemia Hypophosphatemia due to urinary phosphate wasting is the hallmark of XLH. In children, the normal range for serum phosphorus levels is higher than that in adults. Therefore, it is critical to know the normal range at a given age. There have been many instances in which hypophosphatemia was mistaken for a normal value because the clinician was not aware of this fact [7]. Hypophosphatemia is rarely due to dietary phosphate deficiency in developed countries, where the diet is high in phosphate-containing foods. Hypophosphatemia due to inadequate dietary intake was not uncommonly seen in breast-fed premature infants prior to the widespread use of phosphate-containing breast milk fortifiers since human breast milk is relatively low in phosphorus content. However, most hypophosphatemia is probably due to the extracellular-intracellular shifts of phosphate associated with refeeding the malnourished individual, thereby replenishing the intracellular phosphate content. Another example of this compartmental shift phenomenon is in the treatment of diabetic ketoacidosis. Finally, hypophosphatemia due to renal phosphate wasting may occur in children due to a number of rare disorders that can lead to Fanconi syndrome, phosphate deficiency, and rickets. These disorders include cystinosis, Lowe's syndrome, tyrosinemia type I, and certain drugs (such as ifosfamide), heavy metals, and other toxins [8]. These disorders are usually characterized by renal losses of other compounds, including glucose, amino acids, and bicarbonate. XLH is the most common cause of isolated renal phosphate wasting leading to rickets. The inappropriately normal 1,25(OH)2D levels and the lack of renal losses of other minerals distinguish XLH from most other forms of phosphate wasting. In order to document hypophosphatemia secondary to renal phosphate wasting, a 2-hr urine sample is collected after at least a 4-hr fast, with a serum sample obtained after l hr [9]. Phosphorus and creatinine are measured in the serum and urine. The tubular
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Alkaline Phosphatase Alkaline phosphatase activity is high in children with most forms of rickets due to high bone turnover. However, the alkaline phosphatase activity in XLH tends not to be as high as in other forms of rickets, such as nutritional rickets. With treatment, the alkaline phosphatase activity decreases and is a good marker of healing rickets, although alkaline phosphatase rarely normalizes completely. In adults with XLH, alkaline phosphatase activity is often within the normal range.
PTH Although the elevation in PTH in XLH usually occurs as a result of treatment with phosphate, PTH can be mildly elevated at presentation [6]. However, unlike for nutritional rickets, hyperparathyroidism is not a prominent feature of XLH. Manifestations of XLH
In children, XLH is manifested primarily by short stature, lower extremity bowing due to rickets, and dental abscesses. In adults, osteomalacia, bone pain and stiffness, and enthesopathy are common findings.
FIGLIRE 2 Radiographof the lower extremities of a child with XLH demonstrating features consistent with rickets. Bowing of the femur and tibias and widening of the metaphyses are seen. This child was originally misdiagnosed as having metaphyseal dysplasia.
Skeletal Findings In children, XLH is characterized by rickets, although this is not an invariant feature of the disease [13]. The skeletal manifestations are most severe in the lower extremities, and bowing or a knock-knee deformity is common. Radiographically, findings are typical of rickets, with flaring, fraying, and cupping of the ends of the metaphysis of the femur and tibia (Fig. 2). Osteotomies are frequently required to correct the severe bow deformity. Bone mineral density has been reported to be normal or elevated in the axial skeleton (lumbar spine) and decreased in the appendicular skeleton in children and adolescents with XLH [14-18]. The bone density changes persist into adulthood [15,17]. Treatment does not correct the changes in bone mineral density [18]. Decreased joint mobility is seen in most adults with XLH [19]. Despite corrective osteotomies as children, many adults will continue to have an abnormal leg axis [19]. Early degeneration of the knee joints with shedding of the articular cartilage is seen in young adults, and osteochondritis-like lesions are seen in some adolescents [20]. Bone and joint pain, pseudofractures, and enthesopathy (proliferation of bone at sites of attachment of ligaments) are common findings [1,20,21]. Osteomalacia is the primary bone histomorphometric finding in XLH (Fig. 3). The osteoid volume is increased, mineralization lag time is prolonged, and rates of bone formation are decreased [1,22,23]. However, despite the osteomalacia, the trabecular calcified bone volume is
FIGURE 3 Biopsy of adult XLH osteomalacia. Goldner-stained undecalcified sections of iliac crest bone from an adult with XLH (magnification, x 360). Note the excess osteoid accumulation with relatively normal abundance of mineralized bone. (see color plate.)
normal or increased, suggesting an imbalance between the rates of bone resorption and formation [22]. The formation rate of new bone remodeling units is decreased, but the formation component of the turnover cycle is prolonged [23]. These findings suggest that abnormalities of the osteoblasts contribute to osteomalacia [23]. In adults, the degree of osteomalcia, as measured by the osteoid volume on bone histomorphometry, is positively correlated with the degree of bone pain [24].
25. Familial Hypophosphatemia and Related Disorders
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and TMP/GFR, confirming that the hypophosphatemia is due to renal phosphate wasting.
reabsorption of phosphate (TRP), which is the fraction of excreted phosphate that is reabsorbed by the kidney, can then be determined as follows [10]" TRP - 1 - (urine phosphorus x serum creatinine)/(serum phosphorus x urine creatinine). The normal range varies with age, and in children it is between 0.85 and 1.0, depending on the serum phosphorus concentration. From the TRP, the tubular threshold maximum for phosphorus per glomerular filtration rate (TMP/GFR) can be calculated using a nomogram developed by Walton and Bijvoet [11,12] (Fig. 1). The normal range for TMP/GFR in adults is 2.5-4.2 mg/dl [11,12]; it is higher in children. In general, the normal range for TMP/GFR in children is approximately the same as the normal range for serum phosphorus [9]. In XLH, the urine phosphorus levels are high and the serum phosphorus levels low. This~results in a low TRP
Vitamin D Metabolism
Vitamin D levels in XLH differ from those in many other forms of rickets and other causes of hypophosphatemia. 25(OH)D levels are normal in XLH, unlike in nutritional rickets due to vitamin D deficiency in which 25(OH)D concentrations are low. In addition, calcium levels are normal. 1,25(OH)2D is also normal, and this finding bears some discussion. In hypophosphatemia secondary to dietary phosphate deprivation, 1,25 (OH)2D levels are elevated because hypophosphatemia stimulates lcz-hydroxylase in the proximal tubule of the kidney, increasing the conversion of 25(OH)D to 1,25 (OH)2D, leading to elevated levels of 1,25(OH)2D. The lack of elevation in 1,25(OH)2D levels in XLH is part of the underlying defect, the cause of which is unclear.
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F I G U R E 1 Nomogram to calculate the TMP/GFR from the TRP [TRP = 1 - (urine phosphorus • serum creatinine)/(serum phosphorus • urine creatinine)] and the concurrent plasma phosphate concentration. A line is drawn through the plasma phosphate concentration (the left axis) and TRP (the diagonal axis), and the TMP/GFR value is where that line intersects the TMP/GFR axis (the right axis). The interior axes are in SIUs (mmol/L) and the exterior axes are in mg/dl. (Reproduced with permission from Walton and Bijvoet [12]).
25. Familial Hypophosphatemia and Related Disorders
Growth Short stature is a phenotypic hallmark of XLH, although there is great variability in final adult height, which has been reported to be -2.00 to -2.55 standard deviations (SDs) [1,25]. The mean final adult height in females is 150-152.4 cm and in males it is 157.3-158 cm [1,19]. Upper segment length is more normal (-0.91 SDS) compared to lower segment length (-2.59 SDs) [26]. The disease appears to have a greater impact on height in early childhood; children remain approximately 2 SDs below the mean for height after 5 years of age and have a normal pubertal growth spurt [27]. Conventional treatment has a significant positive impact on final adult height [28]. However, some reports refute this finding [29], and the response to therapy can be variable.
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in XLH. Chiari I malformation has also been described [44]. Hearing loss is a common finding in XLH [45-48] and may be more common in adults [47]. Cardiovascular problems, including hypertension and left ventricular hypertrophy, have been described in XLH patients, all of whom also had nephrocalcinosis [49]. The relationship to the disease and/or treatment is unclear [49]. Although psychological problems are common in XLH [19], the vast majority of XLH patients do not have major psychological problems. When they do have psychological problems, these are most likely due to the fact that patients have a chronic, sometimes deforming, disease and they do not appear to be part of the disorder per se.
Dental Findings Dental abscesses are common in children with XLH and start in early childhood. Twenty-five percent of patients developed abscesses of the primary dentition [30]. Dental problems continue into adulthood [1,19], during which more than 85% of patients have reported dental problems, many of whom required dental clearance [1]. Individuals who develop one abscess go on to develop multiple abscesses, indicating that the development of one abscess predicts future abscesses [30]. The primary tooth defect in XLH is in the dentin, whereas the enamel is relatively normal [31,32]. In normal teeth, calcium-phosphate calcospheres form and coalesce, forming the dentin matrix [3]. In XLH, the dentin is undermineralized and characterized by calcospherites that do not coalesce normally and thus are separated by large amounts of interglobular dentin [32,33]. This leads to expansion of the pulp chambers and weakening of the enamel barrier. Abscesses in XLH often occur in the absence of dental caries [32,34]. Since the enamel barrier is weakened, microorganisms can penetrate [32], possibly through microclefts [32] or infractures [34]. Microorganisms pass through the dentinoenamel junction, invading the dentin [32]. Dental taurodontism is seen in males [34,35]. The globular nature of the dentin and undermineralization does not appear to be impacted by therapy [36]. As a result, the impact of therapy on the dental manifestations of XLH, including the occurrence of abscesses, is not significant [30,37]. Other dental findings include a normal rate of dental development and an increased prevalence of ectopic permanent canine teeth in males with XLH [35]. Other Manifestations Ossification of the posterior longitudinal ligament leading to cervical myelopathy [38], ossification of the ligamentum flavum [39], spinal cord compression [40,41], and spinal canal stenosis [42,43] have all been reported
Autosomai Dominant H y p o p h o s p h a t e m i c Rickets A D H R is a rare renal phosphate wasting disorder. Bianchine et al. [50] provided the first description of a family with male-to-male transmission, thereby excluding the more common diagnosis of XLH. The father was a severely affected individual with a windswept deformity (valgus on one side and varus on the other) of the lower extremities. He had two affected daughters and one affected son. Aside from the father's increased tendency to fracture, affected individuals were reported to be similar to patients with XLH. Econs and McEnery [51 ] evaluated a large A D H R kindred with 23 affected individuals who presented with hypophosphatemia for age, normocalcemia, normal renal function, and inappropriately normal calcitriol concentrations. In 6 of 7 patients tested, there was no amino aciduria. In 1 patient, amino acid analysis revealed an increase in urinary leucine and valine excretion but no other renal impairment or glycosuria. None of the patients tested in this study had glycosuria or acidosis. Detailed analysis of this large kindred allowed exploration of the phenotypic variability of this disease in a large number of individuals who all had the same mutation, even before the gene responsible for the disorder was identified. In contrast to XLH, ADHR displays variable penetrance. In one study, patients were classified into two general groups: those who presented with renal phosphate wasting after puberty (group 1) and those who presented with renal phosphate wasting and rickets before puberty (group 2). Patients in group 2 had no history of rickets or lower extremity deformity as children and were first noted to have hypophosphatemia and symptoms after puberty. Patients were included in the second group if they had documented hypophosphatemia in childhood (n = 6) or a history of treatment for rickets or lower extremity deformity as a child and
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documented hypophosphatemia as an adult (n = 3). Nine patients met the criteria for inclusion into group 1 and nine patients met criteria for inclusion into group 2. Five patients could not be placed in either group. In three instances, the age of onset of clinically evident disease could not be verified. In two instances, affected adults were found to have hypophosphatemia on family screening but were asymptomatic. Additionally, two women were identified who were either carriers or had not yet presented with the disease since they had no previous history of rickets and were asymptomatic with a normal serum phosphorus and creatinine. These individuals were not included as affected patients. Patients in group 1, who became clinically affected after the onset of puberty, grew and developed normally. At the onset of clinically evident disease, they complained of bone pain, weakness, and insufficiency and pseudofractures. Indeed, these features are strikingly similar to those of patients with tumor-induced osteomalacia. Of note, all nine of these patients were female and in several instances the disease became evident soon after pregnancy. Nine individuals were identified who presented with clinically evident disease during childhood. Patients in group 2 presented with lower extremity deformities. Radiographs or reports of radiographs were available for six children, all of whom displayed radiographic evidence of rickets. In some cases, affected children had pronounced rickets. Age at presentation was 2 + 0.7 years and ranged from 1 to 3 years. Mean serum phosphorus concentration was 2.57 + 0.39mg/dl, and all patients were hypophosphatemic for age. These patients were clinically indistinguishable from XLH patients. Serum phosphorus concentrations as adults were available from eight of nine individuals in this group. In four individuals, hypophosphatemia persisted into adulthood. In two individuals, adult serum phosphorus concentrations (after therapy) were in the indeterminate range despite marked hypophosphatemia as children. Surprisingly, two individuals presented with renal phosphate wasting, lower extremity deformity, and rickets but later the renal phosphate wasting defect resolved. It remains unclear why some patients present with the disease in childhood and others present with the disorder as adults, and still others appear to maintain carrier status for most, if not all, of their lives. Apparently, individuals who presented as adults were able to compensate for the genetic defect during childhood and adolescence and then lost the ability to compensate. In some instances, the start of clinically evident disease was correlated with a physiologic stress such as pregnancy; however, this was not always the case. As noted previously, all nine individuals with delayed onset of penetrance of the disease were female. Although there are no data to confirm or refute the contention, it is
possible that postpubertal increases in sex steroids may play a role in the delayed onset of penetrance. An additional novel feature of A D H R is the loss of the renal phosphate wasting defect in some individuals. Data available for two patients (both male) indicate that they had renal phosphate wasting and rickets as children but later lost the renal phosphate wasting defect. Although fewer data are available for other members of the kindred, several other males appear to have lost the defect. A D H R is the only hereditary disorder of renal phosphate wasting in which patients may regain the ability to conserve phosphate. The mechanism that underlies the ability of patients to attain normal phosphate homeostasis is not known, but a better understanding of this mechanism may lead to better therapies for hypophosphatemic disorders. In summary, A D H R patients present with isolated renal phosphate wasting and inappropriately normal calcitriol concentrations, similar to patients with XLH. In contrast to XLH, A D H R displays variable age of onset, incomplete penetrance, and loss of the phenotype in at least two instances. H e r e d i t a r y H y p o p h o s p h a t e m i c Rickets with Hypercalciuria In 1985, Tieder et al. [52] described an unusual variant of hypophosphatemic rickets in a Bedouin tribe in which intermarriage had been prevalent for several generations. The affected family members were found to have an unusual metabolic profile. As in XLH, hypophosphatemia with renal phosphate wasting was evident, documented by very low TMP/GFR values. The serum alkaline phosphatase activity was elevated to a range comparable to that seen in XLH. However, other parameters of mineral metabolism were distinctly different from those observed in patients with XLH. Urinary calcium excretion was considerably elevated, and the circulating levels of PTH were suppressed to the low range. Of particular note, circulating 1,25(OH)zD levels were invariably elevated, indicating that the renal pathophysiology of this condition is strictly limited to defective epithelial renal phosphate transport. The normal 1~-hydroxylase trophism of low blood phosphate levels is intact, in striking contrast to XLH, in which the renal defect involves both a disruption of the normal hypophosphatemia-l~ hydroxylase axis and renal tubular phosphate handling.
Mixed Skeletal Phenotype Initial reports of the disorder described a characteristic osteomalacia, but osteopenia is also frequently noted. Osteopenia was striking in the three individuals that we cared for who had the disease, and it appears to be
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evident from an early age. This finding differs from the typical pattern of skeletal disease in XLH, in which increased bone volume is often present, and osteosclerosis may occur as the skeleton matures. Histological bone sections demonstrated evidence of active bone resorption in one study [52], but others have noted decreased numbers of osteoclasts [53,54]. Urinary excretion of the N-telopeptide of type I collagen (Ntx, the bone resorptive marker) was elevated in an individual affected with HHRH under our care compared to that of age-matched controls. It has been suggested that the chronically elevated levels of circulating 1,25(OH)2D may play a role in the development of osteopenia.
Renal Stones In the original report of Tieder et al. [52], renal stones were present only in the adult individual studied and not in the children. In contrast, renal stones have been present in both school-age and adolescent children affected with the condition in our clinic.
Clinical Physiology Other physiologic consequences of persistently elevated 1,25(OH)2D levels include a well-demonstrated state of calcium hyperabsorption and an exaggerated increase in serum phosphate following an oral phosphate load [52]. Renal losses can be significant, however; a short-term balance study in one patient demonstrated negative phosphate balance [53]. No apparent renal hypersensitivity to PTH is evident because injection of PTH has resulted in appropriate decreases in TMP/GFR and increases in urinary cAMP excretion. In follow-up studies of the patient reported by Burnett et al. [2], PTH and circulating 1,25(OH)2D levels decreased appropriately with intravenous calcium infusion, and 1,25 (OH)2D levels increased appropriately with PTH infusion, suggesting that the PTH/Ca regulation of the 10~hydroxylase axis is fully intact in the setting of HHRH.
Relation to Idiopathic Hypercalciuria HHRH has interesting implications regarding the pathophysiology of idiopathic hypercalciuria. Tieder et al [54] discovered that in family members of the originally described Bedouin tribe, absorptive hypercalciuria is a frequent finding. Detailed study of these individuals demonstrated that phosphate wasting and elevations in circulating 1,25(OH)2D occur in this group but to a lesser degree than seen in those with frank HHRH. The serum and urinary calcium levels are comparable between the idiopathic hypercalciuria group and related patients with HHRH. Thus, the biochemical cor-
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relate of the affected skeleton appears to be a more severe degree of phosphate wasting. Although this may seem to be a useful model of idiopathic hypercalciuria, it should be noted that it is not the metabolic profile evident in the majority of patients carrying the diagnosis of idiopathic hypercalciuria in most clinic settings, where renal phosphate wasting is generally not observed [55].
Management and Course HHRH is usually managed by administration of phosphate salts alone. Attention to both the bone disease and hypercalciuria is important. The therapeutic strategy is to decrease the elevated circulating 1,25(OH)2D levels, effecting a decrease in intestinal calcium absorption, and to provide an increase in ambient phosphate levels to enhance skeletal mineralization. Because of the unique pathophysiology, there is little risk of developing hyperparathyroidism, unlike in the management of XLH in which hyperparathyroidism is notoriously problematic. Reports of long-term monitoring are limited, but in the authors' experience improvement in bone pain and height velocity has occurred with therapy. No recurrence of renal stones has occurred in two patients who experienced nephrolithiasis prior to the onset of therapy. Bone mineral density has improved, but one patient continues to have low bone density. Thus, bisphosphonates or other antiresorptive therapies may be important in the long-term management of affected adults, although there are theoretical concerns about using bisphosphonates in the setting of osteomalacia. Generous hydration is important, and avoidance of a high sodium intake is recommended.
A Candidate Gene? Because the normal physiologic response to chronic hypophosphatemia can explain the spectrum of clinical and biochemical findings observed in patients with HHRH, it has been proposed that the defect is specific to the regulation of epithelial renal tubular transport of phosphate. This differs from the pathophysiology of XLH, in which a defect proximal to isolated renal tubular transport is hypothesized, because other features of the disease are not easily explained by a solitary defect in apical phosphate transport. The most abundant renal tubular phosphate transporter in mammalian kidney is the type II Na-Pi cotransporter Npt2. This transporter localizes to the brush-border surface of proximal renal tubular cells, and its expression is regulated by PTH and dietary phosphate. The gene for this transporter has therefore been proposed as a candidate for the site of mutation in HHRH. Thus, two studies have examined the possibility that HHRH maps to the chromosome
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5q35 locus, where Npt2 is located. Jones et al. [56] examined four families, including members of the Bedouin tribe reported by Tieder et al. [52]. No mutation in Npt2 or evidence for linkage to the relevant area could be demonstrated. Van den Heuvel et al. [57] sequenced the entire open reading frame of the N p t 2 gene in affected and unaffected members of another family and excluded a mutation of Npt2 in this region as well. Linkage disequilibrium studies in the Bedouin family also excluded the association of H H R H with chromosome 6p22, the site of Nptl, a lesser abundant renal phosphate transporter. The phenotypic variability in the reported families suggests that H H R H may be the manifestation of different genetic defects. Variable expression within an autosomal recessive or codominant inheritance pattern is likely. It may be possible that some cases represent a twogene disorder with variable penetrance. Interestingly, in one of our (nonconsanguinious) families, asymptomatic hypercalciuria with normophosphatemia was evident in the mother of an affected H H R H boy, whereas the father had normocalciuria and asymptomatic hypophosphatemia. Other candidate genes remain to be examined, including less abundant renal transporters and phosphate transport modulators such as diphor-1 [58] and Pius [59].
Tumor-lnduced Osteomalacia TIO is a hypophosphatemic syndrome with clinical features similar to those seen in XLH. In general, a useful point of clinical distinction is the recognition that TIO is usually an acquired phenotype, in contrast to the inherited hypophosphatemic disorders that tend to manifest by the second or third year of life. There are exceptions to this generality, of course, in that later-onset forms of familial hypophosphatemic rickets clearly occur, and sporadic phosphate wasting disorders such as linear sebaceous nevus syndrome have been reported as a congenital occurrence [60]. Most reports of TIO identify the characteristic hypophosphatemia and renal phosphate wasting observed in patients with XLH [6164]. The serum phosphate levels may be lower than those seen in XLH, but there is considerable overlap of serum 1,25(OH)zD levels between the syndromes. The TIO patient may exhibit more severe symptoms, including bone pain and muscle weakness, whereas these complaints are more tempered in the XLH patient. Adult TIO patients frequently present with fractures and proximal muscle weakness. Elevated serum alkaline phosphatase activity is usually present, in some cases in a range higher than that usually seen in XLH. Most reports indicate normal serum calcium levels, although this parameter has been infrequently described as low. The circulating PTH levels are also variable, although they are usually normal. The
circulating 1,25(OH)2D level is low or normal, and it is often lower than that seen in XLH. Thus, the typical clinical and biochemical phenotype is similar to that seen in XLH, but the degree of severity in the abnormality is often greater.
Pathology A wide variety of pathologic diagnoses have been attributed to TIO-associated tumors. Tumors from most patients are classified as benign tumors of mesenchymal origin. A leading diagnosis in tabulated reviews of the syndrome is hemangiopericytoma, a vascular lesion that contains an abundance of pericytes [62]. Spindle cells are prominent in many reports. Other frequent pathologic descriptions are ossifying and nonossifying fibromas and intraskeletal lesions. It should be emphasized, however, that significant malignancies may be associated with TIO, including prostatic carcinoma, oat cell carcinoma of the lung, and, more commonly, osteosarcoma. Obviously, one concern is that although many of the tumors are considered benign at the time of detection, their natural history is not well documented, and it may well be that over time a malignant transformation may occur. One recent case history is suggestive of this phenomenon [65]. The similarity in appearance of many of these tumors has been noted by Weidner and Santa Cruz. [66], who classified this somewhat heterogeneous group into four distinct morphologic patterns: a mixed connective tissue variant with usual origins in soft tissue, prominent vascularity, osteoclast-like giant cells, osseous metaplasia, and/or poorly developed cartilaginous areas; osteoblastoma-like; ossifying fibroma; and nonossifying fibroma. The latter three groups tend to occur in bone.
Evaluation The occurrence of the XLH phenotype in an acquired setting or in the absence of family history raises the possibility of a diagnosis of TIO. Thus, a careful search for a possible tumor is an important step in the evaluation of such patients. Not only is there the possibility that the hypophosphatemia may signal an occult malignant neoplasm but also it is likely that significant morbidity can be avoided by removal of a causal tumor, whether benign or malignant. One major difficulty is the fact that many of the described lesions are extremely small and not detectable by physical examination or plain radiographic techniques. This feature is not universal, however, because some tumors are quite large. There appears to be a propensity for many of the small tumors to occur in the head and neck, and detailed imaging of the sinuses and jaw areas with computed tomography (CT) or magnetic
7_5. Familial H y p o p h o s p h a t e m i a and Related Disorders
resonance imaging (MRI) has been suggested. Sites within bones are also typical. Pelvic tumors are described; other unusual clinical presentations include plantar warts and tibial stress fractures [67]. Recent success in the localization of small TIO tumors with indium-111 pentetreotide or octreotide scintigraphy suggests that this technique is a useful measure in the evaluation of this syndrome [68]. Technetium-99 methylene disphoshonate scintigraphy has also been employed but tends to demonstrate increased uptake of isotope in areas of active osteomalacia rather than localizing a tumor per se. Indeed, with the increasing awareness of this syndrome, coupled with recent reports describing detection of these tumors using MRI techniques [69], scintigraphy technology, and CT scans, more of these tumors will certainly be identified, and we may discover that they are not as rare as once thought.
Treatment and Clinical Course The clinical course of this disease is dramatically affected by removal of the tumor. In the absence of identification of a tumor, many patients have been treated with the combination of vitamin D metabolites (preferably calcitriol) and oral phosphate salts, employing a strategy similar to that for the treatment of XLH. Given the severity of disease, however, treatment of the condition in this manner does not usually result in an ideal clinical response. Tumor removal, on the other hand, almost always results in a rapid and complete response. Serum phosphate, TMP/GFR, and circulating 1,25(OH)2D levels correct within hours to days of tumor removal [70]. The 1,25(OH)2D levels may actually rebound to and briefly become elevated after removal of the offending tumor. The skeletal changes, because they have developed over time, often require months to correct [71]. Even biochemical markers of bone turnover, such as serum alkaline phosphatase activity, tend to correct well after the renal phosphate wasting and vitamin D metabolism defects are normalized. Nevertheless, in the absence of identification of a tumor, or if resection is not possible, there is usually some benefit to treatment with calcitriol and phosphate. Recently, an individual with an unresectable tumor who could not tolerate oral phosphate medications was described to have successful symptomatic relief when given long-term intravenous phosphate in concert with oral calcium and vitamin D [72]. This therapy was not without complications, however, because central venous catheter-related infection occurred. Thus, intravenous phosphate therapy should only be employed when the promise of benefit from the therapy outweighs the risk of catheter-related complications. Another approach to treatment is the use of octreotide, a synthetic somatostatin analog [73]. A TIO-
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associated tumor was detected by indium-111 octreotide scintigraphy, providing evidence for the presence of somatostatin receptors on the tumor. The patient was then given 50-100gg of subcutaneous octreotide three times daily for 2 weeks prior to surgical resection of the tumor. A dramatic correction of serum phosphate and renal tubular phosphate wasting occurred as well as correction of elevated serum alkaline phosphatase activity and serum osteocalcin. In this patient, circulating 1,25 (OH)zD levels were in the normal range prior to administration of octreotide and remained so during treatment. An unexpected effect of this therapy was a marked hyperparathyroidism, temporally related to octreotide administration, that resolved completely upon tumor resection. There are several anecdotal reports of early diagnoses of TIO with no recognizable tumor but after multiple years of therapy the tumor enlarged and osteosarcoma became evident. Review of early radiographs revealed small, suspicious lesions. Others have had recurrent disease following initial removal of tumor, progressing to death. Thus, it is highly recommended that one establish a reasonable margin of resection that is free of tumor, particularly in tumors with high degrees of atypia or mitotic elements.
Pathophysiology The pathophysiology of TIO has been puzzling for decades. The dramatic response to surgical removal of the tumors suggested that a substance secreted by the tumor has direct effects on the renal tubule resulting in severe phosphate wasting. The subsequent bone disease has been explained by this severe hypophosphatemia. When circulating vitamin D metabolite levels were documented in TIO and in XLH, it became evident that a similar pathway was affected, such that both tubular phosphate transport and vitamin D-l~-hydroxylation activity were aberrant in both syndromes. This finding gave further credence to the hypothesis that a circulating factor may mediate the pathophysiology of XLH as well as TIO. An attempt to document renal phosphate wasting activity was reported in the 1970s. A child with the epidermal nevus syndrome variant of TIO underwent excision of an affected skin lesion and improved following this procedure [74]. Material from the resection was administered to a dog that developed phosphaturia following the infusion. Although this experiment may have suffered from inadequate controls, it began the long quest for tumor-derived substances that could mediate renal phosphate wasting. Difficulties in isolating and purifying a causative material have been hampered by the low abundance of tissue, the very limited amount of source material from the small excised tumors, and the
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limited duration of secretion of material from tumor cells in culture. Nevertheless, with the advent of subtraction RNA technology, several candidate species have recently been identified. A great deal of supportive data have been generated by several groups implicating FGF23, a novel member of the fibroblast growth factor family, as a causative factor [75-77]. This finding is of particular interest because another hypophosphatemic syndrome that manifests the XLH phenotype, ADHR, has recently been found to result from specific mutations in FGF23 that apparently retard its degradation [78]. Others have found that a large glycosylated protein, matrix extracellular phosphoglycoprotein (MEPE), is abundantly secreted from TIO tumors, but an effect of this material on renal phosphate transport has not been clearly documented [79]. On the other hand, when FGF23 was provided to mice via transfected cells overexpressing the material or used in a transgenic overexpressing mouse model, mice developed a syndrome with characteristic features of TIO [76,80]. It is unclear whether FGF23 is the direct agent mediating phosphate transport inhibition at the level of the renal tubular cell or whether some other agent mediates this effect of FGF23. There is preliminary evidence that frizzled-related protein 4 (FRP4) is also secreted from TIO tumors, inhibits phosphate uptake in vitro, and is degraded by PHEX [81-83], although the role of FRP4 is unclear. Finally, smallmolecular-weight substances isolated from TIO tumors have been shown to have direct inhibitory activity on renal phosphate transport as well [84].
Related Syndromes There are other circumstances in which the XLH or TIO phenotype appears secondary to unusual lesions or disease. These are discussed in the following sections.
Epidermal Nevus Syndrome Epidermal nevus syndrome (ENS) is a rare sporadic disorder that may involve severe hypophosphatemic rickets, clinically similar to that seen in TIO [85]. This disorder usually presents early in life and has been noted to be a congenital finding. Multiple types of nevi have been reported, including sebaceous, verrucous, and hyperpigmented ones. There have been surrounding areas of hypopigmentation and characteristic orange-colored nevi. A number of other anomalies may occur. Growth can be severely retarded. Structural central nervous system malformations have been described, including macrocephaly, ventriculomegaly, or hemimegalencephaly. Deafness, cortical blindness, nystagmus, intracranial vascular malformations, and cranial nerve palsies have been reported. Central precocious puberty has also been described.
Unlike the dramatic response to surgical removal of the lesion, there is usually not an impressive response to nevus removal in ENS. Although there are reports of correction of serum phosphate values following excision of epidermal lesions, this has occurred in a minority of patients, and only transient correction has generally been the case. Furthermore, the skeletal lesions in ENS are quite severe and appear to be more complex than those seen in patients with TIO or XLH. Radiographic rachitic deformities of the growth plates are present. In one patient observed by the authors, these deformities responded well to treatment with phosphate and calcitriol. However, skeletal radiographic examination of this patient revealed a severe diminution in bone mass, with cyst-like scalloping seen throughout the diaphyses of long bones. Scoliosis and kyphosis are described. Extreme paucity of trabecular bone was evident on histological specimens from the patient observed by the authors, as well extensive osteoid accumulation suggestive of a near-complete failure to mineralize. In summary, this syndrome appears to manifest hypophosphatemia and renal phosphate wasting, as does TIO and XLH. However, it is not clear that ENS is entirely analogous to these disorders, or whether secretion of phosphaturic substances by cells associated with nevi or their surrounding skin mediate the disease. Multiple anomalies are likely present, and the severity and nature of the bony lesions suggest that a more extensive process is occurring.
McCune-Albright Syndrome McCune-Albright syndrome (MAS) is a chimeric disorder usually characterized by cafr-au-lait pigmentation, fibrous dysplasia of bone, gonadotropin-independent precocious puberty, and occasionally other endocrine disorders. The manifestations are due to a somatic activating mutation in diffuse tissues of Gsct, a subunit of the GTP exchange protein that functions to couple cell membrane hormone receptors with adenylate cyclase. The result is constitutive activation of the cAMP-PKA signaling pathway. The extent of the fibrous dysplasia throughout the skeleton can be quite variable in MAS. Rickets or osteomalacia have also been reported as complications of the syndrome. In cases in which an etiology has been sought, associated hypophosphatemia and renal phosphate wasting have been observed. In a systematic study of 42 MAS patients, hypophosphatemia and renal phosphate wasting were seen in approximately 50% and tended to correlate with the extent of fibrous dysplasia [86]. The renal tubular phosphate wasting in MAS has been considered to result from the presence of the activating mutation in the kidney, generating a PTHlike effect on phosphate handling, or to occur as a result of a circulating material generated by the skeletal lesions.
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This hypothesis was put forward by Dent and Gertner [87] after noting the correction of hypophosphatemic rickets following surgical excision from bone of fibrous dysplasia lesions. The correlation of the incidence of biochemical parameters of renal phosphate wasting with the extent of skeletal involvement supports this hypothesis. It is of interest that the degree of renal phosphate wasting is not as severe as that typically seen in TIO, and presumably this explains the finding that frank rickets is less frequent than hypophosphatemia in individuals with MAS. Other Neuroendocrine Entities
A TIO phenotype has been described in other neuroendocrine entities, such as neurofibromatosis, neurinoma, Schwannoma, and paraganglioma [61,88,89].
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however, no pharmacokinetic data are available to support this measure. By 18-24 months of age, parents can usually supervise the child swallowing the capsule. Phosphate preparations used later in childhood are not convenient in infancy. Phospha-Soda (Fleet's) solution, which contains 127mg of elemental phosphorus per milliliter, is a convenient preparation in this regard. An older child may use Neutra-Phos or Neutra-Phos K powder (250mg elemental phosphorus per packet or capsule) dissolved in water, drinking the solution at intervals through the day. When the child is old enough to chew or swallow a tablet, we prefer K-Phos Neutral, which contains 250mg of elemental phosphorus per tablet. Others have experience with K-Phos No. 2, a urinary acidifier with decreased sodium content compared to K-Phos Neutral.
Monitoring TREATMENT R e c o m m e n d a t i o n s for Medical M a n a g e m e n t of XLH
Treatment in Early Childhood Physician awareness of XLH is an important issue because a correct diagnosis early in the course can be advantageous to the child. Children in affected families should therefore be screened for abnormal serum and urine phosphorus levels and serum alkaline phosphatase activity within the first month of life and at 3 and 6 months. Results suggestive of XLH are an indication for radiographic examination. If rickets is present, therapy with calcitriol and phosphate is initiated. Occasionally, we have carefully followed children with XLH who did not have rickets, but the vast majority of patients do go on therapy. We have seen normal-appearing adults with XLH who never received therapy.
Initial Doses Initial dosing for an infant in the first 2 years of life is generally 0.25 lag once or twice daily, with 250-375 mg of elemental phosphorus daily, provided in two or three dosages. Calcitriol has recently been formulated in solution, which is convenient to administer to infants and small children. Alternatively, the liquid contents of a calcitriol capsule can be extracted using a 1-ml syringe and an 18-gauge needle. Warming the capsule slightly in a microwave oven softens the coating, making the liquid easier to extract. The oil-based liquid can be added to formula or administered directly to the child from the syringe after removal of the needle. We have also orally administered a calcitriol preparation for intravenous use;
Approximately 2 weeks after the onset of therapy, serum calcium and PTH levels and urinary calcium and creatinine excretion should be assessed. The goal is to maintain normal serum calcium and PTH concentrations and normal urinary calcium excretion. In young children, sampling should be repeated at 3-month intervals. In older children, during stable growth periods and in the absence of dose changes or complications, frequency of visits may be reduced to every 3 or 4 months. During active dose changes at any age, however, patients should have laboratory studies monitored monthly. After 1 or 2 years of therapy, and intermittently thereafter, a renal ultrasound study should be performed to assess the development of nephrocalcinosis. If the appearance of the kidneys on these examinations is stable, the frequency of ultrasonographic examination can be decreased. We perform radiographs of the knees after 1 year of therapy and every 2 years thereafter throughout the growing years to ensure that the epiphyseal defect is optimally responsive to therapy. Thus, the radiographic appearance of the epiphyses is an important gauge of medical therapy and can be an important indicator of the need for dosage adjustments.
Adjustments in Dosage In practice, the dosage range in XLH patients under our care is quite wide, dependent on severity of the rickets, the response to therapy, and if complications are encountered. One of the most important principles in the management of XLH is to maintain an appropriate balance of the two types of medication. Excessive calcitriol will lead to the development of hypercalcemia and hypercalciuria, whereas excessive phosphate dosing often results in secondary hyperparathyroidism. In
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general, when an increase in dosage is indicated from the clinical evaluation, and when no complications of treatment are present, balanced increases in calcitriol and phosphate should be employed. Minimal increments in dosages of each agent are prescribed and follow-up laboratory investigation should be performed within 1 month of the change in dose. One particularly recurrent observation in our experience is that after significant correction of the bone disease, perhaps related to optimizing skeletal mineralization, a dose that was appropriate for months to years is suddenly excessive. Thus, the scheduled, continual monitoring of this situation is in the patient's best interest from a therapeutic perspective and to avoid toxicity. Furthermore, it is often necessary to increase dosages of these medications as the pubertal growth spurt begins. Rapid growth during puberty may result in greater mineral demands and worsening of bowing defects so that a transient increase in dosing is often advantageous. Thus, monitoring of patients at 3- or 4-month intervals through puberty until cessation of growth is recommended. In later childhood, we usually use doses of calcitriol that average 0.75 lag per day, rarely exceeding 1.5 lag per day; 10-50ng/kg/day of calcitriol is the usual body weight normalized dosage. However, because responses to therapy are quite variable, dosing is not strictly based on body weight for a given individual. In fact, most patients in our clinic receive calcitriol at the low end of this range (10-25 ng/kg/day) except for transient increases during puberty. Measurement of PTH is a useful guide to therapeutic decisions as well. An increase in calcitriol dosage in response to an elevation in circulating PTH is often a useful maneuver. If suboptimal healing of the epiphsyeal lesions is evident on knee radiographs, an increase in the calcitriol dose in conjunction with an increase in the phosphate dose is usually recommended. Elevated PTH values, without hypercalcemia or hypercalciuria, generally call for an increase in calcitriol alone. In the event of hypercalciuria or hypercalcemia with normal PTH values, borderline vitamin D intoxication may be present, indicating the need for a decrease in the calcitriol dose; if hypercalcemia is severe, temporary cessation of all therapy is recommended. After the growth spurt and achievement of final height, dosages may be lowered to an adult maintenance doses. Treatment of adults is controversial. Adult patients frequently complain of bone pain. Treatment does lessen osteomalacia and bone pain, and it is reasonable to treat patients with this complaint. Additionally, it is advisable to treat patients who have nonunion after fractures or osteotomies since treatment may improve fracture healing. However, in light of the complexity of therapy, possible side effects, and lack of increased risk of fracture in patients without pseudofractures, we generally do not
recommend treatment of assymptomatic patients who do not have pseudofractures. Adults who are treated are prescribed medications based on symptomatology; a dose of 0.75-1.0 lag of calcitriol daily is typical. In children, dosages of elemental phosphorus are 1 g per day on average and rarely exceed 2 g per day. Diarrhea or persistent dose-related abdominal pain or discomfort indicate the need to reassess the dosage schedule and usually reduce the total phosphate dose. The development of secondary hyperparathyroidism is an indication to alter the calcitriol/phosphate balance by decreasing phosphate or increasing the calcitriol dosage. Given the diurnal fluctuation in serum phosphorus levels, variability in kinetics of phosphate absorption, and the baseline hypophosphatemia in fasting patients with XLH, serum phosphorus should not be employed as an indicator of phosphorus demands. Indeed, this may be counterproductive: In the case of decreasing serum phosphorus levels, the physician may be prompted (inappropriately) to increase the phosphate dosage when the underlying reason for the decreasing serum phosphate value is advancing hyperparathyroidism. An increase in phosphate dosing in this case is likely to worsen the hyperparathyroidism, often leading to further renal phosphate losses, with no improvement in serum phosphorus. Administration of phosphate at three or four conveniently spaced intervals throughout the day is recommended. Furthermore, it does not appear to be necessary to awaken the child to provide medication through the night because a diurnal increase in serum phosphorus occurs at night in patients with XLH independent of therapy [6]. Although serum alkaline phosphatase activity usually decreases slightly with therapy, this value rarely normalizes until adult age and usually is not a useful marker of disease at that time. Therefore, although serum alkaline phosphatase activity is of interest to periodically monitor, its value as an indicator of therapeutic response is limited.
Adjunctive T h e r a p i e s Human Growth Hormone
Within the past 10-15 years, there has been an interest in determining whether adjunctive treatment with human growth hormone (hGH) has a positive effect on the treatment of XLH. Initial reports were promising but only demonstrated an increase in growth velocity over a relatively short term, with no long-term outcome data. Recent studies have provided longer term follow-up data. In a study by Baroncelli et al. [90], therapy was continued to adult height in a group of very short children with XLH who were shown to respond to a 3-year treatment course of hGH in an earlier report [91]. Treated patients were administered 0.3 mg/kg/week of
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25. Familial H y p o p h o s p h a t e m i a and Related Disorders
hGH (the dose often used in patients with Turner's syndrome), increased to 0.45 mg/kg/week during pubertal years. The study compared the treated group to a group of taller stature at the outset of the study who remained untreated with hGH and did not show the degree of improvement in height z scores observed in the hGH-treated group. The greatest effect occurred in the first year of treatment; the height z scores at adult final height were actually lower in this group than after the initial 3 years of therapy. In general, those beginning therapy at a younger age demonstrated a greater increment in height z score. No significant side effects were reported. These data are consistent with those of Seikaly et al. [92], who reported an increase in height z scores following 1 year of therapy at relatively high doses (0.56mg/kg/week) in a randomized crossover study. This study may have been contaminated by the "catchdown" growth that often follows cessation of hGH therapy during the control period in subjects who were randomized to receive hGH in the initial phase of therapy. This study also noted an increased effect of treatment on truncal height, thereby potentially exaggerating the disproportionate nature of the skeletal phenotype in XLH. This phenomenon has been reported by Haffner et al. [93], who also documented an accelerated increase in skeletal age during treatment with hGH. On the other hand, Cameron et al. [94] demonstrated that 1 year of therapy with hGH had no significant effect on height or height velocity when prepubertal children were treated with 0.18 mg/kg/week of hGH, a dose typically used in overt growth hormone deficiency. Thus, the cumulative evidence suggests that, as in children with idiopathic short stature, at high-dose therapy for several years an effect on adult height in a selected group of short children with XLH may be possible. However, the significance of this effect may be limited. The underlying skeletal disease does not appear to be significantly effected for better or worse, but a significant risk of further exaggerating trunk/limb disproportion accompanies this approach. There does not appear to be any lasting effect of acute changes in calcium and phosphorus homeostasis that are seen in the early weeks of hGH therapy [95]. Thus, it is difficult to make a case for routine treatment of XLH with hGH.
Other Adjunctive Therapies 24,25(OH)2D3 in addition to standard treatment has been employed with success in enhancing radiographic healing of epiphyseal lesions as well as reducing indices of osteomalacia in adults [96]. This therapy is impressive in its ability to decrease circulating PTH levels, which may be very important in the mediation of disease sever-
ity. This metabolite is no longer readily available. It is possible that other nonhypercalcemic vitamin D analogs would be of use in this setting. Dipyramadole can be an inhibitor of phosphaturia; however, trials using this substance in XLH failed to demonstrate any beneficial effect [97]. Thiazide diuretics or amiloride have been suggested to increase renal calcium reabsorption and to enhance mineralization [98]. The long-term effects of this measure have not been reported. Finally, calcium supplementation may be necessary in some individuals who fail to ingest adequate dietary calcium. Surgery Some patients require surgical correction of severe bowing, regardless of medical intervention. The approach to surgery for XLH is physician dependent, but several guidelines have been established. In general, candidates for osteotomy in childhood should have severe bowing, with the projection that irreversible progression of the defect is unavoidable as growth continues [99]. Children younger than 6 years of age should not generally undergo an osteotomy for correction of XLH because bow defects are not often severe, and aggressive medical therapy has a reasonable chance of correcting the deformity over time [100]. We have seen that healing of osteotomy in young children may be prolonged. Surgical procedures vary: Fixation with plating and stapling have been performed, and external fixation devices have recently been used [101]. We often have an experienced orthopedic surgeon examine children with lower extremity malignment at approximately 10 years of age. Obviously, an orthopedic surgeon with experience in procedures in children with XLH should be consulted before any intervention, and medical expertise throughout the course of surgical intervention and healing is important. Medication dosages may require adjustment due to immobilization-associated hypercalcemia following osteotomy, but therapy should continue to facilitate healing of the osteotomy. Finally, leglengthening procedures have been attempted in a few patients with XLH [25]. Complications Complications of therapy for XLH include hypervitaminosis D, hyperparathyroidism, and soft-tissue calcification, particularly in kidney (Fig. 4). Significant clinical consequences of these complications can usually be avoided with careful monitoring. Treatment is essentially a compromise in this setting: In order to effectively achieve mineralization of the growth plate, the systemic mineral load to the patient must be increased. However, one must not allow this mineral load to become so
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lngrid A. Holm et al. Elevations in circulating PTH occur at night in most individuals with XLH [6].
Soft-Tissue Calcification
FIGURE 4 Renal ultrasound of nephrocalcinosis. Diffuse evidence for calcifications is seen.
excessive that significant soft-tissue calcification or derangement of parathyroid hormone function result.
Hypervitaminosis D Hypervitaminosis D is manifest by hypercalcemia and hypercalciuria and is not frequently encountered using the previously described regimen. Instead, the occurrence of hypervitaminosis D was much more frequent with earlier generations of the therapeutic protocol, particularly when high doses of fat-soluble vitamin D were used and when serum and urine biochemistries were monitored infrequently. Death from unrecognized vitamin D intoxication occurred in the past with treatment of XLH, and the memory of such events has influenced attitudes about medical therapy in surviving family members. The 1~-hydroxylated vitamin D metabolites currently utilized are more polar, are not stored in fat, and have a short half-life. As a result, these metabolites escape the toxic effects more rapidly than native vitamin D. Fortunately, with the availability of 10t-hydroxylated vitamin D metabolites, hypervitaminosis D as a complication of treatment of XLH is much less common today.
Hyperparathyroidism Hyperparathyroidism occurs frequently in XLH; therefore, circulating PTH must be routinely monitored in individuals with the disease, particularly if receiving therapy. The stimulatory effect of treatment with phosphate salts on PTH secretion in XLH is well described [102,103]. One cardinal rule of therapy is that phosphate supplementation should never be given as single therapy. Concomitant use of calcitriol dampens the stimulatory effect of phosphate on PTH secretion and should always be part of the treatment regimen.
Soft-tissue calcification, particularly at the level of the renal medullary pyramids (nephrocalcinosis), can be detected by renal ultrasound examination [104]. Many patients with XLH in our clinics demonstrate nephrocalcinosis within 3 or 4 years of beginning therapy; however, poorly compliant patients are less likely to develop this finding. Most patients develop a modest grade of calcification and do not progress to more severe stages. Occasionally, a slight decrease in severity occurs with time. This experience is similar to that of Kooh et al. [105], who reported an 80% incidence of nephrocalcinosis in patients with XLH and have seen no associated sequelae in patients with nephrocalcinosis present for up to 15 years. A decrease in glomerular filtration rate, however, was reported in one patient with hypertension and nephrocalcinosis [104]. The pathogenesis of the lesion is not clearly understood; some have suggested that increased urinary oxalate occurs with phosphate loading, and that calcium oxylate precipitation is enhanced [106]. Others have shown that the lesions are composed of calcium phosphate precipitates and can be reproduced in the H y p mouse by administration of phosphate [107]. It is also possible that increased PTH levels predispose the tissue to calcification. Other related problems in XLH include calcification of the entheses [108], which is not thought to be treatment dependent. In addition, ocular calcifications [109] and myocardial and aortic valve calcifications [110] have been described.
FAMILY/GENETIC STUDIES XLH as an X-Linked D o m i n a n t Disorder XLH is inherited in an X-linked dominant fashion [111]. Features of XLH that are consistent with X-linked dominant inheritance include the lack of male-to-male transmission and a female-to-male prevalence ratio of approximately 2 to 1. The presence of a gene dosage effect, which is characteristic of many X-linked dominant disorders, is not overtly evident in XLH. One would expect a hemizygous male (who carries one X chromosome and thus only one defective gene) to be more severely affected than a heterozygous female (who carries two X chromosomes, one of which has the defective gene and one of which has a normal gene). In XLH, the assumption has traditionally been that heterozygous females are less severely affected than hemizygous males due to a gene
25. Familial Hypophosphatemia and Related Disorders
dosage effect [3]. In practice, however, it has been very difficult to prove even a minor gene dosage effect in humans [112]. There does not appear to be a gene dosage effect on serum phosphate levels because males and females have similar degrees of hypophosophatemia [113,114]. In one study [112], biochemical parameters, including serum phosphate levels and height z scores, were similar between untreated prepubertal girls (heterozygous) and boys (hemizygous). However, girls appear to respond better to calcitriol and phosphate therapy compared to boys [115]. In addition, radiographic and bone scan findings are less severe in adult women [116], and although the disease can be equally severe in men and women, there tends to be a lower incidence of symptomatic bone disease among women [1]. These findings suggest a gene dosage effect in adults and with treatment but not in untreated prepubertal children. There is also evidence for a puberty-dependent gene dose effect in teeth in individuals with XLH. The tooth phenotype of heterozygous females is between that of hemizygous males and normal [114], and males are more likely to be affected with dental abscesses [30]. However, there is evidence that the gene dosage effect only occurs in secondary dentin (in individuals 15 years of age or older), not in primary dentin (in children younger than 15 years of age) [113]. One explanation for lack of a gene dosage effect could be preferential inactivation of the chromosome carrying the normal X chromosome in females with XLH. However, at least in the blood, there appears to be random X inactivation [117]. This does not rule out the possibility that the X inactivation pattern in the bone, teeth, and/or kidney is not random. Familial v e r s u s S p o r a d i c C a s e s In most studies of individuals with XLH, approximately one-third of cases are sporadic. There are no differences in severity between familial and sporadic cases, although serum phosphate levels are lower in sporadic cases [112]. These findings suggest that genetic anticipation does not occur with XLH. Individuals with the sporadic occurrence of XLH tend to have children with XLH, suggesting the sporadic occurrence represents a new mutation.
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the gene defective in XLH using positional cloning techniques [130]. The name was derived from the homology of PHEX to a family of neutral endopeptidases, including neprilysin (NEP) [131], endothelin converting enzyme-1 (ECE-1) [132], and the Kell antigen [133]. The full-length PHEX cDNA [134,135] consists of a 2247-bp coding region spanning 22 exons [136]. The full-length PHEXis 60% similar to NEP and 57% similar to ECE-1 on the amino acid level [135]. Studies of PHF_.Xin Families with XLH The identification of PHEX made it possible to perform mutational analysis in XLH patients [136-145] and to distinguish individuals with XLH from those who may have ADHR. In these studies, PHEX mutations were identified in 60-95% of FHR patients. Although most of the mutations identified are in the coding region, mutations in the noncoding regions have also been identified [146]. In most studies, PHEXmutations were detected in a smaller percentage of sporadic cases (28-93%) compared to familial cases (71-100%) [136,137,142,143]. This may reflect the fact that it is easier to detect some types of mutations in males than in females (i.e., large deletions can be missed in females if sequencing or SSCP is the mutation detection method). In familial cases, males are usually tested (there is usually an affected male in the family), whereas two-thirds of sporadic cases are female. Thus, the lack of mutation detection in sporadic cases may simply reflect the difficulty in detecting mutations in females. Another possibility is that some of the sporadic cases may have ADHR; however, this has not been the case in a large number of cases [78]. Although a variety of mutations have been detected, suggesting that some of the variability in XLH may be explained by the variety of PHEX mutations, our phenotype-genotype analysis of PHEX in XLH has disputed this hypothesis [138]. There is now a database of PHEX mutations, phenotypes, and authors (http://data.mch. mcgill, ca/phexdb) [147].
MOLECULES, PATHOPHYSIOLOGY, AND LESSONS FROM ANIMAL MODELS M o u s e M o d e l s for XLH
Identification of t h e G e n e Defect in XLH Efforts to understand the defect in XLH led to the localization of the XLH locus to Xp22.1 by linkage analysis in some families [118-129]. PEX, now termed PHEX (phosphate regulating gene with homologies to endopeptidases, on the X chromosome), was identified as
There are two mouse models for XLH mapping to the mouse X chromosome [148]~the Hyp mouse [149] and the Gy mouse [150]. The manifestations of Hyp in the mouse are very similar to those of XLH in humans [149]. Compared to normal mice, the Hyp mouse is characterized by hypophosphatemia due to phosphate wasting,
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a slightly decreased serum calcium level without flank hypocalcemia or secondary hyperparathyroidism, elevated alkaline phosphatase activity, slow growth, rickets, and osteomalacia [4]. Additional phenotypic features are found in the Gy mouse, including sterility in the male, circling behavior, and reduced viability [150].
Phosphate Wasting The Hyp mouse has a defect in renal phosphate transport [151], confined to the renal brush-border membrane [152,153]. PTH is not responsible for the phosphate wasting [154,155], although Hyp mice have higher PTH levels compared to normal mice [156-158], likely due to mild hypocalcemia [157]. Hyp mouse parathyroid glands adapt appropriately to phosphate deprivation [159]. Phosphate wasting in the Hyp mouse results from an ~50% decrease in sodium-dependent phosphate transport (Na-Pi transport) in the brush-border membrane of the proximal tubule [153,160], mostly due to a defect in the high-affinity, low-capacity sodium-dependent phosphate cotransporter NPT2 [161]. There are three classes of high-affinity Na-Pi transporters [162]. The type II transporters (Npt2) are responsible for 84% of the Na-Pi transporter mRNA. NPT2/Npt2 has been cloned [163-166], and the Hyp mouse renal proximal tubule has ~50% decreased mRNA [162,166,167] and protein levels of Npt2 [167]. Phosphate restriction of the Hyp mouse leads to an increase in Npt2 mRNA and protein and subsequently to an increase in Na-Pi transport, as it does in the normal mouse [168]. The increase in Npt2 protein in the Hyp mouse exceeds that of mRNA levels, suggesting that translational or posttranslational mechanisms are involved in the response [169]. The other two Na-Pi transporters, type I (Nptl) and type III (GlvrI and Ram-I), are responsible for 15 and 1% of Na-Pi transporter mRNA, respectively [162]. The Hyp mouse shows decreased expression of Nptl mRNA levels to 78% of normal and increased Glvr-1 mRNA to 145% of normal [162]. NPT2 was ruled out as the genetic defect in XLH with the mapping of NPT2 to human chromosome 5q35 [170,171] and with the mapping of the homologous mouse gene to mouse chromosome band 13B [172]. Furthermore, targeted mutagenesis of Npt2 in the mouse leads to a phenotype in the homozygous mouse that is similar to that of the Hyp mouse in certain respects, with renal phosphate wasting, hypophosphatemia, and increased serum alkaline phosphatase activity [173]. However, unlike the Hyp mouse, the Npt2 knockout mice have appropriate elevations in 1,25(OH)zD levels, leading to hypercalcemia, hypercalciuria, and decreased PTH levels, and they do not have rickets or osteomalacia [173]. Inter-
estingly, the mouse heterozygote for a disruption in the
Npt2 gene has a 50% reduction in Npt2 mRNA, like the Hyp mouse, but has normal Npt2 protein and normal Na-Pi transport activity [162], unlike the Hyp mouse. Bone and Teeth As in humans, Hyp mice demonstrate rickets and osteomalacia. Osteoid thickness and volume are increased, and bone mineral content is decreased [174]. Although hypophosphatemia and low 1,25(OH)2D levels are likely partially responsible for the bone defect in Hyp, there is evidence for a primary defect in bone in the Hyp mouse. Phosphate supplementation of the Hyp mouse leads to improvement but not correction of the bone mineralization defect [175], and although there is further improvement when 1,25(OH)2D is added, osteomalacia persists [176]. Further evidence for an intrinsic bone defect derives from a series of experiments in which normal and Hyp periostea and osteoblasts were transplanted into muscle of normal and Hyp mice [174,177179]. These studies revealed that the impaired mineralization of Hyp cells is improved but not corrected when placed in a normal mouse [174], and that prior exposure to a hypophosphatemic environment does not explain the abnormality [177]. Administration of 1,25(OH)2D or phosphate to Hyp or normal mice improves but does not correct defective bone formation by transplanted Hyp cells [178,179]. As noted by Econs and Francis [180], these studies do not rule out the possibility that a humoral factor present during development led to a permanent defect in bone mineralization, which persisted once the factor was removed. The nature of the bone defect is not clear. Transport of phosphate in cultured osteoblasts is normal [181,182]. Intrinsic abnormalities in osteocalcin or osteopontin, markers of bone mineralization produced in the osteoblast, do not appear to be responsible for the bone defect in the Hyp mouse. In vivo, serum levels of osteocalcin are higher in Hyp mice [183], and 1,25(OH)2D treatment leads to a decrease in osteocalcin in the Hyp mouse, whereas in normal mice osteocalcin increases [183,184]. However, in vitro studies in primary osteoblast cultures derived from Hyp mice demonstrate that there is no difference in osteocalcin production or mRNA levels at baseline or after exposure to 1,25(OH)2D compared to cultures derived from normal mice [185]. These findings suggest that the changes in osteocalcin expression in vivo are due to the associated changes in calcium and/or PTH levels and not to an intrinsic defect in the osteoblast [184]. Osteoblast osteopontin mRNA levels are similar in Hyp and normal mice, although casein kinase II activity and phosphorylation of osteopontin are decreased in the Hyp mouse [186].
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25. Familial Hypophosphatemia and Related Disorders
Like humans with XLH, the Hyp mouse has a defect in dentin formation [187-190] that is not resolved by correcting the hypophosphatemia [191]. Vitamin D Metabolism
Regulation of 1,25(OH)2D metabolism is abnormal in the Hyp mouse due to abnormal renal 25-hydroxyvitamin D-l~-hydroxylase (1-OHase) activity [the enzyme converting 25(OH)D to 1,25(OH)zD] and 25-hydroxyvitamin D-24-hydroxylase (24-OHase) activity [the enzyme converting 25(OH)D to 24,25(OH)zD]. 24-OHase is the first enzymatic step in the inactivation of 1,25(OH)zD. When normal mice are made hypophosphatemic (by phosphate deprivation), 1-OHase activity [192] and subsequently 1,25(OH)2D levels [193] increase, and there is no change in 24-OHase products [193], activity, or mRNA levels [194]. However, in the Hyp mouse, 1,25 (OH)2D levels [193,195] and 1-OHase activity [192] are not elevated; instead, they are similar to those of normal mice that are not phosphate deprived. The levels of 24-OHase products are increased more than twofold in the Hyp mouse compared to the normal mouse [193]. Thus, the normal 1,25(OH)2D levels seen in Hyp represent an inappropriate response to hypophosphatemia. The Hyp mouse response to phosphate deprivation is paradoxical, with a further decrease in 1-OHase activity [192,196] and increase in renal 24-OHase activity, protein, and m R N A levels [193,194,197], leading to even lower 1,25(OH)zD levels [193,195,198]. The Hyp mouse also responds paradoxically to phosphate supplementation with an increase in 1,25(OH)zD levels and decrease in the products of 24-OHase, whereas in phosphatesupplemented normal mice 1,25(OH)2D levels and the products of 24-OHase do not change [193]. Despite the fact that 1-OHase is a mitochondrial enzyme, found in the epithelium of the renal proximal tubule at the site of the phosphate transport defect in the Hyp mouse, the abnormal regulation of 1,25(OH)zD is not due to a primary defect in renal mitochondrial phosphate transport [199]. In the Hyp mouse, the 1,25(OH)2D response to vitamin D and calcium deprivation is appropriate but blunted. Compared to the normal mouse, the Hyp mouse shows less of an increase in 1-OHase activity and less of a decrease in 24-OHase activity [199-201]. The Hyp mouse also has a blunted 1-OHase response to exogenous PTH [202], endogenous PTH stimulated by a low-calcium diet [202], and PTHrP [203]. These findings suggest that the calcium threshold required to stimulate increased 1,25(OH)zD formation may be lower in the Hyp mouse than in normal mice [200]. On the other hand, calcitonin stimulates 1-OHase activity to similar levels in Hyp and normal mice [204].
Although vitamin D metabolism is abnormal in the
Hyp mouse, the renal response to 1,25(OH)zD supplementation is appropriate. 1,25(OH)2D supplementation leads to an increase in calcium and phosphate, no increase in renal phosphate transport [205], and an increase in renal 24-OHase mRNA and activity [194]. The response of bone from Hyp mice to 1,25(OH)2D is abnormal. In vitro, 1,25(OH)zD inhibits bone collagen synthesis in both Hyp mice and normal mice [206]. This 1,25(OH)2D-induced inhibition increases in the presence of a lowered phosphate concentration in normal mice but not in Hyp mice [206]. In addition, Hyp osteoblasts in culture do not respond to physiologic doses of 1,25 (OH)zD with an increase in alkaline phosphatase activity and decrease in cell proliferation, as seen with normal cells, even when there is a normal concentration of phosphate in the medium [207]. Whether or not these findings are due to vitamin D resistance at the level of the bone is not clear. In the adult Hyp mouse, intestinal transport of phosphate and calcium is normal. However, in younger Hyp mice, intestinal transport of phosphate is decreased [208-212] and is associated with a greater than 50% decrease in levels of intestinal calcium-binding protein (CaBP) [213,214]. The lower CaBP levels and decreased calcium absorption in the young Hyp mouse are not explained by differences in the binding affinity or number of vitamin D receptors (VDRs) [212,215], nor by differences in the basal levels of intestinal VDR m R N A [216]. In addition, the Hyp mouse responds normally to 1,25(OH)zD supplementation with increased intestinal absorption of calcium and phosphate and increased duodenal and renal CaBP [214,217]. Instead, the decreased intestinal absorption of calcium and phosphate appears to be due to the defect in 1,25(OH)zD metabolism [4]. The Hyp mouse has decreased nuclear uptake of 1,25(OH)zD in intestinal mucosal cells [218], which is corrected after phosphate supplementation [219]. These findings suggest that hypophosphatemia at least in part explains the vitamin D resistance. Thus, there does not appear to be an intrinsic resistance to vitamin D at the level of the intestine, but instead the apparent resistance appears to be due to low 1,25(OH)zD and phosphate levels. The Location of the Defect in
Hyp
Although there has been controversy as to whether the defect in phosphate transport is intrinsic to the kidney or represents a humoral factor, a number of observations in the Hyp mouse point to a humoral factor. Plasma phosphate levels decrease in normal mice parabiosed to Hyp mice due to increased renal excretion of phosphate in the normal mice [220,221]. In cross-transplantation studies,
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in which normal kidneys are transplanted into Hyp recipient mice or Hyp kidneys are transplanted into normal recipient mice, the phenotype of the recipient mice does not change [222]. In immortalized cells from the S1-$3 segments of Hyp proximal tubule, phosphate transport is intrinsically normal [223,224]. Serum from Hyp mice has been shown to inhibit phosphate uptake in cultured proximal tubule cells [225]. There is evidence that a responsible humoral factor is released by, or modified in, the osteoblast. Media from Hyp mouse primary osteoblast-like cell cultures inhibits phosphate uptake in cultured proximal tubule cells compared to normal mouse bone cell-conditioned media [225]. In studies comparing immortalized osteoblasts from SV40 transgenic Hyp mice and normal mice [226], cultures derived from Hyp mice failed to mineralize in comparison to those from control mice. On the other hand, when using cultures of primary osteoblasts from newborn mice, Hyp cultures were no different from controls with respect to osteocalcin production and the osteocalcin response to 1,25(OH)2D [185]. Furthermore, normal mineralization has been demonstrated in primary osteoblast cultures derived from newborn calvaria in Hyp mice [227]. Thus, whether the osteoblast per se is the site of an intrinsic mineralization defect is controversial. Nevertheless, conditioned medium from Hyp mouse bone marrow cultures induced defects in bone marrow cell cultures of wild-type cells, supporting the hypothesis that there is an intrinsic osteogenic defect [228].
PHEX/Phex Soon after the identification of PHEX, the mouse Phex was cloned [229]. PHEX is highly conserved between mouse and humans, with the mouse Phex cDNA being 91% identical to the human PHEX on the nucleotide level and 96% identical on the amino acid level [135,229-231]. The Hyp mouse has a 3' deletion of Phex, including exons 16-22 [134,230,232] and the 3~ untranslated region [232]. The Gy mouse has a deletion of exons 1-3 and upstream sequences [230], including a partial deletion of the spermine synthase gene, which could explain some of the additional phenotypic features of the Gy mouse [233-235]. The identification of PHEX finally proved that the HYP/Hypdefect was not intrinsic to the kidney. Although little is known about the function of PHEX, there is a great deal of evidence that PHEX does not regulate phosphate transport directly. Phex is a 100-to 105-kDa glycoprotein [236]. PHEX/ Phex is expressed primarily in human and mouse fetal and adult bone [134,229,236-239] and in mouse teeth
[229,236,238,239]. Expression is 2-to 10-fold higher in bone than in other tissues [237]. Phex m R N A expression remained high in brain but decreased to undetectable levels with age in the other tissues [240]. In the femur and calvaira, Phex protein has also been shown to decrease with age [236]. In most [134,135,229] but not all studies [237], no PHEX expression has been detected in adult kidney. Human PHEX expression has also been detected in ovary [134,135] and lung [134,135]. PHEX expression was detected in fetal liver in one study [135] but not in others [134,237], and it has been detected in fetal muscle [134,237]. PHEX is also expressed in the parathyroid glands [241]. No Phex expression is found in male Hyp mice [236]. Phex appears to be a marker of mature osteoblasts and odontoblasts and is associated with matrix mineralization [238,239]. Although Phex m R N A is not detectable in mouse MC3T3-E1 preosteoblasts, m R N A and proteins levels increase with differentiation of the osteoblast and concomitant matrix mineralization [231,239]. Phex expression is downregulated by 1,25(OH)2 D, which inhibits matrix mineralization [239]. By in situ hybridization, Phex m R N A can first be detected in osteoblasts and odontoblasts on Day 15 of mouse embryonic development, which coincides with the onset of matrix deposition in bone [238]. The amount of Phex transcript is decreased in adult bone and nongrowing teeth [238]. Phex protein expression mirrors that of m R N A and is found in osteoblasts, osteocytes, and odontoblasts but not in osteoblast precursors [236]. Of interest, in one study transplantation of bone marrow from normal to Hyp mice led to increased serum phosphorus, increased renal Na-Pi cotransporter gene expression, and decreased alkaline phosphatase activity [242]. In another study, targeted overexpression of Phex in Hyp osteoblasts failed to correct the mineralization defect or the hypophosphatemia [243]. This surprising finding could be secondary to lack of adequate expression in the osteoblasts, but it could also indicate that expression in other tissues is critically important. PHEX expression has been detected in tumor tissue associated with TIO at levels twofold higher than in bone, suggesting an overabundance of PHEX expression in TIO [237]. PHEX is not mutated in these tumors [244]. The PHEX protein has features consistent with the hypothesis that it is a member of the neutral endopeptidase family [237]. PHEX is glycosylated and located on the cell surface with the carboxy domain facing extracellularly [237]. Three missense mutations responsible for HYP have been shown to interfere with the membrane targeting of the mutant PHEX protein [245]. PHEX has also been shown to degrade PTH-derived peptides
25. Familial Hypophosphatemia and Related Disorders
[237,246]. Osteocalcin and phosphate inhibit the degradation of PHEX [246]. The fact that the PHEX protein is a membrane-bound endopeptidase virtually excludes the possibility that PHEX is the circulating phosphaturic factor in the Hyp mouse. Instead, PHEX may activate or inactivate this factor. It has been proposed that the TIO defect and the XLH defect are in the same pathway involving PHEX [247]. The favored hypothesis is that there is a circulating protein ("phosphatonin") [248] that inhibits phosphate reabsorption at the level of the kidney and is normally degraded by PHEX. The absence of PHEX in the male, or haploinsufficiency in the female, leads to decreased amounts of the PHEX protein, less degradation of phosphatonin, and thus greater amounts of phosphatonin, in turn leading to greater inhibition of phosphate transport, resulting in phosphate wasting [61,248]. In this model, the TIO defect leads to overproduction of phosphatonin, in turn leading to hypophosphatemia [249]. Phosphatonin per se has not been identified; thus, this hypothesis has not been proven. An alternative hypothesis is that PHEX activates a protein that normally increases phosphate transport, and that lack of activation of the protein leads to decreased phosphate transport and phosphaturia. However, evidence does not support this hypothesis. Therefore, what is the PHEX substrate? There is evidence that FGF23, the defective protein in ADHR, could be a PHEX substrate. Another possibility is that MEPE [79] is a substrate for PHEX. MEPE is a candidate for the tumor-derived phosphaturic factor in TIO [79,250]. In the normal mouse, Mepe is expressed in osteoblasts, increases during matrix mineralization induced by osteoblasts, and is downregulated by 1,25-(OH)zD3 [250].Targeted disruption of the murine homolog of Mepe, osteoregulin, results in increased bone formation and bone mass [251]. Mepe mRNA levels were observed in bone and osteoblasts derived from Hyp mice, the murine homolog of human XLH. In Hyp mouse bone and osteoblasts, Mepe mRNA levels are increased compared to those of normal mice [250]. However, when Chinese hamster ovary cells that secrete MEPE are implanted into nude mice, there is neither hypophosphatemia nor an increase in renal phosphate wasting [77]. In summary, PHEXis the gene defective in XLH. As a membrane-bound endopeptidase that is primarily expressed in bone and is not expressed in kidney, PHEX must be regulating phosphate transport indirectly. The complete understanding of the roles of PHEX, FGF23, MEPE, and/or another as yet uncharacterized proteins in the pathway that leads to defective phosphate transport and abnormal bone awaits further study.
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Npt2 Knockout Mice Targeted inactivation of the type II Na/Pi cotransporter, Npt2, in mice has provided a provocative animal model for examining the role of phosphate wasting in the development of hypophosphatemic skeletal diseases. Npt2 is the most abundant renal tubular transporter, and its expression is regulated by PTH and by dietary phosphate, the major regulators of total body phosphate economy. Beck et al. [173], constructed a disruptive Npt2 gene, introduced this into stem cells, and bred mice from heterozygous matings to produce mice homozygous for the disrupted allele. The mice were small but grew with comparable velocity to wild-type mice. Survival was poorer than that of wild-type mice--an observation that was attributed to poor feeding--and their reproductive ability was less than that of wild-type animals. Mice heterozygous for the defect were normal in all of these respects. The biochemical phenotype of Npt2 knockout mice is strikingly similar to that seen in HHRH. Renal excretion of phosphate was greater in knockout mice than in wildtype animals, and hypophosphatemia was always present. Despite an intermediate level of renal phosphate clearance in heterozygotes, their serum phosphate levels were normal. A slight but significant hypercalcemia was evident in knockout mice, and PTH levels were suppressed to the low range. Circulating 1,25(OH)2D levels were elevated, as was urinary calcium excretion. Serum alkaline phosphatase activity was elevated. The heterozygous mice demonstrated an elevation in 1,25(OH)2D levels intermediate between homozygous mice and wildtype mice but no significant difference in serum calcium, urinary calcium excretion, or alkaline phosphatase activity. Based on this identical biochemical phenotype to the human disorder HHRH, it seems probable that a disorder resulting from a primary disruption in renal tubular brush-border phosphate transport can result in recognizable hypophosphatemic rickets. However, the skeletal phenotype of this model, although complex, does not have features of rickets or osteomalacia. The bones of 21-day-old homozygous Npt2 knockouts demonstrate delay in epiphyseal maturation and have poorly developed metaphyseal trabeculae. These findings are intermediate in the heterozygous mice. There is evidence of reduced bone turnover as well. Of particular note, by Day 45 the metaphyseal defect is not present, and with age the homozygous knockout mice show increased metaphyseal trabeculae compared to the wild-type mice. Thus, the skeletal defect in Npt2 knockout mice is age dependent and does not recapitulate the findings observed in HHRH. This is in keeping with the recent exclusion of this gene as the site of mutation in several pedigrees with HHRH.
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The Npt2 knockout is instructive, however, in understanding hypophosphatemic osteomalacia. The experiment demonstrates that at least in mice, osteomalacia does not necessarily result from the usual degree of hypophosphatemia typically seen in renal tubular phosphate wasting disorders. Serum phosphate levels in these mice are comparable to those seen in Hyp mice (PHEX deletion mice), and the Npt2 knockout skeletal phenotype differs considerably from the osteomalacia seen in Hyp mice or mice exposed to increased levels of FGF23 [77]. This finding implies that other events are required to generate the osteomalacia or the Hyp phenotype. Furthermore, the pathophysiology of XLH (and Hyp mice) would therefore be predicted to result from a renal abnormality not exclusively involving renal phosphate handling at the brush-border membrane site. The striking differences between PHEX mutations and Npt2 mutations imply that PHEX mutations (although interfering with renal tubular phosphate transport) disrupt other significant functions, suggesting a defect more proximal in a pathway(s) regulating other unidentified functions in addition to brush-border membrane transport. FGF23 The availability of a large ADHR kindred allowed a genomewide linkage screen to be performed without concern for genetic heterogeneity. The genomewide linkage screen revealed that the gene was located on chromosome 12p13 [252]. Further analysis revealed missense mutations in a novel member of the fibroblast growth factor family, FGF23 [78]. Sequencing of FGF23 exons from four ADHR families revealed three missense changes affecting two arginines, which are three amino acids apart. Families 1406 and 1478 shared the same mutation, R176Q (527G>A). Family 2318 had an R179W (535C>T) mutation and family 329 had an R179Q (536G>A) substitution. None of these mutations were observed in normal controls. Studies indicate that FGF23 is transcribed at low levels. Northern blots that included multiple normal tissues failed to show bands. Radioactive in situ hybridization with antisense mouse FGF23 probe on sagittal sections of mouse embryos at different developmental stages was negative. Similar studies using paraffin sections of various tissues and frozen sections of E18.5 tibias were also negative. Using reverse transcriptasepolymerase chain reaction, FGF23 RNA was detected in heart, liver, and thyroid/parathyroid [78]. This technique has also amplified products from thymus [253]. In contrast, a Northern blot made from cancer cell lines displayed bands (after 7 days of autoradiography) at
3.0 and 1.3 kb in a chronic myelogenous leukemia cell line, whereas other tumors expressed either the 3.0- or 1.3-kb bands. In light of the clinical similarity between ADHR and TIO, tissue from tumors that caused TIO as obtained and analyzed for FGF23 expression [75]. Northern blots from five tumors revealed robust expression of both the 3.0- and 1.3-kb bands in all instances and an antibody to the C-terminal portion of FGF23 detected a 32-kDa band on Western blot [75]. These results were subsequently confirmed by others [76,77]. These data implicated FGF23 as the factor previously called phosphatonin that is responsible for the phosphate wasting in this disorder. Additional important evidence implicating FGF23 as phosphatonin derived from studies performed by Shimada et al. [77]. These investigators also found that FGF23 was highly expressed in tumors that cause TIO. They found that intraperitoneal administration of recombinant human FGF23 resulted in increased renal phosphate excretion and decreased serum phosphorus concentrations. In subsequent experiments, they made Chinese hamster ovary cell lines that stably expressed FGF23 and implanted them into nude mice. Compared to controls, these mice manifested renal phosphate wasting, hypophosphatemia, increased alkaline phosphatase activity, and reduced calcitriol concentrations. The mice also displayed rickets and osteomalacia. In essence, these investigators completely reproduced the syndrome of tumor-induced osteomalacia in these mice. Recently, work has been performed to elucidate the mechanism by which the FGF23 mutations in arginine 176 and 179 cause ADHR. Using a C-terminal FGF23 antibody, Western blots of cells transfected with wildtype FGF23 demonstrated bands of approximately 32 and 12 kDa [75]. Further analysis indicated that the 12kDa fragment was a C-terminal cleavage product of the wild-type 32-kDa protein [75]. Similar studies done in all three ADHR mutations yielded only the intact, 32-kDa FGF23 protein [254]. Of note, the mutated arginines form an RXXR motif, which is a recognition sequence for the furin class of proteolytic enzymes [255]. These data indicate that the ADHR mutations may protect the protein from degradation and thereby elevate circulating FGF23 concentrations in ADHR patients. In summary, ADHR is caused by missense mutations in arginines 176 or 179 of FGF23, which appear to protect the protein from degradation. Furthermore, FGF23 is markedly overexpressed in tumors that cause TIO, and FGF23-expressing Chinese hamster ovary cells implanted into nude mice reproduce the TIO syndrome. These studies indicate that FGF23 has a role in the pathogenesis of these disorders.
Z5. Familial Hypophosphatemia and Related Disorders
CURRENT PROBLEMS AND UNRESOLVED QUESTIONS
treatment for XLH if hypophosphatemia results from increased FGF23 concentrations. Are FGF23, MEPE, FRP4, or any other substance secreted by TIO tumors involved in the pathogenesis of other conditions that cause phosphate wasting, such as epidermal nevus syndrome and fibrous dysplasia? Do any of these substances have a role in hypophosphatemia that is often seen after renal transplantation? What role, if any, do these substances play in the normal regulation of phosphate homeostasis? A proposed summary of the interactions of these proteins based on current information is shown in Fig. 5. In addition to these questions, several old questions remain unanswered. Is there a phosphate sensor or sensing mechanism and do aberrations in a sensor or sensing mechanism lead to disease in humans? What is the pathogenesis of the enthesopathy in XLH? If patients do not properly mineralize bone, why do they mineralize (and indeed, ossify) their tendons and ligaments? Finally, can we use this new data to design new therapeutic approaches for phosphate wasting disorders that have less toxicity than current regimens? Such approaches
Directions for Future R e s e a r c h The metabolic bone community has made great strides in understanding the pathogenesis of XLH, ADHR, and TIO. The flurry of new data during the past few years has opened up numerous avenues for further investigation. Much of this research may lead to new therapies for these disorders. Some data suggest that the disorders result from defects along a common pathway, but more data are necessary before a firm conclusion can be made. For example, FGF23 is clearly involved in the pathogenesis in ADHR, but does it have a role in the pathogenesis of XLH? If FGF23 is a P H E X substrate, is it the only substrate or does P H E X have multiple substrates? Does FGF23 cause renal phosphate wasting through one or more of the known F G F receptors or does it work through a novel receptor? The answers to these questions have important therapeutic implications because receptor blockade could be a useful
Phosphaturia Reduced 1,25(OH)2D3synthesis Impaired mineralization
~
n
Active phosphatonin?
!,
MEPE? FRP4? Other Factors?
.4 z
TIO tumors
~
623
i ........... Zn++
!1 . . . . . . . .
Inactive Phosphatonin
PHEX (reduced or lost in XLH)
Nq~mal osteoblast/osteocyte
FIGURE 5 A proposed interaction between FGF23, which may be the Ion-sought phosphatonin, and PHEX in phosphate wasting. In patients with ADHR and TIO tumors, there is increased availability of biologically active FGF23 and consequently phosphaturia, reduced 1,25-D synthesis, and impaired mineralization. FRP4 and potentially other proteins may represent additional phosphatonins. In the normal situation, phosphatonin is presumably cleaved by PHEX; in patients with XLH, PHEX expression is reduced or absent. This may result in impaired phosphatonin degradation and consequently an increased concentration of phosphatonin, leading to the features of XLH. PHEX, a zinc-binding protein, is found in the membrane of normal osteoblasts and osteocytes. TIO tumors also appear to secrete MEPE, FRP4, and other factors, but it remains to be determined whether and how these molecules relate to the pathogenesis of the disorder.
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could include administration of soluble PHEX or other substances that would decrease circulation phosphatonin concentrations or receptor blockade. Clearly, there is great cause for optimism for patients who suffer from these diseases.
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dysregulation in X-linked hypophosphatemia. Pediatr. Nephrol. 13, 607-611. Miyamura, T., Tanaka, H., Inoue, M., Ichinose, Y., and Seino, Y. (2000). The effects of bone marrow transplantation on X-linked hypophosphatemic mice. J. Bone Miner. Res. 15, 1451-1458. Liu, S., Guo, R., Tu, Q., and Quarles, L. D. (2002). Overexpression of Phex in osteoblasts fails to rescue the Hyp mouse phenotype. J. Biol. Chem. 277, 3686-3697. Nelson, A. E., Hogan, J. J., Holm, I. A., Robinson, B. G., and Mason, R. S. (2001). Phosphate wasting in oncogenic osteomalacia: PHEX is normal and the tumor-derived factor has unique properties. Bone 28, 430-439. Sabbagh, Y., Boileau, G., DesGroseillers, L., and Tenenhouse, H. S. (2001). Disease-causing missense mutations in the PHEX gene interfere with membrane targeting of the recombinant protein. Hum. Mol. Genet. 10, 1539-1546. Boileau, G., Tenenhouse, H. S., Desgroseillers, L., and Crine, P. (2001). Characterization of PHEX endopeptidase catalytic activity: Identification of parathyroid-hormone-related peptide 107-139 as a substrate and osteocalcin, PPi and phosphate as inhibitors. Biochem. J. 355, 707-713. Nelson, A. E., Robinson, B. G., and Mason, R. S. (1997). Oncogenic osteomalacia: Is there a new phosphate regulating hormone? Clin. Endocrinol. (Oxford) 47, 635-642. Econs, M. J., and Drezner, M. K. (1994). Tumor-induced osteomalacia--Unveiling a new hormone [Editorial; Comment]. N. Engl. J. Med. 330, 1679-1681. Drezner, M. K. (2000). PHEX gene and hypophosphatemia. Kidney Int. 57, 9-18. Argiro, L., Desbarats, M., Glorieux, F. H., and Ecarot, B. (2001). Mepe, the gene encoding a tumor-secreted protein in oncogenic hypophosphatemic osteomalacia, is expressed in bone. Genomics 74, 342-351. Brown, T. A., Gowen, I. C., Petersen, D. N., Stock, J. L., Tkalcevic, G. T., Vail, A. L., Simmons, H. A., ChidseyFrink, K. L., and McNeish, J. D. (2000). Targeted disruption of "osteoregulin," a novel bone specific gene, results in increased bone formation and bone mass in mice [Abstract 1126]. J. Bone Miner. Res. 15, S170. Econs, M. J., McEnery, P. T., Lennon, F., and Speer, M. C. (1997). Autosomal dominant hypophosphatemic rickets is linked to chromosome 12p13. J. Clin. Invest. 100, 2653-2657. Meyer, R. A. (2001). Elevated mRNA gene expression of fibroblast growth factor 23 (FGF23) in the thymus of X-linked hypophosphatemic (Hyp) mice [Abstract SA129]. J. Bone Miner. Res. 16, $251. White, K. E., Carn, G., Lorenz-Depiereux, B., Benet-Pages, A., Strom, T. M., and Econs, M. J. (2001). Autosomal-dominant hypophosphatemic rickets (ADHR) mutations stabilize FGF-23. Kidney Int. 60, 2079-2086. Seidah, N. G., and Chretien, M. (1999). Proprotein and prohormone convertases: A family of subtilases generating diverse bioactive polypeptides. Brain Res. 848, 45-62.
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1261 Rickets Due to Renal Tubular Abnormalities RUSSELL W. CHESNEYand DEBORAH P. JONES University of Tennessee Health Science Center, Department of Pediatrics, Memphis, Tennessee
INTRODUCTION
however, demineralization in a given disorder may be due to a combination of these mechanisms. These mechanisms are hypophosphatemic osteomalacia, which is the consequence of impaired renal tubular phosphate reabsorption and continued phosphaturia despite the signal of reduced serum phosphate concentration [2]; hyperexcretion of calcium; hyperexcretion of magnesium, which can diminish the availability of both calcium and magnesium for mineralization of osteoid; bicarbonate wasting, which causes metabolic acidosis and reduced bone formation and enhanced bone resorption [4,5]; renal osteodystrophy, which accompanies those renal tubular disorders that progress to renal failure with the result of diminished calcitriol biosynthesis; and disordered vitamin D metabolism, which alters the intestinal absorption of calcium and phosphate [6-10]. Although minor degrees of bone disease are found in virtually every renal tubular disorder, florid bone findings are characteristic of Fanconi syndrome (Fig. 1), all forms of renal tubular acidosis, hypercalciuria with nephrocalcinosis, renal magnesium wasting, and Dent disease. This chapter focuses on the bone features of these syndromes. Other renal tubular disorders causing growth failure and demineralization are discussed in other chapters.
Osteomalacia, with or without rickets, as well as osteopenia are the characteristic bone lesions of numerous renal tubular disorders [1,2]. The renal proximal and distal tubules are responsible for the reabsorption of the divalent minerals calcium, magnesium, and phosphate [1,3]. Indeed, total body phosphate homeostasis is regulated at the level of the renal tubules, especially the proximal tubule [1,3]. The renal proximal tubule is also the tissue location for the enzymatic synthesis of 1,25dihydroxyvitamin D or calcitriol [3]. Of the many regulators of calcitriol synthesis, normal renal proximal cell structure and function are critical to the production of this mineral hormone [3,4]. The renal proximal tubule and distal tubule are the respective sites for the phosphaturic and calcium reabsorptive actions of parathyroid hormone [1,3]. Thus, renal tubular injury and fibrosis and inherited defects of tubular transporter action significantly impact on the regulation of bone formation and resorption. Bone disease is a key feature of many renal tubular disorders in terms of both clinical presentation and disease morbidity [1]. The following clinical aspects of renal tubular disease especially impinge on bone formation and status: These disorders may be hereditary. They may present with a diminished growth rate. Their presentation may include the features of rickets (e.g., wrist and knee widening and bowing). Therapy generally includes replacement of the mineral lost in the urine and the provision of exogenous calcitriol.
FANCON! SYNDROME Osteomalacia with or without rickets is universal in the renal Fanconi syndrome [1]. The renal features of this complex generalized tubulopathy include defects in the reabsorption of ions, organic solutes, and proteins whose molecular weight is less than 50 kDa. Since the proximal tubule is responsible for the bulk transfer of these
At least six fundamental pathophysiologic mechanisms underlie bone disease in renal tubular syndromes;
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FIGURE 1
substances, the molecules excreted in large quantities in Fanconi syndrome include sodium, potassium, bicarbonate, glucose, uric acid, amino acids, other organic acids and ketones, as well as magnesium, calcium, and phosphate [1,3]. Moreover, some forms of Fanconi syndrome may progress to chronic renal insufficiency and the need for dialysis and/or renal transplantation, especially cystinosis, Lowe syndrome, and Fanconi-Bickel disease [6,7].
Etiology There are many causes of Fanconi syndrome, both hereditary (Table 1) and nonhereditary (Table 2). Rather than discuss eacli separate cause of the syndrome, which has been done in standard texts [7,8], this chapter discusses the syndrome from the perspective of its bone disorders, their.pathogenesis, and treatment and focuses on those causes that result in profound bone disease [8-13].
26. Rickets Due to Renal Tubular Abnormalities TABLE 1 Hereditary Disorders Associated with Fanconi's Syndrome Primary of idiopathic (no identifiable associated disorder) Familial Sporadic Hereditary Cystinosis (Lignac-Fanconi disease) or infantile nephropathic cystinosis Lowe's syndrome Hereditary fructose intolerance Tyrosinemia, type I (tryosinosis) Galactosemia Glycogen storage disease (untyped), defect in GLUT-4 Wilson's disease Subacute necrotizing encephalomyelopathy (Leigh's syndrome) Hereditary mitochondrial myopathy with lactic acidemia Other conditions Familial nephrotic syndrome (focal sclerosing glomerulonephritis), defective NHP2 Metachromatic leukodystrophy Hereditary nephritis (Alport's syndrome) Medullary cystic disease
Many renal tubular disorders represent the loss of a single ion channel, dual ion cotransporter, or transporter of an organic solute. Fanconi syndrome differs in that the function of a large variety of transporters is perturbed. It is evident that no genetic process can account for abnormalities in the transport of monovalent and divalent ions, hexoses, organic solutes (amino acids, purines, and fatty acids), peptides, and lowmolecular-weight proteins. Hence, the tubular defect involves major disturbances in protein synthesis, trafficking, and turnover; energetics (e.g., the synthesis of ATP or other high-energy compounds); the impact of toxic metabolites (e.g., galactose-l-phosphate in galactosemia, fructose-l-phosphate in hereditary fructose intolerance, and copper in Wilson disease); and mitochondrial function--both genetic mutations in mitochondrial D N A resulting in Fanconi syndrome and toxin damage (e.g., lead, uranium, and copper) will perturb mitochondrial function and result in diminished ATP synthesis [1,7,14]. D i a g n o s i s of Fanconi S y n d r o m e The child usually presents with growth failure. A urine dipstick test usually shows proteinuria, glucosuria, ketonuria, and an alkaline urine. The diagnosis of Fanconi syndrome is established by measurement of the serum
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TABLE 2 Acquired Disorders Associated with Fanconi's Syndrome Disorders of protein metabolism/excretion Multiple myeloma Benign monoclonal gammopathy Light-chain nephropathy Amyloidosis Sj6gren's syndrome Nephrotic syndrome Immunologic disorders Interstitial nephritis with anti-tubular basement membrane antibody Renal transplantation Malignancy Other renal disorders Balkin nephropathy Paroxysmal nocturnal hemoglobinuria Renal vein thrombosis in newborn infant Vitamin D disorders with secondary hyperparathyroidism Vitamin D deficiency X-linked hypophosphatemic rickets Vitamin D dependency Disorders linked with drug, heavy metal, or other toxin exposure Drug-related causes Outdated, degraded tetracycline Methyl-3-chromone 6-Mercaptopurine Gentamicin and other aminoglycoside antibiotics Valproic acid Streptozotocin Isophthalanilide Ifosfamide Heavy metal exposure Cadmium Lead Mercury Uranium Cis-platinum Other toxin exposure Paraquat poisoning Lysol burn Toluene inhalation (glue sniffing)
and urinary concentrations of the substances lost in the urine: Na, K, HCO3, PO4, amino acids, uric acid, creatinine, glucose, and low-molecular-weight proteins (such as 132-micoglobulin and ceruloplasmin). Commonly, serum potassium, uric acid, phosphate, and bicarbonate
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are reduced. By the measurement of a timed urine collection, one can determine the fractional excretion of any substance measured in urine and serum and for which there also exist a urine and serum creatinine. It may be difficult to measure 24-hr urine because of polyuria in these children. Other useful tests are long bone X-rays to show rickets, osteomalacia, and a delayed bone age. Careful measurements of height and weight show diminished growth and growth velocity. An ophthalmologic examination may disclose cystine crystals in cystinosis and cataracts in galactosemia. The ultimate condition responsible for Fanconi syndrome can usually be diagnosed by selective metabolic studies or by DNA analysis using known molecular probes to show mutant DNA. Association with B o n e D i s e a s e The pathophysiologic mechanisms responsible for this bone disease and the therapeutic approach to these forms of bone disorders are especially relevant in this proximal tubulopathy. Both rickets and osteomalacia, as well as a diminished growth rate, are common in childhood forms of Fanconi syndrome. Table 3 lists the ascribed causes of bone disease.. Several forms of Fanconi syndrome, especially those that result from a single gene defect (Table 1), can present in infancy or young children with growth failure, bowing, fracture, or a waddling gait [1,7,8]. From a radiologic standpoint, these children appear to have nutritional rickets with metaphyseal widening, fraying at the ends of long bones, and osteopenia with prominent trabecular markings. Prominent among these Mendelian disorders are infantile nephropathic cystinosis, Lowe syndrome, tyrosinosis type I, the Fanconi-Bickel form of glycogen storage disease, and pseudovitamin D-deficiency rickets (Prader
type) [1]. The etiology of these disorders includes abnormalities in transport, energetics, synthesis, and metabolism, all of which result in a pervasive proximal tubular deficit. Forms of Bone D i s e a s e Clinical Features
As mentioned previously, the clinical features of the bone disease in Fanconi syndrome are bowing, short stature, enlargement of the wrists, knees, and ankles, bone pain, rnicrofractures, and a waddling gait [1,8]. Skeletal X-rays reveal metaphyseal widening and fraying with the typical features of rickets, thickened trabecular markings with osteopenia, microfractures, and thinning of the cortex of long bones [1]. Patients may also demonstrate varus deformities, valgus deformities, a rachitic rosary, and Harrison's groove. Prominent frontal bossing is sometimes a feature. The findings in Fanconi syndrome patients with rickets and hypophosphatemic osteomalacia are similar to those in other types of rickets, including nutritional vitamin D deficiency and Xlinked hypophosphatemic rickets, particularly since both have hypophosphatemia and abnormalities of vitamin D metabolism [6-11]. Other features, including looser zones, zones of provisional calcification, and failure of ossification of secondary centers of calcification, are also common features in the bone disease of Fanconi syndrome (Fig. 2). Another aspect of Fanconi syndrome that worsens bone disease is the hyperchloremic metabolic acidosis that accompanies the massive bicarbonate wasting that occurs [4,5,8]. The acidosis is difficult to treat (vide infra) and is believed to contribute to growth failure in Fanconi syndrome. Because of undermineralization, failure of ossification of secondary centers such as the metacarpal bones, and growth failure, the bone age in these patients is retarded.
TABLE 3 Fundamental Pathophysiologic Mechanisms That Underlie Bone Disease in Renal Tubular Syndromes
Bone Histology
Hypophosphatemic osteomalacia: Renal phosphate hyperexcretion with less phosphate available for mineral deposition Calcium hyperexcretion: Lesscalcium available for mineral deposition Magnesiumhyperexcretion:Hypomagnesemialeads to lesscalciumand magnesium available for mineralization Bicarbonate wasting: Leads to hyperchloremicmetabolic acidosis, reduced bone formation, and increased bone resorption Renal osteodystrophy: Found in tubular disorders with progressive renal failure, reduces calcitriol synthesis Disordered vitamin D metabolism: Reduces intestinal calcium and phosphate absorption
General findings are those predicted in hypophosphatemic osteomalacia, acidosis, vitamin D deficiency, and, in certain patients, renal osteodystrophy. Although osteopenia is common, the typical lesion is one of osteomalacia [2,6]. Whenever renal failure occurs, bone histology reveals osteitis fibrosa and/or osteomalacia [11,15]. In most studies in animals, inhibition of endochondral bone formation and cartilage cell progression is evident [3,4]. There are few studies using modern histomorphometric analysis of patients with Fanconi syndrome, and most of these patients have an adult form of the disease [2,6]. One study of a child with an autosomal dominant
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form of the disease and renal insufficiency [15] showed low bone turnover with increased osteoclastic activity (unpublished observation) (Fig. 3).
Changes in Bone Mineral Density Studies of bone density in children with Fanconi syndrome have largely consisted of single case reports [15] or have been performed as part of large-scale studies in which a very small number of patients with the syndrome have been shown to have reduced bone mineral content [16,17]. A study of a single patient revealed reduced dualphoton bone density using quantitative computed tomography [15]. Pathogenesis
Role of Hypophosphatemia Since 85-90% of filtered phosphate is reabsorbed by the S1-S3 segments of the proximal tubule by a sodiumdependent cotransport process [8], if there were no other defects in bone, vitamin D metabolism, or other
FIGURE 2 (CONT.)
minerals, the hypophosphatemia characteristic of Fanconi syndrome would result in osteomalacia and/or rickets. The bone lesion of Fanconi syndrome is primarily due to this hypophosphatemic osteomalacia [8-11]. This is also found in animal models of Fanconi syndrome, including the maleic acid model [7]. Hypopho sphatemic osteomalacia is also prominent in several forms of Fanconi syndrome in adults [2,6,7]. The reversal of rickets and osteomalacia following oral phosphate supplemental supports the role of hypophosphatemia in the pathogenesis of the disease [1].
Role of Hypercalciuria
FIGURE 2
Although urinary calcium excretion is increased in Fanconi syndrome, the proximal tubule is not the major site for calcium reabsorption [18]. Calcium reabsorption that does occur in the proximal tubule and ascending limb of the loop of Henle occurs by a passive paracellular mechanism. The regulation of calcium transport occurs via a parathyroid hormone (PTH) and
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FIGURE 3
calcitriol-dependent process in the distal tubule. This process is transcellular and not influenced by sodium. Because Fanconi syndrome affects mainly the proximal tubule, urinary calcium hyperexcretion is not a major feature and hypercalciuria is not a major cause of osteopenia [1,8]. However, hypercalciuria is a major cause of bone disease in several of the calcium and magnesium wasting syndromes discussed later [19]. Role of Hypermagnesuria
Although hypermagnesuria is evident in Fanconi syndrome, its effect on bone disease is not as marked as in renal magnesium wasting, hypercalciuria hypermagnesuria, Gitelman syndrome, or drug-induced magnesuria. The major features of bone disease occur with profound hypomagnesemia, which is relatively uncommon in Fanconi syndrome [19]. Role of Vitamin D Metabolism
Abnormalities of vitamin D metabolism are common in Fanconi syndrome [1]. Nutritional vitamin
D deficiency can present with many features of Fanconi syndrome, including rickets, hypophosphatemia, metabolic acidosis, glucosuria, and amino aciduria with elevated serum PTH values [11,20]. Hepatic fibrosis and cirrhosis evident in untreated galactosemia, Wilson disease, hereditary tyrosinemia, Fanconi-Bickel syndrome, and hereditary fructose intolerance could theoretically impair 25-hydroxylation of vitamin D [1]. Furthermore, fat malabsorption due to impaired bile acid excretion may result in diminished vitamin D absorption and other fat-soluble vitamins [11,16]. In one study of patients with Fanconi syndrome, the circulating values of calcitriol were either reduced or normal but not elevated, as might be anticipated with the occurrence of hypophosphatemia and, sometimes, secondary hyperparathyroidism [12]. Whenever calcitriol values are reduced, bone demineralization appears to be more evident [13]. The reduction in calcitriol synthesis may be associated with impaired proximal tubule cell metabolism or due to changes in the structure of the tubule cell [17]. Since calcitriol is synthesized in the proximal tubule cell mitochondria [16,17], damage to this organelle will also impair calcitriol synthesis. Other studies, however, have only detected reduced calcitriol values in the presence of renal insufficiency [17]. This is particularly the case in cystinosis, a disorder associated with progressive renal failure [7,21]. It is of interest that adults with light-chain nephropathy may develop bone disease due to defective vitamin D metabolism (reflected by low serum calcitriol values) and hypophosphatemic osteomalacia [22]. Role of End-Stage Renal Disease
Many patients with several variants of Fanconi syndrome will develop renal insufficiency progressing to endstage renal disease and renal osteodystrophy [17,21,22] (Fig. 2). This is especially the case with cystinosis [1,17] and in certain patients with Lowe syndrome [8] or Wilson disease [1]. A remarkable biochemical feature of these patients is that with glomerular filtration rate (GFR) values < 15 ml/min/1.73 m 2, these patients become hyperphosphatemic with secondary hyperparathyroidism. The features of renal osteodystrophy are described elsewhere in this book and do not need to be further discussed here. The child with bone disease and possible Fanconi syndrome should be evaluated as outlined previously.
Treatment Oral Phosphate
Although the therapy for osteomalacia/rickets in Fanconi syndrome is determined by the cause of the bone disease, replacement with phosphate is essential [1]. The goal of phosphate-replacement therapy is to
26. RicketsDue to Renal TubularAbnormalities TABLE4
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Phosphate Preparations for Therapy of Phosphaturic Syndromes and Fanconi's Syndrome
Preparation
K-Phos M.F. K-Phos No. 2 K-Phos Neutral Neutra-Phos Neutra-Phos (capsules) Joulie's solutiona
Contents
Phosphate content
Potassium-acid phosphate Sodium-acid phosphate Potassium-acid phosphate Sodium-acid phosphate Potassium-acid phosphate, monobasic Sodium-acid phosphate, monobasic/dibasic Potassium-acid phosphate, monobasic Sodium-acid phosphate, monobasic/dibasic Potassium-acid phosphate, monobasic Sodium-acid phosphate, monobasic/dibasic Sodium phosphate Phosphoric acid
125.6mg/tablet 250 mg/tablet 250mg/tablet 1g/300ml 250 mg/capsule (dissolve in water) 30.4mg/ml
aMust be prepared by pharmacist.
restore serum phosphate to the normal range. Since the proximal tubule is constantly wasting phosphate and since an oral phosphate load will last only a few hours, oral phosphate supplements need to be given 24 hr a day. Phosphate can be given in several forms (Table 4). The principal forms of therapy are Neutra-Phos, which can be given as a capsule or a suspension. In general, oral phosphate should be given four to six times daily. The main side effect of sodium phosphate therapy is diarrhea, which is related to its osmotic load. It should be recalled that sodium phosphate is the main component in a Fleets enema preparation, which is used as a potent cathartic [23]. Measurement of serum phosphate approximately 1 hr postdosing should be done to determine if serum phosphate concentrations are in the normal range for age [8]. Another complication of oral phosphate therapy is a rapid decline in serumionized calcium concentration, which will lead to tetany. As hypocalcemia becomes chronic, secondary hyperparathyroidism will develop accordingly and frequent measurements of serum calcium and phosphate are appropriate. There are a number of both specific and nonspecific therapies for patients with Fanconi syndrome, depending on the etiology of the disease. Specific therapies include cysteamine therapy to reduce intracellular cystine in infantile nephropathic cystinosis, L-thyroxine in cystinosis, and NTBC [(2-(nitro-4triflouromethylbenzoyl)- 1,3-cyclohexanedione)] therapy for hereditary tyrosinemia [24]. Renal transplantation is necessary in cystinosis and a liver-kidney transplant is necessary in hereditary tyrosinemia [1]. Fluid and electrolytes need to be replaced. No therapy is indicated for the
renal loses of glucose, uric acid, low-molecular-weight proteins, and amino acids [24]. Therapy with Minerals
As noted previously, the treatment of nonspecific Fanconi syndrome includes water, sodium bicarbonate (at doses of 10-15 mEq/kg body weight/day), potassium chloride, or, alternately, potassium bicarbonate, citrate, or acetate to treat hypokalemia and metabolic acidosis resulting from both proximal and distal renal tubular acidosis. Calcium carbonate may also be necessary to reverse hypocalcemia in subjects with renal insufficiency. If patients are hypomagnesemic, magnesium oxide therapy may be indicated [25]. Vitamin D Therapy
Treatment with vitamin D should be employed for at least three reasons: Hypocalcemia may lead to secondary hyperparathyroidism [24]; tubular disease may impair the l
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vitamin D [1,12,13,24]; however, vitamin D has been used extensively [24]. RENAL MAGNESIUM WASTING Magnesium depletion is a rare mineral disorder and may be overlooked because it may occur in a complex clinical situation. Renal magnesium wasting can occur as an isolated finding, as part of other clinical disorders, or as part of a primary inherited disorder called Gitelman syndrome [19,26]. This group of inherited conditions results in the excessive excretion of magnesium into the urine despite hypomagnesemia [26,27]. These patients often present with hypercalciuria, nephrocalcinosis, renal stones, and infection, and some patients may progress to renal failure. Recent genetic evidence has defined at least three genes that are responsible for this disorder [19]. In the past, this group of diseases was confusing, particularly since renal magnesium wasting was apparently associated with a heterogeneous array of signs and symptoms. These syndromes result in bone disease, as discussed later. Excessive urinary losses of magnesium are also associated with diabetic ketoacidosis, and hyperaldosteronism and occur following loop diuretics and after the use of several therapeutic agents, including aminoglycoside antibiotics, cyclosporin A, and cisplatin [3]. Hypomagnesemia should be considered when serum Mg 2+ is less than 0.65 mM [26]. Age-dependent upper reference values for urinary magnesium:creatinine ratios are available and should be consulted to make the diagnosis of hypomagnesemia [26]. Etiology Magnesium is transported at several sites along the nephron [3], particularly the ascending limb of the loop of Henle and the distal convoluted tubule. The renal handling of magnesium is quite different from that of calcium and phosphorus, as is the specific localization of this divalent mineral ion within the body. Whereas 99 and 85% of total body calcium and phosphate, respectively, are found in bone, only 50% of magnesium is present. Indeed, 50% of total body magnesium is intracellular, which is remarkably higher than the amount of calcium (1% intracellular). Moreover, magnesium transport is not regulated by the gut, as is the case for calcium, or by the proximal tubule of the kidney, as is the case for phosphate. Hence, magnesium uptake does not downregulate in either intestine or kidney when its plasma concentrations are high [1,3,16]. When magnesium is less available in the diet, magnesium ions can shift out of cells, especially lymphocytes, and into the extracellular
fluid. There also exists an age-dependent normal fractional excretion of the filtered magnesium load [3]. In general, urine magnesium is age dependent and should be evaluated [26]. The transport of magnesium may be transcellular or paracellular [3]. Transcellular magnesium uptake proceeds across the apical and basolateral surfaces of the loop of Henle cell and requires active uphill transport. The paracellular transport of magnesium occurs across the intracellular space between each cell. One of the magnesium wasting conditions, Gitelman syndrome, is due to a defect in the thiazide-sensitive sodium chloride cotransporter [28] that is located in the distal convoluted tubule [3].
Isolated Hypermagnesuria Isolated hypermagnesuria is due to mutations in the Na,K-ATPase 7 subunit in the ascending limb of the loop of Henle [19]. These patients present with isolated magnesium wasting and may have hypocalcemia and bone disease [19].
Michelis-Castrillo Syndrome Michelis-Castrillo syndrome is the hypercalcuric form of hypomagnesemia secondary to a defect in the paracellin-1 protein, which seems to be responsible for paracellular magnesium transport. This mutation prevents the opening of the paracellular space so that magnesium is wasted [19], This syndrome is associated with hypomagnesemia, hypercalciuria, hypocalcemia, nephrocalcinosis, and bone diseage [26,27]: Many of these patients develop progressive renal failure requiring renal replacement therapy, such as dialysis and transplantation [28,29] (Fig. 4). These patients may also have chondrocalcinosis, rickets, and gouty arthritis [27,29]. This syndrome, first described in 1982, may also have ocular findings [30,31]. The bone disease in this syndrome is complex, as indicated later.
Gitelman Syndrome In Gitelman syndrome [32,33], the defect resulting in hypermagnesuria appears to be in the distal tubule [34]. The features of Gitelman syndrome include renal magnesium wasting, hypokalemia, alkalosis, and hypocalcuria [28]. When exogenous oral magnesium ions are provided, a large proportion is wasted into the urine. Although Gitelman syndrome has been classically viewed as a variant of Bartter syndrome, the urinary excretion of magnesium and calcium is completely different in Bartter syndrome, in which there is normal or increased urinary calcium excretion and normal serum magnesium [35].
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FIGURE 4
Forms of Bone Disease
P a t h o g e n e s i s of Bone Disease
As noted previously, the bone disease in renal magnesium wasting is complex [27,29,35,36]. Osteopenia has been found in many patients with excessive magnesuria [27], which is evident on X-rays or bone densitometric analysis. Vertebral compression fractures have also been described, with reduced lumbar spine bone mineral content and density [36]. Another frequently described feature is calcium pyrophosphate dihydrate crystal deposition arthropathy [35]. The impact of bone disease on quality of life has been evaluated. Compared to normal control subjects, patients with Gitelman syndrome have muscle cramps, fatigue, generalized weakness, and muscle paralysis. Demineralization has been described, as has chondrocalcinosis [37]. As patients with Michelis-Castrillo syndrome develop progressive renal failure, they develop renal osteodystrophy [27]. Certain features of this hypomagnesemic with hypercalciuria syndrome with regard to renal osteodystrophy are quite different [27], including X-ray evidence of severe rickets with demineralization and fraying and widening of the metaphyses (Fig. 4). These patients may show stress fractures and a diminished bone age. They do not have evidence of secondary hyperparathyroidism, such as osteitis fibrosa, erosion of the lamina dura, and subperiosteal erosion. In this syndrome, despite severe renal insufficiency, the serum immunoreactive PTH value is normal or low normal. In isolated renal magnesium wasting, a rare autosomal dominant condition with mild hypomagnesemia [38], the main bone effect is chondrocalcinosis [39].
A key feature of magnesium deficiency is related to altered parathyroid gland function, including impairment of PTH secretion [40]. Hypomagnesemia alters end-organ (in this case, bone) responsiveness to circulating PTH and thus contributes to hypocalcemia. Infusion of intravenous magnesium supplements to magnesiumdeficient hypocalcemic patients augments the serum concentrations of PTH. Magnesium is also an important exogenous factor in the action and/or metabolism of vitamin D [40]. This is indicated by the finding that some patients who have hypomagnesemia may respond to calcitriol or l~-hydroxyvitamin D3 only after serum magnesium is normalized [1]. Serum concentrations of calcitriol are frequently reduced in magnesium-depleted patients [40]. Hence, the mineral balance abnormalities may include reduced serum values of magnesium, calcium, calcitriol, and PTH; these can be restored to normal by infusion and/or oral ingestion of magnesium salts. Treatment of H y p o m a g n e s e m i a The goal of ]the treatment of hypomagnesemia is to normalize serum magnesium when there is excessive urinary magnesium excretion. Therapy consists of administration of 1-5 g (50-250mEq) magnesium oxide in three divided doses, depending on age and size. A starting dose of magnesium oxide is 3-6mg of elemental Mg+/kg/24 hr, but in patients with renal Mg + wasting this may need to be increased substantially (2- to 10-fold).
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The goal of treatment of hypomagnesemia is to normalize serum magnesium. Given in daily doses, this will cause the following changes: increased serum and urine magnesium and calcium, reduced serum phosphate, increased urine phosphate excretion, and elevations in calcitriol and PTH [28]. Although the doses of oral magnesium required vary between patients, improved magnesium homeostasis can usually be achieved. Magnesium should be taken three or four times daily in the form of magnesium oxide or magnesium pyrilidine carboxylate since urinary magnesium losses occur continuously. Patients, even if hypomagnesemic, have continuous excretion. Infants who have hypomagnesemic tetany should receive 0.4-0.8 mg/kg of a 50% solution of MgSO4 either intramuscularly or intravenously, with a total of 25-50 mg/kg/dose. For safety reasons, intravenous magnesium should be infused slowly with careful cardiac monitoring, and calcium gluconate should be readily available to deal with arrythmias. Finally, renal magnesium wasting can develop in association with varying degrees of renal insufficiency [40]. These patients may require 0.25-1.0gg of calcitriol along with calcium lactate, carbonate, or acetate.
HYPERCALCIURIA Idiopathic hypercalciuria (IH) refers to increased urinary excretion of calcium as an isolated defect, not in association with known renal tubular defects such as renal tubular acidosis, renal insufficiency, and Barttertype syndrome. That tubular wasting of calcium is associated with renal stone formation has been known for at least 50 years. Diminished bone mineral content in this condition was recognized in the 1970s. The debate about which occurs first, bone disorder or renal disorder, continues. IH was subdivided into the renal leak (or fasting) form or the absorptive form. Although this differentiation was attractive from a pathophysiologic standpoint, it is not currently used clinically because of the difficulty in discriminating between patients and the fact that the same individual might be classified as having either form at different times [41]. In addition, measurement of bone mineral density according to subtype has yielded conflicting results. There is little doubt that this metabolic disorder leads to renal stones and that stone-formers with IH are at risk for bone disease. Hypercalcuria can be defined by age, but in older children it is defined by urinary calcium excretion of >4 mg/kg/24 hr [13].
Disordered Regulation of CalcitriolfVitamin D Receptor When Charles Pak proposed the differentiation of IH into the absorptive and renal hypercalciuric subtypes, the fasting form was thought to represent a primary renal leak caused from increased PTH, which led to increased calcitriol and increased gut calcium absorption [42,43]. The absorptive subtype was attributed to a primary increase in intestinal absorption of calcium and was characterized by low PTH levels. Later, Coe and Bushinsky [44] proposed that altered vitamin D metabolism, possibly through polymorphisms in the gene for the intestinal vitamin D receptor, was the cause of the absorptive form of IH. That increased PTH might cause renal leak or fasting hypercalciuria was supported by the finding of increased urinary excretion of cAMP, the known second messenger for PTH action in the kidney [43]. Subsequent studies, however, failed to implicate increased PTH as the primary cause of fasting hypercalciuria [42].
Dent Disease Dent's disease is an X-linked variety of nephrocalcinosis, with hypercalciuria and low-molecular-weight proteinuria [45]. This disorder is named after Dr. Charles Dent, an expert in genetic renal disorders who led a metabolic unit in London. This condition consists not only of low-molecular-weight proteinuria and hypercalciuria with nephrolithiasis and nephrocalcinosis but also other proximal tubule abnormalities, including aminoaciduria, phosphaturia, and glycosuria. This condition, which is more common in males, may result in metabolic bone disease and progressive renal failure [46]. Patients may also have mild acidosis but no evidence of renal tubular acidosis [47]. This disorder is caused by a mutation in the voltage-gated chloride channel 5 (C1C5) [45,48], particularly in the $3 segment of the proximal tubule [19]. Deletion of the gene for C1C5 in knockout mice results in a murine homolog of Dent disease [49]. The prototype for the chloride channel is found in the electric eel. Mutations in another chloride channel (C1C Kb) are found in Bartter syndrome type III [501. In the endosome of the renal proximal tubule, the process of degradative endocytosis of filtered proteins occurs, which requires the action of an H+-ATPase at the endosome [51]. Whenever a mutation is found in C1C5, the process of endosome acidification cannot occur, which thus prevents the endosomal uptake of low-molecular-weight proteins. It has been speculated that this endosomal degradation of protein within bone may be important in bone formation and may be defect-
26. Rickets Due to Renal Tubular Abnormalities
ive in Dent disease [51]. Hypercalciuria also contributes to reduced mineralization in bone. Several human diseases are due to mutations in the chloride channel family, including Thompson disease, Becker hypotonia, diabetes insipidus, Bartter syndrome type III, as well as Dent disease [45]. The mechanism of hypercalciuria is unclear, but it has been posited that defective proximal tubular endocytosis of calcitropic hormones could result in abnormal calcium metabolism with hypercalciuria [19].
Laboratory Diagnosis of Idiopathic Hypercalciuria Hypercalciuria refers to increased urinary calcium excretion, defined in children as >4 mg/kg/24 hr or, alternatively, as a random urine calcium:creatinine ratio >0.20. Urinary calcium excretion is higher in infants and toddlers younger than 2 years of age. In children, IH presents with hematuria (either gross or microscopic) or as urolithiasis. Occasionally, IH may manifest as enuresis, dysuria, or flank pain. Laboratory investigation for IH includes a timed urine for calcium, creatinine, and sodium. This should be performed when the child is well, on a regular diet, and not during an acute stone episode. High salt intake is associated with elevated urinary calcium excretion. Therefore, reduction of dietary sodium is often the initial recommendation. In some children, dietary salt restriction (2 or 3 g per day) alone results in normalization of urinary calcium excretion and resolution of hematuria. Stapleton recommends dietary calcium restriction (no milk products) as the next diagnostic step in the evaluation of IH. If hypercalciuria continues, then a trial of hydrochlorothiazide (1 or 2 mg/kg/day) for 4 weeks followed by measurement of urinary calcium excretion is useful in proving that the urinary symptoms (i.e., hematuria or lower tract symptoms) are related to the hypercalciuria. Secondary conditions, such as renal tubular acidosis, Dent's disease, hyperparathyroidism, hypervitaminosis D, and systemic disorders such as juvenile rheumatoid arthritis, should be excluded. Stapleton no longer recommends establishing the subtype of IH (absorptive or renal leak) by oral calcium loading test, although this may be of use in research studies. Bone D i s e a s e A s s o c i a t e d with Hypercalciuria
Clinical Characteristics As mentioned previously, IH in children most often presents as hematuria or during the evaluation ofnephrolithiasis. Of 83 children with IH and no history ofurolithiasis or urinary tract infection, hypercalciuria was present
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in 27% [52]. Gross hematuria and a positive family history of renal stones were more likely in the group with IH. The condition has been reported in children throughout the world but is relatively uncommon in African American children. Frequency/dysuria, incontinence/enuresis, and abdominal/back pain are associated symptoms of hypercalciuria in some series [53]. Among the hypercalciuric children reported by Alon and Berenbom [53], the urine calcium:creatinine ratio ranged from 0.22 to 0.45 (mean, 0.32) and the 24-hr urine calcium excretions ranged from 4.49 to 9.2mg/kg (mean, 5.64 mg/kg). At follow-up of 33 children after an average of 4.6 years, no child had a renal stone or recurrent macroscopic hematuria. Pharmacologic treatment was not used, and approximately one-third of the children were said to adhere to a relatively salt-restricted diet. Half of the children remained hypercalciuric on the first urine sample, and one-fourth remained hypercalciuric on the second sample. Only 1 child demonstrated a renal calculus on ultrasound examination. Of those children with persistent hypercalciuria, all responded to a low-sodium and highpotassium diet with normalization of urinary calcium.
Alterations in Bone Mineral Density Decreased bone mineral density has been found among stone-forming patients as well as hypercalciuric patients with both types of hypercalciuria, although findings are conflicting, with some studies failing to demonstrate decreased bone density among the absorptive type [54,55]. When placed on a low-calcium diet, subjects who responded with normalization of urinary calcium tended to have normal bone density, whereas those who continued to excrete excess calcium had decreased vertebral mineral density [56]. Some children with hypercalciuria have decreased bone mineral density as measured by bone densitometry on the lumbar spine. Perrone et al. [57] found that 4 of 20 children with IH had decreased bone mineral density early in their diagnosis and the osteopenia appeared to be progressive in those children with sustained hypercalciuria. Seventy-three Spanish children with IH underwent analysis; 22 children (30%) had osteopenia as defined by a z score o f - 1 or less [58]. Weisenger [42] found decreased lumbar spine bone mineral density in 21 hypercalciuric children with a mean age of 9.3 years. Cortical and total bone mineral density were not decreased.
Histology Although limited, bone biopsy data are available. Malluche et al. [59] performed bone biopsies on 15 recurrent
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stone-formers with hypercalciuria. There was increased osteoid volume and decreased osteoblastic activity and mineralizing osteoid seams. The authors summarized their findings as decreased mineralization of osteoid with no evidence of increased bone resorption. This was supported by the work of Pak and colleagues [60]. Other groups noted increased bone resorption [61,62]. Pathogenesis The possibility that disordered bone metabolism might result in IH has been considered. Increased expression of interleukin- 1 (IL- 1) by monocytes from patients with renal leak and not absorptive IH was reported; however, due to the age of the population, some postmenopausal women may have been included (increased IL-1 production has been proposed to contribute to postmenopausal osteoporosis) [63]. Weisinger et al. [64] found that IL-10~ production had an inverse correlation with bone mineral density in 29 stone-forming hypercalciuric adults, all of whom displayed diminished trabecular bone mineral density. There was no significant increase in the nonstimulated production of IL01[3, IL-6, and tumor necrosis factor
warrants concern, but there is no consensus regarding therapy to prevent bone disease in children. In fact, this issue raised so much controversy that a prospective study could not be designed to the satisfaction of the Southwest Pediatric Nephrology Study Group and therefore was never finalized. As suggested by Stapleton [52], the initial treatment is dietary salt restriction and increased water intake. Alon and Berenbom [53] also increase dietary potassium intake. The addition of thiazide diuretics, which reduce calcium excretion by their action on the Na+-Ca + cotransporter in the distal nephron, may be indicated for diagnostic purposes and in children with urolithiasis. The question often arises as to how long to continue a treatment with serious potential side effects, such as electrolyte disturbances, hyperlipidemia, and photosensitivity [67]. Some use Naqua, whereas others use hydrochlorothiazide. In adults, thiazide diuretics allow recovery of bone mineral content [66], whereas a low-protein and low-salt diet has been shown to lower the stone risk as well urinary calcium excretion [65]. Restriction of protein in a growing child is usually not recommended; thus, most pediatric nephrologists employ either salt-restricted diets or pharmacologic treatment. The use of bisphosphonates in children with IH has not been examined. Careful longitudinal studies are needed before conclusions can be made regarding the need for treatment of the child with IH. Limitation of dietary salt and encouragement to drink plenty of water are logical, reasonable recommendations. The addition of specific therapy such as thiazide diuretics is not universally recommended, but it may be beneficial in the case of chronic hypercalciuria despite conservative measures.
RENAL TUBULAR ACIDOSIS Renal tubular acidosis (RTA) refers to a heterogeneous group of disorders characterized by chronic hyperchloremic metabolic acidosis as a result of abnormal renal tubular handling of bicarbonate or hydrogen ions [68]. RTA is typically subdivided according to the tubular site affected and the underlying pattern. Type I, or distal, RTA results from inadequate net hydrogen ion secretion by the cortical collecting duct alpha intercalated cells. Type II, or proximal, RTA results from impaired bicarbonate reabsorption by the proximal tubule.
Typical Biochemical Abnormalities a n d Diagnosis of RTA RTA can be divided into two major subtypes based on clinical and physiologic manifestations. Proximal RTA
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(pRTA) is characterized by a decreased renal tubular threshold for sodium bicarbonate. The proximal tubule cannot reabsorb bicarbonate when the plasma bicarbonate and filtered load of bicarbonate are normal, resulting in alkaline urine. The fractional excretion of bicarbonate exceeds 15%. pRTA is more often manifest in combination with generalized proximal tubular dysfunction (Fanconi's syndrome) but also may be isolated. Linear growth failure is the most common clinical manifestation, along with hypokalemia and its symptoms. Diagnosis of pRTA relies on demonstrating abnormal bicarbonate excretion when the plasma bicarbonate is increased from low to normal. Measurement of urine pH or the fractional excretion of bicarbonate are common clinical tests. Most children with pRTA require large doses of alkali to maintain a normal plasma bicarbonate. A fractional excretion of bicarbonate {[(Wbicarb/Pbicarb ) • (Pcreat/Wcreat)] • 100} of 15% or more is characteristic of pRTA.
Proximal RTA
Clinical Characteristics pRTA often accompanies Fanconi syndrome but may rarely occur as a transient, isolated condition. When persistent, it is may be acquired or inherited. Clinical manifestations during infancy include polyuria, polydipsia, vomiting, dehydration, muscle weakness, and failure to thrive. Two phenotypes have been described: (i) pRTA with ocular abnormalities and/or mental retardation with an autosomal recessive pattern and (ii) pRTA with an autosomal dominant pattern [68].
mouse model for the Na/H exchanger with pRTA [50]. Mutations in the gene encoding for the Na/bicarbonate cotransporter have been described in autosomal recessive pRTA associated with ocular disease (cataracts, glaucoma, and band keratopathy) [19].
Bone Disease Associated with pRTA Bone disease among individuals with pRTA has been assumed to be the result of chronic acidosis, which induces bone carbonate release and may also cause hypercalciuria. Osteoporosis and osteomalacia have been described in these individuals, although they are not often documented by bone histomorphometric analysis. Recently, bone mineral density and histology were analyzed in a kindred with isolated pRTA [71]. Both affected and unaffected family members underwent analysis. Affected individuals were shorter compared to unaffected individuals; however, none of the affected individuals had hypercalciuria or radiographic evidence of bone disease. Bone mineral density measured in the radius was low in the affected individuals. Bone biopsies revealed thin cortices, normal bone mineralization rates, and no histomorphometric evidence of osteomalacia or osteoporosis. Therefore, members of the kindred with untreated pRTA did have abnormal total bone mass and bone structure. Since these individuals were studied as adults, it is conceivable that there might have been more impressive radiologic or histologic abnormalities present during growth. Distal RTA
Physiology The physiologic defect in pRTA is impaired net reabsorption of bicarbonate. This could arise from any one of the transport systems or enzymes involved in proximal tubular bicarbonate reabsorption and could result from a deficiency of carbonic anhydrase, which allows conversion of intracellular carbon dioxide and water to bicarbonate; abnormalities in the luminal Na/H exchanger; or abnormalities of the basolateral Na/bicarbonate cotransporter [19]. Autosomal mutations of carbonic anhydrase II have been reported in RTA in association with osteopetrosis, mental retardation, and intracranial calcifications [69]. However, these patients manifest both proximal and distal defects [70]. The bone disease associated with carbonic anhydrase deficiency results from the inability of osteoclasts to secrete acid to dissolve bone mineral [681. No human mutations of the Na/H exchanger have been identified; however, this is an attractive candidate for the autosomal dominant form. There is a knockout
Distal RTA (dRTA) is defined as impaired distal hydrogen ion secretion and the inability to lower urine pH to less than 5.5 during systemic acidemia. Linear growth failure, rickets, polyuria, and nephrocalcinosis/ urolithiasis are the most common clinical symptoms of dRTA. Classical tests of distal tubular acidification include the ammonium chloride loading test and measurement of urinary ammonium excretion. Alternatives to these cumbersome tests include arginine hydrochloride infusion, sodium sulfate infusion, and intravenous furosemide tests. Urine pH is measured by pH meter after systemic acidosis is produced in the first test or distal sodium delivery is enhanced producing a luminal negative charge favoring proton secretion in the latter two tests. Recently, the use of the urinary anion gap (UAG) as a method to estimate urinary ammonium excretion has replaced the more cumbersome methods listed previously as a screening test for dRTA [72]. The UAG is calculated using the random concentrations of Na, K, and C1 during acidosis. The difference between [C1] and
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the sum of [Na] and [K] equals the concentration of ammonium plus unmeasured urine anions. This test has been validated in adults and children [73]. If a urine sample from a child with hyperchloremic metabolic acidosis has a negative anion gap ([C1] > {[Na] + [K]}), then gastrointestinal or renal bicarbonate loss should be suspected. Alternatively, if the anion gap is positive, a distal acidification defect is suspected. This is confirmed by using a more stringent test of renal distal acidification. The acetazolamide test has gained popularity as an easy and reliable measure of distal tubular proton secretion in which the urine-to-blood pCO2 gradient is measured after flooding the distal tubule with bicarbonate. Alon et al. [73] described this test as follows" 17 mg/kg of acetazolamide is given as a single oral dose 3 hr after the last meal. The first urine is discarded, after which freshly voided urine is collected in a syringe, capped, and delivered on ice to the laboratory, where the pH and pCO2 are measured. Only urine samples with a pH >7.5 are suitable for analysis. The second and third urine samples are analyzed, and venous samples for pH and pCO2 are collected. A urine pCO2 - blood pCO2 <20 mmHg indicates abnormal distal acidification. A separate form of dRTA is hyperkalemic: In these individuals, impaired ammoniagenesis causes a decrease in acid excretion and is related to aldosterone deficiency or resistance. In children, obstructive uropathy and pseudohypoaldosteronism are common causes of type IV hyperkalemic RTA. Clinical Characteristics dRTA usually presents in infancy with polyuria, vomiting, dehydration, failure to thrive, hypokalemia, and inappropriately alkaline urine during systemic acidosis. dRTA may be inherited as autosomal dominant, which is the less severe form, or autosomal recessive, the more severe, infantile form, which is often accompanied by nephrocalcinosis and may progress to renal insufficiency. The infantile form is also often associated with sensorineural hearing loss [68]. dRTA is characterized by hypercalciuria and hypocitraturia, which likely explains the high prevalence of nephrocalcinosis and renal stones in individuals with dRTA. Physiology In the 0~-intercalated cell of the cortical-collecting duct, hydrogen ions are actively secreted by H-ATPase, which is a multimeric pump in the superfamily of proton ATPases. In order to maintain intracellular pH, bicarbonate is exchanged for chloride at the basolateral cell membrane. Mutations in the anion exchanger have been
reported to account for both autosomal dominant and recessive dRTA [68]. Recently, mutations in the 13 subunit and the noncatalytic subunit of the proton ATPase have been described in individuals with dRTA [19]. Bone Disease Associated with dRTA Osteomalacia is the suspected bone lesion in dRTA, but few studies actually demonstrate this conclusively. Bone disease in dRTA was examined by Brenner et al. [74], who described abnormal skeletal radiographs in only 1 of 44 adults studied. This individual also had azotemia and osteopenia. Vitamin D levels measured in children with dRTA were within the normal range [75]. Occasionally, mild hyperparathyroidism and hypercalciuria are described in adults with dRTA [76]. Bone pain and osteomalacia appear to respond to treatment with alkali [77]. Of 33 patients with dRTA, 9 had metabolic bone disease by X-ray, 4 had osteomalacia, and 5 had osteoporosis; however, the diagnosis was confirmed by biopsy in only 3 patients and was made by radiography alone in 6 patients [78]. A recent study of untreated adult farmers from Thailand with dRTA revealed that all 14 of the affected individuals were shorter than normal and also displayed a reduced body mass index [79]. Seventy-one percent had muscle weakness and 29% had renal stones. Two had previously experienced pathologic fractures. Osteopenia was present radiographically in all, and bone density was decreased: bone mineral density at L2-L4 indicated osteopenia in 29% and osteoporosis in 21%, and that at the trochanter indicated osteopenia in 43% and osteoporosis in 14%. On histomorphometric analysis, bone formation rates were significantly lower (0.02 + 0.02~tm3/day) than the control mean of 0.07 + 0.045 ~tm3/day. Both osteoblastic and osteoclastic numbers/area were decreased compared to controls, but this did not reach statistical significance. Osteoid volume and surface were increased, but no difference in osteoid thickness was detected except in 1 individual whose osteoid thickness was 29.96 ~tm, fulfilling the criteria for osteomalacia. None of the subjects had hypercalciuria, but they were all on a relatively low-salt diet. Although they did display decreased bone mineral density and had symptomatic bone disease (pain), bone formation rates were low rather than high, as was previously suspected. Osteomalacia was not typical. Since none of the subjects were treated, this argues against osteomalacia as a result of chronic acidosis alone. Incomplete RTA may be more common than generally recognized; it was found in 10 of 46 adults referred for investigation of osteopenia/osteoporosis [80]. Individuals referred for suspected osteoporosis underwent ammonium chloride loading and furosemide testing for
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urinary acidification. None of the subjects had overt acidosis or known renal disease, although 5 had previously had renal stones and 4 had hypercalciuria. There were 20 age- and gender-matched controls. Lumbar (L2-L5) bone mineral density was assessed. A score of -1.0-2.5 was definitive for osteopenia, and a score less than -2.5 indicated osteoporosis. Ten subjects had incomplete RTA, as demonstrated by inability to acidify the urine below pH 5.5. The group with incomplete RTA was 12 years younger that the group without RTA. Associated disorders among incomplete RTA included renal stones or hypercalciuria, and 1 subject had medullary sponge kidney disease and 1 had a family history of dRTA. Although the small numbers of this series do not allow broad conclusions, the authors speculated that the subjects with abnormal distal acidification were prone to more rapid bone mineral loss, causing them to present at an earlier age [80].
Treatment Treatment for RTA consists of supplementation of base in the form of Na bicarbonate or a combination of sodium and potassium citrate given in three or four daily doses. Patients with type I typically require 2 or 3 mEq/ kg/day, whereas those with pRTA usually need in excess of 10 mEq/kg/day. Unless overt vitamin D deficiency is observed, supplementation is usually not required since the levels are typically within normal limits. The form of RTA due to carbonic anhydrase II deficiency associated with osteopetrosis has been treated with bone marrow transplantation.
CONCLUSION Renal tubular syndromes are uncommon in children but result in a variety of bone disorders. These bone disorders are important in diagnosis and require therapy. At least six fundamental pathophysiologic mechanisms underlie bone disease in renal tubular syndromes; however, demineralization in a given disorder may be due to a combination of these mechanisms. These mechanisms are hypophosphatemic osteomalacia, which is the consequence of impaired renal tubular phosphate reabsorption and continued phosphaturia despite the signal of reduced serum phosphate concentration; hyperexcretion of calcium; hyperexcretion of magnesium, which can diminish the availability of both calcium and magnesium for mineralization of osteoid; bicarbonate wasting, which causes metabolic acidosis and reduced bone formation and enhanced bone resorption; renal osteodystrophy, which accompanies those renal tubular disorders that progress to renal failure with the result of diminished
calcitriol biosynthesis; and disordered vitamin D metabolism, which alters the intestinal absorption of calcium and phosphate.
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a hypocalciuric variant of the syndrome. Miner. Electrolyte Metab. 18, 43-51. Calo, L., Punzi, L., and Semplicini, A. (2000). Hypomagnesemia and chondrocalcinosis in Bartter's and Gitelman's syndrome: Review of the pathogenetic mechanisms. Am. J. Nephrol. 20, 347-350. Oktenli, C. (2000). Renal magnesium wasting, hypomagnesemic hypocalcemia, hypocalciuria and osteopenia in a patient with glycogenosis type II. Am. J. Nephrol. 20, 412-417. Cruz, D., Shaer, A., Bia, M., Lifton, R., and Simon, D. (2001). Gitelman's syndrome revisited: An evaluation of symptoms and health related quality of life. Kidney Int. 59. Cole, D. (2000). Inherited disorders of renal magnesium handling. J. Am. Soc. Nephrol. 11, 1937-1947. Geven, W., Monnens, L., WiUems, J., Buijs, W., and ter Haar, B. (1987). Renal magnesium wasting in two families with autosomal dominant inheritance. Kidney Int. 31, 1140-1144. Carpenter, T., and Key, L., Jr. (1990). Disorders of the metabolism of calcium, phosphorus and other divalent ions. In Pediatric Textbook of Fluids and Electrolytes (I. Ichikawa, Ed.), pp. 237-268. Williams & Wilkins, Baltimore. Aladjem, M., Barr, J., Lahat, E., and Bistritzer, T. (1996). Renal and absorptive hypercalciuria: A metabolic disturbance with varying and interchanging modes of expression. Pediatrics 97, 216-219. Weisinger, J. (1996). New insights into the pathogenesis of idiopathic hypercalciuria: The role of bone. Kidney Int. 49, 1507-1518. Pak, C., Ohata, M., Lawrence, E., and Snyder, W. (1974). The hypercalciurias: Causes, parathyroid functions and idiopathic criteria. J. Clin. Invest. 54, 387-400. Coe, F., and Bushinsky, D. (1984). Pathophysiology of hypercalciuria. Am. J. Physiol. 247, F 1-F 13. Wills, N., and Fong, P. (2001). C1C chloride channels in epithelia: Recent progress and remaining puzzles. News Physiol. Sci. 16, 161-166. Scheinmau, S. (1998). X-linked hypercalciuric nephrolithiasis: Clinical syndromes and chloride channel mutations. Kidney Int. 53, 3-17. Piwon, N., Gunther, W., Schwake, M., Bosl, M., and Jentsch, T. (2000). C1C-5 CI channel disruption impairs endocytosis in a mouse model for Dent's disease. Nature 408. Jentsch, T., Friedrich, T., Schriever, A., and Yamada, H. (1999). The C1C chloride channel family. Pflugers Arch. 437, 783-795. Wang, S., Devuyst, O., Courtoy, P., Wang, X.-T., Wang, H., Wang, Y., Thakker, R., Guggino, S., and Guggino, W. (2000). Mice lacking renal chloride channel, C1C-5, are a model for Dent's disease, a nephrolithiasis disorder associated with defective receptor mediated endocytosis. Hum. Mol. Genet. 9, 2937-2945. Simon, D., Bindra, R., Mansfield, T., Nelson-Williams, C., Mendonca, E., Stone, R., Schurman, S., Nayir, A., Alpay, H., Bakkaloglu, A., Rodriguez-Soriano, J., Morales, J., Sanjad, S., Taylor, C., Pilz, D., Bren, A., Trachtman, H., Griswold, W., Richard, G., John, E., and Lifton, R. (1997). Mutations in the chloride channel gene, C1CNKB, cause Bartter's syndrome type III. Nature Genet. 17, 171-178. Beyenbach, K. (2001). Energizing epithelial transport with the vacuolar H+-ATPase. News Physiol. Sci. 16, 145-151. Stapleton, F. B. (1994). Hematuria associated with hypercalciuria and hyperuricosuria: A practical approach. Pediatr. Nephrol. 8, 756-761. Alon, U. S., and Berenbom, A. (2000). Idiopathic hypercalciuria of childhood. Pediatr. Nephrol. 14, 1011-1015.
26. Rickets Due to Renal Tubular Abnormalities 54. Lawoyin, S., Sismilich, S., Browne, B., and Pak, C. (1979). Bone mineral content in patients with calcium urolithiasis. Metabolism 28, 1250-1254. 55. Lindergard, B., Collen, S., Mansson, W., Rademark, C., and Rogland, B. (1983). Calcium loading test and bone disease in patients with urolithiasis. Proc. EDTA 20, 460-465. 56. Bataille, P., Achard, J., Fournier, A., Boudailliez, B., Westeel, P., Esper, N., Bergot, C., Jans, I., Lalau, J., Petit, J., Henon, G., Jeantet, M., Bouillon, R., and Sebert, J. (1991). Diet, vitamin D, vertebral mineral density in hypercalciuric calcium stone formers. Kidney Int. 39, 1193-1205. 57. Perrone, H., Marone, M., Bianco, A., Toporovski, J., Malvestiti, L., and Schor, N. (2002). Bone mineral density in hypercalciuric children: A 5 year follow-up [Abstract]. Pediatr. Nephrol., C121. 58. Garcia-Nieto, V., Ferr~ndez, C., Monge, M., de Sequera, M., and Rodrigo, M. (1997). Bone mineral density in pediatric patients with idiopathic hypercalciuria. Pediatr. Nephrol. 11, 578-583. 59. Malluche, H., Tschoepe, W., Ritz, E., Meyer-Sabellek, W., and Massry, S. (1980). Abnormal bone histology in idiopathic hypercalciuria. J. Clin. Endocrinol. Metab. 50, 654-658. 60. Zerwekh, J., Sakhaee, K., Breslau, N., Gottschalk, F., and Pak, C. (1992). Impaired bone formation in male idiopathic osteoporosis: Further reduction in the presence of concomitant hypercalciuria. Osteoporosis Int. 2, 128-134. 61. Pfeferman-Hejlberg, I., Martini, L., Szejnfeld, V., Carvalho, A., Draibe, S., Ajzen, H., Ramos, O., and Schor, N. (1994). Bone disease in calcium stone forming patients. Clin. Nephrol. 42, 175-182. 62. Steiniche, T., Mosekilde, L., Christensen, M., and Melsen, F. (1989). A histomorphometric determination of iliac bone remodeling in patients with recurrent stone formation and idiopathic hypercalciuria. A P M I S 97, 309-316. 63. Pacifici, R., Rifas, L., Teitelbaum, S., Slatapolsky, E., McCracken, R., Bergfeld, M., Lee, W., Avioli, L., and Peck, M. (1987). Spontaneous release of interleukin-1 from human blood monocytes reflects bone formation in idiopathic osteoporosis. Proc. Natl. Acad. Sci. USA 84, 4616-4620. 64. Weisinger, J., Alonzo, E., Bellorin-Font, C., Blasini, A., Rodriguez, M., Paz-Martinez, V., and Martinis, R. (1996). Possible role of cytokines on the bone mineral loss in idiopathic hypercalciuria. Kidney Int. 49, 244-250. 65. Borghi, L., Schianchi, T., Meschi, T., Guerra, A., Allegri, F., Maggiore, U., and Novarini, A. (2002). Comparison of two diets for the prevention of recurrent stones in idiopathic hypercalciuria. N. Engl. J. Med. 346, 77-84. 66. Adams, J., Song, C., and Kantorovich, V. (1999). Rapid recovery of bone mass in hypercalciuric, osteoporotic men treated with hydrochlorothiazide. Ann. Intern. Med. 130, 658-660. 67. Reusz, G. S., Dobos, M., Tulassay, T., and Miltenyi, M. (1993). Hydrochlorothiazide treatment of children with hypercalciuria: Effects and side effects. Pediatr. Nephrol. 7, 699-702.
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68. Rodriguez-Soriano, J. (2000). New insights into the pathogenesis of renal tubular acidosis from functional to molecular studies. Pediatr. Nephrol. 14, 1121-1136. 69. Sly, W., Whyte, M., Sundaram, V., Tashian, R., Hewett-Emmett, D., Guibaud, P., Vainsel, M., Baluarte, H., Gruskin, A., A1Mosawi, M., Sakati, N., and Ohlsson, A. (1985). Carbonic anhydrase II deficiency in 12 families with the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcifications. N. Engl. J. Med. 313, 139-145. 70. Nagai, R., Kooh, S., Balfe, J., Fenton, T., and Halperin, M. (1997). Renal tubular acidosis and osteopetrosis with carbonic anhydrase II deficiency: Pathogenesis of impaired acidification. Pediatr. Nephrol. 11,633-636. 71. Lemann, J., Adams, N., Wilz, D., and Brenes, L. (2000). Acid and mineral balances and bone in familial proximal renal tubular acidosis. Kidney Int. 58, 1267-1277. 72. Rodriguez-Soriano, J., and Vallo, A. (1990). Renal tubular acidosis. Pediatr. Nephrol. 4, 268-275. 73. Alon, U., Hellerstein, S., and Warady, B. A. (1991). Oral acetazolamide in the assessment of (urine-blood) PC02. Pediatr. Nephrol. 5, 307-311. 74. Brenner, R., Spring, D., Sebastian, A., McSherry, E., Genant, H., Palubinskas, A., and Morris, J. R. (1982). Incidence of radiographically evident bone disease, nephrocalcinosis, and nephrolithiasis in various types of renal tubular acidosis. N. Engl. J. Med. 307, 217-221. 75. Chesney, R. W., Kaplan, B., Phelps, M., and DeLuca, H. (1984). Renal tubular acidosis does not alter circulating values of calcitriol. J. Pediatr. 104, 51-55. 76. Coe, F., and Firpo, J. (1975). Evidence for mild reversible hyperparathyroidism in distal renal tubular acidosis. Arch. Intern. Med. 135, 1485-1489. 77. Richards, P., Chamberlain, M., and Wrong, O. (1972). Treatment of osteomalacia of renal tubular acidosis by sodium bicarbonate alone. Lancet 11,994-997. 78. Harrington, T., Bunch, T., and Van den Berg, C. (1983). Renal tubular acidosis: A new look at treatment of musculoskeletal and renal disease. Mayo Clinic Proc. 58, 354-360. 79. Domrongkitchaiporn, S., Pongsakul, C., Stitchantrakul, W., Sirikulchayanonta, V., Ongphiphadhanakul, B., Radinahamed, P., Karnsombut, P., Kunkitti, N., Ruangraksa, C., and Rajatanavin, R. (2001). Bone mineral density and histology in distal renal tubular acidosis. Kidney Int. 59, 1086-1093. 80. Weger, W., Kotanko, P., Weger, M., Deutschmann, H., and Skrabal, F. (2000). Prevalence and characterization of renal tubular acidosis in patients with osteopenia and osteoporosis in non-porotic controls. Nephrol. Dial Transplant. 15, 975-980.
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[27] Hypophosphatasia DAVID E. C. COLE Departments of Labratory Medicine and Pathobiology, Medicine, and Genetics, University of Toronto, Toronto, Canada
Hypophosphatasia (MIM Nos. 146300, 241500, and 241510) is an inherited metabolic bone disease that clarifies the critical role that alkaline phosphatase (ALP) plays in skeletal mineralization. Subnormal activity of ALP in serum (hypophosphatasemia) is the biochemical hallmark and reflects a generalized deficiency of tissue-nonspecific (liver/bone/kidney) ALP isoenzyme (TNALP), with attendant clinical effects of skeletal undermineralization, pathologic fractures, and loss of teeth [1-4].
by a 20-residue signal sequence in the N-terminal portion of the nascent peptide [10-12]. After proteolytic cleavage, internal disulfide bridge formation is catalyzed by periplasmic enzymes; then, partial folding is followed by self-association to form enzymaticaUy active homodimers [13,14]. In the process, the enzyme acquires two zinc ions that are coordinately bound and participate in enzyme catalysis. In the third metal-binding site, zinc or magnesium may be present, but magnesium contributes to maximum enzymatic activity [15-17]. The active pocket may be occupied by an inorganic phosphate anion, a strong competitive inhibitor [15,17,18]. The sporulating organism, Bacillus subtilis, expresses at least two ALPs (phoAIII and phoAIV), which show strong homology [19]. In both E. coli and B. subtilis, as in other prokaryotes, expression of the ALP is regulated as part of an operon, pho, which governs the organism's multigene response to phosphate starvation. In yeast (Saccharomyces cerevisiae), the PH08 gene specifies an ALP containing considerable sequence identity with both the E. coli and human enzymes [20]. The most highly conserved regions are those surrounding the active sites for substrate and metal ligand binding. The N-terminus sequence is rich in basic and hydroxylcontaining amino acids and serves as a signal for translocation to the intracellular lysosomal vacuole, where the active enzyme is localized. The functional yeast enzyme is a dimer that undergoes N-glycosylation at specific asparagine residues [20]--a feature that appears to be common to eukaryotic ALPs.
BIOLOGY OF ALKALINE PHOSPHATASE ALP denotes a group of isoenzymes [orthophosphoric monoester phosphohydrolases (alkaline optimum), EC 3.1.3.1] characterized by optimal hydrolytic activity toward artificial phosphomonoesters at alkaline pH [5]. In vitro measurements of enzyme activity for assessment of bone and liver disease have been a part of clinical laboratory medicine for decades, but there is still some uncertainty about the precise composition of these proteins. Studies in different organisms and molecular sequencing of the structural genes have led to a better understanding of their structure and function [3]. Bacterial and Yeast Enzymes ALP activity is widely distributed among prokaryotic and eukaryotic organisms. The protein from Escherichia coli has been sequenced [6] and the crystal structure mapped [7,8]. Specified by a single gene (PhoA) [9], the 47-kDa protein product is secreted into the periplasmic space of the bacterial envelope, directed there
PediatricBone
Alkaline P h o s p h a t a s e in Higher O r g a n i s m s In many higher animals, genes are related as orthologs to the E. coli enzyme, forming a large family with more
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Copyright 2003, Elsevier Science (USA). All rights reserved.
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David E. C. Cole
than 150 members. In many cases, gene duplication and functional divergence have led to the fixation of multiple enzyme forms. In the fruit fly (Drosophila melanogaster), for example, four loci (Aph-1 to Aph-4) are listed in the electronic database Flybase (http://flybase.bio.indiana. edu:7081/) that encode genetically related enzymes with multiple polymorphic alleles, although only one (Aph-4) has been curated in relation to structure-function relationships. In the mouse genome, there are four ALP paralogs. The tissue nonspecific product, TNAP (Akp2, chr. 4), is widely expressed throughout development [21] and is homologous to genes in rat and human (92 and 84%, respectively; Fig. 1) [22]. The other three paralogs (including one putative pseudogene) are more closely related to one another, consistent with a series of duplication events in the course of vertebrate evolution. The similarity of tissue expression patterns and enzyme functions suggests that several tissue-specific ALP lineages is the more likely evolutionary scenario in higher vertebrates [23,24], but comparative analysis of mouse and human genes suggests otherwise [25]. The mouse intestinal alkaline phosphatase (IAP) genes (Akp3 and Akp4) on chr. 1 [26] are more closely related to the embryonic isoenzyme (EAP or Akp5) and the pseudogene (Akp-Psl) than to its human orthologs, making it more likely that an ancestral tissue-specific gene has undergone duplication and divergence more than once in vertebrate evolution. More detailed comparative studies will likely provide the evidence necessary to support this hypothesis, but the separate origin and divergence of tissue-specific genes has important implications for species-specific effects of deleterious mutations in the tissue nonspecific enzyme that causes hypophosphatasia. Human Isoenzymes and Isoforms In humans, three major isoenzymes have traditionally been identified by biochemical analysis of serum [5,27]: an intestinal enzyme (IAP), an isoenzyme associated with the placenta (PLAP), and the tissue nonspecific alkaline phosphatase (TNAP or TNSALP) with multiple different isoforms. Human PLAP (Regan isozyme) was the first eukaryotic form to be sequenced by analysis of proteolytic fragments. The amino acid sequence was subsequently confirmed by examining the nucleotide sequences for cloned cDNA [28,29]. The PLAP gene is also the only form with significant polymorphisms [29,30]. Of the three common placental alleles, two (ALPpl and ALPp3) differ at 13 sites and 7 of these result in an altered amino acid sequence. A second PLAP-like gene was found in testis and thymus and is occasionally seen in sera from patients with various neoplastic diseases
[5,27,31]. This isoenzyme is referred to as germ cell alkaline phosphatase (GCAP) or Nagao isozyme [32]. Genes for human PLAP and IAP were mapped to chromosome 2q34-37 using somatic cell hybridization and chromosomal exclusion analysis [33]. Since their duplication is likely a recent evolutionary event, it is not surprising that they should be found close together in the human genome [34,35]. Although expression of different IAP proteins during development has been described [36], genomic sequencing has made it unlikely that there is more than one IAP locus [37]. All three tissue-specific genes (PLAP, IAP, and GCAP) and a highly degenerate pseudogene (ALP-PS) are found in a 220-kb stretch of chromosome 2q37.1 (Fig. 2). They are not contiguous but interspersed with other genes. Analysis of gene order, orientation, and structure points to repeated duplications of a three-gene cluster composed of an upstream gene of unknown function (FAM6A), an ancestral tissue-specific ALP, and a downstream gene transcribed in the opposite direction (ECEL1) that encodes another zinc metallopeptidase [37]. The single-copy gene for human TNAP was first mapped to chromosome l p36.1-p34 by in situ studies, although linkage to the nearby Rh blood group locus was known previously [38,39]. Weiss and colleagues [40] cloned and sequenced the cDNA for bone TNAP (bTNAP). Sequence positional identity is shared with orthologs in other mammals, invertebrates, yeast, and prokaryotes, with conservation of key residues and structure-function relationships [8]. The TNAP gene also shares substantial positional identity with the tissuespecific ALP paralogs on chromosome 2 (Fig. 1). Comparison of the cDNA and genomic sequences indicates that there are 12 exons generating a mature m R N A of 2.5 kb [41]. Of these, the 11 translated exons are grouped together, but the untranslated leader exon (1B) is positioned more than 50 kb upstream. This leader exon is flanked on its upstream side by the gene encoding ECE1 (endothelin converting enzyme-l). On the downstream flank a GTPase activating enzyme gene, RAP1GA1, is positioned less than 20 kbs 3t of the TNAP transcription termination site. The upstream ECE1 gene on chromosome 1 shares significant similarity (33% amino acid homology) with the ECEL1 (ECElike-l) gene found downstream of IAP on chromosome 2, suggesting that the ancient bifurcation leading to a cluster of tissue-specific ALP genes distinct from TNAP included a progenitor of the ECE family as well. Both ECE1 and ECEL1 belong to a family of neutral zinccontaining metallopeptidases, and the possibility that these two zinc-dependent enzyme families (ECE and AP) share more than a superficial resemblance may be fertile ground for future studies of ALP regulation at the genomic level.
f mPLAP mTNAP
FVPEKERD PSYWRQQAQE TLKNALKLQK FVPEKERD PSYWRQQAQE TLKNALKLQK
28 28
mPLAP DMQYELNRNN LTDPSLSEMV EVALRILTKN LKGFFLL ~ T N A P DMQYEWRNN LTDPSLSEW EVALQILTKN PKGFFLL&
tlPL.AP
-
?lSLVPEI(EKD PKYWRDQAQE TLKYALELQK I IPVFFEH PDFWNREAAE AL(;AAKKI.QP
28 28
~ T N A P DMOYELNRNNVTDPSLSEMV VVAIQILRKN PEFFLL h P L A P DMKYEIHRDS WSLMEMT EAALRLLSRN PRGFFLF
mPLAP mTNAP
LNTNVAKNVI MFLG GMGVS TVTAARILKG QLHHNTGEET RLEMDKFPFV LNTNVAKNVI MFLG GMGVS TVTAARILKG QLHHNTGEET RLEMDKFPFV
78 78
mPLAP mTNAP
KAKQALHEAV EMDQAIGKAG AMTSQKDTLT W T KAKQALHEAV EMDQAIGKAG AMTSQKDTLT W T A
TVTAARILKG QWINPGEET RLEMDKFPEV 7 8 TVTAARILKG QKKDKLGPEI PLAMDRFPYV 7 7
hTNAP hPLAP
KAKQALHEAV EMDRAI-G SLTSSEDTLT RAYRALTETI MFDDAIEWG QLTSEEDTLS
-----
-----
~ T N A -~- - - -t A I
f T'. L F ! V1.Al
i ! . '.I'
--
I
~ T N A P LNTNVAICNVI MFL h P L A P AQT-AAKNLI I F L
G R I HG G GRILC%G
$
GR+G& G G R I HG
326 326
S
326 322
SHVF TFGGYTPRGN SHVF TFGGYTPRGN
376 376
4
%
%
%
***
t
k
mPLAP mTNAP
ALSKTYNTNA QVPD ALSKTYNTNA QVPD
hTNAP hPLAP
ALSKTYNTNA QVPD GTAT AYLCGVKANE GTVGVSAATE RSRCNTTQGN ALSKTYNVDK HVPD GATAT AYLCGVKGNF QTIGLSAAAR FNQCNTTRGN
mPLAP mTNAP
GTAT AYLCGVKANE GTVGVSAATE RTRCNTTQGN GTAT AYLCGVKANE GTVGVSAATE RTRCNTTQGN
+
*F
***
f 128 128
mPLAP mTNAP
SIFGLAPMVS DTDKKPFTAI LYGNGPGYKV VDGERENVSM VDYAHNNYQA SIFGLAPMVS DTDKKPFTAI LYGNGPGYKV VDGERENVSM VDYAHNNYQA
128 127
hTNAP hPLAP
SIFGLAPMLS DTDKKPETAI LYGNGPGYKV VGGERENVSM MYAHNNYQA SIFGLAPGKA R-DRKAYTVL LYGNGPGYVL KDGARPDVTE SESGSPEYRQ
178 178
mPLAP mTNAP
QSAVPLRHET QSAVPLRHET
-
t
426 426
-
426 421
C
4
EVTSILRWAK DAGKSVGIVT TTRVNH EVTSILRWAK DAGKSVGIVT TTRVNH
P S AAYAHSA P S AAYAHSA
EVTSIIRWAK DAG~SVGIVT TTRVN* EVISVMNRAK KAGKSVGWT TTRVQH
AAYUISA& PA GTYAHTV
D WYSDNEMPPE D WYSDNEMPPE
W Y S D ~ P E 178 WYSDADVPAS 1 7 7
PGEDVAVEA
KGPMAHLLHG VHEQNYIPHV MAYASCIG-GGEDVAVFA KGPMAHLLHG VHEQNYIPHV MAYASCIG--
~ T N A P QsA~PLRHET h P L A P QSAVPLDEET
K G ~ L L A GVHEQNYVPIN MAYAACIG-RGPQAHLVHG VQEQTFIAHV MAFAACLEPY
mPLAP mTNAP
-ALLLPLAV L S L P T L F --ALLLPLAV LSLRTLF---
474 474
474 471
* mPLAP mTNAP
ALSQGCKDIA YQLMHNIKDI DVIMGGGRKY MYPKNRTDVE YELDEKARGT ALSQGCKDIA YQLMHNIKDI DVIMGGGRKY MYPKNRTDVE YELDEKARGT
hTNAP hPLAP
ALSQGCKDIA YQLMHNIRDI DVIMG-Y MYPIWKTDVE YESDEKARGT ARQEGCQDIA TQLISNM-DI DVILGGGRKY MFRMGTPDPE YPDDYSQGGT
mPLAP mTNAP
R L D G L D L I S I WKSFKPRHKH SHYVWNRTEL L--ALDPSRV R L D G L D L I S I WKSFKPRHKH SHYVWNRTEL L--ALDPSRV
DYLLGLFEPG DYLLGLFEPG
276 276
hTNAP hPLAP
W G L D L V D T FJKSEXPBXI(II m T E L L--TLDPHNV DYLLGLFEPG RLDGKNLVQE WLAKR---QG ARYVWNRTEL MQASLDPS-V AHLMGLFEPG
276 272
228 228
f 228 226
t
+
I
ANLDHCAWAG SGSAPSPG-ANLDHCAWAG SGTAPSPG--
-
--
507 507
lt ~ T N A P ANLGHCAPAS S A G S ~ G - P L L U U L YPLSVLP--h P L A P TACDLAPPAG TTDAAHPGRS W P A L L P L L A GTLLLLETAT AP
507 513
LEGEND - k e y a c t i v e site r e s i d u e s # - s i t e o f c a r b o x y - t e r m i n a l cleavage + - v e r i f i e d sites o f N - g l y c o s y l a t i o n f - a d d i t i o n a l c o n s e n s u s sites f o r N - g l y c o s y l a t i o n %
-
311 T
y r i n t e r a c t i n g w i t h t h e a c t i v e site o f t h e m a t c h i n g monomer
FIGURE 1 Amino acid sequences for TNAPs from mouse and man.'~hownare best alignments (by ClustalW) for mouse and human TNAP (mTNAP and hTNAP, respectively), along with mouse and human placental alkaline phosphatase (mPLAP and hPLAP, respectively). Signal sequences are shown in gray, whereas conserved residues involved in metal binding are reverse highlighted and key active site residues participating in catalytic phosphate transfer are distinguished by asterisks [8]. Single underline of the hTNAP sequence (bold) delineates the amino-terminal sequence participating in monomer-monomer interactions. Double underline indicates loop with calcium-binding properties and single overline demarcates the collagen-binding domain within the crown region [59].Although the site of carboxyi cleavage and transfer to the GPI anchor is known to be ~s~~~~in PLAP [49], the site of cleavage in TNAP is speculative.
654
David E. C. Cole
(PsAP)
FAM6A
PLAP
(ECEL1P3)
GCAP
(ECEL1P2)
lAP
(ECEL1PllFAM6B)
ECEL1
FIGURE 2 Schematicof genomic relationships between the human TSAPs and their neighboring genes. In the 219.7-kb region on 2q37.1 [37], a previously unknown gene, FAM6A (with homology to the C termini of the RNase type II family of proteins), is followed by an alkaline phosphatase pseudogene, PsAP (ALPPP), a highly degenerated 160-bp stretch of DNA with 86% homology to exons 1 and 2 of placental alkaline phosphatase (PLAP or ALPP). The placental gene is flanked on both sides by pseudogenes of the ECEL1 family, as is the germ cell alkaline phosphatase (GCAP or ALPPL2). Finally, the single copy of intestinal alkaline phosphatase (IAP or IALP) is flanked upstream by a pseudogene, FAM6B, followed by the single functional copy of ECEL-1 (endothelin-converting enzyme-like peptide 1). Arrows indicate the relative orientation and stippling the presence of an untranscribed or pseudogene. The most parsimonious explanation of this tandem repeat array is the duplication, in toto, of an ancestral trigenic cluster, FAM6A-ALPP-ECEL1, followed by two duplications of the internal digenic tandem repeat, ALP-ECEL1 [37].
A second leader exon (1L) is observed in transcripts expressed by liver cells, but the additional m R N A species in hepatic tissues are likely the result of variable transcription stop sites [42]. In kidney, both leader exons are detected in c D N A prepared from purified T N A P m R N A . The liver-type transcript (1TNAP) is found in renal cortex and medulla, but the b T N A P is found in cortex only [43]. B o n e TNAP E x p r e s s i o n In humans, the tissue specificity of the so-called tissue nonspecific gene is achieved by differential transcription and cotranslational or posttranslational modification so that three major isoforms are readily distinguished~ liver, bone, and kidney (Table 1). Thus, the term liver/ bone/kidney alkaline phosphatase is probably a more accurate description of the enzyme phenotype expressed from the T N A P gene.
Transcription Isolation of m R N A suggests that there is a single mature transcript in bone, but the levels vary considerably between connective tissues, in different developmental states, and under different physiological influences. The 5' end of the gene contains a T A T A box and four S p l - b i n d i n g sites, as well as a stretch of CpG-rich sequence and a number of repeat motifs [41] that may play a role in constitutive expression (Fig. 3). Also present upstream are consensus sequences for hormone response elements, including ones for the vitamin D receptor (VDR)/retinoid acid X receptor (RXR) heterodimer [44], the peroxisome proliferator-activated receptor (PPAR)/RXR heterodimer, and nearby elements putatively recognizing metal-regulated transcription factor-1 (MTF-1) [45].
At the other end (downstream of the stop codon in exon 12) is a 3' untranslated region consisting of approximately 760 bps followed by a consensus polyadenylation/ m R N A cleavage motif [41]. Despite a wealth of sequence information, our understanding of how the various cisacting elements interact to modulate b T N A P expression is limited.
Translation, Self-Association, and Subsequent Modification A portion of exon 2 is untranslated and the single unambiguous start codon is located in the middle of the exon. A leucine-rich, 17-residue signal peptide allows for docking of the bTNAP/ribosome complex with the endoplasmic reticulum (ER), followed by insertion of the nascent peptide into the membrane and secretion into the ER lumen (Fig. 1). Further processing occurs at the C terminus with the recognition of a signal for cleavage and transfer of the mature peptide to a glycosylphosphatidylinositol (GPI) anchor. The anchor is synthesized in the ER membrane separately from the peptide target, and the transfer is a one-step transamidation reaction requiring a target recognition sequence [46-48], activating a carboxyl group (Asp 484 in PLAP [49]), which becomes the final peptide of the mature enzyme [50]. In bTNAP, the homologous position for acceptable co amino acid (G, A, S, C, D, or E preferred) and co+2 amino acid (G, A, or S preferred) residues [51] suggests that Ser 487 in the C-terminal segment SSAG487SLAAGP is a likely acceptor site, but direct experimental verification of this reaction in purified b T N A P has not been reported. As with all ALPs, self-assembly into noncovalently associated homodimers occurs soon after translation, but the relationship of this self-assembly step to the protein folding events and anchoring to the phosphatidylglycan membrane moiety is not known. The emergent
655
2 7. H y p o p h o s p h a t a s i a
TABLE 1
Properties
of Human
Alkaline Phosphatase
bTNP
lsoenzymes
in S e r u m I
ITNAP
lAP
PLAP
§
§ +
GCAP
Heat stability - 1 0 m i n @ 56 ~
§247
- 1 0 m i n @ 65 ~
++
++
Inhibition by ++
+
+_
- L-phenylalanine
- urea
-
-
+
- L-tryptophan
-
-
+
++
++
-
- L-leucine
- L-homoarginine
-
-
-
- levamisole
++
++
-
++
-
- L-p-bromotetramisole
§
Electrophoretic migration - before n e u r a m i n i d a s e t r e a t m e n t
fast
intermediate
slow
variable 3
- altered by n e u r a m i n i d a s e
++
+
-
+
+
+++
Physiological effects - increase in late p r e g n a n c y 4
++
1
A d a p t e d f r o m Cole et al. [1,191] 2 Relative effects scaled f r o m substantial positive ( + + + ) to n o n e ( - ) 3 A t least 15 different P L A P p o l y m o r p h i s m s (total frequency of 35 per 1000 p o p u l a t i o n ) are distinguishable by their differential electrophoretic m i g r a t i o n [216] 4 I s o e n z y m e c o n c e n t r a t i o n s decrease in early p r e g n a n c y due to h e m o d i l u t i o n . In late pregnancy, b T N A P is increased due to increased m a t e r n a l b o n e t u r n o v e r , b u t P L A P f r o m the placenta a n d o t h e r tissues is also a c o n t r i b u t o r to total m a t e r n a l s e r u m activity [115,147,148,194]. Small a m o u n t s I A P n o r m a l l y excreted by the fetal intestine into the a m n i o t i c fluid can sometimes be f o u n d in the m a t e r n a l circulation [217].
66-kDa peptide then undergoes glycosylation during its transport to the Golgi apparatus, becoming an ~80-kDa peptide at the cell surface [52]. As much as 10% of the nascent peptide may escape membrane attachment and be secreted directly [53]. TNAP contains at least five potential sites for N-glycosidic attachment of oligosaccharide side chains [40] (Fig. 1). Two sites--Asn 123 and AsnZ54--are shared with tissue-specific isoenzymes and therefore are unlikely to contribute to the unique properties of TNAP. The Asn 213 site is located between a conserved 13 sheet and adjacent ~ helix, however, and potentially modifies the catalytic properties of the active center [43]. There is no agreement on the precise composition or number of these side chains, but studies of transfected COS-1 cells indicate that at least three sites are used [52]. The enzyme is inactive without the N-glycosylation and experiments with enzymes cleaving O-glycosidic bonds suggest that other carbohydrate moieties may be present [43]. There is also evidence that the same modulators of bTNAP transcription (e.g., retinoic acid) can independently alter the rate and type of glycosylation [54].
P h y s i c o c h e m i c a l P r o p e r t i e s of t h e TNAP M u l t i m e r
Peptide Structure The TNAP gene predicts a sequence of 507 amino acids following cleavage of the signal peptide and removal of the C terminus with GPI attachment. At the heart of the properly folded structure is a conserved metalloenzyme motif that belongs to a large superfamily of enzymes. Metal ions are coordinated around an active site that allows transfer of an anion through a transient covalent attachment to a nucleophilic side chain of a central amino acid. Creating the catalytic peptide core is a conserved 10-stranded 13sheet situated between cz helices [8] consistent with an ~/13/~ sandwich structure (http:// www.biochem.ucl.ac, uk/bsm/cath new/ class3/-40/720/ 10/index.html) that confers membership in an ancient superfamily (http://scop.mrc-lmb.cam.ac.uk/scop/data/ scop.b.d.hg.b.html) that would have arisen during prokaryotic evolution [55]. This family includes phosphopentomutase and phosphoglycerate mutase as well as the members of the sulfatase family [56]. The close
656
David E. C. Cole -805 C4~TCCCCTTC T G C T T C T T C T
-745
TGCGGTAGCC
GGTGGGGGTG CAGAGTCAG~ mlm~~r
685 I
AGAca
AGGGAGGGCA
++++ GCCCACGGGC
~GAGACA
GAGAGAC~ It~w..lei~
GACCCAGAGA
AGGAAGCGGG
GAaACA
-625 G A C G C C A G A G
AGGAAGGCAG
ACAAAGAGAC
GGGTGGAGAC
AAAGACTCCC
ACCAAGAGAC
-565 G C A G A A G G A A
GATGCCGACG
GTAAAGACAA
AACAGGAGAC
GCGCGC~I~
V-AIB~.~al
CGCTGAGAGA
GGAAGGGCTG
GGCTGGGGCA
GCCCGGAGGC
AGAGAGACCG
-445 A G A G T G C G G G
GCGGGCGAGG
GACGCCAGGG
CCGCGTCACC
CCAGCCCGTT
CCTAGCTCCG
-385 C T C C C G G C A G
++++ GGGGCGCCCT
GGCCTCGTGG
CACGACCGGC
++++ CCGCGGGGCG
++++ CGGCM]CTCGG
-325 GCCGGGG.GC.G G G G C C G G G G C
CGGGCTGGGG
AGGGGTTGGG
GCC~.G...C.G....GGGGAGC~...G...G.
~505
II~~~GCT
++++
**********************
++++
++++ -265
,C.GGGCTGCCC GGGCCTCACT_. CGC~CCCCGC GGCCGCCTT'~ AT/X~GGCC~C ~ G T G G T G
- 205 G C C C G G G C C G
CGTTGCGCTC
CCGCCACTCC
GCGCCCGCTA
TCCTGGCTCC
GTGCTCCCAC
-145 G C G C T T G T G C
CTGGACGGAC
CCTCGCCAGT
GCTCTGCGCA
Ggtaaggatt
cgacgctgcc
ccgcgccctg
gttccccagg
gccccagcgg
acgtggtcca
tccccttctg
catcctccgc
tggccccgtg
gttgaacttt
aatggc
FIGLIRE 3 Details of the TNAP 5' flanking region. The sequence is numbered according to Weiss et al. [41]. Nucleotides in the first intron are shown in lowercase. Nucleotides identified as approximate transcription start sites are shown in bold. A minor site is found a short distance upstream (-243 to -246) of the TATA box (double overline and double underline) and the major start site (bold underline). Within the transcribed sequence is a consensus binding site for the Myc complex (bold overline). Within the GC-rich sequence (approximately -250 to -350), there are three copies of an Spl-binding sequence (broken underline) and a fourth is found upstream of a cyclic AMP response element within a sterol regulatory response sequence (asterisks). Two copies of a GATA-binding motif (broken overlines) and core sequences for six AP-2 binding sites (plus signs) are similarly indicated. Also shown is an atypical AP-1 binding site (light gray shading) recognized by the Jun-Fos transcription factors [220, 221] and a single consensus sequence for binding of a PPAR-RXR dimer (white letters on gray background).Tandem repeat consensus sequences for the V D R - R X R dimer are shown as white letters on black background, with the trinucleotide spacer shown in bold. Each VDRE is preceded by a tetranucleotide core sequence (single light overline) for MTF-1. Only selected motifs identified by Matlnspector, a promoter analysis program (http://www.genomatix.de/cgi-bin/ matinspector/matinspector.pl) [222], are shown here.
27. Hypophosphatasia
superposition of the E. coli ALP and E. coli sulfatase peptide folds suggests that they are structurally related, although the sequence homology is less than 10%. Identification of residual arylsulfatase activity in purified ALP provides experimental evidence for this conjecture [57]. Recently, the deduced crystal structure for human PLAP at 1.8 A resolution [58] has been used by Mornet et al. [59] to create a detailed picture of TNAP through molecular modeling. Physically adjacent to the catalytic serine residue (Ser 92) are bound phosphate and water molecules along with two zinc atoms and a magnesium atom. All four cysteine residues of the E. coli sequence are replaced in human ALPs, but two disulfide bridges are maintained (CySlZz-scy184 and CyS472-SCy 480 in TNAP) and a free sulfhydryl cysteine (CySH 1~ is found (Fig. 1). Most but not all amino acid residues forming coordinate ligands with these metals are conserved. It is likely that the basic catalytic mechanism is unchanged, but different allosteric interactions found in mammalian enzymes [60] can be explained by specific structural modifications. The dimer interface is partially conserved, but its sequence varies between PLAP and TNAP isoenzymes. Common to both, however, is the presence of an s-helical N-terminal structural motif (residues 9-25) not found in the E. coli enzyme. This helix interacts extensively with the second monomer and may contribute to the dimerization process. Also important is a conserved tyrosine residue (Tyr TM in TNAP) that may be stabilized by the adjacent N-terminal ~ helix. It is positioned to contribute to the hydrogen-bonding network extending through a conserved histidine (His 437) in the other monomer to one of the zinc atoms (Zn 1) [17] in the opposite active center. There is also a flexible surface loop (residues 405-435) not found in orthologs of single-celled organisms that contributes to the collagen-binding properties of TNAP (Fig. 1) [60,61]. Modeling suggests that this collagenbinding motif is positioned at the top of the molecule relative to its C-terminal anchoring by GPI. It constitutes part of the so-called crown domain (residues 371431) and consists of two small interacting 13sheets at the dimer interface. It is found in all mammalian structures. Of prime importance to the understanding of bTNAP function is the presence of a fourth metal-binding site contained within a novel surface domain (residues 210-289) found only in the mammalian enzymes (Fig. 1). X-ray diffraction of PLAP crystals suggests that either magnesium or calcium might be bound [58], but synchrotron radiation X-ray fluorescence analysis of purified protein is most consistent with calcium [59]. There is also structural evidence for a coordination number of 7 at this site in PLAP and TNAP, favoring calcium over magnesium and other cations [58,59]. In addition, there is physiological evidence for independent calcium-
657
binding properties of the bTNAP protein [62]. Stabilizing or modifying this site in PLAP is an adjacent N-acetylglucosamine linked to the consensus asparagine residue (Asn T M in TNAP) [58], suggesting that different glycosylation patterns could contribute to different metal-binding properties at this site.
Catalytic Reaction Kinetically, ALP can be classified as a serinedependent phosphohydrolase enzyme [15]. In E. coli, substitution of the catalytic serine with the isomorphous residue cysteine by site-directed mutagenesis leads to a peptide with different enzyme properties, confirming the importance of the conserved Ser 92 residue in catalysis [63]. The enzyme complex has an absolute requirement for zinc but displays optimal catalytic activity with addition of magnesium [16]. The catalytic core is a very ancient metalloenzyme motif involving transfer of anion to a key nucleophilic amino acid side chain as an intermediate in the catalytic cascade. Other enzymes with this motif are sulfatases, phosphodiesterases, phosphonohydrolases, and a calcium-dependent ATPase [55]. Based on this evolutionary evidence, it was predicted that ALPs would show some reactivity toward sulfoester and phosphodiester substrates--a prediction that has been born out by direct experimental evidence. Thus, it is not surprising that human bTNAP is considered promiscuous since its catalytic center displays little discrimination among phosphoesters under certain conditions [57,64]. The phosphoester hydrolysis reaction is markedly pH dependent, as the name of the enzyme suggests, since a free hydroxide anion is an important intermediate. At artificially high pH (alkaline optimum), the reaction is rapid and the rate-limiting step is the release of phosphate from the enzyme; therefore, enzyme activity under these conditions is essentially independent of the nature of the substrate compound [65,66]. Kim and Wyckoff [18] analyzed crystal structure data for metal-substituted E. coli ALP to determine the coordination of the two zinc atoms with the bound phosphate group and assigned the conserved arginine residue (Arg 167 in human TNAP) a role in stabilizing the pentacoordinate phosphate transition state (Fig. 1). Although the magnesium requirement for full ALP activity is well recognized [16], only recently has its catalytic role been defined. New crystal structures for the E. coli dimer [17] place the three central metal ions in positions that allow stabilization of the hydroxide anion intermediate that serves as a general base to promote deprotonation of serine and its attack on the cationic phosphorus center of the phosphoester substrate (Fig. 4). The magnesium is positioned by three conserved amino acid residues (Asp 43, Thr 156, and Glu 315 in hTNAP) in an octahedral
658
David E. C. Cole
EoROP
E Znt t..
a|o ,,,
~
.-&.
...~" ~ ' 1
oi.
Argo66
C
..
Ar8166
Serl02 ROP
Asp51 "'~.y8 Thr155 Glu322
"'I'~"Mg
.L''
Asp51
Glu322
]04
PO~ RO"
" ' ~ ' 1 Serl02
Glu322
E~
~
. ~
Scrl02
Glu322
E-P
FIGURE 4 Catalytic reaction for E. coli alkaline phosphatase. In the free enzyme (E), the phosphate-binding site is filled with three water molecules (w). The hydroxyl group of the catalytic serine (Ser1~ participates in a hydrogen-bond chain that extends through a hydroxide ion to the hexacordinate magnesium ion. Formation of the enzyme-substrate complex (E~ROP) involves coordination of the oxygen atom in the phosphoester substrate (R) to zinc with bidentate liganding to the guanidinium group of the conserved Arg 166 residue. With substrate binding, the hydroxide ion accepts the proton from the serine -OH group, generating the Ser-O- nucleophile that attacks the phosphorus atom. With release of the leaving group (RO-), the phosphate becomes bound covalently to Ser1~ Then, a hydroxide anion bound to the highly electrophilic Znl atom in the covalently phosphorylated enzyme (E-P) attacks the phosphorus atom from the opposite side, with regeneration of the serine-OH group and formation of a noncovalently bound enzyme-phosphate complex (E-P). In the final step, a water molecule bound to magnesium contributes a proton to reform the SerOH ~22 side chain with release of the inorganic phosphate product (reproduced with permission from Stec et al. [17]).
configuration that includes three water atoms, one of which becomes the p r o t o n d o n o r to the catalytic serine. The substrate-binding zinc a t o m (Znl) is held in pentacoordinate configuration by bidentate liganding with the 13-carboxyl of a n o t h e r conserved aspartate residue (Asp 32~ in h T N A P ) as well as two histidines (His 324 and His 437 in h T N A P ) . The serine-binding zinc (Zn2) is tetracoordinately positioned with liganding to two aspartate
residues and another conserved histidine (Asp 43, Ser 92, Asp 361, and His 37~ in h T N A P ) .
Physiological Substrates T N A P activity is readily assayed with synthetic substrates (e.g., p-nitrophenyl p h o s p h a t e and 4-methylumbelliferyl phosphate) at p H 10 (Fig. 5). T N A P will
27. Hypophosphatasia
H~C~'O HO
C~o~
phosphotyrosine residues generated by protein kinases appear to be hydrolyzed by a separate series of intracellular phosphatases, but alkaline phosphatase may also be active where it has access to substrate [2,72-74]. Discoveries of endogenously elevated levels of three phospho-compounds (Fig. 5) in hypophosphatasia provided the first indications of the role(s) TNAP [3]. In 1955, identification of increased urinary phosphoethanolamine excretion [75] provided a useful biochemical marker for the inherited disorder. In 1965 and 1971, high levels of pyrophosphate (PPi), an inhibitor of hydroxyapapite crystal formation, were noted in urine and blood [76], suggesting a mechanism for the defective mineralization of hard tissues. In 1985, elevated plasma levels of pyridoxal-5'-phosphate (PLP) were f o u n d ~ a n observation that first indicated an ectoenzyme action for TNAP [2,77,78].
= 0
o.
H+
pyridoxal 5'-phosphate OH
I
O-
I
O"
H2
.c +H3N/ ~C /
O=P--O--P----O I ! O" OH
inorganic pyrophosphate
S%r/o-T _O~N.~ , , . v ~
[
.O--P~---O
J oH
phosphoethanolamine
O"
O"
I
I --o
OH
II O p-nitrophenylphosphate
659
OH CH3
4-methylumbelliferylphosphate
FIGURE 5 Substrates for the TNAP enzyme. Accumulation of endogenous phospho-compounds in hypophosphatasia is taken as one indication of their physiological relevance as natural substrates of TNAP [3]. Increased serum pyridoxal Y-phosphate is a hallmark of hypophosphatasia in man [77,218] and mouse [185,188]. Increased urinary excretion of both inorganic pyrophosphate [76] and phosphoethanolamine [75] is also typical. Of the artificial substrates with alkaline optima, p-nitrophenylphosphate has been widely used since it undergoes a spectral shift with dephosphorylation that is readily detected by simple colorimetry [94]. For a more sensitive fluorimetric assay of TNAP activity in biological fluids, 4-methylumbelliferylphosphate is a commonlyavailable substrate.
hydrolyze most primary alcohol esters as well as pyrophosphate, nucleotide di- and triphosphates, and phosphocreatine, although other enzymes with higher turnover and narrower specificity are thought to be more physiologically relevant [2,67,68]. At physiologically neutral pH, the rate-limiting reaction step is the release of the dephosphorylated compound, which is consistent with the limited repertoire of natural substrates for this enzyme [3,15,65]. If an alcohol group is present in high concentration, ALP may catalyze a phosphate transfer from a phosphoester cosubstrate [18,69,70]. With human serum enzyme, this phosphotransferase activity is apparently highly dependent on saturating concentrations of zinc ion [71]. Intracellular
Endogenous Inhibitors and Allosteric Modifiers Given that the release of inorganic phosphate is a ratelimiting step in TNAP catalysis, it is not surprising that phosphate is also a competitive inhibitor. This property is relevant for the hydrolysis of the endogenous substrate PLP at physiologic concentrations [79]. Because ALP is measured clinically with artificial substrate at pH 10, it is important to account for the endogenous phosphate level in assessing enzyme activity toward natural substrate at physiologic pH. The effects of varying magnesium concentrations within the physiologic range are complex, suggesting that there is more than one role for this ion. Maximal activity of the enzyme in vitro is a monotonic function of magnesium concentration, related solely to occupancy of the third metal-binding site in the active center. With natural substrates, however, magnesium also forms coordinate complexes with diphospho-compounds, including pyrophosphate and phosphodiesters, and likely interferes with their acceptance as substrates. Finally, the presence of a cation-binding site at a distance from the active center [58] may contribute further to the allosteric modulation of TNAP pyrophosphatase activity [80]. Similar arguments apply for the effects of ambient calcium: This cation competes with magnesium and zinc for the third metal site (albeit weakly), it may bind at the fourth site outside the active center, it can alter effective concentrations of natural substrate in the local environment of the active site, and it has independent effects on cell signaling and expression of the TNAP enzyme. Calcium will also bind phosphate, altering the effective concentration of this inhibitor and modulating independent effects of phosphate on cell function. Thus, it should not be surprising that the relationships between extracellular concentrations of calcium and TNAP activity at the cell and tissue level are also complex [81].
David E. C. Cole
660 Membrane Association and Tetramer Formation
In the plasma membrane, T N A P probably exists as GPI-anchored tetramera with 68- to 100-kDa subunits [82]. The complexes have mass values greater than predicted from the primary sequence and vary considerably according to the extent and type of glycosylation. In vitro, treatment with glycosidases inevitably leads to the reduction of this microheterogeneity. Subcellular fractionation procedures suggest that most b T N A P activity occurs in the plasma membrane, and functional studies with intact cells indicate that it is indeed an ectoenzyme
expressed only on the outside surface of the cell [3]. The release of ALP from the cell surface by phosphatidylinositol-specific phospholipase D [83,84] is further evidence that this ectoenzyme is linked to the membrane by a novel covalent linkage between peptide and inositol phospholipid (Fig. 6). This configuration confers high lateral mobility on the membrane-bound enzyme [85,86] and probably contributes to stabilization of the tetrameric configuration [82, 87]. Because of the significant role of phosphatidylinositol in intracellular signaling pathways, this membrane
The various forms of ALP present in vivo and their conversions by in vitro experiments i
i
ii
iiiii
ii i
iiiiiiiiiiii
iiii ii
FIGURE 6 Extracellularforms of alkaline phosphatase present in vivo and generated by in vitro experiments. Alkaline phosphatase may be shed from hepatocyte, osteoblast, or intestinal cells in vesicles as membrane-bound (Mem) tetramers or as soluble dimers (Sol). Solubilization of Mem tetramers by bile acids releasesthe GPI anchorbearing tetramer (Anch). Treatment of the Anch tetramer with phosphatidylinositol-specific phospholipase C (PI-PLC) or D (PI-PLD) yields ALP dimers with phosporylated and nonphosphorylated inositol glycan, respectively, whereas ficin treatment cleaves the C-terminal linkage and yields dimers only (reproduced with permission from Van Hoof and De Broe [94]).
27. Hypophosphatasia attachment and release phenomenon may be associated with important regulatory functions of the cell in response to perturbations of cell homeostasis [86]. B o n e TNAP in Vioo Bone and Extracellular Matrix
Bone ALP is constitutively expressed in the osteoblast and has been localized to the plasma membrane by a variety of histological techniques [2,88]. It is also found in ameloblasts and odontoblasts [89] and in cultured cells with the osteoblast phenotype [88,90,91]. Although it is a GPI-anchored ectoenzyme, it does not appear to be restricted to specific areas of the plasma membrane. Whether it is associated with lipid islands has not been determined. In principle, some enzymes may be recirculated by endocytosis, but significant amounts are released into the extracellular space [92] either by hydrolysis from the GPI anchor or by association with a portion of the membrane that is shed as a matrix vesicle (Fig. 6) [93,94]. Matrix vesicles, critical to the initiation of mineralization, are rich in bTNAP and bind calcium and phosphate [95,96]. The primary form of TNAP appears to be an anchor-intact hydrophobic form, but electrophoretic separation of bTNAP isolated from bone reveals at least two fractions (B1 with GPI anchor and B2 without) whose relative abundance is dependent on whether it derives from cortical or trabecular tissue [97]. Since ALP was first identified in calcifying mammalian cartilage, it has been considered important in bone mineralization (Table 2), but hypophosphatasia mutations are the most compelling evidence for its essential role in skeletal metabolism [4,98]. It has also been suggested that ALP may be essential for normal cell proliferation [99], but fibroblasts from hypophosphatasia patients lacking the enzyme have normal morphology and growth characteristics [3,100].
TABLE 2
bTNAP properties relevant to bone mineralization
1. Expressionby mineralizing osteoblasts and intact release with shedding of mineralizing matrix vesicles [2,91,164] 2. Localizationby GPI anchor to the extracellular space adjacent to the plasma membrane [90] [218,219] 3. Specificcell surface compartmentation regulated by GPI anchor and microdomain association with caveolae [48] 4. Interactionof matrix vesicleTNAP with other (pyro)phosphatases [3,74] 5. Bindingsites for collagenand calcium in the extracellular space at a point distal to the TNAP anchor to the cell [59] 6. Phosphatase activity towards mineralization inhibitors (inorganic pyrophosphate, 13-glycerophosphate)[3]
66 1
Circulation
Circulating ALP activity is almost entirely composed o f T N A P isoforms, although PLAP isoenzyme is found in large amounts in pregnant women and some IAP may appear subsequent to a large meal. Fractionation by isoelectric focusing reveals at least 12 proteins of different isoelectric points and confirms that bone and liver isoforms account for most of the circulating activity [101]. TNAP is readily distinguished from the placental and intestinal isoenzymes by its electrophoretic mobility (Table 1) [94], its sensitivity to inhibition by homoarginine [102] (but not phenylalanine or phenylalanylglycylglycine [103],) and its sensitivity to heat denaturation [94]. It has also been identified in leukocytes [104-106]. The liver and bone isoforms are distinguished electrophoretically and enzymatically. The bTNAP isoform has an apparent isoelectric point of 4.21 [101 ], which is higher than that of the liver isoform but lower than that of the kidney isoform, giving it intermediate mobility on electrophoresis [107]. Much of the residual charge heterogeneity of the bone isoform can be abolished by treatment with neuraminidase, suggesting a variable number of terminal sialic acid residues in situ. The bone isoform is also more sensitive to inhibition by denaturing agents, such as urea and guanidine hydrochloride [108], and to inactivation by heat. It is also differentially bound by wheat germ lectin, presumably by virtue of the oligosaccharide composition. These isoforms are reviewed elsewhere [94,101,109]. During childhood and adolescence, total serum activity is a function of age (Fig. 7), which is in large part reflective of the changing rates of bTNAP secretion from the maturing skeleton into the circulation [5,110,111]. Thus, total ALP can used as an approximate clinical index of skeletal activity, but direct measurement of the bonederived isoform in serum is more sensitive [81,112,113]. During pregnancy, there is significant enhancement of placental isozyme expression in various tissues, contributing to an increase in serum total ALP levels, both in controls and in hypophosphatasia patients [114,115]. The clinical enzymology of serum ALPs is reviewed extensively elsewhere [94].
CLINICAL HYPOPHOSPHATASIA Hypophosphatasia is an inherited disorder of bone mineralization characterized by rachitic changes in childhood or osteomalacia in adulthood, the absence of dental cementum with premature loss of teeth, and decreased ALP enzyme activity. Hypophosphatasia was first identified as a separate entity by Rathbun in 1948 [116], although there are earlier case reports [2]. A wide range of presentations are described, but all patients have
662
David E. C. Cole
Distribution of Sol Bone ALP according to Age
FIGURE 7 Serum total alkaline phosphatase activity as a function of age. Shown are the smoothed agedependent reference ranges, medians, and 5th and 95th percentiles (p05 and p95, respectively). Note that there is a wide variation in the lower limits, particularly in boys, and that levels in both sexes show a variable increase during the pubertal growth spurt (reproduced with permission from Van Hoof and De Broe [94]).
some deficit in serum and tissue enzyme activity toward natural substrates. The birth prevalence ofhypophosphatasia was estimated to be 1/100,000 on the basis of pediatric hospital records [117], but this is likely an incomplete ascertainment because asymptomatic adults and individuals with milder forms of the disorder would have been excluded. From a clinical perspective, most individuals can be classified on the basis of the time of their first presentation~perinatal, infantile, childhood, or adult onset (MIM Nos. 24150, 24151, and 14630). Scriver and Cameron [118] also described a patient with clinical features of the childhood disorder but with normal serum ALP activity toward artificial substrate, and they designated this variant pseudohypophosphatasia.
Clinical P r e s e n t a t i o n Perinatal
In the perinatal form of hypophosphatasia, markedly impaired mineralization occurs in utero. The extremities are shortened, the long bones are deformed, and the cranial vault is poorly mineralized. Polyhydramnios has been observed more frequently in hypophosphatasia pregnancies, and stillbirths are not uncommon [3,119, 120]. Radiographs may show small, sclerotic bones at the base of the skull and a membranous calvaria (Figs. 8A and 8C). Rachitic changes may be evident in the long bones, with irregular extensions of undermineralized cartilage and bone into the metaphyses (Figs. 8B and 8D).
27. Hypophosphatasia
FIGURE 8 Perinatal (lethal) hyposphosphatasia. Typical features seen in radiographs from a 23-week gestation fetus include small sclerotic bones at the base of the skull (A) and largely membranous calvariae. (B) Long bones show severe, patchy demineralization with flared ends and misshapen midshaft architecture, and there are large distinctive notches in the metaphyses. Pedicles of the vertebrae are not visible and the angulation of the one femur is an indication of in utero fracture. Lateral (C) and anteroposterior (D) films of a 38-week gestation fetus show less severe but closely matching features, including small, severely demineralized calvarial plates, thin ribs, and notched long bones. Irregular failure of mineralization in the distal appendicular skeleton is seen at 23 weeks (metacarpals and metatarsals; B) and at 38 weeks (tali; D) gestation.
663
664
David E. C. Cole
The ribs are small, thin, and deformed, and there may be associated pulmonary hypoplasia [121]. Sclerotic patches are also observed in various tubular bones, and (Bowdler) spurs may be seen at the midshafts of the ulna and fibula, creating an overall radiologic appearance that is often diagnostic [122,123]. In the neonate, other features include a high-pitched cry, unexplained fever, anemia, and seizures [2]. In these live births, the outcome depends on the extent of pulmonary and neurological compromise, but demise often occurs within a few days. Infantile
In the infantile form of the disease, overall mineralization and clinical course immediately after birth can be nearly normal, but significant rachitic disease and failure to thrive are evident by 6 months of age [3,124,125]. Thus, most affected newborns do well for a short period of time and then experience a wide variety of problems related to impaired bone growth. Chest deformities and rachitic, flailing ribs are associated with respiratory insufficiency and a predisposition to pneumonia. Hypercalcemia may be marked, explaining a history of irritability, poor feeding, anorexia, vomiting, hypotonia, polydipsia, polyuria, dehydration, and constipation. Changes in parathormone or vitamin D metabolism apparently are not contributing factors, although increased serum PTH has been observed [126-128], perhaps related to renal compromise. Episodes of unexplained fever, tender bones, and hypotonia are also described, and hyperphosphatemia has been reported [129]. Renal function may be impaired by hypercalciuria and nephrocalcinosis, and atraumatic fractures are frequently found [123,124,130]. The anterior fontanel is often enlarged and may bulge. The membranous cranial sutures are also frequently widened, and some degree of ocular prominence due to shallow orbits may be apparent within the first few months of life; blue sclerae are also observed. The head circumference increases more slowly than expected as premature sutural fusion sets in. Radiographs show widespread demineralization and rachitic changes in the metaphyses, but usually with less diaphyseal bowing than would be expected with severe metaphyseal disease [130]. Bowdler spurs are also seen in this form of the condition and are associated with overlying skin dimpling that may persist into adulthood [131]. In infants who survive, there is often spontaneous improvement in mineralization and remission of clinical problems [118,125,130,132], with the exception of craniosynostosis. Although the sutures appear widened and membranous, intense osteoblastic activity indicating imminent synostosis may be detectable by nuclear scintigraphy [133].
Premature loss of deciduous teeth and short stature are also common, but the long-term prognosis is good. Childhood
Childhood hypophosphatasia is a milder condition that often presents as bowing and radiographic changes of rickets in the second and third years of life. Focal bony defects near the ends of major long bones may also be observed and help determine the diagnosis (Fig. 9A) [130]. Other signs of rickets, including beading of the costochrondral junctions, enlargement of the wrists, periarticular tenderness, myopathy [134], and a waddling gait may also be present. Exfoliation of the teeth, often beginning with the incisors and involving minimal root resorption, is also seen. Signs of intracranial hypertension or failure to thrive are typical [2,124,130]. Some long-term calvarial and long-bone deformities are not unusual but tend to improve with time (Fig. 9B). In these patients, regional osteopenia may be observed. On X-ray absorptiometry, increased bone mineral density (BMD) in the femoral neck and radius may be associated with trabecular sclerosis, whereas BMD in the lumbar vertebrae is reduced but within the normal range [135]. One of the more serious concerns in this group is craniosynostosis. Because the calvarial sutures are uncalcified (and therefore radiotransparent), and the typical sutural ridging of craniosynostosis is uncommon, the problem may be difficult to detect on the basis of physical or radiographic signs. All sutures appear to be involved, but ocular prominence due to shallow orbits can be quite marked. Other ocular signs include blue sclerae and keratopathy or conjunctival calcification due to hypercalcemia. In childhood hypophosphatasia, spontaneous remission of bone disease is well-known in adolescence, but the disease may reappear in middle or late adulthood. Adult
The adult form is mild but osteomalacia may be associated with symptomatic pseudofractures, marked bone pain, and increased susceptibility to traumatic fractures and extended breaks at pseudofracture sites [125,136]. The proximal femur is a frequent site of pseudofractures that may progress to complete transverse fractures and loss of mobility (Fig. 10). In this group, a bone scan can be helpful in identifying and clarifying the sources of pain (Fig. 11). There is also a predilection for chondrocalcinosis and marked osteoarthropathy later in life [137]. In such situations, a bone scan may confirm sites of tenderness due to osteomalacic demineralization or microfracture. Affected adults are susceptible to dental disease that may lead to tooth extraction, but they cannot be easily pulled from the alveolus (self-extracted) as the deciduous teeth can be in children.
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FIGURE 9 Radiographic changes in childhood hypophosphatasia. (A) Note the tongues of radiolucency that project into the femoral and tibial metaphyses in this view of the knee from an 18-month-old girl. Irregular sclerotic margins are also visible at this age. (B) By age 9 years, these features have largely disappeared without any medical intervention. This spontaneous improvement is not uncommon, but the disease may remanifest in adulthood with localized bone pain, osteomalacia, and pseudofracture [reproduced with permission from Whyte, M. P. (1994). Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization. Endocr. Rev. 15, 439-461. Copyright 9 The Endocrine Society].
Odontohypophosphatasia In some patients, the only clinical feature is dental disease, and radiologic studies are otherwise normal, although the biochemical findings are generally indistinguishable from those in patients with milder generalized disease [125,138]. The anterior deciduous teeth are more likely to be affected and the most frequent loss involves the incisors [139]. The process is that of relatively painless extrusion and does not invoke periodontal inflammation (Fig. 12A). Dental X-rays show reduced alveolar bone and enlarged pulp chambers and root canals but normal enamel (Fig. 12B) [125,139]. Odontohypophosphatasia should be considered in any patient with a history of early unexplained loss of deciduous teeth or abnormally loose teeth on dental examination.
Pseudohypophosphatasia In this rare variant, first described by Scriver and Cameron [118], patients with typical radiologic features ofhypophosphatasia express an enzyme in the circulation that behaves normally toward artificial substrate but
have increased urinary phosphoethanolamine and pyrophosphate excretion as well as increased serum PLP (Fig. 13) [140] due to decreased enzyme activity toward these endogenous substrates [141]. True examples are rare and must be carefully distinguished from cases in which the elevation of ALP into the normal range is a transient response (e.g., after a fracture) or the biochemical profile has been misinterpreted [2]. Although biochemical studies of parents and proband suggest autosomal recessive inheritance, the molecular defect is not known. L a b o r a t o r y Findings
Clinical Chemistry In the classical forms of hypophosphatasia, total serum ALP activity is distinctly reduced. This does not appear to be the result of increased degradation since the half-life of infused ALP is normal [142,143]. Nor is it due to an endogenous inhibitor since coincubation experiments show no inhibition of exogenous ALP by serum from hypophosphatasia patients [2]. Rather, it reflects a failure of liver and bone to contribute the usual amounts
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FIGURE 10 Femoral fracture in adult hypophosphatasia. Readily visible in this AP view of the left pelvis and proximal femur pelvis (left) is a transverse midshaft fracture. The extensiveperiosteal sclerosiswith persistence of the unmineralized gap in the lateral midshaft cortex suggests that osteomalacic demineralization (Looser zone) preceded the transcortical break and that mineralization is delayed--a typical finding in adult hypophosphatasia [136].
of enzyme activity to the serum. Even in the most severe cases, some degree of ALP activity is detectable in serum. This may be due to the presence of circulating placental or intestinal isoenzyme, true residual activity of the TNAP, or contributions from other phosphomonoesterases (e.g., Y-nucleotidase) to the phosphatase activity measured in vitro. Other tissues that show a deficiency in ALP include granulocytes, bone, liver, kidney, and cultured skin fibroblasts [104,119,144,145]. Although a low serum ALP level is a helpful diagnostic indicator, this is also found in other conditions (Table 3). Attention to age-specific limits (Fig. 7) and proper collection of blood in tubes without a metal-chelating anticoagulant (i.e., EDTA) are important considerations. In early pregnancy, total ALP may be low due to hemodilution but increases to higher than normal levels in late pregnancy [115,146-148] (Table 1). In adult males, cardiac surgery is the most common cause of low ALP [149,150]. The greatest confusion is likely to arise in neonates who have other severe congenital bone disease, such as osteogenesis imperfecta and achondroplasia, or in infants and children whose ALP is probably low on the basis of malnutrition, particularly those with
anemia or vitamin disorders that also cause bone changes (e.g., scurvy) [146]. Urinary phosphoethanolamine (Fig. 5) was the first phosphoester found to be increased in hypophosphatasia [2]. Affected patients show increased concentrations in serum and urine. Quantitation of urinary excretion by amino acid analysis has been a useful confirmatory test for hypophosphatasia [151,152], but it should be recognized that phosphoethanolaminuria has been observed in association with other metabolic bone diseases and some patients with hypophosphatasia may have normal excretion [153,154]. Similarly, most patients with hypophosphatasia have increased serum and urinary concentrations of inorganic pyrophosphate (Fig. 5) [155,156], although the accurate measurement of this metabolite is by no means simple, even in urine [157-159]. Whyte and colleagues found that an increased level of serum PLP is a sensitive marker of hypophosphatasia (Fig. 13) [2,77,78]. Although supplementation with large amounts of pyridoxine (vitamin B6) can be a confounding artifact, B6 loading in hypophosphatasia patients shows a significantly exaggerated response and may be useful in carrier detection [160]. Overall, there appears to be a correlation between circulating ALP activity and PLP levels [161], but vitamin supplements may alter this correlation. Although serum PLP is increased in hyposphosphatasia serum, tissue levels are normal and vitamin B6 nutritional status is undisturbed in affected individuals [78]. In most other bone diseases, in which serum ALP activity may be elevated, serum PLP concentrations tend to be lower than normal, thus providing increased diagnostic discrimination in affected individuals (Fig. 13). An increased circulating level of thiamine pyrophosphate has also been reported for a few patients with hypophosphatasia [162], but there is no indication that this contributes to the pathogenesis.
Histopathology and Pathogenesis In the infant, bone histomorphometry reveals a marked excess of osteoid volume and an osteomalacic pattern of tetracycline labeling in dynamic studies [124]. Bone ALP is usually undetectable and electron microscopy shows otherwise normal subcellular architecture of osteoblasts and their associated matrix vesicles [124,163]. Iliac crest biopsies in adults show less dramatic and more variable changes. The severity of the osteomalacia, as measured by relative osteoid volume, is inversely correlated with the amount of detectable ALP in bone and serum concentrations of ALP activity. In shed teeth, marked deficiency or absence of cementum is a striking characteristic, accounting for the ready loss of teeth [125,139]. This appears to be the result of aplasia since
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FIGURE 1 1 Bone scan findings. (A) Increased uptake of radionuclide is seen in various sites, including the coracoid processes of the scapulae (arrows) and ends of the ribs (top left). Sites of pseudofracture and fracture in weight-bearing bones (asterisks) are also readily visualized. (B) In more distal views, asymmetrical hyperactivity is seen at the knees (top left) and ankles (top right), consistent with variably increased metaphyseal remodeling. In the chest wall (bottom), a small area of increased uptake (arrows) is readily seen in the 10th rib posteriorly. This matched the site of the patient's pain and tenderness to palpation, indicating a new area of pseudofracture, but no corresponding radiographic changes could be seen.
resorption of c e m e n t u m has never been observed. Dentin f o r m a t i o n is delayed and less appears to be formed. Interglobulin dentin and osteodentin have also been observed. It was p r e s u m e d that the severe mineralization defect induced by b T N A P deficiency in perinatal hypop h o s p h a t a s i a should be evident at the very earliest stages of mineralization based in p a r t on reports suggesting that the p r i m a r y site for this event, the matrix vesicle (MV) shed by the mineralizing osteoblast, a p p e a r e d not to contain microcrystal deposits of h y d r o x y a p a t i t e [163]. H o w ever, A n d e r s o n et al. [164] found evidence that needle-like crystals o f h y d r o x y a p a t i t e are deposited within the microvesicle but fail to extend m u c h farther b e y o n d the m e m brane. They speculated that a key role of b T N A P as an ectoenzyme m a y be to r e m o v e extracellular inhibitors that otherwise limit the extension of crystal g r o w t h t h r o u g h the extracellular m e d i u m to nearby matrix elements such as collagen [165]. This would be c o n s o n a n t with evidence that the o u t e r m o s t aspect of the m e m b r a n e b o u n d b T N A P t e t r a m e r contains high-affinity, n o n c a t a lytic sites for b o t h calcium and collagen [59]. FIGURE 12 Dental changes in hypophosphatasia. (A) View of the edentulous maxilla in a child with hypophosphatasia. Note the lack of inflammation and nubbins of unerupted permanent teeth with reduced size of the alveolar ridge due to excess bone resorption. (B) Matching pantomogram shows the premature loss of deciduous incisors and bicuspids without eruption of the matching permanent dentition. Note
also the enlarged pulp chambers and root canals, in addition to hypomineralized areas of dentin within the teeth.
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David E. C. Cole TABLE 3
Other causes of low circulating alkaline phosphatase
Physiological
9 pregnancy(early) Nutritional
9 9 9 9
metaldeficiency (magnesium, zinc) proteincalorie malnutrition vitamindeficiency (B12,C, D) vitaminexcess (D)
Endocrine disease
9 hypothyroidism,hyperthyroidism 9 estrogen replacement therapy in postmenopausal women Other conditions
9 9 9 9
osteogenesisimperfecta achondroplasia severeanemia hyperphosphatemia
Iatrogenic causes
9 bloodcollection with a metal-chelatinganti-coagulant (e.g., EDTA) 9 cardiacsurgery, cardiac bypass 9 transplantation 9 multipleblood transfusions
Much attention has been focused on the potential role of b T N A P as a protein phosphatase or a nucleotide phosphodiesterase, but neither appears to play an important role in the hypophosphatasia defect [67,166]. Of interest is the study of Johnson et al. [74] revealing decreased ATP-dependent Ca 45 precipitation and increased inorganic pyrophosphate in MV fractions from T N A P knockout mice. This led the investigators to assess another ectoenzyme, phosphodiesterase nucleotide pyrophosphatase (PDNP/NTPPPH), which hydrolyzes ATP to generate inorganic pyrophosphate. P D N P / N T P P P H is most likely associated with the plasma cell membrane protein glycoprotein- 1 (PC- 1), another moiety colocalizing with T N A P in osteoblast MV fractions and pericellular matrix. In vitro, T N A P added to T N A P knockout preparations directly antagonized inhibition by PC-1 of MV-mediated Ca 45 precipitation, suggesting that T N A P is key to the action of PC-1 in MV-directed mineralization. Paradoxically, transfection of cells with wild-type T N A P significantly increased osteoblast MV fraction N T P P P H and the specific activity of N T P P P H was twofold greater in MV fractions of osteoblasts from normal versus knockout mice. Johnson et al. concluded that T N A P attenuates PC-1/NTPPPH-induced inorganic pyrophosphate generation that would otherwise inhibit MV-mediated mineralization. However, it remains unclear why this is not neutralized by the paradoxical increase in inorganic pyrophosphate-generating activity that presumably follows TNAP-induced upregulation of PC-1 expression and N T P P P H activity[74].
Molecular Genetics
Mutation Analysis
FIGURE 13 Elevated pyridoxal Y-phosphate (PLP) in hypophosphatasia. Shown are serum concentrations in different groups. In bone patients with Paget's disease, other causes of osteomalacia, or other disease (o), PLP levels are either below the detectable limit (- - -) or within the normal range (less than 100 nmol/liter; hatched area). In patients with hypophosphatasia (1) or odontohypophosphatasia (D), PLP is consistently elevated. Three different samples in a patient (LW) with pseudohyposphatasia confirmed consistently elevated circulating PLP concentrations in this disease (reproduced with permission from Cole et al. [140]).
Greenberg and coworkers [39] first showed linkage between the Rh locus on chromosome lp with infantile hypophosphatasia. Weiss and colleagues [167] provided the first direct evidence of T N A P gene involvement in hypophosphatasia. They identified a homozygous G711A transition mutation in the affected offspring of a consanguineous couple. Although the Ala162Thr substitution is positioned in an extensively conserved domain, mutagenesis of the homologous residue in E. coli demonstrated no loss of activity [168]. When the human mutation was introduced into 3T3 cells, no ALP activity was found, although inactive protein was still immunologically detectable [167]. Transfection experiments in COS-1 cells revealed accumulation of highmolecular aggregates within the ER and failure of the translocation to the cell surface, suggesting that some error of protein folding may occur because of this mutation [52]. Subsequently, other missense mutations have been identified in various recessive forms of hypospho-
27. Hypophosphatasia sphatasia [169], including the founder mutation (G317D) responsible for the perinatal hypophophatasia in Canadian Mennonites [170]. In the past few years, more than 112 mutations have been identified in Japanese, European, and North American kindreds (Fig. 14) [3,98, 171-177]. (An Internet-accessible database can be found at http://www.sesep.uvsq.fr/Database.html.) In 2000, a survey of 65 distinct mutations in hypophosphatasia kindreds indicated that the majority (77%) are missense, with some clustering in exon 6 but otherwise distributed across all 12 exons [98]. Splice-site mutations, small deletions, and frameshift mutations are also known. Mutations affecting the signal peptide and the transcription region have been found, but large deletions have not been identified despite a systematic search by Southern blotting of probands for whom no mutation was detected by Single-strand comformational polymorphism (SSCP) methodology, denaturing gradient gel electrophoresis (DGGE), direct sequencing, or longrange polymerase chain reaction techniques [98,176, 178,179]. Of the 11 mutations discovered more than once, only the E174K mutation is frequent in multiple mild to moderately affected kindreds from North America [180] and Europe [98], whereas 1159delT appears to be recurrent in the Japanese [171,181,182]. The frameshift causes a substitution at Leu 5~ 5 amino acids prior to normal termination, and beyond. An immunoreactive peptide with a molecular weight consistent with the predicted elongation of 80 amino acids can be detected in serum from affected patients, but no mature peptide is detected on the surface of transfected cells and the pep-
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tide has no detectable enzymatic activity [181]. To date, only three of the nine SNPs in the human b T N A P gene are missense. The V505A polymorphism, with an allele frequency of 0.23 in a Manitoba population [170], is a conservative mutation in the C terminus and retains 94% of wild-type activity in cotransfection experiments [183]. The R135H mutation has only recently been described and appears to be uncommon (3 of 168 control alleles) [179]. Although the Y246H mutation [169] could be considered potentially functional [174], transfection studies indicate near-normal enzyme activity toward artificial substrate [175]. In both cases, interactions with other mild or moderately deleterious mutations or tissue-specific trans effects are difficult to rule out. Sequencing of b T N A P appears to detect up to 94% of all mutations, but there are recessive kindreds in whom a second contributory allele has not been identified and dominance has been effectively excluded [178]. To date, no phenotype other than hypophosphatasia has been associated with b T N A P mutations, and there appears to be a reasonable correlation between genotype, biochemical phenotype, and natural history [98]. In many forms of hypophosphatasia, recessive inheritance can be proven by molecular analysis, but the case for the rarer dominant forms is more difficult to make. In an odontohypophophatasia family with an affected father and four affected children, Muller et al. [183] showed inheritance of a single D361V mutation and a dose-dependent dominant negative effect of this mutation in transfected COS-1 cells. Similar analyses of mutations in seven families by Lia-Baldini et al. [178]
FIGURE 14 Map of representative TNAP mutations causing hypophosphatasia. Shown in this schematic are the 12 TNAP exons with their intronic gaps. The untranslated region (exon 1 and part ofexon 2) is hatched and the signal sequence (exon 2) is stippled. Most darkly shaded are the exonic segments responsible for homodimer interfacing (exons 2-4,10, and 11). Also darkly shaded are sequence segments involved in the fourth (calcium) binding site (exons 7 and 9) as well as those constituting the crown domain (exons 11 and 12) containing the collagen-binding site. Most lightly shaded are the segmentsthat contribute to the active site (exons 3,5,6,9, and 10). Mutations described in the Japanese population are shownin italics; those found in patients with mild or moderate forms of hyposphosphatasia (childhood, adult, and odontohyposphosphatasia)are underlined (courtesy of Dr. E. Mornet).
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revealed that two (D361V and G46V) are fully dominant at an enzymatic level, whereas three (A99T, R167W, and N461I) are partially inhibitory. With four other mutations, no dominance was observed, increasing the likelihood that an undetected second mutant allele may be present. Structure-function correlations suggest that all five domains of the enzyme--the active site, active site valley, homodimer interface, crown domain, and calcium site-can be affected adversely by mutation. However, mutations that disrupt protein folding or higher order structure are not uncommon [59]. Although disease severity correlates with enzymatic activity, direct association with enzyme conformation by analysis is still inferential, causing some uncertainty about the consistency of phenotype [184].
alization was progressive and failure of secondary ossification frequent. Histologically, this was matched by arrest of chondrocyte differentiation and hypoplasia of the hypertrophic zones in epiphyseal growth plates. In trabecular bone, marked osteoid thickening was observed, consonant with findings in humans [2,4]. E x vivo studies of cultured osteoclasts and osteoblasts show that cell growth is normal, as is cytokine expression and production of bone-specific proteins, including type I collagen, osteocalcin, and osteopontin. Nodule formation also occurs normally, but osteoblasts from TNAPnull mice fail to mineralize these nodules, whereas cells from the heterozygous animals show a delay in comparison to wild-type cells [189]. This last observation is supportive of the notion that milder mutant genotypes in humans can be associated with a dominant mode of inheritance [175].
Lessons from Mouse Models
There are significant interspecies differences in the expression of ALP isozymes, but the generation of knockout mice has provided important corroboration for the hypophosphatasia phenotype. First produced on an inbred 129/Sv mouse background, homozygous null mutants for TNAP (Akp2) are grossly normal at birth but fail to gain weight normally [185]. By 2 weeks of age, epileptiform seizures occur and death follows the onset of status epilepticus. Partial rescue by pyridoxine and decreased levels of 7-aminobutyric acid, a B6-dependent neurotransmitter, suggest that central deficiency of PLP is the proximate cause of the neuropathy. As expected, PLP levels in brain are low, but this deficiency is also seen in other tissues. Surprisingly, few gross mineralization defects were seen in the homozygotes, although shortened survival severely restricted the observation period. On a C57Bl/6J background, independently generated TNAPnull homozygotes showed similar postnatal weight loss and progressive seizure activity but also thymic hypoplasia, splenic abnormalities, and thinning of the caudal peripheral nerves [186]. By 8 days, hypomineralization and spontaneous fractures were observed, but demineralization continued even after exogenous B6 rescue of the neuropathic phenotype [187]. Fedde et al. [188] conducted a detailed study of the bone biology in both models, which were fed a soft diet with gastric tube supplements. Although there was a decrease in serum ALP activity in the heterozygotes, no gross abnormalities or histopathologic changes were observed at birth. In both sets of homozygotes, the small differences in birth weight increased with time. The biochemical phenotype included increased urinary phosphoethanolamine and inorganic pyrophosphate excretion as well as elevated serum PLP. Skeletal mineralization was indistinguishable from normal at 6 days of age; thereafter, however, hypominer-
Management Medical
There is no known treatment consistently effective in ameliorating the bony disease in this condition [4]. Although temporary improvement has been observed after treatment in some cases, the natural history of spontaneous improvement in others makes it difficult to interpret the response in the absence of a controlled clinical trial. The rarity of this condition and its variability, even within families, may complicate the unbiased assessment of therapeutic trials. The availability of mouse models raises hope for evidence-based therapies in the future. Among the agents that have been tried (without success) are the catalytic metals zinc and magnesium. Although supplemental phosphate might be expected to decrease levels of the endogenous mineralization inhibitor inorganic pyrophosphate [190], phosphate is an inhibitor of the enzyme [79] and it is not surprising that trials of high-dose phosphate have not been consistently helpful [3,123]. An early trial of infusing serum from patients with hyperphosphatasia [191] was promising [143], but subsequent experience has been disappointing [192,193]. During pregnancy, patients and carriers experience an increase in circulating ALP activity [115,194] and a parallel decrease in substrate accumulation, suggesting that methods to induce placental ALP might be of direct benefit to patients, despite the fact that direct intravenous infusion of placental isozyme has not been successful [3]. Traditional therapies for rickets and osteomalacia, such as supplemental vitamin D (or its metabolites), calcium, and phosphate, should be avoided because there is no evidence of primary disturbance in a bone
27. Hypophosphatasia
and mineral metabolism [126]. Restriction of vitamin D in the face of hypercalcemia may be rachitogenic [128], but glucocorticoids, saline diuresis, and calcium restriction may be essential for the control of severe hypercalcemic episodes sometimes seen in infantile disease [3,195]. In one case of infantile hypophosphatasia, calcitonin was used successfully to reduce hypercalcemia, coupled with thiazide treatment to minimize the hypercalciuria [196]. In older children and adults, inflammatory bone and joint disease may respond to nonsteroidal antiinflammatory medication [3,197]. Allogenic bone marrow transplantation (BMT) was performed in a 3-month-old with the infantile form of the disease [198]. Clinical improvement followed, but biochemical hallmarks remained unchanged and the long-term outcome remains to be seen. Other cases have also been treated with BMT, and it is likely that this will be an important therapy in the future for some of the more severe cases [223].
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additional heterogeneity [124], and examples of more than one presentation (e.g., infantile and adult or infantile and childhood) in the same sibship have been reported [151,202]. Allelic diversity is a possible explanation in some cases, but clinical discordance in kindreds with a known mutation is well established, suggesting that modifying effects of nearby loci or other genes are also important. Given the rarity of the condition, the lack of effective treatment, and the potential severity of the recurrent disease in affected families, it is not surprising that patients have become more active in providing selfsupport. For affected individuals, their families, and concerned caregivers in North America, access to such groups as well as additional information can be obtained from the website of the Canadian Hypophosphatasia Contact Group and groups the Magic Foundation http://www.homestead.com./hypophosphatasia.
Prenatal Diagnosis Surgical During childhood, pseudofractures or atraumatic fractures occur not infrequently, and their natural history does not appear to be altered by medication. Orthopedic intervention may be needed, but it does not materially alter the risks for secondary fracture or pseudofracture later. Better results appear to be achieved with the use of medullary rods in the fixation of long bones rather than load-sparing devices, such as screwed plates, since osteomalacic demineralization is frequently found in bone adjacent to such devices, allowing them to work free [136]. There can be increased loading of prosthetics where there is little support from adjacent cortical bone, suggesting that risks of mechanical failure can be reduced by selecting devices that minimize such risks [199]. Craniosynostosis may be severe enough to require surgical intervention and should be followed closely [133]. Expert dental care is especially important for affected children, who may require dentures if there are significant losses of permanent teeth at an early age.
Genetic Counseling Both recessive and dominant modes of inheritance have been observed in different hypophosphatasia kindreds [125,200]. In many families with more severe disease, the frequency of consanguinity and the recurrence rates for infantile hypophosphatasia are clearly indicative of recessive transmission. The suggestion that milder hypophosphatasia may be an autosomal dominant condition with homozygous lethality is in keeping with data on the adult form of the disorder [152,201], but there is
Most of the ALP activity found in human amniotic fluid originates from the fetal intestine [203,204]. Monoclonal antibodies to TNAP may be helpful for analysis of chorionic villus samples [205], but amniotic fluid levels are so low under normal conditions that routine enzyme assays are of little diagnostic value by themselves [119,206]. Most severe forms of perinatal hypophosphatasia can be diagnosed by second-trimester ultrasonography and confirmed by assay of TNAP in cultured amniocytes. Amniotic fluid ALP can be measured with the more sensitive fluorometric assay [119,206,207], but molecular methods probably provide optimal diagnostic discrimination. Linkage analysis has been used to determine transmission of a mutant allele, but direct sequencing of the proband mutation is currently performed more often [98,172,208,209]. Given the high positive predictive value of molecular testing, it has become the method of choice. Nevertheless, the inability to predict the severity of the phenotype is still a major concern. Moore et al. [210] reported two families with four cases of mild dominant hypophosphatasia in which the disorder first manifested in utero as severe long-bone bowing, only to be followed by spontaneous improvement postnatally. A fifth instance of very severe bone dysplasia with a subsequent benign course was reported by Pauli et al.[211]. The spontaneous improvement of long-bone angulation began prenatally and persisted into infancy. It was suggested that this form of the disease (benign prenatal hypophosphatasia) be distinguished from others and added to the differential diagnostic possibilities considered when angulation or bowing of long bones is discovered prenatally.
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Cleidocranial Dysostosis: A Phenocopy? Hypophosphatasia research provides plenty of evidence to support the notion of a very close correspondence between mutant bTNAP genotype and hypophosphatasia phenotype. However, there is reason to believe that cleidocranial dysostosis (CCD) can occasionally present as a phenocopy. Biochemical and bony features typical of hypophosphatasia have been reported in patients with CCD [212,213], but there has been uncertainty about transmission of the biochemical phenotype. Unger et al. [214] observed a case of CCD with persistent hypophosphatasemia, elevated PLP, and increased phosphoethanolamine excretion. In addition to radiologic signs of CCD, a bone spur of the fibula and an overlying dermal dimple characteristic of early onset hypophosphatasia were found [131]. It is speculated that CCD, caused by mutations in the bone-specific transcription factor Cbfal, exerts a downregulatory effect on TNAP sufficient to result in a metabolic and molecular hypophosphatasia phenotype. In support of this concept is in vitro evidence that Cbfal expression is normal in the TNAP knockout mouse [189], but TNAP expression is markedly suppressed in osteogenic cells without full CBFA1 expression [215]. From a clinical perspective, all patients with undefined or atypical hypophosphatasia should be examined for the classic signs of CCD: absence or hypoplasia of the clavicles and pubic rami and multiple pseudoepiphyses of the metacarpals. Detailed molecular studies of CCD/hypophosphatasia patients are another means by which the basic biology of bone ALP can be explored through the clinical investigation of disease in humans.
Acknowledgments I thank M. Whyte, E. Mornet, M. M. Cohen, Jr., L. Canaff, G. Hendy, C. Wei, and S. Unger for helpful advice. J. Evrovski, T. Sukovic, and L. Donnelly provided important research assistance, and C. Honeywell and W. Newman reviewed the manuscript. I am particularly grateful to all of the hypophosphatasia families I have been privileged to know, for they have each taught me something unique about this disease.
References 1. Cole, D. E., and Cohen, M. M., Jr. (1990). Mutations affecting bone-forming cells. In The Osteoblast and Osteocyte (B. K. Hall, Ed.), pp. 431-487. Telford, Caldwell, NJ. 2. Whyte, M. P. (1994). Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization. Endocr. Rev. 15, 439-461. 3. Whyte, M. P. (2000). Hypophosphatasia. In The Genetics of Osteoporosis and Metabolic Bone Disease (M. J. Econs, Ed.), pp. 335-356. Humana Press, Totowa, NJ. 4. Whyte, M. P. (2001). Hypophosphatasia. In The Metabolic and Molecular Bases of Inherited Disease (C. R. Scriver, A. L. Beaudet, W. S. Sly, D. Valle, B. Childs, K. W. Kinzler, and B. Vogelstein, Eds.), 8th ed., pp. 5313-5329. McGraw-Hill, New York.
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and detection of heterozygote carriers within the family. Clin. Genet. 56, 313-317. Moore, C. A., Curry, C. J., Henthorn, P. S., Smith, J. A., Smith, J. C., O'Lague, P., Coburn, S. P., Weaver, D. D., and Whyte, M. P. (1999). Mild autosomal dominant hypophosphatasia: In utero presentation in two families. Am. J. Med. Genet. 86, 410-415. Pauli, R. M., Modaff, P., Sipes, S. L., and Whyte, M. P. (1999). Mild hypophosphatasia mimicking severe osteogenesis imperfecta in utero: Bent but not broken. Am. J. Med. Genet. 86, 434-438. Rubecz, I., Mehes, K., Klujber, L., Bozzay, L., Weisenbach, J., and Fenyvesi, J. (1974). Hypophosphatasia: Screening and family investigation. Clin. Genet. 6, 155-159. Moore, C. A., Ayangade, G., Okolo, P., Bull, M. J., Whyte, M. P., and Bixler, D. (1992). Cleidocranial dysostosis: A natural history study including evaluationof a five-generation family. Proc. Greenwood Genet. Center 11, 91.
214. Unger, S., Mornet, E., Mundlos, S., Blaser, S., and Cole, D. E. C. (2002). Severe cleidocranial dysplasia can mimic hypophosphatasia. Eur. J. Pediatr., 161,623-626. 215. Zhang, Y. W., Yasui, N., Ito, K., Huang, G., Fujii, M., Hanai, J., Nogami, H., Ochi, T., Miyazono, K., and Ito, Y. (2000). A RUNX2/PEBP2alpha A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia. Proc. Natl. Acad. Sci. USA 97, 10549-10554. 216. Harris, H. (1990). The human alkaline phosphatases: What we know and what we don't know. Clin. Chim. Acta 186, 133-150. 217. Bailyes, E. M., Seymour, P. M., Fulton, I., Price, C. P., and Luzio, J. P. (1988). A monoclonal antibody capture assay for intestinal alkaline phosphatase and the measurement of this isoenzyme in pregnancy. Clin. Chim. Acta 172, 267-274. 218. Fedde, K. N., and Whyte, M. P. (1990). Alkaline phosphatase (tissue-nonspecific isoenzyme) is a phosphoethanolamine and pyridoxal-5t-phosphate ectophosphatase: Normal and hypophosphatasia fibroblast study. Am. J. Hum. Genet. 47, 767-775. 219. Bourrat, C., Radisson, J., Chavassieux, P., Azzar, G., Roux, B., and Meunier, P. J. (2000). Activity increase after extraction of alkaline phosphatase from human osteoblastic membranes by nonionic detergents: Influence of age and sex. Calcif. Tissue Int. 66, 22-28. 220. Lian, J. B., Stein, G. S., Bortell, R., and Owen, T. A. (1991). Phenotype suppression: A postulated molecular mechanism for mediating the relationship of proliferation and differentiation by Fos/Jun interactions at AP-1 sites in steroid responsive promoter elements of tissue-specific genes. J. Cell Biochem. 45, 9-14. 221. Peverali, F. A., Basdra, E. K., and Papavassiliou, A. G. (2001). Stretch-mediated activation of selective MAPK subtypes and potentiation of AP-1 binding in human osteoblastic cells. Mol. Med. 7, 68-78. 222. Quandt, K., Frech, K., Karas, H., Wingender, E., and Werner, T. (1995). MatInd and MatInspector: New fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res. 23, 4878-4884. 223. Whyte, M. P., Kurtzberg, J., McAlister, W. H., Mumm, S., Podgornik, M. N., Coburn, S. P., Ryan, L. M., Miller, C. R., Gottesman, G. S., Smith, A. K., Douville, J., Waters-Pick, B., Armstrong, R. D., and Martin, P. L. (2003). Marrow cell transplantation for infantile hypophosphatasia. J. Bone Miner. Res. 18, 624-636.
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[28[ Renal Osteodystrophy: Pathogenesis, Diagnosis, and Treatment BEATRIZ D. KUIZON and ISIDRO B. SALUSKY Department of Pediatrics, UCLA School of Medicine, Los Angeles, California
INTRODUCTION
renal osteodystrophy have histologic evidence of both osteomalacia and hyperparathyroidism; the rate of bone formation in mixed lesions depends on the predominant lesion. Although the type of renal bone disease is primarily determined by serum PTH levels, additional factors that modify bone formation and turnover include calcium, phosphorus, vitamin D analogs, growth hormone (GH), and aluminum (Fig. 1) [1].
Renal osteodystrophy is a disorder of bone and mineral metabolism that has long been recognized as a consequence of renal dysfunction. Disturbances in calcium and phosphorus homeostasis, reduced synthesis of 1,25dihydroxyvitamin D3, altered metabolism of parathyroid hormone (PTH), and impaired renal clearance of PTH fragments and other substances, such as aluminum and 132-macroglobulin, develop during progressive renal impairment. These factors are fundamental in the pathogenesis of renal bone disease. Although it is well established that these skeletal lesions in adults primarily represent disturbances in bone remodeling, in children they occur during longitudinal and appositional bone growth and modeling. Thus, the control of secondary hyperparathyroidism remains a critical element in the care of pediatric patients with renal failure in order to prevent potential serious long-term consequences, such as bone deformities and growth retardation. In this chapter, the pathogenesis, clinical manifestations, and management of renal osteodystrophy in children with chronic renal failure and those treated with maintenance dialysis are discussed.
Disturbances in Bone R e m o d e l i n g Bone remodeling is a complex process by which old bone is resorbed and replaced by new bone at localized sites within the skeleton. Each cycle takes approximately 100-120 days. It is a continuous process throughout life and allows for maintenance and repair of the skeleton. Several hormones (PTH, calcitonin, insulin, GH, thyroid
Spectrum of Renal Osteodystrophy Calcium, Vitamin D 9
L o w turnover
[PTH I
H~h
turnover
v
[Adynamic|
THE SPECTRUM OF RENAL OSTEODYSTROPHY
I Oste~
Renal osteodystrophy represents a spectrum of skeletal lesions that range from high-turnover disorders (osteitis fibrosa and mild lesions of secondary hyperparathyroidism) to low-turnover bone diseases (osteomalacia and adynamic lesion) (Fig. 1) [1]. Mixed lesions of
Pediatric Bone
GH / IGF
i
Normalbone I formation ~
! OsteitisI fibrosa
I Mixed lesion / FIGURE 1 Spectrum of renal osteodystrophy (reproduced with permission from Salusky and Goodman [1]).
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Copyright 2003, Elsevier Science (USA). All rights reserved.
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hormone, 1,25-dihydroxyvitamin D3, glucocorticoids, and sex steroids) and growth factors [insulin-like growth factor- 1 (IGF- 1), fibroblast growth factor, and transforming growth factor-13] are known to regulate bone remodeling. In renal failure, however, PTH is the major factor influencing the rate of bone formation and turnover. Secondary hyperparathyroidism is characterized by increased bone formation rates as determined by double tetracycline labeling. Patients with osteitis fibrosa have much higher bone formation rates (two to four times the upper limit of normal) than those with mild lesions of secondary hyperparathyroidism. Osteoblastic activity is increased, as evidenced by the elevated number of plump and cuboidal osteoblasts along trabecular surfaces. The high rate of bone formation accounts for the greater amount of unmineralized bone or osteoid. Bone resorption is also higher, as demonstrated by increases in the number and size of osteoclasts, the amount of trabecular surfaces lined with osteoclasts, and the number of resorption bays or Howship's lacunae. Fibrous tissue is detected adjacent to the trabeculae and within the marrow space in patients with more advanced secondary hyperparathyroidism, whereas it is absent in those with mild lesions of secondary hyperparathyroidism (Fig. 2). The low-turnover bone lesions of renal osteodystrophy, on the other hand, are characterized by decreased
number of osteoblasts and osteoclasts along trabecular surfaces and markedly reduced or unmeasurable rates of bone formation using double tetracycline labeling. Due to a defect in skeletal mineralization, the amount of osteoid and the proportion of trabecular surfaces covered with osteoid are increased in osteomalacia. In contrast, osteoid volume is normal or low in adynamic bone. Although aluminum toxicity was the major pathogenic factor for both osteomalacia and adynamic bone lesions in the past, recent studies have demonstrated the absence of bone surface stainable aluminum in lowturnover lesions [2,3]. D i s t u r b a n c e s in Longitudinal a n d Appositional Bone G r o w t h In addition to disturbances in bone remodeling, renal osteodystrophy in pediatric patients is associated with alterations in the two types of bone growthmthat is, longitudinal bone growth and appositional modeling. Longitudinal bone growth occurs through endochondral bone formation, the process through which the growth plate cartilage at the proximal and distal ends of long bones is replaced by bone. The bone elongates as the growth plate and bone front progressively move away from the center of the bone. This process continues until the end of puberty, when the growth plate starts to fuse.
FIGURE 2 Osteitis fibrosa is characterized by increased osteoblastic activity leading to greater osteoid volume, increased osteoclast number and resorptive surface, and the presence of peritrabecular surfaces and fibrosis.
28. Renal Osteodystrophy
Appositional modeling, on the other hand, results in the growth of the diameter or width of the long bone. This is achieved by periosteal apposition and by endosteal bone resorption [4]. Disturbances in endochondral bone formation were demonstrated on autopsy material obtained from children with severe osteitis fibrosa approximately three decades ago [5]. Marked chondroclastic erosion and abnormal vascularization of the growth plate cartilage in these patients were thought to contribute to the pathogenesis of growth retardation and slipped capital epiphyses in children with renal bone disease. However, the mechanisms responsible for growth retardation remain poorly understood. Since bone growth and elongation are regulated through a complex autocrine/paracrine system within the growth plate that involves GH, IGF-1, and local growth factors parathyroid hormone related peptide (PTHrP) and the PTH/PTHrP receptor, analysis of the expression of these critical factors in the growth plate may provide meaningful insight regarding the reasons for impaired growth in renal failure [6,7]. Indeed, considerable reductions in growth plate width and PTH/PTHrP receptor expression using in s i t u hybridization were observed in growth plate cartilage of uremic rats with severe secondary hyperparathyroidism but not in animals with mild secondary hyperparathyroidism or adynamic bone induced by calcium supplementation (Fig. 3) [8,9]. Similarly, we found diminished mRNA expression for the PTH/PTHrP receptor in the growth plate cartilage of
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children with osteitis fibrosa compared to those with normal rates of bone formation [10]. In contrast, uremic rats given a high-calcium diet to induce adynamic bone had increased growth plate width associated with reductions in chondrocyte apoptosis, matrix degradation, and angiogenesis [9]. These findings suggest that impaired linear growth in renal failure may be related in part to changes in the regulation of chondrocyte proliferation and differentiation, and these events may involve the PTH/PTHrP receptor. Different types of renal osteodystrophy are thus likely to have a significant impact on endochondral bone elongation in renal failure. GH and IGF-1 have long been recognized as playing key roles in bone growth and elongation. Growth retardation develops despite normal or increased serum GH levels in renal failure, suggesting tissue resistance to the actions of GH in this condition [11,12]. The mechanism underlying GH resistance in renal failure is not well understood. Diminished mRNA expression of the hepatic growth hormone receptor (GHR) and IGF-1 and impaired hepatic GH-mediated signal transduction have been found in uremic animals, and these may be potential mechanisms contributing to growth failure [13,14]. Although recent studies demonstrate that the paracrine/ autocrine effects of IGF-1 on chondrocytes represent the main determinant of postnatal growth, there are limited data regarding the alterations in GH and IGF-1 actions in the growth plate in renal failure [15,16]. Decreased expressions of GHR and IGF-1 have been described in chondrocytes of uremic rats, but these studies did not
FIGLIRE 3 The growth plate in uremic rats givencalcium supplementationto induce adynamicbone (Nx-Ca) was substantially wider than that in control rats (Control) and in uremic rats given regular diet (Nx-C) or phosphorus supplementationto induce severesecondaryhyperparathyroidism(Nx-P). Note the markedincrease in the width of the hypertrophiczone (reproducedwith permissionfrom Sanchezet al. [9]).
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address the role of the underlying bone disease as a modifier of the expressions of GHR and IGF-1 in the growth plate [17,18]. Treatment with GH is widely recommended to improve linear growth in children with renal failure, but the response is substantially less in those undergoing dialysis [19,20]. Whether this difference is due to the underlying bone disease and the alterations in GH and IGF-1 actions associated with certain skeletal lesions is not known. Previous studies also failed to address whether concomitant calcitriol therapy modifies the response to GH, particularly since the antiproliferative effects of calcitriol differ markedly from the trophic actions of GH. Calcitriol exerts dose-dependent inhibitory effects on chondrocyte proliferation in vitro and neither GH nor IGF-1 can overcome these inhibitory effects [21,22]. In rats with severe secondary hyperparathyroidism, we have shown that calcitriol inhibits the expected increases in growth plate width and mRNA expressions for collagen types II and X in growth plate chondrocytes during GH therapy [8]. Moreover, we found diminished linear growth in prepubertal children with end-stage renal disease during high-dose intermittent calcitriol therapy [23]. Overall, these data suggest that calcitriol modifies chondrocyte proliferation and/or differentiation within the growth plate and counteracts the stimulatory effects of GH on chondrocyte activity, thereby contributing to suboptimal GH response. PATHOGENESIS OF RENAL OSTEODYSTROPHY Secondary Hyperparathyroidism Role of Calcium, Phosphorus, and Calcitriol The extracellular calcium concentration is generally considered the main regulator of minute-to-minute release of PTH from the parathyroid gland [24]. Hypocalcemia stimulates whereas hypercalcemia inhibits PTH secretion, and this inverse sigmoidal relationship between PTH and ionized calcium levels is well described in vivo and in vitro [25-27]. PTH secretion in response to hypocalcemia occurs within seconds and maximal secretion can be maintained for 60-90 min. Hypercalcemia, however, does not totally inhibit PTH secretion; this nonsuppressible component may have significant implications in disorders associated with parathyroid hyperplasia [28]. One of the major scientific advances in the past 10 years has been the identification of the G proteincoupled calcium-sensing receptor (CAR), the molecular mechanism through which the main cells of the parathyroid glands and C cells of the thyroid gland can recognize variations in extracellular calcium concentrations and
regulate the synthesis and/or secretion of PTH and calcitonin, respectively [29,30]. However, it is primarily PTH, and not calcitonin, that maintains ionized calcium levels within a narrow physiologic range. The CaR is also expressed in multiple sites in bone, kidney, and intestine, which in addition to being target organs for the actions of calciotropic hormones can directly respond to local changes in extracellular calcium. Recent data demonstrating its presence in tissues that are not otherwise involved in calcium homeostasis suggest that the CaR is als0 integrally involved in the regulation of cell proliferation and differentiation, apoptosis, hormone secretion, and probably other unknown functions [30]. Heterozygous inactivating mutations of the gene encoding the human CaR result in familial hypocalciuric hypercalcemia, and homozygous or compound heterozygous mutations lead to severe life-threatening infantile hyperparathyroidism; activating mutations of the CaR gene are found in hereditary forms of hypoparathyroidism associated with inappropriate hypercalciuria [31-33]. Alterations in calcium homeostasis similar to those observed in families with inactivating CaR mutations have been demonstrated in mice with deletions of the CaR gene [34]. The actions of the CaR are largely mediated through intracellular-free calcium and IP3 but other signaling pathways are likely to be involved as well; however, these remain to be defined. In renal failure, hypocalcemia arises from hyperphosphatemia, diminished intestinal calcium absorption due to calcitriol deficiency, and decreased skeletal response to PTH. Short-term (minutes) hypocalcemia results in reduced CaR activity and thus stimulates PTH secretion. More sustained hypocalcemia increases PTH gene transcription (hours and days) and parathyroid cell proliferation [35-37]. Conversely, suppression of PTH gene transcription by elevations in calcium is mediated in part by negative regulatory elements in the 5' flanking region of the PTH gene [38,39]. In addition, serum calcium regulates PTH mRNA levels in vivo by a posttranscriptional mechanism that involves the binding of parathyroid cytosolic proteins to the Y untranslated region of the PTH mRNA, which then determines the stability and levels of PTH mRNA [40]; the effect of serum phosphorus on PTH gene expression occurs by a similar mechanism. Parathyroid proteins from hypocalcemic animals had increased RNA protein binding as determined by ultraviolet cross-linking studies, whereas decreased binding was seen with proteins from hypophosphatemic animals [40,41]. Two negative calcium-response elements (nCaRE) located far upstream (-2.4 and -3.5kbps) of the human PTH gene have been described. The authors suggested that the conferred negative regulation of transcription by calcium involves the redox factor protein
28. Renal Osteodystrophy
refl [38,42]. As such, decreases in the availability of either 1,25-dihydroxyvitamin D or calcium will promote pre/pro-PTH gene transcription, whereas PTH synthesis will diminish when 1,25-dihydroxyvitamin D or calcium are abundant. Phosphate retention develops when renal function declines below 25-30% of normal [43]. Its role in the pathogenesis of secondary hyperparathyroidism has been well recognized for years. By decreasing serum calcium levels and inhibiting calcitriol production, hyperphosphatemia indirectly contributes to excess PTH secretion and parathyroid gland hyperplasia. Recently, studies have shown that phosphorus directly influences parathyroid gland function. Indeed, uremic animals given dietary phosphorus supplementation developed increased serum PTH levels and parathyroid gland hyperplasia, and these disturbances occurred without changes in the serum levels of both calcium and calcitriol [44,45]. This effect of phosphorus appeared to be posttranscriptional since pre/pro-PTH mRNA levels were not increased in the animals given a high-phosphorus diet [44]. When animals with established secondary hyperparathyroidism were then given a phosphorus-restricted diet, serum phosphorus and PTH levels normalized, but the parathyroid glands remained hyperplastic and no evidence of apoptosis was found [46]. Moreover, no difference in PTH synthesis, parathyroid gland cytosolic PTH, or the parathyroid gland secretory response to calcium was demonstrated, suggesting alterations in PTH exocytosis [46]. It is not known whether there is a specific receptor for phosphorus, but a specific parathyroid cell sodium-dependent phosphate cotransporter (PiT-l) has been identified by Tatsumi et al. [47]. Vitamin D and hypophosphatemia increase PIT-1 mRNA levels; thus, this cotransporter may mediate the actions of phosphate and vitamin D on PTH secretion [47]. Calcitriol, the most potent metabolite of vitamin D, is produced by l~-hydroxylation of 25(OH)D3 in the proximal tubules of the kidney. The gene encoding 1~hydroxylase has been cloned from rat, mouse, and human tissues, and mutations in the gene result in vitamin D-dependent rickets type II [48-50]. PTH, hypocalcemia, and hypophosphatemia stimulate renal l~hydroxylase activity, whereas increased levels of calcium, phosphorus, and calcitriol inhibit enzyme activity. In renal failure, impaired calcitriol synthesis is one of the major factors that contribute to the pathogenesis of secondary hyperparathyroidism. Calcitriol deficiency decreases intestinal calcium absorption, which in turn leads to hypocalcemia and increased PTH secretion. Since calcitriol inhibits pre/pro-PTH gene transcription through the negative vitamin D response elements in the PTH gene, low serum calcitriol levels also promote PTH synthesis and subsequently PTH release. Indeed, sup-
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pression of PTH gene transcription by calcitriol has been described both in vitro in bovine parathyroid cells and in vivo in rats [51-53]. Negative regulatory elements responsible for the suppression of human PTH gene expression have been identified upstream of the PTH gene transcription start site. Demay et al. [54,55] found DNA sequences in the 5' regulatory region (-125 to -101 from the start of exon 1) of the human PTH gene that bind the vitamin D receptor (VDR) and inhibit transcription in response to vitamin D. Moreover, the retinoid X receptor (RXR) was not necessary for binding of the VDR to the downregulatory DNA sequence. Sequences that bind the VDR have also been reported in the 5t flanking region of bovine and avian PTH genes [56-58]. Calcitriol also regulates PTH synthesis via the VDR. Calcitriol upregulates VDR gene expression in the parathyroid gland; such an effect may be important in augmenting the feedback inhibition of PTH gene transcription, particularly since renal failure is associated with reduced binding of VDR to the osteocalcin VDRE and because parathyroid glands of animals and humans with secondary hyperparathyroidism have decreased VDR expression [59-62]. Overall, the inhibitory actions of calcitriol on PTH synthesis and secretion have provided the basis for its widespread use in the management of secondary hyperparathyroidism in patients with renal failure. The long-term use of high intermittent doses of calcitriol, however, can contribute to the development of adynamic bone and poor growth in children with endstage renal disease (ESRD) (vide infra). Finally, it is interesting to keep in mind the studies by Demay et al. on mice with targeted ablation of the VDR. These animals developed hypocalcemia, markedly elevated serum PTH levels, parathyroid gland hyperplasia, and features of rickets at the level of the growth plate cartilage [63]. Dietary calcium and phosphorus supplementation normalized serum calcium and phosphorus levels and prevented the development of rickets despite the absence of VDR [64,65]. Such findings suggest that the actions of vitamin D per se are not required to prevent parathyroid gland hyperplasia. The implications of these findings for the treatment of renal osteodystrophy remain to be elucidated. Set-Point Abnormalities in Renal Failure
Disturbances in the regulation of PTH secretion have long been considered in the pathogenesis of secondary hyperparathyroidism in chronic renal failure. This was based on previous in vitro studies using the four-parameter model, which showed that the set point for calcium-regulated PTH release was increased in parathyroid cells obtained from patients with primary or
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secondary hyperparathyroidism [27,66,67]. In vivo assessments in humans, however, have provided controversial data. Inconsistent results may be due, in part, to differences in methodologies used in the dynamic assessments of parathyroid gland function and in estimating the set point. Earlier in vivo studies of PTH secretion performed in our institution did not demonstrate disturbances in calcium sensing in patients with mild and moderate secondary hyperparathyroidism nor in those with adynamic renal osteodystrophy [68-70]. A higher set point was subsequently found only in patients with severe secondary hyperparathyroidism requiring parathyroidectomy; such findings are consistent with previous data on dispersed parathyroid cells [71]. Lowering of the set point during calcitriol therapy has also been reported in patients with secondary hyperparathyroidism, but we and others found no difference in set point after calcitriol treatment [68,72-74]. Decreased CaR expression is thought to contribute to the elevation in set point in patients with primary or secondary/tertiary hyperparathyroidism from renal failure, as it does in mice with heterozygous or homozygous null mutation of the CaR gene and in humans with inherited inactivating CaR mutations [31-34]. Thus, CaR agonists or calcimimetic agents present a novel approach for the management of these disorders associated with alterations in CaR function. In vitro studies of parathyroid gland function or CaSR are needed in humans with mild to moderate renal failure to assess whether alterations in CaSR expression may account for disturbances in PTH secretion in early renal failure.
Parathyroid Gland Hyperplasia Parathyroid gland hyperplasia is a significant feature of secondary hyperparathyroidism; however, the mechanisms regulating parathyroid cell proliferation and apoptosis in renal failure remain poorly understood. The lack of available parathyroid cell lines has made research in this area difficult. Moreover, little is known regarding the relationship between abnormal parathyroid cell proliferation and excess hormone secretion. Whether disturbances in PTH secretion consequently lead to increased cell division or vice versa remains to be determined [75]. Recent data, however, demonstrate that a primary defect in parathyroid cell proliferation results in altered calcium sensing by the parathyroid glands. Indeed, transgenic mice with primary hyperparathyroidism induced by parathyroid-targeted overexpression of cyclin D1, an oncogene implicated in the pathogenesis of parathyroid adenoma, developed enlarged parathyroid glands associated with increased parathyroid cell proliferation, high bone formation and turnover with cortical bone loss, and higher set point for
calcium-regulated PTH release [75]. Interestingly, expression of the calcium-sensing receptor in the parathyroid glands of these transgenic animals was diminished, consistent with previous observations of parathyroid tissues from patients with primary hyperparathyroidism [75]. Parathyroid cell proliferation is often diffuse and polyclonal (nonneoplastic) in less advanced secondary hyperparathyroidism, whereas the nodular (neoplastic) type of growth is frequently found in patients with severe secondary hyperparathyroidism that is refractory to medical therapy [76]. The monoclonality of parathyroid tumors, which may be attributable to gene polymorphisms, is another mechanism that may modify the degree of parathyroid gland hyperplasia in patients with ESRD. Disturbances in phosphorus, vitamin D, and calcium metabolism are also implicated in the pathogenesis of parathyroid hyperplasia in renal failure. Dusso et al. [77] studied the role of cyclin/cyclin-dependent kinase (Cdk) inhibitor p21 and transforming growth factor-0~ (TGF-~) in mediating the effects of phosphorus on parathyroid cell proliferation [77]. They found that phosphorus restriction may prevent parathyroid hyperplasia in early renal failure by stimulating p21. In contrast, parathyroid hyperplasia induced by phosphorus supplementation is mediated by increasing TGF-ac In a subsequent study, they demonstrated that vitamin D analogs [calcitriol and 19-nor-l,25(OH)zD2] and dietary calcium supplementation controlled PTH secretion and the development of parathyroid gland hyperplasia in uremic animals. These effects were mediated by increasing p21 expression and by inhibiting the expected increase in TGF-cz content [78]. Parathyroids with nodular hyperplasia have more diminished VDR expression than those with diffuse hyperplasia, and VDR density is inversely correlated with parathyroid gland weight [62]. Since vitamin D exerts antiproliferative effects, the reduction in VDR density in parathyroids may contribute to the pathogenesis of parathyroid hyperplasia. Calcium-sensing receptor expression is also reduced in hyperplastic parathyroid tissues obtained from patients with renal secondary hyperparathyroidism [79]. Parathyroid cell proliferation, however, preceded the reduction in CaR expression in experimental uremia [80]. Diminished expression of another putative calcium-sensing protein and its mRNA, CAS (gp330/megalin), has also been reported in parathyroid adenomas [81]. Vitamin D has been shown to modify CaR expression in parathyroid tissue [82]. Indeed, parathyroid CaR mRNA expression was downregulated in vitamin D-deficient animals and treatment of these animals with vitamin D led to a dose-dependent increase in CaR mRNA levels. This finding suggests a possible role of the CaR in the inhibitory effect of vitamin D analogs on PTH [82].
28. Renal Osteodystrophy
Because of the exceedingly low turnover of the parathyroid cells (half-life estimated to be approximately 30 years), regression of parathyroid hyperplasia is difficult. The nonsuppressible component of PTH secretion from an increased number of parathyroid cells can be sufficient to produce hypercalcemia and progressive bone disease in patients with ESRD. Skeletal Resistance to the Calcemic Actions of P T H
Decreased skeletal response to PTH has been described as one of the factors responsible for the development of hypocalcemia and secondary hyperparathyroidism in renal failure. Indeed, decreased calcemic response to PTH stimulation and delayed recovery from EDTA-induced hypocalcemia have been reported in patients with renal failure even when there is substantially increased serum PTH levels [83]. Moreover, bone histomorphometry studies demonstrate that serum PTH levels that are approximately three times the upper limit of normal correspond to normal rates of bone formation and turnover, whereas values less than 150pg/ml or within the normal range correspond to adynamic renal osteodystrophy in patients with ESRD [84,85]. There is limited information, however, regarding the etiology of the skeletal resistance to the actions of PTH in renal failure. Disturbances in vitamin D metabolism are thought to contribute to these changes [86]. In addition, bone biopsies from patients with ESRD showed diminished PTH/PTHrP receptor expression in osteoblasts [87]. Downregulation of PTH/PTHrP receptor mRNA expression was similarly observed in growth plate chondrocytes of rats with severe secondary hyperparathyroidism [8]. These abnormalities may also contribute to tissue resistance to PTH in uremia. Recently, C-terminal PTH (C-PTH) fragments, possibly PTH(7-84), which are retained in renal failure, have been shown to inhibit the calcemic actions of PTH(1-84) and PTH(1-34) in parathyroidectomized animals [88,89]. Indeed, in vitro data demonstrate that human PTH(7-84) inhibit bone resorption through receptors that are different from the PTH/PTHrP receptor and presumably specific for C-PTH fragments [90,91]. A d y n a m i c Renal O s t e o d y s t r o p h y In the past decade, adynamic renal osteodystrophy has become a common skeletal lesion in adult patients with ESRD, although its prevalence has remained unchanged in pediatric patients, affecting approximately 15-20% of those treated with dialysis [84,85,92-95]. Adynamic bone is characterized by reductions in osteoblastic activity and rates of bone formation, and these changes can be due either to direct and specific inhibitory effects
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of systemic factors on osteoblastic function or to indirect changes in osteoblastic activity mediated through PTHdependent mechanisms [96]. In some cases of adynamic bone, osteoblastic function improves over time and the disorder can be considered to be reversible [97]. In other cases, impairments in osteoblastic activity persist, and bone formation and turnover cannot be restored to normal with currently available therapeutic interventions. Disorders associated with long-standing or irreversible reductions in osteoblastic activity and bone formation include age-related or postmenopausal osteoporosis, steroid-induced osteoporosis, idiopathic or surgically induced hypoparathyroidism, and diabetes mellitus [98]. Reversible causes of adynamic renal osteodystrophy include lesions that develop after subtotal parathyroidectomy in patients with ESRD, those due to bone aluminum toxicity, and disorders arising from the use of large doses of calcium-containing medications, vitamin D, and elevated dialysate calcium concentration in patients undergoing peritoneal dialysis [97-101 ]. Adynamic renal osteodystrophy was originally described as a manifestation of bone aluminum toxicity. Aluminum adversely affects the differentiated function of osteoblasts, and it also inhibits osteoblastic proliferation both in vivo and in vitro [102,103]. A portion of the inhibitory action of aluminum on the proliferation of osteoblast-like cells is mediated directly, whereas some of the decrease in osteoblastic activity in vivo may be due to aluminum-induced decreases in PTH secretion. In this regard, aluminum inhibits PTH release from dispersed parathyroid cells in vitro, and serum PTH levels are normal or only minimally elevated in many patients with adynamic renal osteodystrophy due to bone aluminum deposition. Although bone formation may initially be markedly reduced in patients with adynamic renal osteodystrophy due to bone aluminum deposition, osteoblastic activity and bone formation generally increase as aluminum-related bone disease resolves [99].
CLINICAL MANIFESTATIONS Patients with mild to moderate chronic renal failure are generally asymptomatic, although abnormalities in bone histology have been demonstrated. Clinical manifestations are often nonspecific, and biochemical or radiologic findings may not correlate with the severity of the bone disease. The most striking consequences of untreated bone disease in children are severe bone deformities and growth retardation, particularly in those who develop renal failure early in life.
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Proximal myopathy is a recognized feature of renal bone disease. Its onset is insiduous and often presents as muscle pain and weakness. Symptoms may be progressive and disabling, however, and may result in inability to ambulate and, occasionally, confinement to bed or a wheelchair. Serum levels of creatine kinase, aldolase, and transaminases are usually within normal limits, and electromyography is either normal or shows nonspecific changes. Although several factors have been implicated, the presence of severe secondary hyperparathyroidism or aluminum-related bone disease must be considered in the evaluation of these patients. Indeed, improvement in symptoms has been reported during calcitriol or 25 (OH)D3 therapy or following parathyroidectomy in the case of myopathy due to secondary hyperparathyroidism, whereas muscle pain from aluminum-related bone disease improves after aluminum removal with deferoxamine. Response to vitamin D treatment may be attributable, in part, to the presence of vitamin D receptors in skeletal muscle [104]. Bone pain also develops gradually in patients with renal bone disease. Symptoms are more pronounced in those with aluminum-related bone disease or during treatment with recombinant human growth hormone; pain is generally diffuse, nonspecific, and exacerbated by weight bearing and changes in position. Some patients may present with localized pain in the lower extremities, lower back, or hips, whereas others may not specifically complain of bone pain but limit their physical activity. Abnormalities in physical examination are not usually found unless the patient has underlying fractures or slipped epiphysis. Calcific uremic arteriolopathy (calciphylaxis) is a rare but serious condition characterized by painful purple skin lesions that commonly progress dramatically, resulting in extensive ischemic necrosis and ulceration of the skin and underlying tissue [105,106]. Infectious complications and sepsis often lead to death. Calciphylaxis has been described in patients with chronic renal failure, in those treated with dialysis, and following renal transplantation. Nonetheless, it is quite uncommon in pediatric patients. The pathogenesis of the calciphylaxis syndrome remains to be determined. Biopsy specimens demonstrate medial calcification and intimal hypertrophy of subcutaneous arterioles (small vessel disease) [107]. Although calciphylaxis is generally associated with severe secondary hyperparathyroidism and increased calcium-phosphorus ion product, this condition has been reported in patients with biopsy evidence of low-turnover bone disease [108]. As such, parathyroidectomy should only be considered in patients with evidence of secondary hyperparathyroidism.
BIOCHEMICAL DETERMINATIONS Serum calcium concentrations are generally decreased in patients with moderate renal failure. Treatment with calcium-containing phosphate binders, vitamin D sterols, and dialysis raises serum calcium levels. When hypercalcemia occurs, the presence of severe secondary hyperparathyroidism, aluminum-related bone disease, adynamic bone, vitamin D therapy, and the use of large doses of calcium-containing phosphate binders should be considered in the diagnostic workup. Although malignancy and extrarenal sources of vitamin D, such as tuberculosis or sarcoidosis, are uncommon in children, these conditions should be excluded if hypercalcemia persists. Serum phosphorus levels are maintained within normal limits in early renal failure. The normal range for serum phosphorus is age adjusted: Values are quite elevated in the first 3 months of life, ranging from 4.8 to 7.4mg/dl (mean, 6.2mg/dl), but levels decrease to 4.5-5.8 mg/dl (mean, 5.0mg/dl) at 1 or 2 years of age, to 3.5-5.5 mg/dl (mean, 4.4 mg/dl) during childhood, and to adult values by late adolescence [109]. Hyperphosphatemia develops when the glomerular filtration rate (GFR) decreases to approximately 25ml/min unless measures such as dietary phosphorus restriction and use of phosphate-binding agents are employed [43]. Hyperphosphatemia often persists in patients undergoing dialysis because neither peritoneal dialysis nor hemodialysis can completely compensate for the amount of phosphorus absorbed from the diet. Furthermore, vitamin D therapy increases serum phosphorus levels because it enhances intestinal phosphorus absorption. When combined with normal or elevated serum calcium levels, hyperphosphatemia increases the calcium-phosphorus ion product and leads to extraskeletal calcification. Serum total alkaline phosphatase is a biochemical marker of osteoblastic activity that is less helpful in predicting the histologic lesions of renal osteodystrophy, most likely because values include contributions from sources other than bone (liver and intestine). Assays for bone-specific alkaline phosphatase, however, have been shown to be more reliable in this regard, particularly for detecting low-turnover lesions of bone. Low concentrations of bone alkaline phosphatase (_<27U/liter) have been used to exclude high-turnover lesions of bone [110]. Serum PTH, measured by first-generation immunoradiometric assay (IRMA) for PTH, has been widely used as a noninvasive marker for distinguishing patients with low-turnover lesions from those with secondary hyperparathyroidism. In patients with stable mild to moderate chronic renal failure, first-generation PTH levels that are within normal range generally correspond
687
28. Renal Osteodystrophy
to normal rates of bone formation, whereas mildly increased levels suggest the presence of secondary hyperparathyroidism [111,112]. In dialyzed children who are either untreated or receiving small daily oral doses of calcitriol, first-generation PTH levels of approximately three times the upper limit of normal generally correspond to a normal bone formation rate. Levels >250-300 pg/ml are associated with bone biopsy evidence of secondary hyperparathyroidism, whereas values < 150 pg/ml indicate an adynamic bone lesion [85]. When criteria for both firstgeneration PTH and calcium levels are used, we have reported increased specificity and a positive predictive value for identifying patients with either high- or lowturnover lesions. Thus, first-generation PTH >200 pg/ml and serum calcium <10mg/dl excluded all patients with normal or reduced bone formation rates. On the other hand, combined criteria of first-generation PTH < 150 pg/ml and serum calcium level > 10 mg/dl excluded all those with normal or increased bone formation rates [85]. Similar values may not be applicable in dialyzed children receiving intermittent calcitriol therapy since suppression of bone formation may develop despite persistently elevated first-generation PTH levels [113]. This discrepancy may be due to the direct effects of calcitriol on osteoblastic activity. An alternative explanation is that the current IRMA for PTH detects not only the full-length molecule but also large C-terminal fragments, possibly hPTH(7-84), which are retained in renal failure, thus resulting in much higher PTH levels. Indeed, a first-generation PTH assay showed indistinguishable cross-reactivity with hPTH (1-84) and hPTH(7-84), whereas a second-generation PTH assay detected only the full-length peptide [114]. PTH levels measured with this second-generation assay were lower than those measured using first-generation PTH assays in pediatric and adult patients treated with dialysis [ 114-116] (Fig. 4). A PTH(1-84):C-PTH (difference between first- and second-generation PTH) fragment ratio > 1 has been used to predict high or normal bone turnover (sensitivity, 100% ), whereas a ratio <1 has been shown to indicate a high probability (sensitivity, 87.5%) of low bone turnover [116]. These findings, which may have significant implications for the treatment of the different types of renal bone diseases, have not yet been confirmed by other investigations.
[117]. However, there is limited information about the histologic features of bone disease in children and adults with stable renal failure. Previous studies in children with renal disease involved small numbers of study subjects and most did not use double tetracycline labeling of bone. Abnormal bone histology was demonstrated in almost all subjects with moderate chronic renal failure and it was associated with elevated serum levels of Cterminal PTH [118-122]. In children with GFRs <50ml/ min/1.73m 2, Norman et al. [118] and Eke et al. [119] found mixed lesions of renal osteodystrophy (both osteomalacia and hyperparathyroidism) in approximately half of the study subjects, whereas secondary hyperparathyroidism alone was seen in 25-30% of patients. The remaining one-third of patients in the study by Norman et al. had osteomalacia alone despite normal levels of 25hydroxyvitamin D. The predominance of mixed lesions was also reported by Coen et al. [111] and Bianchi et al. [123] in adults with chronic renal failure. However, in another series of 176 adults with GFRs between 15 and 50 ml/min, osteitis fibrosa was the most common histologic lesion observed, followed by normal histology in 25% of subjects [112]. On the other hand, Hodson et al. [124] reported normal bone histology in 21 of 24 children with a G F R >20ml/min/1.73m 2. Similarly, preliminary data in 19 pediatric patients with moderate chronic renal failure (mean calculated GFR, 36ml/min/1.73m 2) from our institution showed that the majority of patients had normal rates of bone formation. Interestingly, the mean serum intact PTH level in patients with normal bone formation rates was within the normal range for this assay, whereas patients with secondary hyperparathyroidism had a mean intact PTH level three times the upper limit of normal [125]. These results are consistent with those reported in adult patients with chronic renal failure, where histologic lesions of osteitis fibrosa were found despite only mildly increased serum intact PTH levels [111,112]. Thus, mild elevations in serum intact PTH levels that are usually associated with adynamic lesions in patients with ESRD treated with maintenance dialysis generally correspond to high-turnover lesions of bone in those with mild to moderate chronic renal failure. These data suggest that there is less skeletal resistance in chronic renal failure than in ESRD. ESRD Treated with Dialysis
HISTOLOGIC MANIFESTATIONS Stable Chronic Renal Failure Abnormalities in bone histology have been described in patients with mild to moderate chronic renal failure
The features of renal osteodystrophy have been more extensively investigated in children and adults with ESRD undergoing regular dialysis. Studies in our institution demonstrate that the prevalence of the different bone lesions has not changed substantially in the past 10 years and histologic lesions of secondary
mi-
--j
FIGURE 4 Serum PTH levels during calcium infusion as measured by a second-generation IRMA (A) were lower than those measured by a firstgeneration IRMA (). in three patients with osteitis fibrosa (A-C) and in two patients with adynamic bone (D, E). (F) Serum PTH levels during calcium infusion in six patients with osteitis fibrosa (reproduced with permission from John et al. [I 141).
28. Renal Osteodystrophy
hyperparathyroidism remain the most common type of lesion of renal osteodystrophy in children treated with either peritoneal dialysis or hemodialysis [84,85,95]. Of the 156 bone biopsies performed at UCLA from 1983 to 1992, osteitis fibrosa was found in 46% of subjects, whereas mild lesion of secondary hyperparathyroidism was present in 17% during treatment with daily doses of oral calcitriol [85]. Serum intact PTH levels were approximately 10 times the upper limit of normal in subjects with osteitis fibrosa, whereas values were less markedly elevated in those with mild lesions of secondary hyperparathyroidism. In contrast, the prevalence of low-turnover bone disease has increased substantially in adult dialysis patients in the past decade. Adynamic renal osteodystrophy accounts for the majority of low-turnover lesions in this population, whereas osteomalacia is seen infrequently [3,92,94,97]. Indeed, half of 259 biopsy specimens from adult patients undergoing regular dialysis showed evidence of adynamic lesions of bone associated with normal or mildly increased serum PTH levels. A greater proportion of patients receiving peritoneal dialysis had lowturnover bone disease than those treated with hemodialysis [94]. In addition, a substantial proportion of biopsies obtained from adult patients immediately prior to dialysis initiation had histologic features of adynamic bone [3,92]. Although the prevalence of adynamic lesions of renal osteodystrophy has remained less than 20% in pediatric patients with ESRD during daily calcitriol therapy, adynamic bone developed in 33% of subjects after they received large intermittent doses of calcitriol for treatment of secondary hyperparathyroidism [113]. Currently, most patients with adynamic renal osteodystrophy do not have evidence of aluminum bone deposition [3,97]. Water-purification methods and avoidance of aluminum-containing phosphate-binding agents have markedly reduced the prevalence of aluminum-related bone disease in patients with ESRD. Other factors, such as large doses of vitamin D analogs, calcium supplementation either from dialysis solutions or from calcium-containing phosphate binders, diabetes, increasing age, and corticosteroid therapy, are thought to contribute to the pathogenesis of adynamic lesions of bone [94]. In children, however, calcium and vitamin D are the most likely factors involved in the development of adynamic bone.
RADIOGRAPHIC FEATURES OF RENAL OSTEODYSTROPHY Subperiosteal erosion is the most common radiographic finding in secondary hyperparathyroidism and
689
it correlates with serum PTH levels and histologic findings of osteitis fibrosa [126]. Subperiosteal erosions can occur at any site, but they are typically found along the radial margins of the middle phalanges of the second and third digits in adults. In contrast, phalangeal involvement may not be present in young children but rather involve the lateral aspect of the distal radius and ulna and the medial side of the proximal tibia [127]. Osteosclerosis is another feature of secondary hyperparathyroidism that results in the "salt-and-pepper" appearance of the skull, "rugger-jersey" spine, and increased density of the pelvis and metaphyses of long bones [127]. Other findings include brown tumors, which are intraosseous soft-tissue masses that appear as welldefined lytic lesions in the metaphyses of long bones, jaws, ribs, and ilium. In severe secondary hyperparathyroidism, the growth plate cartilage may appear widened and poorly mineralized; such findings are similar to those associated with vitamin D-deficiency rickets [127]. Slipped epiphyses and bone deformities, such as genu valgum, genu varum, and enlargement of the wrists, ankles, and medial ends of clavicles, are long-term skeletal consequences of advanced ostetis fibrosa. In addition, periosteal neostosis (new bone formation), intracortical resorption, erosion of the terminal phalanges (acroosteolysis), and the absence of the lamina dura on dental films are also evident in secondary hyperparathyroidism. Radiographic findings of osteomalacia are less specific than those of secondary hyerparathyroidism. Pseudofractures or Looser's zones may be the only distinguishing features of osteomalacia. They appear as wide radiolucent bands that are perpendicular to the long axis of the bone, and they are most commonly found in the medial femoral neck, pubic rami, lateral border of the scapula, and ribs. Other noninvasive imaging techniques such as positron emission tomography (PET) may be potential modalities for detecting bone cellular activity. PET studies of bone using [18F]fluoride ion can differentiate lowturnover from high-turnover lesions of renal osteodystrophy and provide quantitative estimates of bone cell activity that correlate with bone histomorphometry in patients treated with maintenance dialysis [128]. Peripheral quantitative computed tomography (pQCT) is another tool that has recently been shown to selectively measure the densities of cortical and trabecular bone in the appendicular skeleton. However, there is little information on to the usefulness of pQCT for the measurement of bone density in healthy children or in those with metabolic bone disease. We utilized pQCT to measure the densities of cancellous and cortical bone in the distal radius of children with renal osteodystrophy undergoing peritoneal dialysis [129]. Trabecular bone
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Beatriz D. Kuizon and Isidro B. Salusky
density was higher in dialysis patients than in controls, whereas cortical bone density was lower. The latter was inversely related to serum levels of alkaline phosphatase and PTH. These findings suggest that secondary hyperparathyroidism adversely affects cortical bone density but increases trabecular bone [129]. This technique could potentially be used as an additional tool in the diagnosis and treatment of the different subtypes of renal osteodystrophy.
LONG-TERM CONSEQUENCES Skeletal deformities are potentially debilitating manifestations of bone disease in pediatric patients. The skeletal changes of secondary hyperparathyroidism affect the metaphyses and manifest as genu valgum, ulnar deviation of the hands, pes varus, pseudoclubbing, and enlargement of the wrists, ankles, and medial ends of clavicles. Characteristic features in early childhood are similar to those that develop due to advanced vitamin Ddeficiency rickets, such as rachitic rosary and Harrison's grooves [127]. Dental abnormalities (teeth malformations and enamel defects) are also common. In addition, craniotabes and frontal bossing of the skull may be observed in patients who develop renal failure in infancy. Overall, approximately 25-30% of children aged 5-15 years who are treated with maintenance dialysis require surgical correction of severe bone deformities. Slipped epiphysis is another disabling complication of severe secondary hyperparathyroidism in young renal failure patients, although it also develops in primary hyperparathyroidism. Postmortem analysis of the growth plate specimens from children with ESRD showed severe abnormalities of the growth plate cartilage, which predispose to slipping of the epiphyseal growth plate cartilage from the metaphyseal bone and to metaphyseal fractures, even in the absence of trauma [11]. The most common affected sites in younger children are the epiphyses in the proximal and distal femur and distal tibia, whereas the upper femoral and distal forearm epiphyses are more frequently involved in older children [127]. Bone deformities, such as ulnar deviation of the hands, and gait abnormalities may result from epiphyseal slipping of the distal forearm and proximal femur, respectively. Extraskeletal calcification is another complication of renal osteodystrophy that is well described in adults treated with maintenance dialysis [130,131]. Less appreciated is its occurrence in a substantial proportion of pediatric patients with renal failure. However, Milliner et al. [132] found postmortem evidence of soft tissue and vascular calcification in 72 of 120 (60%) children with
ESRD, 43 (36%) of whom had systemic calcinosis (calcification of two or more sites). The most common sites of calcification were blood vessels, lungs, kidneys, heart and coronary arteries, central nervous system, and stomach. Cardiopulmonary calcification contributed to death in some cases. Several factors, including vitamin D therapy, peak calcium-phosphorus ion product, age at onset of renal failure, and male sex, were associated with calcinosis, but treatment with vitamin D sterols, particularly calcitriol, showed the strongest correlation with calcification [132]. These data are consistent with those reported in adult ESRD patients showing a strong association between calcification and disturbances in mineral metabolism. Vascular calcification is prevalent in both adults and children with renal failure, and it is plausible that vascular calcification may contribute to the markedly high mortality rate from cardiovascular disease in ESRD [132,133]. Indeed, recent data show that patients with renal failure have heavily calcified plaques, whereas most subjects with normal renal function have fibroatheromatous plaques [134]. Calcium deposition is a prominent feature of arteriosclerotic lesions, but the process of arterial calcification is not well understood. Recent data indicate, however, that factors normally involved in skeletal metabolism may participate in the localized accumulation of calcium in the arterial wall. Bone morphogenetic protein-2, one of a family of proteins integrally involved in skeletal development, and the bone matrix proteins osteocalcin and osteopontin have each been identified within atherosclerotic lesions in human pathological specimens [135-137]. PTHrP and the PTH/PTHrP receptor are also expressed in arteriosclerotic plaques [138]. Thus, several factors that normally regulate bone and mineral metabolism may be important modifiers of calcium deposition within arteriosclerotic lesions. Several reports indicate that coronary artery calcification detected by electron-beam computed tomography (EBCT) corresponds to the extent and severity of angiographically documented lesions. As such, EBCT may be a useful noninvasive method to screen patients at risk for cardiovascular disease. Using this technique, Braun et al. [139] found higher calcification scores in adult hemodialysis patients aged 28-74 years than in subjects of the same age with normal renal function. Moreover, calcification scores increased after 1 year in patients treated with maintenance dialysis. Whether similar changes occur in younger patients with ESRD is not known. Therefore, we studied the presence and progression of coronary artery calcification by EBCT in 39 dialysis patients aged 7-30 years. None of the 23 patients younger than 20 years old had coronary calcification, whereas 14 of 16 patients between the ages of 20 and 30 years had positive EBCT scans (Fig. 5)--all
28.
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these patients initiated treatment with dialysis as teenagers [140]. The negative EBCT scores in pediatric patients may represent a technical limitation of the application of EBCT or the effect of shorter duration of treatment with maintenance dialysis. Similar findings were described by Eifinger e t al. [141] in patients with ESRD who had been undergoing therapy with dialysis since childhood. In contrast, coronary calcification was detected in only 3 of 60 volunteers aged 20-30 years with no known history of cardiovascular or renal disease. Patients with positive scans were older, had a longer duration of dialysis treatment, higher calcium-phosphorus ion product, and an approximately twofold daily intake of calcium-containing phosphate-binding agents. Calcification scores doubled within a follow-up interval of less than 2 years (Fig. 5) [140]. Serum concentrations of phosphorus and the calcium-phosphorus ion product correlated with the change in calcification scores at follow-up. Thus, it is possible that abnormalities in mineral metabolism play a role in the development of vascular calcifications in patients with ESRD and they may contribute to decreased survival in ESRD [140].
TREATMENT The management of children with renal osteodystrophy is aimed at achieving normal rates of bone formation and turnover, maintaining serum PTH levels that correspond to normal rates of skeletal remodeling, and preventing extraskeletal calcifications. Early diagnosis and appropriate treatment of renal bone disease are essential to prevent the debilitating consequences of this disorder for the growing skeleton.
Dietary Modifications Dietary phosphorus restriction is often necessary to prevent the development and progression of secondary hyperparathyroidism and extraskeletal calcifications in patients with advanced renal failure. In addition, hyperphosphatemia and an elevated calcium phosphorus product have also been reported as independent risk factors for mortality in adult dialysis patients [142]. Treatment goals include maintaining serum phosphorus
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Beatriz D. Kuizon and lsidro B. Salusky
levels within normal limits for age and avoiding calcium phosphorus products higher than 50-60 mg/dl. The average phosphorus intake of children in the United States is approximately 1500-2000mg/day, and 60-70% of the dietary intake is absorbed. Patients who develop hyperphosphatemia are usually instructed to reduce phosphate intake to approximately 800mg/day. Patients treated with dialysis also require dietary phosphorus restriction since the quantity removed by either peritoneal dialysis (approximately 300-400mg/day) or hemodialysis (800rag/treatment) is often insufficient to maintain normal serum phosphorus levels. Strict adherence to such a regimen is often difficult, however, because adequate protein and nutritional intake are necessary for growth, and low-phosphate diets are unpalatable, especially to children. Thus, the additional use of phosphatebinding agents is required to maintain age-appropriate levels in most patients. It is essential to monitor serum phosphorus levels regularly to prevent hypophosphatemia, which may result from aggressive dietary restriction and the use of large doses of phosphate-binding agents. Infants are particularly at risk for hypophosphatemia due to low phosphorus intake, large doses of phosphate binders, higher phosphate removal by peritoneal dialysis probably due to larger surface area, and possibly nutritional repletion [143]. Bone diseases such as osteomalacia and rickets, proximal myopathy, rhabdomyolysis, and congestive heart failure have been reported in patients with severe and persistent hypophosphatemia [143,144]. P h o s p h a t e - B i n d i n g Agents Phosphate-binding agents are widely utilized in the management of hyperphosphatemia in children and adults with renal failure. They reduce intestinal phosphate absorption by forming poorly soluble complexes with phosphorus in the intestinal tract. Aluminumcontaining phosphate binders were frequently used in the past, but long-term treatment led to bone disease, encephalopathy, and anemia. The use of aluminumcontaining phosphate binders should therefore be restricted to hyperphosphatemia associated with hypercalcemia or an elevated calcium phosphorus product since both conditions will be aggravated by calcium-containing compounds. In such cases, the dose of aluminum hydroxide should not exceed 30 mg/kg/day and the lowest possible dose should be taken only for a limited period. Plasma aluminum levels should be monitored regularly. Concomitant intake of citrate-containing compounds should be avoided because they increase intestinal aluminum absorption [145]. Constipation is a common side effect and can be relieved by stool softeners. Calcium-containing phosphate binders are currently prescribed worldwide for the control of hyperphospha-
temia. They also serve as a source of supplemental calcium. Several calcium salts are commercially available, including calcium carbonate, calcium acetate, and calcium citrate. Calcium carbonate is the most often used compound, and studies in adults and children have shown its efficacy in controlling serum phosphorus levels [146,147]. Moreover, administration of calcium carbonate alone lowers serum PTH levels in adult patients with secondary hyperparathyroidism [148-150]. Large doses of calcium carbonate are often required, however, and this may lead to hypercalcemia, particularly in patients treated with vitamin D or those with adynamic bone lesions. In adult patients, studies comparing calcium carbonate and calcium acetate demonstrated that the latter bound twice the amount of phosphorus using equivalent doses, but the incidence of hypercalcemia is inconsistent among studies [151,152]. One pediatric study found no difference in the episodes of hypercalcemia between these two compounds, although the study included only a small number of patients [153]. Although the dose of calcium-containing phosphate binders should be proportional to the phosphorus content of the meal and adjusted to achieve acceptable levels of serum calcium and phosphorus, it is recommended that the amount of elemental calcium should not exceed twice the recommended daily allowance. Phosphate binders that do not contain calcium may be needed to control serum phosphorus levels. Doses should be taken with meals if the calcium salt is used as a phosphate binder and taken between meals if it is given for hypocalcemia. Hypercalcemia is usually reversible with reductions in the dose of oral calcium salts and dialysate calcium concentrations. Using calcium acetate may lessen the oral calcium load because it has a lower calcium content than calcium carbonate (25 vs 40%), but hypercalcemia may still develop. Decreasing the dose of vitamin D and administration of the sterol at bedtime may also minimize hypercalcemia in patients ingesting calcium-containing binders. Other patients may require a combined treatment with calcium salts and aluminum hydroxide to control hyperphosphatemia. Calcium citrate is also an effective phosphate-binding agent; however, it should be used with caution in patients with renal failure because it enhances intestinal aluminum absorption and therefore increases the risk of aluminum toxicity. Calcium ketoglutarate is another phosphate binder that is less calcemic and has additional anabolic effects, but gastrointestinal side effects and the high cost of therapy may limit its use [154,155]. Because of the risks of hypercalcemia associated with the use of calcium salts and toxicity from ingestion of aluminum hydroxide, alternative phosphate binders have been developed and investigated. Sevelamer hydrochloride (Renagel), a calcium- and aluminum-free
28. Renal Osteodystrophy
hydrogel of cross-linked poly(allylamine hydrochloride), has been reported to lower serum phosphorus, calciumphosphorus ion product, and PTH without inducing hypercalcemia in adult patients treated with hemodialysis [156-158]. Thus, sevelamer is a particularly appealing agent since both aluminum toxicity and exogenous calcium loading with its associated complications, such as extraskeletal calcification, may be avoided. In addition, concentrations of serum total cholesterol and lowdensity lipoprotein cholesterol declined whereas highdensity lipoprotein increased during sevelamer treatment [158], and these effects may offer additional benefits in reducing cardiovascular complications in patients with ESRD. Sevelamer may be used as the primary agent, particularly in patients who are prone to develop hypercalcemia and in those requiring large doses of vitamin D, but further studies are warranted to evaluate the longterm effects of this therapy on bone disease in children with renal failure. Other alternative phosphate-binding agents include magnesium, iron, and lanthanum compounds. Magnesium carbonate lowers serum phosphorus levels, but magnesium-free dialysate solutions should be used in patients treated with dialysis to prevent hypermagnesemia [159]. However, large doses result in diarrhea, thereby limiting the use of this compound as a single agent. Iron compounds, such as stabilized polynuclear iron hydroxide and ferric polymaltose complex, are novel phosphate binders that are effective in short-term studies in adults with chronic renal failure [160,161]. Another novel agent, lanthanum chloride hydrate, decreases intestinal phosphate absorption in experimental studies and a clinical trial is currently under way to assess its efficacy and safety [162]. Vitamin D Therapy Calcitriol deficiency has been identified as a major factor in the pathogenesis of secondary hyperparathyroidism, and this provides the basis for the administration of vitamin D sterols to most patients with ESRD. In addition, a potential role of calcitriol in the prevention of growth retardation in pediatric patients has been reported [163]. Several vitamin D derivatives are available but calcitriol, the most active metabolite of vitamin D, is most often used in the United States, whereas 1~hydroxyvitamin D is widely prescribed in Europe and Japan. Both compounds have been shown to suppress PTH secretion and reverse the biochemical, radiographic, and histologic changes associated with highturnover bone disease. Dosage regimens have generally ranged from 0.25 to 1.0~tg/day. Hypercalcemia is the most common side effect at doses >0.5 ~tg/day, which limits the dose that can be given safely. However, hyper-
693
calcemia is usually reversible after a reduction in dose or temporary cessation of the drug. Several studies have documented the efficacy of intermittent calcitriol therapy for the management of secondary hyperparathyroidism both in children and in adults with advanced renal failure [164-167]. The use of intermittent dosing was based on reductions in parathyroid pre/pro-PTH mRNA in experimental animals and in serum PTH levels for up to 96 hr in patients with chronic renal failure after a single administration of calcitriol [52, 168,169]. Despite the greater bioavailability of intravenous calcitriol, prospective studies showed that intermittent oral calcitriol was equally effective as intermittent intravenous calcitriol in reducing serum PTH levels [165,166,170]. Earlier studies found that intermittent calcitriol therapy was better than daily oral calcitriol for decreasing PTH levels; however, most of these studies were uncontrolled, had a small number of patients, compared the effects of different weekly doses of calcitriol, or did not stratify patients based on the PTH levels at the start of the study [164,171,172]. Recent studies comparing the same weekly dose of calcitriol given daily or intermittent did not show greater efficacy of intermittent dosing in the treatment of secondary hyperparathyroidism in children with chronic renal failure and in adult patients with ESRD [173,174]. Moreover, high-dose intermittent calcitriol therapy resulted in marked decline in bone formation rates and development of the adynamic lesion in a substantial proportion of children undergoing peritoneal dialysis [2,113]. Corresponding reductions in serum PTH levels were observed only in patients who received intraperitoneal doses of calcitriol, whereas serum PTH levels remained persistently elevated in those given intermittent oral doses of calcitriol despite significant reductions in bone formation rates on repeat biopsy [113]. These results suggest that calcitriol may directly inhibit osteoblastic activity, independent of PTH, thereby contributing to the development of adynamic lesions. Thus, patients should be closely monitored during intermittent calcitriol therapy to prevent oversuppression of PTH and bone formation. Moreover, calcitriol should be avoided in patients with adynamic lesions. The increasing prevalence of adynamic lesions has raised considerable concern about the long-term consequences of this bone disease on the growing skeleton. Adynamic bone lesions have been associated with more frequent episodes of hypercalcemia, increased fracture risk, and earlier mortality in adult dialysis patients [175-177]. Moreover, impaired linear growth has been reported during intermittent calcitriol therapy in prepubertal children with secondary hyperparathyroidism [23]. z scores for height decreased during 12 months of
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Beatriz D. Kuizon and Isidro B. Salusky
intermittent calcitriol therapy, and the largest decline was observed in those who developed adynamic bone disease. Delta z scores for height correlated with serum PTH and alkaline phosphatase levels during intermittent calcitriol therapy, whereas such relationships were not evident during daily calcitriol therapy [23]. These results suggest that large intermittent doses of calcitriol adversely affect epiphyseal growth plate chondrocyte activity, thereby contributing to linear growth reduction. Based on current data, we recommend initiating vitamin D therapy when serum intact PTH levels are higher than the upper limit of normal (>65 pg/ml) in children with mild to moderate chronic renal failure. The starting dose of calcitriol or alfacalcidol is 0.25-0.5 ~tg/day. These doses are usually well tolerated and are infrequently associated with hypercalcemia, hyperphosphatemia, or accelerated progression of renal failure. Although early reports have shown a more rapid decline in renal function during vitamin D therapy, a substantial number of subsequent studies have reported no adverse effects [112,178-181]. If hypercalcemia or hyperphosphatemia develop, treatment may be withheld temporarily and restarted at a lower dose when values have normalized. Although limited data are available to determine the optimal serum PTH levels for children who have not yet developed ESRD, it may be advisable to maintain PTH levels within the normal range. Oversuppression of PTH levels should be avoided in view of the recent observation that more severe growth retardation develops in children with adynamic bone disease [23]. In contrast, we recommend starting calcitriol therapy in pediatric patients receiving maintenance dialysis when serum PTH levels exceed 300-400 pg/ml. Calcitriol or alfacalcidol may be started at a daily dose of 0.25-0.5 gg and the dose gradually increased in 0.25-to 0.5-gg increments to achieve serum calcium levels between 10.0 and 10.5 mg/dl. Intermittent doses may be considered when serum PTH levels are higher than 500-600pg/ml. The initial dose is 0.5-1 gg three times per week administered by the oral or intravenous route. The intraperitoneal route is currently not approved by the Food and Drug Administration. Decreasing the dialysate calcium concentration in patients receiving dialysis may allow higher doses of calcitriol to be given. Longterm treatment with high pulse doses of calcitriol should be undertaken with caution in prepubertal children with secondary hyperparathyroidism. Suppressing PTH levels below 200 pg/ml should be avoided. Additional studies are needed to define the optimal PTH levels during calcitriol treatment that support normal rates of bone formation and linear growth in pediatric patients with renal osteodystrophy. New vitamin D compounds that retain the suppressive effects on PTH but induce less hypercalcemia and
hyperphosphatemia are being evaluated. However, studies that compare the biochemical and histologic responses of these new compounds with standard therapy (calcitriol or alfacalcidol) have not been performed. 22Oxacalcitriol (OCT) inhibits PTH mRNA expression in vitro and in vivo, and it prevents reduction of vitamin D receptor in the parathyroid glands of rats with renal failure [182]. Short-term treatment with OCT lowered serum PTH levels and bone formation rates without inducing hypercalcemia in experimental animals with either normal or reduced renal function [183]. Longterm administration, however, resulted in hypercalcemia and hyperphosphatemia in uremic dogs [184]. Moreover, although OCT diminished abnormal woven osteoid, lamellar osteoid, and fibrosis, it did not decrease bone formation rates on repeat biopsy [184]. In early clinical trials, OCT controlled secondary hyperparathyroidism, but hypercalcemia prevented further increases in dose in half of the subjects [185,186]. Although controlled studies are needed, preliminary findings involving a small number of patients do not support an advantage of OCT over calcitriol. 1Qt(OH)-vitamin D2 (1 ~D2; doxercalciferol) is a vitamin D analog equipotent to 10~D3 in intestinal calcium absorption and bone calcium mobilization in vitamin Ddeficient rats, but it requires larger doses than 10~D3 to induce hypercalcemia and toxicity in normal rodents [187,188]. Initial clinical-trials demonstrated suppression of PTH levels during treatment with daily or intermittent oral doses of 10r (starting dose, 4 ~g/day or 4 ~tg three times per week) in 24 adult hemodialysis patients with moderate to severe secondary hyperparathyroidism. Serum calcium levels increased moderately, from 8.8 _+ 0.18 to 9.5 _ 0.21mg/dl ( p < 0.001), and treatment had to be stopped only once due to hypercalcemia [189]. In a subsequent multicenter trial involving 80 adults undergoing hemodialysis, treatment with larger intermittent oral doses of l czD2 (10 ~tg three times per week) effectively decreased serum PTH levels with minimal hypercalcemia and hyperphosphatemia [190]. Although these results are encouraging, studies that compare this compound to either calcitriol or 1~D3 are needed. Doxercalciferol has recently been approved for treatment of secondary hyperparathyroidism in the United States. 19-nor- 1ct,25(OH)ED2 (paricalcitol; Zemplar) was initially reported to reduce PTH secretion without changing the concentrations of plasma ionized calcium or plasma phosphorus in rats with renal failure [191]. In addition, parathyroidectomized rats fed either a low-calcium or a low-phosphate diet had fewer increases in plasma calcium or phosphorus levels during treatment with paricalcitol than with similar doses of calcitriol [192]. Results of double-blind, placebo-controlled, randomized clinical
28. Renal Osteodystrophy
trials demonstrated the efficacy of paricalcitol in suppressing PTH, and this was associated with increases in final mean serum calcium levels compared to baseline values, although levels were still within the normal range [193,194]. Similarly, comparison studies against standard treatment regimens and trials using this agent in pediatric patients have yet to be done.
3.
4.
Calcimimetic Compounds Calcimimetic agents are allosteric activators of the CaR in the membrane of parathyroid cells. Short-term administration of NPS R-568 resulted in dose-dependent reductions in both PTH secretion and parathyroid gland proliferation in nephrectomized rats with mild secondary hyperparathyroidism [195]. Such findings were confirmed by a study employing an 8-week treatment with daily oral doses or sustained infusion of NPS R-568 to rats with severe secondary hyperparathyroidism [196]. In preliminary clinical trials of NPS R-568, suppression of PTH was demonstrated in patients with primary hyperparathyroidism after single oral dosing and in adult dialysis patients with mild secondary hyperparathyroidism who received two daily doses of the compound [197]. Ionized calcium levels decreased during treatment, particularly in patients given higher doses, but mean levels were higher than 1.1 mmol/liter and none of the patients developed signs or symptoms of hypocalcemia. In a subsequent study, a 2-week treatment using R-568 lowered PTH levels in adult hemodialysis patients with secondary hyperparathyroidism [198]. Thus, calcimimetic agents may offer a novel approach for the treatment of secondary hyperparathyroidism in patients with renal failure. CaR agonists may be beneficial for those who are refractory to calcitriol or in whom calcitriol treatment is limited by hypercalcemia and/or hyperphosphatemia. Long-term studies are needed to assess its efficacy and safety in inhibiting PTH release in children with ESRD, and since the CaR is expressed in growth plate chondrocytes, calcimimetic drugs may affect bone growth and elongation.
5.
6.
7.
8.
9.
10.
11.
12. 13.
14.
Acknowledgments
15.
This work was supported in part by Grants DK-35423 and RR00865 and the Casey Lee Ball Foundation.
16.
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28. Renal Osteodystrophy of calcitriol in dialysis patients: A randomized prospective trial. Nephron 67, 48-53. 175. Atsumi, K., Kushida, K., Yamazaki, K., Shimizu, S., Ohmura, A., and Inoue, T. (1999). Risk factors for vertebral fractures in renal osteodystrophy. Am. J. Kidney Dis. 33, 287-293. 176. Coco, M., and Rush, H. (2000). Increased incidence of hip fractures in dialysis patients with low serum parathyroid hormone. Am. J. Kidney Dis. 36, 1115-1121. 177. Avram, M. M., Sreedhara, R., Avram, D. K., Muchnick, R. A., and Fein, P. (2000). Enrollment parathyroid hormone level is a new marker of survival in hemodialysis and peritoneal dialysis therapy for uremia. Am. J. Kidney Dis. 28, 924-930. 178. Enomoto-Iwamoto, M., Iwamoto, M., Mukudai, Y., Kawakami, Y., Nohno, T., Higuchi, Y., Takemoto, S., Ohuchi, H., Noji, S., and Kurisu, K. (1998). Bone morphogenetic protein signaling is required for maintenance of differentiated phenotype, control of proliferation, and hypertrophy in chondrocytes. J. Cell Biol. 140, 409-418. 179. Eke, F. U., and Winterborn, M. H. (1984). Effect of low dose lahydroxycholecalciferol on glomerular filtration rate in moderate renal failure. Arch. Dis. Child. 58, 810-813. 180. Christiansen, C., Rodbro, P., Christensen, M. S., and Hartnack, B. (1981). Is 1,25-dihydroxy-cholecalciferol harmful to renal function in patients with chronic renal failure? Clin. Endocrinol. (Oxford) 15,229-236. 181. Tougaard, L., Sorensen, E., Brochner-Mortensen, J., Christensen, M. S., Rodbro, P., and Sorenson, A. W. S. (1976). Controlled trial of l a-hydroxycholecalciferol in chronic renal failure. Lancet 1, 1044-1047. 182. Denda, M., Finch, J., Brown, A. J., Nishii, Y., Kubodera, N., and Slatopolsky, E. (1996). 1,25-Dihydroxyvitamin D3 and 22-oxacalcitriol prevent the decrease in vitamin D receptor content in the parathyroid glands of uremic rats. Kidney Int. 50, 34-39. 183. Hirata, M., Katsumata, K., Masaki, T., Koike, N., Endo, K., Tsunemi, K., Ohkawa, H., Kurokawa, K., and Fukagawa, M. (1999). 22-Oxacalcitriol ameliorates high-turnover bone and marked osteitis fibrosa in rats with slowly progressive nephritis. Kidney Int. 56, 2040-2047. 184. Monier-Faugere, M. C., Geng, Z., Friedler, R. M., Qi, Q., Kubodera, N., Slatopolsky, E., and Malluche, H. H. (1999). 22-Oxacalcitriol suppresses secondary hyperparathyroidism without inducing low bone turnover in dogs with renal failure. Kidney Int. 55, 821-832. 185. Kurokawa, K., Akizawa, T., Suzuki, M., Akiba, T., Ogata, E., and Slatopolsky, E. (1996). Effect of 22-oxacalcitriol on hyperparathyroidism of dialysis patients: Results of preliminary study. Nephrol. Dial Transplant. 11, 121-124. 186. Tsukamoto, Y., Hanaoka, M., Matsuo, T., Saruta, T., Nomura, M., and Takahashi, Y. (2000). Effect of 22-oxacalcitriol on bone histology of hemodialyzed patients with severe secondary hyperparathyroidism. Am. J. Kidney Dis. 35, 458-464.
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187. Sjoden, G., Smith, C., Lindgren, U., and DeLuca, H. F. (1985). 1 Alpha-hydroxyvitamin D2 is less toxic than 1 alpha-hydroxyvitamin D3 in the rat. Proc. Soc. Exp. Biol. Med. 178, 432-436. 188. Sjoden, G. (1985). Effects of vitamin D. A comparison of 1 alpha OHD2 and 1 alpha OHD3 in rats. Acta Orthop. Scand. Suppl. 217, 1-84. 189. Tan, A. U., Jr., Levine, B. S., Mazess, R. B., Kyllo, D. M., Bishop, C. W., Knutson, J. C., Kleinman, K. S., and Coburn, J. W. (1997). Effective suppression of parathyroid hormone by 1 alphahydroxy-vitamin D2 in hemodialysis patients with moderate to severe secondary hyperparathyroidism. Kidney Int. 51, 317-323. 190. Frazao, J., Chesney, R. W., and Coburn, J. W. (1998). Intermittent oral lalpha-hydroxyvitamin D2 is effective and safe for the suppression of secondary hyperparathyroidism in haemodialysis patients, l alphaD2 Study Group. Nephrol. Dial. Transplant. 13 (Suppl. 3), 68-72. 191. Slatopolsky, E., Finch, J., Ritter, C., Denda, M., Morrissey, J., Brown, A., and DeLuca, H. (1995). A new analog of calcitriol, 19nor-l,25-(OH)2D2, suppresses parathyroid hormone secretion in uremic rats in the absence of hypercalcemia. Am. J. Kidney Dis. 26, 852-860. 192. Finch, J. L., Brown, A. J., and Slatopolsky, E. (1999). Differential effects of 1,25-dihydroxy-vitamin D3 and 19-nor- 1,25-dihydroxyvitamin D2 on calcium and phosphorus resorption in bone. J. Am. Soc. Nephrol. 10, 980-985. 193. Martin, K. J., Gonzalez, E. A., Gellens, M., Hamm, L. L., Abboud, H., and Lindberg, J. (1998). 19-Nor-l-a-25-dihydroxyvitamin D2 (paricalcitol) safely and effectively reduces the levels of intact parathyroid hormone in patients on hemodialysis. J. Am. Soc. Nephrol. 9, 1427-1432. 194. Llach, F., Keshav, G., Goldblat, M. V., Lindberg, J. S., Sadler, R., Delmez, J., Arruda, J., Lau, A., and Slatopolsky, E. (1998). Suppression of parathyroid hormone secretion in hemodialysis patients by a novel vitamin D analogue: 19-nor-l,25-dihydroxyvitamin D2. Am. J. Kidney Dis. 32, $48-$54. 195. Wada, M., Furuya, Y., Sakiyama, J., Kobayashi, N., Miyata, S., Ishii, H., and Nagano, N. (1997). The calcimimetic compound NPS R-568 suppresses parathyroid cell proliferation in rats with renal insufficiency. Control of parathyroid cell growth via a calcium receptor. J. Clin. Invest. 100, 2977-2983. 196. Wada, M., Nagano, N., Furuya, Y., Chin, J., Nemeth, E. F., and Fox, J. (2000). Calcimimetic NPS R-568 prevents parathyroid hyperplasia in rats with severe secondary hyperparathyroidism. Kidney Int. 57, 50-58. 197. Antonsen, J. E., Sherrard, D. J., and Andress, D. L. (1998). A calcimimetic agent acutely suppresses parathyroid hormone levels in patients with chronic renal failure. Rapid communication. Kidney Int. 53, 223-227. 198. Goodman, W. G., Frazao, J. M., Goodkin, D. A., Turner, S. A., Liu, W., and Coburn, J. W. (2000). A calcimimetic agent lowers plasma parathyroid hormone levels in patients with secondary hyperparathyroidism. Kidney Int. 58, 436-445.
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I29[ Bone Tumors in Children MARC H. ISLER* and ROBERT E. TURCOTTE*'t *University of Montreal Hopital Maisonneuve Rosemont and Hopital Sainte Justine, Montreal Quebec, Canada t McGill University, Montreal Quebec, Canada
INTRODUCTION
at a variety of clinicians, it seems necessary to highlight this point. Primary musculoskeletal tumors are missed not because they are occult on the physical examination or plain X-ray films but because the possibility of their existence is not considered or they are thought to be too rare to be worth considering. However, the stakes are high enough to justify a disproportionate amount of energy and cost to be spent with the goal of early diagnosis and avoiding inappropriate treatment of sarcomas in general. Early recognition of a primary malignant tumor of bone and the appropriate initial management can make the difference between limb salvage and amputation and the difference between life and death.
Bone tumors are rare, and many clinicians will only see a few during their career. They account for a very small fraction of the malignant and benign lesions of the human body. Although accurate estimates for North America are lacking, estimates from Sweden indicate a yearly incidence of 2.8/1 million for osteosarcoma, 2.26/ 1 million for chondrosarcoma, and 0.87/1 million for Ewing's sarcoma. Benign tumors occur three or four times more frequently than their malignant counterparts. Even to the general orthopedic surgeon, referral for a bone tumor only occurs a few times a year. In the adult, a bone lesion is most likely caused by an infection, trauma, or metastasis of a carcinoma. In the child, metastatic lesions are rarely the initial presentation of disease. Infections are a relatively common source of bone lesions, as are a wide variety of benign bone tumors. Malignant lesions of bone are rare in children, but when they exist they are more likely to be primary. It is also notable that diagnoses such as tendonitis or bursitis are uncommon and the clinician should always consider the possibility of a malignant bone tumor in a child with persistent musculoskeletal pain or a mass. For example, young patients with clinical findings suggestive of an intraarticular abnormality should have high-quality radiographs made to search for epiphyseal lesions such as chondroblastoma, infection, or some other type of lesion. There are no specific symptoms or signs associated with bone tumors other than pain. The most important part of the evaluation of a bone tumor is the recognition of its existence by the clinician. Since this book is aimed
PediatricBone
GENERAL PRINCIPLES OF TREATMENT Bone tumors include a diverse group of pathologic and clinical entities. Their clinical behavior is variable and requires a broad spectrum of treatment. Some of the benign lesions can be observed without any form of intervention; others require en bloc excision followed by complex reconstruction [1]. Curettage involves intralesional removal of the tumor through a window in the bone using curettes to scrape the boundaries of the lesion. High-speed burrs are often used to remove tumor that infiltrates the bone, usually in the context of aggressive lesions such as aneurysmal bone cyst or giant cell tumor. Adjuvants such as phenol or liquid nitrogen are occasionally used to decrease the rate of recurrence.
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Copyright 2003, Elsevier Science (USA). All rights reserved.
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Bone grafting involves packing a lesion with fragments of bone or a substitute. Recently, some of the sources of bone morphogenic proteins have seen increased clinical use to replace or augment traditional methods of bone replacement. Some lesions require bone grafts with structural or even vascular properties. It should be noted that some lesions require much less aggressive treatment than others. For example, eosinophilic granuloma of bone (histiocytosis X) usually heals after simple biopsy and will also respond to intralesional injections of methylprednisolone, whereas aneurysmal bone cyst tends to recur in approximately half of patients treated with aggressive curettage alone, particularly younger patients. When resected, some bones leave little or no disability and are termed expendable. Expendable bones include the clavicle, fibula, ribs, portions of the scapula, small tubular bones of the foot, and the anterior arch of the pelvis. Other sites, such as the distal femur (if the diagnosis and presentation warrant extensive resection), require replacement with a structural bone graft, osteoarticular allograft (cadaver bone), or a metallic prosthetic device. Soft tissue coverage procedures are commonly required for the treatment of sarcomas of the extremities. A few benign bone lesions also have the potential for malignant transformation followed by metastasis [2]. Others may metastasize to the lung while retaining a benign histology but can be lethal because of extensive involvement of the lung parenchyma. Malignant bone tumors are often mistakenly diagnosed as benign, leading in some cases to inappropriate treatment; likewise, overtreatment of benign tumors can also occur, and both may lead to suboptimal results. Malignant lesions require expert diagnosis and treatment in a multidisciplinary tertiary care setting, particularly due to their relative rarity and the potential for cure when properly and aggressively treated.
DIAGNOSIS Diagnostic strategies for benign bone tumors center on the initial clinical and radiographic presentation [3]. Biplanar radiographs are the best method for evaluating any long bone lesion. However, a lesion in a flat bone such as the pelvis may require axial imagery to obtain sufficient information to make a differential diagnosis. Initially, one must determine whether the tumor appears benign radiographically using several parameters. The first step is to determine how the tumor is affecting the bone. A benign tumor generally does not destroy the cortex and extend into the soft tissue. The second step is to determine how the bone is reacting to the tumor. A
slow-growing lesion allows the bone to form a margin in reaction to the neoplastic process. Most benign tumors have a geographic pattern of bone destruction with a sharp zone of demarcation between the tumor and the host bone. A dense, sclerotic margin around the tumor is a characteristic sign of a benign bone tumor. On the other hand, permeative destruction represents a gradual zone of transition and is more common in malignant tumors. The third step is to determine whether periosteal responses are present. If the periosteum has had an opportunity to lay down mature bone in reaction to slow endosteal resorption, a benign process is suggested. The lesion is then described as being expansile. If the periosteal response to the tumor creates a sequential layering of immature bone on its surface (onion-skinning), this suggests a rapidly evolving process, which can occur in both benign and malignant conditions. Codman's triangle represents rapid periosteal elevation with reactive changes and is another sign of an active or, in many instances, a malignant process. The so-called sunburst reaction, a spiculated pattern of subperiosteal bone formation, is another radiographic feature of malignancy. Fourth, one searches for extension of the lesion into soft tissue. This is an ominous sign and suggests a malignant or very rapidly growing benign process. Fifth, the lesion is evaluated for any matrix mineralization. If the tumor is characterized by destruction of bone, then the presence of calcification or ossification will suggest the type of neoplastic process present. For example, a calcified, lytic phalangeal lesion strongly suggests the presence of calcified cartilage, consistent with enchondroma. Finally, the location of the tumor within the bone is also helpful in identifying which type of tumor is likely present. Most benign tumors are metaphyseal; however, giant cell tumor and chondroblastoma are typically epiphyseal [4]. Diaphyseal tumors are rare and typically include fibrous dysplasia and eosinophilic granuloma. Certain benign tumors, such as aneurysmal bone cyst (ABC) and osteoblastoma, are relatively common in the spine, especially in the posterior elements. A solitary lytic lesion in a cortical location suggests the differential diagnosis of osteoid osteoma, subacute infection of bone, or, occasionally, stress fracture. The interpretation of radiographic studies of bone lesions is extensively discussed by Sweet et al. [5,6]. Note that although the experienced clinician or radiologist will be able to correctly identify a bone lesion using the previously discussed guidelines, he or she will regularly see exceptions to the classic patterns. Other imaging studies, such as technetium bone scan, computed tomography (CT), and magnetic resonance imaging (MRI), are important in evaluating bone tumors. Sarcomas in general and bone sarcomas in particular are unlikely to spread through the lymphatic system so
29. Bone Tumors in Children
that enlarged lymph nodes are more likely in the context of an infectious process than in the presence of a primary bone tumor. Hematopoietic spread is the rule and merits lung screening when a malignant bone tumor is suspected. Radioisotope imaging with technetium-99 diphosphonate is very helpful in determining whether the process is monostotic or polyostotic. Certain benign tumors, such as multiple hereditary exostoses, enchondromatosis, nonossifying fibroma, and fibrous dysplasia, can be polyostotic. Osteoid osteoma often takes up radioisotope intensely, and the scan is useful in identifying the location of the nidus as well as in documenting adequate surgical excision. Axial imaging, such as CT or MRI, has greatly aided clinicians in determining the location and extension of bone tumors. CT is particularly precise and helpful in characterizing changes in bone morphology (i.e., bone destruction or formation), whereas MRI is best at showing intramedullary changes, such as edema and tumor extension, as well as extension into the soft tissues. Predicting the resectability of a sarcoma in light of its proximity to neurovascular or joint structures is greatly aided by MRI, and this has allowed surgeons to achieve limb salvage in approximately 85% of malignant bone tumors. When a malignant tumor is suspected, staging studies should be performed to determine if distal spread of the disease has occurred (Fig. 1). Technetium whole-body bone scans screen for occult multifocal skeletal disease. Most important is chest imagery. Although plain radiographs of the lung are useful, the current standard is the chest CT, which offers a higher yield in screening for lung metastases. After adequate staging of the tumor is obtained, biopsy is indicated in most clinical scenarios. This should usually be done by a surgeon experienced in the definitive management of bone tumors since a poorly planned biopsy can have disastrous consequences for the patient. A biopsy can be excisional, open or incisional, or core needles can be used (with or without CT guidance). Some lesions lend themselves to fine needle aspiration, but this technique provides the pathologist with only a cytological specimen and is discouraged in most pediatric institutions. Even core needle techniques should only be used after discussion with the pathologist to weigh the advantages and disadvantages [1,3,7,8]. Open incisional biopsy is the technique most frequently used for bone tumors. The biopsy should be carefully placed in an anatomic site that will allow for the definitive surgical procedure. In addition, meticulous hemostasis must be obtained to avoid a postoperative hematoma. The risk of postoperative hematoma can be eliminated by performing an open incisional biopsy followed by intraoperative examination of a frozen
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FIGURE 1 Technecium bone scan is a sensitive screening tool for asymptomatic foci of disease, a) Pain was the sole complaint of this 12year-old girl with osteosarcoma, b) Bone scan showed increased uptake in the primary lesion and also showed a skip lesion in the proximal femur on the same side. c) Radiographic demonstration of abnormal ossification within the proximal femur consistent with osteosarcoma.
section and then performing the definitive procedure under the same anesthetic. This may not be appropriate if the clinical and radiological diagnosis is unclear or contradicts the findings of the frozen section, for example. Excisional biopsy is appropriate when the tumor can be completely curetted without extending the limits of the lesion so that if the final diagnosis is unexpected, treatment options remain optimal. This has the advantage of sampling the lesion in its entirety, such as in cartilage tumors. Likewise, if the diagnosis of a benign lesion is confirmed, treatment may be complete and a second procedure avoided. Decisions of this type
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should be made by surgeons experienced in the diagnosis and management of bone tumors, with the help of expert pathologists and radiologists. Finally, needle biopsy is occasionally useful for bone tumors when they are located in a relatively inaccessible location (such as the pelvis and spine), and it is usually done under CT guidance.
CLASSIFICATION AND NOMENCLATURE Although it would theoretically be useful to group bone neoplasms according to their biological behavior, there is considerable overlap between and variation within different tumors of the same type. It is probable that eventually enough information about the molecular biology of bone tumors will accumulate to allow a different type of classification, perhaps with a more predictable relationship to the lesion's behavior. Bone tumors are currently classified by the histology of the neoplastic tissue within the lesion and, more precisely, by its histogenesis. Although many different classification systems exist [3,9], we prefer the one proposed by Campanacci [10] because it is complete, allows relatively easy comparison to other existing systems, and attempts to include variations in biological behavior between histologically similar tumors. Histology alone, however, will not allow the clinician to differentiate between entities with similar histology but entirely different natural histories. For example, parosteal osteosarcoma has clinical, radiographic, and prognostic features that are clearly different from those of classic central osteosarcoma. These considerations are essential to the process of formulating a useful classification system. One must also keep in mind that not all histological variations have a clinical impact and thus do not necessarily justify a separate diagnostic category. Each lesion must be considered not only histologically but also from a clinical and radiological perspective to evaluate the unique biological behavior of each lesion on a case-bycase basis. An area of controversy is the classification of lesions such as fibrous dysplasia, multiple hereditary osteochondromatosis, Ollier's disease, and other lesions that could be considered from a perspective of dysplasia rather than neoplasia. As our knowledge of molecular biology increases, these distinctions may become less important. On the other hand, many of the dysplastic entities, particularly those mentioned previously, have a definite oncogenic potential that justifies their inclusion in a complete bone tumor classification (Table 1).
TABLE 1
Classification of Bone Tumours in Children (adapted from Campanacci (ref. 9))
Tissue differentiation Fibrous Benign:
Non-Ossifying Fibroma (NOF) Giant Cell Tumour (GCT) Desmoid
Malignant:
.Fibrosarcoma
Cartilage Benign:
Osteochondroma (OCE) Hereditary Multiple Osteochondroma (HMOCE) Hemimelic Epiphyseal Dysplasia Chondroma Ollier's Disease Chondroblastoma Chondromyxoid Fibroma (CMF)
Malignant:
Chondrosarcoma Mesenchymal Chondrosarcoma
Bone Benign:
Malignant:
Osteoid osteoma Osteoblastoma Fibrous Dysplasia Osteofibrous Dysplasia Osteosarcoma (centromedullary) Periosteal Osteosarcoma
Hematopoietic Benign: Malignant:
Vascular Benign: Malignant:
Neurogenic Benign: Malignant:
Lymphoma Leukemia
Hemangioma Lymphangioma Hemangioendothelioma Hemangiopericytoma
Neurinoma Neurofibroma Ewing's Sarcoma PNET (primitive neuroectodermal tumor)
Adipose Benign: Malignant:
Lipoma Liposarcoma
Mixed Benign: Malignant:
Adamantinoma Malignant Mesenchymoma
(Continued)
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29. Bone Tumors in Children TABLE 1
(Continued)
Notochord
Benign: Malignant:
Chordoma
Tumorlike
Benign:
Solitary (Unicameral) Bone Cyst (UBC) AneurysmaI Bone Cyst (ABC) Giant Cell Reparative Granuloma Eosinophilic Granuloma (Histiocytosis) Myositis Ossificans Brown Tumor of Bone
BACK PAIN AND SPINAL NEOPLASMS IN CHILDREN It is uncommon for a child to complain of back pain. Children's back problems can be classified into three categories: developmental (35%), predominantly caused by kyphosis or, to a lesser extent, scoliosis; traumatic (35%), the majority of which are spondylolysis of L5S1; and spontaneous onset (15%), usually caused by tumor or infection. The remaining 15% may be of a functional nature. Children are often vague with regard to the nature of their complaints as well as the localization of their pain. This is compounded by the fact that they have a limited vocabulary with respect to medical conditions. Night pain and the relief of symptoms with aspirin (or NSAIDs) should lead one to suspect an osteoid osteoma or osteoblastoma [11-13]. The dangerous signs of childhood back pain are persistent pain unrelieved by rest or immobilization, night pain, and increasing pain. In these cases, the clinician must conduct timely investigation and follow-up. Young children who complain of recurrent back pain may have a spinal or intraspinal tumor. Malignant astrocytoma, chordoma, and, rarely, sarcomas must be considered as well as benign tumors, such as dermoid cysts, lipomas, hemangioma, eosinophilic granulomas, osteoid osteomas, osteoblastomas, and ABCs. If a child with idiopathic scoliosis complains of persistent pain, one should exclude causes such as tumor, infection, or spondylolysis [13]. The evaluation of a child with back pain should include a standing anteroposterior and a lateral radiograph of the entire spine. Because children usually do not precisely localize the area of discomfort, the long films used for scoliosis and kyphosis can be helpful. The vertebral canal should be examined for widening of the interpedicular distance, which suggests an intraspinal mass or one
of the various forms of dysraphism. As in the adult, the disappearance of the pedicle (winking owl sign; Fig. 2) and other lytic changes suggest neoplasm or infection. Bone scan is recommended as a routine study if the child exhibits findings suggestive of tumor or infection. A bone scan is excellent for detecting the reactive process associated with recent trauma, repetitive stress, tumor, and inflammatory conditions that involve the spine and/ or pelvis. Single photon emission computed tomography is a more sensitive imaging technique when a stress reaction of bone is suspected [14]. The bone scan is the most sensitive method for detection of a metastatic or primary bone tumor, and in children it is more sensitive than skeletal surveys [15,16]. CT can be helpful for characterizing the nature of a bony lesion once the anatomic location has been identified. MRI is an important aid in evaluating disk problems, lesions contained within the spinal canal, spinal edema, and the relationship between the neural structures and bony vertebral elements. Benign N e o p l a s m s Osteoid osteoma and osteoblastoma are the most common benign tumors of the spine. They typically present with painful scoliosis. The pedicles and posterior elements are the usual location of a small nidus
FIGURE 2 Painful scoliosis in a 9 year old boy. This radiograph shows the "winking owl sign", indicating a destructive lesion of the pedicle at the apex of the scoliotic curve (arrow).
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surrounded by a sclerotic bone response in the case of an osteoid osteoma, whereas osteoblastoma, which is similar but larger, has a lytic component that usually predominates. These changes are best seen on CT performed using thin slices [12,17] (Fig. 3). Osteoblastomas can compress the spinal cord, particularly in the cervical and thoracic spine. The pain associated with osteoid osteoma is classically nocturnal or rest pain that is relieved by aspirin or NSAIDs. Patients with osteoblastoma may have a similar response to NSAIDs. Eosinophilic granuloma involves the vertebral body and causes progressive destruction and gradual collapse. A trivial injury can precipitate a sudden collapse and acute pain [ 18]. Plain films usually reveal the classic radiographic findings of vertebra plana (Fig. 4). M R I can confirm the diagnosis in most cases, but occasionally definitive diagnosis requires biopsy. Paraplegia is rare, although in some cases the tumor herniates posteriorly into the spinal canal, causing spinal cord compression. ABCs may be asymptomatic for a long period of time. A fracture through the weakened bone, collapse, and spontaneous hemorrhage occur after minor trauma or with disease progression. These lesions generally occur in the thoracic, lumbar, or sacral spine. They typically affect the vertebral body but occasionally involve the posterior elements (Fig. 5). Exploration and bone grafting usually resolve the problem.
FIGURE 3 CT scan showing a small well-definedlesion (nidus) and a rim of dense reactive bone around it, characteristic for the diagnosis of osteoid osteoma in this young girl with painful scoliosis.
FIGURE 4 (b) MRI clearly shows a classic vertebra plana in the upper thoracic spine, which was very poorly appreciated on plain radiographs (a).
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FIGURE 5 Aneurysmalbone cyst of the sacrumin an 11 year old girl. These lesions generally occur in the thoracic, lumbar or sacral spine. They typically affect the vertebral body but occasionally involve the posterior elements.
Malignant Neoplasms Primary malignant tumors are rare in the spine, with the most common being Ewing's sarcoma, leukemia, and lymphoma [19]. More commonly, such lesions are metastatic and represent spread from a distant primary site, usually from bone or bone marrow. In children, the most common skeletal metastases to the spine are neuroblastomas and rhabdomyosarcomas. Less common are teratoma, teratocarcinoma, Wilm's tumor, and osteogenic sarcoma [15,19,20]. Patients with neuroblastoma have up to a 70% chance of presenting with metastatic skeletal disease at some time in the course of their treatment [19,21 ]. Spinal involvement in these cases is typical. Diffuse permeative destruction of bone with variable amounts of periosteal reaction is seen, similar to long bone metastases [21]. Rhabdomyosarcoma is the most common soft tissue malignancy in children, and spinal metastases occur in approximately 40% of cases [22]. Lytic destruction of the vertebrae causes structural failure and collapse, sometimes acutely after minor trauma [20,23]. Neurologic complications have been reported, usually after progressive complaints of pain [15,24].
FIBROUS T U M O R S OF BONE N o n o s s i f y i n g Fibroma a n d Fibrous Cortical D e f e c t
Synonyms: Histiocytic fibroma, fibroma, nonosteogenic fibroma, fibrous defect of the cortex, fibrous metaphyseal defect, fibrous xanthoma, and histiocytic
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xanthogranuloma. Nonossifying fibroma (NOF) and fibrous cortical defect (FCD) are histologically identical; they are benign tumors with slightly different characteristics. FCD is asymptomatic and usually solitary, with a predilection for the diaphysis of long bones. It is quite common (Mirra and associates [8] estimated that it occurs in 30-40% of children between 4 and 8 years of age), and it rarely needs treatment. The etiology is unknown. Apart from its location, the dimensions of the lesions are the usual criteria to differentiate the two; a lesion >2cm in diameter is diagnosed as NOF [25]. NOF is usually solitary, but polyostotic N O F does exist. The majority of these lesions are asymptomatic and show a preference for the metaphysis of long bones. Symptoms arise if the lesion is large enough to weaken the bone, and pathologic fracture may occur. This lesion has been considered by some authors as faulty ossification rather than a true neoplasm. Again, it is estimated that approximately 30% of normal growing children have a NOF or a FCD; these are usually detected as incidental findings on radiographs prescribed for other purposes. Eighty percent of NOFs are discovered in patients younger than 20 years old. The male-to-female ratio is estimated to be 1.4:1 [8,25]. NOF accounts for approximately 2% of primary bone tumors undergoing biopsy [3]. Most of these lesions do not cause symptoms, perhaps because of the markedly ossified reactive border that characterizes the typical case. There are three phases in the growth of NOF: early, middle, and regressive. Lesions in the early and middle phases are found in patients younger than 20 years old. They cause symptoms only if the bone is weakened sufficiently for microfractures or stress fractures to occur. After skeletal maturity, these tumors regress and begin to heal; they are rarely a source of consultation in patients older than 35 years of age [8]. Polyostotic (or multicentric) NOF is an unusual form of NOF, with lesions distributed ipsilaterally, arising in a serial fashion in growing children. Although these patients are more likely to need treatment for pathological fracture and ensuing deformity, they are otherwise similar to those with the solitary form [10].
Diagnostic Imagery On plain radiographs, NOF is usually metaphyseal and eccentric in its location; it usually involves the medullary canal and the overlying cortex. The polylobulated appearance of the lesional margins has been likened to soap bubbles. The margins are classically thick and sclerotic but, variably, thinning and even cortical discontinuity may occur (Fig. 6).
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FIGURE 6 Nonossifyingfibroma is usually metaphyseal and eccentric in its location. The polylobulated appearance of the lesional margins has been likened to soap bubbles. The margins are classicallythick and sclerotic. Generally, plain radiographs are diagnostic and additional imaging is not necessary. Technetium bone scan usually shows minimally to mildly increased uptake, which may even be difficult to distinguish from the adjacent growth plate. A CT scan shows additional cross-sectional anatomy and may assist in the diagnosis. It is of particular interest to assess the risk of fracture, which will often influence the choice of treatment (Fig. 7). M R I will generally show a low signal on both T1- and T2-weighted images, reflecting the fibrous tissue content of the lesion, but it is of little help in the clinical setting [26].
Pathology N O F usually is brown, yellow, or grayish and is composed of soft or firm fibrous tissue. Histologically, the lesion comprises fibrous tissue, xanthoma cells, and multiple giant cells. In the early phase, a spindle-cell stroma may also be present. As the lesion matures through the middle and regressive phases, there is increased production of collagen, a decrease in the number of giant cells, and an increase in the number of lipidrich macrophages [8]. Hemosiderin pigment is in evidence throughout the life of the lesion but increases with maturation. The differential diagnosis of these tumors includes brown tumor of hyperparathyroidism, Paget's disease, fibrous histiocytoma, desmoplastic fibroma of bone, osteosarcoma, and osteofibrous dysplasia.
FIGURE 7 In this child with Ollier's disease, increased diameter is obvious in the affected small bones of the hands. Enchondroma in children is often not calcified.
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Treatment Asymptomatic lesions may be observed. Theoretically, a very large lesion may weaken the bone and may need to be treated prophylactically. It has been proposed that a NOF that involves more than 50% of the width of the bone on plain radiographs in two projections or that is more than 33 mm in length may cause fracture and should be treated [27]. After pathologic fracture, the likelihood of spontaneous healing is high. If the lesion requires treatment, local intralesional curettage with bone grafting is sufficient. Increasingly, the morbidity associated with autologous grafting is avoided because allograft tissue is being used with good results [28,29]. Alternatively, healing has been observed after a single injection of methylprednisolone.
Benign Fibrous Histiocytoma This is an adult tumor and will not be described. Giant Cell Tumor
Synonym: Osteoclastoma. Giant cell tumor (GCT) is a benign neoplastic lesion that tends to arise in the metaepiphyseal area of long bones. This usually solitary lesion becomes symptomatic when bone destruction interferes with the mechanical integrity of the bone. It is extremely unusual in skeletally immature patients [251. GCT represents approximately 19% of benign bone tumors and 9% of all primary bone tumors (all ages) [3]. Since GCTs occur after physeal plate closure, they arise more often in females younger than 17 years of age than in males of the same age. This slight female-to-male preponderance holds true at all ages [8]. Age at presentation usually ranges from 18 to 45 years. By far, most GCTs occur around the knee and in the distal radius, where they tend to behave in an aggressive fashion. GCTs can affect the spine, particularly in females in the second and third decades [30]. The clinical presentation typically involves a joint, with pain, swelling, giving way, and weakness being common findings. Also, a hard, painful, and sometimes crepitant mass is found in more than 80% of patients. Muscle atrophy, effusion, and other inflammatory signs may also be present. Pathologic fracture can be the presenting complaint [3,26]. Patients with involvement of the spine or sacrum may have neurologic signs and symptoms. GCTs can undergo spontaneous sarcomatous transformation (fibrosarcoma, osteosarcoma, or malignant fibrous histiocytoma), although this is mostly seen
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after radiation therapy. The rate of malignant transformation is less than 5%, but this is not seen in children [10].
Diagnostic Imagery Plain radiographs are usually diagnostic, but occasionally the lesion may be difficult to see. Its usual characteristics include lytic changes involving the epiphysis with extension to the metaphysis and abutting subchondral bone. It may break through the cortex to form a soft tissue mass [25]. In patients with incomplete closure of the physis, the lesion tends to be centered around this region. CT scanning can further define the extent and severity of bony destruction, cortical thinning, and perhaps the risk of fracture; however, MRI is reported to be more effective in evaluating subchondral cortical penetration, joint involvement, and pathologic fracture [31]. Since soft tissue extension of the lesion is an important variable for staging and treatment planning, MRI is the most useful imagery technique. Radioisotopic bone scanning is useful for detection of multicentric disease, which is rare (< 1%) [3].
Pathology GCT tissue ranges from brownish tan to yellow, with reddish areas of hemorrhage. Areas of necrosis may appear cyst-like. Histologically, a benign GCT contains predominantly osteoclast-like giant cells and oval or spindle-shaped stromal cells. Both cell types have round or oval nuclei, are uniform in size, and have blandly granular chromatin and prominent nucleoli [8]. The giant cells never show mitotic figures; however, the stromal cells can show 5-10 mitoses per 10 high-power fields. The differential diagnosis of GCT includes brown tumor of hyperparathyroidism, malignant fibrous histiocytoma, NOF, chondroblastoma, osteoblastoma, and osteosarcoma. Careful inspection of the complete specimen is mandatory to rule out areas of osteosarcoma. Note that giant cells are present in a number of bone neoplasms but that in GCT they are its characteristic and predominant element, whereas in all other lesions they are only present occasionally, probably as a reactive element whose role is not unlike that of a macrophage [10].
Staging GCTs exhibit wide variations in biologic activity. Some remain local and noninvasive and do not metastasize; others are extremely destructive locally or metastasize to
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the lungs. It is not possible to predict the biologic behavior of a particular tumor on the basis of its histologic appearance. More sophisticated studies, such as flow cytometric analysis of DNA, have not been helpful in predicting whether GCT will metastasize [32]. Campanacci and associates developed a staging system based on a combination of clinical, radiographic, and pathologic findings [10,33]. A stage 1 GCT is defined as one that causes symptoms and appears latent radiographically. A stage 2 GCT may cause symptoms and shows an active radiographic appearance without evidence of metastasis. A stage 3 GCT causes symptoms and has radiographic signs of rapid and invasive growth with extra cortical and subchondral extension. The histologic findings in stages 1-3 are always benign. Several studies have shown this system to be predictive of local behavior. Although their histology will show the same benign characteristics as the primary lesion, pulmonary metastases occur in up to 10% of patients, often in a delayed fashion.
apy should be reserved for patients in whom complete resection is impossible. Follow-up of all patients with GCTs should be prolonged since recurrence is frequent and the appearance of metastatic disease can be delayed for up to 10 years. D e s m o i d Fibroma Synonym: Desmoplastic fibroma As in soft tissue desmoid tumors, this rare lesion is a slowly progressive tumor of well-differentiated mature fibrous tissue, with a tendency to recur [10]. It has been observed at all ages but is usually considered to occur in childhood or adolescence. Any site can be involved, but this lesion most commonly occurs in the ilium and mandible. Males and females are affected equally [7]. Moderate pain, pathologic fracture, and, occasionally, a mass (from expansion of the bone) may be the presenting symptoms. Typical features are the discordance between the size and appearance of the lesion on radiography and a paucity of symptoms (suggestive of very slow progression) [7,10].
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Treatment GCT is most often treated with curettage or intralesional resection, with a significant rate of local recurrence [34]. The rate of recurrence depends on the technique of curettage rather than on adjuvants such as phenol or polymethy! methacrylate (PMMA) cement. Cryotherapy using liquid nitrogen extends the resection margin by circumferential necrosis; however, dermal necrosis and delayed pathologic fracture have been reported with this technique [35]. In some cases, a more aggressive approach involving en bloc resection may be indicated [8]. Although the rate of recurrence associated with this technique is quite low, the rate of complications is higher [36,37]. The risk of recurrence following curettage must be weighed against the risk of complications and a poor functional result. In children, most surgeons use bone graft rather than PMMA cement after curettage of benign bone lesions [25]. Occasionally, GCTs occur in an area such as the sacrum [38], in which resection is extremely difficult or impossible; such tumors have a very high rate of recurrence. Local and meticulous curettage remains the principal treatment of sacral tumors. Most authors agree that radiation therapy should be avoided in the treatment of GCTs because there is a high prevalence of sarcomatous degeneration [4]. Although recent studies have indicated that modern radiation techniques can be used to treat benign GCTs of bone more effectively [39], these studies are in an early stage and the follow-up period is too short to adequately evaluate the prevalence of sarcomatous degeneration. Radiation ther-
Diagnostic Imagery Radiographs show pure osteolysis interrupted by septae in a diffuse, bubbly pattern with thinned cortex. It arises centrally within long bones, usually near the metaphysis, and may be associated with remarkable expansion of the bone. This lesion can be mistaken for telangiectatic osteosarcoma, metastatic renal cell or thyroid carcinoma, and, in the mandible, ameloblastoma [7]. Radioisotopic bone scans tend to show little or no increase in activity [10].
Pathology The gross pathology is typical of desmoid tumors, with firm, compact, whitish connective tissue, whorled texture, and a well-defined boundary. On microscopic examination, mature, compact fibrous tissue is seen, with little or moderate cellularity. The fibroblasts and fibrocytes are small and mature, with only rare mitotic figures. The margins of the lesion are not as well demarcated as they appear on gross examination, tending to blend with surrounding tissue. This lesion must principally be distinguished from low-grade fibrosarcoma, which shows fibroblasts with plump nuclei, pleomorphism, and more mitotic activity.
Treatment Curettage results in approximately 50% recurrence rate, whereas en bloc resection is generally successful.
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Cryotherapy may be useful to decrease recurrence after curettage [7,10]. Fibrosarcoma Fibrosarcoma is composed of cells with a differentiation that is almost exclusively fibroblastic, producing a stroma of reticular and collagen fibers. Estimated to represent approximately 2% of primary malignant bone tumors, it is 10 times rarer than osteosarcoma. It occurs equally in males and females and at any age. It tends to be localized in the following sites in order of decreasing frequency; the distal femur, the proximal tibia, the proximal femur, the proximal humerus, and the pelvis. Nearly half of reported cases involve the knee region, 20% involve the proximal limbs, and 20% involve the axial skeleton. It primarily affects the metaphysodiaphyseal segment of the long bones, and it is almost never seen in the immature skeleton. Secondary fibrosarcoma is generally found in adults following radiation therapy. Pain is the most common complaint, often preceding pathologic fracture. Swelling is unusual and suggests a high grade of fibrosarcoma.
Diagnostic Imagery Osteolysis is the dominant finding on radiography, and it is associated with aggressive signs, including cortical destruction, permeative changes, and soft tissue invasion. Periosteal reaction is not usually significant. The radiographic picture is extremely variable, however. The lesion is medullary (two-thirds of cases) or periosteal (one-third of cases), affecting all parts of long bones.
Pathology Tumoral tissue is whitish, compact, and firm (lower grades with better differentiation); it is less firm in higher grade lesions with higher cellularity, more vascularity, and necrosis. Cystic or myxoid areas may be present. Within a given lesion, the grade of malignancy tends to be uniform, similar to its soft tissue counterpart. The distinction between aggressive benign lesions of desmoid fibroma and low-grade fibrosarcoma is not always clearcut, as is seen in soft tissue lesions. By definition, a fibrosarcoma does not change in grade during its course of evolution. The typical orientation of the collagen fibers in this tumor has been called a herringbone pattern.
Treatment Wide resection is the mainstay of treatment, either by limb salvage or by amputation depending on grade and
tumor extension. Chemotherapy is of unknown benefit, but it is more likely to be used as an adjuvant in younger patients. Radiation has little effect on fibrosarcoma of bone and is reserved for palliative treatment in exceptional cases [10,40]. Malignant Fibrous H i s t i o c y t o m a Malignant fibrous histiocytoma of bone is an aggressive tumor mainly seen in adults. Although several reports note its appearance in the second to fifth decades, the patients reported appear to be skeletally mature and a only small proportion are younger than 20 years of age. The reader is referred to texts dealing with adult bone tumors for more details [41,42].
CARTILAGE-FORMING TUMORS Chondroma
Synonym: Enchondroma Chondroma is a lesion of mature hyaline cartilage that may be located centrally within the bone (enchondroma) or may arise either in or beneath the periosteum (periosteal or cortical chondroma, respectively). Intraarticular and soft tissue variants are even more unusual, but they do exist. Malignant transformation is rare in the solitary lesion but occurs more frequently in polyostotic forms. Chondroma has been reported to account for 25% of benign bone tumors and 12% of biopsy-proven primary bone tumors [1]. Chondroma is detected throughout life. Patients with multiple lesions are usually 10-30 years old at the time of diagnosis, with peak prevalence occurring during the third decade of life. There is no sex predilection for skeletal chondroma. Enchondroma is quite common in the small bones of the hands and feet. In one study, 58% of the lesions occurred in these areas [8]. Indeed, enchondroma is the most common primary tumor in the bones of the hand. Asymptomatic enchondromas are often found incidentally on radiographs. Atypical enchondromas are characterized as such because of their association with a history of pain. A fracture is sometimes seen in the area of enchondroma, perhaps because of previous weakening of the bone or simply as an incidental finding. Occasionally, a patient has multiple enchondromatosis or Ollier's disease. A patient who has multiple enchondromas and angiomas of the soft tissues has Maffuci's syndrome. Sarcomatous transformation of the enchondromas can occur in association with both of these syndromes, but malignant transformation of solitary enchondroma is rare, occurring in fewer than 1% of patients [8].
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Periosteal chondroma is a rare, benign cartilaginous tumor that arises from the periosteal tissues. It is the periosteal counterpart of medullary enchondroma. Additional names for this lesion are parosteal chondroma and juxtacortical chondroma. The signs and symptoms of periosteal chondroma are similar to those of solitary enchondroma. Pain is the most common symptom, but the lesion is usually found on an incidental basis. It rarely, if ever, converts to a malignant lesion.
Diagnostic Imagery Classically, enchondroma is a long, oval lesion that is located centrally in the tubular portion of the bone. It usually has a lobulated appearance grossly and typically is sharply demarcated. Calcifications may be present in the matrix of the lesion, but in young children they are less likely. Expansion of the surrounding cortex is uncommon unless the lesion occurs in a small bone, such as the bones of the hands or feet, or in the fibula (Figs. 7 and 8). Radiographic features of malignant transformation are more likely to be found in multiple enchondromatosis and are a manifestation of the increased biologic activity, as evidenced by increased endosteal scalloping, pathological fracture, or increased size. Radioisotopic scans are not a reliable way to assess malignant transformation. MRI is occasionally helpful in assessing the nonmineralized component of the lesion, whereas CT scans can demonstrate whether the pattern of calcification
is heterogeneous or if there is increased bone resorption [3]. The differential diagnosis includes epidermoid cyst in the distal phalanx and fibrous dysplasia, NOF, simple bone cyst, chondroblastoma, chondrosarcoma, chondromyxoid fibroma, and bone infarction elsewhere. Usually, these lesions can be excluded on the basis of radiographic examination. If not, biopsy may be necessary. Although bone infarcts can occur in children in the context of storage diseases and hemoglobinopathies, chondrosarcoma is extremely rare and, when present, is likely to be a rare variant called mesenchymal chondrosarcoma, which is easily differentiated from classic chondrosarcoma on radiological and histological examination.
Pathology Enchondromas are composed of well-circumscribed nodules of benign, hyaline cartilage cells. Cellularity is low and binucleate cells are absent. The chondrocytes have small condensed nuclei [43]. Nuclear atypia (generally minimal) is variable depending on the location. Signs compatible with low-grade chondrosarcoma in a femoral location, for example, may frequently be found in a benign cartilage tumor located in the small bones of the hands and feet. It is thus important for the clinician, radiologist, and pathologist to reach a consensus that takes into account all the information available [2]. Although this principle is particularly true when evaluating a cartilage tumor in the adult, it bears repeating as a general principle and further highlights the need for referral of many of these cases to tertiary multidisciplinary centers.
Treatment
FIGURE 8 a) Surface enchondromas affect both ends of this boy's humerus, b) The lesion is clearly demarcated by the saucer-shaped depression in the cortex (CT image).
Typical enchondromas that are asymptomatic can be observed with serial radiography. An atypical, progressive, or symptomatic enchondroma should be diagnosed by incisional biopsy and treated locally with marginal or intralesional resection, keeping in mind that sampling error is a potential pitfall with cartilage tumors. Notwithstanding that chondrosarcoma is extremely rare in the pediatric population, the tissue should be analyzed carefully for any histologic changes suggestive of malignancy. Defects created in large and weight-bearing bones should be grafted with either autologous or allograft material [4,7]. Bone grafting may not be necessary after curettage of an enchondroma of the hand [8,44]. Periosteal chondroma is a benign lesion. Marginal or wide local resection is the procedure of choice. However, if the lesion is excised incompletely, it may recur. Bone
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grafting may or may not be necessary, depending on the extent of the local resection. If imaging studies and the clinical situation indicate the potential for malignant transformation, biopsy is mandatory. However, no case of malignant degeneration of a solitary enchondroma has been reported in children. Metachondromatosis and Other Developmental Syndromes Variations of enchondromas can occur on the surface of bone, in soft tissue, and in joints, usually attached to an epiphysis. Syndromes such as metachondromatosis are characterized by the association of enchondromas and osteocartilaginous exostoses, and these are often atypical in their orientation and location.
Ollier's Disease
Synonyms: Dyschondroplasia and multiple enchondromatosis Multiple enchondromatosis may be considered an inborn error of osseous development rather than a classic neoplasm, but histologically it resembles solitary enchondroma. From a physiological standpoint, the fact that masses of cartilage can be found in the metaphysis may suggest it is a disease of the growth plate. The origin of the cartilage lesions is unclear. The cartilage in the metaphysis may be immature hypertrophic cells left behind by the advancing growth plate [9], or the cartilage cells may be derived from the cambium layer of the periosteum [45]. This latter theory may explain cartilage tumors in the midshaft of a long bone. Radiographic examination shows a radiolucent defect in the metaphysis or diaphysis of the involved bone. Frequently, the defect appears as a large cystic area that is trabeculated and irregularly calcified. Depending on the size and location of the cartilage accumulations, growth may be altered, leading to shortening or angulation or both (Fig. 9). Usually, the lesions are unilateral, and the lower extremities are affected more often than the upper extremities. Maffuci's syndrome is by definition an association between multiple enchondromas and hemangiomas of the soft tissues. It is extremely rare, and in most large series of multiple enchondromas (Ollier's disease), only 3-5% are associated with hemangiomas [46]. The associated hemangiomas are recognized radiographically by their calcified phleboliths. Malignant Potential of Enchondromatosis Long-term follow-up of patients with multiple enchondromas, of which most did not have hemangiomas
FIGURE 9 Altered longitudinal growth may be seen with multiple enchondromatosis (Oilier disease).
(Ollier's disease) and some did (Maffucci's syndrome), allowed the rate of malignant degeneration and other cancers to be determined from life table analyses. An estimated 25% of patients with Ollier's disease will develop malignancy by the age of 40, whereas malignant degeneration is almost a certainty in patients who have Maffucci's syndrome. Periodic surveillance of the brain and abdomen for occult malignant lesions may be indicated in patients who have enchondromatosis, especially with hemangiomas (Maffuci's syndrome). Of interest, there is no evidence of hereditary transmission or a familial tendency for this syndrome. In one study, all but one of the skeletal sarcomas were located on the side of the body that had the most enchondromas. Another observation was that in all patients, when the organ in which a malignant lesion eventually developed was anatomically paired, the cancer always occurred on the side that was predominantly involved with enchondromas. The tendency to develop secondary chondrosarcoma appeared to be greater in the axial skeleton than in the appendicular skeleton [47-49].
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Osteochondroma and Hereditary Multiple O s t e o c h o n d r o m a t o s i s Synonyms: Osteocartilagineous exostosis and hereditary multiple exostoses or diaphyseal aclasia, respectively. These lesions are aberrant developmental abnormalities formed of bone and cartilage that occur at the periphery of the physeal plates. They can be found on any bone but are more frequent near the most active growth plates. Distinguishing features include continuity of the cortex and marrow space with the underlying bone, a cartilage cap with histology of the physeal tissue, and progressive centrifugal ossification. During periods of active growth they tend to enlarge, whereas further growth after skeletal maturity suggests malignant transformation. These lesions are bony masses that can become symptomatic from mechanical interference with periarticular structures such as nerves and tendons [50], fracture [51], infrequently from chronic injury to vascular structures (pseudoaneurysm) [52], and rarely from malignant degeneration. Solitary osteochondromas account for 40% of all benign bone tumors. They affect all ages but are usually diagnosed in childhood. Males are affected twice as often as females. Most are found around the knee, shoulder, or hip. If the scapula is affected, the patient may complain of painful "snapping" of the shoulder. Osteochondromatosis is rare. Age at the time of diagnosis ranges from birth to adolescence, but most are diagnosed before the age of 5. A mass, pain after trauma, angular deformity, short stature, and limb length inequality are common modes of presentation. The lesions can affect any long bone, the pelvis, ribs, the scapula, and the distal ends of the proximal and distal phalanges [53]. This is an autosomal dominant disorder with variable penetrance. Approximately 70% of cases are inherited, whereas 30% of patients present the syndrome without a family history of multiple exostoses. Schmale et al. [54] demonstrated that the rate of penetrance of the responsible gene was approximately 100% in 23 pedigrees with an adequate multigenerational history. There was no evidence of a substantial reduction of penetrance in female subjects.
Diagnostic Imagery These lesions are generally metaphyseal, show continuity of the cortex and the medullary cavity with the underlying bone, and tend to grow perpendicular and then away from the adjacent epiphysis (Fig. 10). Largebased exostoses are termed sessile, whereas those with thin stalks are described as pedunculated. Especially notable for multiple exostoses, there is deficient remodeling of the
FIGURE 10
A solitary osteochondroma of the pedunculated type.
bone (diaphyseal aclasia) resulting in widening of the diaphyseal extremities (Fig. 11). Calcifications may be seen in the "cap" of cartilaginous tissue at the periphery of the exostosis, which is called Sisson's sign. Radionuclide bone scans and skeletal surveys are both useful modalities for detecting multiple lesions. CT scan is usually diagnostic since it precisely documents the contiguity of cortex and medullary cavity that differentiates this lesion from both myositis ossificans and parosteal osteosarcoma (Fig. 12). MRI is useful for the quantification of the thickness of the cartilage cap [55]. A cartilage cap thicker than 1 cm is considered suggestive (if not diagnostic) of malignant transformation (usually into chondrosarcoma). Cartilage thickness >2 cm is more worrisome, especially if myxoid changes are present on MRI or if Sisson's sign is positive on radiographs or CT.
Pathology Resected specimens consist of normal appearing bone with a more or less regular cartilage cap. Progressive maturation of mature chondrocytes arranged in parallel
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FIGURE 1 1 a) The deformation of forearm bones is evident in this case of hereditary multiple osteochondromatosis. It is mainly the result of the space-occupying lesions, and reactive remodelling of the adjacent bone. b) Deficient remodelling of the metaphyses has given rise to the synonym Diaphyseal Aclasia, and may in severe cases interfere with longitudinal growth of the affected bone. This is often most striking about the knee.
FIGURE 12 a) This osteochondroma presents contiguity of the cortex and medullary cavity with the underlying bone which is well shown by CT. This is the best imaging modality to differentiate this entity from both (b) myositis ossificans, where a soft tissue gap separates the two structures, and (c) parosteal osteosarcoma, in which the tumor is in intimate contact with the surface of the cortex, but without communication with the marrow space.
clusters resembles the physeal pattern of ossification, with irregular cartilage cores protruding into the trabeculae. The marrow spaces are normal and merge imperceptibly with those of the underlying bone.
Treatment
Biopsy is rarely required. Surgery may be required for deformity, interference with joint function, pathologic fracture, and to rule out or treat malignant degeneration
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(usually in adults). Biopsy is mandated by pain without mechanical explanation, enlargement of the lesion(s) after skeletal maturity, and new areas oflysis within preexisting lesions. Fracture of an osteochondroma does not predispose to malignant degeneration. Patients and their families need counseling on surveillance for malignant change as well as genetic counseling. Some patients may opt for sterilization procedures to avoid transmission of the disease.
Chondroblastoma
Synonym: Codman's tumor Chondroblastoma was named by Jaffe and Lichtenstein. Typically, it is an epiphyseal tumor that occurs in adolescents. It represents approximately 5% of all benign bone tumors. Chondroblastoma is slightly more common in boys (64%) than in girls [4,25,56,57]. Few patients are older than 20 years of age at presentation, and the vast majority of cases occur in the second decade of life. Some controversy regarding the clinical presentation and recommended treatment is apparent in the literature. The average age of patients with an open epiphysis is 12 years, that of patients with a closing epiphysis is 15 years, and that of patients with a closed epiphysis is 20 years. Although many authors have reported that approximately two-thirds of these lesions occur in the proximal humerus, a large series (72 patients) from the Rizzoli Institute [57] found that the proximal end of the humerus was the most common location (18), with the proximal end of the femur (15), distal end of the femur (15), and proximal end of the tibia (12) being other frequently involved sites. Though not as frequent, chondroblastoma of the talus and the innominate bone have also been reported, usually in adult patients. Patients typically complain of joint pain adjacent to the lesion, the duration of which can vary from a few weeks to more than 1 year. A universal finding istenderness on direct palpation of the involved bone and most have a measurable, but usually slight, loss of motion in the adjacent joint. Whether joint effusion is evident depends at least partly on the location of the tumor. Joint effusion around the knee is detectable in 50% of patients with chondroblastoma, whereas effusion in the hip or shoulder area is not usually detectable [25,56,57]. Many of these patients are erroneously diagnosed with chronic synovitis months or years before the correct diagnosis is made. Although involvement of the epiphyseal plate occurs in some patients, it is usually not a clinical problem, perhaps because most of these patients have little growth remaining [57].
Diagnostic Imagery The plain radiograph is diagnostic in most cases. This epiphyseal abnormality is radiolucent, usually with a sclerotic border, tending to expand into the metaphysis if untreated, and it can be quite destructive (Fig. 13). Classically, there are small foci of matrix calcification [25,56]. According to the Rizzoli Institute study [57], patients with chondroblastoma can be classified into three groups based on the radiological status of the adjacent epiphyseal plate. Group 1 includes patients with open epiphyses (a wide, well-defined radiolucent epiphysis), group 2 patients haves a closing epiphyseal plate (thin and irregular), and group 3 patients present with a closed epiphyseal plate. The lesions can also be separated into radiographic stages indicative of the lesion's activity: latent, active, and aggressive. Latent lesions are confined to bone, with a well-defined, complete reactive rim of bone around the lesion. Active lesions are either confined to bone with an incomplete reactive rim or penetrate the cortex, albeit with a surrounding rim of reactive periosteal bone. Aggressive lesions have a poorly defined
FIGURE 13 Chondroblastoma. This epiphyseal lesion is radiolucent, usually with a sclerotic border, and can be quite destructive. Classically there are small foci of matrix calcification.
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29. Bone Tumors in Children
margin with minimal or absent intraosseous or periosteal reaction. The distribution of the different radiographic stages for tumor activity is similar for the three subgroups regarding the status of the epiphysis. CT, as an adjunct to plain radiographs, helps to define the bony changes and to detect recurrence. These lesions tend to be very active on bone scan.
Pathology On study of gross pathology, one notes a grayish-pink lesion, which may have zones of hemorrhage or necrosis. Operative findings include partial invasion of the articular cartilage in 20% of cases, whereas frank penetration of the articular cartilage by the tumor is rare (but mechanical damage may ~occur from subchondral bone destruction and trauma). Synovitis is usual. It is present with intraarticular tumor but is also noticeable to some degree in most other patients if arthrotomy is performed during treatment [58]. Histological features include chondroblasts with an indentation or a longitudinal groove in the nucleus. There is a predominance of roundish or polyhedral cells of varying size with occasional mitoses (but no atypical mitoses), multinucleated giant cells of varying number, and foci of chondroid matrix. From studies using electron microscopy, histochemical analysis, and tissue culture [10,58], the predominant cell has been found to be most similar to the epiphyseal chondrocyte, with four distinguishing morphological features: a nucleus that tends to be multilobulated, large nucleoli, blunted cytoplasmic microvillous processes, and a continuous dense band of nuclear substance along the inner nuclear membrane. The multinucleated giant cells are of the reactive type. Approximately 25% of patients in a large series had histological features of a secondary aneurysmal cyst within the chondroblastoma without relation to the status of the growth plate [58] or apparent relation to the aggressivity of the lesion.
Treatment and Prognosis The recommended treatment of chondroblastoma includes a biopsy to document the histology followed by curettage. Intralesional curettage can be combined with local adjuvant such as cryotherapy or phenol. The defects created by this lesion often require bone grafting with either autogenous or allogeneic bone. The occasional patient with a marked synovial reaction in the context of a known chondroblastoma should not have a synovectomy because the synovitis resolves after curettage of the tumor. Rarely, intraarticular tumor
extension occurs and simple excision of the synovial nodules is likely to be effective. There is no evidence that chondroblastoma heals spontaneously, and early surgical treatment is recommended to avoid extension of the lesion across the epiphysis, through the cortex, or into the joint. Thorough curettage is crucial; thus, the operative approach should permit adequate exposure of the lesion, using an intraarticular approach and even dislocation if necessary. Despite the fact that significant subchondral erosions are typically produced by this tumor, arthrodesis or joint reconstruction are indicated in only a minority of patients. Overall, the prognosis is good for most patients. The rate of local recurrence is 10-14% [57,58]. Treatment of recurrences generally follows the same guidelines as that for primary tumors. Careful preoperative imaging is required for optimal surgical results. This lesion is one of the few benign tumors that has a small risk of associated pulmonary nodules [58]. The pulmonary metastases have the same benign histologic appearance as the primary lesion. If pulmonary nodules occur, they should be removed whenever possible. C h o n d r o m y x o i d Fibroma First described by Jaffe and Lichtenstein [59] in 1948, chondromyxoid fibroma is the least common of the benign cartilage tumors, accounting for less than 1% [4,59,60].Males are affected approximately twice as often as females. Age at presentation varies from 10 to 30 years. Most patients complain of dull aching pain, sometimes of several years' duration. Swelling (or a mass) is the second most common symptom. Occasionally, the lesion is discovered as an incidental finding. Approximately 10% of the cases reported by the Mayo Clinic presented with a pathologic fracture [4]. Typically, it affects the metaphysis of long bones, of which 30% involve the proximal tibia. It affects the distal femur almost as frequently, followed by the foot and pelvis. When located in the foot, it tends to be more central, involves most of the bone involved, and looks more aggressive than in other sites. When the iliac bone is involved, it also tends to be larger but is otherwise typical. In the long bones, the location is usually metaphyseal at a variable distance from the physis. In some cases, the lesion may transgress the physis and involve both epiphysis and metaphysis. No cases of multicentric disease have been reported [4,25,59-61].
Diagnostic Imagery Classically, the plain radiograph demonstrates a lytic eccentric lesion with a sharply circumscribed zone of
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rarefaction delineated by marginal sclerosis, thinned or destroyed cortex, and, rarely, fine matrix calcifications. The cortical outline is absent in 8% of cases [4]. Some expansion of the bone may occur as periosteal apposition follows continual endosteal resorption. In a small bone, this may produce fusiform expansion of the entire bone. Pseudotrabeculation is a characteristic sign in this lesion and is an image created by the residual ridges on the periphery of the lesion (Fig. 14). The usual diameter of the lesion varies from 1 to 10 cm. As with calcifications, a periosteal reaction is unusual. Nuclear bone scintigraphy will consistently show abnormal activity with chondromyxoid fibroma [62]. With MRI, the chondroid tissue, myxoid tissue, and normal hyaline articular cartilage appear as an intermediate to high signal density both in proton density and T2weighted images. The fibrous tissue has variable vascularity, which can be demonstrated with gadolinium enhancement [63]. The differential diagnosis includes nonossifying fibroma, aneurysmal bone cyst, enchondroma, and chondroblastoma.
Pathology As the tumor is exposed by the surgeon, it may have a fibrous appearance or resemble hyaline cartilage. Myxoid foci, hemorrhages, cystic areas, and calcific deposits may be found. A matrix with a liquid mucinous quality is suggestive of chondrosarcoma [4]. Surgically and grossly, chondromyxoid fibroma is characterized by its sharp demarcation from surrounding bone. Lobulations of the tumor are frequent and match the corrugations of the residual bony cavity. Histological findings include a lobular-appearing tumor containing cells with stellate or spindle-shaped features in a myxomatous or chondroid matrix. The larger lobules often demonstrate increasing peripheral cellularity. The stroma surrounding and separating the lobules contains oval to spindle-shaped cells accompanied by various amounts of mulinucleated giant cells and scattered blood vessels. Microscopic variation is often observed in different fields of a given tumor. Calcification and hyaline cartilage are less often seen. Occasionally, bizarre or atypical nuclei are present, but mitotic activity is rare or absent. The differential diagnosis includes chondroblastoma, chondroma, chondrosarcoma, and chondroblastic osteosarcoma [4,64,65]. Although not yet useful from a clinical standpoint, simple clonal aberrations have been reported in chondromyxoid fibroma [66,67].
Treatment and Prognosis Curettage is the treatment of choice for most cases of chondromyxoid fibroma, usually followed by grafting with autogenous or allograft bone. The use of strut (structural) grafts has been reported, as has the use of polymethylmethacrylate cement. In some cases, en bloc excision has been performed for this tumor. With curettage, a recurrence rate of 12.5-25% has been reported [4,64,68], whereas with resection recurrence is less likely [61,62,65] but may involve greater morbidity. Recurrence of this relatively aggressive benign tumor should generally be treated in a similar manner as primary lesions. Chondrosarcoma
FIGURE 14 Pseudotrabeculations are a characteristic sign of chondromyxoid fibroma, and represent residual ridges after deep endosteal scalloping.
This malignant cartilage tumor is extremely rare in children [69]. Classic chondrosarcoma occurs in adults older than 30 years of age and is estimated to account for 11% of all malignant bone tumors in all age groups. Most are primary, but some arise from an enchondroma or an osteochondroma. Primary chondrosarcomas favor the pelvis or proximal femur. They present with local swelling, pain, and a mass that tends to be hard.
29. Bone Tumors in Children
Mesenchymal Chondrosarcoma This rare subtype of chondrosarcoma was originally described in 1959 by Lightenstein and Bernstein [70]. It is even less common than clear-cell chondrosarcoma, which even in adults is a very rare tumor (it can be considered as the malignant counterpart to chondroblastoma and occurs almost exclusively in the femoral head). Mesenchymal chondrosarcoma most commonly arises in the bone but has also been found in the soft tissues [71]. Pain, mass, and, occasionally, pathologic fracture are presenting symptoms. Systemic signs such as asthenia and fever have been noted in some patients. In a review of 111 patients with mesenchymal chondrosarcoma, Nakashima and associates [72] reported that the 5-year survival rate after wide resection was 60% and the 10-year survival rate was 25%. In this group of patients, 57 of whom were female and who ranged in age from 4 to 74 years, the ribs, spine, pelvis, and femur were the most common osseous sites.
Diagnostic Imagery On plain radiographs, classic chondrosarcoma presents as a radiolucent lesion with calcifications and variably, reactive bone formation. Common findings include fusiform expansion of the shaft and cortical thickening as well as matrix calcification and signs of cortical destruction. The loss of a homogenous pattern of calcification, the appearance of new endosteal erosions, or the increased size of an enchondroma on serial radiographs
721
are signs suggestive of malignant degeneration, as is the presence of new or progressive pain. In contrast, mesenchymal chondrosarcoma is more likely to resemble osteosarcoma in its localization and its appearance. Aggressive lytic changes, periosteal reaction, soft tissue extension, and (reactive) bone formation predominate, whereas matrix calcifications are not always present (Fig. 15).
Pathology The usual dimensions of a periosteal chondroma are rarely >3 cm, whereas chondrosarcomas rarely measure <5 cm. Gross pathological examination is characterized by lobules of variable dimension (from a few millimeters to several centimeters). The matrix varies from a firm hyaline appearance to a more myxoid stroma that is highly suggestive of malignancy. It is often difficult to differentiate low-grade chondrosarcoma from chondroma, and expert consultation is necessary. Sampling error can frequently be misleading, and correlation with clinical data such as location, symptomatology, and radiological evaluation is essential. Mesenchymal chondrosarcoma is unlikely to be confused with classic chondrosarcoma by the pathologist, particularly in the context of multidisciplinary consultation (i.e., diagnosis is made with knowledge of the clinical and radiological features of the case). However, if the cartilaginous component is not sampled by the biopsy, a small, round, blue cell tumor may be diagnosed. It is composed of nodules of benign-appearing
FIGURE 15 MesenchymalChondrosarcoma in bone creates a destructive lesion with reactive changes and aggressive periosteal changes, an image easilyconfused with osteosarcoma, a much more common lesion, a) X-ray; b) CT
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Marc H. lsler and Robert E. Turcotte
cartilage within a background of undifferentiated small, round cells. The undifferentiated small, round cell component may have the histologic appearance of a hemangiopericytoma.
Treatment Wide surgical resection is the only proven curative modality for classic chondrosarcoma; adjuvants such as chemotherapy and radiotherapy have not been effective. Local recurrence is likely if margins are not adequate. Mesenchymal chondrosarcoma is also treated by wide or radical surgical resection. Chemotherapy may be considered depending on the clinical presentation, but its role is controversial. Radiation has not been used routinely for sarcomas of this type [25,69].
The main radiographic feature is extensive thickening (sclerosis) of the involved cortex, secondary to the progressive deposition of reactive mature lamellar bone by the periosteum. These findings are reversible after cure of the disease and are also absent if no periosteum is present (e.g., in the femoral neck). Although often spectacular, sclerosis represents an accessory finding. The actual tumor is a small lytic defect (<1 cm) within the reactive bone called the nidus. Typically, the nidus shows a small calcification in its center. The nidus may be difficult to identify, especially in cancellous bone or near an articular surface where the sclerotic reaction can be absent (Fig. 16). In conjunction with a typical clinical presentation, CT scan with thin slices will likely show the nidus clearly and is then considered diagnostic. Technetium bone scan is 100% sensitive to osteoid osteoma and thus serves as an
BONE-FORMING TUMORS Osteoid Osteoma Osteoid osteoma is a relatively frequent benign tumor of bone, accounting for 14% of all bone tumors in some series. It can occur at any age, but 75% involve patients ages 5-20 years. Males are three times more likely to be affected than females. Although any bone can be affected, this tumor shows a predilection for the ends of long bones. Its neoplastic nature is unclear, and by definition it is nonprogressive. Symptoms caused by osteoid osteoma are among the most characteristic of any tumor. Severe pain is the predominant feature and is typically worse at night and unrelated to the level of activity. Interestingly, pain is often not alleviated by the use of opiates but is markedly relieved by the use of salicylates and other inhibitors of prostaglandins. Referred pain and muscle wasting are frequent findings. Location near a joint can result in effusion that may suggest arthritis or another articular pathology. In the spine, the lesion is usually located in the posterior elements (pedicle, lamina, and spinous process) and will classically present with painful scoliosis. The lesion can then be found in the neural arch of the apical vertebra on the concave side [73,74].
Diagnostic Imagery Although occasionally present on the endosteal or periosteal surface of long bones, osteoid osteoma is typically an intracortical lesion. Differential diagnosis includes mainly stress fracture and subacute (Brodie's) abscess since other cortical lesions tend to be larger.
FIGURE 16 a) Osteoid osteoma is characterized by a small nidus surrounded by dense reactive bone. b) Fine slices of CT scan are the most reliable means of demonstrating the nidus and can be considered diagnostic in most cases.
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excellent tool to screen for this and other lesions. A focal increase in uptake of radioisotope can be very precise but is not considered specific for osteoid osteoma. MRI can be helpful, but sometimes the important edema noted on T2-weighted images hides the nidus [74,75].
Pathology Macroscopically, the nidus is seen as a small red lesion that is sometimes referred to as a cherry-red spot. Histologically, the lesion consists of tightly woven osteoid trabeculae lined by osteoblasts. Loose connective fibrovascular tissue is found between the immature trabeculae. This histological appearance is indistinguishable from that of osteoblastoma. These lesions are richly vascularized and innervated. They have been shown to produce high levels of prostaglandins [73,75].
was reported to be useful and consists of removing the entire nidus percutaneously under CT guidance. The risk of fracture remains significant due to the size of the defect created, meriting restrictions of activity for several weeks if not months. Thermal ablation techniques have been developed using either a laser or a microwave probe (radiofrequency ablation). Like percutaneous drilling, these techniques allow a core biopsy to be performed and at the same time precise localization of the nidus with CT guidance. However, the size of the defect is 2 or 3 mm, thus rendering the risk of fracture extremely low. Since the diameter of necrosis produced by the techniques approximates the size of the nidus, the rate of success is at least as good as that with traditional surgical techniques and probably better than that with medical treatment. Both procedures can be performed on an outpatient basis under general or regional anesthesia and do not require activity restriction [73,76,77].
Treatment Historically, surgery has been the mainstay of treatment. It is now known that only the nidus needs to be removed because the sclerotic reaction is accessory and resolves after successful removal of the nidus. Because the nidus is small and difficult to locate, careful preoperative planning is essential to avoid morbidity from removal of excessive bone and, more important, to increase the chance of successful removal of the nidus. Several methods have been devised to locate the nidus, including the insertion of a needle or the injection of dye under CT guidance immediately before surgery to mark the site of the nidus. Another method involves the intravenous injection of radioisotope (99Tc) a n d the use of a gamma probe during surgery to precisely locate the nidus. Tetracycline has also been used less successfully to mark the area of increased bony turnover. En bloc excision of the nidus with radiographic and pathologic confirmation of its removal or shaving bone until the nidus is exposed for curettage have both been successful in eradicating the disease in approximately 85% of cases. These approaches may weaken the bone, requiring internal fixation or at least prolonged activity restriction to avoid fracture. Apart from the usual risks of surgery, this morbidity led to attempts to treat the condition medically with prolonged courses of nonsteroidal antiinflammatory medication and to treat it with alternate, percutaneous methods of nidus removal. With NSAID treatment, more than 80% of patients can be cured of the disease after an average of 4 years. This is applicable to patients who are completely relieved of their symptoms and who tolerate the medication well. Recently, minimally invasive techniques have been developed to minimize morbidity and avoid the need for prolonged medication. Percutaneous core drilling
Osteoblastoma This benign aggressive osteoblastic lesion accounts for only 1% of all primary bone tumors. Males are affected twice as often as females. Most patients are younger than 30 years old. Pain of long duration is the usual complaint and is not readily relieved by salicylates or NSAIDS, in contrast to osteoid osteoma. Rest pain is often present. Infrequently, the patient may be asymptomatic. When the spine is affected, the patient presents with painful scoliosis, similar to osteoid osteoma. On physical examination, muscular atrophy of the affected limb may be found. Bony tenderness and a palpable mass are common. As noted previously, spinal lesions can be accompanied by scoliosis, but neurological signs of compression have only rarely been reported [73,78,79].
Diagnostic Imagery In addition to the spine and sacrum, for which this tumor has a marked predilection, any long bone can be involved. In order of decreasing frequency, the femur, tibia, and skull are relatively common sites. The metaphyseal and diaphyseal segments of long bones are usually involved. By definition, the lytic lesion is >2 cm (to distinguish it from osteoid osteoma), with variable reactive or tumoral bone formation. In general, there is less sclerotic change in the surrounding bone than occurs with osteoid osteoma. The destructive nature of the lesion can be extensive, and aneurysmal or cystic changes can occur. The periosteal reaction surrounding the soft tissue extension of the more aggressive lesions may be discontinuous (Fig. 17).
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lesions seem to behave like low-grade osteosarcomas after recurrence.
Fibrous Dysplasia
FIGURE 17 Osteoblastoma is among the most frequent primary lesions of the spine in children. Destruction of bone is variable and is accompanied by neoplastic bone formation.
Bone scans will show an intense increase in activity with most lesions. CT scans help to show details of bone destruction and further qualify the aggressive nature of the tumor, whereas MRI is useful to delineate soft tissue extension, especially spinal extension. Both these modalities can show fluid levels if aneurysmal cyst degeneration develops [78,79].
Pathology The histopathology of this lesion is usually indistinguishable from that of osteoid osteoma. However, some lesions show more aggressive characteristics, with a spectrum that can even mimic low-grade osteosarcoma. (This spectrum is mirrored by variations in radiological and clinical behavior.) Interconnected trabeculae of bone exist in a fibrovascular stroma, with osteoblasts and osteoclasts rimming the trabecular surfaces. The cells retain cytologically benign characteristics. Areas of the tumor may resemble giant cell tumor, aneurysmal bone cyst, or both. A key feature used to differentiate this lesion from osteosarcoma is the absence of evidence of infiltration or sequestration of normal bone. In osteoblastoma, the lesion merges with the adjacent bone [73,79-81].
Treatment Whenever possible, en bloc excision is preferred since the rate of recurrence is significant. This is particularly true for expendable bones. The risk of recurrence after intralesional excision is 200, partly related to soft tissue extension in difficult anatomical locations such as the spine. After curettage, most lesions require either bone grafting or cementation. Radiation therapy should be avoided if possible, but it may be required for some spinal locations [73,78,79]. Careful follow-up is mandatory since recurrence is a problem and some
Fibrous dysplasia (FD) is a condition in which defective maturation of osteoblasts results in abnormal bone formation. This poorly organized bone is of poor structural quality and may lead to deformation or pathologic fracture of the involved bone. Extension of the process is mediated by increased osteoclastic activity and bone resorption [10,82]. Its name was proposed' by Jaffe and Lichtenstein in 1942 [83], and it represents approximately 1% of primary bone tumors. It is diagnosed most commonly in adolescents, although approximately one-fourth of the lesions are discovered in adults (age range, 5-30 years). Younger age at diagnosis carries a worse prognosis, with more extensive involvement and a higher prevalence of fractures or bone pain. The monostotic form is six times more common than the polyostotic form. It occurs slightly more often in females than in males, but females account for the majority of cases of McCune-Albright syndrome (MAS). Polyostotic disease is present in 25% of patients. The monostotic form of FD is usually an incidental finding, whereas polyostotic disease is more likely to cause pain, deformation, and loss of function. The most common sites of monostotic involvement by order of decreasing frequency are the maxilla, proximal femur, tibia, humerus, ribs, skull, radius, iliac bone, and cervical spine. The proximal femur is a site of predilection in polyostotic disease. Any bone can be involved, but hands, feet, and vertebrae are rarely involved. The distribution is unilateral in most cases of polyostotic FD including MAS. The distribution can be multifocal, with involvement of a lower limb and its hemipelvis or the upper limb (monomelic form), or it can be hemicorporal. In long bones, centromedullary disease begins in the metaphyseal region and extends toward the diaphysis. The open growth plate represents a true barrier to disease progression. Twenty percent of cases will present an association with pigmented skin lesions (caf6 au lait with "coast of Maine" borders) often located near the bone lesions. MAS is a combination of polyostotic FD, caf~ au lait skin pigmentation, and endocrinopathies, and it is the most severe manifestation of this spectrum of disease. The combination consisting of precocious puberty, polyostotic disease, and caf6 au lait markings on the skin constitutes the findings of classic Albright's syndrome [84-86].
29. Bone Tumors in Children
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Mazabraud syndrome is a rare combination of intramuscular myxomas and polyostotic FD, with usual skin lesions. The myxomas tend to grow and require resection, tend to recur, and can exhibit malignant transformation. Females are affected in the majority of cases [87]. Sarcomatous transformation of the lesions of F D can occur with or without prior irradiation. The incidence of this complication is 0.3% for monostotic disease and 4% in MAS. In order of frequency, osteosarcoma, fibrosarcoma, chondrosarcoma, and malignant fibrous histiocytoma (MFH) have all been described. The facial bones and the skull are most often affected, followed by the proximal femur. Progressively increasing pain, rest pain, a mass, and progressive lysis and soft tissue extension are hallmarks of sarcomatous transformation in FD. Therapeutic irradiation increases the risk of malignant transformation. A case of osteosarcoma has been described that occurred when bone was lengthened through a F D lesion. These cases generally have a poor prognosis and should be treated promptly and aggressively [82,88-90]. (see chapter 21.)
Etiology F D is a rare sporadic affliction featuring defective maturation of bone in one or more locations of the skeleton. It is absent at birth. A characteristic mutation of the GNAS1 gene is associated with the 20q 13 gene locus and is responsible for the abnormal maturation of not only osteoblasts but also other target tissues, resulting in a spectrum of skin pigmentation and endocrinopathies [82].
FIGURE 18 Classicalhallmarks of fibrous dysplasia include a lytic centromedullary lesion in a diaphyseal or metaphyseal location, with well-defined mildly sclerotic margins, a matrix of intermediate, homogenous ('ground-glass') density, endosteal scalloping (resorption). Fracture may occur with minimal trauma, as in this 14 year old who felt pain and heard a cracking sound as he swung a baseball bat.
Diagnostic Imagery In most cases of FD, the radiological picture is sufficiently typical to make a diagnosis. Biopsy may be required for those cases with less than typical radiological signs or incompatible history and physical examination. Classical hallmarks of F D include a lytic centromedullary lesion in a diaphyseal or metaphyseal location with well-defined mildly sclerotic margins, a matrix of intermediate, homogenous (ground-glass) density, and endosteal scalloping (resorption) (Fig. 18). The affected bone may demonstrate an increase in diameter. Some lesions may also present more distinct areas of matrix calcification; this is more frequent in flat bones. Repeated microfractures in affected bone are caused by its progressively worse mechanical qualities and result in progressive deformation of bone. Weight-bearing segments are affected, resulting in tibial bowing (lateral or anterolateral) and, classically, a varus deformity of the proximal femur (Sheperd's crook) or the so-called parrot's beak deformity of the femoral neck.
Pseudarthrosis can occur, although in the context of adequate immobilization bone healing occurs in a normal time frame. Rarely, spinal involvement is characterized by wedging and matrix alterations and can lead to thoracolumbar kyphosis in children. The radiological image can vary according to age. Purely lytic lesions are seen in young children. In the skeletally mature, stabilization of the disease process is usually seen (although major varus deformity can explain progression) and occasionally involution of the lesions is seen, with progressive sclerosis of the margins. Some lesions involute to a cystic form [10,82]. CT scan can be useful, particularly in flat bones, to define the extent of disease more precisely, but it is of limited diagnostic value. M R I can be useful, like CT, in evaluating the extent of maxillofacial disease. Radioisotope bone scans can be used as a screening tool and may detect lesions not visible on plain radiographs, but false-negative studies are not infrequent.
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Marc H. lsler and Robert E. Turcotte
Gallium is a more sensitive marker than technecium. The finding of multiple lesions in a hemicorporeal distribution is suggestive of MAS [82,91].
Pathology The medullary cavity is widened, with replacement of normal tissue by firm, pale yellow fibrous tissue. Abnormal woven bone formation is dispersed within the fibrous stroma in irregular shapes evocative of Chinese characters and notable for the absence of peripheral osteoblastic rimming.] The fusiform cells of the stroma may be swollen but otherwise show no atypia, and they are often arranged in a storiform disposition. Variably, there may be foamy macrophages, multinucleated giant cells, myxoid areas, and areas of chondral differentiation (with or without calcifications) [10].
Differential Diagnosis Although most cases are simple to diagnosis, FD may resemble other fibrous lesions as well as simple bone cysts (vs involutional change in FD), enchondromas in the young child, eosinophilic granuloma, and osteofibrous dysplasia (ossifying fibroma).
Treatment Treatment of FD remains challenging in both children and adults. The typical patient who has a large, painful monostotic or polyostotic lesion usually benefits from intramedullary fixation of that osseous segment. A biopsy is required in most surgical situations. In general, intralesional resection (curettage) of these lesions is not indicated because it may lead to recurrence and worsen the already poor mechanics of the diseased bone. It is useful to consider treatment in terms of biomechanical correction rather than tumor eradication. This can involve simple prophylactic fixation to avoid deformation and relieve pain (no single form of fixation is considered superior, although some form of intramedullary fixation is often used) or realignment of a deformed bone using osteotomies and fixation. Although some authors have proposed a 90 ~ varus deformity of the femoral neck as a threshold for surgical correction, it appears that both correction and fixation are easier and more likely to be successful if performed when the cervicodiaphyseal angle is approximately 120 ~. Nonsurgical (i.e., orthopedic) treatment of fractures in FD tends to result in residual deformity, which in turn alters the biomechanical situation and predisposes to further fracture, deformation, and pain. Bracing and cast immobilization are used as temporary measures to
relieve pain while awaiting definitive surgical treatment [10,92]. Recently, however, biphosphonates have been used with some success to relieve bone pain and in some cases to reverse osteolysis. The underlying assumption is that osteoclastic activity is increased in FD, is a mechanism of disease progression, and thus is at least partially responsible for the pain and functional handicap associated with the disease. Since biphosphonates have been successfully used for a variety of conditions in which increased osteoclastic activity is a common denominator, it is logical to think they may have applications in FD [93].
Prognosis The overall prognosis for a patient with FD depends on the severity of involvement, which is related both to the involvement of individual bones and to the number of lesion sites [94,95]. The prognosis is good for a typical child who has a monostotic lesion because the pain can usually be mitigated with prophylactic internal fixation. Although the development of secondary osteosarcoma in a FD segment of bone has been reported in the literature, it is a rare complication [4,95]. The development of osteosarcoma from FD is demonstrated radiographically by progressive erosion of bone or clinically by a soft tissue mass or both, and also by increasing bone pain. The method of treatment should be selected carefully according to the severity of involvement and the radiographic and clinical findings for each patient. O s t e o f i b r o u s Dysplasia
Synonyms: Osteitis fibrosa, fibrous dysplasia-like lesion, intracortical fibrous dysplasia, congenital fibrous defect of the tibia, and ossifying fibroma. This lesion is a congenital dysplasia that involves the tibia or, less often, the fibula, almost exclusively. It consists of fibrous and osseous tissue, has a progressive course in most cases, and has been referred to as a hamartoma. It has often been confused with FD, but age, site of involvement, clinical course, radiographic features, and histology all have distinguishing features [96]. Diagnosis is usually made before the age of 5 years (range, 1-35 years). It may be diagnosed in the newborn [97]. Males are affected more than females (3:2 ratio). In almost all cases the tibia is affected, with some ipsilateral fibular involvement. Bilateral involvement of the tibiae has been reported. The middiaphyseal region is the most commonly affected region of the tibia, whereas in the fibula it usually involves the distal third. Patients present because of deformation and cosmetic changes caused by bowing and thickening of the subcutaneous tibia. Fatigue fracture may cause pain.
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Diagnostic Imagery This lesion is characterized by eccentric intracortical bone destruction with variable expansion and bowing of the bone. Thinning of the cortex may be accompanied by fracture, usually undisplaced. Within the bone, a sclerotic margin is usual. The medullary canal is often obliterated to some degree. The lesion may have the appearance of multiple bubbles. The density of the matrix varies from purely lytic to an intermediate density with a ground-glass appearance (Fig. 19).
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A zonal architecture can be seen if the biopsy allows study of the full thickness of the lesion. The center of the lesion is exclusively fibrous, and as one moves toward the surface of the lesion progressive ossification can be seen, with a progressive decrease in the density of the fibrous stroma. Although the origin of this disease is poorly understood, evidence suggests that there is a clonal and possibly neoplastic origin. It is also notable that trisomy 7 and trisomy 12 have been demonstrated in specimens of both osteofibrous dysplasia and adamantinoma [98].
Pathology Grossly, the lesion is composed of compact tissue of a whitish, yellowish, or reddish color under normalappearing periosteum but thinned cortex. The consistency is often gritty. The histology consists of well-differentiated fibrous tissue embedding bony trabeculae with osteoblastic rimming. The density of the fibrous tissue is variable, often with a whorled (or storiform) pattern. Occasional multinucleate giant cells can be seen. The bony trabeculae are of woven bone in the center of the lesion, with progressive maturation to lamellar bone at the periphery.
Differential Diagnosis This lesion may occasionally resemble nonossifying fibroma on histological grounds, especially in the proximal tibia; however, the diagnosis should be clear on clinical and radiographic basis. More commonly, it is confused with FD. Most important, this lesion must be differentiated from a malignant lesion, adamantinoma, especially if the lesion causes pain or shows progression, especially after puberty.
FIGURE 19 Osteofibrous dysplasia, a) The radiograph illustrates tibial location, bowing, intracortical location of the lesion, well defined sclerotic borders, all typical features, b) On MRI the intracortical location of the lesion is indicated by the thick arrow and obliteration of the medullary canal (thin arrow) can also be observed.
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Treatment This is a subject of considerable controversy. Campanacci and Laus [96] stated that surgical treatment should be avoided or at least delayed as long as possible because spontaneous resolution is sometimes observed and because recurrence after curettage is frequent and may favor extension of the disease process. They noted that when fracture occurred, simple immobilization almost always resulted in healing of the fracture. They limited surgical indications to 1) forms of the disease that are so extended that they considerably expand and/or weaken the bone, in which case it is best to use tangential resection by extraperiosteal approach with bone grafting; 2) the presence of pseudarthrosis, in which case they advocated solid ostosynthesis associated with strong bone grafting, and 3) accentuated tibial bowing, which should be corrected by osteotomy when patients are 10-12 years of age. On the other hand, some evidence indicates that this lesion is a precursor for adamantinoma, in which case a more aggressive approach may be warranted. Although current recommendations are conservative, it is important to note that when the clinical course is not favorable, biopsy is justified. If an incisional biopsy does not demonstrate the epithelial nests typical of an adamantinoma, then curettage should be done to allow complete histopathological examination of the tumor to rule out sampling error. Furthermore, patients with osteofibrous dysplasia should be followed indefinitely or until regression of the lesion occurs [96,99].
Osteosarcoma Osteosarcoma is defined by the production of osteoid by malignant cells. This must be differentiated from the metaplastic or reactive bone formation often seen in tumors. Osteosarcoma is the second most common malignant primary bone tumor after myeloma and comprises 20% of tumors. The population-based incidence is estimated to be 2 cases per 1 million per year. It is most frequent during the second decade of life, and males are more frequently affected than females (3:2 ratio). Any bone can be affected by osteosarcoma, although it has rarely been reported in the small bones of the hands and feet. It typically involves the metaphyseal area of long bones, and more than 50% of all cases of osteosarcoma involve the knee. Osteosarcoma is usually a primary lesion, although secondary osteosarcoma has been shown to arise on preexisting benign lesions, such as in FD, bone infarction, and Paget's disease. Radiation therapy, however, is the most common cause of secondary osteosarcoma and carries a poor prognosis. Secondary osteosarcoma rarely, if ever, occurs in children.
Primary osteosarcoma can be subdivided into categories based on histological differentiation and other features. In children, almost all cases are of the classic high-grade centromedullary variety and mostly chondroblastic in differentiation. The periosteal variant is rare but typically affects teenagers in the diaphyseal area, producing a high-grade chondroblastic surface tumor. Telangiectatic subtypes present cystic change, can occur at any age, and may carry a worse prognosis. Symptoms at presentation include pain of rapid evolution, swelling or a mass, muscle wasting, and, depending on the location, joint effusion. When the tumor is large, distension of the skin and enlargement of the superficial veins can be seen. Pathologic fracture can occur, either on presentation or as a complication during treatment, and it significantly complicates the situation [100,101]. Laboratory studies are mostly normal, but erythrocyte sedimentation rate can be elevated although not specific. Lactate dehydrogenase and serum alkaline phosphatase elevation may be negative prognostic indicators.
Diagnostic Imagery The radiographic appearance of central high-grade osteosarcoma is usually one of mixed blastic and lytic changes involving the metaphyseal portion of a long bone. In 7% of cases, the diaphysis of long bone is involved. The margins of the lesion are usually poorly defined and the cortex is commonly disrupted with tumor extending into the adjacent soft tissues. The periosteum can be elevated by the tumor, and deposition of nonneoplastic bone results in the classical sign known as the sunburst reaction or Codman's triangle (Fig. 20). Formation of bone within the lesion is a classic and relatively common finding. Violation of the epiphyseal plate or invasion of adjacent joint or capsule and ligaments are not rare with osteosarcoma. Bone scan will be very active and is a useful screening tool for multifocal disease. Chest CT is used to rule out pulmonary metastasis, the most common mode of distant spread of this disease, as it is for most sarcomas. MRI is an important local staging modality because it allows precise appraisal of the medullary extension of the disease as well as the extraosseous soft tissue extension. This modality has allowed surgeons to correctly assess tumor extension and safely perform limb-sparing surgery in the majority of cases (Fig. 21) [101-103].
Pathology The histological picture is one of highly malignant sarcomatous stromal cells producing variable amounts of osteoid. Mineralization of the matrix is highly
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FIGURE 21 Osteosarcoma. MRI is the modality of choice for local staging as it defines the tumor extension both in the medullary cavity (short wide arrow) and in the soft tissues (long narrow arrow).
lesion with indistinct borders. Macroscopically, the tumor reveals large cystic blood-filled cavities with multiple septae. These septae are lined by malignant cells and lack a true endothelial lining. Osteoid production is often minimal and merits thorough pathological examination. Giant cells of the benign type are frequently seen. The main differential diagnosis is with aneurysmal bone cyst. Treatment is the same as that for conventional osteosarcoma. FIGURE 2 0 a) Osteosarcoma is often accompanied by aggressive forms of periosteal reaction. The lower arrow indicaties a Codman's Triangle, indicative that rapid growth of the lesion is elevating the periosteum. The upper arrow shows patchy increases in bone density, suggestive of neoplastic bone formation, b) The spiculated type of periosteal reaction creates a sunburst effect.
variable. Other types of tissue formation can occur, such as the formation of chondroid matrix in the chondroblastic subtype, and these characteristics can predominate. Even generous biopsy samples do not always reflect the bone-forming portions of the lesion. Areas of necrosis are frequently seen. The histological subtypes carry a similar prognosis and are treated in a similar fashion [100,104]. Telangiectatic osteosarcoma is a rare variant that represents less than 3% of all osteosarcomas. It is characterized radiographically by the existence of a large cyst-like
Treatment
Osteosarcoma is usually treated with neoadjuvant chemotherapy for high-grade tumors followed by local wide resection. In approximately 85% of cases, limbsparing surgery can safely be performed (Fig. 22). Following local control, chemotherapy is continued for several months, depending on the protocol. Low- grade osteosarcoma is treated by wide surgical resection alone. When the patient presents with lung metastases, these are resected, if feasible, at the same time as the local disease. This aggressive approach results in a 5-year disease-free survival (DFS) rate of approximately 40%, whereas the patient without lung metastases can expect a 5-year DFS of 75%. The best prognostic indicator is the extent of tumor necrosis after neoadjuvant chemotherapy, with >90% necrosis being a favorable response.
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FIGURE 22 As in all sarcomas of the extremities in children, limb salvage surgery for osteosarcoma is possible in most cases, a) This 15 year old boy carries a voluminous soft tissue mass which is the manifestation of the advanced osteosarcoma affecting his humerus. (shown in Figure 20b and 21) b) Postoperative radiograph after resection and reconstruction using a massive allograft.
Radiation therapy has not been useful except for palliative treatment in rare cases [100,101,105,106].
HEMATOPOIETIC Leukemia Leukemia is a malignant neoplasm of the hematopoietic stem cells that usually involves bone marrow diffusely. These cancers are classified as acute, which is typified by blasts or immature cells, or chronic, in which the cells are mature or well differentiated. They are also classified according to the cell line of the malignant cells as myelogenic (myeloid cell line) or lymphocytic (lymphoid cell line). Thus, leukemias are called acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), or chronic lymphocytic leukemia (CLL). A fifth subtype is hairy cell leukemia (so called because of characteristic circulating lymphocytes with "hairy" cyto-
plasmic projections). Lastly, any leukemia can occur outside of the bone marrow (rare) and may then be named granulocytic sarcoma, or called a chloroma. ALL is a disease of childhood and A M L can occur at all ages, whereas CLL and C M L only occur in adults. Children present with bone pain, often fleeting and migratory, or polyarthralgia of a similar nature. Diffuse marrow replacement leads to asthenia, bleeding disorders, and decreased immune response to injury and infection. Apart from painful swelling at the involved site, lymphadenopathy and visceromegaly can be found. Anemia and other bloodline anomalies can be demonstrated on a complete blood count. Bone marrow studies are diagnostic, but various genetic and molecular techniques are used to determine the subtype of the disease.
Diagnostic Imagery In children, lesions tend to occur in areas of rapid growth, such as the proximal tibia, distal femur, distal
29. Bone Tumors in Children radius, and ulna. The lesions may be focal lytic lesions (50%) or areas of calcifications representing infarction, but metaphyseal bands are characteristic (if not specific) and are due to interference with normal bone formation adjacent to the growth plate (Fig. 23). Periosteal reaction and limited permeative changes can also occur. Pathologic fracture can occur, and in advanced disease a generalized osteopenia can be seen. Bone scan can show multifocal disease.
Treatment Biopsy is only required if complete blood count or marrow aspiration are not diagnostic. These patients are managed by a pediatric hematooncologist. Lymphoma Lymphoma of bone is rarely seen as a primary tumor, even in adults. Hodgkin's lymphoma and other
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lymphomas combined account for approximately 10% of malignant bone lesions. The vast majority of these occur in patients older than age 20, but primary lymphoma of bone does occur in children. When non-Hodgkin's lymphoma is limited to bone, it is also known as reticulum sarcoma of bone. Many clinical features of this disease are similar to those of Ewing's sarcoma, such as permeative bone lytic changes (Fig. 24), soft tissue mass, and the presence of systemic symptoms. However, the location of lymphoma may or may not be typical for Ewing's sarcoma. The differential diagnosis is occasionally difficult using only immunohistochemical data, and correct specimen handling is crucial to avoid inappropriate diagnosis and treatment. Lymphoma may also resemble eosinophilic granuloma and leukemia. The detection of specific genetic translocations is becoming part of the diagnostic strategy for these and other tumors. Staging studies include bone marrow aspiration, chest and abdomen CT, and whole body gallium scans. Treatment includes chemotherapy and usually radiotherapy, to which this tumor responds predictably, often with complete restitution of bony morphology [107,108].
VASCULAR Hemangioma Hemangioma of bone is a benign lesion that is more often latent than active. Although it most commonly affects adults in the fourth to sixth decades, children can be affected, most often in a vertebral location. Males and females are affected equally. Congenital hemangiomatosis is a rare variant of this tumor, affecting both soft tissues and bone in an aggressive infiltrative fashion. These are usually a mixture of capillary and cavernous hemangiomas. Limb length discrepancy is not unusual and is caused by increased flow to adjacent growth plates. Only a small percentage of these patients have direct bone involvement. Massive osteolysis or Gorham's disease is also a rare variant that can affect children. Its relation to hemangioma is unclear but generally accepted, and its clinical course is unpredictable. Some cases show a self-limited course, whereas others have significant morbidity from bone destruction. It can affect any bone. Vertebral involvement can cause pain, vertebral collapse, and occasionally neurologic compromise.
Diagnostic Imagery FIGURE 23 metaphysealbands are a characteristic sign of bone involvement in leukemia.
After the spine, the craniofacial bones, femur, humerus, and hands and feet are the usual locations, in
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FIGURE 2 4 Although rare, primary lymphoma of bone does occur, as in this 12 year old boy who presented with progressive pain and swelling of the knee. Although Ewing's sarcoma must be considered in the differential diagnosis, permeative lytic changes of bone such as these are not quite typical, and epiphyseal location is decidedly unusual for Ewing's sarcoma, a) X-Ray, b) CT
order of frequency. In the vertebra, the lesion first affects the body and then extends into the pedicles. The socalled corduroy effect is created by residual vertical bony trabeculae after lysis by hemangioma expansion (also called a jailhouse vertebrae) (Fig. 25). Axial imagery of the same changes has been described as honeycombing (Fig. 26).
FIGURE 26 Honeycomb patterns can be seen in hemangiomas with axial imagery (CT).
FIGURE 25 Vertebral hemangioma classically results in vertical striations leading to terms such as "corduroy vertebra" and the "jailhouse effect". Mixed sclerotic and lytic changes occur in other sites, such as in this femur.
Congenital hemangiomatosis is characterized by disproportionately long, slender bones with a box-like appearance at the ends, calcified phleboliths, and poorly delineated increased vasculature on angiography. Radioisotopic bone scan shows little increase unless the bone is directly involved. When massive osteolysis (Gorham's disease) occurs, massive lysis can be seen crossing a joint space.
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Treatment
Pathology
Biopsy is rarely required since many of these lesions are latent and plain radiographs are diagnostic. If a neurologic deficit occurs from vertebral collapse, anterior resection with or without preoperative embolization is indicated. Painful lesions without neurological compromise may be addressed surgically or by intralesional sclerotherapy in selected cases. Systemic treatment has not been successful. Surgical treatment of congenital hemangiomatosis is usually unrewarding, often leading to loss of function and amputation. Some of these cases have been successfully treated by percutaneous sclerotherapy [1,109].
In viable areas, solid masses of tumor appear graywhite, glistening, moist, and translucent. Necrotic areas are liquefied, gray, or pus-like. On histological examination, the tumor is remarkably cellular, with little intercellular stroma, regular sheets, and cords of monotonously similar small, round, blue cells with round to oval nuclei, slightly granular cytoplasm, and indistinct cell outlines. A specific chromosomal translocation has been identified for Ewing's sarcoma [t(11;22) (q24;q12)] and is being increasingly used for diagnosis. Also, it is hoped that sensitive screening techniques for abnormal protein products of this translocation will serve to detect minimal residual disease after treatment.
NEUROGENIC
Ewing's S a r c o m a a n d Primitive Neuroectodermal Tumor This small, round, blue cell tumor is one of the most lethal of bone sarcomas. It accounts for approximately 6% of all malignant tumors, affecting patients in the first to third decades; however, the majority of patients are immature. This is the second most frequent malignant tumor in the teenage group. Males are twice as likely to be affected as females. It rarely affects blacks and Asians [1,110]. Although the first description of this tumor was provided in 1921, the cell of origin is still unclear. It is thought to arise from the nonhematopoietic elements of the medullary cavity. Any bone may be involved, but the diaphysis of long bones (particularly the femur) is the most common site. The pelvis and other fiat bones are affected in 40% of cases [1,111]. Pain and swelling, a palpable tender mass, dilated veins over the tumor, and febrile symptoms (fever and elevated ESR) are frequent findings [25].
Treatment The surgeon who performs open drainage of an abscess must send nonnecrotic material for histopathological examination since its clinical and radiological presentation may be difficult to distinguish from that of
Diagnostic Imagery Radiographs often show a large destructive lesion involving the entire shaft of a long bone. Lyric changes predominate, but variably reactive bone formation can be seen. Permeative lysis leads to thickened blurred cortices and plurilamellar periosteal elevation (onion skin). On MRI, extensive edema and bone marrow extension are the rule and a large soft tissue extension of the tumor is often seen (Fig. 27). Bone scan is used for systemic staging to screen for multifocal disease and chest CT is used to screen for lung metastases. Up to 30% of patients will present with distant metastases, signifying a very poor prognosis [25]. Bone marrow aspiration is a standard part of staging studies for Ewing's sarcoma.
FIGURE 27 Diffusepermeative bone destruction, diaphyseal location and significant soft tissue mass are highly suggestive of Ewing's sarcoma. This 6 year old girl presented with a limp, followed by fever, asthenia and a pelvic mass. MRI revealed extensive invasion and destruction of the left ilium with a voluminous soft tissue mass. The large arrow indicates displacement of the bladder by the tumour mass, while the small arrow shows the joint involvement. Incisional biopsy showed Ewing's sarcoma.
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Ewing's sarcoma. Likewise, it is prudent to obtain culture specimens when performing open biopsy of bone tumors. Multiagent neoadjuvant chemotherapy and a combination of surgery and/or radiation therapy has improved the 5-year rate of DFS from 10% to more than 40% (overall). Patients with localized disease have a 60-70% chance of being disease free at 5 years. In general, pelvic lesions fare worse and distal appendicular lesions fare better. The recent addition of modalities such as intensified chemotherapy with bone marrow transplantation and total body irradiation may improve results.
ADIPOSE Lipoma In adults, lipoma of bone is an extremely rare benign bone tumor. It is very common as a soft tissue tumor in adults and as such is not uncommon in children (but solid soft tissue masses in children should be viewed with suspicion and approached with appropriate staging and biopsy to rule out sarcoma). To the best of our knowledge, lipoma of bone has never been reported in children. It is worth noting that intraspinal lipomas constitute 1% of all spinal axis tumors. They may be the cause of neurological symptoms, and this is their usual mode of presentation. The epidural space and, occasionally, the intradural elements are involved. They occur equally in both sexes and affect all ages. They cause remodeling of the vertebrae that presents radiographically as a radiolucent lesion with sclerotic borders and, occasionally, vertical trabeculation. The sacrum is the most likely site. Conservative resection using careful microsurgical dissection yields good results [71,112,113].
because the histological appearance was thought to be similar to that of ameloblastoma (of the maxillary bones). Pain is the presenting complaint in almost every case, although some patients note a slowly developing mass or thickening of the subcutaneous bone. Pathologic fracture may occur [96,119-121].
Diagnostic Imagery Multiple osteolytic lesions are seen within the cortex, surrounded by reactive bone and associated with variable extension into either the medullary cavity or, less frequently, the soft tissues. The borders of the lesion are well defined in some areas and typically vague in others. The contours of the bone may be enlarged, the cortex thinned, and less frequently cortical destruction can occur (Fig. 28). Periosteal reaction is not usually seen.
MIXED Adamantinoma This slowly evolving cancer is very rare and accounts for only 0.1-0.3% of all primary bone tumors. Although some authors suggest it is only found in adults, teenagers can be affected by this lesion. Furthermore, there is a controversial link between ostofibrous dysplasia (a childhood disease) and adamantinoma [114-118]. The mean age at diagnosis is 30 years (range, 10-70 years), and males are twice as often affected as females. The tibia is by far the most frequent site, but adamantinoma has been reported to occur in the fibula, femur, mandible, humerus, ulna, and radius. The diaphysis is usually affected. The term adamantinoma was chosen
FIGURE 28 Adamantinomais almost always localized to the tibia, and has a radiological presentation which can be easily confused with that of osteofibrous dysplasia, or even fibrous dysplasia. In addition to those signs which ressemble osteofibrous dysplasia, adamantinoma usually has one or more areas with more aggressive lytic changes and cortical destruction (arrow).
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The differential diagnosis between FD, osteofibrous dysplasia, and adamantinoma can be difficult [120,122-124]. Although the radiographic picture is similar to that of osteofibrous dysplasia, one area of the lesion is typically more aggressively lytic, destroying the cortex, and often corresponding to the area of maximal pain [115,117,124].
Malignant M e s e n c h y m o m a By definition, this primary sarcoma of bone presents differentiation in two or more tissue types (not including fibroblastic tissue). Few cases have been reported, all of which have presented elements of liposarcoma and osteosarcoma. It resembles osteosarcoma in its clinical and radiological presentation and is probably best managed as osteosarcoma [127].
Pathology Macroscopic examination of these tumors reveals a lobulated, well-defined mass of gray or white tissue containing variable spicules of bony trabeculae. The histology of the lesion is variable from one specimen to another and within the same specimen. According to Weiss and Dorfman [125], several aspects are seen. First, cords and islands of cells similar to basal cell carcinoma (the basalioid aspect) are seen with spindling at the center and cubic or cylindrical palisading at the periphery. This feature resembles ameloblastoma of the jaw. Similar cells can also be seen without peripheral palisading and with reticulin fibers surrounding single cells, and occasionally a whorled pattern (the spindlecellular aspect) is suggestive of fibrosarcoma. Cubic cells may also line tubular or alveolar cavities in single or multiple layers (the tubular aspect), and this may suggest either adenocarcinoma or hemangioendothelioma. Less often, nodules of squamous cells can be seen (the squamocellular aspect). These cellular aggregates are easy to distinguish from the surrounding bland fibrous stroma, characterized by dense connective tissue. Immature osteoid can also be seen around the neoplastic cells or within the stroma. To further confuse the diagnosis, adamantinoma, ossifying dysplasia, and FD may be present in the same patient. Since many of these lesions have overlapping features, incisional biopsy in the wrong part of the lesion may fail to demonstrate the pathognomonic features of adamantinoma [96,120,121,126].
Treatment Although adamantinoma is less likely to occur in children, pain and progressive resorption of bone merit biopsy in the most aggressively lytic area of the lesion. If negative for adamantinoma, complete sampling of the lesion by curettage should be performed; if adamantinoma is confirmed, wide resection is the treatment of choice. Inadequate resection leads to recurrence and metastasis. The course of this lesion is indolent, so followup should be prolonged for at least 10 years. Patients who develop metastases may survive for several years [124].
NOTOCHORD Chordoma Chordoma is a rare tumor of the midline axial skeleton occurring exclusively in adults.
TUMOR-LIKE Unicameral Bone Cyst
Synonyms: Solitary bone cyst, simple bone cyst, and solitary unicameral bone cyst This benign cystic lesion of bone can be active or latent. Its origin and neoplastic nature are poorly understood and controversial. Some authors view it as a developmental anomaly (related to a variety of hypothetical defects), whereas some believe it is related to a benign tumor that produces osteolytic factors. Although adults are occasionally diagnosed with simple bone cyst, this lesion is typically pediatric and some adult cases may represent the involutional stage of another disease (e.g., FD). The most common complication of bone cyst is that it causes fracture, without which the lesion would not cause symptoms. Rarely, limb length discrepancy has been observed due to growth plate involvement. Males may be slightly more affected than females. People of any age may be affected, but 90% of patients are younger than 20 years of age. Younger age is associated with more active disease. Sixty to 75% of these lesions present with fracture after minimal trauma (e.g., swinging a baseball bat). Fractures heal readily with simple orthopedic treatment, are often undisplaced or incomplete, and tend to occur in the proximal humerus or, less frequently, the proximal femur. Eighty percent of unicameral bone cysts (UBCs) occur in one of these two locations. Contrary to early reports, it has been shown that spontaneous healing of the cyst after fracture only occurs in a minority of patients (15%). Diagnostic Imagery Plain radiographs are usually diagnostic and further staging studies are not required in the typical case. The
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lesion is centromedullary lytic without any matrix of note. The borders of the lesion are well defined and slightly lobulated in some cases, with expansion of the bone suggesting interference with metaphyseal remodeling (Fig. 29). These lesions are thought to arise in the subphyseal area, and if they do not progress significantly the physis can be observed to migrate away from the lesion, with the result that the cyst can be found in a subphyseal location (usually in younger children) or in a diaphyseal area. Some of the more active lesions can aggress the physeal plate, penetrate the epiphysis, and cause growth arrest (Fig. 30). Growth problems may also be related to traumatic factors, but this subject has not been extensively studied [128-134]. When radiographs are taken soon after fracture through the cyst, one may observe a fragment of the bone in the distal aspect of the cyst (the fallen fragment or fallen leaf sign). Periosteal reaction is only seen in response to trauma. Thinning of the cortex is the rule, and fracture healing is accompanied by cortical thickening in most cases. However, this may be a temporary phenomenon and does not indicate healing of the cyst. MRI will help to differentiate UBC from aneurysmal bone cyst (ABC), in particular to show fluid levels in the latter. These represent the sedimentation of blood cells from serum, which usually occurs in ABC but is only found in UBC when hemorrhagic, such as after a fracture.
Pathology The cyst is filled with straw-colored fluid unless traumatized and hemorrhagic. The cyst wall is lined with a thin layer of fibrous tissue. Flattened spindle cells are present. The pathologist must be informed of the presence of fracture and its age to avoid misinterpreting reactive bony changes (sometimes confused with osteosarcoma).
Treatment The gold standard of treatment is the method of methylprednisolone injection described by Scaglietti et al. [134]. This method includes careful atraumatic aspiration of the fluid for qualitative (visual), quantitative, and cytological analysis. If the fluid appears clear or straw colored, an equivalent volume of methylprednisolone (40 mg/cc) is injected after opacification to delineate possible septations of the cyst. If the fluid is not typical, biopsy is preferred. With this technique, an average of two or three injections yields an 85% chance of healing the cyst. The interval between injections appears to be important and should be no more than 3 months for optimal results. Traditionally, curettage and bone grafting were done, with higher rates of recurrence and significant morbidity.
FIGURE 2 9 Unicameral bone cyst. Note the radiolucent aspect of the cyst, cortical thinning, increase in diameter and the fallen leaf sign.
They are usually done only when biopsy is indicated. Alternative percutaneous methods have been described, but the literature does not allow for valid comparison. Follow-up of 2 years or more is required to rule out recurrence or persistence of active disease. It should be remembered that the goal of treatment is to avoid repeated fractures in the growing child. A n e u r y s m a l B o n e Cyst ABC is a rare, benign, usually solitary tumor representing approximately 10% of bone lesions. More than half occur in large tubular bones, and approximately 30% occur in the spine. Patients usually complain of insidious rest pain, which is exacerbated by activity. It has a tendency to local recurrence [135,136]. In the appendicular skeleton, the most common sites of involvement are the proximal humerus, femur, tibia, and ilium. The lesion may extend to the growth plate, and disruption of normal growth is not unusual. This can be related to the presence of the lesion adjacent to the
29. Bone Tumors in Children
FIGURE 30 (arrows).
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Plainradiographs (a) and MRI (b) show transgression of the physis by this unicameral bone cyst
growth plate, soliciting enhanced blood flow and stimulation of the physis, but more commonly destruction of a segment of the physis by the tumor (or its treatment) causes progressive angular deformity (depending on the age of the child).
Diagnostic Imagery Plain radiographs of an ABC typically show a metaphyseal, eccentric, lytic lesion with a thinned cortex and expansion of the contour of the bone (Fig. 31). The periosteal reaction is variable and may occasionally suggest an aggressive tumor. In some cases, a Codman's triangle or onion-skinning can be observed [136]. The lesion may extend to the growth plate and in some cases extend into the epiphysis. Spinal lesions originate in the posterior elements (Fig.5) and secondarily involve the vertebral body [137]. Rarely, ABC may occur as a surface lesion, usually in the diaphysis. Nuclear bone scan may be useful to assess the degree of activity within a lesion that appears invasive radiographically [3,4]. On MRI, marked inflammation in the surrounding bone can be observed, and in some instances it may be difficult to differentiate from osteosarcoma [135]. Telangiectatic osteosarcoma should always be considered in the differential diagnosis of ABC, as should UBC, nonossifying fibroma, and chondromyxoid
FIGURE 31 Plainradiographs of an Aneurysmalbone cyst typically show a metaphyseal, eccentric, lytic lesion with a thinned cortex and expansion of the contour of the bone.
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fibroma. Axial imagery (both MRI and CT) will show fluid levels in most cases of ABC (Fig. 32).
Pathology Gross pathological examination of surgical specimens reveals anastomosing cavernous spaces filled with unclotted blood. An eggshell thin layer of subperiosteal new bone delimits the lesion. Microscopically, the cavernomatous spaces lack a true vascular endothelial lining. Rather, they are surrounded by a lining of compressed mesenchymal cells. Solid portions include a fibrous stroma, multinucleated giant cells, and, occasionally, a lace-like pattern of osteoid trabeculae. Small fragments of bone may be sequestered by the destructive process. This variable histology has similar features with both UBC and telangiectatic osteosarcoma [138,139]. A primary ABC is one in which there is no evidence of any other underlying bone neoplasm. Eighty percent of patients with a primary ABC are between 10 and 20 years of age at diagnosis. It occurs throughout childhood, with a slightly higher prevalence in adolescents than in preadolescents. It is unusual in patients in their 30s or older [135,139]. By definition, a secondary aneurysmal bone cyst is associated with another bone lesion, although careful histopathological examination of surgical specimens may be required to discover the primary element. It is estimated that 66% of ABCs are secondary in nature. Primary neoplasms reported in association with secondary ABCs include chondroblastoma, osteoblastoma, and giant cell tumor.
Treatment Treatment of ABCs generally consists of curettage and bone grafting. Internal fixation may be required to avoid or treat fracture, although in younger children
orthopedic treatment is often adequate. The presence of a pathologic fracture may render local control less predictable. Local adjuvants, such as liquid nitrogen or phenol, have been used by some authors [140]. As noted previously, the rate of local recurrence is high [135], and multiple recurrences may threaten an adjacent growth plate or joint surface and eventually necessitate more extensive resection. Although some authors have suggested injection of steroids as an alternate form of treatment, others have found that this method is ineffective and may even accelerate the destructive activity of the lesion [3,4,134].
Eosinophilic G r a n u l o m a a n d L a n g e r h a n s Cell Histiocytosis
Synonyms: Histiocytosis X and Langerhans cell granulomatosis Langerhans cell histiocytosis (LCH) describes a spectrum of disease involvement that has been characterized by three syndromes: Letterer-Siwe syndrome, HandSchuller-Christian syndrome, and eosinophilic granuloma [141,142]. All of these syndromes involve proliferation of histiocytes. Symptoms vary according to the different systems involved. LCH is a benign, nonneoplastic lesion of bone probably arising from the reticuloendothelial system. It occurs from infancy to the sixth decade but peaks between 5 and 10 years of age. Any bone can be involved, but the skull is the most common site. Usually, the child complains of a solitary painful focus, and a palpable or visible mass may be noted. With a vertebral lesion collapse may occur, creating the classic radiographic vertebra plana. Letterer-Siwe disease is severe and is seen in younger patients who present with hepatosplenomegaly, pulmonary disease, and anemia, with or without dermatitis. These children are usually acutely ill and may be immune
FIGURE 32 Axialimagerysuch as a) CT, and b) MRI will show fluid fluid levels in most cases of ABC.
29. Bone Tumors in Children
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deficient. The disease progresses along a malignant course that may necessitate chemotherapy. The longterm prognosis is poor [142]. Hand-Schuller-Christian disease generally affects children between the ages of 3 and 12 years. These children may present with hepatosplenomegaly, exophthalmus, diabetes insipidus, mastoiditis, and dermatitis. A small percentage of these children may also have a malignant course over a longer period of time. More commonly, they have a less aggressive form of the disease. One of the complications is diabetes insipidus, which is extremely difficult (if not impossible) to reverse and is best detected early [141-143]. Eosinophilic granuloma is a more limited disease process. Usually, it is characterized by a solitary bone lesion. Multiple bone lesions are not usually accompanied by visceral disease nor is the prognostic indication the same. However, children who have eosinophilic granuloma do require careful staging to rule out other sites of involvement. Appropriate staging (of this as well as the other forms of LCH) includes chest CT, a total-body technetium bone scan or skeletal series (including radiographs of the skull and pelvis), urine osmolality tests, complete and differential blood cell counts, and liver enzyme studies. Children who have multisystem disease require more aggressive management, often including steroid-based chemotherapy, under the care of a pediatric hematooncologist [3,141-143].
Diagnostic Imagery The radiographic picture of eosinophilic granuloma is primarily that of a cystic lesion of bone but with a very inflamed, aggressive-appearing reaction to the lesion [144]. It truly is the great imitator among pediatric bone tumors and, like osteomyelitis, should be included in all differential diagnoses in children, especially those younger than 12 years of age. The dominant radiographic hallmark is a permeative lesion associated with lucency. Small lesions of Ewing's sarcoma are occasionally diagnosed as LCH, proving that it is important to perform a biopsy before implementing the definitive surgical treatment. In the skull, it may present a series of "punched-out" lesions that may be quite large. In the long bones, the osseous reaction is impressive; however, typically there is no large soft tissue mass associated with this lesion, which helps to distinguish it from malignant tumors of bone such as Ewing's sarcoma. As noted previously, the skull is the most common site, but other osseous sites include the pelvis, proximal femur, spinal column, ribs, hands, and feet (Fig. 33). Classic involvement of the spine with vertebral collapse is referred to as vertebra plana and has a coin-on-edge appearance [3,144].
FIGURE 33 a) The skull is the most common site of Langerhans Cell Histiocytosis (LCH), the lesions showing a "punched out" appearance (arrows) b) Pelvic bones are another common site of LCH. c) LCH can affect any bone; this toddler had a solitary eosinophilic granuloma of the proximal femur (arrow), which healed after biopsy.
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Pathology
References
Curettage of eosinophilic granuloma typically reveals soft lesional tissue that is gray, pink, or yellow. On histology, the pathognomonic features include foci of proliferating histiocytic cells, often with ill-defined cytoplasmic boundaries and oval or indented nuclei. Mitoses are common, and varied numbers of eosinophils, lymphocytes, and neutrophils are present. Occasionally, the round cell histologic appearance can be quite difficult to distinguish from that of other inflammatory conditions such as osteomyelitis, from round cell tumors such as Ewing's sarcoma, and from primitive neuroectodermal tumor, especially on frozen section. Immunohistochemical techniques may be helpful in excluding other diagnoses, and cultures should always be performed at the time of biopsy [143,145,146].
Treatment Incisional biopsy is almost always curative, and simple nonaggressive curettage allows predictable healing. Corticoid injection has been reported to be successful and is of particular interest for those patients with multiple lesions (after biopsy of one site) or for lesions difficult to access surgically [8,147]. Low doses (400-1000rad) of radiation have been used successfully for lesions in the spine, skull, sacrum, and pelvis [134]. The survival rate for Letterer-Siwe syndrome is low, and that for Hand-Schuller-Christian syndrome is 90% [143,148]. Solitary eosinophilic granuloma is always curable. The prognosis for any particular child is related to the extent of involvement rather than the age at presentation. Other lesions usually develop within 6 months of diagnosis, if at all. As mentioned previously, these children should be evaluated and followed by a pediatric hematooncologist in collaboration with an orthopedic surgeon.
CONCLUSION The treatment of benign bone tumors needs to be individualized on the basis of the known natural history of the lesion and its biologic behavior. There are several potential pitfalls, but they can be minimized with a careful systematic approach to these tumors. When in doubt, however, consultation with an experienced orthopedic oncologist will help to determine the best approach for the patient. Doing so will minimize the risk of undertreatment or overtreatment.
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Index
A AAL, see Acute lymphoblastic leukemia c-Abl, fetal mineral metabolism and regulation, 280 Achondrogenesis types and diagnosis, 395-396 Acute lymphoblastic leukemia (ALL) survival, 416 pediatric osteoporosis, 416-417 sequelae, 417 ADHR, see Autosomal dominant hypophosphatemic rickets Adrenomedullin osteoblast effects, 228-229 receptors, 228-229 structure, 228 tissue distribution, 228 A G C 2 , 16 Aggrecan A G C 2 gene, 16 glycosaminoglycan attachment region, 16 hyaluronan binding, 16 structure, 16 Alkaline phosphatase (TNAP) bacteria and yeast enzymes, 652 bone and extracellular matrix distribution, 661 bone expression regulation assembly, 654-655 promoter, 654-656 transcription, 654-656 translation, 654 bone formation marker, 342, 661 catalytic reaction, 657-658 circulation, 661 defects, see Hypophosphatasia developmental changes, 344-345 functions, 569, 661 glycosylation, 655 human isoforms, 652 inhibitors and allosteric modifiers, 659 knockout mouse, 670 membrane association and tetramer formation, 66 osteoblast marker, 52-53
premature infant levels, 348 sequence homology between mouse and man, 653 structure, 655-657 substrates, 658-659 vitamin D deficiency rickets levels, 350-351 X-linked hypophosphatemia levels, 606 Amylin bone volume effects, 227-228 gene, 225 osteoblast effects, 226-227 osteoclast effects, 225-226 receptor, 227 synthesis, 225 AN, see Anorexia nervosa Androgen, bone formation regulation, 124 Anorexia nervosa (AN) pediatric osteoporosis and management, 417-419 prevalence, 417-4 18 APECED, see Autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy Appendicular skeleton, skeletogenesis overview, 92 apical ectodermal ridge formation and maintenance, 92-94, 96 limb bud positioning and induction, 92-93 limb identity determination, 93 limb patterning, 93-96 zone of polarizing activity, 95 Asphyxiating thoracic dysplasia (ATD), radiography, 393 ATD, see Asphyxiating thoracic dysplasia Autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy (APECED), features, 489 Autosomal dominant hypophosphatemic rickets (ADHR) clinical features, 184, 607-608 FGF23 defects, 184, 612, 622 prospects for study, 623-624 treatment strategies, 184 unifying hypothesis for renal phosphate wasting, 186
745
Axial skeleton skeletogenesis overview, 88 patterning genes, 91-92 sclerotome differentiation, 90-91 somite formation, 88-90 heredity, 607
B BAG-75, see Bone acidic glycoprotein-75 Basic multicellular unit (BMU), bone remodeling, 44 BFR/BS, see Bone formation rate per bone surface B G L A P , 24 B G N , 18
Bicarbonate, parathyroid hormone and renal reabsorption effects, 159-160 Biglycan B G N gene, 18 knockout mouse phenotype, 19 processing, 18 structure, 18 Biopsy, bone histomorphometry, see Histomorphometry, bone sample processing, 360, 362 site for collection, 359-360 tetracycline labeling, 360-361 trocar, 360 Bisphosphonates fibrous dysplasia management, 531-532 osteogenesis imperfecta management, 457-460, 463 pediatric osteoporosis management, 428 BMPs, see Bone morphogenic proteins Bone acidic glycoprotein-75 (BAG-75), features, 26 Bone age assessment atlas technique, 329, 333 comparison of atlas and bone-specific methods, 333-334 Fels hand-wrist technique, 332-333
746 Bone age assessment ( c o n t i n u e d ) initial considerations in maturation assessment chronological versus maturational time, 325-327 indicators of maturity, 325-328 variability in maturation, 325-326, 328 sexual dimorphism, 326, 328 size versus maturity, 326, 329 uneveness of maturation, 326 Oxford method, 329-330 reliability of different systems, 333 Roche-Wainer-Thissen technique, 332 Tanner-Whitehouse method, 330-333 Bone biopsy, s e e Biopsy, bone Bone, composition, 1 Bone formation endochondral, s e e Endochondral ossification growth factors activating mutations, 123-124 fibroblast growth factors FGF-2, 124 FGF-18, 124 growth hormone, 121-122 insulin-like growth factor-I, 121-122 insulin-like growth factor-II, 122 receptor knockout studies, 123 intramembranous, s e e Intramembranous ossification leptin regulation, 127 markers alkaline phosphatase, 342 clinical and research value, 352 developmental changes, 344-345 discharge to two years of age, 576 two years onwards, 578 Ehlers-Danlos syndrome type VI studies, 351 growth disorders and growth prediction, 349-350 interpretation, 346-347 nutritional rickets levels, 552-553 osteocalcin, 342 osteogenesis imperfecta studies, 347-348 premature infants, 348 corticosteroid therapy studies, 348-349 preterm infant skeletal health over time birth to hospital discharge, 574-575 procollagen type I propeptides, 342 renal osteodystrophy studies, 350 rickets studies, 350-351 urinary excretion, 341 osteoblasts in longtitudinal growth control, 124-125 osteoclast differentiation and function control, 127-130 prenatal, s e e Skeletogenesis renal osteodystrophy disturbances, 680-681 sex hormone roles, 124 thyroid hormone regulation, 124 Bone formation rate per bone surface (BFR/ BS), bone histomorphometry, 365 Bone Gla protein, s e e Osteocalcin
Index Bone histomorphometry, s e e Histomorphometry, bone Bone mineral density (BMD), s e e a l s o Peak bone mass assessment, s e e Dual-energy X-ray absorptiometry; Magnetic resonance imaging; Peripheral quantitative computed tomography; Quantitative computed tomography; Quantitative ultrasound; X-ray radiogrammetry Fanconi's syndrome changes, 637 idiopathic hypercalciuria changes, 643 pediatric osteoporosis, 402-403 Bone modeling, fibrous dysplasia, 520-521 Bone morphogenic proteins (BMPs) apical ectodermal ridge maintenance, 94 bone formation initiation, 97-99 chondrogenesis induction, 100 limb patterning, 95-96 osteoblast differentiation role, 126 skull patterning, 87 Bone osteochondrodysplasias, s e e Skeletal dysplasias Bone remodeling coupling of bone formation and resorption, 340 definition, 340 fibrous dysplasia, 522-524 marker interpretation, 340-341 Bone resorption osteoclastic defects, 473-477 markers clinical and research value, 352 collagen pyridinium cross-links, 343-344 corticosteroid therapy studies, 348-349 developmental changes, 344-345 Ehlers-Danlos syndrome type VI studies, 351 growth disorders and growth prediction, 349-350 hydroxylysine glycosides, 344 hydroxyproline, 342-343 interpretation, 346-347 nutritional rickets levels, 552-553 osteogenesis imperfecta studies, 347-348 premature infants, 348 preterm infant skeletal health over time birth to hospital discharge, 574-575 discharge to two years of age, 576 two years onwards, 578 renal osteodystrophy studies, 350 rickets studies, 350-351 tartrate-resistant acid phosphatase, 344 urinary excretion, 341 Bone sialoprotein I B S P gene, 25 osteoblast marker, 52-53 RGD sequence, 26 structure, 25-26 Bone tumors adamantinoma imaging, 734 pathology, 735 treatment, 735
anueurysmal bone cyst imaging, 737 pathology, 738 treatment, 738 back pain and spinal neoplasms benign neoplasms, 707-708 imaging, 707 malignant neoplasms, 709 chondroblastoma imaging, 718 pathology, 719 treatment, 719 chondroma imaging, 714 malignant potential, 715 Ollier's disease, 715 pathology, 714 treatment, 714 chondromyxoid fibroma imaging, 719-720 pathology, 720 treatment, 720 chondrosarcoma imaging, 721 pathology, 721 treatment, 722 classification and nomenclature, 706-707 curettage, 703 desmoid fibroma imaging, 712 pathology, 712 treatment, 712-713 diagnosis, 704-706 eosinophilic granuloma imaging, 739 pathology, 740 treatment, 740 Ewing's sarcoma imaging, 733 pathology, 733 treatment, 733-734 fibrosarcoma imaging, 713 pathology, 713 treatment, 713 fibrous dysplasia differential diagnosis, 726 etiology, 725 imaging, 725 pathology, 726 prognosis, 726 treatment, 726 giant cell tumor imaging, 711 pathology, 711 staging, 711-712 treatment, 712 hemangioma imaging, 731-732 incidence, 703 treatment, 733 incidence, 703 leukemia imaging, 732
Index treatment, 732 lipoma, 735 mesenchymoma, malignant, 736 lymphoma imaging, 731 treatment, 731 nonossifying fibroma imaging, 709 pathology, 710 treatment, 711 osteoblastoma imaging, 723 pathology, 724 treatment, 724 osteochondroma imaging, 716 pathology, 716 treatment, 717 osteofibrous dysplasia differential diagnosis, 727 imaging, 727 pathology, 727 treatment, 727 osteoid osteoma imaging, 722 pathology, 723 treatment, 723 osteosarcoma imaging, 728 pathology, 728-729 treatment, 729 treatment principles, 703-704 unicameral bone cyst imaging, 735-736 pathology, 736 treatment, 736 Bone volume per tissue volume (BV/TV), bone histomorphometry, 363 Breast milk, see Lactation Bruck syndrome clinical features, 407-408, 449 pediatric osteoporosis, 407-408 BV/TV, see Bone volume per tissue volume
C Calbindin-D, placental calcium transport role, 280, 286-287 Calcitonin, fetal mineral metabolism and regulation, 277-278 Calcitonin gene-related peptide (CGRP) aging changes, 218 birth changes, 569 calcium response, 219-220 discovery, 217 forms, 220 functions, 220, 222 gene, 220 knockout effects in fetus, 277 osteoblast effects, 222 osteoclast effects, 218-219 osteoclast effects, 220, 222 placental calcium transport role, 283
pregnancy levels, 252 receptors, 223 renal, loL-hydroxylase stimulation, 196 synthesis, 217-218 Calcitriol, X-linked hypophosphatemia management, 613-614 Calcium absorption, 138 birth changes, 569 blood calcium, 294-295 bone histomorphometry of deficiency, 368 breast milk concentration, 261 concentrations in body fluids, 135 deficiency in vitamin D deficiency rickets, 546-548 extracellular homeostasis fluxes and balance, 137-139 fetal mineral metabolism and regulation calcium sources, 293-294 overview, 272-273 functional overview, 135 intake and peak bone mass effects, 240-241 intracellular homeostasis, 135 lactation dietary effects on mineral metabolism and breast milk production, 262 metabolism, 256-258 nutrition childhood and adolescence, 139 factors affecting balance, 139 total and ionized calcium, 135, 137 parathyroid gland growth regulation, 145 parathyroid hormone homeostasis role, 148-149 renal reabsorption effects, 156-157 secretion regulation, 143-144 pediatric osteoporosis prevention, 427 placental transfer, 295 placental transport requirements, 280 energetics, 280 calbindin-D role, 280, 286-287 active transport initiation, 281 maternal regulation, 281 assessment, 281-282 calcitonin, 283 fetal regulation molecular mechanisms, 286-288 parathyroid hormone, 283 parathyroid hormone-related protein, 283-288 pregnancy dietary effects on mineral metabolism and skeletogenesis, 254 metabolism, 250-251 vitamin D, 283 renal handling fetus, 290-291 overview, 138, 139 skeletal mineralization, 295-296 vitamin D in homeostasis, 201 Calcium deficiency rickets biochemical abnormalities, 551-555 bone histomorphometry, 368
747 clinical presentation, 550-551 epidemiology, 548 historical perspective, 542-543 prevention, 558-560 radiological diagnosis, 555-557 treatment, 557-558 vitamin D metabolite levels, 554-555 Calcium-sensing receptor (CaSR) activation, 141 calcium binding, 140-141 fetal mineral regulation, 289-290 function, 140 knockout effects in fetus, 273, 275-276, 289-290 magnesium binding, 142 mutations, 492, 500 signal transduction, 142 structure, 140 Campomelic dysplasia, features, 390, 392-393 Camurati-Engelmann disease (CED) clinical features, 479 radiography, 479-480 transforming growth factor defects, 479480 Carbonic anhydrase, osteopetrosis deficiency, 475 Cartilage-hair hypoplasia (CHH), diagnosis, 390 Cartilage, see also Hyaline cartilage histologym in skeletal dysplasia, 395-396 Cartilage link protein 1 C R T L 1 gene, 16 function, 16 structure, 16 Cartilage oligomeric matrix protein (COMP) functions, 22 gene, 22 knockout mouse phenotype, 23 ligands, 23 structure, 22-23 CaSR, see Calcium-sensing receptor Cathepsin K bone matrix degradation, 60 pycnodysostosis defects, 473-474, 477 Cbfal, see R u n x 2 Cerebral palsy (CP) clinical features, 413 management, 414 pediatric osteoporosis, 412-4 14 prevalence, 412 C H A D , 20 CHH, see Cartilage-hair hypoplasia Child abuse, differentiation from bone fragility conditions, 428-430, 450 Chondroadherin C H A D gene, 20 structure, 20 Chondroblastoma imaging, 718 pathology, 719 treatment, 719 Chondrocyte, see Endochondral ossification Chondrodysplasia punctata ConradiHfinermann, features, 389
748 Chondrodysplasia punctata, diagnosis, 393-394 Chondroma imaging, 714 malignant potential, 715 Ollier's disease, 715 pathology, 714 treatment, 714 Chondromyxoid fibroma imaging, 719-720 pathology, 720 treatment, 720 Chondrosarcoma imaging, 721 pathology, 721 treatment, 722 Chronic renal failure, see also Renal osteodystrophy hyperparathyroidism, 500 Classic metaphyseal lesion (CML), child abuse, 429 Cleidocranial dystosis hypophosphatasia phenocopy, 672 CML, see Classic metaphyseal lesion Codman's tumor, see Chondroblastoma COL1A1, 2-3 COL1A2, 2-3 COL5A2, 7 COL9A1, 10 COL9A2, 10 COL9A3, 11 COLIOA1, 14 COLllA1,12-13 C O L l l A 2 , 13
Cole-Carpenter syndrome, features, 449 Collagen type I assembly, 3-5 collagen type V interactions, 8 chain processing, 2 C-propeptide cleavage, 4-5 C-telopeptide, 2 COL1A1 gene, 2-3 COL1A2 gene, 2-3 developmental changes, 344-345 fibril formation and molecular packing, 5-6 gene mutations, see Osteogenesis imperfecta hydroxylation, 3-4 lysyl oxidase interactions, 5-6 messenger RNA processing, 3 osteoblast marker, 52-53 propeptides as bone formmation markers, 342 structure, 1-2, 5-6 tissue distribution, 1 Collagen type II assembly, 10 chain properties, 8-9 COL2A1 gene, 8 fibrils, 10 forms, 8 processing, 9 propeptides, 9 proteases, 9 tissue distribution, 8
Index
type IIB isoform, 9 Collagen type III C-propeptide, 2 Collagen type V chains, 6-8 collagen type I interactions, 8 COL5A1 gene, 6-7 COL5A2 gene, 7 C-propeptide, 7-8 C-telopeptide, 7 ligands, 7-8 processing, 8 structure, 6-7 Collagen type IX assembly, 11 chain properties, 10-11 cross-linking, 11 COL9A1 gene, 10 COL9A2 gene, 10 COL9A3 gene, 11 structure, 10-11 Collagen type X chain properties, 14 COLIOA1 gene, 14 functions, 15 NC1 domain, 14-15 proteolytic cleavage, 15 supramolecular forms, 15 Collagen type XI assembly, 13 chain properties, 12-13 COll l A 1 gene and alternative splicing, 12-13 COll 1A2 gene and alternative splicing, 13 fibril growth, 13-14 structure, 11-12, 14 COMP, see Cartilage oligomeric matrix protein Condensation, skeletal, 97-99 Cone-shaped epiphyses, 392 Core width, bone histomorphometry, 363 Cortical width, bone histomorphometry, 363 Corticosteroid therapy bone histomorphometry, 372 bone metabolism marker studies, 348-349 pediatric glucocorticoid-induced osteoporosis dose-dependence, 425-426 fracture rate, 425 pathogenesis, 42 treatment, 426 CP, see Cerebral palsy CT, see Quantitative computed tomography CRTL1, 16
CT, see Quantitative computed tomography CYP24 knockout mouse phenotype, 203 substrate specificity, 584 vitamin D metabolism, 196-198, 583-584 CYP27 knockout mouse phenotype, 202 vitamin D metabolism, 193-195, 544, 584
CYP27B 1 deficiency and rickets clinical presentation, 585-586 laboratory features, 586 nomenclature, 585 linkage analysis, 587 molecular genetics, 587-589 mutation types, 589-590 phenotypic variation, 58 treatment, 592 gene cloning, 584-585 knockout mouse phenotype, 203-205 structure and function, 589-591 vitamin D metabolism, 195-196, 583-585
D DBP, see Vitamin D-binding protein DCN, 18 Decorin D C N gene, 18 developmental changes in growth plate, 18 knockout mouse phenotype, 19 processing, 18 structure, 18 Dent's disease, features, 642 Dentin matrix acidic phosphoprotein-1 DMP1 gene, 26 structure, 26 Dentinogenesis imperfecta, 444, 451 Desmoid fibroma imaging, 712 pathology, 712 treatment, 712-713 Dgyvve-Melchior-Clausen syndrome, radiography, 391-392 Diabetes mellitus, pediatric osteoporosis, 424 Distal renal tubular acidosis (dRTA) bone disease, 646 clinical characteristics, 645-646 diagnosis, 644-645 physiology, 646 treatment, 647 Dlx-5, osteoblast regulation, 55 DMD, see Duchenne muscular dystrophy DMP1, 26 dRTA, see Distal renal tubular acidosis DSPG3, 20 Dual-energy X-ray absorptiometry (DXA) advantages and limitations, 316-317 applications, 307-308 calibration, 305-306 challenges to interpreting bone mass measurements in childhood and adolescence, 303-304 dosimetry, 306, 308 pediatric osteoporosis, 402 precision versus accuracy, 303, 306 preterm infant skeletal health over time birth to hospital discharge, 572-575 discharge to two years of age, 575-576 two years onwards, 577-578
749
Index principles, 305 reference data, 306-307, 309, 318-319 Dubow-Bailey rod complication rate, 461 osteogenesis imperfecta management, 461 Duchenne muscular dystrophy (DMD) clinical features, 414 management, 415 pediatric osteoporosis, 414-415 DXA, see Dual-energy X-ray absorptiometry
E EDS, see Ehlers-Danlos syndrome Ehlers-Danlos syndrome (EDS) clinical features, 408-409 pediatric osteoporosis, 408-409 types, 408 Ehlers-Danlos syndrome type VI, bone metabolism marker studies, 351 Enl, limb patterning, 96 Endochondral ossification lengthening of bones, 120, 339 perichondrial cells, 120 skeletogenesis chondrocyte maturation, 103-104 differentiation, 99-101 growth and proliferation, 101-102 overview, 79-81 stages, 119-120 ENS, see Epidermal nevus syndrome Epidermal nevus syndrome (ENS), hypophosphatemia syndrome association, 612 Epiphycan D S P G 3 gene, 20 structure, 20 tissue distribution, 20 Eroded surface per bone surface (ES/BS), bone histomorphometry, 365 ES/BS, see Eroded surface per bone surface Estrogen bone formation regulation, 124 fetal mineral metabolism and regulation, 279-280 osteoclast regulation, 62-63 Ewing's sarcoma imaging, 733 pathology, 733 treatment, 733-734 Exercise peak bone mass effects, 239-240 pediatric osteoporosis prevention, 426-427
F Fanconi's syndrome bone disease bone mineral density changes, 637 clinical features, 535 histomorphometry, 636-637
pathophysiology, 636-637 diagnosis, 635-636 etiology, 634-635 renal defects, 633-634, 638 treatment minerals, 639-640 vitamin D, 639-640 FD, see Fibrous dysplasia Fels hand-wrist technique, bone maturity assessment, 332-333 Fetal bone development, see Pregnancy; Skeletogenesis Fibroblast growth factor (FGF) axial skeleton formation role, 90 bone formation regulation receptor knockout studies, 123 activating mutations, 123-124 FGF-2, 124 FGF-18, 124 chondrocyte proliferation regulation, 101-102 FGF23, see also Phosphatonin autosomal dominant hypophosphatemic rickets defects, 184, 612, 622 defects in autosomal dominant hypophosphatemic rickets, 184tumor-induced osteomalacia role, 185 tumor-induced osteomalacia role, 612 unifying hypothesis for renal phosphate wasting, 186 limb formation induction and patterning, 93-94 receptors, 122-123 skull patterning, 87 STAT signaling, 102 types, 122 Fibromodulin F M O D gene, 19 knockout mouse phenotype, 19 structure, 19 Fibrosarcoma imaging, 713 pathology, 713 treatment, 713 Fibrous dysplasia (FD) bone histomorphometry, 372 clinical features extraskeletaI lesions, 513-516 radiographic features, 511-513 skeletal lesions, 510-511 diagnosis, 530-531 differential diagnosis, 726 etiology, 725 G N A S 1 mutation consequences, 510, 517-518 history of study, 509-510 molecular diagnostics, 531 mutability of R201 codon, 517 types, 516-517 imaging, 725-726 pathogenesis excess cyclic AMP downstream effects, 527-529
fibrous tissue features, 527 GsoLexpression in bone, 526-527 mosaicism and viability of cells and tissues, 529-530 pathology, 726 bone mineralization, 521-522 bone modeling, 520-521 deposition and internal structure of bone, 521 bone remodeling, 522-524 bone tumors, 526 comparison with other fibroosseous lesions, 525-526 growth of lesions, 525 hyaline cartilage deposits, 525 imprinting, 519-520 phenotypic variability determinants site and time of origin of mutated clone, 518-519 somatic mosaicism, 518 prognosis, 726 size and viability of mutated clone, 519 vascularity of lesions, 524-525 treatment, 726 bisphosphonates, 531-532 endocrine imbalances, 530-531 prospects, 533 surgery, 532 gene therapy, 532-533) medical therapy, 532 FMOD,
19
Formation period (FP), bone histomorphometry, 365 c-Fos, osteoclast differentiation role, 128 FosB, osteoblast regulation, 55-56 FP, see Formation period Fra-1, osteoblast regulation, 55-56 FzGF, see Fibroblast growth factor
G Galectin-3, osteoblast marker, 54 GATA3, mutation in hypoparathyroidism, deafness, and renal anomalies syndrome, 491-492 G c m 2 , knockout mouse, 490-491 GCT, see Giant cell tumor Gene therapy fibrous dysplasia, 532-533 osteogenesis imperfecta, 64, 462-463 GH, see Growth hormone Giant cell tumor (GCT) imaging, 711 pathology, 711 staging, 711-712 treatment, 712 Gitelman's syndrome, features, 640 Glucocorticoid-induced osteoporosis, see Corticosteroid therapy GNAS1
Cushing's syndrome mutations, 515 fibrous dysplasia mutation
750 GNAS1
Index (continued)
consequences, 510, 517-518 mutability of R201 codon, 517 types, 516-517 Gso~isoforms, 516 imprinting, 519-520 molecular diagnostics, 531 protein expression in bone, 526-527 pseudohypoparathyroidism mutations, 492-495 structure, 516 gp 130, signaling in osteoclast differentiation, 129 Growth hormone (GH) bone formation regulation, 121-122 deficiency and bone metabolism marker studies, 349-350 deficiency and pediatric osteoporosis, 421-423 fibrous dysplasia excess, 514-515 osteogenesis imperfecta management, 460-461 renal osteodystrophy disturbances, 680-681 Turner syndrome management, 421 X-linked hypophosphatemia management, 614-615 GRP, see Calcitonin gene-related peptide G y mouse, see X-linked hypophosphatemia
H Hemangioma imaging, 731-732 treatment, 733 Hereditary dihydroxyvitamin D-resistant rickets, see Vitamin D-dependent rickets type II Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) bone histomorphometry, 369 candidate genes, 186, 609-610 clinical features, 186, 608-609 course, 609 idiopathic hypercalciuria relationship, 609 laboratory findings, 608-609 management, 609 mixed skeletal phenotype, 608-609 renal stones, 609 HHRH, see Hereditary hypophosphatemic rickets with hypercalciuria Histiocytosis X, see Eosinophilic granuloma Histomorphometry, bone advantages, 359 biopsy, see Biopsy, bone calcium deficiency, 368 corticosteroid therapy, 372 Fanconi's syndrome, 636-637 fibrous dysplasia, 372 hereditary hypophosphatemic rickets with hypercalciuria, 369 idiopathic hypercalciuria, 643 idiopathic juvenile osteoporosis, 371-372 indications in pediatrics, 372-373
microscopy, 362 nomenclature, 362-363 normal pediatric bone development, 366-367 osteogenesis imperfecta types I, III, and IV, 369-370 types V and VI, 370-372 type VII, 371 osteomalacia, 367-368 osteopetrosis, 372 parameters dynamic formation parameters, 364-365 static formation parameters, 364 static resorption parameters, 365 structural parameters, 363-364 renal osteodystrophy, 372 reproducibility, 365 vitamin D deficiency, 368 X-linked hypophosphatemic rickets, 368-369, 372 Homocystinuria genetic defects, 409-410 pediatric osteoporosis, 409-410 treatment, 410 H o x genes axial skeleton patterning, 91-92 digit patterning, 96 limb patterning, 92, 94-95 skull patterning, 87 Hsp47, collagen interactions, 4 HSPG20
Hurler disease, radiography, 394 Hyaline cartilage composition, 1 fibrous dysplasia deposits, 525 Hyaluronan aggrecan binding, 16 degradation prior to condensation, 98 H 1oL-Hydroxylase, see CYP27B 1 24-Hydroxylase, see CYP24 25-Hydroxylase, see CYP27 hydroxylysine glycosides bone resorption markers, 344 circadian variation, 344 developmental changes, 344-345 Hydroxyproline bone resorption markers, 342-343 developmental changes, 344-345 H y p mouse, see X-linked hypophosphatemia Hypercalcemia, see Parathyroid disorders Hypercalciuria, see also Idiopathic hypercalciuria Fanconi's syndrome, 637 Hypermagnesuria Fanconi's syndrome, 638 isolated, 640 Hyperparathyroidism, see Parathyroid disorders Hyperphosphatemia pseudohypoparathyroidism, 187 renal insufficiency, 186-187 tumoral calcinosis, 187 Hypocalcemia, see Parathyroid disorders
Hypomagnesemia bone disesease forms, 641 etiology, 640 Gitelman's syndrome, 640 isolated hypermagnesuria, 640 Michillis-Costello syndrome, 640 pathogenesis, 64 treatment, 641 Hypoparathyroidism, see Parathyroid disorders Hypophosphatasia alkaline phosphatase mutation analysis, 668-670 cleidocranial dystosis as phenocopy, 672 clinical presentation adult, 664 childhood, 664 infantile, 664 odontohypophosphatasia, 665 perinatal, 662, 6 6 4 pseudophosphatasia, 665 definition, 651 genetivc counseling, 671 histopathology, 666 laboratory findings, 6 6 5 - 6 6 6 management medical, 670-671 surgical, 671 mouse models, 670 prenatal diagnosis, 671 prevalence, 661-662 psathogenesis, 666-668 Hypophosphatemia, see also Autosomal dominant hypophosphatemic rickets; Hereditary hypophosphatemic rickets with hypercalciuria; Tumor-induced osteomalacia; X-linked hypophosphatemia Fanconi's syndrome, 637 fibrous dysplasia, 515
I 25 Idiopathic hypercalciuria (IH) bone disease bone mineral density changes, 643 clinical charactaristics, 643 histomorphometry, 643 pathogenesis, 644 definition, 642 Dent's disease, 642 diagnosis, 642 forms, 642 treatment, 644 vitamin D receptor defects, 642 Idiopathic juvenile osteoporosis (IJO) bone histomorphometry, 371-372 clinical presentation, 410 osteogenesis imperfecta differential diagnosis, 407, 410-411,450 pediatric osteoporosis, 410, 412
IBSP,
Index
treatment, 412 IGFs, s e e Insulin-like growth factors IH, s e e Idiopathic hypercalciuria IHH, s e e Indian hedgehog IJO, s e e Idiopathic juvenile osteoporosis Immobilization, pediatric osteoporosis association, 415-4 16 Indian hedgehog (IHH) chondrocyte proliferation regulation, 102 osteoblast development role, 47, 126 parathyroid hormone-related protein signaling interactions, 104 Insulin-like growth factors (IGFs) bone formation regulation IGF-I, 121-122 IGF-II, 122 developmental role, 101 parathyroid hormone effects, 156 protein intake effects, 242 Intramembranous ossification overview, 120, 121 sclerosing bony dysplasias, 478-480 skeletogenesis, 78-79, 99
I
Jansen's disease, features, 500-501 JRA, s e e Juvenile rheumatoid arthritis Juvenile rheumatoid arthritis (JRA), pediatric osteoporosis, 417
physiological changes, 259-260 effects on senile osteoporosis, 260-261 vitamin D levels, 258-259 Langerhans cell histiocytosis, s e e Eosinophilic granuloma osteoporosis, 260 Leptin, bone formation regulation, 127 Leukemia, s e e a ls o Acute lymphoblastic leukemia imaging, 730 treatment, 730 Limbs, s e e Appendicular skeleton Lipoma, bone, 734 Lipoprotein receptor-related protein-5 (LRPS) gene polymorphisms and peak bone mass, 239 osteoblast regulation, 56 LRP5, s e e Lipoprotein receptor-related protein-5 Lumican function, 19 structure, 19 Lymphoma imaging, 731 treatment, 731 Lysyl hydroxylases, types and genes, 3-4 deficiency, s e e Bruck syndrome Lysyl oxidase, collagen interactions, 5-6
M K Kearns-Sayre syndrome (KSS), hypothyroidism, 489-490 KSS, s e e Kearns-Sayre syndrome
L Lactation bone growth promotion in infants, 261-262 dietary effects on mineral metabolism and breast milk production calcium, 262 general nutrition, 263-264 magnesium, 263 phosphate, 263 vitamin D, 263 zinc, 263 mineral concentrations in breast milk, 261 mineral metabolism calcium, 256-258 magnesium, 258 overview, 255-256 phosphate, 258 zinc, 258 bone turnover, 258 parathyroid hormone levels, 258-259 parathyroid hormone-related protein levels, 259 skeletal changes in mother
Macrophage colony-stimulating factor (M-CSF), osteoclast regulation, 61-62, 128-129 Magnesium breast milk concentration, 261 fetal mineral metabolism and regulation, 274 lactation dietary effects on mineral metabolism and breast milk production, 263 metabolism, 258 parathyroid hormone and renal reabsorption effects, 156-157 placental transport, 288 pregnancy dietary effects on mineral metabolism and skeletogenesis, 255 metabolism, 251 Magnetic resonance imaging (MRI) bone trabeculation evaluation, 316 challenges to interpreting bone mass measurements in childhood and adolescence, 303-304 child abuse, 429 dosimetry, 308 microscopy, 315-316 precision versus accuracy, 303 principles, 315 reference data, 318-319 MAPC, s e e Multipotent adult progenitor cell MAR, s e e Mineral apposition rate
751 Marfan syndrome fracture risk, 409 gene mutations, 409 pediatric osteoporosis, 409 MAS, see McCune-Albright syndrome Matrilins genes, 21 structures, 21-22 tissue distribution, 21 types, 21 Matrix Gla protein knockout mouse phenotype, 25 M g p gene, 25 processing, 25 structure, 25 tissue distribution, 25 Matrix metalloproteinases (MMPs), bone matrix degradation, 60-61 Maturation, s e e Bone age assessment; Puberty McCune-Albright syndrome (MAS), s e e a l s o Fibrous dysplasia hypophosphatemia syndrome association, 612-613 M-CSF, s e e Macrophage colony-stimulating factor Mechanostat theory, cumulative model of bone development, 403-405, 419 Megalin, knockout mouse phenotype, 202-203 MELAS syndrome, hypothyroidism, 489-490 Melorheostosis 13ig-h3 downregulation, 479 clinical features, 479-479 radiography, 478-479 M E N 1 , parathyroid tumor mutations, 498 M E N 2 , parathyroid tumor mutations, 499 Mesenchymal stem cell (MSC) osteoblast differentiation, 44-50 skeletal disease therapy prospects, 64-65 Metabolic bone disease of prematurity, s e e Preterm infants M f h l , sclerotome differentiation role, 90 M G P , 25 m i , osteoclast differentiation role, 128 Michillis-Costello syndrome, features, 640 Gitelman's syndrome syndrome, 640 Mineral apposition rate (MAR), bone histomorphometry, 365 Mineralization lag time (Mlt), bone histomorphometry, 365 Mineralizing surface per bone surface (MS/BS), bone histomorphometry, 365 Mlt, s e e Mineralization lag time MMPs, s e e Matrix metalloproteinases Morquio syndrome, features, 393-394 MRI, s e e Magnetic resonance imaging MS/BS, s e e Mineralizing surface per bone surface MS/OS, s e e Mineralizing surface per osteoid surface MSC, s e e Mesenchymal stem cell Msx-2 chondrogenesis regulation, 101 osteoblast regulation, 55
7 5 2-
Index
Multipotent adult progenitor cell (MAPC) osteoblast differentiation, 44, 46-47 skeletal disease therapy prospects, 64-65
N Nail-patella syndrome, features, 391 N-cadherin, condensation role, 97-98 NCAM, s e e Neural cell adhesion molecule NCoA-62, vitamin D receptor coactivation, 199 Neural cell adhesion molecule (NCAM), condensation role, 97-98 NF-KB, s e e Nuclear factor-KB NHE3, parathyroid hormone effects, 159-160 NOF, s e e Nonossifying fibroma Noggin, sclerotome differentiation role, 90 Nonaccidental injury, s e e Child abuse Nonossifying fibroma (NOF) imaging, 709 pathology, 710 treatment, 711 Notch, axial skeleton formation role, 89 Nuclear factor-KB (NF-KB) ectodermal dysplasia with osteopetrosis signaling defects, 477 osteoclast differentiation role, 128
O Octreotide, tumor-induced osteomalacia management, 611 Odontohypophosphatasia, features, 665 OF45, osteoblast marker, 54 OI, s e e Osteogenesis imperfecta Ollier's disease, features, 715 Oncogenic hypophosphatemic osteomalacia, s e e Tumor-induced osteomalacia OPG, s e e Osteoprotegerin OPPG, s e e Osteoporosis pseudoglioma syndrome OS/BS, s e e Osteoid surface per bone surface Osteoblast adrenomedullin effects, 228-229 amylin effects, 226-227 Osteoblast activation-resorption bone formation sequence, 44 antibodies in precursor marker studies, 48-50 bone metabolism marker interpretation of activity, 346-347 bone modeling, 339-340 calcitonin gene-related peptide effects, 222 criteria, 50 commitment of stem cells, 45-46 colony-forming units-fibroblasts, 44-45, 64 diseases, 63-64 fate, 43 functions, 43
hormones, growth factors, and cytokines in regulation, 56-58 longtitudinal bone growth control, 124-125 markers, 52-54 ontogeny mesenchymal stem cells, 44-50 multipotent adult progenitor cells, 44, 46-47 osteogenesis imperfecta defects, 453-454 parathyroid hormone effects, 154-155 plasticity of precursors, 46-47 developmental control genes, 47-48, 126 postulated transitions in lineage with changes in self-renewal capacity, proliferation, and differentiation, 51-52 R u n x 2 regulation, 47, 54-55, 99, 125-126 transcription factor regulation, 54-56 Osteoblast surface per bone surface, bone histomorphometry, 364 Osteoblastoma imaging, 723 pathology, 724 treatment, 724 Osteocalcin BGLAP gene, 24 bone formmation marker, 342 circadian variation, 344 developmental changes, 344-345 osteoblast marker, 46, 52-53 structure, 25 Osteochondroma imaging, 716 pathology, 716-717 treatment, 717-718 Osteoclast activation-resorption bone formation sequence, 44 activity cycle, 43 amylin effects, 225-226 bone modeling, 339-340 bone resorption defects, 473-477 bone resorption mechanism degradation of bone organic matrix, 44, 60-61 degradation product processing, 61 demineralization, 60 extracellular resorption zone establishment, 60 calcitonin effects, 218-219 calcitonin gene-related peptide effects, 220, 22 diseases, 63-64 markers, 43 morphological features, 59-60 multinucleation and function, 63 origins, 61 regulation of activity and differentiation, 61-63, 128-130 transcription factors in differentiation, 128 Osteoclast surface per bone surface, bone histomorphometry, 365 Osteocyte markers, 53-54, 58
mechanosensors and signal transducers, 58-59 parathyroid hormone effects, 155 Osteofibrous dysplasia differential diagnosis, 727 imaging, 727 pathology, 727 treatment, 729 Osteogenesis imperfecta (OI) anesthesia risks, 452-453 birth complications, 452 bone histomorphometry types I, III, and IV, 369-370 types V and VI, 370-372 type VII, 371 bone metabolism marker studies, 347-348 cardiovascular involvement, 451 clinical manifestations, 443-444 connective tissue alterations, 451-452 dentinogenesis imperfecta, 444, 451 differential diagnosis child abuse, 428-430, 450 idiopathic juvenile osteoporosis, 407, 410-411,450 endocrine changes, 451 gene mutations, 407, 443,445-447 heredity, 407, 445 hypophosphatasia, 450 incidence, 406, 443 laboratory findings, 450 life expectancy, 453 molecular diagnostics, 449 neurological involvement, 450-451 ocular changes, 452 osteoblasts in longtitudinal bone growth control, 124-125 pathophysiology metabolic activity of bone, 454-456 osteoblast defects, 453-454 osteoid formation and mineralization, 453 pediatric osteoporosis, 406-407 renal involvement, 451 respiratory problems, 451 treatment, 407 bisphosphonates, 457-460, 463 gene therapy prospects, 64, 462-463 growth hormone therapy, 460-461 historical perspective, 457 orthopedic management, 461 physical therapy, 461-462 types Bruck syndrome, 449 classification schemes, 444-445 Cole-Carpenter syndrome, 449 osteoporosis pseudoglioma syndrome, 448-449 overview, 406-407 type I, 445-446 type II, 446-447 type III, 447 type IV, 447 type V, 447-448 type VI, 448
Index type VII, 448 Osteoid osteoma imaging, 724 pathology, 725 treatment, 725 Osteoid surface per bone surface (OS/BS), bone histomorphometry, 364 Osteoid thickness, bone histomorphometry, 364 Osteomalacia bone histomorphometry, 367-368 definition, 401 Osteomodulin gene, 19 structure, 20 tissue distribution, 20 Osteonectin functions, 23 knockout mouse phenotype, 24 ligands, 24 S P A R C gene, 23-24 structure, 24 Osteopenia, definition, 401 Osteopetrosis adult forms, 475-476 bone histomorphometry, 372 carbonic anhydrase deficiency, 475 ectodermal dysplasia with osteopetrosis, 477 forms, 474-475 primary spongiosa, 474 progressive form, 476-477 Osteopontin functions, 26 knockout mouse phenotype, 27 ligand-binding sites, 27 osteoblast marker, 52-54 posttranslational modification, 27 S P P 1 gene, 26 structure, 26-27 tissue distribution, 26 Osteoporosis effects on senile osteoporosis lactation, 260-261 pregnancy, 253-254 lactation, 260 pediatric, see Pediatric osteoporosis pregnancy, 253 Osteoporosis pseudoglioma syndrome (OPPG) clinical features, 408,448-449 gene mutations, 408, 448 Osteoprogenitor cell bone nodule assays, 50 criteria, 50 fates, 50-51 glucocorticoid effects, 57 osteoblast development markers, 52-54 osteocyte development markers, 52-54 plasticity, 51 Osteoprotegerin (OPG) fetal mineral metabolism and regulation, 280 osteoclast regulation, 129 parathyroid hormone effects, 153 Osteosarcoma
imaging, 727 pathology, 727 treatment, 729 Oxford method, bone maturity assessment, 329-330
P Parathyroid disorders hypercalcemic disorders calcium-sensing receptor mutations, 500 chronic renal failure and hyperparathyroidism, 500, 682 clinical manifestations, 496 gene defects, 485-486 Jansen's disease, 500-501 parathyroid tumors autosomal dominant hyperparathyroidism syndromes, 499-500 chromosome lp gene mutations, 499 M E N 1 mutations, 498 M E N 2 mutations, 499 multiple hit hypothesis, 497 oncogene activation, 496-497 P R A D 1 mutations, 497-498 R b mutations, 499 treatment, 496 Williams syndrome, 501 hypocalcemic disorders clinical features, 488 gene defects, 485-486 hypoparathyroidism autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy, 489 Blomstrand's disease, 495-496 calcium-sensing receptor mutations, 492 DiGeorge syndrome, 490 familial syndromes, 492 GCM protein defects, 490-491 hypoparathyroidism, deafness, and renal anomalies syndrome, 491-492 mitochondrial disorders, 489-490 parathyroid hormone gene mutatations, 489 pseudohypoparathyroidism, 492-495 treatment, 488 X-linked recessive hypoparathyroidism, 489 Parathyroid gland anatomy, 139-140 calcium-sensing receptor activation, 141 calcium binding, 140-141 function, 140 signal transduction, 142 structure, 140 development, 140 fetal mineral regulation, 289-291 growth regulation
753 calcium, 145 phosphate, 145-146 vitamin D, 145-146 hyperplasia in renal osteodystrophy, 684 knockout effects in fetus, 273,275-276, 289-290 magnesium binding, 142 mutations, 492, 500 tumors, see Parathyroid disorders Parathyroid hormone (PTH) assays, 146-148 biosynthesis, 143 birth changes, 569 bone effects formation, 154-156 resorption, 153-154 CYP24 regulation, 198 evolution, 143 fetal mineral metabolism and regulation, 274-275 fetal thymus production, 290 gene structure and function, 485,487 knockout effects in fetus, 273-274, 291-292 lactation levels, 258-259 mineral ion homeostasis, 148-149 nutritional rickets levels, 551 osteoblast regulation, 56-57 osteoclast regulation, 61 peripheral metabolism, 146 placental calcium transport role, 283 pregnancy hyperparathyroidism, fetal response, 293 hypoparathyroidism, fetal response, 293 levels, 252 processing, 143 regulation of synthesis and secretion calcium, 143-144 phosphate, 144 uremia, 144-145 vitamin D, 144 renal, loL-hydroxylase stimulation, 196 renal actions bicarbonate reabsorption, 159-160 calcium reabsorption, 156-157 magnesium reabsorption, 156-157 phosphate reabsorption, 157-159 vitamin D metabolism, 160 secretion physiology, 140 sequence homology between species, 142 Parathyroid hormone receptor activating mutations, 151 carboxyl-terminal hormone receptor, 152-153 knockout effects in fetus, 274-275, 278-279, 290-291 ligands binding and activation, 150-151 types, 487-488 osteoblast marker, 52-54 osteocyte marker, 54, 58 regulation gene expression, 152 protein, 152 resistance, see Pseudohypoparathyroidism
754 Parathyroid hormone receptor (continued) signaling, 149-151 tissue distribution, 488 types, 149 Parathyroid hormone sensitivity in renal osteodystrophy, 685 Parathyroid hormone, X-linked hypophosphatemia levels, 606 Parathyroid hormone-related protein (PTHrP) bone effects, 156 bone formation role, 223-224 chondrocyte maturation inhibition, 103-104 development role, 142-143, 223-224 discovery, 223 fetal mineral metabolism and regulation, 278-279 gene structure and function, 487-488 Indian hedgehog signaling interactions, 104 knockout effects in fetus, 274-275, 278-279, 292 knockout mouse phenotype, 224 lactation levels, 259 placental calcium transport role, 283-288 pregnancy levels, 252 receptor, 103, 142, 223-224 tissue distribution, 142, 223 P a x genes, sclerotome differentiation role, 90-91 PBM, see Peak bone mass Peak bone mass (PBM) bone biochemical markers in puberty, 237-238 definition, 235 determinants gene polymorphisms, 238-239 heredity, 238, 403 nutrition calcium, 240-241,427 protein, 241-242 physical activity, 239-240 development of bone mass, 235 fracture risk reduction, 235, 242-243 phosphate renal reabsorption in puberty, 236-237 time of attainment, 235-236 variance, 236 Pediatric osteoporosis bisphosphonate therapy, 428 child abuse differentiation from bone fragility conditions, 428-430 definition, 401 diagnosis, 402-403 differential diagnosis, 405 idiopathic juvenile osteoporosis bone histomorphometry, 371-372 osteogenesis imperfecta differential diagnosis, 407 mechanostat in pathogenesis, 403-405 prevention, 426-428 primary osteoporosis Bruck syndrome, 407-408 Ehlers-Danlos syndrome, 408-409 homocystinuria, 409-410 idiopathic juvenile osteoporosis, 410, 412
Index Marfan syndrome, 409 osteogenesis imperfecta, 406-407 osteoporosis pseudoglioma syndrome, 408 overview of causes, 405-406 prospects for study, 430 secondary osteoporosis anorexia nervosa, 417-419 cerebral palsy, 412-414 corticosteroid therapy, 425-426 diabetes mellitus, 424 Duchenne muscular dystrophy, 414-415 growth hormone deficiency, 421-423 hyperprolactinemia, 424 hyperthyroidism, 423-424 iatrogens, 426-427 immobilization and limb disuse, 415-416 leukemia, 416-417 overview of causes, 405, 412 puberty disorders, 419-420 rheumatologic disorders, 417 Turner syndrome, 420-421 Peptidyl prolyl cis-trans isomerase, collagen interactions, 4 Peripheral quantitative computed tomography (pQCT) advantages and limitations, 318 applications, 311-312 challenges to interpreting bone mass measurements in childhood and adolescence, 303-304 dosimetry, 308 precision versus accuracy, 303, 311 principles, 311 reference data, 318-319 Perlecan functions, 21 H S P G 2 gene, 20 structure, 20 tissue distribution, 20-21 Phex disease mutation, see X-linked hypophosphatemia endopeptidase activity, 183-184 function, 620-621 gene structure, 617 homology between mouse and humans, 620 mutation analysis, 183 osteoblast marker, 54, 620 osteocyte marker, 54, 58 positional cloning, 183 substrates, 621 tissue distribution, 183, 620 unifying hypothesis for renal phosphate wasting, 186 Phosphate absorption, 173-174 binding agents, 692 birth changes, 569 breast milk concentration, 261 extrarenal soft tissue handling, 174 fetal mineral metabolism and regulation, 273-274 homeostasis regulation overview, 604 lactation
dietary effects on mineral metabolism and breast milk production, 263 metabolism, 258 parathyroid gland growth regulation, 145-146 parathyroid hormone homeostasis role, 148-149 renal reabsorption effects, 157-159, 177 secretion regulation, 144 placental transport, 288 pregnancy dietary effects on mineral metabolism and skeletogenesis, 255 metabolism, 251 preterm infant supplementation, 578 renal transport, see also Sodium/phosphate cotransporter overall tubular transport, 176 puberty, 236-237 rates, 176 regulation developmental changes, 177-178 dietary intake response, 177 parathyroid hormone, 157-159, 177 sodium/phosphate cotransporter, 177 vitamin D, 178 tubular localization of reabsorption, 176, 177 tissue distribution, 173 transport matrix vesicle function, 175-176 osteoblast function, 174-175 osteoclast function, 174 Phosphate, X-linked hypophosphatemia management, 613-614 Phosphatonin identification as FGF23, 185, 622 tumor-induced osteomalacia secretion, 185 Physical activity, see Exercise P i t x l , limb identity determination, 93 P K A , see Protein kinase A Placental mineral transport calcium active transport initiation, 281 calbindin-D role, 280, 286-287 energetics, 280 fetal regulation assessment, 281-282 calcitonin, 283 parathyroid hormone, 283 parathyroid hormone-related protein, 283-288 vitamin D, 283 maternal regulation, 281 molecular mechanisms, 286-288 requirements, 280 magnesium, 288 phosphate, 288 pQCT, see Peripheral quantitative computed tomography P R A D 1 , parathyroid tumor mutations, 497--498 Pregnancy
Index calcitonin levels, 252 dietary effects on mineral metabolism and skeletogenesis calcium, 254 general nutrition, 255 magnesium, 255 phosphate, 255 vitamin D, 255 zinc, 255 fetal development, s e e Skeletogenesis hyperparathyroidism, fetal response, 293 hypoparathyroidism, fetal response, 293 lactation, s e e Lactation mineral fluxes from mother to offspring, 249-250 mineral metabolism bone turnover, 251-252 calcium, 250-251 magnesium, 251 phosphate, 251 zinc, 251 parathyroid hormone levels, 252 parathyroid hormone-related protein levels, 252 placental mineral transport, s e e Placental mineral transport skeletal changes assessment, 252-253 effects on senile osteoporosis, 253-254 osteoporosis of pregnancy, 253 overview, 292-293 vitamin D levels, 252 PRELP gene, 19 structure, 19 Prenatal bone development, s e e Skeletogenesis Preterm infants alkaline phosphatase levels, 348, 574 body composition and growth, 567 bone mass in neonates, 567-568 definition, 567 metabolic bone disease of prematurity biochemical features, 571 clinical manifestations, 571 history of study, 569-571 prevention, 578-579 radiological features, 572 mineral homeostasis physiological changes at birth, 569 nutrition and bone health, 573, 577-579 skeletal health over time birth to hospital discharge bone mass, 572-575 linear growth, 572 bone turnover, 574-575 discharge to two years of age bone mass, 575-576 bone turnover, 576 fractures, 576-577 linear growth, 575 fractures, 575 two years onwards bone mass, 577-578
bone turnover, 578 fractures, 578 linear growth, 577 skeletogenesis, s e e Skeletogenesis Procollagen C-endoproteinase, types and enhancers, 5 Prolactin hyperprolactinemia and pediatric osteoporosis, 424 Prolyl 4-hydroxylase collagen type I processing, 3 subunits, 3 Protein disulfide isomerase, collagen as substrate, 4, 14 Protein intake and peak bone mass effects, 241-242 Protein kinase A (PKA) chondrogenesis induction signaling, 100 Proximal renal tubular acidosis (pRTA) bone disease, 645 clinical characteristics, 645 diagnosis, 644-645 physiology, 645 treatment, 647 pRTA, s e e Proximal renal tubular acidosis PSACH, s e e Pseudoachondroplasia Pseudoachondroplasia (PSACH), radiography, 390-391 Pseudohypoparathyroidism clinical features, 494 G N A S 1 mutations, 492-495 types, 492 Pseudophosphatasia, features, 665 PTH, s e e Parathyroid hormone PTHrP, s e e Parathyroid hormone-related protein Pu-1 osteoclast differentiation role, 128 Puberty, s e e a l s o Peak bone mass age at menarche, 336 bone biochemical markers, 237-238 bone metabolism markers, 345 disorders and pediatric osteoporosis constitutional delay of puberty, 420 precocious puberty, 420 initial considerations in maturation assessment chronological versus maturational time, 325-327 indicators of maturity, 325-328 sexual dimorphism, 326, 328 size versus maturity, 326, 329 uneveness of maturation, 326 variability in maturation, 325-326, 328 mechanostat model and sexual dimorphism in skeletal development, 419-420 phosphate renal reabsorption, 236-237 secondary sexual events in males, 337 self-assessment of pubertal status, 335-336 Tanner staging breast development, 334 genitalia development, 334 limitations of clinical evaluations, 335 pubic hair development, 334
755 Pycnodystostosis cathepsin K defects, 473-474, 477 radiography, 391-392, 477-478 Pyk2, osteoclast regulation, 130 Pyridinium cross-links bone resorption markers, 343-344 developmental changes, 344-345 Ehlers-Danlos syndrome type VI levels, 351
Q Quantitative computed tomography (QCT) advantages and limitations, 317-318 applications, 308-312 challenges to interpreting bone mass measurements in childhood and adolescence, 303-304 dosimetry, 308, 311 precision versus accuracy, 303, 311, 317 principles, 308 reference data, 318-319 Quantitative ultrasound (QUS) advantages and limitations, 314, 318 applications, 314-315 challenges to interpreting bone mass measurements in childhood and adolescence, 303-304 equipment, 313 precision versus accuracy, 303, 314 principles, 312-313 reference data, 318-319 QUS, s e e Quantitative ultrasound
R Radiogrammetry, s e e X-ray radiogrammetry RANKL fetal mineral metabolism and regulation, 280 osteoclast regulation, 62, 129 parathyroid hormone activation, 153 RARot, s e e Retinoic acid receptor e~ R b , parathyroid tumor mutations, 499 Renal osteodystrophy adynamic renal osteodystrophy, 685 bone histomorphometry, 372 bone metabolism marker studies, 350 bone remodeling disturbances, 679-680 clinical manifestations, 685-686 clinical spectrum, 679-680 Fanconi's syndrome, 638 histologic manifestastations hemodialysis treatment, 687, 689 stable chronic renal failure, 687 laboratory findings, 686-687 long-term consequences, 690-691 management calcimimetic agents, 695 dietary modification, 691-692 phosphate-binding agents, 692 vitamin D therapy, 693-695 pathogenesis
756
Index
Renal osteodystrophy (continued) parathyroid gland hyperplasia, 684 parathyroid hormone sensitivity, 685 secondary hyperparathyroidism, 682 set-point abnormalities in renal failure, 683-684 radiographic features, 689-690 Renal tubular acidosis, see Distal renal tubular acidosis; Retinoic acid receptor oL(RARo0, chondrogenesis inhibition, 101 Rickets, see also Autosomal dominant hypophosphatemic rickets; Calcium deficiency rickets; Hereditary hypophosphatemic rickets with hypercalciuria; Osteomalacia; Vitamin D deficiency rickets; X-linked hypophosphatemia classification, 54 definition, 541-542 epidemiology, 541 1oL-hydroxylasedeficiency, see Vitamin D-dependent rickets type I renal tubular abnormalities in etiology, see Distal renal tubular acidosis; Fanconi's syndrome; Hypomagnesemia; Idiopathic hypercalciuria; Proximal renal tubular acidosis vitamin D receptor mutations, see Vitamin D-dependent rickets type II Roche-Wainer-Thissen technique, bone maturity assessment, 332 R u n x 2 , osteoblast regulation, 47, 54-55, 99, 125-126
S Scleraxis, sclerotome differentiation role, 91 Sclerosing bony dysplasia, see also specific disorders
classification, 473-474 gene defects, 473-474 osteoclastic bone resorption defects osteopetrosis, 474-477 overview, 473-474 pycnodystostosis, 477 prospects for study, 480-481 sclerosteosis, 480 transforming growth factor defects Camurati-Engelmann disease, 479-480 melorheostosis, 478--479 overview, 478 Sclerosteosis, features, 480 Sclerotome, differentiation, 90-91 Shh, see Sonic hedgehog Skeletal dysplasias, see also specific diseases clinical spectrum, 376 clinical work-up biochemical investigations, 393-394 cartilage histology, 395-396 history, 376, 389 imaging, 390-393
molecular diagnostics, 396 overview, 375 physical examination, 376, 389 incidence, 375 international nosology and classification, 375, 377-388 prenatal diagnosis, 396-398 Skeletogenesis, see also Pregnancy appendicular skeleton apical ectodermal ridge formation and maintenance, 92-94, 96 limb bud positioning and induction, 92-93 limb identity determination, 93 limb patterning, 93-96 overview, 92 zone of polarizing activity, 95 axial skeleton overview, 88 patterning genes, 91-92 sclerotome differentiation, 90-91 somite formation, 88-90 bone metabolism markers, 344-345 calcium accretion rate, 249, 271,567-568 definition, 78 endochondral ossification chondrocyte maturation, 103-104 differentiation, 99-101 growth and proliferation, 101-102 overview, 79-81 fetal adaptive goals, 271-272 initiation of bone formation condensations, 97-99 epithelial-mesenchymal interactions, 97 overview, 96 intramembranous ossification, 78-79, 99 mineral metabolism and regulation in fetus calcitonin, 277-278 calcium, 272-273 magnesium, 274 parathyroid hormone, 274-275 parathyroid hormone-related protein, 278-279 phosphate, 273-274 sex steroids, 279-280 vitamin D, 275-277 overview, 77-78 skull cartilaginous neurocranium, 82-83 cartilaginous viscerocranium, 83-84 dermal neurocranium, 83 dermal viscerocranium, 84-85 patterning genes, 86-87 skull cell embryonic origins, 85-86 sutures, 85 synchondroses, 85 subperiosteal ossification, 78 timing and sequence in humans, 81-82 Skull skeletogenesis cartilaginous neurocranium, 82-83 cartilaginous viscerocranium, 83-84 dermal neurocranium, 83
dermal viscerocranium, 84-85 patterning genes, 86-87 skull cell embryonic origins, 85-86 sutures, 85 synchondroses, 85 SLRPs, see Small, leucine-rich, interstitial proteoglycans Small, leucine-rich, interstitial proteoglycans (SLRPs) biglycan, 18-19 chondroadherin, 20 classification, 17 collagen binding, 17 decorin, 18-19 epiphycan, 20 fibromodulin, 19-20 structure, 17 transforming growth factor-J31 binding, 17-18 Sodium/phosphate cotransporter parathyroid hormone effects, 158-159 signaling in regulation, 181 type I, 179 type IIa gene, 179 knockout mouse, 621-622 mechanism, 179 membrane traffic in fast regulation, 180-181 regulation, fast versus slow mechanisms, 180 structure, 179-180 type III, 180 Somite, formation, 88-90 Sonic hedgehog (Shh) limb patterning, 95 sclerotome differentiation role, 90 S O S T , sclerosteosis defects, 480 S o x genes, chondroblast differentiation role, 100 S P A R C gene, 23-24 S P P 1 , 26 c-Src, osteoclast regulation, 129 Subperiosteal ossification, definition, 78 Sutures, skeletogenesis, 85, 120 Synchondroses, skeletogenesis, 85
T Tanner staging, puberty breast development, 334 genitalia development, 334 limitations of clinical evaluations, 335 pubic hair development, 334 Tanner-Whitehouse method, bone maturity assessment, 330-333 Tartrate-resistant acid phosphatase bone resorption markers, 344 developmental changes, 344-345 T b x genes, limb identity determination, 93 Tetracycline, bone labeling, 360-361 TGFq3), see Transforming growth factor-13 TH, see Thyroid hormone
757
Index
Thrombospondins genes, 22 knockout mouse phenotype, 23 ligands, 23 structure, 22-23 subfamilies, 22 thrombospondin-5, s e e Cartilage oligomeric matrix protein tissue distribution, 22 types, 22 Thymus, fetal parathyroid hormone production, 290 Thyroid hormone (TH) bone formation regulation regulation, 124 hyperthyroidism and pediatric osteoporosis, 423-424 TIO, s e e Tumor-induced osteomalacia TIP39 parathyroid hormone homology, 143 receptor, 143 TNAP, s e e Alkaline phosphatase Trabecular number, bone histomorphometry, 363 Trabecular thickness, bone histomorphometry, 363 Transforming growth factor-J3 (TGF-[3) clinical features, 184-185, 610 course, 611 evaluation, 610-611 FGF23 role, 612 limb patterning, 96 osteoblast regulation, 56-57, 126 parathyroid hormone effects, 156 pathology, 610 pathophysiology, 611-612 phosphatonin secretion, 185 prospects for study, 623-624 related syndromes epidermal nevus syndrome, 612 McCune-Albright syndrome, 612-613 sclerosing bony dysplasia defects Camurati-Engelmann disease, 479-480 melorheostosis, 478-479 overview, 478 sclerosteosis, 480 small, leucine-rich, interstitial proteoglycan binding, 17-18 treatment, 185-186, 611 Tumor-induced osteomalacia (TIO), unifying hypothesis for renal phosphate wasting, 186 Turner syndrome growth hormone therapy, 421 pediatric osteoporosis, 420-421
U Ultrasound, s e e a l s o Quantitative ultrasound prenatal diagnosis of skeletal dysplasias, 396-398 Uremia, parathyroid hormone secretion regulation, 144-145
V VDDR-I, s e e Vitamin D-dependent rickets type I VDDR-II, s e e Vitamin D-dependent rickets type II VDR, s e e Vitamin D receptor Versican, function, 16 Vitamin D bone histomorphometry of deficiency, 368 fetal kidney synthesis, 290-291 fetal mineral metabolism and regulation, 275-277 food fortification, 559 hepatic 25-hydroxylation by CYP27, 193-195, 544, 584 hypervitaminosis D, 615-616 knockout mouse studies CYP24, 203 CYP27, 202 CYP27B 1,203-205 megalin, 202-203 vitamin D receptor, 205-207 vitamin D-binding protein, 202 lactation levels, 258-259 metabolism Fanconi's syndrome, 638 overview, 193, 543, 583 preterm infant supplementation, 578 sunlight role, 193, 543-544 24-hydroxylation by CYP24, 196-198, 583-584 metabolites and bioactivity, 207-208 osteoclast regulation, 61, 201 parathyroid gland growth regulation, 145-146 parathyroid hormone metabolism effects, 160 secretion regulation, 144 pediatric osteoporosis prevention, 427 phosphate effects, 178 physiological actions, 201 placental calcium transport role, 283 pregnancy levels, 252 dietary effects on mineral metabolism and skeletogenesis, 255 prospects for study, 208-209 renal, 1oL-hydroxylation by CYP27B 1, 195-196, 583-585 renal osteodystrophy management, 693-695 Vitamin D-binding protein (DBP), knockout mouse phenotype, 202 Vitamin D-dependent rickets type I (VDDR-I) clinical presentation, 585-586 CYP27B 1 mutation types, 589-590 laboratory features, 586 linkage analysis, 587 molecular genetics, 587-589 nomenclature, 585 phenotypic variation, 589 treatment, 592
Vitamin D-dependent rickets type II (VDDR-II) clinical presentation, 593 history of study, 592-593 laboratory features, 593 ligand-binding negative phenotype, 594, 597 ligand-binding positive phenotype, 594 molecular genetics, 594 nomenclature, 592-593 treatment, 597-598 vitamin D receptor mutations, overview, 596-597 Vitamin D deficiency rickets biochemical abnormalities, 551-555 bone histomorphometry, 368 bone metabolism marker studies, 350-351 clinical presentation, 548-550 epidemiology age of onset, 544 breast-fed infants, 544-545 calcium deficiency role, 546-548 developed countries, 546 factors influencing vitamin D production in skin, 544 genetic susceptibility, 546 geographic distribution, 545-546 vitamin D metabolism, 543-544 historical perspective, 542-543 prevention, 558-560 radiological diagnosis, 555-557 treatment, 557-558 vitamin D metabolite levels, 553-554 Vitamin D receptor (VDR) coactivators and corepressors, 199-200, 593 defects in idiopathic hypercalciuria, 642 domains, 198, 593 gene polymorphisms and peak bone mass, 238-239 knockout mouse phenotype, 205-207, 276-277 ligand affinity, 198-199 mutations and rickets, s e e Vitamin D-dependent rickets type II nonclassical effects, 200 phosphorylation, 199 response elements and target genes, 200 retinoid X receptor heterodimerization, 198, 593
W Wall thickness, bone histomorphometry, 364 Williams syndrome, features, 501 Wnt chondrogenesis inhibition, 101 functional groups, 100-101 limb formation induction, 93 limb patterning, 96 osteoblast differentiation role, 126 sclerotome differentiation role, 90 skull patterning, 87
758
Index
X XLH, see X-linked hypophosphatemia X-linked hypophosphatemia (XLH) biochemical findings alkaline phosphatase, 606 hypophosphatemia, 604-605 parathyroid hormone, 606 vitamin D metabolism, 605 bone histomorphometry, 368-369, 372 clinical features dental findings, 607 growth, 606-607 overview, 181-182, 603, 607 skeletal findings, 606 familial versus sporadic cases, 617 gene dosage effect, 61 6-617 incidence, 603 mouse models bone and teeth manifestations, 618-619 Gy, 182-183, 617-618 Hyp, 182-183, 617-620 phosphate wasting, 618 vitamin D metabolism, 619 PHEX
endopeptidase activity, 183-184
genotype-phenotype correlations, 617 mutation analysis, 183, 617 positional clonir/g, 183, 617 tissue distribution, 183, 620 prospects for study, 623-624 related syndromes epidermal nevus syndrome, 612 McCune-Albright syndrome, 612-613 treatment complications of therapy hyperparathyroidism, 615-616 hypervitaminosis D, 615-616 soft-tissue calcification, 615-616 dosage adjustments, 613-614 early childhood, 613 growth hormone, 614-615 initial doses, 613 monitoring, 613 overview, 184 surgery, 615 vitamin D analogs, 615 unifying hypothesis for renal phosphate wasting, 186 X-linked recessive hypoparathyroidism, features, 489
X-ray radiogrammetry advantages and limitations, 316 applications, 305 challenges to interpreting bone mass measurements in childhood and adolescence, 303-304 dosimetry, 308 precision versus accuracy, 303, 305 radiographic absorptiometry, 304-305 reference data, 318-319 tubular bone measurement, 304
Z Zinc lactation breast milk concentration, 261 dietary effects on mineral metabolism and breast milk production, 263 metabolism, 258 pregnancy dietary effects on mineral metabolism and skeletogenesis, 255 metabolism, 251
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ISBN 0-12-286551-0 90038
CHAPTER 15, PLATE 1 magnification 200x.
Tetracycline double label in cancellous bone. Original
CHAPTER 15, PLATE 2 Wall thickness in cancellous bone. The reversal line is difficult to visualize directly with the staining used here, but is indicated by the abrupt change in the orientation of lamellae under polarized light. Wall thickness is the mean distance between this line and the nearest bone surface. Original magnification 200x.
CHAPTER 15, PLATE 3 Osteomalacia. Mineralization disorders are characterized by the accumulation of osteoid (upper panel) and blurring of the tetracycline labels (lower panel). Original magnification 200x.
CHAPTER 15, PLATE 4 Paratrabecular fibrosis due to secondary hyperparathyroidism in a 2-year old girl with a vitamin D receptor defect. Original magnification 200x.
CHAPTER 15, PLATE 5 Accumulation of osteoid and hypomineralized periosteocyctic lesions in X-linked hypophosphatemic rickets. Boy, 15 years. Original magnification 200x.
CHAPTER 15, PLATE 6 Patterns of lamellation in healthy control (upper left; girl, 13 years), OI type I (upper right; boy, 11 years), OI type V (lower left; boy, 6 years) and OI type VI (lower right; boy, 6 years). Original magnification 250x.
CHAPTER 15, PLATE 7 Differences between OI type I (upper panel; boy, 9 years) and IJO (lower panel; boy, 8 years). Bone metabolic activity is high in OI, with many osteoid seams and a very cellular bone marrow. In contrast, there is low bone turnover in IJO. Correspondingly, osteoid seams are scarce and many fat cells are present in the bone marrow. Original magnification 250x.
CHAPTER 15, PLATE 8 Fibrous dysplasia of bone. Simultaneous biopsies from unaffected (upper left) and affected (upper right) bone tissue in a 12-year-old boy. Original magnification 40x. The lower panels show the border between affected and unaffected bone tissue. The difference between non-affected lamellar bone and affected woven bone is clearly visible under polarized light (lower right). Original magnification 100x.
CHAPTER 15, PLATE 9 Osteopetrosis. Cancellous bone from a healthy control (girl, 15 years) and a patient with osteopetrosis (boy, 15 years). The material with light green staining represents calcified cartilage. There is a large number of multicellular osteoclasts.
CHAPTER 16, FIGURE 15 Examples of architectural disturbances at the metaphyses. (Left) Metaphysis of a long bone of a fetus (28 weeks) with thanatophoric dysplasia type 1. The width of the proliferating, columnar chondrocyte zone (between the arrows) is dramatically reduced; column formation is barely recognizable. There is a dense fibroosseous band just proximal to the growth zone that correlates with a cupped appearance of the metaphysis on radiographs. (Right) Metaphysis of a long bone of a fetus (33 weeks) with hypophosphatasia. The defect in alkaline phosphatase activity impairs terminal differentiation of the proliferating chondrocytes to hypertrophic chondrocytes. Therefore, column formation is exuberant (some columns can be followed almost to the bottom of the figure). Magnification, approximately 20x.
CHAPTER 16, FIGURE 16 Examples of different patterns of changes in chondrocytes and cartilage matrix in epiphyseal cartilage of fetuses with achondrogenesis type 1A (left) and type 1B (middle) and fibrochondrogenesis (right). (Left) The cartilage matrix in achondrogenesis type 1A is smooth and homogeneous and thus near normal. The chondrocytes have irregular sizes, and in some vacuolization of the cytoplasm can be recognized. Also, some chondrocytes display eosinophilic inclusions (which would show better after PAS staining). (Middle) The cartilage matrix in achondrogenesis type 1B does not have a smooth ground-glass pattern but shows instead coarse collagen fibers that tend to coalesce around the chondrocytes. Some of the chondrocytes show a limited pericellular (territorial) zone with some preservation of matrix. (Right) Fibrochondrogenesis. With this conventional hematoxyline and eosine staining, the main abnormality visible is the spindle-shaped (fibroblast-like) chondrocytes that tend to be grouped in nests separated by fibrous strands.
CHAPTER 18, FIGURE 6 Dentinogenesis imperfecta (DI). Teeth of affected individuals appear transparent due to abnormal dentin. Enamel is normal. The severity of the DI has no relation to the severity of the skeletal involvement in the case of OI.
CHAPTER 18, FIGURE 10 Microscopic bone changes under bisphosphonate treatment. Cortical width is significantly increased after 2 years of treatment with intravenous pamidronate, as seen in this pair of iliac crest biopsies stained with toluidine blue. The baseline biopsy is shown on the left.
CHAPTER 21, FIGURE 1 The facies fibrodysplastica. Note the prominent frontal bossing and malar prominence, associated with widening and elongation of the midface, and depression of the nasal bridge.
CHAFFER 21, FIGURE 9 Caf6 au lait macules in the skin of a child with MAS. Note that the macules arrest at the midline, which is a common but not an obligatory feature. Also note the breast bud development induced by precocious puberty.
CHAPTER 21, FIGURE 10 (A) Mutations at codon 201 demonstrated by sequencing of the relevant PCR-amplified region of exon 8. CGT--,CAT transition (left, asterisk) results in the R201H mutation; CGT--,TGT transition (right, asterisk) results in the R201C mutation. (B) Selective amplification of the mutated allele by PCR in the presence of PNA oligos blocking the amplification of the normal allele [85]. This method allows the demonstration of low amounts of the mutated genotype (low numbers of mutated cells). (C) Reverse transcriptase-PCR analysis of normal and mutated stromal cell strains. Only the normal genotype is demonstrated in normal cells; both the normal and mutated alleles are expressed in FD samples [13].
CHAPTER 21, FIGURE 12 Sharpey fibers and osteoblast cell shape in fibrous dysplasia. (Top) H&E section demonstrating multiple bundles of collagen (Sharpey fibers) running perpendicular to the trabecular surface into the adjacent fibrous tissue. (Bottom) Undecalcified plastic section of FD bone. The better resolution of plastic sections allows one to discern retracted osteoblasts along the osteoid surface. The processes of these cells outline round features that represent cross sections of Sharpey fibers (arrows).
CHAPTER 21, FIGURE 13 Osteomalacic change in FD bone. (a, b) Samples of the same biopsy of an FD affected iliac crest processed separately for paraffin embedding after decalcification (a) and for undecalcified methyl metacrylate embedding (b). (a) A paraffin section stained with H&E in which the total amount of bone plus osteoid was imaged in fluorescence. (b) A plastic section stained with von Kossa. The paucity of mineralized bone, but not that of total bone matrix, is readily apparent. (c, d) Transmitted and polarized light views of a plastic section of FD demonstrating the huge excess of osteoid and the woven texture of the osteoid.
CHAPTER 21, FIGURE 14 Histological patterns of FD. (a) The parallel arrangement of FD trabeculae commonly seen in jawbones, expressing a local modeling drift. Osteoblasts in these structures are always located on homologous sides, and large numbers of conjoined osteocyte lacunae are seen (hyperosteocytic bone). (b) Note the excavation of FD trabeculae from within (tunneling resorption; arrows). (c) The conventional "Chinese writing," which is mainly the result of extensive tunneling resorption of primary FD bone. (d) Intracortical erosion by FD tissue of the prominent vascularity (arrows), also seen in e. (f) Detail of an irregular FD trabecula extensively excavated from within.
CHAPTER 21, FIGURE 15 Images from a 24-year-old patient with polyostotic FD and hypophysphatemia, low 1,25(OH)2D3, and secondary hyperparathyroidism. Hyperparathyroidism-induced changes include a prominent pattern of tunneling resorption, unusually high numbers of osteoclasts (arrows), and the formation of solid clusters of osteoclasts (bottom). bv, blood vessel.
CHAPTER 21, FIGURE 17 Strains of stromal cells derived from normal bone marrow or from FD were transplanted ectopically into the subcutaneous tissue of immunocompromised mice using hydroxyapatite/tricalcium phosphate particles as a carrier. Eight weeks later, normal bone and hematopoietic marrow with adipocytes formed in transplants of normal cells (a, b), and abnormal bone and fibrous marrow depleted of hematopoiesis and adipocytes formed in transplants of FD cells (c, d). Undecalcified methyl-metacrylate embedding; Goldner's stain, hac, hydroxyapatite carrier.
CHAPTER 25, FIGURE 3 Biopsy of adult XLH osteomalacia. Goldner-stained undecalcified sections of iliac crest bone from an adult with XLH (magnification, x360). Note the excess osteoid accumulation with relatively normal abundance of mineralized bone.
CHAPTER 29, FIGURE 2 2 a
(a) Osa clinical presentation.
